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extremely low birth weight infants (elbw) pose a heavy resource burden in perinatal healthcare. while it is inevitable that elbw infants would have relatively long neonatal intensive care unit (nicu) length of stay (los), there may be opportunities for improving and standardizing care, as unexplained variation in los has been described for premature infants. depending on the patient, hospital, and payer, there may be financial considerations that could influence practice in such a manner to discharge such infants sooner or later. for these reasons, after baseline characteristics that contribute meaningful increments in explanatory power are accounted for, los may be considered a quality of care measurement. in order to use elbw los as a quality measurement amongst hospitals, we need to reveal the impact of practice variation among providers via a model employing inclusion / exclusion criteria and baseline patient characteristics that are broadlyaccepted by relevant stakeholders. previous studies on modeling length of stay for premature infants have been limited due to being confined to one or a few centers or concerned only certain level nicus. a population - based study in sweden evaluated moderately preterm infants 3034 weeks. a study of 30 nicus in the uk included a broad group of premature infants including those 23 to 32 weeks gestation and with birth weight up to 3000 grams. our aim was to characterize los for the smallest, most vulnerable infants who draw especially heavily on nicu resources, across wide levels of hospital care. the purpose of this study was to modellos for elbw infants in order to examine provider variation and thus to inform quality assessmentwithin a population - based dataset. because both widely heterogeneous patient care needs and patient care in more than one discrete system of care can obscure the true contribution of various independent risk factors to los, we studied elbws who survived and were cared for from birth or shortly after birth at a single institution until discharge to home. in particular, we considered : 1) type of risk adjustment model, 2) the potential for bias from excluding patients who died before discharge, and 3) the potential for bias from excluding patients who were transferred to a lower level hospital for convalescent care. we used data from the california perinatal quality care collaborative (cpqcc), a voluntary collective of 129 nicus that report demographic and clinical data into a central repository. more than 90% of nicus in california participated in this collaborative between january 1, 2008, and december 31, 2010. member nicus collect data on their patients in a prospective manner, and each record undergoes a variety of range, logic, and missing data checks. the period of analysis included infants cared for in cpqcc nicus for the three years, 2008 to 2010. inclusion criteria were infants with birth weight 4011,000 grams who were cared for at a cpqcc hospital from birth or within the first day after delivery, until discharge to home. exclusion criteria were patients who died, those with congenital anomalies, and infants who had any surgery except circumcision. the primary outcome ofinterest was length of stay (los), defined as the number of days from admission to discharge. for infants those infants not cared for at a cpqcc hospital on the first day, the first day of hospital stay at a cpqcc hospital counted as day one. the following baseline characteristics were considered in risk adjustment : infant characteristics (sex, birth weight, gestational age, small for gestational age (sga)), maternal and intrapartum variables (maternal age, race / ethnicity, prenatal care, location of birth (inborn / outborn), antenatal steroid use, multiple birth, mode of delivery, antenatal conditions), and delivery room variables (5 minute apgar scores). in considering sga (defined as birth weight = 30 weeks. as birth weight = 30 weeks). we chose 30 weeks as the cut point because the 3rd percentile of 1,000 gram infants is approximately 30 weeks. the rationale was to distinguish small size by infants developmental stage in terms of gestational age. previous research has shown a survival advantage for sga neonates of = 30 weeks. as birth weight = 30 weeks). we chose 30 weeks as the cut point because the 3rd percentile of 1,000 gram infants is approximately 30 weeks. the rationale was to distinguish small size by infants developmental stage in terms of gestational age. previous research has shown a survival advantage for sga neonates of < 31 weeks associated with cesarean delivery. we examined collinearity among the model variables under consideration, as collinearity may influence estimation and inference for the primary parameters and lead to a loss of power, considering variance inflation factors and tolerance (see appendix and table e1 for details on this selection process). from this analysis, we included birth weight and sga, but not gestational age as explanatory variables in models. skewness in the empirical distribution with a long positive side tail suggested that the use of the non - parametric test (kruskal - wallis) would be preferable when comparing los between different levels in each risk factor. as the los of each infant cared for within a hospital may not be independentfrom that of another infant in the same hospital, even after adjusting for the fixed risk factors under study, we accounted for this hospital - level clustering by using generalized linear mixed models with hospital of care accounted for as a random effect. we used a randomly selected 50% of the elbw cohort (n=1,006) for model building, with the remaining 50% serving as a validation set. further details of model selection are noted in the appendix and online table e2. in our review of the literature as described in the appendix, these seven models were tested for goodness of fit and other characteristics. model 6, a generalized linear model with negative binomial distribution and model 2, a linear mixed model using the log scale were considered the two optimal los models. the results of primary analyses and tests for biases (described below) were similar for both models, and model 2 formed the basis for the remainder of the study (see appendix). in model 2 we use a mixed model to account for clustering within nicus and we use the log transformation of los to reconcile the non - normal distribution that results from the very long los of some elbws with modeling assumptions. having selected the linear mixed model using log transformation as the preferred solution to estimating risk adjustedlos, the model was applied to the whole study cohort. we started from an unconditional means model, then added infant - level risk factors. final risk factor selection for the model was determined by backward selection (alpha < 0.05 significance level). the model diagnostics and validation of model assumptions were checked again in the final model. when residuals for the final model fitting were examined, there were two extreme outlier observations (with significantly higher dffits and cook s distance statistics). these two extreme data points were excluded from analysis in order to optimize model fit. as a linear mixed model does not generate an r statistic, we estimated r by using the same covariates in a model without random effects. based on infant - level estimates, risk adjusted los at each hospital was computed in the following fashion : (a) for each infant, estimate an expected los(b) obtaina calibration factor and apply to infant expected los. the calibration factor was obtained by the overall ratio of (sum of actual los / sum of expected los) across the whole study cohort, as described in previous studies in order to center the estimate around 1.0.(c) sum infants actual los and their expected los obtained in the previous step by hospital(d) divide hospitals actual los sum by expected los sum to obtain o / e ratio(e) multiply hospitals o / e ratio by overall average los (a) for each infant, estimate an expected los (b) obtaina calibration factor and apply to infant expected los. the calibration factor was obtained by the overall ratio of (sum of actual los / sum of expected los) across the whole study cohort, as described in previous studies in order to center the estimate around 1.0. (c) sum infants actual los and their expected los obtained in the previous step by hospital (d) divide hospitals actual los sum by expected los sum to obtain o / e ratio (e) multiply hospitals o / e ratio by overall average los in order to examine variation by hospital, we estimated risk - adjusted observed to expected los ratios for each hospital. in order to facilitate root cause analysis of a nicu s infants with excessive los, outliers for los at the individual patient level were identified based on studentized residuals outside of the 2 to + 2 range. as infants who died were excluded from the analysis, we wanted to examine the possibility that hospitals that may have shorter mean los could have higher rates of mortality, thereby having a less acutely ill surviving population of infants that would be biased toward shorter los. to examine this potential bias on average los the mortality rate was computed from an established cpqcc algorithm, a modification of a vermont oxford network model, using all elbw infants excluding delivery room death in the cpqcc dataset between 20082010 (n=7,270). observed to expected mortality ratios were computedand their relationship to hospital los was evaluated using weighted correlation. in this regression model, each hospital was assigned a weight of the number of eligible patients cared for at that hospital. another potential source of bias could be the exclusion of infants who are transferred out to lower levels of care. when elbw infants cared for at level iii centers are transferred out for feeding and growing at lower levels of care, it is possible that the infants remaining in that nicu may present more complex cases who could be biased toward longer los. similar to the analysis of mortality, we considered the relationship of transfer - out rates for feeding and growing with average los by hospital for nicus with weighted correlation. all statistical analyses were conducted using sas version 9.3 (sas, cary, nc). there were 2,012 elbw infants in the eligible cohort cared for in 111 hospitals (figure e1 in online supplementary materials), with an average birth weight of 830 grams (sd 126) and gestational age 27.2 weeks (sd 2.0). the majority of patients were cared for in community level nicus (64%), with 32% of infants cared for in regional nicus, 1% in intermediate nicus, and 4% in non - classified nicus. the median los was 79 days, with a range of 23 to 219 days (table e3 in online supplementary materials). infants who had antenatal steroid exposure and were born by cesarean delivery had shorter mean los. over the 3-year study period, los variation between years was not statistically significant (p=0.11). the results of the risk adjusted los model are shown in table 1, and the distribution of observed and predicted los is shown in figure 1. birth weight, gender, sga, ethnicity, antenatal steroid use, fetal distress, maternal hypertension, and 5 minute apgar score were significant risk factors for longer los. the random hospital intercept was strongly significant, indicating significant variation in los between hospitals. the model without hospital included as a random effect had r of 0.45. in order to assess the possibility of bias for nicus that had higher mortality rates leading to shorter los, we investigated the relationship between risk - adjusted mortality to the mean risk - adjusted los in hospitals (figure 2). the mean mortality rate at the nicu level was 15.3% with interquartile range : 8.7% to 22.4% (risk adjusted mortality rate mean 16.0%, interquartile range 9.9% to 23.5%). the regression fit between los and risk adjusted mortality rate was poor with a slope of 0.00351and r = 0.0207, indicating no relationship between mortality rate and length of stay by nicu. as our study included only infants who were discharged home, another potential bias that we considered was that hospitals that transferred more infants to lower levels of care for feeding and growing may have higher los. the average transfer - out rate for growth and discharge planning was 7.5% overall for elbw infants, with range between 0 to 31%. the mean adjusted los for nicus with transfer out rate < 10% (n=96 nicus) was 80.7 days (interquartile range 79.1 83.0) while the mean adjusted los for nicus with transfer out rate greater than or equal to 10% (n=15 nicus) was 82.5 days (interquartile range 80.4 83.5). figure 3 displays the relationship between transfer - out rate and risk - adjusted los. overall, the impact of transfer - out rate on our risk - adjusted los appeared limited. after risk adjustment, the mean nicu los among 27 nicus with more than 20 eligible patientswas 81.3 days with an interquartile range of 80.781.8 days and a total range of 78.283.7 days. for the 84 centers with 20 eligible infants, the mean adjusted los was 81.8 days with a hospital interquartile range of 78.783.7 days and a total range of 54.0104.5 days. if these smaller nicus were considered a single unit, the observed to expected ratio would be 1.003. at the patient level, there were 91individual outliers identified of which 56 had observed los exceeding predicted. using a large population dataset, we developed a risk adjustment model for nicu los for elbw infants, for estimating individual patient los, and for comparing nicu variation. the methodology employed in this study also forms a basis for further development of los models for other nicu sub - populations. there was wide variance in los by birth weight, by gestational age, and other factors (table e3). these differences should be noted as various stakeholders may consider los to be a benchmark of quality of care, highlighting the need for the development of optimal risk adjustment models. because of the non - normality of los, we tried several forms of transformation as well as different conditional distribution models to fit the data. the log - scale model was chosen as the primary model due to favorable model characteristics. we made certain exclusions in order to get a group of elbw infants that would best represent the typical los of a patient in this nicu subgroup. as deaths can occur soon after birth and all the way up to several weeks or months of age, the noise in this group of patients would make assessment of nicu los difficult. however, by excluding deaths, there was the possibility that nicus that perform poorly in survival could be perceived as having lower los, as their sickest patients would not be counted in the analysis. in a study by cotten examining infants born at or before 28 weeks gestational age, there was noted to be an inverse relationship of los and mortality, with centers having less infants with prolonged los more likely to have higher mortality. in contrast, across 111 nicus that included regional, community, and intermediate nicus, and caring for a few to many elbw patients, we found less concern for this potential bias, as there was no correlation between los and risk - adjusted mortality. in the study by cotten, the cohort included births during 1998 to 2001, and mortality rate was higher overall at 28.5% vs. 15.3% in our cohort. although we have addressed one aspect of the question of how deaths are incorporated into analyses on los, this is a complex issue that will require ongoingstudy to develop optimal strategies for assessment, as well as study in other settings where the death to los relationship may differ. on the other hand, nicus that transfer many of their less acute patients to nicus with lower level of care could falsely have an increased los, as their sickest patients would be over - represented in the analysis. some centers may have a networkof several lower level nicus at outlying hospitals for transferring convalescing infants, whereas other nicus may not have that capacity. within our heterogeneous group of nicus, we did not find a significant relationship between risk - adjusted los and transfer - out rates for feeding and growing, alleviating this concern. however, as our analysis only concerned elbw infants, these potential biases would need to be further tested for other nicu populations. further work in evaluating models that include transferred patients, both transferred in to a higher level of care, as well as those transferred out to lower level of care may increase the potential to assess larger systems of care. there may be several limitations to our analysis in regard to the data available for modeling. there may be some socio - demographic factors that were not accounted for in the dataset, such as family income. although we accounted in part for severity of illness by including variables such as birth weight and apgar score in the model, other measures obtained at time points such as admission to the nicu or in the early hospital course could potentially strengthen the model fit. on the other hand, as antenatal and delivery room management could impact both severity of illness and length of stay, the ultimate purpose of the los assessment may guide inclusion of such variables in modeling. in our random effects model, the hospital intercept was highly significant, indicating that there are opportunities for reducing the variation of elbw los across nicus. when the hospital effect was removed from the model, the explanatory power of the model (r = 0.45) was comparable to that reported in a previous study of nicu all patient diagnosis - related groups, a classification system for inpatient hospital care. as our model incorporated both clinically and statistically significant maternal and neonatal factors for risk adjustment, it may be useful as a basis for assessing and comparing nicu los for an individual center across time, as well as between centers. we have developed a patient - level risk - adjusted model for assessing los for elbw infants, which additionally accounts for clustering of outcomes by nicu (hospital). our study also allows hospitals to assess their risk adjusted los and will give the ability for each nicu to identify infants whose los are extreme and could serve as a starting point for root cause analysis of excessive los. this work forms the basis for developing models for los for other nicu sub - populations. further research will focus on optimal methods to compare hospitals in los for the purpose of quality improvement, public reporting, and optimizing payment structures. this work also forms the foundation for studying the relationship of los to short- and long - term clinical outcomes, and future research on how family resources and competency, nicu processes, and hospital factors such as level of care may influence risk - adjusted los. discharged home with eligibility criteria (inborn or outborn admitted within 24 hours from birth) transfer or discharge home cases where eligibility criteria were not met | objectiveto develop a length of stay (los) model for extremely low birth weight (elbw) infants.study designwe included infants from the california perinatal quality care collaborative with birth weight 4011,000 grams who were discharged to home. exclusion criteria were congenital anomalies, surgery, and death. los was defined as days from admission to discharge. as patients who died or were transferred to lower level of care were excluded, we assessed correlation of hospital mortality rates and transfers torisk adjusted los.resultsthere were 2,012 infants with median los 79 days (range 23219). lower birth weight, lack of antenatal steroids, and lower apgar score were associated with longer los. there was negligiblecorrelation between risk - adjusted los and hospital mortality rates (r = 0.0207) and transfer - out rates (r = 0.121).conclusionparticularly because elbw infants have extended hospital stays, identification of unbiased and informative risk - adjusted los for these infants is an important step in benchmarking best practice and improving efficiency in care. |
numerous common human diseases and phenotypic traits are believed to arise from a combination of genetic and environmental factors. the unravelling of the genetic predisposition to complex traits is a major challenge, and it could lead to better prevention, diagnosis and treatment of disease. recently, advances in genotyping technologies, reduction in genotyping costs and the availability of data regarding genome - wide sequence variation through the international hapmap project and 1000 genomes project have made genome - wide association studies (gwas) possible. gwas have emerged as a powerful tool for identifying genetic variants associated with complex traits. in the past few years, more than 500 loci have been found to be associated with human common diseases and traits (1). gwas have proven to be much more successful than linkage studies, which were underpowered to detect variants of modest effect (2), and candidate gene studies, which are non - systematic and biased due to our limited knowledge of the biological pathways implicated in disease pathogenesis (3). gwas are based on the common disease common variant (cdcv) hypothesis (4), which states that relatively common genetic variants (maf > 5%) of relatively low penetrance are important contributors to the genetic susceptibility to common diseases. well - powered gwas, which capture a substantial majority of common variation in the genome, have been now conducted for many common diseases. however, for the majority of these diseases, common variants explain only a small proportion of heritability (5), due to small individual effect sizes. it has been estimated that only 13% of all identified susceptibility loci have odds ratios (or) above 2, and only 1% have or above 10 (6). for example, if we consider a total estimated sibling recurrence risk ratio (s) of 510 for rheumatoid arthritis (ra) (7), 15 for type 1 diabetes (t1d) (8), 1735 for crohn 's disease (cd) (9) and 3 for type 2 diabetes (t2d) (10), their established susceptibility loci would contribute 3347%, 55.6%, 1012.6% and 11.9% of the total heritability, respectively (table 1). table 1.established susceptibility loci for ra, t1d, cd and t2dchromosomesnppositionregion / generaforsreferencerheumatoid arthritis 1p36rs38907452553624tnfsfr140.681.121.003(16) 1p13rs2476601114377568ptpn220.101.941.068(16) 1p13rs11586238117263138cd2, cd580.241.131.003(16) 1q23rs12746613161467042fcgr2a0.121.131.002(16) 1q31rs10919563198700442ptprc0.871.141.002(16) 2p16rs1303123761136129rel0.371.131.004(16) 2p14rs93473465595586spred20.491.131.004(16) 2q11rs10865035100835734aff30.471.121.003(16) 2q32rs7574865191964633stat40.221.161.004(16) 2q33rs1980422204610396cd280.241.121.002(16) 2q33rs3087243204738919ctla40.561.151.005(16) 3p14rs1331559158556841pxk0.091.291.007(16) 4p15rs87404026108197rbpj0.301.141.004(16) 4q27rs6822844123509421il2, il210.821.111.002(16) 5q11rs685921955438580ankrd55, il6st0.791.281.009(16) 5q21rs26232102596720c5orf130.681.141.004(16) 6p21hla1.800(70,71) 6q21rs548234106568034prdm10.331.101.002(16) 6q23rs10499194138002637tnfaip30.731.101.002(16) 6q23rs6920220138006504tnfaip30.221.221.008(16) 6q23rs5029937138195151tnfaip30.041.401.006(16) 6q25rs394581159482521tagap0.701.101.002(16) 6q27rs3093023167534290ccr60.431.131.004(16) 7q32rs10488631128594183irf50.111.191.003(16) 8p23rs273634011343973blk0.251.121.003(16) 9p13rs281237834710260ccl210.341.101.002(16) 9q33rs3761847123690239traf1, c50.431.131.004(16) 10p15rs21042866099045il2ra0.731.091.001(16) 10p15rs47503166393260prkcq0.811.151.003(16) 11p12rs54038636525293traf60.861.141.002(16) 12q13rs167854257968715kif5a0.621.101.002(16) 20q13rs481048544747947cd400.751.181.005(16) 22q12rs321825337544810il2rb0.261.091.001(16)type 1 diabetes 1p31rs226924164108771pgm10.191.101.001(15) 1p13rs2476601114377568ptpn220.142.051.104(15) 1q31rs2816316192536813rgs10.821.121.002(15) 1q32rs3024505206939904il100.831.191.004(15) 2p25rs1534422126407412p250.461.081.001(15) 2q12rs917997103070568il18rap0.781.201.005(15) 2q24rs1990760163124051ifih10.601.161.005(15) 2q33rs3087243204738919ctla40.551.141.004(15) 3p21rs33346345611ccr50.881.181.003(15) 4p15rs10517086260855114p150.301.091.002(15) 4q27rs17388568123132492il20.261.261.011(15) 5p13rs689793235874575il7r0.731.121.002(15) 6p21mhc3.058(72) 6q15rs1175552790958231bach20.471.131.004(15) 6q22rs9388489126698719c6orf1730.451.171.006(15) 6q23rs6920220137973068tnfaip30.221.091.001(15) 6q25rs1738074159465977tagap0.561.091.002(15) 7p15rs7804356268916657p150.761.141.003(15) 7p12rs494808851027194cobl0.951.301.002(15) 9p24rs70206734291747glis30.501.141.004(15) 10p15rs115946566,62015il2ra0.751.191.005(15) 10p15rs127224956137289il2ra0.891.591.015(15) 10p15rs9474746430456prkcq0.811.101.001(15) 10q23rs1050954090023033c10orf590.721.331.015(15) 11p15rs6892138800ins0.712.301.096(15) 12p13rs47638799910164cd690.371.091.002(15) 12q13rs229223956482180erbb30.341.311.018(15) 12q13rs167853657979190multiple0.721.121.002(15) 12q24rs3184504111884608sh2b30.491.281.015(15) 14q24rs14657886926359914q240.711.161.004(15) 14q32rs49003849849895114q320.291.091.002(15) 15q25rs382593279235446ctsh0.681.161.005(15) 16p13rs1270871611179873clec16a0.651.231.009(15) 16p12rs124442682034257216p120.301.101.002(15) 16p11rs478808428539848il270.581.161.005(15) 16q23rs72028777524724516q230.101.131.001(15) 17p13rs16956936763369217p130.871.091.001(15) 17q12rs229040038066240ormdl30.511.151.005(15) 17q21rs72211093877028617q210.651.051.001(15) 18p11rs189321712809340ptpn20.171.131.002(15) 18q22rs76336167531642cd2260.471.161.006(15) 19q13rs4251054720848119q130.841.161.003(15) 20p13rs2281808161055120p130.641.111.002(15) 21q22rs1120320343836186ubash3a0.431.131.004(15) 22q12rs57530373058172222q120.391.101.002(15) 22q13rs22954137591318c1qtnf60.431.111.003(15) xq28rs2664170153945602xq280.321.161.005(15)crohn 's disease 1p31rs1146580467475114il23r0.932.501.025(14) 1p13rs2476601114179091ptpn220.901.311.005(14) 1q23rs2274910159118670itln10.681.141.004(14) 1q24rs92868791711288571q240.241.191.006(14) 1q32rs115843831992024891q320.701.181.005(14) 2q27rs3828309233845149atg16l10.531.281.015(14) 3p21rs319799949696536mst10.271.201.007(14) 5p13rs461376340428485ptger40.131.321.010(14) 5q31rs21889621317987045q310.431.251.013(14) 5q33rs11747270150239060irgm0.091.331.008(14) 5q33rs10045431158747111il12b0.711.111.002(14) 6p22rs690842520836710cdkal10.781.211.006(14) 6q21rs77460821065419626q210.291.171.005(14) 6q27rs2301436167357978ccr60.461.211.009(14) 7p12rs1456893502402187p120.681.201.007(14) 8q24rs15513981266092338q240.621.081.001(14) 9p24rs107586694971602jak20.351.121.003(14) 9q32rs4263839116606261tnfsf150.681.221.008(14) 10p11rs175824163532765610p110.351.161.005(14) 10q21rs1099527164108492znf3650.391.251.012(14) 10q24rs11190140101281583nkx2 - 30.481.201.008(14) 11q13rs792789475978964c11orf300.391.161.005(14) 12q12rs1117559338888207lrrk2, muc190.021.541.005(14) 13q14rs37641474335592513q140.221.251.010(14) 16q12rs206684749321280nod20.023.991.147(14) 17q21rs287250735294289ormdl30.471.121.003(14) 17q21rs74416637767727stat30.571.181.007(14) 18p11rs254215112769947ptpn20.151.351.014(14) 21q21rs17361351572709121q210.571.181.007(14) 21q22rs76242144439989icoslg0.391.131.004(14)type 2 diabetes 1p13p11rs10923931120319482notch20.111.091.001(73) 1q32rs340874212225879prox10.561.071.001(18) 2p23rs78009427594741gckr0.611.061.001(18) 2p21rs757859743586327thada0.901.151.002(73) 2p16rs24302160438323bcl11a0.461.081.001(74) 2q26rs7578326226728897irs10.641.111.002(74) 3p25rs180128212368125pparg0.851.231.005(73) 3p14rs460710364686944adamts90.761.101.002(73) 3q13q21rs11708067124548468adcy50.771.121.002(18) 3q27rs4402960186994381igf2bp20.311.111.002(73) 4p16rs100101316343816wfs10.591.141.004(73) 5q13rs445705376460705zbed30.261.081.001(74) 6p22rs1094639820769013cdkal10.331.091.002(73) 7p21rs219134915030834dgkb / tmem1950.471.061.001(18) 7p15rs86474528147081jazf10.501.081.001(73) 7p15rs460751744202193gck0.21.071.001(18) 7q32rs972283130117394klf140.551.071.001(74) 8q22rs89685496029687tp53inp10.481.061.001(74) 8q24rs13266634118253964slc30a80.681.121.003(73) 9p21rs1081166122124094cdkn2a / b0.841.171.003(73) 9q21rs1329213681141948chchd90.931.111.001(74) 10p13rs1277979012368016cdc123/camk1d0.181.111.002(73) 10q23rs501548094455539hhex / ide0.591.101.002(73) 11p15rs23344991653428dusp80.411.081.001(32) 11p15rs2313622648047kcnq10.521.081.001(74) 11p15rs22378922796327kcnq10.341.421.031(73) 11p15rs521917366148kcnj110.391.151.005(73) 11q13rs155222472110746centd20.881.141.002(74) 11q21rs1083096392348358mtnr1b0.301.091.001(73) 12q14rs153134364461161hmga20.11.11.001(74) 12q14q21rs796158169949369tspan8/lgr50.271.061.001(73) 12q24rs7957197119945069hnf1a0.851.071.001(74) 15q25rs1163439778219277zfand60.61.061.001(74) 15q26rs804268089322341prc10.221.071.001(74) 16q12rs805013652373776fto0.381.211.009(73) 17cen q21.3rs75721033170628hnf1b (tcf2)0.381.101.002(73) xq28rs5945326152553116dusp90.211.271.011(74)ra, rheumatoid arthritis ; t1d, type 1 diabetes ; cd, crohn 's disease ; t2d, type 2 diabetes ; raf, risk allele frequency in controls ; or, odds ratio.sibling recurrence risk ratio (s) was calculated using the formula : where q is the risk allele frequency, p = 1 q, and is the genotype relative risk under the additive model. established susceptibility loci for ra, t1d, cd and t2d ra, rheumatoid arthritis ; t1d, type 1 diabetes ; cd, crohn 's disease ; t2d, type 2 diabetes ; raf, risk allele frequency in controls ; or, odds ratio. sibling recurrence risk ratio (s) was calculated using the formula : where q is the risk allele frequency, p = 1 q, and is the genotype relative risk under the additive model. explaining this missing heritability ' of complex diseases (1113) is an area of active research, and there are likely to be multiple contributing factors. part of the explanation is likely to be an underestimate of the contribution made by the types of variants targeted by gwas. for instance, it might be that there are large numbers of variants of very small effect, which early gwas were underpowered to detect, yet to be found. this idea is supported by the observation that meta - analyses of published gwas are discovering a substantial number of new susceptibility loci (1425). in addition, for most loci, causal variants and potential independent additional markers within the region have not been identified yet. new ways of analysing the genetic architecture of complex traits using gwas data are suggesting that indeed a large proportion of heritability can be explained by common variants and that larger gwas will yield many more validated loci for complex traits (26,27). of course, gwas only interrogate a portion of the types of variation that could underlie disease risk. analysis of gwas data has been mainly focused on single nucleotide polymorphisms (snps), but there are other types of genetic variation, such as structural variants, that have not been studied in depth. however, recent studies of common (maf > 5%) copy number variants (cnvs) have shown that they seem unlikely to account for a substantial proportion of the missing heritability (28). similarly, the analysis of gene environment and gene gene interactions (epistasis) might improve the fraction of heritability explained by loci documented thus far. several epistatic interactions have been indentified in humans [e.g. between the ret protooncogene and endothelin receptor type b genes in hirschsprung disease (29), the interleukin 4 receptor variants and interleukin 13 promoter variants in asthma (30) and the alpha- and beta - adrenergic receptors in congestive heart failure (31) ], although they have not been replicated. however, this phenomenon has not been thoroughly explored through large - scale analysis of genome - wide snp interactions, first due to the fact that current sample sizes are underpowered to detect modest interaction effects and secondly due to the paucity of sample collections with genetic and detailed environmental exposure data. complex patterns of inheritance, such as parent of origin effects (32), as well as inherited epigenetic modifications of the genome, the presence of phenotype heterogeneity in the cohorts used in the first wave of gwas, or even an initial over - estimation of the heritability of complex traits (33) can also contribute to the missing heritability. while the above - mentioned plausible contributors seem unlikely to play a substantial role in explaining missing heritability, rare variants are increasingly thought to account for a large proportion of it (3436). contrary to the cdcv hypothesis, the multiple rare variant (mrv) hypothesis argues that the summation of the effects of low - frequency polymorphisms, each conferring an intermediate increase in risk (i.e. incompletely penetrant, but greater than those observed for common variants), can explain a significant proportion of the genetic susceptibility to common diseases and traits. some studies analysing rare variants using gwas data have been carried out, but these have proven to be underpowered to detect robust associations. re - sequencing approaches are more suitable for rare variant analysis, and, as these are becoming more cost - effective and new analysis methods are being developed (37,38), they will soon be applied to large - scale studies of rare variants. indeed, several targeted sequencing studies have already proven successful for the identification of associations between rare variants and some human diseases and disease - related phenotypes (3943). the same argument can also be extended to other forms of genetic variation, and it has been recently proposed that rare cnvs may be responsible for some fraction of the missing heritability of complex traits (44,45). it has been recently proposed that gwas signals that have been credited to common variants could instead reflect the effect of mrvs. synthetic association ' signals in gwas, by occurring more often in association with one of the alleles of a common tag snp (fig. 1), which would thus synthetically confer an increased risk for disease this might also mean that the causal variants could be megabases away from the common variants detected in gwas, and that the real effect size could be much stronger than that implied by the common tag snp. if true, the synthetic association hypothesis would suggest that follow - up studies from gwas hits should encompass a much larger region than the linkage disequilibrium region surrounding the detected common variant (6). figure 1.simplified view of genetic variation at the nod2 locus, a well - documented example of a synthetic association. the left - hand side shows a genealogical tree representing six snps in this region after discarding rare recombinant events. the right - hand side shows the resulting haplotypes and their population frequencies (48), with coloured circles representing common gwas snps, and starbursts representing previously identified low - frequency coding variants responsible for association between nod2 and cd. while none of the gwas snps is strongly correlated with any individual causal allele, the three coding variants create a synthetic association because they cluster by chance on the side of the tree marked by the green gwas snp (rs2076756). simplified view of genetic variation at the nod2 locus, a well - documented example of a synthetic association. the left - hand side shows a genealogical tree representing six snps in this region after discarding rare recombinant events. the right - hand side shows the resulting haplotypes and their population frequencies (48), with coloured circles representing common gwas snps, and starbursts representing previously identified low - frequency coding variants responsible for association between nod2 and cd. while none of the gwas snps is strongly correlated with any individual causal allele, the three coding variants create a synthetic association because they cluster by chance on the side of the tree marked by the green gwas snp (rs2076756). there are very few documented examples showing that mrvs may be responsible for a common variant gwas signal (47). it therefore seems sensible to evaluate this hypothesis in the broader context of human disease genetics, including historical study designs, functional annotations of gwas regions and experiments in human populations with diverse ancestry. while sequencing experiments currently underway or in planning will ultimately resolve the role of synthetic association, the balance of evidence available today is already illuminating. one line of evidence that suggests that synthetic associations do not underlie many reported gwas associations is provided by linkage scans that have been conducted in the past. the genetic model that underpins synthetic association (allelic heterogeneity caused by several low - frequency variants with larger effects than commonly seen in gwas) is highly tractable by linkage analysis, which combines information from all causal variants at a particular locus. this relationship is highlighted by the widely replicated linkage between the nod2 gene and cd, which is driven by three independent, low - frequency causal variants (4850) which cause a synthetic association signal in gwas of cd (fig. nod2 is the exception that proves the rule that, despite many attempts, very few replicable linkages to complex diseases have been discovered (51). this dearth of findings is informative when considering the likelihood of synthetic associations because it rules out a class of genetic models from playing a substantial role in complex disease. power calculations comparing a large - scale linkage scan (52) with the largest gwas considered by dickson. (46) show that only a small fraction of the genetic models which can give rise to synthetic associations would not be detected by linkage. furthermore, the scenario where synthetic associations could have escaped linkage comprises models with a small number of causal variants with genotype relative risk < 2.5 (53). while these observations do not entirely rule out synthetic associations, they seriously confine the parameter space in which they might exist. in addition, comparisons of even modest linkage signals with gwas regions have shown only a few overlaps, and even these are largely driven by atypically large effects like the mhc in autoimmunity. in addition, attempts to explicitly use linkage information to boost the power of gwas (54) have not been successful. this contrast between largely overlapping genetic models that linkage and synthetic association are well powered to detect and almost completely non - overlapping results from linkage and gwas strongly suggests that synthetic associations do not underlie many gwas signals. another prediction made by the synthetic association hypothesis is that the most significantly associated common variant identified by gwas might be located several megabases away from the underlying low - frequency functional variants. the empirical properties of linkage disequilibrium between low - frequency and common variants are not fully understood, although the complete 1000 genomes project (http://www.1000genomes.org/) will soon provide information necessary to evaluate this question directly. nevertheless, two indirect pieces of evidence suggest that most gwas hit snps are within a few hundred kilobases (and many within tens of kilobases) of their tagged functional alleles. first, a large number of gwas signals across a variety of traits are nearby to genes previously established to cause mendelian forms of the same trait (55). for example, 8 of 10 proteins involved in the th17-differentiation signalling pathway have been associated with one or more auto - inflammatory diseases (56). as with many aspects relating to the evaluation of the prevalence of synthetic associations, deeper sequence data sets will be needed to fully answer the question of the distance between gwas tag snps and causal variants, but these early patterns imply that the tag snp often resides in the proximity of the relevant functional genomic element. under the synthetic associations model, common variant signals reflecting single or multiple rare alleles are unlikely to be consistent across populations of different ancestry. this is based on the fact that many of these rare variants would have arisen recently and will therefore not be shared across diverged populations. for example, a study from early 2010 clearly demonstrated that common variant signals for t2d are reproducible and have similar effect sizes across east asian populations including chinese, malays and asian - indians in singapore (57). in fact, t2d - associated variants have been found to be associated with disease in diverse populations (ranging from african - americans to chinese) by several studies (5862). similarly, in ra, the stat4 locus, as an example, has shown reproducible association with disease in the usa (63), uk (64), spanish, swedish, dutch (65), korean (66), colombian (67), japanese (68) and greek (69) populations. although synthetic associations explaining common gwas signals for complex polygenic traits are certainly plausible and can occur under specific circumstances (e.g. nod2 in cd), results from studies thus far suggest that these scenarios are actually a rarity. the idea that mrvs at a particular locus may be associated with complex traits of interest has been around for over a decade. we are now starting to accrue a growing body of empirical evidence in support of this hypothesis. the field of complex trait genetics has over the last few months engaged in discussions on the controversial topic of synthetic associations, but it transpires that there is little evidence to support this as a widespread scenario. empowered by advances in sequencing technologies, attention is currently shifting towards the comprehensive study of low - frequency and rare variants. resources such as the 1000 genomes project and emerging large - scale studies like the uk10k project will undoubtedly facilitate the examination of variants at this end of the allele frequency spectrum. in parallel, improved strategies for accurate imputation and powerful analysis of low - frequency and rare variants in aggregate are being further developed and fine - tuned to the needs of these next generation truly genome - wide scans for association. g.o. is funded by the european union (marie curie ief fellowship pief - ga-2009 - 235662). are supported by the wellcome trust (wt088885/z/09/z, wt089120/z/09/z). funding to pay the open access charge | genome - wide association studies (gwas) have successfully identified a large number of genetic variants associated with complex traits, but these only explain a small proportion of the total heritability. it has been recently proposed that rare variants can create synthetic association ' signals in gwas, by occurring more often in association with one of the alleles of a common tag single nucleotide polymorphism. while the ultimate evaluation of this hypothesis will require the completion of large - scale sequencing studies, it is informative to place it in the broader context of what is known about the genetic architecture of complex disease. in this review, we draw from empirical and theoretical data to summarize evidence showing that synthetic associations do not underlie many reported gwas associations. |
tuberculosis (tb) remains a major cause of morbidity and mortality in developing countries affecting millions of people worldwide (1,2). skeletal tb, constituting 1020% of all the extrapulmonary tb (etb), is a well - recognized clinical condition that is easily diagnosed and managed with an excellent outcome (2). however, the occurrence of multifocal skeletal involvement, which is defined as osteoarticular lesions that occur simultaneously at two or more locations in the skeletal system, is exceptional and constitutes < 5% of all skeletal tb cases, even in countries where tb is endemic (36). radiological examinations, including conventional roentgenography, magnetic resonance imaging (mri) and computed tomography (ct) scans, can be used to assist the diagnosis of multifocal skeletal tb (4,5,7). however, the diagnosis and treatment of multifocal skeletal tb are frequently delayed due to the rarity and vague symptoms of the disease, thus allowing progression to severe deformities and functional deficits (3,5,6). in the present study, a case of atypical disseminated multifocal skeletal tb unusual radiological images were observed, which were more compatible with a hematological malignancy or metastatic disease rather than an infectious disease. despite widespread osteoarticular involvement, a 19-year - old male patient was admitted to subei people 's hospital of jiangsu (yangzhou, china) on 5th september 2011 with a 2 month history of recurrent fever and intermittent thoracic back pain. the recurrent fever did not occur at specific times and the temperature did not exceed 38.0c. the patient also reported limited mobility, decreased appetite and weight loss, but did not experience chills or night sweats. the patient presented a normal general physical condition, with a body temperature of 37.3c. a physical examination revealed slight tenderness over the mid - thoracic spine, lumbar spine and left sacroiliac joint. no paraspinal swelling or spasms were observed, while the sensation, muscle strength and muscle tone of the lower extremities were normal. laboratory examinations showed a normal white blood cell count [6.610/l ; normal range (nr), 3.59.510/l ] and normal levels of hemoglobin (120 g / l), human leukocyte antigen - b27 (78 ; nr, 0145) and rheumatoid factor (5.0 iu / ml ; nr, 014 iu / ml). in addition, tests for autoantibodies, immunoglobulin, tb antibodies and tumor - associated antigens were negative. the erythrocyte sedimentation rate (esr) was 75 mm / h, which was higher than the normal range (015 mm / h), and the c - reactive protein level was normal (10 mg / l ; nr, 010 mg / l). human immunodeficiency virus (hiv) and purified protein derivative (ppd) tests were negative. further imaging analysis showed normal abdominal ultrasound findings and x - rays of the chest, thorax, lumbosacral region and pelvis. ct reconstruction of the sacroiliac joint indicated multiple round hypodense lesions with a hyperdense center, as well as some cortical destruction and soft tissue swelling in the lumbar and sacral vertebrae, and the left ilium regions (fig. 1). a contrast - enhanced ct scan of the chest, with omnipaque (1.5 ml / kg ; ge healthcare life sciences, shanghai, china) as the contrast enhancement agent, demonstrated an irregular patchy hypodense signal in the left lung lobe with a patchy translucent shadow inside, which was enhanced heterogeneously (fig. 2). a chest ct scan showed multiple round hypodense lesions with higher central density in the thoracic vertebrae, ribs and sternum (fig. 3). furthermore, a ct reconstruction of the thoracic spine indicated bone destruction with partial cortical perforation (fig. mri scans revealed multiple round ring signal shadows with a continuous finishing border from th12 to s4 in the anterior, central and posterior elements, but not in the end plate. in addition, sagittal t1-weighted and t2-weighted images revealed a hypodense signal in the internal of the lesions and a slightly hypodense signal in the peripheral region, which was accompanied by soft tissue swelling, with visible ring enhancement and soft tissue enhancement following intravenous administration of gadolinium (ge healthcare life sciences) (fig. 5). based on the symptoms and all the aforementioned examinations, lymphoma, metastatic disease or bone tb since the ct and mri findings did not allow for a final diagnosis, a ct - guided biopsy from the lesions in the th7 vertebral, left iliac and adjacent left lung using an 8-gauge needle (yangzhou city jiangzhou medical instrument co., ltd., yangzhou, china) was performed to confirm the diagnosis. however, no evidence of granulomata, necrosis, lymphoma or other malignancies was observed. subsequently, an open biopsy was performed in order to establish a pathological diagnosis, during which fish - shaped soft tissue adhesion with surrounding tissues under the lamina of th7 and local sequestrum formation were observed. part of the surrounding soft tissues and bone lesions were removed for pathological examination and culturing. upon hematoxylin and eosin staining of the tissue and visualization under the olympus bx43 microscope (olympus corporation, tokyo, japan), a large amount of necrosis, epithelioid cells, langerhans giant cells and dead bone 6), which are characteristics of tb (2,5). the results of acid - fast bacilli culturing (sinobest biotech co., ltd., shanghai, china) were negative ; however, a polymerase chain reaction performed by shanghai genechem co., ltd. postoperatively, the patient was administered a common quartet anti - tb chemotherapy regimen (2,5), including isoniazid (300 mg / day), rifampicin (600 mg / day), pyrazinamide (750 mg / day) and ethambutol (750 mg / day ; all shanghai sine pharmaceutical co., ltd., the patient 's symptoms greatly improved after 1 month of anti - tb treatment and the esr decreased to 65 mm / h. at the final follow - up on 11th march 2013, the patient was free of symptoms and the esr had decreased to the normal value. mri scans obtained following 20 months of anti - tb therapy showed that the range of the extensive abnormal signal intensity had become smaller, as compared with that before anti - tb treatment was initiated (fig. informed consent was obtained from the patient 's family prior to the publication of the present case. tb remains a major cause of morbidity and mortality worldwide, and is responsible for ~1.6 million deaths per year (1,4). according to the world health organization, ~8.8 million new cases of tb occur every year worldwide (5,8). multifocal skeletal tb has rarely been reported in non - immunocompromised patients and in patients with normal pulmonary findings, particularly in china (5,9). the spine is the most commonly affected site, of which the upper lumbar and lower thoracic regions are the most frequently involved, comprising 50% of all multifocal skeletal tb cases (36). the average number of vertebrae that are affected by multifocal tb, as seen radiologically, is 3.0 in children and 2.53.8 in adults (10). in the present case, there were multiple round hypodense lesions in the ribs, sternum, multi - segment thoracic, multi - segment lumbar and sacral vertebrae, and ilium regions, accompanied by some adjacent soft tissue swelling. a positive ppd test is a fundamental finding in tb patients ; however, a negative test is not sufficient to exclude the disease. although radiological findings do not firmly establish the diagnosis in the case of a negative ppd result, imaging remains the most useful diagnostic tool in such cases (5,6,10). the characteristic findings of spinal tb include destruction of two adjacent vertebral bodies, destruction of the intervening disc, and the occurrence of paravertebral and epidural abscesses (7,11). skeletal tb does not present any specific features on radiological images in the early stages of the disease, thus mimicking metastatic tumors or certain primary osseous lesions, such as eosinophilic granuloma, particularly if multiple destructive lesions are present (5,7,12). plain radiography is of little help unless the collapse of the disc margin or vertebra body appears (47). in addition, ct scans are unable to show the early alterations in the vertebral marrow and to differentiate an infectious disease from neoplastic involvement (47). by contrast, mri has been shown to be a more sensitive and specific imaging modality that may guide the diagnosis at early stages of skeletal tb. the typical mri patterns are abnormal signal intensities appearing hypointense or isointense on a t1-weighted image of the affected vertebral bodies and discs and hyperintense on a t2-weighted image of the osseous, and soft - tissue changes with heterogeneous enhancement of the vertebral body (47,11). overall, the sensitivity and specificity of mri for tb may be 100 and 88.2%, respectively (5,7). in the present case, the mri features included multiple round hypodense lesions with finishing borders in the thoracic, lumbar and sacral vertebrae, which was not sufficient evidence for the diagnosis of tb ; on the contrary, these observations were suggestive of hematological malignancies, such as lymphoma or multiple myeloma, or metastatic diseases. the common characteristics of end - plate disruption and high signal intensity of the intervertebral disc were not demonstrated. therefore, the presentation of the tb case reported in the current study is considered unusual. to the best of our knowledge, the present study is the first to report an extremely rare case of multifocal osteoarticular tb with multiple round hypodense lesions and a surprisingly negative ppd test result. the etb and disseminated forms of the disease are usually more frequent in certain patients, such as those with a much older age, malnutrition and hemodialysis, as well as immunocompromised and in particular hiv - infected individuals (5,6). however, the current patient was a healthy 19-year - old male with no signs of systemic involvement and a negative hiv status. this demonstrated the diagnostic difficulties of the atypical forms of tb. in patients with skeletal tb, the onset of symptoms is generally insidious, without general alarming signs, such as fever, night sweats, toxicity or extreme weakness. although ct and mri are reliable techniques that help establish a diagnosis, biopsy or ct - guided needle aspiration remain essential for accurate diagnosis and adequate treatment. the advantage of needle aspiration is that it is a less invasive technique ; however, only a small number of specimens can be obtained, particularly when the lesions are sclerotic, and thus the diagnosis rate is only 81.0% in children (13) and 91.3% in adults (14). hao (15) concluded that the diagnostic accuracy of ct - guided thoracic spinal biopsy using a 16-gauge needle was 90.5% overall, as the diagnostic accuracy was significantly lower for the middle thoracic spine (90.0%) compared with that for the lower spine (97.6%), as well as lower for sclerotic lesions (81.3%) compared with that for lytic lesions (96.4%). similarly, in the present study, the ct - guided aspiration using an 8-gauge needle was not able to confirm the diagnosis, possibly due to the limited amount of tissue acquired and the histological nature of the lesion itself, resulting in the use of open biopsy to establish the diagnosis. multidrug anti - tb therapy including isoniazid, rifampin, ethambutol and pyrazinamide advocated for an approximate duration of 1218 months usually leads to complete resolution of tb (2,5). medical treatment is highly effective when administered appropriately, as performed in the present study. the mean time to diagnosis is reported to be 1619 months after the presentation of symptoms (11). ringshausen (12) described the fatal case of a patient with spinal tb, who was mistakenly irradiated for suspected metastatic lung cancer of the spine. the authors claimed that the most common cause in delayed diagnosis is failure to consider this disease (12). thus, tb should be considered in all patients with multiple round hypodense lytic lesions of the skeletal system, especially in countries endemic for tb, in order to avoid delays that may result in irreversible damage and in a high mortality rate. in conclusion, the diagnosis of multifocal skeletal tb is difficult for various reasons, requiring careful consideration and numerous examinations. a high index of suspicion for the possibility of tb appearance has to be maintained in any patient with multiple round hypodense lytic lesions of the skeletal system in countries endemic for tb. in addition, mri is particularly valuable in the diagnostic and follow - up procedures. | tuberculosis (tb) of the musculoskeletal system is a rare clinical condition. multifocal bone involvement is extremely rare and difficult to recognize. thus, due to the diverse and atypical clinical manifestations of multifocal skeletal tb, the disease is easy to misdiagnose. in the present study, a rare case of atypical disseminated multifocal skeletal tb was reported, which exhibited uncommon findings in radiological images that were more suggestive of a hematological malignancy or metastatic disease. in conclusion, the diagnosis of this condition by conventional diagnostic methods is challenging. the importance of ct - guided needle biopsy and open biopsy in the diagnosis of skeletal tb was emphasized. |
laparoscopic liver surgery is now being performed by select groups worldwide. the louisville consensus conference on laparoscopic liver surgery suggested a role for laparoscopic liver resections for lesions in segment 2 to 6. we herein report a male patient undergoing laparoscopic liver resection for a giant right lobe liver hemangioma. a 45-year - old male patient with no known medical risk factors presented to the outpatient department with complaints of right upper quadrant pain restricting his regular activity. a multidetector computerized tomography (mdct) of the abdomen showed a giant hemangioma (18 cm in greatest diameter) arising from segments 5 and 6 of the liver [figure 1 ]. the feeding vessel to the hemangioma was from the anterior branch of right hepatic artery (rha). the right anterior portal pedicle (rapp) was seen abutting the hemangioma supero - medially. (a) computerized tomography showing the giant hemangioma arising from segment 5 and 6 of the liver, (a reconstructed image showing extent of hemangioma.) (b) arterial phase showing the main feeding artery from the anterior branch of the right hepatic artery, (c) portal venous phase showing relation of the anterior portal pedicle to the hemangioma an open entry was achieved with a 10-mm port at the umbilicus for laparoscopic vision and four additional ports were placed [figure 2 ]. at laparoscopy, a giant hemangioma of 18 cm 12 cm was seen to arise from segments 5 and 6 of the liver, reaching up to the iliac fossa on the right side with displacement of the gall bladder medially to midline, in line with the falciform ligament [figure 3a ]. the gall bladder fundal retraction was achieved using a grasper through a port in the right midclavicular port. c, camera port (10 mm) ; rhw, right hand working port (12 mm) ; left hand working port (5 mm) ; lr, liver retractor port (10 mm) ; gbf, gall bladder fundal retraction port (5 mm) (a) laparoscopic view of the giant hemangioma, (b) laparoscopic image showing the bulldog clamp across the anterior branch of the right hepatic artery, (c) line of demarcation on the liver after clamping the anterior branch of the right hepatic artery, (d) laparoscopic view of the enucleation plane the calot 's triangle was dissected and the cystic artery was clipped and divided. the right anterior branch was selectively dissected, looped and occluded with a bulldog clamp [figure 3b ]. the line of demarcation of the anterior segment became clearly evident and was associated with shrinkage of the hemangioma by one - third of its size [figure 3c ]. resection was initially performed in the plane of enucleation medially [figure 3d ]. during the course of this dissection, cranially, a transverse line of transection was marked at the summit level of the hemangioma. the transverse transection plane thus chosen was based on pre - operative planning from the reconstruction from mdct evaluation and intraoperative ultrasound (ious). the hepatic venous tributary running from segment 6 crossed this line as confirmed by ious. the 5-mm port in the epigastrium was exchanged for a 12-mm port to facilitate the use of the 4 habib laparoscopic probe. california, usa), 60 watts setting, was used during liver parenchymal transection with the laparoscopic 4 habib probe by choosing a 2 cm depth of application of rf prongs along the line of transection with parenchymal division performed with straight scissors [figure 4a ]. to prevent injury to the retroperitoneal structures, the posterior 1 cm depth of the parenchymal division was achieved by two firings of an endo gia stapler with 60 mm, white reloads (autosuture) [figure 4b ]. (a) laparoscopic 4 habib probe transection in progress, (b) final transaction surface, (c) morselled specimen an indigenously prepared endobag (urobag) cut to appropriate size with a prolene 2 - 0 suture placed as a pursestring along its open end was then passed into the abdomen through the 12 port. the bag was placed in the right upper abdomen and two 2 - 0 prolene, interrupted sutures were placed on the anterior leaf of the open end of the bag and sutured to the anterior wall of the abdomen. one grasping foreceps held the posterior leaf of the open end of the bag. this suturing technique facilitated in bagging the large specimen comfortably. the cystic duct was clipped, the gallbladder was dissected off from the liver bed, placed in the same endobag and the pursestring suture at the mouth of the bag was tightened. under laparoscopic guidance, the pursestring suture was held and the mouth of the bag was delivered through the umbilical port site. re - laparoscopy was performed, hemostasis was ensured, a subhepatic 24 f tube drain was placed and ports were withdrawn. the patient was started orally the same evening and discharged from hospital on the third post - operative day. an open entry was achieved with a 10-mm port at the umbilicus for laparoscopic vision and four additional ports were placed [figure 2 ]. at laparoscopy, a giant hemangioma of 18 cm 12 cm was seen to arise from segments 5 and 6 of the liver, reaching up to the iliac fossa on the right side with displacement of the gall bladder medially to midline, in line with the falciform ligament [figure 3a ]. the gall bladder fundal retraction was achieved using a grasper through a port in the right midclavicular port. c, camera port (10 mm) ; rhw, right hand working port (12 mm) ; left hand working port (5 mm) ; lr, liver retractor port (10 mm) ; gbf, gall bladder fundal retraction port (5 mm) (a) laparoscopic view of the giant hemangioma, (b) laparoscopic image showing the bulldog clamp across the anterior branch of the right hepatic artery, (c) line of demarcation on the liver after clamping the anterior branch of the right hepatic artery, (d) laparoscopic view of the enucleation plane the right anterior branch was selectively dissected, looped and occluded with a bulldog clamp [figure 3b ]. the line of demarcation of the anterior segment became clearly evident and was associated with shrinkage of the hemangioma by one - third of its size [figure 3c ]. resection was initially performed in the plane of enucleation medially [figure 3d ]. during the course of this dissection, cranially, a transverse line of transection was marked at the summit level of the hemangioma. the transverse transection plane thus chosen was based on pre - operative planning from the reconstruction from mdct evaluation and intraoperative ultrasound (ious). the hepatic venous tributary running from segment 6 crossed this line as confirmed by ious. the 5-mm port in the epigastrium was exchanged for a 12-mm port to facilitate the use of the 4 habib laparoscopic probe. california, usa), 60 watts setting, was used during liver parenchymal transection with the laparoscopic 4 habib probe by choosing a 2 cm depth of application of rf prongs along the line of transection with parenchymal division performed with straight scissors [figure 4a ]. to prevent injury to the retroperitoneal structures, the posterior 1 cm depth of the parenchymal division was achieved by two firings of an endo gia stapler with 60 mm, white reloads (autosuture) [figure 4b ]. (a) laparoscopic 4 habib probe transection in progress, (b) final transaction surface, (c) morselled specimen an indigenously prepared endobag (urobag) cut to appropriate size with a prolene 2 - 0 suture placed as a pursestring along its open end was then passed into the abdomen through the 12 port. the bag was placed in the right upper abdomen and two 2 - 0 prolene, interrupted sutures were placed on the anterior leaf of the open end of the bag and sutured to the anterior wall of the abdomen. one grasping foreceps held the posterior leaf of the open end of the bag. this suturing technique facilitated in bagging the large specimen comfortably. the cystic duct was clipped, the gallbladder was dissected off from the liver bed, placed in the same endobag and the pursestring suture at the mouth of the bag was tightened. under laparoscopic guidance, the pursestring suture was held and the mouth of the bag was delivered through the umbilical port site. the umbilical port was extended to 3 cm. re - laparoscopy was performed, hemostasis was ensured, a subhepatic 24 f tube drain was placed and ports were withdrawn. the patient was started orally the same evening and discharged from hospital on the third post - operative day. the role of laparoscopic liver resection for liver tumors is unclear at present.[25 ] the louisville consensus statement suggests laparoscopic liver resection as an option for lesions in the left lateral and inferior segments of the right lobe. the concerns with regard to laparoscopic liver resections are many. thirdly, there is a lack of tactile feedback, which is critical in evaluating the margin of resection, particularly in malignant tumorus. fourthly, the ideal technique for parenchymal transection during laparoscopic liver resection is not yet standardized. lastly, retrieval of a large specimen may require a large incision, which defeats the primary objective of keeping the procedure minimally invasive. lesions reaching the hilar structures, in particular, pose technical problems with the laparoscopic approach. we elected to perform the resection laparoscopically in our patient because of a suitable location of the tumour, namely segment 5 and 6, with a large exophytic component. also, a good triphasic mdct with reconstruction provided excellent anatomical delineation, facilitating appropriate planning of vascular control and line of parenchymal transection. enucleation has been reported for smaller hemangiomas. in giant hemangioms such as in this report, the cross - sectional area for enucleation is likely to be large and visualization of the entire enucleation plane and achieving blood - less dissection could pose problems. although control of the feeding vessel with subsequent shrinkage of the tumour could facilitate enucleation, choosing, a transverse line of transection cranially in our patient kept the transection surface to the minimum. a blood - less transection was achieved medially in the enucleation plane and cranio - laterally with the laparoscopic 4 habib probe in our patient. others have reported on the use of a laparoscopic habib probe for blood - less liver parenchymal transection. our technique of bagging the specimen is very suitable for solid organs, particularly large specimens. we have used the same technique for laparoscopic retrieval of other solid organs such as spleen or distal pancreas before. in our present report, because there was no concern of studying margins during histopathological examination, the specimen could be morselled and retrieved. in conclusion, laparoscopic resection is feasible in giant liver hemangiomas located in the inferior segments of the right lobe of the liver. laparoscopic 4 habib probe is an important tool in the armamentarium of liver transection methods. | experience with laparoscopic liver resections is limited. laparoscopic resection of a variety of liver lesions has been reported and is considered appropriate for lesions in the left lateral segment and inferior segments of the right lobe. herein, we report a 52-year - old male patient who underwent a laparoscopic resection of giant liver hemangioma with the use of a laparoscopic 4 habib probe. |
spinal cord injury (sci) is the most devastating form of neurological injury, disturbing the normal sensory, motor, and/or autonomic functions. the annual incidence of sci in the usa is about 40 cases per million of population. sci is caused principally by trauma (e.g. traffic injuries, falls, and violence). the risk of death is highest in the first year after sci and varies by level and severity of injury. cardiovascular disease is one of the principal causes of death.3, 4 in the usa, diseases of the circulatory system are the most common causes of death. in norway, the main causes of death following the experience of a sci are pneumonia / influenza, ischemic heart disease, and urogenital disease. in patients with acute cervical scis (rather than thoracolumbar scis), cardiovascular deficits, including severe bradycardia, asystole, and loss of peripheral vascular tone, are known complications.5, 6 acute autonomic imbalances developing after disruption of the sympathetic pathway (located in the cervical cord) are thought to trigger such cardiovascular events.5, 6, 7 although autonomic dysfunction developing after sci can have fatal consequences, including cardiac arrest, many clinicians do not recognize such risks, and the conditions are often misdiagnosed. here a 26-year - old male pedestrian was admitted to hospital via the emergency room after a traffic accident. he was mentally alert and complained of weakness and numbness of both the upper and lower extremities. on physical and neurological examination, no external injuries were evident except for an abrasion on the left arm. the baseline electrocardiogram (ecg) revealed sinus bradycardia (57 beats / min, fig. 1). initial chest and abdominal computed tomography (ct) did not reveal any abnormality or trauma - related injury. a skull fracture, with a small subdural hemorrhage at the right frontal convexity was evident on brain and facial ct. spinal ct revealed acute fractures at the c67 bilateral articular facets and the right - side lamina of c6 (fig. 2b). on the first day of admission, the patient underwent microscopy - assisted corpectomy and discectomy with anterior interbody fusion. postoperatively, the patient remained in the icu for monitoring. on the fifth postoperative day (pod), a pneumonic infiltration with pleural effusion was evident on a follow - up chest x - ray (fig. 3). bronchoscopy was performed, and a large volume of purulent secretion was evident in the dependent portion. the patient was transferred to the division of pulmonology for further respiratory treatment in an icu. antibiotics were commenced, ventilator support put in place, and a tracheostomy performed. during the icu stay, intermittent bradycardia (3055 beats / min) was evident, without specific symptoms ; his vital signs were stable. intravenous atropine (0.25 mg) was given twice to treat severe bradycardia ; the heart rate was recovered in a few seconds. echocardiography revealed no specific abnormality, and the left ventricle exhibited normal systolic function (ejection fraction 60%). on the 22nd pod, chest ct was scheduled for differential diagnosis of a pulmonary thromboembolism. during a positional change prior to imaging in the ct suite, the heart rate fell to 23 beats / min, and blood pressure was not palpable at the carotid artery. after immediate cardiac massage over 1 min, the patient was resuscitated and exhibited stable vital signs. holter monitoring was performed on the day after cardiac arrest (fig. 4). however, 26 pauses were evident, the longest of which was 17.9 s. a temporary cardiac pacemaker was initially placed because of the patient 's poor general condition and the risk of infection. on the 31st pod, the patient was transferred to the department of rehabilitation after ventilator weaning on the 90th pod and was discharged 2 months later. cardiovascular complications, such as bradycardia, arterial hypotension, and loss of peripheral vascular tone, are common complications of acute sci.3, 4 such cardiovascular phenomena are usually observed in patients with upper thoracic or cervical scis. bradycardia is the most common form of dysrhythmia developing after sci, especially in the acute phase of injury. several studies have shown that almost all patients with complete cervical scis will develop bradycardia, and 16% will suffer cardiac arrest.5, 9 cardiac dysfunction usually commences within 35 days after injury, and it typically resolves in 68 weeks.8, 10 although bradycardia developing after acute sci is usually temporary, there is a real risk of cardiac arrest, and adequate management is required. treatment of bradycardia in sci patients generally involves administration of oxygen, atropine, inotropes, and aminophylline. temporary and permanent pacemakers have been placed in patients who are refractory to general treatment and experience life - threatening events such as cardiac arrest.10, 11 we recommend that patients with acute scis should be admitted to icus or similar high - care settings in order to facilitate acute cardiopulmonary management ; this is especially important if a patient has a high - level sci or exhibits hemodynamic instability. several studies have shown that icu monitoring and aggressive medical management improve the outcomes of patients with acute scis.12, 13 additionally, such patients can develop multiple (cardiovascular, respiratory, and urinary) complications, trophic skin changes, and heterotopic ossification. hence, a multidisciplinary icu approach is needed to minimize various problems. however, most clinicians do not recognize the risk of severe bradycardia that can trigger cardiac arrest and often misdiagnose the conditions that then would develop. in our case, the patient was transferred from the department of orthopedics to the division of pulmonology because he developed respiratory complications and thus required further icu care after surgery. the patient usually self - recovered and was stable, and showed no specific symptoms. on the 22nd pod, a ct to explore the possibility of pulmonary thromboembolism was planned because the hypoxia was persistent and the d - dimer level was elevated. however, the patient went into cardiac arrest during a positional change in the ct suite located outside the icu. consequently, a cardiac pacemaker was inserted after asystole was confirmed by 24-h holter monitoring. disruption of the supraspinal sympathetic pathways, inducing parasympathetic dominance, by a cervical sci, is the major cause of cardiovascular instability.15, 16 the preganglionic sympathetic nerve fibers exit the spinal cord at the first - to - fourth thoracic vertebrae (t1t4) and are controlled by higher centers via messages transmitted through the cervical spinal cord. however, the parasympathetic system is controlled by the vagus nerve, which originates in the medulla. thus, the extent of sympathetic dysfunction is related to the location and severity of the sci. any increase in vagal reflexes, as might be induced by tracheal suctioning, a hypoxic episode, or a change in posture, can precipitate a bradycardia event. other studies have shown that the sympathetic hypoactivity gradually improves within 46 weeks after injury.5, 16 the proportion of sci patients requiring a pacemaker ranges from 9% to 17%.5, 10, 18 percutaneous cardiac pacing is only a temporary tool employed in emergency situations. such a pacemaker is associated with risks of injury, skeletal muscle contraction, and pacing failure, and it suffices only until permanent transvenous cardiac pacing can be established. cardiac pacemaker implantation is indicated for patients with high - level scis and continuing symptomatic bradycardia and they do not respond to medical management or suffer recurrent cardiac arrests. sanghvi described a patient with a traumatic c7d1 dislocation who experienced continuous severe bradycardia and cardiac arrest for 2 months after injury. gilgoff reported a patient with a c2 sci causing complete quadriplegia ; the patient experienced symptomatic bradycardia for 21 months after injury despite intensive medical management. clinical improvement was evident after insertion of a cardiac pacemaker. in conclusion, patients who suffer scis are at risk of severe bradycardia and cardiac arrest attributable to dysfunction of the autonomic nervous system. the risks are highest during the acute phase of injury and in patients with upper thoracic or cervical scis. monitoring in an icu, careful assessment of autonomic dysfunction, and aggressive medical management, in addition, procedures that may increase vagal reflexes should be very carefully performed after prior preparation for a possible emergency situation that may develop very rapidly. | bradycardia is the most common form of dysrhythmia developing after disruption of the sympathetic pathway by a spinal cord injury (sci), and it can have fatal consequences, including cardiac arrest. here, we report a case of cardiac arrest developing after cervical sci attributable to sympathetic hypoactivity. a 26-year - old male pedestrian was admitted after a traffic accident. radiologically, fractures were apparent at the c67 bilateral articular facets, and cord contusion with hemorrhage was evident at c47. during his stay in icu, intermittent bradycardia was noted, but the symptoms were not specific. on the 22nd postoperative day, the patient was taken to the computed tomography suite for further evaluation and experienced cardiac arrest during a positional change. after immediate cardiac massage, the patient was resuscitated. we scheduled holter monitoring, which detected 26 pauses, the longest of which was 17.9 s. the patient underwent cardiac pacemaker insertion. no further cardiac events were noted. |
the royal society academia - industry international conference 2014 focussed on the topic of gpcr structure, function, drug discovery and crystallography and was held on september 12 in chicheley hall, uk. this conference brought together 20 renowned experts in gpcr research and drug discovery spanning europe, australia and north america. approximately half of the attendees were from academia and half from industry (see fig. 1attendees of the conference (listed from left to right) : front row isabel moraes, fiona marshall, gebhard schertler and patrick sexton ; second row shweta singh, irina tikhonova, tom ceska, roland seifert, simon hodgson, daniel fourmy and alexander heifetz ; back row ian storer, mike bodkin, vadim cherezov, christofer tautermann, christopher tate, vsevolod gurevich and peter hunt. chicheley hall itself can be seen in the background (the photograph was taken by richard law) attendees of the conference (listed from left to right) : front row isabel moraes, fiona marshall, gebhard schertler and patrick sexton ; second row shweta singh, irina tikhonova, tom ceska, roland seifert, simon hodgson, daniel fourmy and alexander heifetz ; back row ian storer, mike bodkin, vadim cherezov, christofer tautermann, christopher tate, vsevolod gurevich and peter hunt. chicheley hall itself can be seen in the background (the photograph was taken by richard law) gpcrs are a large family of integral membrane proteins that have enormous physiological and biomedical importance. since gpcrs are involved in mediating cell signalling processes, they are implicated in many diseases and are the targets of numerous therapeutic drugs. this is borne out by the fact that 60 % of all prescription drugs today target gpcrs (schneberg. 2004), developed for just 50 established gpcr targets out of the 800 known members of the gene family. the importance of gpcrs was recently highlighted with the nobel prize for chemistry 2012 being awarded to two eminent gpcr researchers, prof. there remains an ongoing need to better understand the interplay between structure and function of these receptors to advance our scientific knowledge and capacity to more effectively harness therapeutic capabilities. as a result however, there is still a sizeable gap between ongoing academic research and the needs of the pharmaceutical industry. a major reason for this is that academic and industrial scientists have too few productive opportunities to meet and interact, particularly to establish cross - discipline collaborations. the intimate atmosphere of such a small conference provided a unique opportunity to stimulate the generation of new networks and partnerships between academia and industry, and to promote current gpcr research and its applications to drug discovery. the invitation of scientists representing structural biology, protein engineering, pharmacology as well as computational and medicinal chemistry provided an interdisciplinary core to enable fruitful discussion and debate. schertler pointed out that during 18 years at the mrc lab in cambridge, he had a continuous string of collaborations with industry partners from small and large pharma that created a very valuable network of complementary expertise. this collaborative network led to breakthroughs in the expression and purification of difficult membrane protein targets, and later this network was an important ingredient in the formation of the drug discovery spin out company heptares therapeutics. schertler s earlier industry partners became the drivers of this gpcr - oriented company with their expertise on target selection, business models and intellectual property (ip) complemented perfectly the expertise of the mrc academic partners. the spin out company has in the meantime grown from three post docs to about 80 people, and it is able to tackle the most difficult gpcr targets with resources that would otherwise not have been accessible in europe from any funding agency. the basis of good industry academia collaboration is a clear agreement about the goals of the collaboration. very often selecting a pre - competitive goal allows the academic partner to fully publish the results and gives the industry partner a significant edge in accessing emerging technologies. the example of heptares therapeutics illustrates that a spin out company can generate resources that would neither be available inside a company or from public funding agencies. if the spin out is able to reach milestones and refinance, then it can become a powerful tool to drive the application of new technology and lead to a change in the research culture in industry. individuals have to commit to a longer - term perspective, which is aimed at changing the scientific landscape. this scientific environment is essential for large companies that exist and are established to be able to recruit an excellent workforce and for small companies to pick up competitive innovation. in multinational companies, the academic and technology environment can start to dictate the location of departments inside a large organisation. from this follows that for a stable business development, the academic environment is as essential to the company as the in - house research activities. one of the most significant advances in previous years has been the structural advances, both in terms of stabilising protein conformational states to make them more amenable to x - ray crystallography, but also in new technology such as x - ray free electron lasers which have the potential to accelerate structural biology not just for gpcrs but for many integral membrane protein drug targets. a significant portion of the meeting discussed these advances and also how they had been used in recent drug - discovery programs within both industry and academia. another focus in recent times has been the concept of ligand bias. it has become clear that individual gpcrs can exist in multiple receptor conformations and can elicit numerous functional responses, both g protein- and non - g protein - mediated. this has led to the discovery that different ligands can stabilise distinct subsets of receptor conformations that can traffic stimulus to diverse functional outputs with varying prominence, a concept referred to as biased signalling (also known as functional selectivity, stimulus bias or ligand - directed signalling). in principle, biased signalling can result in the development of more efficacious and safer drugs, but there are some unresolved questions regarding the best system in which to assess these aspects. the structural information alongside the realisation of biased signalling has also been explored with in silico modelling, and it was demonstrated at the meeting that this can give very useful insight into underlying properties of signalling control. the final section of the meeting focussed on how to best resolve problems in the future, including modelling processes at a higher level. in the following sections, we expand on these discussions in more detail. it is apparent from numerous studies that the stability of the gpcr - ligand complex in detergent solution is an important parameter that will dictate the success of any crystallisation trials (tate 2012). although high thermostability alone does not guarantee the formation of diffraction - quality crystals, if the gpcr - ligand complex is too unstable, then crystals may not form or, if they do, they may diffract only poorly. the majority of gpcr structures have been determined from crystals of the receptor bound to a high - affinity antagonist, which usually binds with kd or ki values in the range of 10 pm to 10 nm. however, if a ligand binds to a receptor only with low affinity and/or the receptor is unstable in detergent, then it may still be possible to obtain a structure if the receptor is thermostabilised. a method developed to thermostabilise gpcrs uses systematic scanning mutagenesis coupled to a thermostability assay performed on the detergent - solubilised mutant receptors to identify specific thermostabilising mutations (tate 2012). each mutation usually imparts 13 c improvement in thermostability to the receptor, although the most highly stabilising mutation found improved thermostability of the agonist - bound conformation of the adenosine a2a receptor by 14 c. once the single thermostabilising mutations have been identified, then the best thermostabilising mutations can be combined to make an optimally stable receptor (shibata. the methodology has been applied to many different gpcrs, in either an antagonist - bound conformation or an agonist - bound conformation, which have been subsequently crystallised and their structures determined. 2014) was a 2.1- resolution structure of the 1-adrenergic receptor (1ar) bound to cyanopindolol (see fig. 2) and the crystals grown in lipidic cubic phase, although without requiring fusions of the receptor to either t4-lysozyme (t4l) or apocytochrome b562ril (bril). the structure showed the presence of an intramembrane sodium ion that was in the identical position to the intramembrane sodium ion in the adenosine a2a receptor that is known to act as an allosteric antagonist. in contrast, the na ion in 1ar does not appear to affect the transition between the inactive and active states of the receptor.fig. 2crystal structures of 1ar bound to novel chemotypes developed by fragment screening and hit optimisation. structures of the ligand binding pocket are depicted with only portions of selected transmembrane helices (h3, h5, h6 and h7) shown with the side chains (green) and ligands (yellow) depicted in sticks : a cyanopindolol, pdb code 2vt4 ; b compound 19, pdb code 3zpq ; c compound 20, pdb code 3zpr figure adapted from the publication (christopher. 2013) crystal structures of 1ar bound to novel chemotypes developed by fragment screening and hit optimisation. structures of the ligand binding pocket are depicted with only portions of selected transmembrane helices (h3, h5, h6 and h7) shown with the side chains (green) and ligands (yellow) depicted in sticks : a cyanopindolol, pdb code 2vt4 ; b compound 19, pdb code 3zpq ; c compound 20, pdb code 3zpr figure adapted from the publication (christopher. 2013) a significant advantage of the thermostabilised receptors is their use in drug discovery, which for the first time opens up the opportunities for structure - based drug design (congreve. the thermostabilised receptors are readily purified and crystallised on a routine basis, which facilitates co - crystallisation with fragments and lead compounds. this was recently demonstrated for the 1ar where a fragment screen performed by surface plasmon resonance (spr), followed by minimal hit optimisation, produced nm - affinity antagonists with novel scaffolds that were readily co - crystallised with the receptor (christopher. the thermostabilisation of gpcrs is a central platform in heptares therapeutics, resulting in numerous crystal structures. another structural approach gaining momentum is the application of x - ray free - electron lasers (xfel) to gpcrs. structural studies of gpcrs, and other biomedically relevant membrane proteins and complexes, are hampered by challenges related to growing sufficiently large crystals capable of withstanding radiation damage and yielding high - resolution data at synchrotron sources. the recent introduction of a new generation of x - ray sources, x - ray free electron lasers (xfels), producing ultra - bright pulses of coherent x - rays with an ultrashort duration, holds the promise to advance our understanding of structure and function of these challenging targets. a novel approach using a membrane mimetic gel - like matrix known as lipidic cubic phase (lcp) for growth and delivery of membrane protein microcrystals for data collection by serial femtosecond crystallography (sfx) at xfels (liu. microcrystals are delivered to the intersection point with an xfel beam in random orientations using a specially designed lcp injector (weierstall. the injector allows adjusting lcp flowrate in a wide range to match the xfel pulse repetition rate and, thus, minimises crystal consumption. lcp - sfx uses highly intense sub-50-fs xfel pulses to overcome radiation damage and collect room temperature high - resolution data from sub-10-m crystals. protein consumption is reduced by two to three orders of magnitude compared to previously used liquid injectors, making the lcp - sfx method attractive for structural studies of challenging membrane and soluble proteins, and their complexes (see fig. 3schematic summary illustrating serial femtosecond crystallography of gpcrs with using lipidic cubic phase for microcrystal growth and delivery schematic summary illustrating serial femtosecond crystallography of gpcrs with using lipidic cubic phase for microcrystal growth and delivery results demonstrating the great utility of this approach were highlighted at the meeting. they included the structure of the human delta - opioid receptor bound to a bi - functional peptide ligand (fenalti. 2014), the structure of a major gpcr signalling complex (unpublished data) and the first novel gpcr structure solved entirely by the lcp - sfx approach (unpublished data). in the future, this method could lead to the development of an efficient gpcr structure - based drug design pipeline by removing the major obstacles, such as the difficulties in preparation of large amounts of homogeneous and stable protein and growing sufficiently large crystals for a large number of different protein - ligand complexes. another advantage of xfels is the ability to freeze protein motion in time and obtain structures of unstable intermediates, advancing our knowledge about the signal transduction mechanism in gpcrs. it was also noted that there are many signalling complexes in the cell that have component parts that have some flexibility as part of the structure necessary for function. quite often crystallography can stumble because only very small crystals are attainable, with very weak diffraction, and this can be due, in part, to some partial disorder in a biologically relevant part of the molecule. the crystallographic solution up to now has been to delete or modify the flexible regions of the protein in order to create constructs that are more ordered so that crystals of significant size could be grown. with diffraction from microcrystals now possible, the structures of these more challenging biological assemblies are within reach. in terms of function, the human 2-adrenergic receptor (2ar) is probably the best - studied gpcr at the molecular, cellular and physiological level (seifert 2013). the 2ar was of critical importance for the development of current models of receptor activation including biased signalling. recent research has also shown for this system that ligand bias can depend on the system studied (native versus recombinant) (seifert 2013) and also on the (patho)physiological state (healthy versus diseased), a feature sometimes also referred to as dynamic bias (michel. accordingly, the analysis of ligands at receptors such as the 2ar has become much more complex, requiring multidimensional approaches. the 2ar constitutes an important drug target ; agonists for this receptor are used for treatment of bronchial asthma and chronic - obstructive lung disease most strikingly, the use of 2ar agonists alone in asthma is associated with increased mortality. receptor desensitisation and activation of deleterious non - canonical signalling pathways, i.e. pathways different from the canonical gs pathway, could contribute to this situation. moreover, specific 2ar polymorphisms may be associated with decreased responsiveness to certain ligands, and the use of racemic 2ar ligands may be problematic. specifically, the distomeric (not therapeutically active) ligands may contribute to drug toxicity (seifert and dove 2009). based on these concerns, non - canonical signalling pathways, receptor polymorphisms and pure 2ar stereoisomers were examined in a pluridimensional signal transduction matrix. this matrix included studies with recombinant 2ar and native 2ar expressed in human neutrophils (see fig. 4). human neutrophils constitute a relevant cell type for inflammation in bronchial asthma that can be readily isolated in substantial numbers. in neutrophils, the 2ar exerts anti - inflammatory effects by inhibiting chemoattractant - stimulated superoxide radical formation. steroisomers of fenoterol were used as model ligands because these ligands have already been shown to exhibit functional selectivity (seifert and dove 2009).fig. the concept of functional selectivity requires that a given gpcr is analysed in multiple different assays. gpcr - ga fusion proteins ensure a defined 1:1 stoichiometry of receptor and g - protein and allow analysis of gdp / gtp turnover, measured in the gtpase activity assay, and effector system activation, measured in the adenylyl cyclase (ac) assay, with high sensitivity. the measurement of -arrestin recruitment is accomplished in hek cells stably transfected with fusion proteins of the 2ar linked to a luciferase fragment and -arrestin linked to the complementary luciferase fragment. upon binding of an agonist, human neutrophilic granulocytes constitute a physiologically relevant model system for the 2ar. in these cells, the 2ar couples to ac (isoform 9), resulting in an increase in the second messenger camp. the 2ar inhibits formyl peptide receptor (fpr)-mediated activation of the neutrophilic nadph oxidase (nox) that generates reactive oxygen species (ros). it is generally assumed that this inhibition is mediated via camp, but an increasing number of studies indicate that the inhibition is actually camp independent. a major goal of current pharmacological research is the development of individualised pharmacotherapy that takes into account individual genetic polymorphisms. at the level of gpcrs, therefore, as a model receptor, the 2ar was analysed because for this gpcr several polymorphisms are already known, but assignment of specific polymorphisms to defined disease entities is controversial. after obtaining consent from volunteers and completing a questionnaire, a small sample of blood (48 ml) was drawn from healthy male and female subjects. a fraction of the blood was used to sequence the 2ar gene to identify known (and unknown) 2ar polymorphisms. the remainder of the blood was used to isolate human neutrophils and assess the pharmacological profile of the 2ar with several standard ligands according to the signalling paradigm shown in (a). additionally, the impact of sex, age, smoking and allergy history on 2ar pharmacology is evaluated. the study fills a gap in the field because it provides data on the pharmacological properties of gpcr polymorphisms in a physiologically relevant context. the results of the study will be submitted for publication to a peer - reviewed journal in spring 2015 a multidimensional analysis of the 2ar in native and recombinant systems. the concept of functional selectivity requires that a given gpcr is analysed in multiple different assays. gpcr - ga fusion proteins ensure a defined 1:1 stoichiometry of receptor and g - protein and allow analysis of gdp / gtp turnover, measured in the gtpase activity assay, and effector system activation, measured in the adenylyl cyclase (ac) assay, with high sensitivity. the measurement of -arrestin recruitment is accomplished in hek cells stably transfected with fusion proteins of the 2ar linked to a luciferase fragment and -arrestin linked to the complementary luciferase fragment. upon binding of an agonist, human neutrophilic granulocytes constitute a physiologically relevant model system for the 2ar. in these cells, the 2ar couples to ac (isoform 9), resulting in an increase in the second messenger camp. the 2ar inhibits formyl peptide receptor (fpr)-mediated activation of the neutrophilic nadph oxidase (nox) that generates reactive oxygen species (ros). it is generally assumed that this inhibition is mediated via camp, but an increasing number of studies indicate that the inhibition is actually camp independent. a major goal of current pharmacological research is the development of individualised pharmacotherapy that takes into account individual genetic polymorphisms. at the level of gpcrs therefore, as a model receptor, the 2ar was analysed because for this gpcr several polymorphisms are already known, but assignment of specific polymorphisms to defined disease entities is controversial. after obtaining consent from volunteers and completing a questionnaire, a small sample of blood (48 ml) a fraction of the blood was used to sequence the 2ar gene to identify known (and unknown) 2ar polymorphisms. the remainder of the blood was used to isolate human neutrophils and assess the pharmacological profile of the 2ar with several standard ligands according to the signalling paradigm shown in (a). additionally, the impact of sex, age, smoking and allergy history on 2ar pharmacology is evaluated. the study fills a gap in the field because it provides data on the pharmacological properties of gpcr polymorphisms in a physiologically relevant context. the results of the study will be submitted for publication to a peer - reviewed journal in spring 2015 in aggregate, these studies revealed that most reported ligands are biased towards canonical gs signalling (reinartz. this is particularly evident for ligands with large n - alkyl substituents, suggesting that these ligand domains, through constrained mobility of transmembrane helices, impede with coupling to gi proteins and -arrestin. in principle however, the seifert group did not identify any ligand with bias towards gi or -arrestin. with regard to polymorphisms, the pharmacological profile of the 2ar with respect to inhibition of superoxide radical formation was assessed. overall, substantial variability in the pharmacological profile of the 2ar in neutrophils was noted, but no association of the pharmacological 2ar profile with a specific polymorphism emerged. thus, at the time being, there is no evidence for the notion that 2ar polymorphisms can be used to optimise asthma therapy. to summarise this aspect, our increase in knowledge of the 2ar has resulted in a situation that renders future research more complicated. most importantly, it is not anymore sufficient to determine a single parameter for a receptor such as gs - mediated adenylyl cyclase activation. rather, multiple parameters have to be determined including non - canonical gi- and -arrestin signalling. it is important to analyse the 2ar not only in recombinant but also in native systems. it will also be very informative to resolve crystal structures of the 2ar in complex with various ligands and coupling partners to understand the molecular basis of functional selectivity (seifert and dove 2009). the glp-1 receptor represents a good model system for studying class b receptor function (koole. glp-1 is a key incretin peptide that promotes insulin secretion in response to nutrient ingestion, but also has a range of other actions including preservation of b - cell mass, reduction in gastric emptying and reduction in appetite that make it a desirable target for treatment of type ii diabetes. class b secretin - like receptors, like many other gpcrs, are pleiotropically coupled to a spectrum of both g - protein - dependent and -independent signalling pathways, and while camp production is the best characterised signalling endpoint for these receptors, physiological and therapeutic responses are the product of the integrated signalling response from all activated pathways. moreover, the different contacts that are made between distinct ligands and their respective receptor can engender unique receptor conformations that give rise to distinct signalling profiles. this behaviour can be observed through differences in activation of second messengers, but also through changes to how receptors are desensitised and down - regulated. biased signalling is further complicated when allosteric drugs are considered, as conformational preferences of the receptor when allosteric and orthosteric (endogenous ligand) sites are co - occupied may be different than when either site is individually occupied. biased signalling is particularly relevant to receptor systems that have multiple endogenous ligands, and where exogenous mimetics are used clinically, as is often seen with class b receptors. clear (ligand - directed) bias for both peptides and small molecule agonists of the glp-1 was demonstrated (koole. 2013a, b), and this is consistent with earlier studies of pituitary adenylyl cyclase - activating polypeptide type 1 receptor (pacapr) receptors and unclassified class b members like hctrs, suggesting that this is likely to be a common feature of agonist activation of class b receptors. transmembrane helical packing and conformational transition involved in receptor activation are assumed to involve key hydrogen bond networks formed around polar residues in the transmembrane helices. the importance of conserved polar residues in class b receptors at the glp-1 receptor has been recently studied (koole. this work has revealed networks of interaction that differentially contribute to global receptor activation and biased signalling. in particular, there appear to be two key interaction networks, one at the base of the receptor that may serve a similar role to the d[e]ry motif in class a receptors to maintain an inactive state ; the second is located in the mid - region of the helical core and plays a critical role in pathway specific signalling, in a ligand - dependent manner. mutation of residues within the central network has identified differences in how ligands propagate activation transition for individual signalling pathways and that distinct ligands utilise only subsets of the network for signal propagation providing initial insight into molecular mechanisms for biased signalling. the concept of biased signalling has also been explored in the cholecystokinin receptor-2 (cck2r, which also binds the digestive hormone gastrin) (magnan. 2011, 2013) and is a gpcr for which pharmaceutical companies and academic laboratories have successfully developed non - peptide ligands, mostly antagonists. since cck2r is a potential target in several pathologies of the central nervous system (anxiety, panic attacks), of the gut (peptic ulcer disease) and of neuroendocrine cancers, the effects of a series of such ligands on stimulation of phospholipase - c and as well as on recruitment of non - visual arrestins and stimulation of receptor internalisation have been studied (see fig. 5schematic representation of the cck2r which can adopt two distinct conformational states upon cck activation. the cck2r state couples to phospholipase - c activation and the cck2r state recruits -arrestins. this figure also shows that gv150013x, a competitive antagonist on cck2r, is inefficient to inhibit recruitment of -arrestins by cck2r because of steric hindrance at the orthosteric binding site schematic representation of the cck2r which can adopt two distinct conformational states upon cck activation. the cck2r state couples to phospholipase - c activation and the cck2r state recruits -arrestins. this figure also shows that gv150013x, a competitive antagonist on cck2r, is inefficient to inhibit recruitment of -arrestins by cck2r because of steric hindrance at the orthosteric binding site several ligands stimulating phospholipase - c with efficacies reaching up to 50 % of that of cck but which were inefficient to stimulate -arrestin1/2 recruitment and receptor internalisation have been identified (magnan. these ligands, which were initially considered as full antagonists at the cck2r, are therefore more accurately described as antagonists on the -arrestin - dependent signalling pathway of this receptor, but are partial agonists of the g - protein - dependent signalling pathway. extending these studies to ligands that are antagonists of g - protein - dependent pathways, it was discovered that the antagonist termed gv150013x (n-(+)-[1-(adamant-1-ylmethyl)-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1h-1,5-benzodiazepin-3-yl]-n-phenylurea) (see fig. 5) could not inhibit -arrestin1/2 recruitment and cck - induced cck2r internalisation (magnan. schild plot analysis of antagonist activity of gv150013x on cck - induced phospholipase - c activation indicated that this molecule competitively inhibited the cck effect. this information led the team to dock gv150013x in the orthosteric binding site of the modelled cck2r, which had been previously validated by site - directed mutagenesis. the docking study predicted that the absence of effect of gv150013x on cck2r recruiting -arrestins was due to the presence of a bulky adamantane moiety in the ligand. gv150013x competitively inhibited cck - induced g - protein - dependent pathway whereas it was inefficient on the -arrestin - dependent pathway. these data suggested that the cck2r could adopt two distinct conformations upon cck activation and these two conformations are distinguishable at the binding site level. fourmy s lab therefore performed a site - directed mutagenesis study of the cck2r orthosteric binding site with a focus on amino acids presumably in contact with the adamantane moiety of gv150013x. in parallel, the team synthesised an analogue of gv150013x in which the adamantane moiety was substituted by a methyl group : 1-(2,3,4,5-tetrahydro-1-methyl-2,4-dioxo-5-phenyl-1h - benzo[b]diazepin-3-yl)-3-phenylurea, termed gv - ch3. pharmacological studies with cck2r mutants and with gv - ch3 consistently demonstrated that the prediction of modelling and docking study was correct : the absence of effect of gv150013x on recruitment of -arrestins to the cck2r was due to a steric hindrance within the binding site which impedes binding of the antagonist (magnan. 2013). overall, this area of research shows how pharmacological analysis of gpcr signalling combined with molecular modelling of gpcrs and chemistry of ligands can be used to analyse the origin of biased signalling. such a strategy together with forthcoming determination of gpcr structures in complex with various signalling proteins (g proteins, -arrestins) opens the possibility of rational drug design of biased ligands. one of the biased - agonism pathways involves arrestins, and they themselves present an alternative line of investigation. arrestins specifically bind active phosphorylated gpcrs, precluding further g protein activation and channelling the signalling to g - protein - independent pathways (gurevich and gurevich 2006). based on the elucidation of an arrestin structure and key functional elements, special arrestins to channel cell signalling in a desired direction were constructed (gurevich and gurevich 2012). enhanced phosphorylation - independent arrestin mutants were designed by disrupting key stabilising intra - molecular interactions that hold arrestins in a basal conformation (gurevich and gurevich 2012). enhanced arrestin-1 was shown to compensate for the defects of rhodopsin phosphorylation in vivo, prolonging the survival of mutant rod photoreceptors, improving their light sensitivity and speeding up photoresponse recovery (song. 2009). while the compensation with the first - generation enhanced mutant was only partial, new more powerful phosphorylation - independent forms of arrestin-1 rhodopsin - specific arrestin-1 is the prevalent form, so it is clear that one needs to target this subtype to compensate for disease - causing defects of rhodopsin phosphorylation. however, activating mutations in many gpcrs underlie various human disorders (schneberg. since the two non - visual arrestins are fairly promiscuous, interacting with hundreds of gpcr subtypes, and most cells express 525 different gpcrs, only one of which is a mutant, to use a compensational approach, one needs receptor - specific non - visual arrestins. to this end, the elements of non - visual arrestins that determine their receptor preference were identified (vishnivetskiy. 2011), and on the backbone of the most promiscuous non - visual subtype, arrestin-3, mutants with high (> 50-fold) receptor specificity were created (gimenez. this finding showed that targeting individual receptors with engineered non - visual arrestins is feasible. arrestins interact with numerous partners, organising multi - protein complexes and recruiting them to particular sub - cellular compartments (gurevich and gurevich 2014a, b). this creates the potential of constructing signalling - biased arrestins that activate or inhibit certain pathways without affecting others. recently, gurevich and colleagues designed an arrestin-3 mutant that acts as a silent scaffold : it binds all kinases in the c - jun n - terminal kinase (jnk) activation cascade, but does not promote its phosphorylation (breitman. this mutant was shown to act in a dominant - negative fashion, suppressing jnk activation in the cell (breitman. multi - functionality of arrestins suggests that parts acting on particular pathways can be separated and used to modify cell signalling. indeed, a small element of arrestin-3 that acts as a mini - scaffold, promoting jnk activation in vitro and in cells, has been identified (zhan. since arrestins play a role in numerous signalling pathways, targeted mutagenesis can yield arrestin - based molecular tools to tell the cell what to do in a language it can not disobey (gurevich and gurevich 2014a, b). despite many examples of successful marketed drugs that modulate the function of gpcrs, there remain a large number of potential therapeutically relevant gpcrs that are regarded as difficult to drug effectively. methods used to analyse the gpcr s ligand binding sites with a view to designing ligands were reviewed and included a discussion on the importance of water molecules for mediating interactions between ligands and receptor. heptares use their star technology to generate thermostabilised gpcrs which can be used for biophysical studies, fragment screening and determination of x - ray structures. examples were presented from different gpcrs showing that the most potent ligands act to displace high energy or an overview of class b and class c structures recently solved at heptares (see fig. 6) was also presented. the corticotropin releasing factor receptor 1 (crf1) x - ray structure identified a novel allosteric binding pocket deep within the transmembrane domain and illustrated why finding ligands for the orthosteric pocket has been challenging. the structure of class c metabotropic glutamate 5 receptor (mglu5) was shown and provided an explanation for the tight structure - activity relationships (sars) and pharmacology mode switching which have been observed for this receptor. the conclusions were that structure - based design could in theory now be applied more broadly across the gpcrome.fig. 2014) complex with cp-376395 from the class b receptor crf1 and overlays of a selection of ligands from class a receptor structures present in the pdb. the observed ligand binding positions demonstrate the spectrum of binding modes across gpcr classes ranging from extracellular orthosteric to deeper intracellular allosteric sites crystal structure of the class c mglu5-mavoglurant receptor (bennett. 2014) complex with cp-376395 from the class b receptor crf1 and overlays of a selection of ligands from class a receptor structures present in the pdb. the observed ligand binding positions demonstrate the spectrum of binding modes across gpcr classes ranging from extracellular orthosteric to deeper intracellular allosteric sites antihistamines are one of the most well - studied drugs (simons and simons 2011). indeed, it is now more than a century since the discovery of histamine as an important biogenic substance and more than 50 years since the production of the first antihistamine drugs (e.g. chlorpheniramine, diphenhydramine, hydroxyzine). h1r antagonists (antihistamines) are widely used in the treatment of a broad range of allergic diseases like rhinitis, conjunctivitis, urticaria and non - allergic disorders like pruritus and insomnia (simons and simons 2011). the first generation of h1r antagonists that had been introduced in the period of 19421980 demonstrated considerable side effects. poor selectivity for the h1r and the ability to cross the blood brain barrier (bbb) interfering with the histaminergic transmission (sedation) were among the most unwanted side effects. recent studies have shown an increase in the number of allergic diseases, currently affecting more than 30 % of the world population (qin 2007). hence, there is an urgent need for more effective and safe anti - allergic drugs. the second - generation h1r antagonists, introduced in the early 1980s, had notable advantages such as being significantly more selective and non - sedating due to the lack of the ability to cross the bbb. these second - generation, non - sedating h1r antagonists have been widely used in the treatment of allergic conditions but still demonstrated some cardiotoxic side effects, e.g. induction of torsades - de - pointes arrhythmias. this has recently led to the development of third - generation h1r antagonists where both sedative and cardiovascular side effects were addressed (oppenheimer and casale 2002 ; canonica and blaiss 2011). the crystal structure of the first - generation h1r antagonist doxepine bound to h1r was solved in 2011 (shimamura. 2011). doxepine has been associated with a large number of different side effects that can be rationalised by its lack of h1r selectivity and being a potent binder of h2r, some members of muscarinic, serotonic and -adrenergic gpcr subfamilies and also of some protein kinases. due to the lack of doxepine selectivity, the need for additional crystal structures of h1r bound to second and third generation of antihistamines that will rationalise the selectivity dr. moraes reported for the first time the solution of two additional holo h1r crystal structures bound to the highly selective second- and third - generation h1r antagonists : cetirizine (gillard. 2002) and fexofenadine (sharma. 2014), respectively (unpublished data ; see fig. cetirizine and fexofenadine are about 600-fold more selective for h1r compared with a wide panel of gpcrs and ion channels (gillard. this work has resulted from collaboration between evotec ltd and the membrane protein laboratory (mpl imperial college london).fig. 7crystal structure of h1r bound to highly selective second- and third - generation antihistamines : cetirizine (left) and fexofenadine (right) crystal structure of h1r bound to highly selective second- and third - generation antihistamines : cetirizine (left) and fexofenadine (right) these new structures provide insights into potency and selectivity, the key challenges for the design of new generation of h1r antagonists and provide a significant contribution to potentially aid computational guided structure - based drug discovery of new antihistamine drugs targeting h2, h3 and h4 receptors where crystal structures are still absent. structural information in conjunction with careful modelling can provide greater insight into the location and functional relevance of druggable binding locations including both orthosteric and allosteric sites. several recent structural publications have provided greater clarity on the binding modes and kinetics of existing drugs in both orthosteric sites, such as bronchodilator tiotropium binding to muscarinic m3 receptor (tautermann. 2013), and allosteric binding sites, such as anti - viral maraviroc which acts as a negative allosteric modulator of chemokine receptor ccr5 (kruse. provision of knowledge of this type should begin to assist in design of more subtype selective ligands, especially when combined with leading edge computational techniques such as homology modelling (storer. an area where increased structural knowledge could be especially impactful is in the improved design and optimisation of selective ligands. selectivity between subfamily members of gpcrs has proved essential yet challenging in some instances, a notable example being the design of highly selective serotonin (5-ht) 5-ht2c - receptor agonists (see fig. 2014) receptor by pyrimido[4,5-d]azepines modelling of binding and activation of 5-ht2c (storer. 2014) receptor by pyrimido[4,5-d]azepines agonism of the 5-ht2c receptor has therapeutic implications in a number of important disease areas including cns disorders and obesity. however, the design of ligands that are selective over agonism of the closely related 5-ht2b receptor, which has been linked with irreversible heart valvulopathy, has proved highly challenging (storer. furthermore, ligand - protein interaction features that lead to receptor agonism versus antagonism versus inverse agonism are not well understood despite numerous examples of subtle ligand structural changes driving pronounced differences in functional efficacy of gpcrs (storer. this is further complicated in instances where the same ligand exerts a different functional effect in different isoforms of the same receptor. this has been demonstrated by differential effects in the human isoform compared to the equivalent preclinical species receptors, hampering both in vivo efficacy and safety studies. an illustrative example is the histamine h4 receptor antagonist program where an inverse agonist of the human h4-receptor was a partial agonist of rat and an antagonist of dog h4 receptor, complicating the interpretation of preclinical in vivo studies (mowbray. therefore, a greater appreciation for the structural basis for these effects could ultimately assist in prediction and systematic avoidance of similar issues in future programs. biased signalling is of current interest to both academia and the pharmaceutical industry based on the hypothesis that it could provide improved therapeutic benefit whilst avoiding undesirable activities that unbiased signalling of certain receptors has historically caused (tautermann 2014). this has provided motivation to revisit some gpcr targets that were previously either poorly drugged or deemed undruggable due to lack of therapeutic index over adverse events (correll and mckittrick 2014). however, despite advances in biology and chemistry providing assay methods to measure bias and clear examples of biased ligands emerging, a deeper structural understanding of protein conformational changes and interactions that lead to differential receptor signalling is still in its infancy but clearly of keen interest to the medicinal chemistry community (violin. ccr4 appears to have three different binding sites for peptide agonist and small molecule ligands (see fig. complexity was further demonstrated in that two classes of chemically distinct small molecule ligands bind to different sites, both of which are allosteric modulators and also display different functional signalling (procopiou. 9homology model of the ccr4 receptor, with putative multiple binding sites for peptide agonists and small molecule allosteric ligands homology model of the ccr4 receptor, with putative multiple binding sites for peptide agonists and small molecule allosteric ligands this complex picture of ccr4 resulted from extensive chemistry and pharmacological studies, and in hindsight it is clear why traditional screening approaches such as binding or whole cell studies did not clearly identify the range of different ligands and sites. in one such screening approach, it was rationalised that the intracellular ligand binding site (helix 8) in ccr4 was likely modified owing the proximal chemical modification required in the commonly used flipr format. whilst the individual targets are well tractable individually, the design of single molecules that fit and potently antagonise both receptors, and have added properties of broader selectivity and intranasal or oral drug properties, is extremely time consuming and challenging (procopiou. 2011) (see fig. 10).fig. 10one of the low - energy conformations of the extended dual h1h3r ligands docked into the homology model of h1 one of the low - energy conformations of the extended dual h1h3r ligands docked into the homology model of h1 thus, structurally enabled approaches to difficult gprc targets should improve the tractability for chemistry by the direct identification of novel multiple ligand binding sites and subsequent chemical optimisation of small molecule ligands to drugs. this is an opportunity for drug discovery in a class of gpcr targets not easily accessible by previous methods. whilst many gpcr targets are chemically tractable resulting in multiple ligands and successful drugs, there are still many gpcr targets which are not amenable to traditional approaches, such as focussed or high throughput screening, or where the natural ligands are unsuitable starting points for the design of oral drugs owing to their properties (e.g. large, lipophilic or peptidic). today, gpcr modelling is widely used in the structure - based drug discovery process. the availability of structural information on the binding site of a targeted gpcr plays a key role in rationalisation, efficiency and cost - effectiveness of drug design. x - ray crystallography, a traditional source of structural information, is not currently feasible for every gpcr or gpcr - ligand complex. this situation significantly limits the ability of crystallography to impact drug discovery for gpcr targets in real - time and hence there is an urgent need for other practical alternatives. gpcr modelling is widely used as a practical alternative in the absence of crystallographic data, but can also provide much more useful information. today, it can address such key issues like gpcr flexibility and dynamics, ligand kinetics (kon / koff rates), prediction of water positions and their role in ligand binding, calculation of the free energy of binding (affinity) and prediction of the effects of mutations on ligand binding, etc. some of these modelling approaches were reviewed in this meeting. the role of flexibility and dynamics in the development of antipsychotic drugs and recent pharmacological studies revealed that clinically effective antipsychotic agents act by binding to several bioamine receptors (roth. in particular, the interaction with the serotonin (5-ht2a and 5-ht6) and dopamine (d2 and d3) receptors (group 1) induces cognition - enhancing effects, while the histamine (h1), 5-ht2c and 5-ht2b receptors (group 2) modulation causes unwanted side effects (selvam. the attempts to develop drugs based on the one - target - one - disease paradigm have been limited (allen and roth 2011). as a result, there remains an urgent need for innovative approaches to develop new effective multi - target agents that improve patients health while reducing care costs. ideal candidates should selectively target disease - active members of the family (group 1) while not binding to members responsible for undesired side effects (group 2). this is a very challenging goal to achieve, as the residues forming the orthosteric binding pocket, i.e. the binding site of endogenous ligands, are conserved within the receptor family, thus causing the recognition of a drug by many members. a strategy to overcome this issue is to design allosteric drugs targeting a less conserved allosteric site, which modulates the orthosteric site, or to design bitopic drugs, which bind to both allosteric and orthosteric sites. the recently released crystal structures of several bioamine receptors in complex with orthosteric and allosteric ligands enable the selectivity issue of antipsychotic drugs to be addressed at the molecular level. thus, structural snapshots provided by crystallography can be used to explore receptor motions using computer simulations. it is conceivable that allosteric and bitopic modulators interact with binding pockets that exist only in a subset of the receptor conformational space. computer modelling can contribute to their identification by providing detailed insights into motions and interactions in the entire protein family and subsequently unravelling complex relationships in generated data within a reasonable time and at low cost. in academia, this approach has been undertaken with some promising results. for example, in the tikhonova group, a computational protocol combining concepts from statistical mechanics and chemoinformatics have been developed to explore the flexibility of the bioamine receptors and identify geometrical and physicochemical properties that characterised the conformational space of the bioamine receptor family (selvam. 2013). figure 11 illustrates the molecular modelling steps undertaken to identify the unique pharmacophoric features of disease - active receptors.fig. 11structure - based computational protocol for selective polypharmacology figure adapted from a recent publication (selvam. 2013) structure - based computational protocol for selective polypharmacology figure adapted from a recent publication (selvam. 2013) the bioamine receptors of groups 1 and 2 have been subjected to molecular dynamics simulations in a realistic environment. the results of simulations show substantial flexibility and its variability across the members of the receptor family. using molecular probe mapping technique combined with the volume - based tanimoto similarity measurements, similar and different geometrical and physicochemical properties were shown across the conformational space of the receptor family and the unique pharmacophoric features of disease - active receptors (group 1) were highlighted. the unique features are then linked with mutational and ligand structure - activity relationship data and tested in retrospective screening. the combination of techniques used gives an efficient method to identify unique properties of the disease - related proteins on the reduced diverse conformational space and represents a novel application of existing computational methods for the investigation of structural reasons for selective polypharmacology (selvam. this protocol can be now exploited by industry for other protein families, involving in cancer and infectious diseases, which require a multi - target approach. in an industry setting, evotec ltd uses a hierarchical gpcr modelling protocol (hgmp) that has been developed in conjunction with the university of oxford to support structure - based drug discovery programs (see fig. the hgmp generates a 3d model of gpcr structures and its complexes with small molecules by applying a set of computational methods. hgmp involves homology modelling, followed by md simulation and flexible ensemble docking, to predict binding poses and function of ligands bound to gpcrs. the hgmp includes a large set of unique plugins to refine the gpcr models and exclusive scoring functions like the gpcr - likeness assessment score (glas) to evaluate model quality (heifetz. armed with a pairwise protein comparison method (pros) used to cluster the structural data generated by the hgmp and to distinguish between different activation sub - states. recently, the capabilities of hgmp have been extended by the addition of gpcr biased ligand tools. the optimisation of hgmp has been performed by evotec ltd in real drug discovery projects.fig. 12 a hgmp workflow and b a model of 5-ht2c (in red) produced by the hgmp workflow. the water molecules and ions are omitted from the figure for clarity a hgmp workflow and b a model of 5-ht2c (in red) produced by the hgmp workflow. the water molecules and ions are omitted from the figure for clarity the performance of hgmp in gpcr drug discovery projects such as mch-1r for obesity treatment (heifetz. 2012, 2013b) and the 5-ht2c for the treatment of metabolic disorders (tye. 2011 additionally, the hgmp technology was used in the solving of the two h1r crystals structures bound to the second- and third - generation antihistamines : cetirizine (gillard. 2002) and fexofenadine (sharma. 2014), respectively, as described by dr. another area that has received increasing attention over recent years has been the role of water networks and their elucidation by md simulations (tautermann. 2014) or the a2ar (liu. 2012) with resolutions of 1.8 ], it has become clear that gpcrs often exhibit conserved water networks, which extend from the extracellular side to the intracellular surface. this conserved solvent network has been implied to be crucial for signalling (nygaard. 2009) and a highly solvated conserved allosteric sodium binding site close to the conserved npxxy motifs of class a gpcrs is postulated to be involved in -arrestin signalling (fenalti., the consideration of water molecules in gpcr ligand design has been shown to be crucial (bortolato. 2013) because several crystal structures show water - mediated ligand - protein interactions (congreve. therefore, a ligand always has to gain more free energy from binding to the receptor than the removal of water actually costs. several methods are available which enable the very crude and quick estimation of the energy penalty for water displacement, and most efficiently they are used for growing a bound ligand (bortolato. they can help medicinal chemists to decide if a certain sub - pocket of the receptor can be explored by hydrophobic moieties or if a displaced water has to be substituted by an entity which has to mimic the hydrogen bond network. obviously, these quick methods are not thorough in a sense that the estimates of the free energy are physically sound, but often they are sufficiently good for a go / no - go decision. in order to get a (formally) correct estimate for the change in the free energy of binding upon ligand modification, methods like thermodynamic integration have to be applied (christ and fox 2014). these more accurate methods all rely on long md simulations of the receptor, and therefore they can also capture reorganisations in the solvent network and induced effects in the binding pocket. one step further beyond assessing the free energy of ligand binding is the estimation of binding kinetics of ligands. water networks also play a crucial role here as well, as the breaking of ligand - receptor hydrogen bonds usually ends up with solvated hydrogen bond acceptor and donor functional groups. as the energy barrier of the reaction determines the rate constant, the direct interaction of water molecules with the ligand - protein hydrogen bonds is decisive for the dissociation rate. if the hydrogen bond is solvent accessible, the breaking of the bond does not require high energy because the re - hydration occurs simultaneously. if the hydrogen bond is buried, a breaking leads to a high energy penalty because acceptor and donor do not find immediate new interaction partners and are in unfavourable solvation states (schmidtke. currently, the in silico prediction of association / dissociation rates is only possible for very small fragments rather than drug - like molecules. therefore, the state - of - the - art is still the explanation of experimental off - rates of drug - like molecules rather than the prediction of them. recently, the duration of action of tiotropium on the human muscarinic m3 receptor (hm3r) was studied (tautermann. 13) by methyl does not reduce the pki strongly, but it has a major effect on the receptor half - life of the molecule. long md simulations (> 2 s) were used to investigate the differences in the bound species, especially focussing on the water network. tiotropium forms a double hydrogen bond to n6.52 in hm3r (fig. 13, left panel), and during the simulation no water comes close (blue solid surface). when substituting the hydroxy - group by methyl (methyl ligand), water density is observed directly above the (single) hydrogen bond (green mesh), corresponding to a large number of md snapshots where n6.52 is (partly) solvated this comes along with a significantly widened exit channel of hm3r, and some md snapshots even show water inserting in the hydrogen bond, as displayed in fig. thus, the explanation for the unexpected change in off - rates is the fact that the shielded hydrogen bond in bound tiotropium becomes solvent exposed when modifying the ligand. this observation was only possible through long md simulations, and an explanation by the static x - ray structure would not have been possible. to summarise, the deep understanding of water networks within gpcrs has proven to be essential if one wants to understand gpcr signalling as well as gpcr ligand binding and dissociation. md simulations are a very valuable tool to derive physically meaningful parameters such as free energy differences or solvent maps because they capture the dynamics of the systems, which is crucial especially for flexible proteins such as gpcrs.fig. 13 left comparison of the water (oxygen) densities in the tiotropium and the methyl ligand. the water densities of the tiotropium md are displayed as blue solid surfaces ; densities in methyl - ligand mds are shown as green mesh. the significant extra density in the methyl - ligand md is marked by a green ellipse. top right chemical structure of tiotropium (r = oh) and the methyl - ligand (r = ch3). bottom right a snapshot of the md simulation with the methyl ligand, where water inserts into the ligand - protein hydrogen bond (in contrast to the tiotropium md, where such water - mediated hydrogen bonds are never observed)figure adapted from the recent publication (tautermann. 2015) left comparison of the water (oxygen) densities in the tiotropium and the methyl ligand. the water densities of the tiotropium md are displayed as blue solid surfaces ; densities in methyl - ligand mds are shown as green mesh. the significant extra density in the methyl - ligand md is marked by a green ellipse. top right chemical structure of tiotropium (r = oh) and the methyl - ligand (r = ch3). bottom right a snapshot of the md simulation with the methyl ligand, where water inserts into the ligand - protein hydrogen bond (in contrast to the tiotropium md, where such water - mediated hydrogen bonds are never observed)figure adapted from the recent publication (tautermann. 2015) an essential part of computational modelling is to re - evaluate predictions often in light of new structural data. researchers at novartis horsham had performed a large amount of work exploring the binding sites of allosteric inhibitors of the chemokine receptor cxcr2, and several interesting things emerged after re - analysing this work in the light of crystallographic data (salchow. it had been determined at novartis horsham that antagonists were acting at an allosteric binding site, and the patent literature around the time of the work suggested that the cxc chemokines could have a binding site close to the intracellular face of the receptor. one of the key residues in this binding site was proposed to be k320 as the cxcr2 antagonists commonly had an acidic functionality of some description within them and this residue was asparagine in the related cxcr1 receptor over which many of the cxcr2 antagonists had selectivity. the mutagenesis experiments that were undertaken did show that residues at the intracellular end of the tm domains had an influence on the binding and/or potency of the antagonists whilst one proposed as critical within the tm domain did not show any influence. however, the expected overlay of the antagonists based upon their ligand - only overlays was not reproduced in the effects seen against the various mutant receptors. the residues proposed for mutagenesis, and the interpretation of their effects, were very dependent upon the model used to create the homology model for cxcr2 at the time, and so a review of how the latest gpcr crystal structures could have influenced this project was presented.fig. 14 a the proposed binding modes for the quinoxaline (yellow carbons) and urea (cyan carbons) series with the influential mutated residues in cxcr2 shown in cpk. the consistent influence of k320 locates the acidic functionality in the antagonists, yet the varying effects of d143 on representatives of these two series suggest that the hydrophobic groups are located differently. b overlay of the cxcr1 nmr structures from the pdb (code 2lnl) n320 is shown in green cpk and influential mutants from our experiments in purple cpk. the protein backbones, in ribbon representation, are coloured from n (blue) to c (red) termini a the proposed binding modes for the quinoxaline (yellow carbons) and urea (cyan carbons) series with the influential mutated residues in cxcr2 shown in cpk. the consistent influence of k320 locates the acidic functionality in the antagonists, yet the varying effects of d143 on representatives of these two series suggest that the hydrophobic groups are located differently. b overlay of the cxcr1 nmr structures from the pdb (code 2lnl) n320 is shown in green cpk and influential mutants from our experiments in purple cpk. the protein backbones, in ribbon representation, are coloured from n (blue) to c (red) termini the nmr structures for the related cxcr1 receptor (as reproduced in fig. 14b) consistently show that the critical n320 residue is not close to the other influential residues, and it would have been hard to rationalise the mutagenesis from this template. the ccr5 crystal structure shows distortion in the tm7/helix 8 region and the critical k320 residue was mutated to a glu, but overall the helical alignment compared to rhodopsin was very similar and so would not have provided any benefit compared to the rhodopsin template used originally. the 2ar - gs - protein structure was a marvellous achievement (rasmussen. 2011), but for the purposes of acting as a template for the intracellular binding area of the antagonists, it is likely to be too different to cxcr2 to be useful. the most relevant structure could have been the cxcr4, but here again the tm7/helix 8 region is distorted in the crystal and a cxcr2 homology model was created by using a chimeric template of this receptor and the tm7/helix 8 region from an earlier 2ar structure (rasmussen. interestingly, this model had a related cxcr2 antagonist bound in the tm domain and the major learning point from the current gpcr symposium was just how much influence changes within the tm domain affected the intracellular coupling and vice versa. a conclusion drawn from the symposium with respect to the work completed at nibr horsham would be that more mutagenesis information from across the whole of the cxcr2 protein would have been needed in order to fully understand the binding area of these allosteric antagonists. finally, network - based approaches, evolutionary algorithms and predictive modelling, all areas that will become more prevalent in the future, were discussed. omics age has brought with it huge quantities of data around diseases, targets, compounds and their effects. interactomes can be built with the ultimate goal to understand disease networks and how they are influenced by the changes in small molecules and their properties. the polypharmacology associated with current typical and atypical anti - psychotics is complex, and as an example, the question of how do we go about designing a novel anti - psychotic given the tools and data we have access to today was raised. the opensource chembl space polypharmacology network viewer (fechner. 2013) was introduced as an interactive way to review the rich pharmacology accessible in the chembl database and identify some good starting points for drug design. the experimental polypharmacology associated with the hits can be complemented using target prediction ligand similarity - based approaches such as the similarity ensemble approach (sea ; keiser. 2009) or broad panel - based predictive modelling approaches (ghosh and jones 2014). predictive modelling approaches were also used to build protein target qsars that in combination with pharmacophore triplet compound similarity were used to develop a multi - objective scoring function. given a small organic fragment, an automated evolutionary design algorithm using reaction vectors was used to grow a molecule by simultaneously optimising the multi - parameters required for the targeted phenotype polypharmacology (patel. the reaction vector design approach was extended to whole reaction sequences and ultimately reaction networks. a gpr38 reaction network was built which exemplified that the chemistry phase space around a hit could be readily expanded to that of closely accessible molecules. 15automated multi - objective compound design using reaction vectors (26k reaction db and 93k reagents) starting from piperidine and using four objectives : similarity to haloperidol and ziprasidone pharmacophores, dopamine d2, 1b adrenergic and histamine qsar models. the tri - cyclics generated appeared similar to known anti - pyschotics, chlorpromazine and fluphenazine automated multi - objective compound design using reaction vectors (26k reaction db and 93k reagents) starting from piperidine and using four objectives : similarity to haloperidol and ziprasidone pharmacophores, dopamine d2, 1b adrenergic and histamine qsar models. the tri - cyclics generated appeared similar to known anti - pyschotics, chlorpromazine and fluphenazine it was also shown how gpcr targets and their interacting partners could be identified from genome wide association studies (gwas) using network analysis followed by the analysis of compound gene expression data to complement target disease gene expression as a strategy for network - based drug design. multiple algorithms have been developed that in combination with omics data and gpcr structure - based design make a powerful arsenal for today s drug designer. all participants agreed that gpcr research and drug discovery can benefit greatly from the collaboration between academia and industry. the effectiveness of such collaborations for gpcr research and drug discovery was widely described and exemplified during this conference : the non - profit gpcr consortium (http://gpcrconsortium.org) described by vadim cherezov ; the collaboration between mrc laboratory and heptares mentioned by gebhard schertler and chris tate ; the consortium between evotec ltd, oxford university uk and the membrane protein laboratory at diamond light source, uk described by alexander heifetz ; and many other successful networks like the adhesion - gpcr consortium (agc) or glisten (gutierrez - de - teran 2014) (gpcr - ligand interactions, structures, and transmembrane signalling : a european research network). there was broad agreement with schertler s and ceska s comments that most ventures between academia and industry are dependent on track record and trust and that individuals have to commit to a longer - term perspective which is aimed at changing the scientific landscape. there was also agreement that meetings between academia and industry such as this conference are very useful to learn about developments within each other s areas of expertise and to share the challenges whilst forging new links and networks. for future meetings of this type, it was proposed to also include a broader mix of younger - generation scientists early in their career in order to maximise benefit to a broader community. | g - protein coupled receptors (gpcrs) are the targets of over half of all prescribed drugs today. the uniprot database has records for about 800 proteins classified as gpcrs, but drugs have only been developed against 50 of these. thus, there is huge potential in terms of the number of targets for new therapies to be designed. several breakthroughs in gpcrs biased pharmacology, structural biology, modelling and scoring have resulted in a resurgence of interest in gpcrs as drug targets. therefore, an international conference, sponsored by the royal society, with world - renowned researchers from industry and academia was recently held to discuss recent progress and highlight key areas of future research needed to accelerate gpcr drug discovery. several key points emerged. firstly, structures for all three major classes of gpcrs have now been solved and there is increasing coverage across the gpcr phylogenetic tree. this is likely to be substantially enhanced with data from x - ray free electron sources as they move beyond proof of concept. secondly, the concept of biased signalling or functional selectivity is likely to be prevalent in many gpcrs, and this presents exciting new opportunities for selectivity and the control of side effects, especially when combined with increasing data regarding allosteric modulation. thirdly, there will almost certainly be some gpcrs that will remain difficult targets because they exhibit complex ligand dependencies and have many metastable states rendering them difficult to resolve by crystallographic methods. subtle effects within the packing of the transmembrane helices are likely to mask and contribute to this aspect, which may play a role in species dependent behaviour. this is particularly important because it has ramifications for how we interpret pre - clinical data. in summary, collaborative efforts between industry and academia have delivered significant progress in terms of structure and understanding of gpcrs and will be essential for resolving problems associated with the more difficult targets in the future. |
the most significant and debilitating complication of herpes zoster is herpetic neuralgia that accompanies and may persist in 10 - 15% of all zoster patients, particularly those over 60 years of age. herpes zoster (hz), a sporadic infection with an estimated lifetime risk of 10 - 20%, occurs in all age groups, especially among individuals who have had varicella any time in the past, but its prevalence almost doubles in each decade past the age of 50 years. the most significant and debilitating complication of hz is herpetic neuralgia that accompanies and may persist in 10 - 15% of all zoster patients, particularly those over 60 years of age. cranial and peripheral nerve palsies, encephalitis, pneumonitis, acute retinal necrosis, encephalitis, myelitis, and secondary bacterial infection are other well - known complications of hz. the described cases had an uncommon complication of spontaneous tooth exfoliation after trigeminal hz, of which only about 30 cases have been reported before 2006 in the english literature.[138 ] this 80-year - old hiv - negative male had spontaneous loss of teeth from right upper jaw about 10 days after severe tooth pain, gingival swelling / ulceration, and cutaneous vesicular eruptions over right cheek, suggestive of hz, for which he took analgesics only. he had few loose or missing teeth previously but not in the right upper quadrant. the skin and gingival lesions had healed in about 2 weeks, but he still had paresthesia over the right cheek. oro - cutaneous examination [figure 1a and b ] showed hyperesthesia, scarring, and milia over right facial skin innervated by maxillary division of right trigeminal nerve, slight deviation of upper lip, and edentulous right upper alveolar ridge due total loss of teeth. there was minimal periodontitis and few missing teeth in lower jaw, but no oral lesions, gingival ulceration / necrosis, blood, purulent exudates, granulation or loose teeth were noted. x - ray of the jaw and orthopantograph (opg) showed complete loss of teeth in right upper quadrant and no necrosis or other abnormality of the alveolar bone [figure 1c and d ]. systemic examination, complete blood counts, serum biochemistry, urinalysis, and chest x - ray were essentially normal, and he had no other predisposing factors for immune suppression. the oral prophylaxis performed, and the patient was provided with artificial removable dentures later. (a) post - herpes zoster scarring and milia formation over the skin innervated by maxillary branch of right trigeminal nerve. (b) unilateral exfoliated right upper incisors, canine, premolar, and molar teeth leaving him edentulous in right upper quadrant. no alveolar bone necrosis is noted this 55-year - old hiv - negative female was hospitalized for painful vesicular lesions over right cheek, right half of tongue and buccal mucosa innervated by right mandibular nerve, and corresponding gingival swelling. although she had toothache 2 days prior to onset of skin lesions, there was no blood, purulent exudates, granulation, or loose teeth. she had mild generalized periodontitis and few missing teeth, but teeth in right lower quadrant were intact. systemic examination, complete blood counts, serum biochemistry, urinalysis, and chest x - ray showed no abnormality, and there were no predisposing factors for immune suppression. clinical diagnosis of hz was made, and she was treated with intravenous acyclovir (500 mg 8 hourly / d) for initial 2 days, followed by oral acyclovir (800 mg 5 times / d) for the next 1 week and was sent home. oro - cutaneous examination on return visit 2 weeks later showed hyperesthesia and hyperpigmentation of facial skin over right mandibular area, slight hypo-/hyperpigmentation of lower lip, and edentulous right lower alveolar ridge due total loss of teeth [figure 2a and b ]. orthopantograph showed complete loss of teeth in right lower quadrant and no necrosis or other abnormality of the alveolar bone [figure 2c ]. the oral prophylaxis performed. (a) post - herpes zoster hypopigmentation and hyperpigmentation over the skin innervated by mandibular branch of right trigeminal nerve. (b) edentulous alveolus showing healing after exfoliation of all teeth in right lower quadrant. (c) orthopantograph shows total loss of right mandibular teeth but no alveolar bone necrosis this 45-year - old hiv - negative male presented with spontaneous loss of few teeth from right lower jaw about 3 weeks after severe gingival / tooth pain and vesiculo - ulcerative eruptions over tongue and skin over right mandibular region, suggestive of hz. the skin and gingival lesions had healed in about 2 weeks, but he still felt abnormal sensations over the healed skin eruption area. there was hyperpigmentation and hyperesthesia over right facial skin innervated by mandibular division of right trigeminal nerve, loss of right lower incisors, canine, premolar teeth, exposed portions of the empty tooth sockets in the alveolar bone, and root surfaces of the exfoliated teeth, but no loose teeth were found [figure 3 ]. x - ray of the jaw showed empty sockets corresponding to lost teeth and no necrosis or other abnormality of the alveolar bone. systemic examination, laboratory work - up, and chest x - ray were essentially normal, and there were no predisposing factors for immune suppression. (a) post - herpes zoster hyperpigmentation ending abruptly in the midline over the skin innervated by mandibular branch of right trigeminal nerve. (b) unilateral empty tooth sockets from the exfoliated right lower incisors, canine, and premolar teeth ending abruptly in the midline are clearly visible. this 80-year - old hiv - negative male had spontaneous loss of teeth from right upper jaw about 10 days after severe tooth pain, gingival swelling / ulceration, and cutaneous vesicular eruptions over right cheek, suggestive of hz, for which he took analgesics only. he had few loose or missing teeth previously but not in the right upper quadrant. the skin and gingival lesions had healed in about 2 weeks, but he still had paresthesia over the right cheek. oro - cutaneous examination [figure 1a and b ] showed hyperesthesia, scarring, and milia over right facial skin innervated by maxillary division of right trigeminal nerve, slight deviation of upper lip, and edentulous right upper alveolar ridge due total loss of teeth. there was minimal periodontitis and few missing teeth in lower jaw, but no oral lesions, gingival ulceration / necrosis, blood, purulent exudates, granulation or loose teeth were noted. x - ray of the jaw and orthopantograph (opg) showed complete loss of teeth in right upper quadrant and no necrosis or other abnormality of the alveolar bone [figure 1c and d ]. systemic examination, complete blood counts, serum biochemistry, urinalysis, and chest x - ray were essentially normal, and he had no other predisposing factors for immune suppression. the oral prophylaxis performed, and the patient was provided with artificial removable dentures later. (a) post - herpes zoster scarring and milia formation over the skin innervated by maxillary branch of right trigeminal nerve. (b) unilateral exfoliated right upper incisors, canine, premolar, and molar teeth leaving him edentulous in right upper quadrant. this 55-year - old hiv - negative female was hospitalized for painful vesicular lesions over right cheek, right half of tongue and buccal mucosa innervated by right mandibular nerve, and corresponding gingival swelling. although she had toothache 2 days prior to onset of skin lesions, there was no blood, purulent exudates, granulation, or loose teeth. she had mild generalized periodontitis and few missing teeth, but teeth in right lower quadrant were intact. systemic examination, complete blood counts, serum biochemistry, urinalysis, and chest x - ray showed no abnormality, and there were no predisposing factors for immune suppression. clinical diagnosis of hz was made, and she was treated with intravenous acyclovir (500 mg 8 hourly / d) for initial 2 days, followed by oral acyclovir (800 mg 5 times / d) for the next 1 week and was sent home. oro - cutaneous examination on return visit 2 weeks later showed hyperesthesia and hyperpigmentation of facial skin over right mandibular area, slight hypo-/hyperpigmentation of lower lip, and edentulous right lower alveolar ridge due total loss of teeth [figure 2a and b ]. orthopantograph showed complete loss of teeth in right lower quadrant and no necrosis or other abnormality of the alveolar bone [figure 2c ]. the oral prophylaxis performed. (a) post - herpes zoster hypopigmentation and hyperpigmentation over the skin innervated by mandibular branch of right trigeminal nerve. (b) edentulous alveolus showing healing after exfoliation of all teeth in right lower quadrant. (c) orthopantograph shows total loss of right mandibular teeth but no alveolar bone necrosis this 45-year - old hiv - negative male presented with spontaneous loss of few teeth from right lower jaw about 3 weeks after severe gingival / tooth pain and vesiculo - ulcerative eruptions over tongue and skin over right mandibular region, suggestive of hz. the skin and gingival lesions had healed in about 2 weeks, but he still felt abnormal sensations over the healed skin eruption area. there was hyperpigmentation and hyperesthesia over right facial skin innervated by mandibular division of right trigeminal nerve, loss of right lower incisors, canine, premolar teeth, exposed portions of the empty tooth sockets in the alveolar bone, and root surfaces of the exfoliated teeth, but no loose teeth were found [figure 3 ]. x - ray of the jaw showed empty sockets corresponding to lost teeth and no necrosis or other abnormality of the alveolar bone. systemic examination, laboratory work - up, and chest x - ray were essentially normal, and there were no predisposing factors for immune suppression. (a) post - herpes zoster hyperpigmentation ending abruptly in the midline over the skin innervated by mandibular branch of right trigeminal nerve. (b) unilateral empty tooth sockets from the exfoliated right lower incisors, canine, and premolar teeth ending abruptly in the midline are clearly visible. hz of trigeminal nerve manifests as painful vesicular eruptions of the skin and mucosa innervated by the affected nerve division. its major complications vary from temporary prodromal odontalgia to permanent tooth nerve damage, devitalized teeth, abnormal development of permanent teeth, root resorption, muscle paralysis, multiple ocular and ear complications, prolonged neuralgia, trigeminal trophic syndrome, and occasionally encephalitis. the pain of trigeminal zoster frequently simulates toothache, not uncommonly leading to tooth extraction. other oral complications like exfoliation of teeth and alveolar bone necrosis or osteomyelitis of maxilla or mandible have been rarely described in dermatology literature. osteonecrosis following hz often presents as painless exfoliation of teeth in the involved area approximately 6 weeks after the vesicular eruptions when the acute phase has subsided. the precise pathomechanism(s) of these oral complications eventually leading to tooth loss and necrosis of the alveolar bone, periodontal tissue membrane, nerve supply, and adjacent gingival tissue remain largely uncertain. the pre - existing local periodontal disease does not appear to influence the tooth loss. although the majority of the described cases are aged over 50 years, had severe hz, were suffering from lymphoproliferative or other malignancies, hiv infection, receiving corticosteroids or chemo-/radiotherapy, the conditions well - known for increased incidence and severity of hz, few of the reported cases have been young, immunocompetent, and otherwise healthy individuals suggesting that factors like old age, pre - existing systemic illnesses too seem not to precipitate this rare complication. it has been also postulated that it could result from intense vasoconstriction, endarteritis, or vasculitis with ischemia of the blood supply that may be caused by the virus migrating along the trigeminal neurovascular bundle. it is possible that the varicella zoster virus invades the sympathetic nerves that accompanying the blood vessels to the alveolar bone causing vasoconstriction and hampered blood supply or that systemic viral infection perhaps injures odontoblasts and cause degenerative tissue changes / pulp necrosis. another plausible explanation is that by direct extension of neural inflammation and edema, the adjacent alveolar artery may get compressed in the narrow bony channels and resultant poor tissue perfusion. the postulation of local vasculitis caused by varicella zoster virus leading to infarction and ischemia of the periodontal tissue and alveolar bone has limited evidence despite well - documented vascular damage it causes in the central nervous system. interestingly, early anti - viral medication too does not appear to prevent this complication as is evident in our case-2. our patients were immunocompetent, never used tobacco, and had only minimal pre - existing local periodontal disease, suggesting that the hz infection perhaps remains the primary determinant for exfoliation of teeth / bone necrosis. we feel that the described cases will make dermatologists aware of this rather unusual trigeminal hz complication described mainly in the literature on oro - dental diseases. spontaneous tooth exfoliation after trigeminal herpes zoster is a rare complication that rarely finds mention in dermatology literature. | the most significant and debilitating complication of herpes zoster (hz) is herpetic neuralgia that accompanies and may persist in 10 - 15% of all zoster patients, particularly those over 60 years of age. the described 3 cases had an uncommon complication of spontaneous tooth exfoliation after trigeminal hz that rarely finds mention in dermatology literature. |
the main and ultimate goal of nursing services is to provide the clients with quality care in order to improve the outcomes of the services given to the patients and society. nurses, as the major force of treatment and care team in educational and treatment centers, should be accountable for the quality of the care given by them from an ethical and legal approach. on the other hand, approach of the nurses, as the valuable source to give services to and have communication with the patients, concerning the quality of the given care is of great importance. improvement of care quality is a constant challenge for nurse managers in hospitals in health care and treatment centers as it reflects the access level to the best outcome of health, which is the quality of care given to the patients. therefore, the given services should be effective, efficient, and economical. with regard to the increased cost of medical services, constant promotion of nursing care quality is essential to increase clients ' satisfaction. meanwhile, nursing care quality includes patients ' access to physical and psychosocial needs fulfillment, their satisfaction with the given care, nurses ' accountability for the given care, and patients ' trust to receive comprehensive and multidimensional care. it is evaluated in physical, psychosocial, and communicational dimensions. among the factors influencing the dimensions of quality care given to the patients is nurses ' occupational stress, which results from their exposure to patients ' pain and suffering, administration of interventional procedures such as cardiopulmonary resuscitation, the concerns of and problems with other health team members, inappropriate physical work condition, work shifts, etc. results showed that nurses working in icu undergo more stress compared to the nurses in other wards, as they have various and numerous roles. they have the highest work load and are more involved in interventions and care, have more working hours by the patients ' bed, and are in increased contact with the patients, compared to other health team members. therefore, the effect of work - related stress on them is so high. on the other hand, it should be noted that most of the nurses are female who have specific physical conditions and play additional roles as a mother, a spouse, and as a home maker, in addition to their multifunctional clinical role. therefore, existing stress in the work environment can negatively affect their function in giving care to the patient in long term. so, many studies have been conducted on education of stress management, reporting somehow positive results, to reduce and tame occupational tensions and their effect on nurses ' stress, quality of life, and mental health. stress management is a trial to increase individuals ' adaptation with the environment or a trial to prevent negative outcomes resulting from pressuring conditions, through detection of negative thoughts, cognitive reconstruction, muscular relaxation, time management, anger management, problem - solving skills, and efficient communication skills. as mentioned before, icu forms stressful conditions for the nurses due to its environmental condition and heavy professional responsibilities. meanwhile, nurses who have proper mental and psychological condition and can manage the existing stress are needed to provide efficient and effective nursing care and for giving proper clinical services to the patients. with regard to the importance of stress management, the present study aimed to investigate the effect of stress management program on the quality of nursing care. this is a two - group, three - stage clinical trial (registered in irct2015022621246n1) with a before, immediately after, and 1 month after intervention test, which was conducted to investigate the effect of independent variable of stress management program on the dependent variable of nursing care quality from the viewpoint of nurses working in icus. after obtaining permission from the vice - chancellery for research in isfahan university of medical sciences, the researcher entered the study environment, and after presenting her letter of introduction and explaining about the study goals to the authorities of the center, and obtaining their approval as well as informed consents from the subjects, she started sampling by random stratified sampling method. with regard to nurses ' dispersion in three icus, the subjects were selected from icu1 (n = 20), icu2 (n = 28), and icu3 (n = 22) for the study and control groups. then, the subjects completed the questionnaire and were randomly assigned to study and control groups by random numbers table and through random allocation. inclusion criteria were being a female nurse with work experience of at least 1 year in the icu, having a bachelor 's degree or higher, permanent or casual employment status, being totally interested in taking part in the study, no history of attending a similar program simultaneously, obtaining a score of 150 in holmes and rahe scale, not being affected by a diagnosed acute or chronic physical disease, mental disease, or drug abuse, and not being pregnant. exclusion criteria were being absent for more than two sessions of the study, incidence of unexpected stressful and critical events during the study (a divorce, death, and other critical events), and loss of interest to remain in the study. stress management intervention program was prepared based on existing research and studies, and was held by a psychiatric nurse (researcher) in the form of ten 90-min sessions twice a week in the intervention group [table 1 ]. the control group participants were asked just to attend in pretest, posttest, and follow - up stages. in order to control the group sessions effect on the study group and follow synchronization issues, the control group also underwent group sessions on just professional issues. content of 10 stress management program sessions in brief questionnaires were completed immediately after and 1 month after the intervention by the two groups, and at the end, prepared educational packages were given to both groups. data were collected by a demographic checklist and nursing care quality assessment tool in physical, psychosocial, and communicational dimensions. the first section was associated with personal characteristics (marital status, number of children, education level) and professional characteristics [work experience, work experience in icu, work shifts, over work, employment status, monthly income, extra jobs (out of nursing profession), and satisfaction with the quality of sleep ]. the second section was the standard tool of nursing care quality scale from the viewpoint of nurses that was adopted in the us in 1975 for the quality of patients ' care and is used to evaluate the care quality in the dimensions of structure, process, and outcome. its validity and reliability were investigated in tabriz, iran in 2003 and were modified according to iranian culture (cronbach alpha = 0.80). this questionnaire has three (psychosocial, physical, and communicational) dimensions from the viewpoint of nurses, which are scored by a three - point likert 's scale (never = 0, sometimes = 1, often = 2). data were analyzed by independent t - test, chi - square test, fisher 's exact test, analysis of variance (anova), and least significant difference (lsd) post hoc test in spss18 (ibm software version 18.0. this research project has approval of the ethics committee of isfahan university of medical sciences. all possible ethical issues addressed as explained above and the participants signed a written informed consent. this research project has approval of the ethics committee of isfahan university of medical sciences. all possible ethical issues addressed as explained above and the participants signed a written informed consent. results showed that intervention and control groups were almost identical concerning demographic variables [table 2 ]. comparison of frequency distribution of subjects age, number of children, work experience, and overtime in study and control groups independent t - test showed no significant difference in the mean scores of nursing care quality between the two groups before intervention. meanwhile, the nursing care quality mean scores were significantly higher in the study group compared to the control group, immediately after and 1 month after the intervention (p 0.05). table 4 shows a significant difference in the mean score changes in all dimensions immediately after and 1 month after the intervention in both study and control groups, compared to before intervention (p < 0.05). comparison of subjects overall nursing care quality mean scores in study and control groups comparison of nursing care quality mean scores in physical, psychosocial, and communicational dimensions in subjects immediately after and 1 month after the intervention compared to before intervention between the study and control groups the present study aimed at investigation of the effect of stress management program on the quality of nursing care. findings showed that overall mean score of nursing care quality and the scores in all dimensions significantly increased after intervention in the study group compared to before intervention (p < 0.001), which reveals the positive effect of this program on nursing care quality. results of the present study and various studies show that nurses who are able to manage their stress give quality care. alavi arjmand., in a study on the effect of a stress management program on occupational stress and work it reveals the effect of education of stress management skill on reduction of nurses ' work family conflict and improvement of the nursing care given by them. the findings of the present study also showed that mean score of nursing care quality was significantly higher immediately after and 1 month after the intervention compared to before intervention (p < 0.05), which shows the positive effect and longevity of stress management program on all dimensions of nursing care quality. hamid., in a study on the effectiveness of cognitive - behavioral stress management on female nurses ' job burnout, showed that this stress management was effective on improvement of job burnout signs and that the mean scores were significantly higher immediately after intervention and in the follow - up stages, compared to before intervention. in this way, stress management led to improvement of patient care quality through enabling nurses to fight against job burnout signs. the findings of the present study showed a significant difference in mean scores of nursing care quality in physical, psychosocial, and communicational dimensions between the three time points in the study group (p < 0.05). in a study on the effect of communication skills on the patients ' nursing care quality, showed that mean scores of nursing care quality in physical, psychosocial, and communicational dimensions increased at various time points after intervention, possibly due to the integration of human existence. various study results showed that in some cases of nursing care administration, care of physical dimension has been less noticed among nurses, compared to other dimensions. meanwhile, the behaviors in this dimension are more touchable and observable, compared to other dimensions, and consequently, promotion of nursing care quality in this dimension is more notable, compared to other dimensions. in the present study, the researcher observed a positive effect on the physical dimension of patients ' nursing care through a stress management program that provided education of healthy lifestyle, physical exercise, and adequate rest. its score increased from 69.8 to 74.7, showing a significant difference (p < 0.05). with regard to psychosocial dimension, it can be noted that cognitive - behavioral stress management program was also adopted in the present study. the findings showed a positive effect and significant association in this dimension of nursing care quality. in a meta - analysis study of kim on the effect of cognitive - behavioral job stress management, a significant effect of this type of intervention on improvement of stress management skills and psychosocial function of employees was observed. with regard to communicational dimension of nursing care quality as one of the important aspects of nursing care, in making communication with the patients, nurses should provide the patients with comfort and support through an efficient communication by consideration of patients ' concerns, understanding them, and being empathetic during giving care. in the present study, the content of stress management program improved nursing care quality with its efficient education of communication skills. patient communication improvement showed that communication skills and efficient communication with the patient could highly affect the quality of patient care. therefore, stress management program affects all dimensions of patient care quality with its comprehensive coverage. findings of the present study showed that mean score of nursing care quality was significantly higher in the study group after intervention compared to before intervention (p < 0.05). in the study of hosseini, the effect of stress management education on nurses ' job burnout was investigated in two hospitals in hamedan, iran, and the results showed a significant reduction in mean score of job burnout in the study group after intervention, compared to control group (p < 0.001). as stress resulting from occupational tensions can affect nurses ' nursing care quality, it can be mentioned that based on the findings of the present study, stress management skill could control stress and, consequently, increase nursing care quality. all previous studies and our results showed not only the effect of stress management program on nursing care quality, but also its stable longevity through time. the results showed that administration of stress management program leads to an increase in nursing care quality scores ; therefore, intervention played an effective role in improvement of nursing care quality among nurses. the health and nursing mangers can take a positive step toward promotion of nursing care quality as indicated by the results of the present study, and education of stress management skills in the form of seminars and continuing education sessions, as well as practical workshops can help in obtaining clients ' satisfaction. personal differences, low and limited sample size in icu, limited length of study and lack of long - term follow - up, and exclusion of male nurses from the study population were among the limitations of the present study. it is suggested to minimize these limitations in future studies and take more samples and have longer follow - up to modify the present study defects in order to firmly report stress management 's positive effects on nursing care quality through prevention, modification, and reduction of stress and its effects on peers. | background : high level of stress in intensive care unit nurses affects the quality of their nursing care. therefore, this study aimed to determine the effects of a stress management program on the quality of nursing care of intensive care unit nurses.materials and methods : this study is a randomized clinical trial that was conducted on 65 nurses. the samples were selected by stratified sampling of the nurses working in intensive care units 1, 2, 3 in al - zahra hospital in isfahan, iran and were randomly assigned to two groups. the intervention group underwent an intervention, including 10 sessions of stress management that was held twice a week. in the control group, placebo sessions were held simultaneously. data were gathered by demographic checklist and quality patient care scale before, immediately after, and 1 month after the intervention in both groups. then, the data were analyzed by student 's t - test, mann whitney, chi - square, fisher 's exact test, and analysis of variance (anova) through spss software version 18.results:mean scores of overall and dimensions of quality of care in the intervention group were significantly higher immediately after and 1 month after the intervention, compared to pre - intervention (p < 0.001). the results showed that the quality of care in the intervention group was significantly higher immediately after and 1 month after the intervention, compared to the control group (p < 0.001).conclusions : as stress management is an effective method to improve the quality of care, the staffs are recommended to consider it in improvement of the quality of nursing care. |
improvement of respiratory support and pharmacotherapy for heart failure has brought extension of life span to patients with duchenne muscular dystrophy (dmd). however, the incidence of cardiomyopathy increases with age ; 100% of patients have cardiac involvement by adulthood.1 some kinds of arrhythmia cause fatal matters as well as worsening heart failure. some dmd patients will need device therapy such as a permanent pacemaker2,3 or an implantable cardioverter - defibrillator (icd).4 icd and cardiac resynchronization therapy have not been established in dmd patients. however, it has been reported that these therapies are beneficial to cardiomyopathy patients with systolic dysfunction.5,6 a 32-year - old man with dmd was admitted to the hospital because of worsening dyspnea and general fatigue. the diagnosis of dmd was made by muscle biopsy when he was 5 years old. he had received ventilation support for respiratory failure by a volume - controlled ventilator from 20 years of age. complete left bundle branch block (clbbb) appeared in electrocardiogram from 3 years before this admission. at the same time, echocardiography revealed abnormal left ventricular wall motion and dyssynchrony. electrocardiogram showed bradycardia with mobitz type ii second - degree atrioventricular block (figure 1a). his blood pressure was 90/50 mmhg, heart rate was 30 bpm, and oxygen saturation was 97% with room air. echocardiography showed global mild left ventricular systolic dysfunction with dyssynchrony (ejection fraction : 45%). the results of laboratory tests showed elevation of plasma levels of creatine phosphokinase (843 u / l), troponin t (0.073 ng / ml), and b - type natriuretic peptide (bnp) (871.0 pg / ml). the device generator was implanted under the greater pectoral muscle with the assistance of a plastic surgeon because his subcutaneous tissue had very poor growth. biventricular pacing was begun immediately after implantation of the crt - d (figure 2). his ventilation support was continued with the same conditions as those before crt - d implantation. his bnp level (196.0 pg / ml) was improved 7 months after crt - d implantation. furthermore, his bnp level was decreased to 116.0 pg / ml after 20 months, and he remains asymptomatic. he is free from complaint and hospitalization for worsening heart failure after crt - d implantation. dmd is an x - linked recessive disease that has an incidence of approximately one in 3,500 male births.1 this disease results from mutations in the dystrophin gene on chromosome xp21. consequently, the absence of dystrophin results in the development of skeletal muscle weakness, cardiomyopathy, and respiratory muscle weakness. the incidence of cardiomyopathy increases with age. approximately 25% of dmd patients have cardiomyopathy by 6 years of age, and 59% are affected between 6 and 10 years of age. by adulthood, 100% of patients have cardiac involvement.1 cardiomyopathy is due to replacement of myocardium by connective tissue or fat, and there is both cardiac dysfunction and arrhythmia. it has been reported that biventricular pacing is superior to conventional right ventricular pacing in patients with atrioventricular block and left ventricular systolic dysfunction.5 our patient had clbbb with left ventricular dyssynchrony. furthermore, cardiac function was expected to deteriorate after that. moreover, the ventricular pacing was indispensable for atrioventricular block. therefore, cardiac resynchronization therapy was suitable for the treatment of our patient, while crt - d decreases the risk of heart failure events compared with an icd alone in patients with a low ejection fraction and wide qrs complex.6 however, the benefit of an icd has not been established in dmd patients. but we could not deny the fatal ventricular arrhythmia, because this disease was a progressive disease. in addition, we thought that the reoperation for upgrade of pacemaker would not be easy for him because of his severe skeletal anomaly such as scoliosis and the risk of general anesthesia. therefore, crt - d was thought to be preferable for our patient as a primary prevention strategy. myotonic dystrophy types 1 and 2, emery dreifuss muscular dystrophy, and limb girdle muscular dystrophy (lgmd) type 1b are generally associated with conduction disease.4 these types of muscular dystrophy need to be frequently implanted with a pacemaker. dreifuss muscular dystrophy and lgmd type 1b because of the risk of bradycardia and ventricular arrhythmia,7 while dmd, becker muscular dystrophy, and lgmd types 2c2f and 2i are generally associated with a dilated cardiomyopathy.4 progression of cardiac dysfunction increases the frequency of sudden death. crt - d has a good possibility of the prevention of progressive heart failure and sudden death. although device therapy must be chosen according to individual conditions, crt - d may become one of the therapies for arrhythmia and heart failure in patients with myopathy. | a 32-year - old man with duchenne muscular dystrophy (dmd) was admitted to the hospital because of worsening dyspnea and general fatigue. he had received medication therapy for cardiomyopathy with heart failure and home mechanical ventilation for respiratory failure. an electrocardiogram on admission showed intermittent third - degree atrioventricular block. echocardiography showed global mild left ventricular systolic dysfunction with dyssynchrony (ejection fraction : 45%). he underwent implantation of a cardiac resynchronization therapy defibrillator. his b - type natriuretic peptide level was improved after cardiac resynchronization therapy defibrillator implantation, and he remains asymptomatic. the incidence of cardiomyopathy increases with age. by adulthood, 100% of patients have cardiac involvement. |
supplementary material is available for this article at 10.1007/s13659 - 011 - 0031 - 7 and is accessible for authorized users. | three new ent - kauranoids, isorosthornins a - c (13), and a new natural product, dihydroponicidin (4), together with five known ones were isolated from the aerial parts of isodon rosthornii. the structures were determined by means of extensive spectroscopic analysis. all diterpenoids isolated were evaluated for their cytotoxicity against hl-60, smmc-7721, a-549, mcf-7, and sw480 cell lines, and compounds 5 and 7 showed significant inhibitory effects on all cell lines. electronic supplementary materialsupplementary material is available for this article at 10.1007/s13659 - 011 - 0031 - 7 and is accessible for authorized users. |
since the early 1980s an intensive clinical research on larynx preservation has been exploring potential reliable alternatives to mutilating surgery defined as the complete removal of the voice box (i.e. a total laryngectomy). combined chemotherapy and radiation therapy have played a major role in this clinical research on larynx preservation with either induction chemotherapy before irradiation or concurrent chemo - radiotherapy. several randomized trials have been carried out and larynx preservation has been validated as a reliable option. the accumulation of papers on this topic over the past decades has led to consider sometimes that surgery was disappearing from the therapeutic arsenal for moderately and advanced larynx and hypopharynx cancers. if larynx preservation is an undisputable advance in the management of laryngopharyngeal malignancies, surgery should not be considered as an obsolete treatment and only indicated for salvage. this situation may be explained by an overuse of the concept of " the standard of care is... " that does not pay enough attention to the potential gap between the selection of patients enrolled in clinical trials and the daily practice. the purpose of this review is to consider the current place of surgery in the armamentarium for larynx and hypopharynx cancer in the era of larynx preservation. actually the main milestone in larynx cancer surgery was the first total laryngectomy by billroth in 1873. at the very beginning of the 20th century the first case of larynx cancer treated by radiation therapy was reported in paris. for a long period of time surgery (either partial or radical) and radiation therapy in the 1970s, the german school pioneered the endoscopic laser microsurgery. at the same time, serial imaging (ctscan and mri) provided tremendously precise data on the local extension of the tumor allowing fine - tuning the choice of surgical procedures adapted to each situation. very recently transoral robotic surgery appeared as a new tool for endoscopic surgery to be evaluated. in the early 1980s chemotherapy, with the platinum compounds, became an additional option and in particular opened the era of larynx preservation clinical trials. induction chemotherapy (with the cisplatin-5 fluorouracil regimen) followed by radiation therapy in case of a reduction of at least 50% of the primary tumor size was the first experimental approach for larynx preservation. from published randomized trials (1 - 5) carried out in the us (va trial) and in europe (gettec trial and eortc 24891 trial), it appeared that this approach did not compromise the ultimate disease control and survival when compared with the conventional treatment (total laryngectomy and postoperative radiation therapy). a recent french trial (gortec 2000 - 01) compared a more intensive induction chemotherapy regimen (adding docetaxel to the conventional cisplatin-5 fluorouracil regimen). in this trial the larynx preservation rate (around 70%) was significantly higher with the triplet induction chemotherapy than with the doublet one but without statistical difference in survival (6). concomitant chemo - radiotherapy (radiotherapy delivered either concurrently or alternatively with chemotherapy) was thereafter compared to induction chemotherapy. one trial in the us (rtog 91 - 11) assessed concurrent chemo - radiotherapy (7, 8). if concurrent chemo - radiotherapy provided significantly higher larynx preservation (84%) than induction chemotherapy or radiation therapy alone, overall survival, progression - free survival and laryngectomy free survival did not significantly differ. one european trial compared (9) alternating chemo - radiotherapy and induction chemotherapy (eortc 24954) but there was no significant difference between both arms as regards survival and larynx preservation. more recently preliminary data suggested that induction chemotherapy followed by concurrent chemo - radiotherapy (10), radiotherapy with concurrent cetuximab (11) or induction chemotherapy followed by radiotherapy and concurrent cetuximab (12) should also be considered for larynx preservation altered fractionation has also been reported as to provide better results than conventional irradiation (13). there is no doubt that this clinical research has shifted some paradigms in the treatment of larynx and hypopharynx cancer leading to a declining use of surgery (14). one study evaluated the parallel evolution of care and of outcome of larynx cancer in the us (15). in this study on 158,426 larynx cancers, it appeared that from mid 80s to mid 90s the use of chemo - radiotherapy increased and the use of surgery decreased while survival progressively decreased over this period of time. in this survey, such data generated a discussion on the reliability and justification of larynx preserving strategies (17). however it must be stressed that a similar survey conducted in the european union has found on the contrary a decrease in mortality due to larynx cancer between 1970 and 2003 (18) despite an increasing popularity of larynx preservation protocols. most probably the discrepancies between the published trials in terms of eligibility criteria, primary endpoints selection and definition and secondary endpoints have generated some confusion. in particular " larynx preservation " was assessed in some studies only as " larynx in place " without capturing other data such as tracheotomy or feeding tube. in other studies a very restrictive definition was used compiling " larynx in place, no residual tumor, no tracheotomy, no feeding tube. " in addition survival was included in the primary endpoint (survival with a functional larynx in place) in few studies. two groups of experts have tried to clarify these issues in defining the guidelines for larynx cancer management (19) or for larynx preservation clinical trials (20, 21). obviously the place of surgery either as the initial treatment or as a salvage procedure must be regularly clarified. the head and neck surgeon has at his disposal an impressive number of partial procedures : for the supraglottic area : endoscopic laser microsurgery, hyoepiglottectomy, supraglottic laryngectomy, laterally extended supraglottic laryngectomy and supracricoid partial laryngectomy with cricohyoidopexyfor the glottic area : endoscopic laser microsurgery, cordectomy, frontolateral laryngectomy, frontal anterior laryngectomy, hemilaryngectomy and supracricoid partial laryngectomy with cricohyoidoepiglottopexyfor the hypopharynx : endoscopic microsurgery, lateral partial pharyngectomy, supracricoid hemilaryngopharyngectomy, supracricoid hemilaryngopharyngectomy for the supraglottic area : endoscopic laser microsurgery, hyoepiglottectomy, supraglottic laryngectomy, laterally extended supraglottic laryngectomy and supracricoid partial laryngectomy with cricohyoidopexy for the glottic area : endoscopic laser microsurgery, cordectomy, frontolateral laryngectomy, frontal anterior laryngectomy, hemilaryngectomy and supracricoid partial laryngectomy with cricohyoidoepiglottopexy for the hypopharynx : endoscopic microsurgery, lateral partial pharyngectomy, supracricoid hemilaryngopharyngectomy, supracricoid hemilaryngopharyngectomy in this list of surgical procedures the surgeon may select the most appropriate one on the basis of the endoscopic and radiologic respective features. when properly selected and performed this surgery insures the local control in more than 90% of the cases (22). the choice between both options is based on various parameters relating to the patient (age, occupation, compliance, whishes), to the tumor (size, macroscopic aspect, history of precancerous changes). whatever the treatment the disease is controlled in the vast majority of the cases, the most frequent carcinologic event during the follow - up is the appearance of metachronous cancer. there is no indication for adding chemotherapy to surgery or to radiation therapy. these cases are not in the frame of larynx preservation, the place of partial surgery is undisputable in this situation. when surgery is selected for the initial treatment, most of patients presenting with advanced larynx and hypopharynx carcinoma are offered total laryngectomy. however either endoscopic or open partial surgery may be still considered in selected cases. endoscopic laser microsurgery has been reported for rather advanced tumors of the larynx (23 - 28) or of the hypopharynx (29, 30). most of the time an open surgery of the neck (for nodal clearance) and a postoperative irradiation were associated to this endoscopic microsurgery. most of the time this surgery has been indicated for t2 diseases aiming to get better local control than with other partial procedures. however there are some reports of such a surgery performed for more advanced cases (31 - 34). it is undisputable that these indications are quite rare and only available for much selected patients and tumors and for highly experienced teams, but they do exist. first there is a semantic point that must be repeated each time organ preservation is discussed. organ preservation means and only means that the entire organ is still in place whether it is still functioning or not. keeping in place an organ that does not function any longer is of very limited interest for the patients. to this extend acute or late toxicity of non - surgical treatments may compromise the function of the preserved organ. what is meaningful actually is to preserve the function whether the organ is entirely preserved or not. that why partial surgery as an alternative to a total laryngectomy when feasible is also a larynx preservation strategy. to be honest it must be recognized that postoperative complications may also occur and compromise the function (as said above, selection of patient and expertise are prerequisites). it must be underscored that this distinction between larynx preservation and larynx function preservation is not always made even in published randomized trials. this distinction is also to be taken into account in the current ambience of treatment intensification that may generate substantial toxicity compromising definitively the larynx function. larynx function preservation is not the only goal when treating an advanced larynx or hypopharynx cancer. what patients are expecting first is to be cured and to survive long (35, 36). this means that we have to propose an alternative to a total laryngectomy pending this alternative may provide disease control and survival at least as good as initial radical surgery does. this means that the best way to assess a larynx preservation trial is to select a primary endpoint mixing the organ and function preservation, the local control and survival. it must be underscored that to date if survival was not compromised in the larynx preservation trials, none of the non surgical approaches has provided a better survival than initial radical surgery. in addition in trials comparing different approaches the difference in terms of larynx preservation did not translate into any difference in terms of overall disease - free and laryngectomy - free survival. this must be honestly and clearly explained to the patient before deciding a larynx preservation protocol as all patients are no willing to trade off chances of cure for keeping their larynx in place. this presentation is not accurate : larynx preservation is a priori a non - surgical approach that may turn into a surgical indication at any time if necessary. actually both survival and ultimate disease control have been maintained in the larynx preservation trials thanks to adequate salvage surgery. salvage surgery may be indicated : 1) after induction chemotherapy in patients who do not respond enough at the primary site, 2) after irradiation delivered after induction chemotherapy in case of residual local disease, 3) after concurrent chemo - radiotherapy in case of residual local disease or 4) during the follow - up in case of relapse. before the clinical research on larynx preservation was initiated, salvage surgery for irradiation failure had known major improvement in terms of postoperative morbidity and mortality thanks to the use of reliable flaps (in particular the major pectoralis muscular or myo - cutaneous flaps) for covering pharyngeal sutures and for protecting vessels. from the experience with induction chemotherapy we have learned that performing salvage surgery after induction chemotherapy was not a concern either for the quality of surgical margins or as regards postoperative morbidity (2). there are fewer data on salvage surgery after concurrent chemo - radiotherapy for larynx preservation. in the rtog-11 study the postoperative morbidity after salvage surgery was acceptable (37). however in the daily practice head and neck surgeons often face some concerns. during the phase of acute toxicity (epidermitis, mucositis, edema etc.) the difficulties come from the ability of detecting and assessing the local extension of a residual disease at the primary site or in the neck. salvage surgery may be compromised due to the uncertainties in evaluating surgical margins even with the use of frozen sections and due to potential healing problems as well. during the phase of late toxicity (fibrosis, sclerosis, edema etc.) surgery may be compromised by the difficulties of dissecting fibrotic tissues, by an increase rate of postoperative complications (early complications for healing or late complications such as stenosis and stricture). the risk of increased rates of acute and late toxicity must be thoroughly considered when discussing treatment intensification as in addition to compromising tolerance and compliance to treatment it could also compromise salvage surgery. but there is about no data of partial surgery for larynx preservation failure. in the absence of robust argument for or against partial surgery, we must be pragmatic. in case of persistent disease after chemotherapy, radiotherapy, concurrent chemo - radiotherapy or concurrent bio - chemotherapy in patients for whom the initial surgical indication would have been a total laryngectomy, radical salvage surgery is the logical proposal. for recurrent diseases during the follow - up, most of the time radical salvage surgery is the best option, however in case of late recurrence partial salvage surgery may be proposed in selected cases according to the local extension, the absence of severe tissue sequel and according to the length of the free interval. the other issue is the discussion of a systematic neck dissection in case of initial extensive nodal context. this attitude has been often advocated in the us and is certainly a " security option " but the risk of increasing late toxicity has been reported (38). larynx function preservation is one of the major advances that have been achieved over the past decades. however the indications of larynx preservation, whatever the protocol (based on the selection of patients with induction chemotherapy or based on concurrent chemo - radiotherapy) are limited to precise clinical situations. when partial surgery is feasible the surgical indication must be maintained. in case of very infiltrative transglottic tumors or in case of tumors extending through the cartilage, upfront radical surgery must be indicated. larynx preservation is indicated between these two clinical situations when tolerance and compliance to treatment seem acceptable. however the patients enrolled in larynx preservation protocols must be informed that surgery could be indicated during the process of these protocols. finally it must be underscored that as there are different treatment options, the best way to select the most appropriate one is a multidisciplinary decision making that is the golden standard of care. | over the past decades, randomized clinical trials have assessed and validated the concept of larynx preservation. this new concept has obviously modified the treatment algorithm for laryngopharyngeal squamous cell carcinoma. however surgery for larynx and hypopharynx cancer remains indicated in many cases. initial partial surgery is indicated for early diseases. this surgery may be performed endoscopically or openly. the results are excellent in terms of local control and function. transoral robotic surgery is under evaluation. initial radical surgery is indicated for advanced diseases in case of very infiltrative tumor, in case of cartilage destruction or when tolerance and/or compliance to chemotherapy - based approached is questionable. larynx preservation is to be discussed between these two situations. in randomized trials evaluating the different larynx preservation strategies, a substantial number of larynxes could be preserved without compromising disease control or survival. the best approach in terms of quality of function preservation, overall acute and late toxicity, disease control and survival is still a matter of clinical research. it must be stressed that salvage surgery is a definite part of these larynx preservation protocols in order to maintain the ultimate disease control. this discussion underscored the need of a multidisciplinary decision making and the need of a coordinated clinical research. |
nowadays, many different extensions of fss are known : l - fuzzy sets (l - fss), interval - valued fuzzy sets (ivfss), vague sets (vss), intuitionistic fuzzy sets (ifss), interval - valued intuitionistic fuzzy sets (ivifss), linguistic fuzzy sets (lfss), type-2 fuzzy sets (t2fss), type - n fuzzy sets (tnfss), and fuzzy multisets (fmss). an interesting extension of fss is the hesitant fuzzy set (hfs) in which the membership degree of an element is defined as a set of possible values. hfss are quite suitable to deal with the situation where we have a set of possible values, rather than a margin of error (as in ifss) or some possibility distribution on the possible values (as in t2fss). later, a number of other extensions of hfss have been developed such as dual hesitant fuzzy sets (dhfss), generalized hesitant fuzzy sets (g - hfss), and hesitant fuzzy linguistic term sets (hfltss). however, hfs and its recent generalization g - hfs have their inherent drawbacks, because they express the membership degrees of an element to a given set only by crisp numbers or ifss. in many practical decision making problems, the information provided by decision makers might often be described by fss instead of crisp numbers or by other fs extensions instead of ifss. this makes decision makers uncomfortable to provide exact crisp values or just ifss for the membership degrees. such a problem can be avoided by using the higher order hfs (hohfs) introduced originally by the author in for describing membership degrees. the hohfs is fit for the situation where the decision makers have a hesitation among several possible memberships for an element. the hohfs is the actual extension of hfs encompassing not only fss, ifss, vss, t2fss, fmss, and hfss, but also the recent extension of hfss, called g - hfss. since soft set theory was first initiated by molodtsov in as a new mathematical tool to deal efficiently with uncertainty and imprecision, many authors have become interested in applying this theory in various fields such as decision making, data analysis, and forecasting recently. moreover, a growing number of studies focus on the combination of fs and some extensions of fss with soft set. among them are, for instance, fuzzy soft set, intuitionistic fuzzy soft set, interval - valued fuzzy soft set, vague fuzzy soft set, and multifuzzy soft set. the rationale behind this study is that on the one hand we tend to propose a more general extension of fuzzy soft set encompassing the existing ones, and on the other hand we try to model those situations where the existing kinds of fuzzy soft set are not applicable. as mentioned above and it will be discussed later, the hohfs encompasses most of the fuzzy extensions as special cases, and therefore the combination of hohfs with soft set contains most of the existing kinds of fuzzy soft set as special cases, too. one of more interesting feature of the higher order hesitant fuzzy (hohf) soft set which will be proposed in this contribution is that we can correspond each parameter to levels with different dimensions, the situation that can not be modeled by the other kinds of fuzzy soft sets, specially by multifuzzy soft set. to make this clear, assume that x = { x1, x2, x3, x4, x5 } is the universe set indicating the set of five kinds of colored drawing paper for engineering. suppose the parameter set a = { e1, e2, e3 } is a set of three criteria to evaluate the performance of these papers, where e1 stands for thickness which includes three levels : thick, average, and thin, e2 stands for color which consists of three levels : red, green, and blue, and e3 stands for ingredient which is made from three levels : cellulose, hemicellulose, and lignin. but if this decision problem is reconsidered slightly different from the original problem as e1 stands for thickness which includes three levels : thick, average, and thin, e2 stands for color which consists of three levels : red, green, and blue, and e3 stands for ingredient which is made from two levels : cellulose and hemicellulose where the level lignin is not considered, then, obviously, this situation can not be handled not only by the existing kinds of fuzzy soft set theory, but also by multifuzzy soft set one in which all the parameters have to possess levels with the same dimension. to deal with such cases an extension of hfss, which is referred to as the higher order hfs (hohfs), is introduced in section 2. section 3 presents hohf soft sets and discusses the properties of hohf soft sets. in section 4, we apply the proposed hohf soft sets to multicriteria decision making problems involving parameters whose levels have different dimensions. this section is devoted to review the basic definitions and notions of fuzzy set (fs) and its new generalization which are originally referred to by farhadinia as the higher order hesitant fuzzy set (hohfs). actually, the hohfs is a generalization of hesitant fuzzy set (hfs) introduced by torra. an ordinary fuzzy set (fs) a in x is defined as a = { x, a(x):x x }, where a : x and the real value a(x) represents the degree of membership of x in a. definition 1. a generalized type of fuzzy set (g - type fs) on x is defined as (1)a~={x, a~(x):xx }, where (2)a~:x (). here, () denotes a family of crisp or fuzzy sets that can be defined within the universal set. a generalized type of fuzzy set (g - type fs) on x is defined as (1)a~={x, a~(x):xx }, where (2)a~:x (). here, () denotes a family of crisp or fuzzy sets that can be defined within the universal set. it is noteworthy that most of the existing extensions of ordinary fs are special cases of g - type fs, for instance, (i)if () =, then the g - type fs a~ reduces to an ordinary fs;(ii)if () = () denoting the set of all closed intervals, then the g - type fs a~ reduces to an ivfs;(iii)if () = () denoting the set of all ordinary fss, then the g - type fs a~ reduces to a t2fs;(iv)if () = l denoting a partially ordered lattice, then the g - type fs a~ reduces to a l - fs. if () =, then the g - type fs a~ reduces to an ordinary fs ; if () = () denoting the set of all closed intervals, then the g - type fs a~ reduces to an ivfs ; if () = () denoting the set of all ordinary fss, then the g - type fs a~ reduces to a t2fs ; if () = l denoting a partially ordered lattice, then the g - type fs a~ reduces to a l - fs. a hesitant fuzzy set (hfs) on x is symbolized by (3)h={x, h(x):xx }, where h(x), referred to as the hesitant fuzzy element (hfe), is a set of some values in denoting the possible membership degree of the element x x to the set h. let x be the universe of discourse. a hesitant fuzzy set (hfs) on x is symbolized by (3)h={x, h(x):xx }, where h(x), referred to as the hesitant fuzzy element (hfe), is a set of some values in denoting the possible membership degree of the element x x to the set h. example 3. if x = { x1, x2, x3 } is the universe of discourse, h(x1) = { 0.2,0.4,0.5 }, h(x2) = { 0.3,0.4 }, and h(x3) = { 0.3,0.2,0.5,0.6 } are the hfes of xi (i = 1,2, 3) to a set h, respectively. then h can be considered as a hfs ; that is, (4)h={x1,{0.2,0.4,0.5},x2,{0.3,0.4},iiiiiiiix3,{0.3,0.2,0.5.0.6}}. if x = { x1, x2, x3 } is the universe of discourse, h(x1) = { 0.2,0.4,0.5 }, h(x2) = { 0.3,0.4 }, and h(x3) = { 0.3,0.2,0.5,0.6 } are the hfes of xi (i = 1,2, 3) to a set h, respectively. then h can be considered as a hfs ; that is, (4)h={x1,{0.2,0.4,0.5},x2,{0.3,0.4},iiiiiiiix3,{0.3,0.2,0.5.0.6}}. as can be seen from definition 2, hfs expresses the membership degrees of an element to a given set only by several real numbers between 0 and 1, while in many real - world situations assigning exact values to the membership degrees does not describe properly the imprecise or uncertain decision information. thus, it seems to be difficult for the decision makers to rely on hfss for expressing uncertainty of an element. to overcome the difficulty associated with expressing uncertainty of an element to a given set, the concept of higher order hesitant fuzzy set (hohfs) is introduced here to let the membership degrees of an element to a given set be expressed by several possible g - type fss. a higher order hesitant fuzzy set (hohfs) on x is defined in terms of a function that when applied to x returns a set of g - type fss. a hohfs is denoted by (5)h~={x, h~(x):xx }, where h~(x), referred to as the higher order hesitant fuzzy element (hohfe), is a set of some g - type fss denoting the possible membership degree of the element x x to the set h~. in this regards, the hohfs h~ is also represented as (6)h~={x,{h~(1)(x),,h~(lx)(x)}:xx }, where all h~(1)(x),,h~(lx)(x) are g - type fss on x. let x be the universe of discourse. a higher order hesitant fuzzy set (hohfs) on x is defined in terms of a function that when applied to x returns a set of g - type fss. a hohfs is denoted by (5)h~={x, h~(x):xx }, where h~(x), referred to as the higher order hesitant fuzzy element (hohfe), is a set of some g - type fss denoting the possible membership degree of the element x x to the set h~. in this regards, the hohfs h~ is also represented as (6)h~={x,{h~(1)(x),,h~(lx)(x)}:xx }, where all h~(1)(x), if x = { x1, x2, x3 } is the universe of discourse, (7)iiih~(x1)={h~(1)(x1)=(0.2,0.4),h~(2)(x1)=(0.5,0.3)},iiih~(x2)={h~(1)(x2)=(0.3,0.4)},iiih~(x3)={h~(1)(x3)=(0.3,0.2),h~(2)(x3)=(0.1,0.3),iiiiiiiiiiiiiiiiih~(3)(x3)=(0.5,0.4) }, are the hohfes of xi(i = 1,2, 3) to a set h~, respectively, where g - type fss h~(k)(xi)=(ki,ki) are intuitionistic fuzzy set (ifs) such that 0 ki, ki 1 and 0 ki + ki 1 for k = 1,2,, then h~ can be considered as a hohfs ; that is, (8)h~={x1,{(0.2,0.4),(0.5,0.3)},x2,{(0.3,0.4)},iiiiiiiix3,{(0.3,0.2),(0.1,0.3),(0.5,0.4)}}. if x = { x1, x2, x3 } is the universe of discourse, (7)iiih~(x1)={h~(1)(x1)=(0.2,0.4),h~(2)(x1)=(0.5,0.3)},iiih~(x2)={h~(1)(x2)=(0.3,0.4)},iiih~(x3)={h~(1)(x3)=(0.3,0.2),h~(2)(x3)=(0.1,0.3),iiiiiiiiiiiiiiiiih~(3)(x3)=(0.5,0.4) }, are the hohfes of xi(i = 1,2, 3) to a set h~, respectively, where g - type fss h~(k)(xi)=(ki,ki) are intuitionistic fuzzy set (ifs) such that 0 ki, ki 1 and 0 ki + ki 1 for k = 1,2,, lxi and i = 1,2,, |x | = 3. then h~ can be considered as a hohfs ; that is, (8)h~={x1,{(0.2,0.4),(0.5,0.3)},x2,{(0.3,0.4)},iiiiiiiix3,{(0.3,0.2),(0.1,0.3),(0.5,0.4)}}. in view of definition 4, it is easily deduced that each hohfs becomes a t2fs if all its g - type fss are the same and are in the form of t2fs. that is, if h~(1)(x)==h~(lx)(x)=:h~(x) for any x x, then the hohfs h~={x, h~(x):xx } reduces to a t2fs. it is noteworthy that the notions of interval - valued hesitant fuzzy set (ivhfs) and interval type-2 fuzzy set (it2fs) both are special cases of hohfss. a hohfs h~={x, h~(x):xx } reduces to an ivhfs, when all g - type fss h~(1)(x),,h~(lx)(x) for any x x are considered as closed intervals of real numbers in. furthermore, an ivhfs h~={x,[hl~(x),hr~(x)]:xx } reduces to an it2fs, when all intervals satisfy [h~l(1)(x),h~r(1)(x)]==[h~l(lx)(x),h~r(lx)(x)]=:[h~l(x),h~r(x) ] for any x x. recently, hfss are extended by intuitionistic fuzzy sets (ifss) and referred to them as generalized hesitant fuzzy sets (g - hfss). it is stated in that fss, ifss, and hfss are special cases of g - hfss. obviously, a g - hfs h~={x, h~(x):xx } is also an special case of hohfs where all g - type fss h~(1)(x),,h~(lx)(x) for any x x are considered as ifss. this implies that hohfss are more useful than g - hfss to deal with decision making, clustering, pattern recognition, image processing, and so forth, when experts have a hesitation among several possible memberships for an element to a set. having introduced hohfes of a hohfs h~={x, h~(x):xx }, we now turn our attention to the interpretation of hohfs h~ as the union of all hohfes ; that is, (9)h~=h~h~{h~ } which is of fundamental importance in the study of hohfs aggregation and the higher order hesitant fuzzy soft matrix that will be introduced within the next parts of the paper. we give the definition of operation on hohfss based on its counterpart on hohfes as follows : (10)h~1h~2=h1~h1~,h2~h2~{h1~h2~}=h1~h1~,h2~h2~{hh1~h1~,hh2~h2~{hh1~hh2~ } }, where the last operation stands for the operation on g - type fss. we give the definition of operation on hohfss based on its counterpart on hohfes as follows : (10)h~1h~2=h1~h1~,h2~h2~{h1~h2~}=h1~h1~,h2~h2~{hh1~h1~,hh2~h2~{hh1~hh2~ } }, where the last operation stands for the operation on g - type fss. note that if there is no confusion, hohfs operator, hohfe operator, and g - type fs operator are denoted by the same symbol. notice in the case that g - type fss in hohfss are reduced to crisp values, the g - type fs operator in (10) is not the usual sum between real numbers because 0.6 + 0.5 = 1.1, and indeed the g - type fs operator is the algebraic sum where 0.6 0.5 = 0.6 + 0.5 0.6 0.5 = 0.3. thus, the hohfs operator is performed as the well - known hfs operator [29, 30 ]. if the operation on g - type fss has a property, denoted by (pg)~, then the operation on hohfes has the counterpart of property (pg)~, denoted by (p)~. let h~1=h~h1h~1{hh1~ } and h~2=h~h2h~2{hh2~ } be two hohfes. if the operation on g - type fss has a property, denoted by (pg)~, then the operation on hohfes has the counterpart of property (pg)~, denoted by (p)~. proofthe proof is obvious from definition 6. suppose that, for example, the operation on g - type fss is commutative ; that is, for any g - type fss hh1~h1~,hh2~h2~, the following equality holds : (11)hh1~hh2~=hh2~hh1~ (property (pg)~). then, one can see from definition 6 that (12)h~1h~2=hh1~h1~,hh2~h2~{hh1~hh2~}iiiiiiiiiii=hh1~h1~,hh2~h2~{hh2~hh1~}iiiiiiiiiii = h~2h~1 (property (p)~). this means that the operation on hohfes is accordingly commutative.by similar reasoning, one can easily prove that the hohfe operator inherits all the properties of the g - type fs operator. suppose that, for example, the operation on g - type fss is commutative ; that is, for any g - type fss hh1~h1~,hh2~h2~, the following equality holds : (11)hh1~hh2~=hh2~hh1~ (property (pg)~). then, one can see from definition 6 that (12)h~1h~2=hh1~h1~,hh2~h2~{hh1~hh2~}iiiiiiiiiii=hh1~h1~,hh2~h2~{hh2~hh1~}iiiiiiiiiii = h~2h~1 (property (p)~)., one can easily prove that the hohfe operator inherits all the properties of the g - type fs operator. as a corollary of theorem 7, it can be observed that hohfs operators inherit subsequently all operational properties of g - type fs operators. let h~1=h~1h~1{h1~ } and h~2=h~2h~2{h2~ } be two hohfss. if the operation on hohfes has a property, denoted by (p)~, then the operation on hohfss has the counterpart of property (p)~, denoted by (p)~. let h~1=h~1h~1{h1~ } and h~2=h~2h~2{h2~ } be two hohfss. if the operation on hohfes has a property, denoted by (p)~, then the operation on hohfss has the counterpart of property (p)~, denoted by (p)~. example 9. let h~1=h~1h~1{h1~ } and h~2=h~2h~2{h2~ } be two hohfss with hohfes in the form of ifss. it can be shown for any ifss hh1~,hh2~ that (13)iiiiihh1~hh2~=hh2~hh1~ ; (property (pg1)~),iiiiihh1~hh2~=hh2~hh1~ ; (property (pg2)~),(hh1~hh2~)=hh1~hh2~, >0 ; (property (pg3)~),i(hh1~hh2~)=hh1~hh2~, >0 ; (property (pg4)~),(1+2)hh1~=1hh1~2hh1~,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii1,2>0 ; (property (pg5)~),iiiiihh1~(1+2)=hh1~1hh1~2,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii1,2>0 ; (property (pg6)~), where (see e.g.,) (14)hh1~hh2~=hh1~,hh1~hh2~,hh2~iiiiiiiiiiiii=hh1~+hh2~hh1~hh2~,hh1~hh2~;hh1~hh2~=hh1~,hh1~hh2~,hh2~iiiiiiiiiiiii=hh1~hh2~,hh1~+hh2~hh1~hh2~;hh1~=1(1hh1~),(hh1~), >0;hh1~=(hh1~),1(1hh1~), >0. accordingly, from corollary 8, it is deduced that (15)iiiiiih1~h2~=h2~h1~ ; (property (p1)~),iiiiiih1~h2~=h2~h1~ ; (property (p2)~),i(h1~h2~)=h1~h2~, >0 ; (property (p3)~),i(h1~h2~)=h1~h2~, >0 ; (property (p4)~),(1+2)h1~=1h1~2h1~,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii1,2>0 ; (property (p5)~),iiiih1~(1+2)=h1~1h1~2,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii1,2>0 ; (property (p6)~). let h~1=h~1h~1{h1~ } and h~2=h~2h~2{h2~ } be two hohfss with hohfes in the form of ifss. it can be shown for any ifss hh1~,hh2~ that (13)iiiiihh1~hh2~=hh2~hh1~ ; (property (pg1)~),iiiiihh1~hh2~=hh2~hh1~ ; (property (pg2)~),(hh1~hh2~)=hh1~hh2~, >0 ; (property (pg3)~),i(hh1~hh2~)=hh1~hh2~, >0 ; (property (pg4)~),(1+2)hh1~=1hh1~2hh1~,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii1,2>0 ; (property (pg5)~),iiiiihh1~(1+2)=hh1~1hh1~2,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii1,2>0 ; (property (pg6)~), where (see e.g.,) (14)hh1~hh2~=hh1~,hh1~hh2~,hh2~iiiiiiiiiiiii=hh1~+hh2~hh1~hh2~,hh1~hh2~;hh1~hh2~=hh1~,hh1~hh2~,hh2~iiiiiiiiiiiii=hh1~hh2~,hh1~+hh2~hh1~hh2~;hh1~=1(1hh1~),(hh1~), >0;hh1~=(hh1~),1(1hh1~), >0. accordingly, from corollary 8, it is deduced that (15)iiiiiih1~h2~=h2~h1~ ; (property (p1)~),iiiiiih1~h2~=h2~h1~ ; (property (p2)~),i(h1~h2~)=h1~h2~, >0 ; (property (p3)~),i(h1~h2~)=h1~h2~, >0 ; (property (p4)~),(1+2)h1~=1h1~2h1~,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii1,2>0 ; (property (p5)~),iiiih1~(1+2)=h1~1h1~2,iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii1,2>0 ; (property (p6)~). in the following, we first review briefly the concepts of soft set and fuzzy soft set. to do this, it may be helpful to recall that the sets x, (x), and e are referred to as the initial universe set, the power set of x, and a set of parameters, respectively. is called a soft set over x, where f is a mapping given by f : a e (x). a pair (f, a) is called a soft set over x, where f is a mapping given by f : a e (x). indeed, a soft set over x is not a set but a parameterized family of subsets of x. example 11. suppose that x = { x1, x2, x3, x4 } is a set of houses under consideration and a = { e1, e2, e3 } is a set of parameter, where e1 = expensive, e2 = beautiful, and e3 = in the good location. let (16)f(e1)={x1 }, f(e2)={x1,x3 }, f(e3)={x2,x4}. we use here the soft set (f, a) to describe the attractiveness of the houses. in this regard, f(e1) stands for houses (expensive) whose function value is the set { x1 }, f(e2) means houses (beautiful) whose function value is the set { x1, x3 }, and f(e3) implies houses (in the good location) whose function value is the set { x2, x4}. suppose that x = { x1, x2, x3, x4 } is a set of houses under consideration and a = { e1, e2, e3 } is a set of parameter, where e1 = expensive, e2 = beautiful, and e3 = in the good location. let (16)f(e1)={x1 }, f(e2)={x1,x3 }, f(e3)={x2,x4}. we use here the soft set (f, a) to describe the attractiveness of the houses. in this regard, f(e1) stands for houses (expensive) whose function value is the set { x1 }, f(e2) means houses (beautiful) whose function value is the set { x1, x3 }, and f(e3) implies houses (in the good location) whose function value is the set { x2, x4}. definition 12 (see). a pair (f,a) is called a fuzzy soft set over x where (x) is the set of all fuzzy subsets of x. let f:ae(x). a pair (f,a) is called a fuzzy soft set over x where (x) is the set of all fuzzy subsets of x. example 13. we can describe the attractiveness of the houses under the fuzzy circumstances using the fuzzy soft set (f,a), where (17)f(e1)={x11,x20,x30,x40},f(e2)={x11,x20,x31,x40},f(e3)={x10,x21,x30,x41}. this example shows that if we let the corresponding membership degrees of all elements e e a be zero, then fuzzy soft sets (f,a) and (f,e) bear the same meaning and have the unified forms. we can describe the attractiveness of the houses under the fuzzy circumstances using the fuzzy soft set (f,a), where (17)f(e1)={x11,x20,x30,x40},f(e2)={x11,x20,x31,x40},f(e3)={x10,x21,x30,x41}. this example shows that if we let the corresponding membership degrees of all elements e e a be zero, then fuzzy soft sets (f,a) and (f,e) bear the same meaning and have the unified forms. let f~:e~(x). a pair (f~,e) is called a higher order hesitant fuzzy (hohf) soft set over x where ~(x) is the set of all higher order hesitant fuzzy subsets of x. in this regard, for any e e, we denote (18)f~(e):={xh~f~(e)(x):xx}iiiiiiii={x{h~f~(e)(1)(x),,h~f~(e)(lx)(x)}:xx }, where all h~f~(e)(1)(x),,h~f~(e)(lx)(x) are g - type fss on x. that is to say, that the higher order hesitant fuzzy (hohf) soft set over x can be denoted by (19)(f~,e)=xxx{h~f~(e)(1)(x),,h~f~(e)(lx)(x)}. we hereafter denote all hohf soft sets over x by hohfs(x) a pair (f~,e) is called a higher order hesitant fuzzy (hohf) soft set over x where ~(x) is the set of all higher order hesitant fuzzy subsets of x. in this regard, for any e e, we denote (18)f~(e):={xh~f~(e)(x):xx}iiiiiiii={x{h~f~(e)(1)(x),,h~f~(e)(lx)(x)}:xx }, where all h~f~(e)(1)(x),,h~f~(e)(lx)(x) are g - type fss on x. that is to say, that the higher order hesitant fuzzy (hohf) soft set over x can be denoted by (19)(f~,e)=xxx{h~f~(e)(1)(x),,h~f~(e)(lx)(x)}. we hereafter denote all hohf soft sets over x by hohfs(x). suppose that x = { x1, x2, x3, x4 } is a set of color cloths under consideration and a = { e1, e2, e3 } is a set of parameter where e1 = color which consists of red and blue, e2 = ingredient which is made from wool, cotton, and acrylic, and e3 = price which can be considered as high and low. in this situation, we should consider a higher order hesitant fuzzy soft set (f~,e) as follows : (20)f~(e1)={x1{(0.1,0.2),(0.3,0.2)},x2{(0.2,0.3),(0.3,0.1)},iiiiiiiix3{(0.3,0.3),(0.4,0.6)},x4{(0.1,0.3),(0.3,0.1)}},f~(e2)={x1{(0.5,0.2),(0.1,0.2),(0.3,0.2)},iiiiiiiiiiiiiix2{(0.2,0.4),(0.6,0.1),(0.3,0.1)},iiiiiiiiiiiiiix3{(0.3,0.3),(0.4,0.4),(0.4,0.6)},iiiiiiiiiiiiiix4{(0.1,0.3),(0.3,0.1),(0.2,0.5)}},f~(e3)={x1{(0.5,0,2),(0.4,0.1)},x2{(0.3,0.7),(0.6,0.1)},iiiiiiiix3{(0.4,0.4),(0.4,0.6)},x4{(0.8,0.2),(0.2,0.5) } }, where the above g - type fss, denoted by (,), are all in the form of ifs on x. suppose that x = { x1, x2, x3, x4 } is a set of color cloths under consideration and a = { e1, e2, e3 } is a set of parameter where e1 = color which consists of red and blue, e2 = ingredient which is made from wool, cotton, and acrylic, and e3 = price which can be considered as high and low. in this situation, we should consider a higher order hesitant fuzzy soft set (f~,e) as follows : (20)f~(e1)={x1{(0.1,0.2),(0.3,0.2)},x2{(0.2,0.3),(0.3,0.1)},iiiiiiiix3{(0.3,0.3),(0.4,0.6)},x4{(0.1,0.3),(0.3,0.1)}},f~(e2)={x1{(0.5,0.2),(0.1,0.2),(0.3,0.2)},iiiiiiiiiiiiiix2{(0.2,0.4),(0.6,0.1),(0.3,0.1)},iiiiiiiiiiiiiix3{(0.3,0.3),(0.4,0.4),(0.4,0.6)},iiiiiiiiiiiiiix4{(0.1,0.3),(0.3,0.1),(0.2,0.5)}},f~(e3)={x1{(0.5,0,2),(0.4,0.1)},x2{(0.3,0.7),(0.6,0.1)},iiiiiiiix3{(0.4,0.4),(0.4,0.6)},x4{(0.8,0.2),(0.2,0.5) } }, where the above g - type fss, denoted by (,), are all in the form of ifs on x. definition 16. a higher order hesitant fuzzy soft set (f~,e) over x is called a null higher order hesitant fuzzy soft set over x denoted by 0~, if for any e e(21)f~(e):={xh~f~(e)(x):xx}={x{0~,,0~}:xx }, where 0~ is the zero g - type fs on x. a higher order hesitant fuzzy soft set (f~,e) over x is called a null higher order hesitant fuzzy soft set over x denoted by 0~, if for any e e(21)f~(e):={xh~f~(e)(x):xx}={x{0~,,0~}:xx }, where 0~ is the zero g - type fs on x. definition 17. a higher order hesitant fuzzy soft set (f~,e) over x is called an absolute higher order hesitant fuzzy soft set over x denoted by 1~, if for any e e(22)f~(e):={xh~f~(e)(x):xx}={x{1~,,1~}:xx }, where 1~ is the one g - type fs on x. a higher order hesitant fuzzy soft set (f~,e) over x is called an absolute higher order hesitant fuzzy soft set over x denoted by 1~, if for any e e(22)f~(e):={xh~f~(e)(x):xx}={x{1~,,1~}:xx }, where 1~ is the one g - type fs on x. definition 18 (see). consider two hohfss h~1=h~1h~1{h1~ } and h~2=h~2h~2{h2~ } on x = { x1, x2, xn}. the definition of distance measure for hohfss is given by (23)d(h~1,h~2)=1ni=1ndh(h~1(xi),h~2(xi)), where for each xi x(24)dh(h~1(xi),h~2(xi)) = max{maxh~1h~1minh~2h~2d(h~1(xi),h~2(xi)),iiiiiiiiiiiiiiiminh~1h~1maxh~2h~2d(h~1(xi),h~2(xi))}. here, d(,) is a distance measure defined on g - type fss. consider two hohfss h~1=h~1h~1{h1~ } and h~2=h~2h~2{h2~ } on x = { x1, x2, xn}. the definition of distance measure for hohfss is given by (23)d(h~1,h~2)=1ni=1ndh(h~1(xi),h~2(xi)), where for each xi x(24)dh(h~1(xi),h~2(xi)) = max{maxh~1h~1minh~2h~2d(h~1(xi),h~2(xi)),iiiiiiiiiiiiiiiminh~1h~1maxh~2h~2d(h~1(xi),h~2(xi))}. here, d(,) is a distance measure defined on g - type fss. notice that to have a correct comparison between two hohfss h~1 and h~2, the two following cases should be taken into account. the first case occurs if all g - type fss in each hohfes of hohfs can be arranged increasingly (or decreasingly) by the use of a partial order. another case occurs when all g - type fss in each hohfes of hohfs can not be arranged increasingly (or decreasingly) using a partial order. in view of the above arguments, we shall represent the definition of a higher order hesitant fuzzy soft subset as follows. (f~,e) is said to be a higher order hesitant fuzzy soft subset of (g~,e) if (i)(in the ordered case) for all e e and for all x x it holds (25)h~f~(e)(k)(x)h~g~(e)(k)(x), 1klx, where stands for a partial order defined on g - type fss;(ii)(in the disordered case) for all e e and for all x x one has (26)dh(h~f~(e)(x),1~)dh(h~g~(e)(x),1~), where dh(,) is that given in definition 18 and 1~ is the absolute higher order hesitant fuzzy soft set over x. suppose that (f~,e),(g~,e)hohfs(x). (f~,e) is said to be a higher order hesitant fuzzy soft subset of (g~,e) if (i)(in the ordered case) for all e e and for all x x it holds (25)h~f~(e)(k)(x)h~g~(e)(k)(x), 1klx, where stands for a partial order defined on g - type fss;(ii)(in the disordered case) for all e e and for all x x one has (26)dh(h~f~(e)(x),1~)dh(h~g~(e)(x),1~), where dh(,) is that given in definition 18 and 1~ is the absolute higher order hesitant fuzzy soft set over x. (in the ordered case) for all e e and for all x x it holds (25)h~f~(e)(k)(x)h~g~(e)(k)(x), 1klx, where stands for a partial order defined on g - type fss ; (in the disordered case) for all e e and for all x x one has (26)dh(h~f~(e)(x),1~)dh(h~g~(e)(x),1~), where dh(,) is that given in definition 18 and 1~ is the absolute higher order hesitant fuzzy soft set over x. the inclusion relation between (f~,e) and (g~,e) is denoted by (f~,e)~(g~,e). definition 20. two higher order hesitant fuzzy soft sets (f~,e),(g~,e) are said to be higher order hesitant fuzzy soft equal sets if and only if (f~,e)~(g~,e) and (g~,e)~(f~,e). two higher order hesitant fuzzy soft sets (f~,e),(g~,e) are said to be higher order hesitant fuzzy soft equal sets if and only if (f~,e)~(g~,e) and (g~,e)~(f~,e). definition 21. (fc~,e) is said to be a higher order hesitant fuzzy soft complementary set of (f~,e) if for all e e and for all x x(27)h~fc~(e)(k)(x)=(h~f~(e)(k)(x))c, 1klx, where (h~f~(e)(k)(x))c means the complement of the corresponding g - type fss h~f~(e)(k)(x). (fc~,e) is said to be a higher order hesitant fuzzy soft complementary set of (f~,e) if for all e e and for all x x(27)h~fc~(e)(k)(x)=(h~f~(e)(k)(x))c, 1klx, where (h~f~(e)(k)(x))c means the complement of the corresponding g - type fss h~f~(e)(k)(x). union of (f~,e),(g~,e)hohfs(x), denoted by (f~,e)~(g~,e), is defined for all e e as (28)(m~,e) = (f~,e)~(g~,e) = xxx{(h~f~(e)(1)(x)h~g~(e)(1)(x)),,(h~f~(e)(lx)(x)h~g~(e)(lx)(x))}. union of (f~,e),(g~,e)hohfs(x), denoted by (f~,e)~(g~,e), is defined for all e e as (28)(m~,e) = (f~,e)~(g~,e) = xxx{(h~f~(e)(1)(x)h~g~(e)(1)(x)), intersection of (f~,e),(g~,e)hohfs(x), denoted by (f~,e)~(g~,e), is defined for all e e as (29)(n~,e) = (f~,e)~(g~,e) = xxx{(h~f~(e)(1)(x)h~g~(e)(1)(x)),,(h~f~(e)(lx)(x)h~g~(e)(lx)(x))}. intersection of (f~,e),(g~,e)hohfs(x), denoted by (f~,e)~(g~,e), is defined for all e e as (29)(n~,e) = (f~,e)~(g~,e) = xxx{(h~f~(e)(1)(x)h~g~(e)(1)(x)), ((f~,e)~(g~,e))c=(f~,e)c~(g~,e)c:=(fc~,e)~(gc~,e), ((f~,e)~(g~,e))c=(f~,e)c~(g~,e)c:=(fc~,e)~(gc~,e). proof part (1). in this regard, ((f~,e)~(g~,e))c=(m~,e)c:=(mc~,e). on the other hand, from definition 22 one gets (30)((f~,e)~(g~,e))c = (mc~,e) = xxx{(h~f~(e)(1)(x)h~g~(e)(1)(x))c,,(h~f~(e)(lx)(x)h~g~(e)(lx)(x))c } = xx(,((h~f~(e)(lx)(x))c(h~g~(e)(lx)(x))c)})1x(((h~f~(e)(lx)(x))c(h~g~(e)(lx)(x))c){((h~f~(e)(1)(x))c(h~g~(e)(1)(x))c),,iiiiiiiiiiiiiiiiiiiiiiiii((h~f~(e)(lx)(x))c(h~g~(e)(lx)(x))c)})1) = xxx{(h~fc~(e)(1)(x)h~gc~(e)(1)(x)),,(h~fc~(e)(lx)(x)h~gc~(e)(lx)(x)) } = (fc~,e)~(gc~,e) = (f~,e)c~(g~,e)c [proved ]. in this regard, ((f~,e)~(g~,e))c=(m~,e)c:=(mc~,e). on the other hand, from definition 22 one gets (30)((f~,e)~(g~,e))c = (mc~,e) = xxx{(h~f~(e)(1)(x)h~g~(e)(1)(x))c,,(h~f~(e)(lx)(x)h~g~(e)(lx)(x))c } = xx(,((h~f~(e)(lx)(x))c(h~g~(e)(lx)(x))c)})1x(((h~f~(e)(lx)(x))c(h~g~(e)(lx)(x))c){((h~f~(e)(1)(x))c(h~g~(e)(1)(x))c),,iiiiiiiiiiiiiiiiiiiiiiiii((h~f~(e)(lx)(x))c(h~g~(e)(lx)(x))c)})1) = xxx{(h~fc~(e)(1)(x)h~gc~(e)(1)(x)),,(h~fc~(e)(lx)(x)h~gc~(e)(lx)(x)) } = (fc~,e)~(gc~,e) = (f~,e)c~(g~,e)c [proved ]. part (2). then (1)((f~,e)~(g~,e))~(k~,e)=(f~,e)~((g~,e)~(k~,e)), (2)((f~,e)~(g~,e))~(k~,e)=(f~,e)~((g~,e)~(k~,e)), (3)(f~,e)~((g~,e)~(k~,e))=((f~,e)~(g~,e))~((f~,e)~(k~,e)), (4)(f~,e)~((g~,e)~(k~,e))=((f~,e)~(g~,e))~((f~,e)~(k~,e)). then (1)((f~,e)~(g~,e))~(k~,e)=(f~,e)~((g~,e)~(k~,e)), (2)((f~,e)~(g~,e))~(k~,e)=(f~,e)~((g~,e)~(k~,e)), (3)(f~,e)~((g~,e)~(k~,e))=((f~,e)~(g~,e))~((f~,e)~(k~,e)), (4)(f~,e)~((g~,e)~(k~,e))=((f~,e)~(g~,e))~((f~,e)~(k~,e)). ((f~,e)~(g~,e))~(k~,e)=(f~,e)~((g~,e)~(k~,e)), ((f~,e)~(g~,e))~(k~,e)=(f~,e)~((g~,e)~(k~,e)), (f~,e)~((g~,e)~(k~,e))=((f~,e)~(g~,e))~((f~,e)~(k~,e)), (f~,e)~((g~,e)~(k~,e))=((f~,e)~(g~,e))~((f~,e)~(k~,e)). let (f~,e)hohfs(x), let 1~ be the absolute higher order hesitant fuzzy soft set over x, and let 0~ be the null higher order hesitant fuzzy soft set over x. then (1)(f~,e)~(f~,e)=(f~,e), (2)(f~,e)~(f~,e)=(f~,e), (3)(f~,e)~1~=1~, (4)(f~,e)~1~=(f~,e), (5)(f~,e)~0~=0~, (6)(f~,e)~0~=(f~,e). let (f~,e)hohfs(x), let 1~ be the absolute higher order hesitant fuzzy soft set over x, and let 0~ be the null higher order hesitant fuzzy soft set over x. then (1)(f~,e)~(f~,e)=(f~,e), (2)(f~,e)~(f~,e)=(f~,e), (3)(f~,e)~1~=1~, (4)(f~,e)~1~=(f~,e), (5)(f~,e)~0~=0~, (6)(f~,e)~0~=(f~,e). (f~,e)~(f~,e)=(f~,e), (f~,e)~(f~,e)=(f~,e), proofthe proof is straightforward. in what follows, we are interested to present an application of hohf soft set theory in a multiple criterion decision making problem. in order to solve decision making problems involving hohf soft sets, an approach is proposed based on the concept of -level higher order hesitant fuzzy soft set. | = m. the higher order hesitant fuzzy soft matrix ~ is defined in the concrete form of (31)f~=(e1 emx1h~f~(e1)(x1) h~f~(em)(x1)xnh~f~(e1)(xn) h~f~(em)(xn))ii=[h~f~(ej)(xi)]nm. we denote all higher order hesitant fuzzy soft matrix ~ over x by hohfsm(x). | = m. the higher order hesitant fuzzy soft matrix ~ is defined in the concrete form of (31)f~=(e1 emx1h~f~(e1)(x1) h~f~(em)(x1)xnh~f~(e1)(xn) h~f~(em)(xn))ii=[h~f~(ej)(xi)]nm. we denote all higher order hesitant fuzzy soft matrix ~ over x by hohfsm(x). as can be seen from definitions 27 and 14, there exists a bijective mapping from hohfs(x) to hohfsm(x), implying that any higher order hesitant fuzzy soft set (f~,e)hohfs(x) can be equivalent with its higher order hesitant fuzzy soft matrix ~hohfsm(x). this equivalent relationship allows us to identify any (f~,e)hohfs(x) with its ~hohfsm(x), interchangeably some arithmetic operations on hohfsm(x) can be defined as follows : (32)f~g~=[h~f~(ej)(xi)h~g~(ej)(xi)]nm, f~g~=[h~f~(ej)(xi)h~g~(ej)(xi)]nm, iiiif~=[(h~f~(ej)(xi))]nm, >0,iiiif~=[(h~f~(ej)(xi))]nm, >0, where the right - hand - side operations are that considered on g - type fss in definition 6. some arithmetic operations on hohfsm(x) can be defined as follows : (32)f~g~=[h~f~(ej)(xi)h~g~(ej)(xi)]nm, f~g~=[h~f~(ej)(xi)h~g~(ej)(xi)]nm, iiiif~=[(h~f~(ej)(xi))]nm, >0,iiiif~=[(h~f~(ej)(xi))]nm, >0, where the right - hand - side operations are that considered on g - type fss in definition 6. accordingly, from definition 28 and corollary 8, it can be deduced for any ~,~hohfsm(x) that (33)iiiiiiiif~g~=g~f~,iiiiiiiif~g~=g~f~,iii(f~g~)=f~g~, >0,iii(f~g~)=f~g~, >0,i(1+2)f~=1f~2f~, 1,2>0,iiiiiif~(1+2)=f~1f~2, 1,2>0. the latter results can be employed whenever some experts are invited to conduct evaluation for one decision making problem. in such a situation, we are required to use aggregation operators on hohfsm(x) given by experts to obtain a collective ~hohfsm(x). however, dealing with the aggregation methods based on multiexperts ' higher order hesitant fuzzy soft matrix is beyond the scope of this paper. before introducing the concept of the -level higher order hesitant fuzzy soft set, we define a threshold vector over parameter set e as (e) = ((e1),, (em)) where (ej)=h~(ej) (j=1,,m). obviously, the hohfes h~(ej) (j=1,,m) are changed dependently on the different parameter set e. consider again the higher order hesitant fuzzy soft matrix ~ given in the concrete form of ~=[h~f~(ej)(xi)]nm. in order to get reasonably a hohfe for each parameter ej e from ~ we define (34)h~(ej)agg = agg(h~f~(ej)(x1),,h~f~(ej)(xn)),iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiieje(j=1,,m), where agg() is an aggregation operator on hohfes. in view of the latter setting, the threshold vector over parameter set e is defined as (35)agg(e)={agg(ej), j=1,,m}iiiiiiiiiiii={h~(ej)agg, j=1, a real function i : hohfe(x) hohfe(x) is called the inclusion measure of the hohfes h~f~(e)(x)={h~f~(e)(k)(x)}k=1lx, h~g~(e)(x)={h~g~(e)(k)(x)}k=1lx, and h~k~(e)(x)={h~k~(e)(k)(x)}k=1lx, if i(,) satisfies the following properties. (2)i(h~f~(e)(x),h~g~(e)(x))=1 iff (36)h~f~(e)(x)~h~g~(e)(x) iff h~f~(e)(k)(x)h~g~(e)(k)(x),iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiik=1,,lx, where the last inclusion operator is that introduced on g - type fss.(3)if h~f~(e)(x)~h~g~(e)(x)~h~k~(e)(x), then i(h~k~(e)(x),h~f~(e)(x))i(h~g~(e)(x),h~f~(e)(x)) and i(h~k~(e)(x),h~f~(e)(x))i(h~k~(e)(x),h~g~(e)(x)). let us now define the following two - valued function i(,) on g - type fss h~f~(e)(k)(x) and h~g~(e)(k)(x) as (37)i(h~f~(e)(k)(x),h~g~(e)(k)(x)) = { 1,if h~f~(e)(k)(x) h~g~(e)(k)(x), k=1,,lx,;0,otherwise, and then, the following function is an inclusion measure of the hohfes h~f~(e)(x)={h~f~(e)(k)(x)}k=1lx and h~g~(e)(x)={h~g~(e)(k)(x)}k=1lx given by (38)i(h~f~(e)(x),h~g~(e)(x))=1lxk=1lxi(h~f~(e)(k)(x),h~g~(e)(k)(x)). a real function i : hohfe(x) hohfe(x) is called the inclusion measure of the hohfes h~f~(e)(x)={h~f~(e)(k)(x)}k=1lx, h~g~(e)(x)={h~g~(e)(k)(x)}k=1lx, and h~k~(e)(x)={h~k~(e)(k)(x)}k=1lx, if i(,) satisfies the following properties. (2)i(h~f~(e)(x),h~g~(e)(x))=1 iff (36)h~f~(e)(x)~h~g~(e)(x) iff h~f~(e)(k)(x)h~g~(e)(k)(x),iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiik=1,,lx, where the last inclusion operator is that introduced on g - type fss.(3)if h~f~(e)(x)~h~g~(e)(x)~h~k~(e)(x), then i(h~k~(e)(x),h~f~(e)(x))i(h~g~(e)(x),h~f~(e)(x)) and i(h~k~(e)(x),h~f~(e)(x))i(h~k~(e)(x),h~g~(e)(x)). let us now define the following two - valued function i(,) on g - type fss h~f~(e)(k)(x) and h~g~(e)(k)(x) as (37)i(h~f~(e)(k)(x),h~g~(e)(k)(x)) = { 1,if h~f~(e)(k)(x) h~g~(e)(k)(x), k=1,,lx,;0,otherwise, and then, the following function is an inclusion measure of the hohfes h~f~(e)(x)={h~f~(e)(k)(x)}k=1lx and h~g~(e)(x)={h~g~(e)(k)(x)}k=1lx given by (38)i(h~f~(e)(x),h~g~(e)(x))=1lxk=1lxi(h~f~(e)(k)(x),h~g~(e)(k)(x)). i(h~f~(e)(x),h~g~(e)(x))=1 iff (36)h~f~(e)(x)~h~g~(e)(x) iff h~f~(e)(k)(x)h~g~(e)(k)(x),iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiik=1,,lx, where the last inclusion operator is that introduced on g - type fss. if h~f~(e)(x)~h~g~(e)(x)~h~k~(e)(x), then i(h~k~(e)(x),h~f~(e)(x))i(h~g~(e)(x),h~f~(e)(x)) and i(h~k~(e)(x),h~f~(e)(x))i(h~k~(e)(x),h~g~(e)(x)). definition 30. we define the (e)-level higher order hesitant fuzzy soft set of ~hohfsm(x), denoted by l(~,(e)), as follows : (39)l(f~,(ej))={xix i((ej),h~f~(ej)(xi))>0},iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiieje(j=1,,m), where i(,) is the inclusion measure introduced by (38). we define the (e)-level higher order hesitant fuzzy soft set of ~hohfsm(x), denoted by l(~,(e)), as follows : (39)l(f~,(ej))={xix i((ej),h~f~(ej)(xi))>0},iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiieje(j=1,,m), where i(,) is the inclusion measure introduced by (38). as a result from definition 30 and (35), the agg(e)-level higher order hesitant fuzzy soft set of ~hohfsm(x) is characterized by (40)l(f~,agg(ej)) = { xix i(h~(ej)agg, h~f~(ej)(xi))>0}. once the (e)-level higher order hesitant fuzzy soft set of ~ is achieved, we calculate the inclusion measure values i((ej),h~f~(ej)(xi)) for ej (j = 1,, m) and xi (i = 1,, n) given by (38) to rank all the alternatives. the optimal decision is then selected as xk if (41)j=1mi((ej),h~f~(ej)(xk)) = maxi=1,,n{j=1mi((ej),h~f~(ej)(xi))}. it is noteworthy to say that if hohfes h~f~(ej)(xi) reduce to ifss, then the inclusion measure values i((ej),h~f~(ej)(xi)) are coincided to choice values ci of xi (i = 1,, now we are in a position to present an algorithm for solving higher order hesitant fuzzy soft set based decision making problem by using (e)-level higher order hesitant fuzzy soft sets initiated in this study. input the higher order hesitant fuzzy soft matrix ~ over a finite initial universe x and a finite parameter set e. input the higher order hesitant fuzzy soft matrix ~ over a finite initial universe x and a finite parameter set e. step 2. compute the threshold vector (e) = ((e1),, (em)) corresponding to ~. compute the threshold vector (e) = ((e1),, (em)) corresponding to ~. step 3. calculate inclusion measure value of all alternatives to determine their ranking and then selecting the optimal decision by the use of (41). calculate inclusion measure value of all alternatives to determine their ranking and then selecting the optimal decision by the use of (41). to illustrate the idea of algorithm 31, let us take into consideration the following example which addresses example 15 with more details. suppose that x = { x1, x2, x3, x4 } is a set of color cloths under consideration and a = { e1, e2, e3 } is a set of parameter where e1 = color which consists of red and blue, e2 = ingredient which is made from wool, cotton, and acrylic, and e3 = price which can be considered as high and low. in this situation, we should consider a higher order hesitant fuzzy soft set (f~,e) as follows : (42)f~(e1)={x1{(0.1,0.2),(0.3,0.2)},x2{(0.2,0.3),(0.3,0.1)},iiiiiiiiiiiiix3{(0.3,0.3),(0.4,0.6)},x4{(0.1,0.3),(0.3,0.1)}},f~(e2)={x1{(0.5,0.2),(0.1,0.2),(0.3,0.2)},iiiiiiiiiiiiix2{(0.2,0.4),(0.6,0.1),(0.3,0.1)},iiiiiiiiiiiiix3{(0.3,0.3),(0.4,0.4),(0.4,0.6)},iiiiiiiiiiiiix4{(0.1,0.3),(0.3,0.1),(0.2,0.5)}},f~(e3)={x1{(0.5,0,2),(0.4,0.1)},x2{(0.3,0.7),(0.6,0.1)},iiiiiiiiiiiiix3{(0.4,0.4),(0.4,0.6)},x4{(0.8,0.2),(0.2,0.5) } }, where the above g - type fss, denoted by (,), are all in the form of ifs on x. suppose that x = { x1, x2, x3, x4 } is a set of color cloths under consideration and a = { e1, e2, e3 } is a set of parameter where e1 = color which consists of red and blue, e2 = ingredient which is made from wool, cotton, and acrylic, and e3 = price which can be considered as high and low. in this situation, we should consider a higher order hesitant fuzzy soft set (f~,e) as follows : (42)f~(e1)={x1{(0.1,0.2),(0.3,0.2)},x2{(0.2,0.3),(0.3,0.1)},iiiiiiiiiiiiix3{(0.3,0.3),(0.4,0.6)},x4{(0.1,0.3),(0.3,0.1)}},f~(e2)={x1{(0.5,0.2),(0.1,0.2),(0.3,0.2)},iiiiiiiiiiiiix2{(0.2,0.4),(0.6,0.1),(0.3,0.1)},iiiiiiiiiiiiix3{(0.3,0.3),(0.4,0.4),(0.4,0.6)},iiiiiiiiiiiiix4{(0.1,0.3),(0.3,0.1),(0.2,0.5)}},f~(e3)={x1{(0.5,0,2),(0.4,0.1)},x2{(0.3,0.7),(0.6,0.1)},iiiiiiiiiiiiix3{(0.4,0.4),(0.4,0.6)},x4{(0.8,0.2),(0.2,0.5) } }, where the above g - type fss, denoted by (,), are all in the form of ifs on x. the aim here is to select one of color cloths depending on its performance evaluated with respect to all parameters. the tabular representation of the above higher order hesitant fuzzy soft matrix ~ is given in table 1. to perform the second step of algorithm 31, we need to compute a proper threshold vector over parameter set e. first of all we evaluate the efficiency of two well - known aggregation operators which are called the arithmetic average operator (aao) and the geometric average operator (gao). based on the aggregation aao, the threshold vector over parameter set e is defined as (43)aao(e)={aao(ej), j=1,,m}iiiiiiiiiiii={h~(ej)aao, j=1,,m }, where (44)h~(ej)aao=1ni=1nh~f~(ej)(xi)iiiiiiiiiiiiiiiii={1ni=1nh~f~(ej)(1)(xi),,1ni=1nh~f~(ej)(lx)(xi)}. needless to say that the operator on h~f~(ej)(k)(xi) (k=1,,lx) is that defined on g - type fss. in this example, operator considered on ifss is given for ifss h~f~(ej)(k)(xi)=(ik, j,ik, j), i=1,,n by (45)1ni=1nh~f~(ej)(k)(xi):=(k, j,k, j)iiiiiiiiiiiiiiiiiiiiiiii=(1i=1n(1ik, j),i=1nik, j). furthermore, based on the aggregation gao, we have (46)h~(ej)gao = (i=1nh~f~(ej)(xi))1/n = { (i=1nh~f~(ej)(1)(xi))1/n,,(i=1nh~f~(ej)(lx)(xi))1/n }, where for ifss h~f~(ej)(k)(xi)=(ik, j,ik, j), i=1,,n, we define (47)(i=1nh~f~(ej)(k)(xi))1/n:=(k, j,k, j)iiiiiiiiiiiiiiiiiiiiiiiiiiiiii=(i=1nik, j,1i=1n(1ik, j)). assume that (48)h~(ej)(k) aao = 1ni=1nh~f~(ej)(k)(xi):=(k, j,k, j)iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii=(1i=1n(1ik, j),i=1nik, j),h~(ej)(k) gao = (i=1nh~f~(ej)(k)(xi))1/n:=(k, j,k, j)iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii=(i=1nik, j,1i=1n(1ik, j)). then, for any xi x(49)i(h~(ej) aao, h~f~(ej)(xi))=0,i(h~(ej) gao, h~f~(ej)(xi))=1, where i(,) is the inclusion measure given by (38). assume that (48)h~(ej)(k) aao = 1ni=1nh~f~(ej)(k)(xi):=(k, j,k, j)iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii=(1i=1n(1ik, j),i=1nik, j),h~(ej)(k) gao = (i=1nh~f~(ej)(k)(xi))1/n:=(k, j,k, j)iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii=(i=1nik, j,1i=1n(1ik, j)). then, for any xi x(49)i(h~(ej) aao, h~f~(ej)(xi))=0,i(h~(ej) gao, h~f~(ej)(xi))=1, where i(,) is the inclusion measure given by (38). proofthe proof is straightforward from the fact that for any indices i, j, k(50)(ik, j,ik, j)(1i=1n(1ik, j),i=1nik, j),(ik, j,ik, j)(i=1nik, j,1i=1n(1ik, j)). k(50)(ik, j,ik, j)(1i=1n(1ik, j),i=1nik, j),(ik, j,ik, j)(i=1nik, j,1i=1n(1ik, j)). the above proposition demonstrates that the threshold vectors over parameter set e defined in accordance with the aggregations aao and gao are not efficient, because indifferent inclusion measure values for the whole alternatives xi x do not allow us to have a promising judgment about optimal decision. in order to overcome adverse effect of indifferent inclusion measure values, we use, but we are not limited to, median operator instead of aao and gao. let us denote any ifs h~f~(ej)(k)(xi) by h~f~(ej)(k)(xi)=(ik, j,ik, j) for 1 i n, 1 j m, 1 k lx., nonmembership degree) of all alternatives in x is arranged in increasing order, and then x(i) is referred to as the alternative with ith largest membership degree (resp., nonmembership degree). in this regards, we define (51)h~(ej)(k)med:=(medk, j,medk, j), where (52)medk, j={((m+1)/2)k, j, if m is odd;12((m/2)k, j+(m/2 + 1)k, j),if m is even,medk, j={((m+1)/2)k, j, if m is odd ; 12((m/2)k, j+(m/2 + 1)k, j), if m is even. now, the threshold vector med(e) can be obtained as represented in tabular form in table 2. by applying the third step of algorithm 31 this paper proposed a more general extension of fuzzy soft set that not only encompasses the existing ones as special cases, but also can be applied to model those situations where the existing kinds of fuzzy soft set are not applicable. the proposed higher order hesitant fuzzy soft set provides us with a multicriteria decision making method to deal with problems involving parameters with different - dimensional levels. as future work, we consider the study of aggregation methods based on multiexperts ' higher order hesitant fuzzy soft matrix where some experts are invited to conduct evaluation for one decision making problem. | the main goal of this contribution is to introduce the concept of higher order hesitant fuzzy soft set as an extension of fuzzy soft set that encompasses most of the existing extensions of fuzzy soft set as special cases. furthermore, this new concept provides us with a method for dealing with multicriteria fuzzy decision making problems which are difficult to explain in other existing extensions of fuzzy soft set theory, especially when problems involve parameters with different - dimensional levels. |
this study supports the main characteristics of a new genus gabonibacter timonensis strain marseille - p3388 (csur p3388) ; a new member of the gabonibacter genus and porphyromonadaceae family, that was isolated from a stool sample of a healthy 47-year - old pygmy woman. |
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two forms (genetic and sporadic) of alzheimer s disease involve main common events : neuronal damage and amyloid plaques accumulation in the human brain. neuronal changes in the end - stage patient brain are diffuse and widespread and neuropathological autopsy findings include significant neuron death an interesting suggestion about this phenomenon is that neuronal cells, which are involved with learning and memory, may synthesize specific substances associated with these cognitive activities and that dysfunction of the blood brain barrier (sohrabji 2007 ; zipser 2007) may cause selective loss of these specific cells (arshavsky 2006). the extracellular space in brain is infiltrated by blood borne -amyloid peptide 142, a neurotoxic peptide that interferes with the cells vital mechanisms and ultimately kills them (atwood 2003a ; swerdlow 2007). this additional supporting evidence for blood brain barrier disruption in alzheimer s disease, which is related to the accumulation of -amyloid peptide ; the featured lesion of the disorder (pluta 1996). another set of evidence suggests that alzheimer s disease may have an autoimmune root, where especially neuronal cells are targeted after opening blood brain barrier (dandrea 2005). the first description of the neuropathology in the patient brain suffering from alzheimer s disease by alzheimer in 1907 presented cerebrovascular alterations of endothelium especially in small vessels (blood brain barrier vessels) (kalaria 1999, 2002 ; grammas 2000 ; humpel and marksteiner 2005 ; dede 2007) that have not received much attention (pluta 2000). the onset of alzheimer s disease is unknown and symptoms develop slowly during patient life. in the end stage of this disorder, the etiology and important risk factors for alzheimer s disease are not well known (spires and hyman 2005 ; pluta 2006a). it is believed that major causes for this disorder include diseases of old age such as transient focal and complete brain ischemic (kalaria 2000 ; pluta 2006a) and head (nimmo 2004) injuries. single and most probably multiple brain ischemic injuries can develop changes similar to those observed in alzheimer s disease (pluta 2006a). some investigators have presented the hypothesis that the primary event in the onset of alzheimer s disease is ischemic injury of brain (kalaria 2000 ; pluta 2004b, 2006a). according to this rival theory the blood brain barrier vessels are damaged (kalaria 1999 ; dede 2007) and at the same time cause the neurotoxic -amyloid peptide to be released from circulatory system into brain parenchyma (pluta 1996) and subsequently react with ischemic neurons (koistinaho 2002) as secondary cause of neuronal death (figure 1). the precursor protein for -amyloid peptide can exist as both a membrane cell surface receptor and a soluble form, mostly in circulating blood. blood platelets carry large amounts of -amyloid peptide and its precursor (chen 1995). the prolonged opening of ischemic blood brain barrier to -amyloid peptide (pluta 2003, 2005 ; pluta 2006) and exposure to the potentially neurotoxic peptide probably initiates amyloid plaques formation / maturation (kawai 1990 ; dickstein 2006) with further additional neuronal cell death (koistinaho 2002) and massive neuronal network destruction (iwasaki 2006). according to the ischemic theory of alzheimer s disease, brain barrier vessels are impaired, which causes the neurotoxic -amyloid peptide to be released from blood and subsequently infiltrate the neuronal cells and brain parenchyma (pluta 2004a, 2004b, 2006a, 2006b, 2007a, 2007b). as a late effect, there will be a narrowing of the small vessels lumen leading to secondary ischemic brain episodes as a vicious cycle (pluta 2006b, 2007b) in which the neuronal network will be completely impaired. in this review we present data that supports the role of ischemia in neuronal death and ischemic blood in the ischemic brain, the main neuropathological focus is concentrated on structural changes in hippocampus because this part of the brain is selectively vulnerable to ischemic- and alzheimer - type injury. in some cases, complete disappearance of vulnerable pyramidal neurons in the ca1 area was noted 7 days after ischemia (butler 2002). moreover, one third of cases with experimental brain ischemia did not present full disappearance of pyramidal cells in ca1 region during short - term survival (sadowski 1999 ; pluta 2000). these animals developed complete death of all neurons of ca1 sector in late stages (pluta 2000, 2002a, 2002b). some studies presented marked structural changes in neurons considered to be resistant to ischemia such as : in sector ca2, ca3, and ca4 of hippocampus and dentate gyrus (pluta 2000). these areas presented acute ischemic changes in neurons from 1 to 12 months following ischemic injury (pluta 2000). recently was found that pathological structural processes in ischemic neurons continue well beyond the acute stages of insult (pluta 2000, 2002a). in these situations, enduring ischemic blood brain barrier dysfunction (pluta 2006b, 2007b) probably leads to enhanced ischemic neuronal vulnerability to -amyloid peptide (figure 1) (table 1) (koistinaho 2002). some studies show that astrocytic apoptosis may contribute to the pathogenesis of different disorders such as ischemic brain and alzheimer s disease (takuma 2004 ; pluta 2006a). astrocytic reactions that are noted in the ischemic brain and alzheimer s disease are swelling, astrogliosis, and astrocytosis (bernaudin 1998 ; stoltzner 2000). in brain ischemia, some experimental studies noted the early import of different epitopes of amyloid precursor protein from brain parenchyma and circulatory system to the astrocytes and in the late stages the export of the neurotoxic c - terminal of amyloid precursor protein and -amyloid peptide from dead astrocytes to the brain tissue (table 1) (pluta 1994b ; pluta 2002b, 2004a, 2004b). at early stages of brain ischemia and reperfusion, the n - terminal of amyloid precursor protein (pluta 1994b) may be synthesized by cells derived from the vascular endothelium that became fragmented after injury (badan 2004) this idea is supported by the expression and production of -secretase in ischemic brain (wen 2004 ; sun 2006 ; zhang 2007). furthermore presenilin expressed in ischemic brain (tanimukai 1998 ; pennypacker 1999 ; pluta 2001) is involved in the metabolism of amyloid precursor protein to produce -amyloid peptide through the -secretase complex (wolfe 1999)., amyloid precursor protein is cleaved at the n - terminal of the -amyloid peptide domain by protease called -secretase. in the second step, endothelial cells change shape and size over time to become incorporated into the amyloid plaques in a close spatial relationship with damaged or dead astrocytes. the same study shows that c - terminal of amyloid precursor protein accumulates in neurons after focal ischemia and as the infarct progresses, the c - terminal of amyloid precursor protein immunoreactive elements become increasingly larger in the core even though the neurons are dying and the core becomes largely acellular (badan 2004). the same and other studies suggest that c - terminal of amyloid precursor protein and -amyloid peptide noted in microglia could be due to the phagocytosis of dead neurons remnants containing c - terminal of amyloid precursor protein and -amyloid peptide by microglia (figure 1) (table 1) (badan 2004 ; dandrea 2004). moreover there are reports showing that astrocytes but not microglial cells can take up -amyloid peptide (table 1) (matsunaga 2003 ; wyss - coray 2003 ; pluta 2006a). other work shows that c - terminal of amyloid precursor protein induces the death of astrocytes whereas the loss of neurons is a secondary consequence of the neuronal dependence on astrocytes for antineurotoxic -amyloid peptide protection (figure 1) (table 1) (abramov 2003 ; pluta 2006a). the localization of some fragments of amyloid precursor protein in astrocytes may be of significant relevance to alzheimer s disease in which chronic astrocytosis is thought to play a key role in the evolution of amyloid plaques. brain ischemia mediated a number of vessel events which are open blood brain barrier and tight junctions, diffuse leakage across necrotic vessel walls, and vasoconstriction (petito 1982 ; mossakowski 1993, 1994 ; pluta 1994a, 2006 ; wisniewski 1995 ; gartshore 1997 ; shinnou 1998 ; lippoldt 2000 ; ueno 2002 ; pluta 2003, 2005). until one year following brain ischemia - reperfusion injury brain gray and white matter areas contained single isolated and scattered sites of horseradish peroxidase extravasations (table 2) (mossakowski 1994 ; pluta 1994a, 2006 ; pluta 2003, 2005). microscopic studies of different ischemic brain sections revealed diffuse and focal staining of tracer. penetrating vessels across different brain structures displayed horseradish peroxidase immunoreactivity in walls. in hippocampus, cortex, basal ganglia, and thalamus and cerebellum, after short - term survival (7 days) following brain ischemia, experimental animals presented with gray and white matter perivascular immunoreactivity for all epitopes of amyloid precursor protein (table 2) (pluta 1994b). on the contrary, following long - term survival of animals, staining only for the toxic c - terminal of amyloid precursor protein and to the -amyloid peptide was observed (pluta 1997b ; pluta 2000, 2003, 2005). multiple and abundant c - terminal of amyloid precursor protein and -amyloid peptide staining deposits embraced and/or adjoined the capillaries. diffuse deposits of c - terminal of amyloid precursor protein and -amyloid peptide like puff of smoke were also observed. immunoreactivity inside capillaries with a halo of c - terminal of amyloid precursor protein staining in the surrounding parenchyma (pluta 2005) suggested diffusion of c - terminal of amyloid precursor and -amyloid peptide out of the vascular compartment (table 2). in any case, the halo of -amyloid peptide staining in the perivascular space suggests that -amyloid peptide can easy cross small vessel walls. the above suggestions are supported by intravenous injection of human -amyloid peptide 142 into ischemic animals, which accumulated this peptide in the ischemic brain gray and white matter vessels walls and in perivascular space (table 2) (pluta 1996, 1997a, 1999). brain barrier receptor - mediated transport (deane 2003) and by blood brain barrier permeability caused by ischemic injury (pluta 1996, 1997a, 1999, 2000) or -amyloid neurotoxicity on ischemic blood brain barrier structure (table 1) (thomas 1996 ; fiala 1998 ; farkas 2003 ; paris 2004a, 2004b). the abnormal staining for apolipoproteins e, a1, and j was noted perivascularly (table 2) (kida 1995 ; pluta 2000). extracellular apolipoproteins e and j deposits were heavily labeled by antibody to apolipoprotein a1, stronger than by apolipoprotein e antibody (kida 1995). they were stained stronger by antibody to apolipoprotein e than apolipoprotein j (kida 1995). it is of interest to notice that perivascular deposits of apolipoproteins colocalize with deposits of different fragments of amyloid precursor protein (kida 1995). apolipoprotein e can modulate and promote the aggregation of soluble -amyloid peptide into the fibrillar conformation. apolipoprotein j is implicated in transport of -amyloid peptide across the blood brain barrier. the main role of apolipoproteins e, a1, and j in controlling the levels of soluble -amyloid peptide in the extracellular / perivascular space of brain as well as their influence on amyloid plaques formation is suggested. these data demonstrate additive effects of apolipoproteins on influencing -amyloid peptide deposition around blood vessels and that they play an important role in regulating extracellular brain -amyloid peptide metabolism independent of -amyloid peptide synthesis. another function, especially for apolipoprotein e in brain, is the suggested clearance of ischemic tissue probably by reverse transport of remnants into blood (pluta 2000). these observations indicate that perivascular apo - lipoproteins e, a1, and j accumulation after ischemia may represent a secondary injury factor that could exacerbate healing of ischemic brain. in different times following ischemic brain injury platelets occurring in aggregates were noted to adhere to the endothelium lining of ischemic blood ischemic platelets contained pseudopodia, which made strong contact with the endothelium. as an effect of these alterations the no - reflow phenomenon was noted (mossakowski 1993 ; pluta 1994c ; pluta 2003). moreover, platelets were observed on the abluminal side of vessels (table 2) (pluta 1994c ; pluta 2003, 2005). these kind of changes occurred in arterioles, capillaries, venules, and veins independently of survival time following brain ischemia. some data suggest that ischemic brain injury results in an increase number of platelet - leukocytes complexes (ishikawa 2004) in the circulatory system (ritter 2005). another study presented increased platelet - leukocyte - endothelium interactions after local brain ischemia (ishikawa 2004). a growing body of evidence has suggested that white cells can play an important pathological role in ischemic brain (caceres 1995 ; gidday 2005). matrix metalloproteinase-9 from white cells recruited to the ischemic brain tissue promotes further white cell recruitment (table 2) to the same brain areas in a positive feedback manner and causes secondary blood brain barrier vessels identified leukocytes adhering to the endothelium of capillaries and venules (caceres 1995). endothelial changes and white cells accumulation and adherence also reflect a no - reflow phenomenon. studies of ischemic microvessels from brain presented vasospasm during recirculation time (wisniewski 1995 ; ohtake 2004). the pathological hallmarks of vasospasm included undulations of basement membrane and endothelium with an increased number of endothelial microvilli (pluta 1991). some damaged endothelial cells presented rupture of endothelium membranes (caceres 1995). other studies of ischemic endothelium included an increased number of deep crater - like pits, endothelium microvilli, and enlarged junctional ridging (pluta 1991). as an effect of presented pathology, aggregating platelets developed microthrombi attached to the vessel wall, which caused a continuous supply of constrictors such as -amyloid peptide (chen 1995 ; thomas 1996). current results suggest that platelets are the main ischemic cause in reperfusion injury, not only through aggregation and thrombus formation, but also through involvement in inflammation processes with leukocytes (nishijima 2004). due to the fact that soluble -amyloid peptide causes vascular constriction (thomas 1996;niwa 2001) and endothelial cell injury (table 1) (thomas 1996), a role for soluble -amyloid peptide in vasospasm and blood brain barrier changes has been suggested. during recirculation following ischemic brain injury, islets of necrotic endothelium in capillaries was found (petito 1982 ; mossakowski 1994 ; pluta 1994a). brain barrier vessels characterize diffuse leakage of different blood elements (petito 1982 ; mossakowski 1994 ; pluta 1994a). this phenomenon is probably due to senescent endothelium, which is triggered during reperfusion and is accelerated by -amyloid peptide (mossakowski 1994). senescent endothelial cells are a common hallmark of vessel aging (erusalimsky and kurz 2005) and are probably also accelerated by ischemic insults (mossakowski 1994). another problem during recirculation, senescent endothelial cells are covered with -amyloid peptide ischemic vessel walls where -amyloid peptide acts as antiangiogenic factor (paris 2004a, 2004b). ischemic episodes and -amyloid peptide harmful influence on pericytes (table 1) (lupo 2001 ; anfuso 2004) and astrocytes (table 1) can regulate blood brain barrier vessel angiogenesis and can disrupt the normal blood brain barrier function (ramsauer 2002). the incidence of alzheimer s disease cases is gender - related (pluta 2006a) and the risk of alzheimer s disease in women is significantly greater than in men. the cumulative risk for alzheimer s disease in women is greater because of a lack of estrogen (figure 2) after menopause, which has been proposed as a risk factor for alzheimer s disease among women (pluta 2006a). brain barrier function control through intercellular junction proteins (kang 2006) and/or intracellular transport elements and through protective effects on the cellular components of barrier such as endothelial, pericytes, and astrocytes cells (yang 2005), cells which are vulnerable to aging and ischemia (table 1) influence in the context of normal blood brain barrier activity (figure 2) (sohrabji 2007 ; zipser 2007). brain barrier can expose brain parenchyma to different elements of blood (table 2) that directly and/or indirectly harm neuronal cells (table 1) and potentate other neuropathological processes. age - related cascades in different areas of the brain can have far reaching consequences for cognitive ability and affect. most investigators have taken the approach of studying estrogen influence on neuropathological events related to alzheimer s disease (figure 2) (simpkins 1997 ; dubal 1998 ; shi 1998 ; chi 2002, 2005). (simpkins 1997 ; dubal 1998 ; shi 1998 ; chen 2001 ; chi 2002 ; yang 2005), but the precise mechanism of their protection is not clearly understood. these hormones may preserve neuronal health (chen 2001) by maintaining the integrity of the blood brain barrier (figure 2) (chi 2002, 2005). another possibility is that estrogen attenuates overexpression of amyloid precursor protein messenger rna (shi 1998). the protective effects of estrogens are seen in all of the neurovascular unit components including vascular endothelial cells, astrocytes, microglia, and neurons (chen 2001 ; yang 2005). additionally postischemic estrogens reduce hypoperfusion and secondary ischemic episodes following brain ischemia (mccullough 2001). prevention of brain ischemia and treatment ischemic episodes may have significant implications for alzheimer s disease therapy. in view of the earlier observations that cognitive decline is progressing after brain ischemia, there is the distinct possibility that we can prevent this decline by targeting the slowly progressing pathology that follows ischemic injury by aiming at molecular events now shown to change following brain ischemia. -amyloid peptide is the main element of the fibrils in senile plaques in the alzheimer s disease brain. a soluble form of -amyloid peptide has been found in plasma (mehta and pirttila 2002). a main vascular source for both the amyloid precursor protein and soluble -amyloid peptide there is a growing body of evidence indicating that > 90% of soluble -amyloid peptide found in the circulatory system is stored in platelets body (chen 1995). acute and chronic ischemic neuronal death and ischemic neurons with enduring blood brain barrier leakage (pluta 2003, 2005 ; pluta 2006) and platelets in the blood brain barrier vessels perivascular space (table 2) (pluta 1994c ; pluta 2003) with neurotoxic epitopes of amyloid precursor protein deposition are involved in the gradual maturation of injurious process in ischemic gray and white matter (table 1), which lead to progressive dementia and alzheimer s disease (pluta 2007c). we propose the role for brain ischemic injury as an alternative etiology / hypothesis of alzheimer s disease that triggers repetitive microischemic insults which form the basis for development of main neuropathological processes in alzheimer s disease (pluta 2007c). the profile of neuronal and blood brain barrier pathology noted in ischemic brain shares a commonality with the same alterations in alzheimer s disease. brain barrier changes are involved in amyloid plaques formation (ujiie 2003). in alzheimer s disease, the decreased length of cerebral capillaries correlates well with neuropathology and clinical features of dementia (attems and jellinger 2004 ; bailey 2004). islets of regressed, senescent (minamino 2004) and degenerated ischemically blood brain barrier vessels (petito 1982 ; mossakowski 1994 ; pluta 1994a) probably act as seeds - core for the amyloid plaques. thus, this data support theory that chronic ischemic blood brain barrier permeability can cause angiopathy, vascular senescence (minamino 2004), neuroinflammatory response (nimmo 2004), and amyloid plaques, which also occur in the alzheimer s disease brain. brain barrier vessels with insufficient repair (atwood 2003b ; dickstein 2006), vascular regression, and senescence, and aberrant and insufficient angiogenesis represent new pathogenic mechanism(s) involved in maturation of alzheimer s disease and final development of amyloid plaques which are unrelated to primary ischemic neuronal death (ishimaru 1996 ; van groen 2005). | the development of neuronal death and amyloid plaques is a characteristic feature of ischemic- and alzheimer - type dementia. an important aspect of neuronal loss and amyloid plaques are their topography and neuropathogenesis. this review was performed to present the hypothesis that different fragments of blood - borne amyloid precursor protein are able to enter the ischemic blood brain barrier. chronic disruption of the blood brain barrier after ischemic injury was shown. as an effect of chronic ischemic blood brain barrier injury, a visible connection of amyloid plaques with neurovasculature was observed. this neuropathology appears to have similar distribution and mechanisms to alzheimer s disease. the usefulness of rival ischemic theory in elucidating the neuropathogenesis of amyloid plaques formation and neuronal death in alzheimer s disorder is discussed. |
laser in situ keratomileusis (lasik) is the most common and popular procedure performed for the correction of refractive errors in the last two decades. we report a case of traumatic flap displacement with flap folding which occurred 3 years after lasik was performed. previous literature suggests that vision prognosis would be closely related to proper and prompt management of traumatic flap displacement with flap folding 3 years after lasik. a 23-year - old female presented to our hospital who had undergone uneventful lasik in both eyes 3 years prior. unfortunately, she had suffered a blunt trauma in her right eye in a car accident. a late onset of corneal flap displacement was found with upper and lower portion of the flap being folded inside the corneal bed. surgical intervention for debridement with subsequent reposition of corneal flap was performed as soon as possible in the operating room. a bandage contact lens was placed, and topical antibiotic and corticosteroids were given postoperatively. two days after the operation, the displaced corneal flap was found to be well attached smoothly on the corneal bed without folds. the best - corrected visual acuity was 6/6 with refraction of 0.75 d to 1.0 d 175 in her right eye 1 month later. we reviewed a total of 19 published cases of late - onset traumatic flap dislocations or displacements after lasik with complete data from 2000 to 2014. traumatic displacement of corneal flaps after lasik may occur after blunt injury with specific direction of force to the flap margin, especially tangential one. according to the previous literature, late - onset traumatic flap displacement may happen at any time after lasik and be caused by various types of injuries. traumatic flap displacement, which is known as one of the complications of lasik, may occur in the early or late postoperative period. the procedures of lasik, used worldwide to correct a wide range of refractive errors, include creating a corneal flap, lifting it, ablating the stromal bed by excimer laser and repositioning the corneal flap at the end. however, the wound healing between corneal flap and stromal bed should be diminished to maintain corneal transparency, which leads to weak flap adhesion. lamellar wound healing is confined to the flap margin without suture to avoid astigmatism.1 since central wound healing does not increase over time, risks of flap displacement or dislocation could be expected after face trauma. postoperative flap dislocation happens within the first 24 hours after surgery in approximately 1%2% of patients, mostly due to eye - rubbing or eyelid motion such as squeezing or blinking.2,3 in comparison to the early - onset flap dislocation, late traumatic flap dislocation arises more than 1 week after the surgery. the increasing incidence of late traumatic flap dislocation in recent years may be related to the popularity that lasik has gained in the last two decades, which is performed by using either microkeratome or femtosecond laser. here, we report a case of traumatic flap displacement after a severe car accident, in a patient who had lasik performed 3 years prior using microkeratome. we also reviewed all previous literature published on medline about late - onset flap dislocation and displacement after previous lasik that required surgical reposition. a 23-year - old female presented to our hospital who had undergone uneventful lasik in both eyes 3 years prior. the patient had suffered a blunt trauma in her right eye in a severe car accident. multiple and irregular lacerations with active bleeding in the right upper eyelid were noted in the emergency room. direct ophthalmoscope revealed a late - onset corneal flap displacement with the flap being folded in the superior and inferior portion. in addition, the bare stroma was exposed. the connection between the nasal hinge of the corneal flap and the cornea was still intact (figure 1). surface debris was thoroughly scraped by a sterile surgical sponge with continuous irrigation of bss. the margin of the corneal flap which was still in situ was then demarcated, followed by dissection of the central portion from the stromal bed. finally, the whole corneal flap was refloated by a lasik spatula. to expose the whole stromal bed, meanwhile, the superior and inferior folds of corneal flap were unfolded and flattened using a sterile surgical sponge, followed by vigorous irrigation with bss at the end. in addition, the epithelial cells and remnants adherent to the underside of the corneal flap and stromal bed were carefully removed. the corneal flap was repositioned and stretched using surgical sponges in order to avoid folds and wrinkles. at last, a bandage soft contact lens was placed on the cornea for protection (figure 2). topical antibiotic (0.3% ciprofloxacin) and corticosteroids (1.0% prednisolone acetate) were given postoperatively. two days after the operation, the displaced corneal flap was found to be well attached smoothly on the stromal bed without folds and wrinkles. the best - corrected visual acuity was 6/6 with refraction of 0.751.0175 in her right eye at 1 month. four months later, the uncorrected vision of her right eye was 6/6 with residual refraction of + 0.250.25d175. the corneal flap was clear and smooth in situ without epithelial ingrowth (figure 3). informed consent was obtained from the patient for publication of this case report and accompanying images. the ethics committee of did not require written informed consent be obtained from the patient because lasik remains the most common and popular procedure for the correction of the refractive errors. it has been increasingly used worldwide for nearly 20 years since it was approved by the us food and drug administration in 1998. a new technique using femtosecond laser instead of microkeratome to make corneal flap has also been developed. although less wound healing between corneal flap and stromal bed enhances corneal interface transparency, lifetime flap adhesion weakness could lead to increased risk of late traumatic flap complications. due to our interest in the prevalence of traumatic flap displacement after lasik, we reviewed several reports published in the previous ophthalmic literature with complete data from 2000 to 2014 on cases of late - onset traumatic flap dislocations or displacements after lasik (table 1).415 we reviewed a total of 19 cases of patients with an average age of 35 years, of whom 52.6% were male. regarding the causes of injury, over 26% of injuries were due to sport - related activities, followed by work - related accidents (15.7%), animal injuries (10.5%) and failure of airbags during car accidents (10.5%). regarding the mechanisms of trauma, the cases of blunt trauma were more common than those of sharp trauma (table 2). the interval between lasik and injury episode varied from 10 days to over 14 years, while the vision after trauma varied from counting finger to 20/20. of all these 19 cases, patients with ots grade 2 accounted for 54.55% (table 3).16 the interim between injury and surgical intervention was a maximum of 2 days. although the uncorrected vision at last visit was generally excellent with an average of 20/25, the patients who experienced car accidents had a relatively poor average final vision of 15/20. the vision prognosis of sharp injury was worse than that of blunt trauma with statistical significance (table 2). besides, patients with other eye injuries such as hyphema or eyelid laceration accounted for 15.7% of all 19 cases, and had ended up with a poorer average final vision of 20/30. furthermore, 31.6% of patients had developed complications even after surgical interventions, though they had an average final vision of 20/25 in the last visit. among them, only 15.7% had undergone further treatment and ended up with an average final vision of 20/25. of all 19 cases, 94.74% of patients had ended up with an ots grade 1 after proper treatment (table 3). as for the flap condition after trauma (table 4), flap dislocation was revealed in 73.7% of cases, which was similar to the finding of 71.1% in xiao study which analyzed all the cases in the people s republic of china.17 in our review, all the cases with corneal flap dislocation had received flap replacement (table 5), while only 55.6% of cases in xiao study had undergone the same treatment. lack of ophthalmic doctors skilled in refractive surgery could be one possible factor.17 during flap replacement surgery, epithelial scraping had been performed to avoid epithelial ingrowth or residual debris in 63.2% of patients, which led to dissemination of lamellar keratitis at last. at the end of the surgery, up to 73.7% of patients were covered with bandage contact lens to make the folded flaps more flat and then smoothly settle on the stromal beds and remain in situ (table 5). after lasik surgery, the whole processes and exact mechanisms of wound healing at the interface of corneal flap stromal bed and around the free margins of corneal flap remain unclear. less and delayed wound healing at the stromal interface is believed to keep cornea transparent, leading to weaker adhesion in the central part compared to the surrounding epithelial rim margin, which was confirmed by the surgical experience during retreatment of lasik. schmack found that the wound margin of lasik flap heals by producing peripheral hypercellular fibrotic stromal scar, which is about 28.1% as strong as normal corneal stroma. in contrast, the central and paracentral hypocellular primitive stromal scar is only 2.4% as strong as normal corneal stroma, which means it is 10 times weaker then peripheral margin scar.18 kato studied corneal wound healing in an animal model. their work on immunohistochemical staining and electron microscopy following lasik showed that up to 9 months after the procedure, wound healing continued to be disorganized and delayed at the interface region.19 the corneal flaps have free margins about more than 270 without any sutures and anchors except the hinge area, which is attached to the corneal flap smaller than 90. any shearing forces, which are tangential to the corneal flap regardless of mechanism and direction, may induce breaking of not only the loose adhesion between corneal flap and stromal bed but also the connection between the free margins and peripheral cornea. these tangential shearing forces may drag the corneal flap away from the corresponding stromal bed and peripheral cornea, leading to the dislocation / stria / split. according to the appearance of corneal flap folding and stria, the direction of shearing forces could be speculated. the pictures of the corneal flap dislocation in our patient showed a larger upper portion of the corneal flap compared to the lower portion. furthermore, there was a part of the upper portion cornea folding between the stromal bed and cornea itself. besides, the patient had multiple lacerations over upper eyelids. these are all hints that the strong shearing forces came from up to down. femtosecond laser - assisted lasik also involves a corneal flap, which may have risks of late - onset traumatic flap dislocation if the force causing blunt trauma comes from a proper angle. however, there are no available studies comparing the late complications of these two different methods of lasik. contrary to the lasik, smile, the newest refractive surgery, involves smaller degrees of free margins and cap incision despite the same interface between the corneal cap and stromal beds. we deduced that even shearing forces can cause breaking of the loose adhesion between corneal cap and stromal bed, and the counterforce of cornea with non - free margin could resist the force, therefore preventing the corneal cap from moving. however, further studies and mechanical models are required to prove that smile might cause less complication after trauma. because the corneal flaps never heal after lasik, eye protection should be used before participating in dangerous sports such as basketball and also in work such as gardening. once traumatic accidents occur, careful and detailed examinations should be undertaken under slit lamp as soon as possible. if dislocation / stria / split of corneal flaps is identified during emergent conditions, corneal flap reposition should be considered immediately. thorough irrigation and scraping off the debris by sponges between corneal flaps and stromal beds should not be ignored. previous experiences suggest that the vision prognosis would be good if proper managements and treatments are followed. | backgroundlaser in situ keratomileusis (lasik) is the most common and popular procedure performed for the correction of refractive errors in the last two decades. we report a case of traumatic flap displacement with flap folding which occurred 3 years after lasik was performed. previous literature suggests that vision prognosis would be closely related to proper and prompt management of traumatic flap displacement with flap folding 3 years after lasik.case presentationa 23-year - old female presented to our hospital who had undergone uneventful lasik in both eyes 3 years prior. unfortunately, she had suffered a blunt trauma in her right eye in a car accident. a late onset of corneal flap displacement was found with upper and lower portion of the flap being folded inside the corneal bed. surgical intervention for debridement with subsequent reposition of corneal flap was performed as soon as possible in the operating room. a bandage contact lens was placed, and topical antibiotic and corticosteroids were given postoperatively. two days after the operation, the displaced corneal flap was found to be well attached smoothly on the corneal bed without folds. the best - corrected visual acuity was 6/6 with refraction of 0.75 d to 1.0 d 175 in her right eye 1 month later.literature reviewwe reviewed a total of 19 published cases of late - onset traumatic flap dislocations or displacements after lasik with complete data from 2000 to 2014.conclusiontraumatic displacement of corneal flaps after lasik may occur after blunt injury with specific direction of force to the flap margin, especially tangential one. according to the previous literature, late - onset traumatic flap displacement may happen at any time after lasik and be caused by various types of injuries. fortunately, good visual function could mostly be restored with immediate and proper management. |
hereditary angioedema (hae) is an autosomal dominant disease characterized by recurrent episodes of potentially life - threatening angioedema (1). the clinical features of the disease include recurrent nonpruritic edema of skin and submucosal tissues associated with pain syndromes, nausea, vomiting, diarrhea, and life - threatening airway swellings (2). the fundamental abnormality in patients with hae is a mutation of the c1 inhibitor (c1-inh) gene, which results in the decreased synthesis of functional c1-inh antigenic protein. two forms of hae have been described : in type i hae patients have low c1-inh antigenic protein and functional activity (85% of cases), while patients with type ii hae have normal or elevated protein but low c1-inh function (15% of cases) ; in patients with a normal c1-inh gene, hae is often referred to as type iii hae. over 200 mutations have been reported in unrelated patients (3,4). while hae is known to be inherited in an autosomal dominant form, 25% of cases we report a case of type i hae with a newly recognized de novo mutation of the c1-inh gene. a 38-year - old woman presented to our emergency department with abdominal pain and nausea that had persisted for three hours. the patient had experienced recurrent episodes of skin edema, sometimes followed by vomiting with abdominal pain, around her menstrual period for 13 years. she had no family history of hereditary diseases. the patient 's white blood cell (wbc) count and platelet count were 23,200/mm and 434,000/mm, respectively. her red blood cell count and creatinine, blood urea nitrogen, glucose, lipid, and transaminase levels were normal. the complement studies revealed a markedly reduced c4 level of 1 mg / dl and the c1-inh level was decreased to 5.53 mg / dl (normal 15 - 35 mg / dl) with an activity level of < 25% (normal 80 - 125%). these laboratory data and the clinical features were compatible with a diagnosis of type i hae ; however, the patient had no family members with a history of similar episodes of recurrent edema or abdominal pain. to establish the diagnosis, we performed a dna analysis of the c1-inh gene in the patient. we asked the center for research, education, and treatment of angioedema (create) to perform a gene analysis. the patient was examined for c1-inh gene mutations by the direct sequencing of polymerase chain reaction (pcr) products. the screening of each exon for small mutations was performed using a single strand conformation polymorphism (sscp) assay. the presence of large mutations presences was then investigated using an multiplex ligation - dependent probe amplification (mlpa) assay. a direct dna sequencing analysis of the pcr products of the c1-inh gene revealed that the patient was heterozygous for a single base pair transposition of t to c at nucleotide 4429 in exon 4 (fig. 2), which led to an amino acid substitution of leu at codon 188 by pro. the leu to pro substitution was considered to be a mutation because none of 50 healthy individuals carried this substitution (data not shown). this mutation has not previously been reported. direct dna sequencing of the polymerase chain reaction (pcr) product of exon 4 from the patient identified a heterozygous, single - base substituteon (t to c) at nucleotide position 4429 (arrow). this mutation predicts substitution of leu (ctg) at codon 188 by pro (ccg). in 1888, william osler presented the first complete clinical description of hae, which occurred over 5 generations in one affected family (6). donaldson and evans noted that a biochemical abnormality, the absence of c1-inh, was responsible for hae in 1963 (7). it is located on chromosome 11q11-q13.1, and consists of 8 exons, with most introns containing multiple alu repeat sequences (8). an electronic database of the reported mutations is available (9). the most common defect is a single base pair mutation. it can occur throughout the gene and no correlation has been found between the genotype and the phenotype (10). our patient had recurrent episodes of skin edema and abdominal pain, low c4 titers with decreased functional and antigenic levels of c1-inh. as this patient had no family history of angioedema, the differential diagnosis of sporadic hae and acquired angioedema was necessary. a genetic analysis offers the clearest information for the diagnosis of such cases (11). the dna analysis revealed that the patient had a novel mutation of t to c at nucleotide 4429 in exon 4 of the c1-inh gene, which was consistent with a diagnosis of sporadic hae type i. the patient was therefore carrying a de novo mutation. although we had hoped to examine her family for mutations of the c1-inh gene (to determine whether or not the present case was sporadic) her family members declined the examination due to their lack of symptoms. since approximately 10% of individuals carrying the defective gene are asymptomatic, a dna analysis of the patient 's parents should be performed (12). however, it is sometimes misdiagnosed or overlooked, as a shown by a review of the japanese literature revealed that the mean interval between the onset of symptoms and the diagnosis of hae was 19 years (13). the patient had episodic edema of the extremities and facial skin and recurrent gastrointestinal attacks that occurred in association with her menstrual period. although these were typical symptoms of hae, she had visited our hospital more than 20 times and had only been diagnosed with gastroenteritis. we described a case of type i hae with a novel mutation of t to c at nucleotide 4429 in exon 4 of the c1-inh gene. when a clinician suspects c1-inh deficiency, even in patients with no family history, we recommend that, when possible, physicians investigate c4, c1 inhibitor function and c1 inhibitor antigenic protein. | we describe a patient with hereditary angioedema type i. the patient had experienced recurrent abdominal pain around the time of her menstrual period for 13 years. a laboratory examination showed reduced functional and antigenic levels of c4 and c1 inhibitor (c1-inh). to establish a diagnosis, we carried out a dna analysis of the patient 's c1-inh gene. we determined that the patient was heterozygous for a single base pair transposition of t to c at nucleotide 4429 in exon 4, which had not been reported in the literature. as the patient had no family history of hereditary diseases, it was considered to be a de novo mutation. |
developmental dysplasia of hip (ddh) is a common condition presenting to a pediatric orthopedic surgeon. early management is of utmost importance to achieve normal development of hip and prevent residual acetabular dysplasia (rad). in infants below 6 months of age, pavlik harness is the gold standard of treatment. once the child achieves walking age, treatment becomes more extensive. in the management of age group 18 - 24 months, majority of surgeons agree on open reduction and hip spica and a supplementary procedure like a proximal femoral osteotomy is usually not necessary in this age group.12 this study aims to recognize situations where an additional procedure may be required in a patient with ddh between 18 and 24 months to prevent a possible acetabular dysplasia. 35 children, of which 22 were girls and 13 were boys with aged between 18 and 24 months with unilateral ddh were operated between 2002 and 2007 at our institute. all patients received conservative treatment prior to their presentation at our center. all the patients presenting to us who had dislocations were included and those with mere subluxations or dysplasia were excluded from the study. radiological preoperative assessment included the extent of dislocation, acetabular index (ai), size of femoral ossification nucleus, sourcil and the tear drop development. the hip joint was approached superficially between sartorius and tensor fascia lata when the sartorius was detached from anterior superior iliac spine to improve exposure. the deep dissection was between the hip abductors and rectus where the later was detached from the anterior inferior iliac spine (aiis) to improve the exposure. the acetabular labrum was identified and care was taken to keep it intact and undamaged. the transverse acetabular ligament identified and incised in order to remove the inferior obstacle to reduction. the acetabulum was cleared of fibrocartilaginous tissue and femoral head was reduced into the acetabulum. the hip was abducted and adducted and the zone of abduction and adduction in which the femoral head remains reduced in the acetabulum (maximum safe abduction taken as 60) was determined. this zone is termed the safe zone. in cases when per - operative safe zone 20 (nine children) an additional pelvic procedure was performed in the form of dega 's acetabuloplasty. no separate incision was required and same incision was extended posteriorly to an adequate extent and used for pelvic osteotomy. the orientation of the osteotomy is first marked on the lateral cortex of the ilium. the direction of the osteotomy is curvilinear when viewed from the lateral cortex, starting just above the anterior inferior iliac spine, curving gently cephalad and posteriorly to reach a point superior to the midpoint of the acetabulum, and then continuing posteriorly to end approximately 1 - 1.5 cm in front of the sciatic notch. a guide wire is inserted under fluoroscopic control at the most cephalad point of the curvilinear marking line, directed caudally and medially to ensure that the osteotomy will exit at the appropriate level just above the horizontal limb of the triradiate cartilage. a straight 0.25 or 0.5-inch osteotome is used to perform the bone cut, which extends obliquely medially and inferiorly, paralleling the guid wire to exit through the inner cortex just above the iliopubic and ilioischial limbs of the triradiate cartilage, leaving the posterior one - third of the inner cortex intact. if predominantly anterior coverage is desired, the medial (inner) cortex is cut over the anterior and middle portion, leaving only the posterior sciatic notch hinge intact. if more lateral coverage is desired, more of the medial cortex is left intact, resulting in a posteromedial hinge based on the posteromedial inner cortex and the entire sciatic notch. all patients were given one and a half hip spica (spica extending just above the malleoli on the affected side and above the knee on the unaffected side) after surgery with the hip in 30 flexion, approximately 20 of internal rotation and 20 - 30 of abduction for 12 weeks, with one change at 6 weeks under general anesthesia (ga). the primary senior surgeon was present in all cases of postoperative spica application to ensure maintenance of reduction and utmost care was taken in molding the posterolateral aspect of the hip region. they were all followed up for a minimum period of 2 years (range 2 - 7 years). postoperatively only x - rays were done and an overlap of the proximal femoral epiphysis on the ischial tuberosity was considered to be a good indicator for an adequate reduction. magnetic resonance imaging and computed tomography (ct) were not performed because of cost factor as well as the amount of radiation involved in case of a ct. the hip joint was approached superficially between sartorius and tensor fascia lata when the sartorius was detached from anterior superior iliac spine to improve exposure. the deep dissection was between the hip abductors and rectus where the later was detached from the anterior inferior iliac spine (aiis) to improve the exposure. the acetabular labrum was identified and care was taken to keep it intact and undamaged. the transverse acetabular ligament identified and incised in order to remove the inferior obstacle to reduction. the acetabulum was cleared of fibrocartilaginous tissue and femoral head was reduced into the acetabulum. the hip was abducted and adducted and the zone of abduction and adduction in which the femoral head remains reduced in the acetabulum (maximum safe abduction taken as 60) was determined. this zone is termed the safe zone. in cases when per - operative safe zone 20 (nine children) an additional pelvic procedure was performed in the form of dega 's acetabuloplasty. no separate incision was required and same incision was extended posteriorly to an adequate extent and used for pelvic osteotomy. the orientation of the osteotomy is first marked on the lateral cortex of the ilium. the direction of the osteotomy is curvilinear when viewed from the lateral cortex, starting just above the anterior inferior iliac spine, curving gently cephalad and posteriorly to reach a point superior to the midpoint of the acetabulum, and then continuing posteriorly to end approximately 1 - 1.5 cm in front of the sciatic notch. a guide wire is inserted under fluoroscopic control at the most cephalad point of the curvilinear marking line, directed caudally and medially to ensure that the osteotomy will exit at the appropriate level just above the horizontal limb of the triradiate cartilage. a straight 0.25 or 0.5-inch osteotome is used to perform the bone cut, which extends obliquely medially and inferiorly, paralleling the guid wire to exit through the inner cortex just above the iliopubic and ilioischial limbs of the triradiate cartilage, leaving the posterior one - third of the inner cortex intact. if predominantly anterior coverage is desired, the medial (inner) cortex is cut over the anterior and middle portion, leaving only the posterior sciatic notch hinge intact. if more lateral coverage is desired, more of the medial cortex is left intact, resulting in a posteromedial hinge based on the posteromedial inner cortex and the entire sciatic notch. all patients were given one and a half hip spica (spica extending just above the malleoli on the affected side and above the knee on the unaffected side) after surgery with the hip in 30 flexion, approximately 20 of internal rotation and 20 - 30 of abduction for 12 weeks, with one change at 6 weeks under general anesthesia (ga). the primary senior surgeon was present in all cases of postoperative spica application to ensure maintenance of reduction and utmost care was taken in molding the posterolateral aspect of the hip region. they were all followed up for a minimum period of 2 years (range 2 - 7 years). postoperatively only x - rays were done and an overlap of the proximal femoral epiphysis on the ischial tuberosity was considered to be a good indicator for an adequate reduction. magnetic resonance imaging and computed tomography (ct) were not performed because of cost factor as well as the amount of radiation involved in case of a ct. group a - those who underwent only open reduction and adductor tenotomy (n = 26) [figure 1 ] and group b - those who underwent additional dega 's acetabuloplasty (n = 9) [figure 2 ]. in group a - 19 cases did well with good femoral and acetabular development, seven cases had rad with ai of 35 and above at the end of 18 months [figure 3 ]. in group they had no symptoms like waddling, limp, shortening or any pain after 6 months of spica removal. suction drain got stuck in one case which necessitated spica change under ga in immediate postoperative period. perineal edema in nine cases in the immediate postoperative period which subsided in a week 's time. (a) x - ray pelvis showing both hip joints in a 20 months old girl (case 1) showing preoperative acetabular index 35 degrees. peroperative safe zone 35. no additional procedure done (b) intraoperative photograph showing that acetabulum is cleared (c) postoperative x - ray pelvis with both hips frog leg lateral view showing well contained hip (d) x - ray pelvis with both hips anteroposterior view showing no rad after 26 months 23 months old boy (case 2) (a) x - ray pelvis with both hip joints anteroposterior view showing preoperative acetabular index 42, preoperative safe zone 20 open reduction and dega 's osteotomy done (b) peroperative clinical photograph showing tagged sartorius and rectus with cleared acetabulum (c and d) fluoroscopic views showing dega 's osteotomy and 3 years followup of dega 's osteotomy showing acetabulum index 20 and consolidation of osteotomy (e) clinical photograph of same patient showing psis levels after 24 months a 21 month old girl (case 3) (a) x - ray pelvis both hips frog leg lateral view showing dysplasia left hip with acetabular index 36 (b and c) peroperative photographs showing ligamentum teres and iliopsoas cut respectively. safe zone 25 and open reduction done (d) x - ray pelvis showing both hips anteroposterior view after 24 months showing rad, no tear drop development, inadequate sourcil, ai - 35 all patients with rad were advised a pelvic osteotomy. out of them six patients underwent a secondary procedure out of which five were doing well at a later followup and one was lost to followup. classically, ddh in children between 18 and 24 months is treated by open or closed reduction followed by hip spica immobilization. according to a study by zionts and macewen (1986),4 secondary procedures on the hip were indicated only when residual subluxation was noted after bracing had been discontinued and the child had resumed walking. prereduction ai appeared to have little or no influence on the need for secondary procedures. moreover, the center edge (c - e) angle of wiberg was considered of little value in children who were 40 and a per - operative safe - zone < 20 increases the need for supplementary pelvic osteotomy in age group of 18 to 24 months because in such cases, the remodeling capacity of the acetabulum is unable to overcome the dysplasia and to form a relatively normal acetabulum. | background : developmental dysplasia of hip (ddh) is a common condition presenting to a pediatric orthopedic surgeon. there is a consensus on the surgical treatment of children with ages ranged from 18 to 24 months where majority agree on open reduction and hip spica. open reduction was done with an additional pelvic procedure wherever required to get better results and prevent residual acetabular dysplasia (rad) and early osteoarthritis.materials and methods:35 children with unilateral ddh were operated between 2002 and 2007 at our institute. open reduction was performed in all using the standard anterior approach and peroperative test for hip stability was done. nine children got an additional pelvic procedure in the form of dega acetabuloplasty. all were followed up for a minimal period of 2 years (range 2 - 7 years).results : no hip got redislocated. at the end of 18 months, there were seven cases of rad with acetabular index (ai) of 35 and above. these were all from the group where open reduction alone was done.conclusion:we feel that a preoperative ai of > 40 and a per - operative safe - zone < 20 increases the need for supplementary pelvic osteotomy in age group of 18 to 24 months because in such cases, the remodeling capacity of the acetabulum is unable to overcome the dysplasia and to form a relatively normal acetabulum. |
osteoarthritis (oa) is a chronic degenerative disorder of multifactorial etiology characterized by the loss of articular cartilage, hypertrophy of bone at the margins, subchondral sclerosis, and range of biochemical and morphological alterations of the synovial membrane and joint capsule.1 pathological changes in the late stage of oa include softening, ulceration, and focal disintegration of the articular cartilage. synovial inflammation also may occur.23 typical clinical symptoms are pain, particularly after prolonged activity and weight - bearing ; whereas stiffness is experienced after inactivity.2 it is probably not a single disease but represents the final end result of various disorders leading to joint failure.12 it is also known as degenerative arthritis, which commonly affects the hands, feet, spine, and large weight - bearing joints, such as the hips and knees.12 most cases of oa have no known cause and are referred to as primary oa.3 primary osteoarthritis is mostly related to aging.12 it can present as localized, generalized, or as erosive oa.34 secondary osteoarthritis is caused by another disease or condition.4 osteoarthritis is the second most common rheumatologic problem and it is the most frequent joint disease with a prevalence of 22% to 39% in india.13 oa is more common in women than men, but the prevalence increases dramatically with age.125 nearly, 45% of women over the age of 65 years have symptoms while radiological evidence is found in 70% of those over 65 years.245 oa of the knee is a major cause of mobility impairment, particularly among females.25 oa was estimated to be the 10 leading cause of nonfatal burden.24 self report surveys may not accurately estimate oa as there could be unknown cases in the community.6 there are few studies of oa that have used a radiological classification of disease. x - ray findings do not always match symptoms, but prevalence based on radiography is probably a reasonable population estimate.7 oa of the knee is more prevalent as per the literature available.7 therefore, for finding the current burden of oa and its association with lifestyle related factors, it was essential to undertake such a study on the prevalence of knee oa in indian population. this study was performed to find the prevalence of knee oa in the indian population and the factors associated with it by conducting a survey at community level in selected sample geographical areas. the target population was from five sites or sample groups. to have geographical representation from all over india, the following sites were selected ; (1) north hilly dehradun / nainital, (2) central agra, (3) western pune + pimpri, (4) south bengaluru / hyderabad, (5) east kolkata. each site comprised one metropolis, one small city, one block headquarters (town), and five villages from that block. the world health organization technical report series 919, the burden of musculoskeletal conditions at the start of the new millennium containing the community - oriented program for control of rheumatic diseases (copcords) bhigwan data on the prevalence of rheumatoid arthritis (ra) and oa among the indian population was taken as a reference for sample size estimation. this was a community - based study and it estimated a total prevalence of 5.5. this sample was further divided into five sites equally (936 from each site). therefore, the total sample size was 5000. the further division of sample within each site was done proportionate to the population. this evaluation study was conducted using the household as the primary sampling unit of the quantitative survey. this method was used to ensure no bias and equal age and sex composition in the sample. the exclusion criteria were as follows : age < 40 years, ra, inflammatory arthritis, bilateral end stage, knee oa, unable to walk without aids, systemic lupus erythematosus, polyarthralgia, previous history of fracture of lower limb or spine, any other surgical or medical condition that severely limits subjects functional ability. structured questionnaire was in the local language and consisted of the following sections informed consent, demographic profile age and sex, socioeconomic profile education, occupation, income, housing conditions, type of work and lifestyle related information, physical parameters height and weight, family history about oa and osteoporosis, respondents history related to osteoporosis presence of symptoms, whether diagnosed already and taking treatment, if not diagnosed, since when symptoms present. this was followed by an x - ray investigation of both the knee joints in two views anteroposterior view and lateral view. the present study shows a prevalence of 28.7% in the overall sample [table 1 ]. the prevalence was higher in villages (31.1%) and big cities (33.1%) as compared to towns (17.1%) and small cities (17.2%) [table 2 ]. osteoarthritis status of indian population site wise distribution of study sample the association of gender and oa of this study is in congruence with the available literatures on knee oa. oa of the knees was found to be more prevalent in females (31.6%) than in males (28.1%). the study found that the prevalence of oa knees increased with increase in body mass index (bmi). knee oa prevalence was significantly (p = 0.007) low in underweight people (28%) as compared to normal weight and obese participants (33%). prevalence was found to be highest in people who are overweight and/or obese [table 3 ]. the prevalence was highest among the age group of 60 and above and lowest in people in the age group of 4050 years (p = 0.001) [table 4 ]. although statistically significant (p = 0.0001), a cause effect relationship can not be derived. this is so because the unemployed group may include people who were retired. in such cases, prevalence was lowest among participants who worked as daily wage workers / laborers (22.2%). prevalence was highest in participants who have a sedentary lifestyle followed by participants with a physically demanding lifestyle and active lifestyle. this difference was statistically significant (p = 0.001) showing that the prevalence of oa was lowest in participants who had a fairly active physical activity level [table 5 ]. since the study recorded the current level of physical activity, it may be possible of having oa that may be more due to age rather than lifestyle. oa prevalence was found to be significantly more (p = 0.001) in participants who used western toilet (42.1%) as compared to those who used indian toilet (29.7%) or both types (38.8%), but it reflects more a condition of difficulty to use indian toilet than a predisposition to oa. bmi and osteoarthritis prevalence of osteoarthritis with age activity level and osteoarthritis prevalence was higher in participants who do not exercise (83.9%) compared to participants who exercise (36.0%). although the questionnaire gathered information on the type of exercises done, there was no significant difference in the prevalence of oa among different exercise groups. oa indeterminately occurs in elderly age group.125 oa occurs commonly in females above 45 years of age while before 45 years, it is common in males.25 the studies done on females for identifying the relation between estrogen and the prevalence of oa in menopausal age showed contradictory results. the prevalence of oa is available for the usa and european populations, but there are scare studies done in other regions.5 in 1990, it was the 10 leading cause of nonfatal diseases contributed 2.8% years of disability.7 an estimated prevalence of symptomatic oa is 18% in females and 9.6% in men.27 in global burden of diseases, in 2000, it was the 4 leading cause of years lived with disability (yld) leading to 3% yld.3 the copcord study also showed a higher prevalence in urban as compared to the rural prevalence of oa in bangladesh.89 in a study done in beijing 's urban population10 and wuchuan 's rural population,11 it was observed that wuchuan men had a prevalence ratio (pr) 2.5, 95% confidence interval (ci) (1.6 - 3.8) and symptomatic knee oa (pr 1.9, 95% ci 1.3 - 2.9). a chinese cohort study showed two to three times higher bilateral knee prevalence as compared to a framingham study.11 in an observational study done in rural tibetan region, the prevalence of knee pain was 25% and significantly associated in 50 years as compared to younger people.12 similarly, a study done by muraki. on japanese population in symptomatic and radiographically confirmed knee oa cases, it was evidenced to have higher prevalence in two mountain regions as compared to rural and urban population.13 in a house - to - house survey done by salve. in south delhi among 260 perimenopausal women, the prevalence of oa was found to be higher in lower socioeconomic than higher socioeconomic population.14 a study done by sharma. had similar results, but with lesser prevalence than this study.15 recently, a cohort study done by martin. showed that bmi is positively associated with knee oa in women and suggested that more active individuals have lower risk of knee oa.16 in a metaanalysis done by blagojevo., it showed that bmi is a risk factor for oa.17 the various modifiable risk factors are repetitive movement of joints, obesity, infection, and injuries. the occupational physical activities include monotonous motions and great forces such as kneeling, squatting on joints,1819202122232425 climbing,26 and heavy weight lifting. kellgren showed that the first - degree relatives of probands had twice higher risk than others. the genetic oa and progression study by riyazi. in multiple sites showed the evidence of familial hereditability of oa of hand, hip, and spine, but not in knee.27 this study has evidenced a large percentage of subthreshold population, that is, k - l grade 1 which is considered as borderline or doubtfull as far as oa diagnosis is considered. awareness program should be initiated at community level which is needed for the prevention of oa of knee at early age. we would like to suggest that a longitudinal cohort study can be planned which, in long run, will prove the impact of physical activity, habits, and lifestyles. stress on early diagnosis on the onset of symptoms should be encouraged among the general population. studies to understand how many people with symptoms of oa seek medical advice are required to understand the treatment seeking behavior and pain tolerance associated with oa. | background : among the chronic rheumatic diseases, hip and knee osteoarthritis (oa) is the most prevalent and is a leading cause of pain and disability in most countries worldwide. its prevalence increases with age and generally affects women more frequently than men. oa is strongly associated with aging and heavy physical occupational activity, a required livelihood for many people living in rural communities in developing countries. determining region - specific oa prevalence and risk factor profiles will provide important information for planning future cost effective preventive strategies and health care services.materials and methods : the study was a community based cross sectional study to find out the prevalence of primary knee oa in india which has a population of 1.252 billion. the study was done across five sites in india. each site was further divided into big city, small city, town, and village. the total sample size was 5000 subjects. tools consisted of a structured questionnaire and plain skiagrams for confirmation of oa. diagnosis was done using kellgren and lawrence scale for osteoarthritis.results:overall prevalence of knee oa was found to be 28.7%. the associated factors were found to be female gender (prevalence of 31.6%) (p = 0.007), obesity (p = 0.04), age (p = 0.001) and sedentary work (p = 0.001).conclusions : there is scarcity of studies done in india which has varied socio geographical background and communities. we conducted this study for analyzing the current prevalence of oa in different locations. this study has evidenced a large percentage of population as borderline oa ; therefore, it depends mainly on the prevention of modifiable risk factors to preserve at ease movement in elderly population through awareness programs. |
in the united states, 4 to 5 million patients undergo abdominal operations each year., 5% of the general population has an umbilical or epigastric hernia, making ventral hernia repair one of the most commonly performed surgical procedures. approximately 90 000 this is attributed to a variety of reasons, including the loss of muscle strength in the anterior abdominal wall, and the prevalence of comorbidities that lead to increased intraabdominal pressure. in addition, advanced medical care has increased life expectancy in industrialized countries. by 2025, 25% of the united states population is estimated to be over the age of 65. while the presence of a ventral hernia in a young adult is considered an indication for repair, there may be some concerns about the safety of elective ventral hernia repair in the elderly population. laparoscopic ventral hernia repair has proven to be a safer and more effective procedure than open ventral hernia repair. whereas laparoscopic procedures were once avoided in the geriatric population, they are now considered a safe approach to manage the surgical needs of this growing population. the goal of this communication is to evaluate the safety and efficacy of laparoscopic ventral hernia repair in patients who exceed 65 years of age. the study retrospectively compared 155 patients of all ages who choose to undergo elective laparoscopic ventral hernia repair between july 2001 and july 2006 at michigan state university / kalamazoo center for medical studies. patients were classified according to their age into 2 groups, group a consisted of patients 65 years old. medical comorbidities and the general medical condition of all patients were evaluated and optimized before surgery. patients with pulmonary or cardiac comorbidities had a pneumoperitoneum established to the pressure of 12 mm hg instead the standard of 15 mm hg. all repair procedures were performed by a single surgeon (the first author) using the same operative technique and perioperative protocols. a baseline comparison was made between the 2 groups for patient demographics (including weight, height, and body mass index), american society of anesthesia score (asa), medial comorbidities, types of hernia, operative time, and the length of hospital stay. descriptive statistics were computed for all variables. to assess the difference between the 2 age groups of hernia patients, the t test was computed for the quantitative variables while the chi - square test and fisher 's exact test were used for categorical variables. all statistical analyses were done using sas version 9.1 (cary, nc) at 5% level of significance. descriptive statistics were computed for all variables. to assess the difference between the 2 age groups of hernia patients, the t test was computed for the quantitative variables while the chi - square test and fisher 's exact test were used for categorical variables. all statistical analyses were done using sas version 9.1 (cary, nc) at 5% level of significance. weight and body mass index where significantly higher in the younger age group (group a). there was no difference in the american society of anesthesia (asa) score between the 2 groups (table 1). the older age group had an increased number of previous hernia repairs (mean number of previous repairs, 0.72 vs. 0.61). lysis of the resultant adhesions may explain the increase in the operative time (mean or time, 125 minutes vs. 97 minutes). another reason for the longer operative time in older patients is the relatively small working space created by the lower pneumoperitoneum pressure used in patients with cardiac problems (which is more common among older patients). there was no significant difference in the length of stay between the 2 groups (table 2). no significant difference was noted between the 2 groups regarding the number of hernia defects, size of the defects, or the surface area of the mesh required to close the defects. incisional hernia was the most common type of hernia between the 2 groups followed by umbilical hernia ; however, the prevalence of incisional hernia was significantly higher in the older age group, which can be explained by the longer past history of medical disease (lead time effect) (table 2). associated comorbidities were significantly more prevalent in the older age group, with hypertension being the most common (79% in group b vs. 31% in group a), followed by cardiac problems (42% in group b vs. 6% in group a). on the other hand, obesity (bmi>30) was more prevalent among younger patients (37% in group a vs. 21% in group b). other comorbidities included pulmonary diseases, diabetes, hypothyroidism, and the presence of other hernias (inguinal, hiatal, and others). these comorbidities were more or less equally distributed between the 2 groups (table 3). all procedures were completed laparoscopically except for those in 4 patients (3 in group a and 1 in group b) due to extensive adhesions. the postoperative course was unremarkable except in 6 patients (all of them are from the younger age group) who developed a prolonged ileus secondary to extensive adhesiolysis. in addition, one patient in the older group developed a postoperative pulmonary embolism that was treated medically with anticoagulants. the most common complication was postoperative seroma formation (11% in group a, and 13% in group b). in most cases, these seromas were transient and resolved with conservative management ; a few cases did require aspiration. complications in both age groups not statistically significant. with a mean follow - up of 8 months (range, 1 to 52), 4 cases of recurrence were noted, 3 occurred in the younger group and only one in the older group. two of these cases (one of each group) were attributed to morbid obesity along with the lack of adequate transfascial sutures. one case of recurrence was secondary to persistent seroma, and one case was secondary to mesh infection that mandated mesh removal (table 4). ventral hernias are a common problem seen in the elderly population. in the united states, complications related to hernias are one of the most common causes of emergency surgery in patients above 50 years old. ventral hernias are the second most common cause of intestinal obstruction, representing 10% to 15% of cases. the geriatric population is more prone to develop electrolyte and acid base imbalances from intestinal obstruction. the management of these complications is significantly more difficult in elderly patients with relatively high morbidity and mortality. during the early days of laparoscopic surgery the rationale was that older patients have depressed myocardial and pulmonary functions that could be further compromised by a pneumoperitoneum. furthermore, laparoscopic procedures used to require significantly longer operative times when compared with open procedures, thus exposing the patients to the risks of prolonged anesthesia. however, with the increased experience of laparoscopy these concerns have proven to be inaccurate. the adverse effects of a pneumoperitoneum can be reduced by keeping the pressure below 12 mm hg, which allows adequate distension of the abdomen with minimal effects on cardiac and pulmonary function. in addition, the advances in both surgical techniques and available laparoscopic tools have reduced the operative time of most laparoscopic procedures to levels comparable to those of open procedures. furthermore, the safety profile of the currently available anesthetics is better than the earlier predecessors, which allows safe laparoscopy even in patients with comorbid conditions like liver cirrhosis. this has encouraged surgeons to perform more surgeries through the laparoscopic approach, especially when dealing with the elderly population. laparoscopic ventral hernia repair has proven to be a safe and effective alternative to the open approach. the recurrence rate following laparoscopic ventral hernia repair is reported to be 4.3%, compared with the recurrence rate of 41% to 52% after suture repair, and 12% to 32% recurrence for open mesh repair. in addition, the patient gets to avoid the large abdominal incisions with the resultant pain, limitation of activity, and the inflammatory, hormonal, and metabolic stress of surgery. recent health care advances have allowed patients to live longer and maintain a higher level of activity even through advanced age, making them both more tolerant to surgery and more liable to hernia complication. although the risks of open ventral hernia repair may exceed those of untreated ventral hernias, the risks associated with laparoscopy are significantly lower. based on this experience, the benefit of avoiding severe, life - threatening complications, such as incarceration and strangulation, exceed those of elective repair of ventral hernias through the laparoscopic approach in addition to the improvement in the quality of life of these patients. this report demonstrates the feasibility, adequacy, and efficacy of laparoscopic ventral hernia repair in the elderly population. the decision to perform or not to perform an elective hernia repair should depend on the patient 's condition and comorbidities regardless of the chronological age of the patient. | introduction : ventral hernias are common surgical problems in the geriatric population. although ventral hernias are electively repaired in younger patients, the safety and efficacy of elective laparoscopic hernia repair in the geriatric age group is not well documented in the literature.methods:a review of 155 patients undergoing laparoscopic ventral hernia repair was undertaken. the patients were classified according to their age into 2 groups, group a (n=126) for those who are 65 years old and group b (n=29) for those who are > 65 years old. the patient demographics, comorbidities, hernia characteristics, and operative and postoperative data were compared.results:younger patients were found to have a significantly increased bmi, while the older group had an increased number of comorbidities. no difference was found in the complication or recurrence rates between the 2 groups.conclusion:elective laparoscopic ventral hernia repair in senior citizens is safe and feasible in our experience. we believe that the decision to perform an elective hernia repair in this patient population should be based on the general condition of the patient rather than the patient 's chronological age. |
west nile virus (wnv) is an arthropod borne neurotropic single stranded rna flavivirus. the virus is introduced into the host by the vector (culex mosquito) during its blood meal. wnv originated in the middle east and africa but in recent years has reached the united states. from 1999 to 2011, a total of 31,414 cases of wnv were reported in the united states. the 2012 incidence of wnv neuroinvasive disease was 0.3 per 100,000 in the united states. approximately 45% of wnv cases were reported in texas, usa. seasonal outbreaks occur annually but are often quite focal and unpredictable in size and location. the majority of those infected with wnv are asymptomatic and only 20 - 30% will present with flu like symptoms. less than 1% of individuals will eventually develop neurological deficits. immunocompromised and elderly patients are more prone to developing wnv meningitis or encephalitis compared to the general population. wnv and other arboviral infections should be considered in the differential diagnosis of aseptic meningitis or encephalitis, especially if the geographical area is prone to wnv infection. the definitive diagnosis of wnv meningoencephalitis is a combination of clinical suspicion and csf serology. we are presenting a very rare case of a immunocompromised patient that developed opsoclonus myoclonus syndrome following wnv encephalitis. forty - eight year old caucasian female presented with a sudden onset of altered mental status after being found unresponsive. she was confused with intermittent bouts of alertness / lethargy and unintelligible responses to questioning. her medical problems included endometrial cancer that was treated with a total abdominal hysterectomy with bilateral salpingectomy and postoperative chemotherapy. the endometrial adenocarcinoma was a stage 2 according to the international federation of gynecology and obstetrics (figo) staging : 8 cm tumor, grade 2, no lymph node involvement, and < 50% invasion of the myometrium and cervical stroma. the patient was currently in remission after receiving chemotherapy (paclitaxel and carboplatin) and dexamethasone. initial vital signs on admission included a rectal temperature was 41.1 degrees celsius, tachycardia (heart rate of 108), stage 1 hypertension (blood pressure of 140/61) and tachypnea (respiratory rate of 34). pertinent physical examination findings revealed a morbidly obese female that was alert but not oriented to person, place or time and only able to answer yes or no to questioning. her pupils were 3 mm bilaterally, equal round and reactive to light with intact extraocular movements and no nystagmus noted. no motor or sensory deficits to light, touch, pain, position sense or vibration noted. initial laboratory work - up (table 1) was unremarkable except hyponatremia and thrombocytopenia. brain computed tomography (ct) on admission was negative for any acute pathological abnormalities. the patient was started on ceftriaxone 2 g iv daily, vancomycin 2 g iv twice daily, and acyclovir 1.5 g iv every 8 hours due to our high suspicion of encephalitis. a lumbar puncture with cerebrospinal fluid (csf) analysis and csf serology (table 2) was consistent with aseptic meningitis. magnetic resonance imaging (mri) of the brain was unremarkable for any acute intracranial abnormalities. vancomycin and acyclovir were discontinued and fluconazole 400 mg po daily was added to the management on the sixth hospital and ceftriaxone was continued until the tenth hospital day. by the seventh hospital day, we noted involuntary random movements of her eyes (see video 1) and generalized jerking movements (myoclonus), mostly in lower extremities. an electroencephalography (eeg) showed a severe and diffuse slowness, consistent with encephalopathy. she was placed on fosphenytoin 100 mg iv every 8 hours without any improvement. on the eighth hospital day, her gcs decreased to 3 and was subsequently intubated. the platelet count continued to decrease during the hospitalization and eventually reached its lowest count of 2110/ul by the tenth hospital day. the west nile virus (wnv) antibody igm and igg were noted to be positive on the twelfth hospital day. after determining that the encephalitis was due to the wnv we continued supportive care and discontinued the fluconazole. they diagnosed her to have opsoclonus myoclonus syndrome (oms) induced by the wnv meningoencephalitis. paraneoplastic antibodies anti - yo, anti - hu, anti - ri and anti - nmdar were negative. a percutaneous endoscopic gastrostomy tube (peg) and tracheostomy were performed on hospital day 26. the patient continued to have improvement in her neurological status and was subsequently discharge to a long term acute care (ltac) facility for further rehabilitation. she underwent rehabilitation for six months and eventually had complete resolution of her neurological deficits on follow visit. opsoclonus - myoclonus syndrome (oms) is a rare neurological disorder associated with chaotic multidirectional eye movements, myoclonus and less frequently cerebellar ataxia. the eye movements are characterized by multidirectional, high amplitude, arrhythmic and conjugate ocular saccadic intrusions without intersaccadic latency causing oscillopsia. it should not be confused with nystagmus which is usually unidirectional and has a slow component. myoclonus may affect any part of the body and may be evoked by action, intention, auditory and visual stimuli. the pathogenesis of opsoclonus is unknown but, one hypothesis suggests that opsoclonus is due to damage of the omnipause cells in the nucleus raphe interpositus of the pons. this prevents unwanted saccades by inhibiting burst neurons in the paramedian pontine reticular formation and rostral interstitial nucleus of cajal. another explanation suggests that opsoclonus may be generated by disinhibition of the oculomotor region of the fastigial nucleus by a process that interferes with the inhibitory projections of the purkinje cells of the deep cerebellar nuclei to the fastigial nuclei. the pathogenesis is not fully understood, however, an autoimmune pathophysiology has been proposed. autoimmune dysfunction of purkinje cells in the dorsal vermis and subsequent disinhibition of oculomotor fastigial region seems to be the most probable mechanism. however, other common causes of opsoclonus include paraneoplastic, viral brainstem encephalitis and drugs. the onconeuronal antibodies associated with oms are anti - ri antibodies (gynecologic cancers) and, less frequently, anti - hu, anti - yo, and anti - ma-2 antibodies. paraneoplastic diseases associated with oms include small - cell lung, ovarian and breast cancer. most infectious disease causes involve the central nervous system or a post infection immune mediated process. the post infectious mechanism has been described as a dysfunction of the cell mediated and humoral immune response. this immune mediated process was postulated because the presence of autoantibodies and the response of oms to immunosuppressive therapy. the specific microorganism is rarely identified however oms has been reported to occur after infection with enterovirus, borrelia burgdorferi, streptococcus, coxsackie virus, epstein - barr virus, mumps or west nile virus. opsoclonus myoclonus syndrome can result in significant long term neurological sequelae if either untreated or undertreated. therapy options have been directed towards the suppression of antibody formation and the removal of the autoantibodies. several therapeutic attempts can be utilized to remove the autoantibodies in patients with oms, such as ivig, plasmapheresis, and immunoadsorption. however there is no available data from clinical trials that suggest a definitive treatment strategy. because the opsoclonus - myoclonus syndrome is so rare, there are no randomized controlled trials to provide guidelines for either symptomatic or disease modifying treatments. according to current medical literature, only two other case reports have reported opsoclonus myoclonus syndrome induced by west nile virus encephalitis. | west nile virus (wnv) is an arthropod borne neurotropic single stranded rna flavivirus with < 1% developing presenting with neurological disease. immunocompromised and elderly patients are more prone to developing wnv meningitis or encephalitis. definitive diagnosis of wnv meningoencephalitis is a combination of clinical suspicion and cerebrospinal fluid (csf) serology. forty - eight year old caucasian female presented with a sudden onset of altered mental status after being found unresponsive. she was confused with intermittent bouts of alertness / lethargy and unintelligible responses to questioning. her medical problems included endometrial cancer that was in remission after undergoing a total abdominal hysterectomy with bilateral salpingectomy and postoperative chemotherapy with paclitaxel and carboplatin. pertinent physical examination revealed muscle strength that was significantly decreased, nuchal rigidity and + 2 pitting edema of both lower extremities. computed tomography and magnetic resonance imaging of the brain were negative for any intracranial pathology. csf analysis was consistent with aseptic meningitis with all csf serology being negative except for positive wnv antibody. a few days after being admitted she developed involuntary random movements of her eyes and generalized jerking movements (myoclonus). this was determined to be opsoclonus myoclonus syndrome (oms) induced by the wnv meningoencephalitis. she then received five consecutive days of plasmapheresis with a significant improvement in her neurological status. opsoclonus - myoclonus syndrome (oms) is a rare neurological disorder associated with chaotic multidirectional eye movements, myoclonus and less frequently cerebellar ataxia. oms affects as few as 1 in 10,000,000 people per year. the pathogenesis is not fully understood with the majority of cases of opsoclonus - myoclonus syndrome being idiopathic. according to current medical literature there have only been two previous case reports of opsoclonus myoclonus syndrome associated with wnv encephalitis. |
bariatric surgery still remains the most effective treatment for morbid obesity, leading to a reduction of comorbidities in the long term. roux - en - y - gastric bypass (rygb) is one of the most frequently performed procedures in the united states and is also widely practiced in europe [24 ]. calcium and vitamin d deficiencies, which frequently arise after surgery, may be overlooked because of their slow development. however, they may give rise to secondary hyperparathyroidism (2hpt) as the body attempts to counter the deficits. obviously, sufficient dietary calcium and vitamin d are necessary to prevent this condition, which can be difficult to achieve because of the bypassed proximal part of the small intestine. this is the obvious reason for the recommendation of supplying adequate amounts of dietary calcium and vitamin d after surgery [68 ]. long - lasting 2hpt for years is believed to be an important contributing factor for increased bone turnover and consequent bone loss. indeed, epidemiologic data have shown that gastric surgery can lead to a state of 2hpt with an increased risk of osteoporotic fractures [911 ]. besides inadequate dietary intake of calcium to compensate for suboptimal calcium absorption, changes of bowel habits after gastrointestinal surgery may also interfere. it is of note that morbidly obese patients frequently have fecal incontinence due to a rise in intra - abdominal pressure with compression and stretch of the pelvic floor and of the muscles and nerves around the anal sphincter complex and in the vicinity of the supporting endopelvic fascia. after bariatric surgery, many patients initially experience less fecal incontinence due to substantial weight loss and associated improvements of intra - abdominal mechanics [13, 14 ]. for obvious reasons, patients may even have a lowered frequency and hardened consistency of defecation in comparison to the situation prior to the operation. nevertheless, more frequent and looser stools are common in operated patients [1517 ]. studies in laboratory animals elaborating on intestinal calcium absorption have shown that intestinal calcium kinetics depends on intestinal transit time. similar observations were found after rygb due to the inability of the small intestine to bind and absorb calcium. to our knowledge, no previous studies were published about interactions between bowel habits after surgery and changes of calcium metabolism. the aim of the present study is to investigate relationships between semiquantified stools scores according to patient reports, compliance for the daily use of extra calcium, and calcium metabolism. patients were eligible for the study if laboratory data were available at baseline and at 11 to 13 months after surgery. a total of 103 patients, 27 males and 76 females, were eligible, and these were invited to discuss the purpose of the study ; all consented to participate. the objectives of the study were (1) to evaluate bowel habit changes after surgery ; (2) to evaluate relationships between (1) and laboratory assessments ; (3) to evaluate relationships between (1) and (2) with the daily use of a formulation, containing 1 g of elementary calcium and 880 to 1000 iu vitamin d3. the medical ethical committee of rijnstate hospital, arnhem, the netherlands approved the study. all patients underwent surgery in a single center for bariatric surgery (rijnstate hospital alysis zorggroep, the netherlands). laparoscopic rygb was performed by 2 dedicated bariatric surgeons who used standard techniques. according to protocol, patients with a bmi > 40 kg / m received a 45 cm biliopancreatic limb, and a 100 cm alimentary limb, while patients with a bmi > 50 kg / m received a 150 cm alimentary limb. 20 patients received a 150 cm limb and 83 patients received a 100 cm limb. 22.2% of all males received a 150 cm limb versus 18.8% of all females, meaning no statistical difference in sex distribution. patients were advised to take daily multivitamins in addition to omeprazol 40 mg (once daily for 6 months) and low molecular heparin sc (fraxiparine 5700 iu once daily for 6 weeks) ; patients were advised to take a daily calcium / vitamin preparation (cad 1000/880 sachets, containing calcium citrate and vitamin d3) or, if they refused to take cad, to try a different formulation containing calcium carbonate and vitamin d3 (calcichew 500/400 chewing tablets b.i.d.). after written informed consent, each participant was requested to complete a questionnaire about his / her bowel habits prior to and at 12 months after surgery. fecal frequency was scored on a five - point scale ranging from less than twice a week (1 point), every two days (2 points), once a day (3 points), twice a day (4 points) to more than twice a day (5 points). in addition, fecal consistency was scored on a five - point scale ranging from watery stools (5 points), watery to solid stools (4 points), normal stools (3 points), firm stools (2 points) to hard stools with concomitant constipation (1 point). summation of fecal consistency and frequency scores yielded a fecal score (fs). compliance in taking additional calcium and vitamin d3 was arbitrarily defined as daily use with the maximal exception of one day per week. patients who reported taking the supplement for 5 or less days a week were categorized as noncompliant. for the current analysis, we recorded (1) compliance with regular use of calcium and vitamin d3 containing formulations, (2) the number of patients who requested to switch from liquid formulations (sachets) to chewing tablets, and (3) the number of patients who used laxatives or constipating agents, at least weekly. calcium, albumin, vitamin d, and pth at baseline (before surgery) and at 12 months after surgery were used in the final analysis of the study. all data are reported as mean 1sd unless otherwise specified ; p 25% for the entire group of 103 patients and exceeded more than half in 83 participants (81%). ewl of the total group at 12 months had decreased by (mean 1sd) 63.5 19.5%. the bmi of the whole group decreased from 48.2 6.2 kg / m before surgery to 33.9 5.5 kg / m at 12 months. serum calcium concentrations corrected for albumin were normal in 101 patients (98%) ; vitamin d insufficiency (6.8 pmol / l) in 36 patients (35%), see table 2. notably, none of the patients were regular laxative users, and 79 out of 103 patients (77.7%) reported permanent changes of their bowel habits. each patient estimated stool frequency and consistency on two 5-point scales ranging from stools less than twice a week to more than twice a day and from watery to hard stools. the summation of each score (frequency plus consistency scores) yielded a fecal score (fs). memorized stool frequency and consistency data before surgery and at 12 months are listed in table 3(a) and the distribution of fecal scores (fs) (from 2 to 10 points) in table 3(b). each score disclosed a significant change comparing frequency of stools (f), consistency of stools (c), and the fecal score (fs). in general, there was a significant shift towards more frequent and less consistent stools, which overall had resulted in higher fecal scores for (f : p <.05, c : p <.0001, and fs : p <.01). laboratory assessments disclosed no significant changes of corrected calcium and vitamin d levels (calcium before surgery 2.31 mmol / l and at 12 months 2.34 mmol / l, vitamin d before surgery 50.4 nmol / l and at 12 months 45.0 nmol / l), while mean pth levels increased from 5.1 to 6.1 pmol / l (p =.02). before surgery, 10 patients had increased pth levels, while 4 of them (40%) had also increased pth levels at 12 months. before surgery, 32 out of 93 patients had normal pth levels but developed raised pth levels at 12 months (34.4%) (ns). pth levels compared for each domain of stool habits (frequency (f), consistency (c), and fecal score (fs)) per tertile at 12 months, showed no significant changes for f and c. however, pth levels rose significantly along with tertile fs (rr 30.5, ci 6.2149.2, p <.001), see table 4. at 12 months, calcium and vitamin d levels were similar between patients with an alimentary limb of 100 cm (n = 83) and 150 cm (n = 20) (100 cm : calcium : 2.31 mmol / l, vitamin d 46 nmol / l and 150 cm : calcium : 2.33 in addition, there was a trend towards higher pth levels after inclusion of all patients using two - sided student t - test : 100 cm : pth : 5.6 pmol / l and 150 cm : pth 7.9 pmol / l (p =.001). pth levels were not significantly significant after inclusion of compliant calcium / vitamin d3 users (logistic regression) (rr 3.2, ci 0.911.8, p =.08). at 12 months, there were 36 patients (35%) with increased pth levels. 24 of these patients had a 100 cm alimentary limb (67%), and 12 patients had a 150 cm alimentary limb (33%). according to the definition used in the current study, pmol / l) were found in 21 patients who belonged to this group compliant for suppletion (26%) against 15 patients who belonged to the 21 remaining noncompliant suppletion users (71%) (rr 14.3, ci 3.656.5, p <.001). in the subgroup of 36 patients with high pth levels, 21 patients (58%) were compliant suppletion users (pth : 9.2 2.8 pmol / l) and 15 patients (42%) were not compliant (pth : 9.9 2.7 pmol / l) (ns). the reasons for noncompliant suppletion use were being principally against regular use of any prescribed drug, frequently forgetting, and taste aversion against calcium formulations. 69 of the 82 compliant suppletion users (84%) had decided to switch from sachets (liquid formulation) to chewing tablets, while the remaining 13 patients (16%) continued to take sachets. the main reasons for changing from sachets to tablets were nonpalatability reported by 54 patients (78%) and/or the inability to drink large volumes reported by 30 patients (43%). the results of the current study showed significant changes of bowel habits after surgery, which went into the direction of a higher stool frequency, less consistency, and consequent higher fecal scores. there were no correlations with either frequency or consistency and pth levels, while fecal score and pth levels were positively correlated. there was also an interaction between compliance for calcium / vitamin d3 suppletion and pth levels, but it should be emphasized that the current results are nothing but associations, which implicates that prospective intervention studies are needed to clarify how these factors interact. moreover, it is entirely unclear whether additional calcium and vitamin d supplements are beneficial in suppressing pth levels and ultimately affect incident osteoporotic fractures in later life. an additional uncertainty is the effect of omeprazol 40 mg (once daily for 6 months) on calcium metabolism after surgery. indeed, proton pump inhibitor therapy increases the risk to skeletal fracture in the long term, possibly by inhibiting intestinal calcium absorption [21, 22 ]. obviously, prospective randomized clinical trials should be long lasting as osteoporotic fractures usually occur much later in life. elevation of pth, even in the presence of unchanged corrected calcium and vitamin d levels, is compatible with 2hpt. in fact, rygb serves as a model for 2hpt exclusively due to inadequate calcium absorption. a study on calcium metabolism of patients who had undergone rygb and matched controls showed that the patients needed an extra amount of 750 mg calcium per day to maintain similar pth levels. these calculations were made at 9 months after surgery, meaning that calcium metabolism was still influenced by skeletal calcium effluxes due to high bone turnover [23, 24 ]. yet, no calcium balance studies have been published that deal with operated patients with a stable body weight to provide information on the optimal amount of dietary calcium. notably, too much extra dietary calcium may be harmful because of calcium - related constipation. it has been shown previously that calcium salts bind to free fatty acids and fecal bile acids in the colonic lumen, forming calcium soaps. in other words, calcium suppresses intestinal motility indirectly through binding at the colon level leading to less free fatty acids and to bile acids that are toxic to the colonic mucosa to provoke frequent and watery stools. it is an open question whether increased pth levels due to chronic calcium malabsorption after rygb are relevant to future skeletal health. bone loss after bariatric surgery can be partly ascribed to the direct and secondary effects of rapid weight loss and partly to 2hpt - related bone loss. moreover, shortly after bariatric surgery, there will be substantial bone loss, which is obviously driven by the catabolic state of the ongoing weight reduction and not by 2hpt. most of this type of rapid bone loss occurs at the hip and ceases with stabilization of body weight. the loss of bone mineral density after surgery occurs mainly at the hip and pelvis, varying between 5 and 10% [23, 24, 26 ]. however, these analyses were performed around 12 months after surgery, during rapid bone loss. thus far, there are no data, for example, after a decade available in the literature. most of the available data come from histological studies of operated patients with a jejunoileal bypass, showing that full - blown osteomalacia was common after this particular procedure. however, jejunoileal bypass surgery is fraught with many long - term complications, among which is vitamin d deficiency, and the operation has therefore been abandoned. since no histology data are available from obese patients after rouxeny bypass surgery, the jejunoileal bypass bone samples are unique and could help to understand some of the skeletal health issues that should be dealt with. in a comparative histomorphometry study in 21 patients 314 years after intestinal bypass surgery for obesity, osteomalacia was found in one - third of all patients. biopsies of those without osteomalacia disclosed a marked reduction of trabecular thickness, partly as the result of 2hpt and partly because of insufficient osteoblastic synthesis. this picture differs from that of age - related bone loss and postmenopausal osteoporosis which share loss of density rather than thickness of trabecular plates. biopsies from 16 middle - aged males after partial gastrectomy with billroth ii anastomosis revealed high bone turnover (leading to bone loss) due to body weight reduction after - surgery and/or 2hpt. finally, there is a histological proof for insufficient osteoblastic recruitment and activity, probably due to deficits of unidentified nutrients resulting from the malabsorption caused by bypassing critical parts of the small intestines. in a bone biopsy study among 41 patients after partial or total biliopancreatic bypass, it was found that defective mineralization and a decrease of bone formation rate were present in spite of serum 25-hydroxyvitamin d concentrations being normal. these biopsy data underscore the concept that in one way or another essential nutrients are needed to support bone formation. this study has several limitations, mainly related to its retrospective and cross - sectional design. however, it underlines the need for more prospective intervention studies in gastric bypass patients, even though this procedure is considered the gold standard, suggesting safety. in the meantime, it remains advisable to consider bone mineral density in the assessments of risk factors prior to surgery, particularly in women. in conclusion, 12 months after gastric bypass surgery, there was a positive relationship between bowel habits (i.e., the summation of stool frequency and consistency scores) and pth levels with an interaction for the compliance with calcium / vitamin d suppletion. the current study underscores the importance of professional management of bowel habits and tailor - made calcium / vitamin d suppletion. long - term prospective intervention studies are critical to evaluate efficacy as well as side effects and should be undertaken in the near future. | background. calcium malabsorption after bariatric surgery may be harmful to skeletal health and demands for optimal skeletal management. methods. 103 patients were evaluated retrospectively at 12 months after surgery. the evaluation included a questionnaire about stool frequency and consistency and laboratory assessments. results. 103 patients, 27 males and 76 females, were included in the study. 83 patients had an alimentary limb of 100 cm and 20 patients one of 150 cm. at 12 months after surgery, 77.7% reported changes of bowel habits, albumin adjusted calcium levels were normal in all but 2 patients, and pth levels were increased in 35%. correlations between semiquantified bowel scores (fecal scores) and data from the laboratory demonstrated increasing pth values along with more frequent and softer / watery stools (rr 30.5, ci 6.2149.2, p <.001). there was a trend for higher pth levels in patients with an alimentary limb of 150 cm. normal pth levels were more frequently found in case of calcium and vitamin d3 use (rr 14.3, ci 3.656.5, p <.001). conclusion. this study demonstrates interrelationships between semi - quantified fecal scores, pth levels, and the compliance of taking calcium / vitamin d3 suppletion. however, prospective randomized studies are necessary to show causal relationships. |
imaging findings, even with computed tomography (ct), of such tumors are rarely described in literature. f-18 fluorodeoxy glucose positron emission tomography / computed tomography (f-18 fdg pet / ct) is emerging as a useful modality in assessment of genito - urinary malignancies. f-18 fdg pet / ct findings in penile leimyosarcoma have not been reported in literature. we present here a case of penile leiomyosarcoma with emphasis on findings on f-18 fdg pet / ct with histopathology correlation. a 47-year - old male presented with a palpable penile mass, with rapid growth over a period of 1 month. he was examined at an outside center and an incision biopsy of the tumor was done that revealed penile leiomyosarcoma. he was later referred to our institute, a tertiary care center, for further management. on examination of the patient at our institute, the patient was found to have 2 2 cm a typical nodular lesion in the proximal penile shaft. review of the histopathology examination after biopsy revealed tumor composed of spindle cells with hyperchromatic nuclei, inconspicuous nucleoli, and frequent mitoses. tumor cells also stained positive for smooth muscle antigen (sma) consistent with diagnosis of leiomyosarcoma of penis [figures 1c and 1d ]. as a part of staging, the patient was subjected to f-18 fdg pet / ct examination 2 months after the biopsy (this delay was due to late reporting of the patient to our institute from the peripheral center). the ct scan revealed moderate fdg uptake in the periphery of an ill - defined heterogeneously enhancing soft tissue lesion 2.9 2.2 cm in size involving corpora cavernosa on the left side of the distal penile shaft. this mass showed slightly thick, peripheral rim enhancement on the contrast - enhanced ct images corresponding to an area of increased f-18 fdg uptake, with an internal homogeneous region of low density, compared to the adjacent normal shaft of the penis, representing residual disease [figures 1a and 1b ]. taking into consideration possible microscopic lymph node metastases total penectomy and bilateral inguinal lymphadenectomy was planned and the patient was counseled. however, the patient refused to undergo lymphadenectomy and only total penectomy was done. on follow - up after 8 months, the patient was found to be disease free. (a) contrast - enhanced axial ct through the penis demonstrates a low attenuating lesion in the corpora of the penis with peripheral enhancement. (b) f18-fdg pet image fused with corresponding ct demonstrates increased fdg uptake in the periphery of an ill - defined lesion within the corpora of the penis. (c) histopathological examination shows tumor composed of spindle cells with hyperchromatic nuclei, inconspicuous nucleoli with frequent mitoses (d) histopathological examination shows positivity for smooth muscle antigen penile cancer is a rare neoplasm and accounts for approximately 0.4% of all male malignancies. the most common primary malignant neoplasm of the penis is squamous cell carcinoma, constituting more than 95% of cases, followed by metastatic neoplasms of the prostate, bladder, rectum, kidney, and testis, as well as those spreading by direct extension from the adjacent structures. it accounts for 1020 % of all malignancies in males in asia, africa, south america, and it has a prevalence of only 1% in western countries. predisposing factors are phimosis, cigarette smoking, and human papilloma virus (hpv) infection. other tumors of the genitourinary tract include sarcoma, melanoma, basal cell carcinoma, and lymphoma. sarcomas are uncommon penile neoplasms, which include epithelioid sarcoma, kaposi sarcoma, leiomyosarcoma, and rhabdomyosarcoma. in general, very little literature exists that discusses the management of penile leimyosarcoma, due to the rarity of disease. role of routine lymphadenectomy is controversial as few authors have suggested that regional lymph node dissection is usually not indicated, since nodal metastases are uncommon. in our case lymphadenectomy was planned ; however, patient refused to undergo lymphadenectomy and was managed only with total penectomy. the ct findings of penile leiomyosarcoma are also rarely reported in literature in which the tumor may appear as a mass showing a relatively thick peripheral rim enhancement with internal regions of homogeneous low density, compared to the adjacent normal shaft of the penis, (similar to findings in our case) or as a soft tissue mass. fdg uptake has been described in penile squamous cell carcinomas and f-18 fdg pet / ct has been shown to be useful in detection of lymph nodal and distant metastases in penile carcinoma. f-18 fdg uptake has also been described in leiomyosarcomas and tumoral f-18 fdg uptake correlates well with the grade of the tumor in case of leiomyosarcomas. f-18 fdg pet / ct plays an important role in diagnosis, staging, treatment response monitoring, and follow - up in patients with sarcomas. this is the first case demonstrating f-18 fdg pet / ct findings in a case of penile leimyosarcoma. | penile cancer is a rare entity accounting for only 0.4% all male malignancies. penile leiomyosarcomas are even rarer with only around 35 cases reported in literature. we report a rare case of penile leiomyosarcoma illustrating f-18 fluorodeoxy glucose (fdg) positron emission tomography / computed tomography (pet / ct) features and histopathology correlation. |
imaging characteristics are usually helpful in the differentiation of various cardiac masses with high probability. the differential diagnosis of aortic thrombus like tumor and vegetation is crucial for avoiding unnecessary surgical intervention. non - invasive imaging methods would be useful in the diagnosis of suspected cardiac mass, because they may provide earlier diagnosis and more accurate assessment of cardiac mass. we recently met a patient who had a native aortic valve thrombosis mimicking papillary fibroelastoma. a 57-year - old female was referred to our hospital because of dyspnea worsening for 2 days. she had no hypertension, diabetes, or prior spontaneous abortion. at presentation, physical examination revealed that arterial pressure was 140/90 mmhg, heart rate was 110 beats / min, and respiration was 20 breaths / min. mild pitting edema was noted in the right lower extremities. she was found to have both a lupus anticoagulant and antibodies to beta2-glycoprotein i of igg, type. igg and igm anticardiolipin antibodies, antinuclear antibodies, double - stranded dna antibodies, and venereal disease research laboratory tests were negative. procoagulant work up including protein c, protein s, antithrombin, urine homocystine assays were normal. the twelve lead electrocardiogram demonstrated sinus tachycardia and non - specific st - t segments changes in precordial leads. color doppler and duplex - scan ultrasonography of the lower limbs presented deep venous thrombosis in right common femoral vein, superficial femoral vein, and popliteal vein. transthoracic echocardiography (tte) showed a dilated right ventricle associated with severely depressed systolic function (supplementary movie 1 and 2). computed tomography (ct) with contrast confirmed a diagnosis of pulmonary embolism via the disclosure of clots at the bifurcation of the common pulmonary artery and in its main branches (fig., the aortic valve mass, located at the non - coronary cusp, resembling papillary fibroelastoma was discovered incidentally during chest ct (fig. however, the aortic mass was not diagnosed initially with tte due to poor acoustic windows. four hours later, her vital signs were unstable ; heart rate was 110 beats / min, blood pressure was 90/60 mmhg, and she was breathing at a rate of 26/min. because of persistent right ventricular dysfunction and progressive clinical deterioration, thrombolytic therapy were immediately performed. after initial treatments, a repeat echocardiography the following day revealed clear improvement in the right ventricular (rv) dysfunction with normal rv dimension. subsequently, a transesophageal echocardiogram (tee) was performed for evaluation of the aortic valve, revealing a 1 cm sized mobile cylindrical mass in the aortic valve. it attached without stalk to the non - coronary cusp annulus of the aortic valve and to extend into the ascending aorta. the aortic valve was tricuspid, without dystrophic calcification, and had no functional abnormality. in addition, there was no intracardiac shunt such as patent ductus arteriosus or atrial septal defect. the aortic valve mass was initially considered as a thrombus according to the presence of deep vein thrombosis and pulmonary embolism. after 2 weeks anticoagulation, however, a repeat tee revealed that there was still a mass over 1 cm long at the non - coronary cusp of the aortic valve (fig. 1c and d, supplementary movie 3 and 4). because a size change of the mass was not found after 2 weeks anticoagulation, we concluded that the aortic valve mass was thought to be a papillary fibroelastoma rather than thrombus.1) therefore, surgical resection was considered as the best treatment option in this patient with mobile mass to prevent fatal embolic complications and to confirm pathologic findings. gross inspection of cardiac mass showed a red - colored gelatinous lobulated mass measuring 1.4 0.9 cm. in the surgeon 's opinion, the histology of specimen revealed an organized thrombus with no evidence of papillary fibroelastoma (fig. she was diagnosed with antiphospholipid antibody syndrome based on her clinical presentation and laboratory findings. we repeated the lab tests after 12 weeks, which confirmed the diagnosis of antiphospholipid antibody syndrome. thrombosis in a native aortic valve is rarely reported and may result in clinically severe complications such as acute myocardial infarction, peripheral ischemia, cardiogenic shock and sudden death.2)3) native aortic valve thrombosis may originate in heart valve disease, a hypercoagulative state or an auto - immune disease.4)5) antiphospholipid antibody syndrome (aps) is an important cause of thromboembolic disease and fetal loss in the general population. cardiac manifestations in the aps include valvulopathy, coronary artery disease, intracardiac thrombus and pulmonary hypertension.6) however, intracardiac thrombus is a rare complication of the aps and can occur in any cardiac chamber.7) recent study have reported that the prevalence of intracardiac thrombus was less than 5% in the aps.8) differential diagnosis of aortic thrombus like tumor and vegetation is crucial for avoiding unnecessary surgical intervention. non - invasive imaging methods would be useful in the diagnosis of suspected cardiac mass, because they may provide earlier diagnosis and more accurate assessment of cardiac mass. recently, advanced imaging techniques improve the sensitivity and specificity.9) however, it is usually difficult to differentiate aortic thrombus from tumors, and especially papillary fibroelastoma which is the most common valvular tumor. most papillary fibroelastomas are asymptomatic, but the lesions are recognized as a cause of embolisms. echocardiogram usually demonstrates a small, mobile, pedunculated or sessile valvular or endocardial mass, which on many occasions flutters or prolapses into the cardiac chambers during systole or diastole.1) as the tumors are small and attached to moving valves, they are usually not seen on ct or magnetic resonance images. the differential diagnosis of a papillary fibroelastoma includes other cardiac tumors, thrombus and vegetations. thrombus can be differentiated by an irregular or lobulated shape, laminated apperance, microcavitations and absence of a pedicle.10) although imaging tools are continually being developed, they may be clinically challenging to diagnose a cardiac mass. this case highlights the limitation of imaging tools for cardiac mass to lead a diagnosis. even though, in the presence of the characteristic imaging, clinical decision making with imaging modalities should be performed cautiously. apical 4-chamber view reveals a markedly dilated right ventricle with depressed systolic function at initial presentation. short - axis view of transesophageal echocardiogram shows a mass filling the non - coronary sinus of valsalva. long - axis view of transesophageal echocardiogram at 136 rotation presents a mass just behind the non - coronary cusp. | the differential diagnosis of cardiac mass is important in determining the therapeutic plan and avoiding unnecessary surgical intervention. non - invasive imaging methods would be useful in the diagnosis of suspected cardiac mass, because they may provide earlier diagnosis and more accurate assessment of cardiac mass. native aortic valve thrombosis is a rare disorder and difficult to differentiate from a tumor, and in particular, a papillary fibroelastoma. thus, the clinical decision making with imaging modalities should be performed cautiously. we recently met a female patient who had a aortic valve mass resembling papillary fibroelastoma in normal native valve. the patient underwent a surgical resection and the pathologic finding showed an organized thrombus with no evidence of papillary fibroelastoma. |
as with nearly every technological advance, the development of genomics holds out the promise of fruitful military applications. mehlman and li persuasively and, i believe, correctly argue that the appropriate bioethical framework for the use of genomic science by the military should be derived primarily from two overriding principles. first comes the traditional (though often disputed) bioethical principle of paternalism. while paternalism in the usual medical context may well be a problematic notion, it is clear in the military context that commanders bear a responsibility for the health and safety of those under their command. proportionality normally applies only to engagements in which civilian casualties can be expected, and states that a military action is permissible only if the harm to civilians and civilian objects is proportional to the military advantage which directly results from that action. expanding the principle to our own troops, we say that a soldier can be compelled to undergo a risky procedure only if the harm which can be expected to befall her is proportional to the military advantage which can be expected to result. proportionality, in mehlman and li 's extended sense, thus requires that, for every proposed genomic intervention, we estimate both the harms to friendly forces and the military advantage gained against our enemies. without the ability to gauge these two quantities, it will be impossible to evaluate whether or not an action is indeed proportional. the difficulty of estimating proportionality has, for example, been raised by schmitt and thurnher, leading them to doubt (due to the context sensitivity and subjectivity of military advantage) whether proportionality calculations could ever be accurately executed by an autonomous robotic weapons system. in this commentary, in fact, i think it is quite likely that situated as it is in the intersection between military ethics and traditional medical ethics, and illuminated by many studies of prior successes and failures of medical research by the military these two criteria are exactly those which ought to be used to guide discussion of the ethical use of genomics by the military. rather, i wish to explore in detail one facet of their framework : the estimation of harms and benefits in the context of genomics. unlike several of the case studies that they discuss, i argue that there are significant in - principle (not just research - based or in - practice) problems with the connection between genotypes and phenotypes. these issues will, at the very least, make incredibly difficult any proper assessment of the proportionality of genomic intervention by the military. at worst and i will argue that there are good reasons to think that we are indeed at worst i want to stress that i do not intend this to be an argument against mehlman and li 's framework. on the contrary, as i have said above, i believe that mehlman and li have set the bar in precisely the correct place. that bar, however, is significantly higher than it may seem on a first reading. justifying any complex military use of genomics will be and should be, for precisely the reasons that i will outline here a difficult, if not impossible, enterprise. the fundamentals of my critique are drawn from recent work in both biology and the philosophy of biology. but first, a brief introduction. an organism is born with a particular genome the contents of its dna or genetic code. the genotype, however, is only one of the resources that produce the traits of the organism that we actually see its phenotype. of course, it is phenotypic traits with which we are actually concerned, as these are the environmentally expressed characteristics, behaviors, morphologies, and so forth that actually matter to the organism during its life. the determination of this relationship is one of the most difficult open problems in contemporary biology. an organism 's development, the environment into which it is born and in which it later lives, and interactions between vast networks of genes make it difficult to precisely trace the connection between the simple possession or absence of a particular gene and the possession or absence of some corresponding phenotype in a one - to - one way. as many commentators have put it, the traditional metaphor of genes as a this has led to the reconceptualization of this connection as the genotype phenotype map (or gp map)the relation from genotypes to phenotypes, which includes all the complex, non - linear interactions of development, genes, and environment. for some traits in some systems, this complexity can be worked around. in mendel 's original study of pea plants (which we all learn in our high school biology classes), mendel 's data give the distinct impression that the very simple traits of peas with which he was concerned (green v. yellow peas or smooth v. wrinkled skin) can be predicted accurately by merely determining whether or not the plant at issue possesses a particular gene. however, to understate the issue, a soldier is not a green, wrinkly pea. a dod report referenced by mehlman and li cites as traits of interest phenomenally complex phenotypes such as susceptibility to post - traumatic stress disorder, the ability to tolerate conditions of sleep deprivation, dehydration, or prolonged exposure to heat, cold, or high altitude, or the susceptibility to traumatic bone fracture, prolonged bleeding, or slow wound healing. later, mehlman and li describe genetic variants associated with coolness under fire (p. 28) as a target for possible military genomic screening. assuming that coolness under fire is a concept sufficiently well defined to count as a phenotype in the first place, it will clearly be a massively complicated one. for the sake of argument, let 's say that we are able to ignore entirely the influences of development and environment on coolness under fire. this is obviously implausible, but we will be able to make trouble even without considering these factors. to get an idea of the complexity of the genetic regulatory networks that produce traits, they describe a single network which controls the differentiation of two body layers in the embryo of the sea urchin. the resulting diagram contains more than 40 genes, in a highly interconnected, robust, and self - regulating network. and this is a network for a single step in the development of a highly simplified and well - understood model organism. inference of gene networks in mammals is even more difficult, confounded by more complex regulatory architecture, higher post - transcriptional modification, and the need to refer not only to gene expression data, but also perturbation (knock - down or over - expression) experiments. thus far, we 've seen a variety of in - practice concerns with the estimation of the gp map but as of yet, nothing of an in - principle sort, nothing that would lead us to believe that it is impossible for sufficient research into human systems to solve the problem. after all, if davidson can describe the genomic regulatory networks for sea urchin embryo development, is it not merely a difference in degree between these networks and those present in humans ? why is trouble in the gp map a difficulty for us here in the first place ? for this, we must return to the concept of proportionality. if we are to accurately estimate either the harm that might befall our own troops or the military advantage that would result from a particular genomic intervention, we clearly must know what phenotypic effects intervening on a particular gene will have in a particular population. and this problem, then, just is the problem of understanding the gp map. without the ability to translate from genetic to phenotypic effects, estimations of both halves of the proportionality calculus will be hard to come by. they argue that the military must be mindful of relying too heavily on the results of genomic tests that have not been adequately validated (p. 31), mentioning particularly the popular media 's tendency to overstate the significance and potential power of genomic results. they explore, then, in some detail, the question of how the military ought to move forward in cases where there is a compelling reason to deploy a genomic technology which hasn't been fully tested, drawing an extensive analogy to off - label use of vaccines against chemical and biological weapons, as deployed fairly extensively in the gulf war and since (pp. this analogy, however, fails to capture the way in which these problems with the gp map are disconcerting for ethical evaluations of military genomic technologies. in the vaccine case, we have a straightforward instance of an absence sufficient testing data an intervention which could readily be shepherded through the traditional clinical trial procedure but has not been, as a result of a lack of time, funding, interest from pharmaceutical companies, or what have you. i think the kinds of concerns already mentioned make it clear that mehlman and li understate the problems in testing and validation for genomic interventions of any real complexity. but for the remainder of the paper, i wish to briefly pursue a more troublesome argument. given the kinds of in - principle difficulties present in the notion of the gp map, i find it likely that it is in fact impossible to obtain enough knowledge concerning the connections between genotype and interesting phenotypes to successfully evaluate proportionality. a plethora of current theoretical and experimental work has indeed improved our knowledge of the gp map. as chronicled by pigliucci, studies from computer science have increased our understanding of modularity and network structure, gene networks are far better understood experimentally than they once were, and rna folding has served as a fruitful model for more complex gp relationships. but while this work indicates that progress has been made in simple systems, there is room for skepticism beyond such cases. pigliucci argues that a truly satisfactory empirical understanding of gp relations in complex organisms may [be ] forever beyond our grasp because of practical epistemic limitations. what are these practical epistemic limitations ? in short, while a minor gap in our empirical understanding of the gp map may only constitute an in - practice experimental difficulty, a large enough number of these minor gaps rises to the level of inability in principle to make accurate predictions. two such twins, who have (almost) identical genotypes, serve as a natural experiment that can isolate and thus estimate the contribution to a disease (or other phenotype) arising solely from that disease 's genetic basis. after statistical analysis in which the study authors estimated the contribution of the genome to some 20 different diseases, they discovered that most sequenced patients would not gain any useful information, since their risk of disease, even with full knowledge of their genome, would be similar to that of the general population. in the best - case scenario, the majority of patients might be alerted to one or a few disease risks. further, this study was in search of disease phenotypes and on the basis of our genetic studies of cancer, for example, we have reason to think that many disease phenotypes are much less complex than those singled out by mehlman and li as of potential military interest. and it is not difficult to see why it is that, for the foreseeable future, [empirical charting of the gp map ] seems feasible only for simple instances of gp. estimation of the gp map requires input from studies of gene networks and evolution, from development, from physiology and morphology, from ecology, from environmental studies, from behavior, and even, in humans, from sociology and studies of culture. as boudry and pigliucci put it, once we start talking about complex organisms, particularly those characterized by flexible developmental trajectories, all bets are off. it is thus likely that, despite the many advances in our understanding of the gp map in recent years, the shape of this map for organisms of any significant complexity not to mention humans is so intricate as to be, at best, far, far beyond our current capacities for estimation, and at worst (which is the more likely outcome) forever beyond our knowledge. mehlman and li have done a great service in laying out and carefully arguing for a comprehensive approach to understanding the ethics of the use of genomic science by the military. indeed, in at least some cases where the phenotypes at issue are relatively simple the collection of genomic data by the military and screening of our armed forces has the potential to improve health outcomes for our troops, a goal which we all should support. the obstacles to this simpler kind of program are of an in - practice sort ; it would require expensive data collection and analysis, a problem that the military is particularly well poised to solve. but when it comes to phenotypes that are much more complex than this phenotypes like coolness under firedifficulties of an in - principle sort arise. the complexity of the relationship between genotypes and phenotypes makes it unlikely that it is possible for us to obtain enough data to accurately estimate the potential harm to our troops and the potential military advantage that would accrue from a genomic intervention. it will, therefore, be impossible to estimate the proportionality of interventions like these. on mehlman and li 's framework, then, we can not determine, for many traits (many of the traits in which the military seems to be most interested, at least according to the documents which mehlman and li cite), whether or not such an intervention is ethical. far from a problem with their analysis, however, i believe that mehlman and li have offered a framework that encourages an appropriate level of caution regarding these types of experiments. for complex, human phenotypes, it should be difficult to ethically justify such an intervention, and it is a virtue of their ethical framework that it lets us see precisely why. | mehlman and li offer a framework for approaching the bioethical issues raised by the military use of genomics that is compellingly grounded in both the contemporary civilian and military ethics of medical research, arguing that military commanders must be bound by the two principles of paternalism and proportionality. i agree fully. but i argue here that this is a much higher bar than we may fully realize. just as the principle of proportionality relies upon a thorough assessment of harms caused and military advantage gained, the use of genomic research, on mehlman and li 's view, will require an accurate understanding of the connection between genotypes and phenotypes accurate enough to ameliorate the risk undertaken by our armed forces in being subject to such research. recent conceptual work in evolutionary theory and the philosophy of biology, however, renders it doubtful that such knowledge is forthcoming. the complexity of the relationship between genotypic factors and realized traits (the so - called gp map) makes the estimation of potential military advantage, as well as potential harm to our troops, incredibly challenging. such fundamental conceptual challenges call into question our ability to ever satisfactorily satisfy the demands of a sufficiently rigorous ethical standard. |
larvae were collected opportunistically from tammany creek, nez perce co., i d (46 21.51.51 n, 117 03.32.05 w), a perennial, undammed creek approximately 13 m in width and roughly 10 km in length. tammany creek drains into the snake river approximately 770 km from the confluence of the snake and columbia rivers. we surveyed the study area (1 km in length) on foot and collected odonates by drag net from 15 february 2012 to 12 march 2012. upon collection, larvae were transported to lewis - clark state college and housed in 50-ml plastic vials with a 3-mm - diameter dowel for perching. larvae were measured with a millimeter rule for total body length and head width and maintained on a photoperiod of 14:10 (l : d) h cycle at an ambient air temperature of 22c. vivida head 24.5 mm, body 619 mm ; a. palmata head 48 mm, body 2335 mm. behavior trials were conducted in 250-ml containers with filtered tap water and a singular 3-mm - diameter perch. one shrimp was placed in the container, as alternative prey, 1 min after simultaneously placing both odonates in the container. vivida head 34 mm, body 1117 mm ; a. palmata head 48 mm, body 2335 mm. to evaluate whether the observed behaviors were species specific, we tested a. palmata and ar. animals used in congeneric trials (not diagrammed) were matched for total body length as closely as possible (31 ar. vivida, a previously undescribed luring behavior was observed by either a. palmata or ar. that luring behavior was demonstrated, the a. palmata attempted, unsuccessfully, to capture the shrimp prior to the luring behavior. luring behavior was initiated only when the odonates had entered into the field of view of one another. once the a. palmata and ar. vivida had oriented toward each other, a brief period (3060 s) of no visible movement ensued. in four of five trials d) sequence of points in luring abdominal movement by a. palmata during a. palmata (large) versus ar.. abdominal movement to the side and exposure of the abdomen of a. palmata to ar. vivida changed position to place the abdomen of a. palmata directly in the field of view (fig. this behavior consisted of 710 movements of the abdomen within approximately 2 s (fig. (a d) sequence of points in luring abdominal movement by a. palmata during a. palmata (large) versus ar.. abdominal movement to the side and exposure of the abdomen of a. palmata to ar. ar. vivida changed position to place the abdomen of a. palmata directly in the field of view (fig. this behavior consisted of 710 movements of the abdomen within approximately 2 s (fig. vivida luring a. palmata was observed. the following sequence of events details that observation (fig. 2a).the abdomen was then fully moved to the opposite side of the body in a slow motion (2 s) and held on the other side of the body for approximately 8 s (fig. 2b).step 2 was repeated to the initial side of luring. at this point, ar. vivida positioned its abdomen directly behind its head and slowly continued movement toward head of a. palmata. vivida attempted to capture a. palmata at its head.upon being struck, a. palmata slowly retreated.ar. vivida continued attempts at luring a. palmata, with rapid abdominal movements to the initial side for approximately 5 s. while luring, ar. vivida slowly approached the retreating a. palmata.the sequence was broken as a. palmata used abdominal thrusts to propel itself away and over ar. the abdomen was then fully moved to the opposite side of the body in a slow motion (2 s) and held on the other side of the body for approximately 8 s (fig. step 2 was repeated to the initial side of luring. at this point, ar. vivida positioned its abdomen directly behind its head and slowly continued movement toward head of a. palmata. vivida continued attempts at luring a. palmata, with rapid abdominal movements to the initial side for approximately 5 s. while luring, ar. the sequence was broken as a. palmata used abdominal thrusts to propel itself away and over ar. vivida. in the other 13 of 18 a. palmata versus ar., a. palmata actively pursued the shrimp prey item, catching and consuming the prey without attacking the ar. vivida positioned itself at the bottom of the chamber and remained motionless for the entirety of the trial. vivida pursuing the prey. in all of these trials, the a. palmata captured and consumed vivida attempting to consume the prey item after the a. palmata had caught and began consumption. in this instance, the ar. vivida was consumed by the a. palmata following consumption of the prey. in one trial, ar. vivida actively attacked and consumed the prey, and a. palmata remained motionless in the chamber. in every congeneric trial vivida, 12 a. palmata versus a. palmata), there were no instances of attack, luring, or attempted consumption of the congeneric by either animal. in our trials, luring occurred only when a head - to - head orientation was present. this is the same orientation described in the well - known agonistic behavior of many larval odonates (corbet 1999). however, in contrast to previously described agonistic behaviors, predatory luring occurred between different odonate families with a different repertoire of movements (figs. 1 and 2) and typically ended in consumption of the prey. in trials where luring was not displayed a caudal attack by either species typically resulted in one of two possible scenarios : 1) removal of lamellae of ar. vivida and subsequent retreat and avoidance of further attack by prey and 2) prey being grasped and subsequent retaliatory bites and aggression resulting in injury to the predator (e.m., personal observations). these two scenarios, we believe, are the reasons for the head - to - head orientation that has been demonstrated as critical for the luring to occur. the caudal luring by the predator caused the lured individual to change orientation slightly to ensure that a successful attack was achieved. the exaggerated slow swaying of the caudal region caused the lured individual to change orientation. the subsequent rapid movements of the caudal region resembled that of the preferred prey in this environment, the freshwater shrimp, which further provoked the lured individual to investigate. the movement of the lured individual from the head - to - head orientation to the more vulnerable position of focusing on the lure provided the predator an opportunity to strike at the prey. we believe that more instances of predatory luring would have been observed had the time duration of the trials been longer than 7 min. all animals used in the trials were nave, not having been involved in any trials previously. we can not comment on the experience of the animals in the field before capture ; however, all animals were housed individually for 3 weeks before trials were conducted. the luring demonstrated by ar. vivida, even though consumption was not achieved (although a strike at the head of the much larger a. palmata did occur), may indicate that this behavior can be used in situations other than predation. vivida is similar to the agonistic behavior of other coenagrionidaen odonates (rowe 1992), several differences exist. 3) the behavior displayed by ar. vivida to physically lure the larger a. palmata close to the ar. vivida versus a. palmata with closely matched sizes will help elucidate whether this behavior is predatory or used to ward off potential predators. although there was no luring of a. palmata by other a. palmata, similarity of ar. the possibility of predation by a congeneric may induce the larger dragonfly to lure the smaller but morphologically similar damselfly to a position in which a fatal attack is much easier and much more likely. vivida and a. palmata at this locale creates the possibility that large larvae are likely to encounter smaller conspecifics. | organisms in the order odonata are highly predatory insects that have a wide distribution globally. to date, there has been zero evidence that odonates employ luring as a means of prey acquisition. however, in this study, we show that aeshna palmata larvae use abdominal movements to lure larval argia vivida, subsequently consuming the lured organism. we also present findings of a similar behavior from larval ar. vivida in an attempt to lure larval a. palmata within striking distance. |
the cerebral cortex of mammals, including humans, displays oscillatory spindle activity (1015 hz) across the lifespan. in adults, sleep spindles, a specific type of spindle activity, occur exclusively during non - rem sleep [14 ] and have been implicated in memory retention and skill learning [57 ]. in humans, sleep spindles first appear 49 weeks postterm and become more prominent and frequent over the next several months. there exists another type of spindle activity called spindle bursts that is phenomenologically similar to sleep spindles in that they share a similar frequency range, duration, and spindle - shaped waveform. for example, spindle bursts predominate early in development : they are readily observed in premature human infants and are thought to disappear soon after birth. in rats, spindle bursts have been recorded in sensorimotor and visual cortex soon after birth through at least the end of the second postnatal week [8, 10, 11 ]. in addition, unlike sleep spindles, spindle bursts are not exclusive to non - rem sleep but rather can occur across the sleep - wake cycle. finally, spindle bursts are most closely associated with self - generated or evoked activity in the sensory periphery ; for example, manually stimulating the limb of a rat or human, during sleep or wakefulness, elicits spindle bursts in sensorimotor cortex [8, 9, 11, 12 ]. across all sensory modalities, however, self - generated activity in the sensory periphery is the predominant trigger of spindle bursts during early development. in the visual system, for example, retinal ganglion cells fire spontaneously before eye opening, producing waves of retinal activity that provide downstream afferent input to visual brain areas [10, 13, 14 ]. in the auditory system, cochlear spiral ganglion cells fire bursts of activity before the onset of hearing that provide downstream afferent input to the auditory system (although spindle bursts have not yet been recorded in auditory cortex, it is likely that they occur) [1517 ]. by providing substantial sensory experience during early development, retinal waves and cochlear bursts shape and refine functional links between the sensory periphery and developing brain networks [14, 18, 19 ]. in the developing sensorimotor system, spontaneous activity takes the form of myoclonic twitching, which is characterized by jerky movements of the forelimbs and hindlimbs, tail, whiskers, and eyes during rem sleep [8, 9, 11, 20, 21 ]. in neonatal rats, sensory feedback from twitching limbs triggers activity throughout the nervous system and has been hypothesized to contribute to the development of sensorimotor circuits [8, 11, 20, 22, 23 ]. moreover, just as retinal waves trigger spindle bursts in visual cortex, sensory feedback from twitching limbs (i.e., reafference) triggers spindle bursts in somatosensory [8, 2426 ] and motor [11, 12 ] cortex. twitches, however, differ from retinal waves in two fundamental ways : first, although retinal waves are transient features of development, twitches persist into adulthood [28, 29 ] and second, although retinal waves seem to occur independently of behavioral state, twitches are an exclusive feature of rem sleep. to reiterate, twitches trigger spindle bursts in sensorimotor cortex [8, 11, 12, 24, 25 ], occur exclusively during rem sleep, originate prenatally [9, 31 ], and persist into adulthood [28, 29 ]. curiously, however, spindle bursts have not been reported during rem sleep in adults. one possible explanation for this absence is that twitches in adults are somehow prevented from triggering spindle bursts in sensorimotor cortex. given how reliably twitches activate the cerebral cortex in early development, this possibility requires that a mechanism emerges at some point between infancy and adulthood that gates twitch - triggered reafference. for example, there is evidence in adult cats of spinally mediated sensory gating that is stronger during rem sleep than during wakefulness. however, even during rem sleep, this gating mechanism only partially blocks sensory feedback. therefore, it remains possible, if not likely, that twitches continue to trigger spindle bursts in the adult sensorimotor cortex. figure 1 presents local field potentials (lfps) in infants rats recorded from the hindlimb region of sensorimotor cortex in relation to twitches detected in the hindlimb electromyogram (emg) (data from). at postnatal days (p) 4 and 810, when background lfp activity is low, hindlimb twitches trigger easily discernible cortical spindle bursts. indeed, because of the low background activity at these ages, visual inspection of the lfp alone that is, without guidance provided by the hindlimb emg allows one to confidently state when a spindle burst occurred. also, the lfp alone is sufficient to predict with high confidence when hindlimb twitches occurred. in contrast, by p12, lfp background activity has increased such that spindle bursts are now much harder to detect. in this case, the lfp alone is not sufficient to predict with high confidence when hindlimb twitches occurred. given this dramatic increase in background activity between p810 and p12, we expect spindle bursts to disappear further into the background over the ensuing days and weeks. but the precise relationship between twitches and spindle bursts is revealed using a twitch - triggered averaging method. in the case of sleeping infant rats at p4, p810, and p12, averaging lfp power in relation to hindlimb twitches reveals increased activity in the hindlimb region of sensorimotor cortex within ~150 ms of a twitch (figure 2(a)). time - frequency spectrograms reveal that these peaks in twitch - related power occur at spindle burst frequency (figure 2(b)). note that, at p4, the spectrogram displays a discrete hotspot immediately after hindlimb twitches. by contrast, at p12, there is increased background activity in the spindle frequency range ; nonetheless, a concentrated twitch - related hotspot in spindle frequency is still apparent. these results suggest that, with increasing age, spindle bursts are harder to detect because they are obscured by increases in background lfp activity. event - triggered averaging, such as that illustrated in figure 2, is routinely used to reveal hidden or obscured cortical events and oscillations. for example, cognitive neuroscientists use event - related potentials (erps) to reveal cortical activity associated with sensory, motor, or cognitive process (figure 3 ;). because eeg electrodes reflect ongoing activity from thousands of neurons, the resulting signals are necessarily noisy. as a result, an individual event (e.g., a flash of light) may not be evident in the raw eeg signal. however, by averaging over many event - triggered trials, random noise that is not associated with neural processing of the stimulus cancels out, leaving behind a distinct and stereotyped cortical pattern the erp that reflects underlying neural processes. we propose that twitch - related spindle bursts have not yet been detected in the activated eeg of human adults during rem sleep because event - triggered averaging would be necessary to observe them. undoubtedly, there are many published studies in human and nonhuman adult animals in which cortical activity recorded across behavioral states can be related to peripheral motor activity, including twitching (e.g., see). critically, to our knowledge, no study in adults has specifically used twitches for event - triggered averaging of cortical activity in somatotopically related areas of sensorimotor cortex. species - typical and behaviorally relevant body parts the bill of a platypus, the star appendage of a star - nosed mole, and the digits of a raccoon have magnified representations in sensorimotor cortex. the degree of magnification reflects the innervation of these peripheral morphological features as well as their use. if twitches are functionally important for the development and maintenance of sensorimotor circuits [8, 23 ], then we expect the quantity and patterning of twitching to reflect an appendage 's innervation density, biomechanics, and behavioral importance across species [23, 36 ]. like other primates, humans are a highly visual species that execute hundreds of thousands of saccadic eye movements (3 per second) during the day. interestingly, much like the skeletal muscles that control the limbs, the extraocular skeletal muscles that move the eyes twitch during rem sleep. in fact, the resulting rapid eye movements give rem sleep its name. moreover, much like the limbs of the body, proprioceptive reafference from eye movements in rats and humans is relayed to sensorimotor cortex [39, 40 ]. since twitches of all skeletal muscles studied thus far trigger spindle bursts [8, 11, 21 ] because rapid eye movements are a prominent feature of rem sleep in human adults, the sensorimotor system that controls eye movement could be an ideal model for exploring the contributions of spindle bursts to the calibration, maintenance, and repair of neural networks. whereas humans, which are diurnal, rely heavily on the visual system for spatial navigation, rats, which are nocturnal, rely heavily on the whiskers. the whiskers of rats and other rodents are controlled by an elaborate set of extrinsic and intrinsic striated muscles [41, 42 ]. importantly, in infant rats, these muscles twitch during rem sleep and reafference from twitches activates the whisker thalamus and barrel cortex. spindle bursts are readily detected in the infant rat 's barrel cortex, occurring immediately after twitching ; the developmental onset of whisker - related cortical activity could contribute to the refinement of the somatotopic map in barrel cortex. whisker twitching has also been observed in adult rats but has not been systematically investigated. therefore, similar to rapid eye movements in humans, we expect whisker twitching and associated spindle bursts to reflect the outsized role of this sensory modality in rats. extrapolating to other species, highly specialized sensorimotor appendages should exhibit relatively high rates of twitching. the bill contains a high density of electro- and mechanoreceptors and, consequently, a large proportion of the platypus ' cerebral cortex is devoted to processing sensory input from the bill [46, 47 ]. as we would expect, platypuses exhibit vigorous twitches of the bill (as well as the eyes and limbs) during rem sleep, even as adults [48, 49 ]. the star - nosed mole uses a specialized set of 22 facial appendages that it uses like a tactile fovea to forage for food. similar to the bill of the platypus, the star appendages contain a high density of somatosensory receptors and, consequently, a large proportion of this animal 's cerebral cortex is devoted to each appendage. although there is no information on the sleeping habits of the star - nosed mole, we predict that it will be found to exhibit high rates of twitching in the star appendages during rem sleep, as well as twitch - associated spindle bursts in sensorimotor cortex. it has been argued that local changes in cortical slow - wave activity can be triggered by learning processes that differentially involve specific brain regions. similarly, after the development of a new waking skill or in response to changes in the sensory periphery (e.g., due to injury), perhaps twitches contribute to the process of learning, adaptation, or recovery of function. for example, in one study, human adults wore goggles during the day that decreased their visual field to 5 degrees. when these subjects were observed over several days during sleep, the duration of rem sleep was unaffected, but the frequency and amplitude of rapid eye movements increased significantly. because the changes in rapid eye movements were a transient response to a waking perturbation of visual experience, these findings suggest to us that twitching in this case in the form of rapid eye movements contributes to the process of visual adaptation. this experimental approach that is, the manipulation of waking motor experience in human and non - human subjects could provide the opportunity to explore the contributions of twitching and twitch - related spindle bursts to learning and adaptation in health and disease. until recently, twitches were widely perceived as functionless byproducts of dreaming, providing little reason to search for their neural consequences. only with the discovery of twitch - related neural activity in infant rats, it has become apparent that twitches could play a functional role in sensorimotor development, akin to spontaneous activity in other sensory systems. it is the persistence of twitching into adulthood, in humans and other mammals, that raises the intriguing possibility that twitching has much more to reveal to us about its functional contributions to neural plasticity within the sensorimotor system. although the idea that twitch - related spindle bursts persist into adulthood is speculative, it can easily be tested using new or even perhaps existing recordings. if evidence of twitch - related spindle bursts is found, there will be a strong basis for expanding our understanding of the functions of spindle activity during non - rem sleep about which we currently know a lot to include rem sleep as well. | sleep spindles are brief cortical oscillations at 1015 hz that occur predominantly during non - rem (quiet) sleep in adult mammals and are thought to contribute to learning and memory. spindle bursts are phenomenologically similar to sleep spindles, but they occur predominantly in early infancy and are triggered by peripheral sensory activity (e.g., by retinal waves) ; accordingly, spindle bursts are thought to organize neural networks in the developing brain and establish functional links with the sensory periphery. whereas the spontaneous retinal waves that trigger spindle bursts in visual cortex are a transient feature of early development, the myoclonic twitches that drive spindle bursts in sensorimotor cortex persist into adulthood. moreover, twitches and their associated spindle bursts occur exclusively during rem (active) sleep. curiously, despite the persistence of twitching into adulthood, twitch - related spindle bursts have not been reported in adult sensorimotor cortex. this raises the question of whether such spindle burst activity does not occur in adulthood or, alternatively, occurs but has yet to be discovered. if twitch - related spindle bursts do occur in adults, they could contribute to the calibration, maintenance, and repair of sensorimotor systems. |
this malignancy is a public health burden in most industrialized countries and a most common cause of cancer - related deaths in the world (2). the incidence and mortality rate of this cancer is increasing in iran and it is predicted that iranian population may be experiencing an acceleration of its burden in future (3 - 5). a surgical resection is the only curative modality for localized colon cancer. it is the primary treatment modality for crc, and outcome is most closely related to the extent of disease at presentation. the location of the primary tumor may also have prognostic significance. for each stage, cancers arising at or below the peritoneal reflection (rectosigmoid and rectum) have a worse five - year survival rate than those arising more proximally (6). furthermore, within the rectum, distal cancers have a worse prognosis than more proximal lesions. over the last 50 years, a gradual shift toward right - sided or proximal colon cancers has been observed (5). the survival rates of crc vary throughout the world due to quality of care, including surgical techniques, which is a predictor of outcomes for local recurrence and cure rates (7, 8). crc that has metastasized, or spread, to the regional lymph nodes carries a worse prognosis and a higher risk for recurrence. studies showed that 15 to 30 percent of patients diagnosed with node - negative colorectal cancer experience disease recurrence within 2 - 5 years (9, 10). this high recurrence rate and a higher incidence of colorectal cancer in subjects at high risk of being crc, highlights a healthcare opportunity for surveillance or interventions to reduce the morbidity associated with crc (11). the aim of this study was to evaluate the incidence of colorectal cancer recurrence and survival rate within 5 years, after being affected by this cancer, with considering the effects of other clinical and demographic factors. this was a historical cohort study in which all patients whose primary colorectal cancer were resected only by the one expert staff of colorectal surgeon or under his supervision and registered in the taleghani hospital, tehran, since 21st mar, 2004 to 20th mar, 2013 entered to the study. the necessary data such as demographic, age, gender, family history of crc, site and size of tumor, stage of tumor, operation details, histological results, treatment method, etc. had been extracted from medical records. all patients were followed for prognosis, mortality status and recurrence after the surgery (by repeated periodic visits, colonoscopy and telephone contact). survival time was calculated from the date of surgery registration until death or the end of the study. due to protocol of crc management, for patients with stages ii - iii and some symptomatic stage iv, a colectomy (surgical removal of the tumor and regional lymph nodes and nearby tissues) were done and then chemotherapy based on 5-fluorouracil was offered to patients younger than 75 years with stage iii or iv colon cancer or complicated stage ii. postoperative chemoradiation was offered only to those with stage ii or stage iii of rectosigmoid cancer when the local clearance was in doubt and preoperative chemoradiation was given to those with fixed t4 lesions. chi - square test and kaplan meier analysis have been done to find the prognosis factors, associated to survival and recurrence. p values of less than 0.05 were considered statistically significant. all analysis carried out, using spss software, version 17. a total of 107 patients (51.2% men) who underwent resection for colorectal cancer during the study period, entered to this study. 23.4% of patients had positive family history of crc, and 53.8% were sporadic crc. the mean age was 53.50 12.68 years (range 24 - 76 years), and the survival rate was 73.8% with mean survival time of 142.17 21.60 month. the recurrence rate after surgery was 5.7% and all of them were alive till the end of study. region of recurrences were : five patients (83.3%) near to site of anastomosis or previous tumor and one patient exactly at site of anastomosis. the demographic and clinicopathological factors associated with survival time after surgery were presented in tables 1 and 2. according to log - rank test, regional lymph nodes, distance metastasis and adjuvant therapy were the prognosis factors of survival in crc patients after surgery, respectively. patients with n1 regional ln, m0 level of metastasis and adjuvant therapy had higher survival. also, there was no significant association between type of surgery and risk of death in patients under study (table 3). the demographic and clinicopathological factors associated with risk of recurrence are presented in tables 4 and 5. according to the results, this study revealed that, in patients who underwent resection for colorectal cancer, the survival rate is high and the recurrence rate was just 5.7%. the evaluation of post - surgical survival of colorectal cancer is necessary due to comparing results together and then reevaluates cause of differences (kind of surgery, experience of surgeons, doing chemotherapy or chemo radiotherapy and its regimens, quality of post - surgical care and surveillance). there was no correlation between bmi and crc survival in this study, which is different from many similar previous researches (12, 13). no age and gender relationship were observed with survival of crc patients in this study, however some iranian studies indicated that colorectal cancer occurs at a younger age (14). this statistics is better than other sites in iran and other countries (15, 16), and similar to some asian studies (17), which could be due to improvement of exactness and expertise of surgeon or better adjuvant therapy or difference stages in the patients who were operated. however, the effectiveness of these treatments in the lower stages needs to be evaluated. the number of examined lymph nodes and distance metastasis statistically predicted the survival, which is similar to study that indicated the risk of death was higher in patients with distant metastasis (18). tumor size, grade of tumor and some other clinical factors were not associated to survival, which is in contrast to western studies (19). the incidence of crc is lower in iran compared to western countries ; however, its burden has been increasing in recent years (20, 21). in addition, five year survival rate of crc has been reported to be lower than the world (22, 23). it seems that among the prognostic factors explored so far, the most important are those that relate to early diagnosis of cancer. so, primary detection is feasible since efficient screening modalities are available (24). due to limited data about survival and recurrence of colorectal cancer after surgery and its risk factors in different areas of iran, our study would be helpful to improving surveillance (pre and post - surgical), survival and recurrence of crc and at least better managements and control of its risk factors. the evaluation of incidence and duration of colorectal cancer recurrence (where site, stage at time of presentation and site of recurrence) could conduct us to the better planning of post - surgical follow up, reevaluation of kind of surgery, adjuvant and neoadjuvant chemotherapy and chemoradiotherapy, in order to increase the survival of patients. the limitation of this study is low sample size of patients who underwent resection for colorectal cancer. it needs to conduct multicenter study in order to merge the patient s dataset and increase reliability of results for future researches in iran. | background : colorectal cancer (crc) is a common malignancyworldwide and its outcome is most closely related to the extent of disease at presentation. early diagnosis of an asymptomatic recurrence increases the likelihood of a complete surgical resection.objectives:the aim of this study was to evaluate the incidence of colorectal cancer recurrence and survival rate within 5 years, after surgery.patients and methods : during the 9-year period since 21st mar, 2004 to 20th mar, 2013, patients whose primary colorectal cancer were resected in taleghani hospital, tehran, iran were selected in a historical cohort. the necessary data such as demographic, age, gender, family history of crc, site and size of tumor, stage of tumor, operation details, histological results, treatment method, histopathologic, etc. were collected. then the recurrence and survival of colorectal cancer within 5 years after operation and their risk factors were evaluated. p value less than 0.05 were considered significant. all analysis was done using spss software.results:a total of 107 patients underwent resection for colorectal cancer during the study period, with mean age of 53.50 12.68 years (range 24 - 76 years), survival rate of 73.8% (rectum 70.0% and colon 75.9%), and mean survival time of 142.17 21.60 month. the recurrence rate of crc patients, during five years after surgery was 5.7%. regional lymph nodes, distance metastasis and adjuvant therapy were significant prognosis factors of survival after surgery.conclusions:the rate of recurrence in iranian patients was low, which could be due to improvement of exactness and expertise of surgeons or better adjuvant therapy. the significant association between survival and adjuvant therapy clarifies this finding. early diagnosis and primary detection could increase the rate of survival. |
with their improved esthetics, physical properties, better bonding systems, curing refinements, and environmental concerns, resin composites are now widely used for the direct restoration of both anterior and posterior teeth rather than amalgam. resin composites have been classified according to various characteristics, such as size, content, and filler type, and the physical and mechanical properties of the materials. nanotechnology, known as molecular nanotechnology or molecular engineering, is the production of functional materials and structures, at a range of 0.1 - 100 nm, by various physical and chemical methods. a nanohybrid is a hybrid resin composite with nanofiller in a prepolymerized filler form, whereas nanofill is a composite resin that is composed of both nanomers and nanoclusters. the success of dental restorations depends on their compressive, diametral tensile and flexural strength, wear and fracture resistance, and polish retention. moreover, the esthetics of restorative materials should mimic the appearance of natural teeth, which is directly related to color matching and color stability. however, when exposed to the oral environment, restorative composites have a tendency to discolor. intrinsic factors, such as the resin matrix of composites and incomplete polymerization, have a considerable influence on color stability. this is usually attributed to chemical degeneration of the filler - resin bond and the solubility of the resin matrix. extrinsic factors such as adsorption or absorption of extrinsic stains, on the other hand, are still a major problem with esthetic restorations. the degree of color change is affected by a number of factors, including water sorption, chemical reactivity, dietary and smoking habits, bad oral hygiene, and surface smoothness of the restoration. besides the composition of the materials, the characteristics of the particles and the finishing and polishing procedures have a direct effect on surface smoothness and susceptibility to extrinsic staining. roughening of the surface caused by wear and chemical degradation may also affect the gloss and consequently increase extrinsic staining. water sorption may cause softening of the resin matrix, degradation of resin, reduction of stain resistance, and changes in translucency. the longevity and esthetic appearance of tooth - colored dental restorative materials greatly depend on the quality of the finishing and polishing techniques used. high - quality finishing and polishing improve both the esthetics and the longevity of composite restorations, whereas rough, poorly polished surfaces contribute to staining, plaque accumulation, gingival irritation, recurrent caries, and discoloration of the restoration. the aim of this in vitro study was to evaluate the effect of drinks on color stability and surface roughness of nanocomposites. in this context, laboratory tests were performed to assess the performance of nanocomposites in direct contact with different substances that are part of the daily diet. the null hypothesis is that the drinks (red wine, grape juice, and acai juice) have no effect on susceptibility to staining and the surface roughness of nanocomposites resins. two commercially available resin composite products, chosen for their different types of filler particles, were used in this study [table 1 ]. the commercial brand name, composition, and manufacturer of the materials used in the study forty circular samples were prepared 2.5 mm thick and 6 mm in diameter. uncured resin composite samples were prepared by condensing them into a teflon ring mold in two increments according to the manufacturer 's instructions. after inserting the second increment of material into the mold, a polyester strip was pressed onto the surface of the mold with a glass plate in order to obtain a flat surface without bubble formation ; excess material was extruded by pressing a glass plate onto the mold with a 500-g weight on top over the resin composite / matrix ensemble, producing specimens with a smooth flat surface. the specimens were light polymerized for 20 s on each increment using conventional quartz halogen tungsten light (optilight plus / gnatus). the distance between the light source and the specimen was standardized using a 1-mm glass slide. the specimens were taken out of the mold immediately after the light - curing cycle and were stored in distilled water for 24 h. the specimens were finished and polished successively with the sof - lex system (3 m espe, st. paul, mn, usa), color - coded from dark (coarse) to light (superfine) : coarse (5 s), medium (5 s), fine (20 s), and superfine (20 s). each disk was used twice in a low - speed hand piece ; they were rinsed briefly in distilled water (10 s) and dried with paper towels between each grit. the specimens were polished with felt discs (fgm, joinville, sc, bra) and diamond excel paste (fgm, joinville, sc, bra) for 20 s. the specimens were then washed with distilled water for 30 s, dried with paper towels and immersed in distilled water for 24 h at 37c. specimens were randomized to four groups according to the storage solution : (1) distilled water (control group) ; (2) acai juice ; (3) grape juice ; and (4) red wine. the ph of the solutions was verified using a ph meter (pht ; t-1000, tekna ind / com ltda, so bernardo do campo, sp, brazil), which indicated that the ph did not change during the period of treatment. all the procedures were carried out by the same operator at room temperature (25c) and relative humidity of 50%. type, batch number, ph, and manufacturers of the immersion solutions after this treatment, the specimens were evaluated for surface roughness. the average surface roughness (ra, m) was measured with a surface profilometer (surftest sj-301, mitutoyo, japan) using a tracing length of 1.25 mm and a cutoff of 0.25 mm to maximize filtration surface waviness, and a measuring speed of 0.5 mm / s using tools 301, mitutoyo, japan. each specimen was measured 3 times at different locations and in different directions near the center of the specimen, and the average roughness, ra, was derived from these readings. a calibration block was used periodically to check the performance of the profilometer, and all test procedures were performed by only one operator. color values were recorded in sequence using a digital spectrophotometer (vita easyshade, vita zahnfabrik, bad sckingen, germany). the spectrophotometer measured the tooth color based on the cielab color space system, which allows the color to be determined in three - dimensional space. l represents the value (lightness or darkness). the a value is a measure of redness (positive a) or greenness (negative a). the b value is a measure of yellowness (positive b) or blueness (negative b). the color difference (e) between the color coordinates was calculated by applying the formula e = [(l) + (a) + (b) ] in order to compare values before and after the storage treatment. three measurements were taken with the active point of the spectrophotometer in the center of each specimen, and thus the instrument automatically averaged the three readings for each specimen, which was then used for the overall data analysis. the roughness and the color were measured before (baseline) and 1, 2, 4, 8, and 12 weeks after storage in the different liquids, and after repolishing. for each group, the specimens were kept immersed in the respective solutions for 4 h daily over a period of 12 weeks. these solutions were renewed daily and after the immersion period, the specimens were washed and stored in distilled water. at the end of the periods of immersion, the values of the optical properties and surface roughness were tabulated and subjected to statistical analysis (spss for windows, version 13.0 ; spss inc., chicago, il, usa) using the paired t - test, one - way analysis of variance, and the tukey test and the independent sample t - test (p 0.05). the specimens in distilled water did present differences after 8 weeks of immersion (p = 0.002). a decrease in staining was observed after repolishing in all groups (acai juice, p = 0.001 ; grape juice, p = 0.000 ; wine, p = 0.007), except the distilled water group (p > 0.05) ; the average staining for the distilled water group was much lower than that in the other groups throughout the experiment. when the filtek z350 xt groups were compared at each time point, there was no statistically significant difference between the distilled water group and the acai juice group (p = 0.17), although there are numerical differences in the averages ; these groups showed less staining compared with the other groups. there was also no significant difference between the grape juice and red wine groups (p = 0.51), however, they presented a statistically significant difference compared with the distilled water and acai juice groups (p = 0.000), which stained less. after repolishing, there was a decrease in the mean staining at 12 weeks, although the pattern of the differences between the groups remained (p = 0.000) [table 3 ] ; water and acai juice stained less and grape juice and red wine stained more. after repolishing, the staining was similar to the reading at the 2 week for the distilled water and acai groups. for grape juice and red wine, means and standard deviations for the color difference (e) between the specimens immersed in different beverages for evolu - x, acai juice (p = 0.008), grape juice (p = 0.003), and red wine (p = 0.003) presented statistically significant changes in the color of the resin from the 2 week ; there was no change in the control group (p > 0.05). the color for the distilled water group changed after 12 weeks (p = 0.016). there was a decrease in the staining after repolishing for the grape juice (p = 0.001) and red wine groups (p = 0.000), however, the distilled water and acai juice groups did not have a statistically significant decrease in staining (p > 0.05), even though the average value of staining in these groups was much less than in others. there was no significant difference between the grape juice and red wine groups in the first 4 weeks (p > 0.05) ; however, the difference was statistically significant compared with the distilled water and acai juice groups (p = 0.000). from week 8, the red wine group showed more staining than the grape juice group (p = 0.000). however, there was no significant difference between the distilled water and acai juice groups (p > 0.05) and they showed less staining than the other groups. after repolishing, the mean amount of staining decreased compared with the values at 12 weeks, although the pattern of the differences between the groups remained the same (p = 0.000) [table 3 ] ; water and acai juice stained less and grape juice and red wine stained more. after repolishing, the staining pattern for the acai juice group was similar to that of the 1 week. for the distilled water, grape juice and red wine groups, the staining pattern was similar to that at the 2 week. comparing the two resins, from the 1 week it was found that the evolu - x specimens immersed in grape juice stained less than the filtek z350 xt specimens (p = 0.014). this pattern continued in weeks 2 (p = 0.020), 4 (p = 0.024), 8 (p = 0.017), and 12 (p = 0.011) and after repolishing (p = 0.002). samples of evolu - x resin immersed in red wine differed from the filtek z350 xt samples after 2 weeks (p = 0.026) and after repolishing (p = 0.002). for the acai juice group, there was a difference between the composites (p = 0.000) after week 12 and after repolishing (p = 0.011). the filtek z350 xt specimens showed a difference in the roughness of the red wine group after 12 weeks of immersion (p = 0.009), but this difference was reversed after repolishing. after 8 weeks of immersion, there was no difference in the roughness of the samples immersed in different media (p > 0.05). after 12 weeks, the samples immersed in red wine had greater roughness than the samples immersed in grape juice (p = 0.005), and this difference was reversed after repolishing [table 4 ]. means and standard deviations for surface roughness measurements for the specimens immersed in different beverages for evolu - x, there was a statistically significant increase in the roughness of the red wine group from the 1 week (p = 0.03) and the acai juice group from the 2 week of immersion (p = 0.039). after 2 weeks of immersion, there was a difference in roughness when the different groups were compared (p = 0.001). however, there was no significant difference between the distilled water and grape juice groups ; the acai juice and red wine groups also did not show a statistically significant difference (p > 0.05). comparing the surface roughness of the two resins, evolu - x had lower roughness than filtek z350 xt (initial, p = 0.002 ; 1 week after, p = 0.08 ; 12 weeks after, p = 0.025) when immersed in distilled water. in the acai juice group, the roughness of filtek z350 xt was lower than evolu - x after repolishing (p = 0.000). in the grape juice group, filtek z350 xt was less rough in the 4 week (p = 0.02). in the red wine group, filtek z350 xt was less rough in the 1 week (p = 0.007) and at week 8 (p = 0.000). there were statistical differences on staining susceptibility and the surface roughness of nanocomposites resins. according to color differences in esthetic restorations, three different intervals were used to distinguish changes in color values : e 3.3, easily observed, not clinically acceptable. the filtek z350 xt and evolu - x resins immersed in distilled water showed clinically acceptable color changes throughout the study. this observation confirms that water sorption itself did not alter the color of composites to a significant extent because distilled water has no colorant components. however, both the filtek z350 xt and evolu - x resins immersed in distilled water showed statistically significant changes in color in weeks 8 and 12, respectively. the filtek z350 xt samples immersed in acai juice showed clinically acceptable color changes in the first 8 weeks, but at week 12 the color changes were not clinically acceptable. the evolu - x samples immersed in acai juice showed clinically acceptable staining detectable only by a skilled person (1.0 3.3 for grape juice and red wine ; when these color changes are easily observed, they are not clinically acceptable and replacement of restorations is required. many studies have demonstrated that resin - based composites are susceptible to staining by common beverages, especially red wine and grape juice. in our study, red wine caused the highest discoloration of the restorative materials, followed by grape juice, acai juice, and distilled water. foods that are rich in anthocyanins, such as blueberries, red grapes, red wine, and acai juice have a strong color. anthocyanins are water - soluble vacuolar pigments that may appear red, purple, or blue, according to the ph. it has been reported previously that alcohol causes some degradation on the surface properties of resin composites. a rougher, degraded surface provides an extensive surface area for adsorption of pigments, thereby leading to more staining. in addition, it is likely that the lower ph of the grape juice affected the surface of the composite resin, increasing absorption of pigment. throughout the study, evolu - x resin showed less staining at the various times and in different immersion media compared with filtek z350 xt. the susceptibility of the resins to staining may be attributed to the composition of the materials and the characteristics of the particles. the hydrophilicity and degree of water sorption of a resin matrix could affect the staining susceptibility of resin composites. if the resin composite can absorb water, then it can also absorb other fluids, which results in discoloration. water sorption occurs mainly by direct absorption into the resin matrix, whereas glass filler particles do not absorb water into the bulk of the material, but can adsorb water onto the surface. water sorption of the resin composite may decrease the life of the restoration by expanding and plasticizing the resin component, hydrolyzing the silane, and causing the formation of microcracks. these microcracks or interfacial gaps at the interface between filler and matrix, allow stains to penetrate and cause discoloration. in this study, the advantages of nanofills did not seem to render them more stain resistant as described in other studies. however, studies have shown that nanofill resins stain more than nanohybrid resins and microhybrid resins. the type of resin matrix used can be a major contributor to the discoloration of the resin composite. it was found that water uptake in bisphenol - a glycidyl dimethacrylate - based resins increased from 3% to 6% as the proportion of triethylene glycol dimethacrylate (tegdma) increased from 0% to 1%, respectively. this study demonstrated that filtek z350 xt, which includes tegdma, was the most susceptible to staining. absorption of alcohol molecules contained in beverages into the resin matrix could result in softening of the surface of the composite. this explains the change in surface roughness of the resin when immersed in red wine. despite the statistically significant increase in roughness at some points during this study, studies by berger. and antonson. showed that the average surface roughness of a resin nanoparticulate after polishing using the sof - lex system was 0.1 and 0.08, respectively. yeh. showed that the surface roughness of a nanohybrid resin when immersed in distilled water was 0.1. thus, with the knowledge that roughness values between 0.06 and 0.1 are within the standard range for these composites, the change in roughness observed in this study does not seem to be relevant. there is a hypothesis that staining occurs in the most superficial layer of the composite. thus, repolishing can possibly removing discoloration, and prevent premature replacement of the restoration. in the current study, repolishing reduced the discoloration caused by immersion in colored beverages, but was not sufficient to remove the discoloration. they concluded that staining caused by coffee and red wine was superficial and 20-m wear was sufficient to remove the discoloration. the solutions tested in this study do not represent all the substances to which restorative materials may be exposed in the oral environment, therefore additional studies are necessary to investigate the color stability of composite resin - based materials. according to study, it was concluded that : colored drinks promoted color change of filtek z350 xt and evolu - x composites during the studyboth filtek z350 xt and evolu - x resins showed clinically unacceptable color change after immersion in grape juice and red wine, even after repolishing, indicating a need for replacement of the restorationrepolishing was effective in reducing the color change of the composites under study, although always to a clinically acceptable levelin general, evolu - x composite showed less color change than filtek z350 xt composite in the period evaluated and after repolishingthe increase in roughness was reversed after repolishing. colored drinks promoted color change of filtek z350 xt and evolu - x composites during the study both filtek z350 xt and evolu - x resins showed clinically unacceptable color change after immersion in grape juice and red wine, even after repolishing, indicating a need for replacement of the restoration repolishing was effective in reducing the color change of the composites under study, although always to a clinically acceptable level in general, evolu - x composite showed less color change than filtek z350 xt composite in the period evaluated and after repolishing the increase in roughness was reversed after repolishing. | objective : the aim was to evaluate the color and surface roughness of nanoparticle (c1) and nanohybrid (c2) composites after immersion in distilled water, acai juice, grape juice and red wine and repolishing.materials and methods : after recording the initial surface roughness and color, the specimens were divided into four groups according to the storage solution. the specimens were reassessed after immersion for 1, 2, 4, 8, and 12 weeks and after repolishing.results:the results showed that after 2 weeks, there were statistically significant changes in color of both resins in all groups, with the exception of the specimens stored in distilled water (p > 0.05). only 12 weeks of immersion in red wine changed the roughness of composite c1 (p = 0.009).conclusions : red wine produced the greatest color change in nanocomposites, followed by grape juice. acai juice made the color unacceptable clinically only after 12 weeks. repolishing reduced the color change in all groups. |
as the leading cause of death in korea, cancer has been the country s major public health concern since 1983. over 210,000 patients were newly diagnosed with cancer in korea, and one in four deaths is due to cancer. although the cancer registration system in korea is highly efficient and can provide nationwide cancer statistics within a relatively brief period, a lag time of at least 2 years is required for collection, compilation, and analysis of the data on a specific year. for planning and implementation of comprehensive cancer control programs, it is important to assess the number of new cases and deaths that are expected to occur during the current year. in this study, we report the projected cancer incidence and mortality for the year 2015 based on data from the 1990s through 2013. the korean ministry of health and welfare initiated a nationwide, hospital - based cancer registry, the korea central cancer registry (kccr), in 1980. the history, objectives, and activities of the kccr have been documented in detail elsewhere. incidence data from 1999 to 2012 were obtained from the korea national cancer incidence database (knci db). cancer cases were classified according to the international classification of diseases for oncology, third edition, and converted according to the international classification of diseases, 10th edition (icd-10). the cancer sites included in this study were (1) all cancers combined and (2) the 24 common cancers as follows : lip, oral cavity, and pharynx (c00-c14), esophagus (c15), stomach (c16), colon and rectum (c18-c20), liver and intrahepatic bile ducts (liver) (c22), gallbladder and other parts of the biliary tract (gallbladder) (c23-c24), pancreas (c25), larynx (c32), lung and bronchus (lung) (c33-c34), breast (c50), cervix uteri (c53), corpus uteri (c54), ovary (c56), prostate (c61), testis (c62), kidney (c64), bladder (c67), brain and central nervous system (c70-c72), thyroid (c73), hodgkin lymphoma (c81), non - hodgkin lymphoma (c82-c85, c96), multiple myeloma (c90), leukemia (c91-c95), and others. population data from 1993 to 2014 were obtained from the resident registration population, as reported by statistics korea. data on the mid - year population, as of july 1 of the respective years, were analyzed. however, for the year 2015, we used population data as of december 31, 2014 because the mid-2015 resident registration population was not yet available at the time of analysis. due to the time required for data collection, compilation, quality control, and analysis, the incidence and mortality data for a specific year are usually available 2 - 3 years later. therefore, to provide an estimate of the nation s current cancer burden, we attempted to project, ahead of 2015, the expected number of new cancer cases and deaths in korea for the year. linear regression models were used to assess time trends and projections. based on the observed cancer incidence data from 1999 to 2012, a linear regression model was fitted to age - specific rates by 5-year age groups against the observed years. the estimated number of new cancer cases was calculated from the projected age - specific rates for 2015 by multiplying the rates by the projected 2015 agespecific population size. for thyroid and prostate cancer, which showed significant curvilinear trends, we used a square - root transformation when fitting a linear regression model and converted the predicted values back to the original scale. to predict the cancer mortality rate in 2015, we first performed a joinpoint regression analysis on the data available for 1993 - 2013 to determine the year when significant changes occurred in cancer mortality trends according to sex and cancer site. a joinpoint regression describes changes in data trends by connecting several different line segments on a log scale at joinpoints. 4.0.1, http://surveillance.cancer.gov/joinpoint) from the surveillance research program of the us national cancer institute. after identifying the year during which significant trend changes occurred through joinpoint regression analysis, a simple linear regression model was fitted to the last line segment for estimation of age - specific cancer mortality rates in 2015. similar to the method used for the projection of cancer incidence, the number of new deaths in 2015 was then predicted by multiplying age - specific cancer mortality rates by the year s age - specific population. we summarized the results using crude rates (crs) and age - standardized rates (asrs) of cancer incidence and mortality. a total of 280,556 new cancer cases are anticipated in 2015 (table 1, fig. 1) with more female (n=142,809) than male (n=137,747) cancer patients expected in the coming year. the projected crs per 100,000 of all sites combined in 2015 are 541.9 and 561.3 in men and women, respectively. moreover, the projected asrs per 100,000 of all sites combined are 361.1 and 353.1, respectively (table 2). in men, the five leading primary sites of cancer are expected to be the stomach (cr, 94.1 ; asr, 61.3), colon and rectum (cr, 92.4 ; asr, 60.7), lung (cr, 69.5 ; asr, 44.7), prostate (cr, 58.3 ; asr, 38.1), and liver (cr, 50.1 ; asr, 32.6), accounting for 67.3% of all newly diagnosed cancers in 2015. in women, the five leading primary sites are expected to be the thyroid (cr, 214.2 ; asr, 150.4), breast (cr, 76.5 ; asr, 50.5), colon and rectum (cr, 57.2 ; asr, 31.0), stomach (cr, 43.8 ; asr, 24.3), and lung (cr, 31.3 ; asr, 15.8), accounting for 75.3% of all newly diagnosed cancers (fig. thyroid cancer alone is projected to account for approximately 38.2% (54,491 cases) of incident cases in women in 2015. the five most common cancer sites expected in 2015 by sex and age group are shown in table 3. leukemia and thyroid cancer are expected to be the most common forms of cancer in both sexes for the 0 - 14 and 15 - 34 age groups. stomach cancer is predicted to be the most prevalent in men aged 35 - 64 years, whereas lung cancer is expected to be more frequent in men aged 65 and over. thyroid cancer is predicted to be the most common cancer in women 35 - 64 years of age, whereas colorectal cancer is expected to be the most prevalent in women aged 65 and over. these projections indicate that the incidences of stomach, lung, liver, colorectal, and prostate cancers will increase gradually with age for men. in women, the age - specific incidence rates of stomach, colorectal, liver, lung, and cervical cancers denote a rising trend in these cancers with age ; however, the incidence of breast and thyroid cancer in women is expected to level off after the late 40s and early 50s, respectively (fig. it is estimated that 76,698 cancer deaths will occur in korea during 2015 (table 1, fig. the projected crs per 100,000 of all sites combined in 2015 for men and women are 187.0 and 114.6, respectively, whereas the projected asrs per 100,000 of all sites combined are 121.7 and 54.5, respectively (table 4). the predicted five leading cancer sites causing mortality in men are lung (cr, 50.1 ; asr, 31.9), liver (cr, 31.5 ; asr, 20.4), colon and rectum (cr, 20.5 ; asr, 13.4), stomach (cr, 18.9 ; asr, 12.2), and pancreas (cr, 11.1 ; asr, 7.2). during the same time period, lung cancer (cr, 19.0 ; asr, 8.5) is projected to be the leading cancer site in women, causing mortality, followed by the colon and rectum (cr, 15.0 ; asr, 6.6), liver (cr, 11.1 ; asr, 5.2), stomach (cr, 10.7 ; asr, 4.7), and pancreas (cr, 10.1 ; asr, 4.5). as a result of a continuous decrease in stomach cancer deaths, stomach was first predicted to be the fourth - common cancer death site in women. the predicted age - specific mortality rates of the selected cancers for males and females in 2015 are shown in further detail in fig. 3. when examined by age, korean men and women aged 60 and over are expected to have the highest mortality rates from lung cancer. a total of 280,556 new cancer cases are anticipated in 2015 (table 1, fig. 1) with more female (n=142,809) than male (n=137,747) cancer patients expected in the coming year. the projected crs per 100,000 of all sites combined in 2015 are 541.9 and 561.3 in men and women, respectively. moreover, the projected asrs per 100,000 of all sites combined are 361.1 and 353.1, respectively (table 2). in men, the five leading primary sites of cancer are expected to be the stomach (cr, 94.1 ; asr, 61.3), colon and rectum (cr, 92.4 ; asr, 60.7), lung (cr, 69.5 ; asr, 44.7), prostate (cr, 58.3 ; asr, 38.1), and liver (cr, 50.1 ; asr, 32.6), accounting for 67.3% of all newly diagnosed cancers in 2015. in women, the five leading primary sites are expected to be the thyroid (cr, 214.2 ; asr, 150.4), breast (cr, 76.5 ; asr, 50.5), colon and rectum (cr, 57.2 ; asr, 31.0), stomach (cr, 43.8 ; asr, 24.3), and lung (cr, 31.3 ; asr, 15.8), accounting for 75.3% of all newly diagnosed cancers (fig. thyroid cancer alone is projected to account for approximately 38.2% (54,491 cases) of incident cases in women in 2015. the five most common cancer sites expected in 2015 by sex and age group are shown in table 3. leukemia and thyroid cancer are expected to be the most common forms of cancer in both sexes for the 0 - 14 and 15 - 34 age groups. stomach cancer is predicted to be the most prevalent in men aged 35 - 64 years, whereas lung cancer is expected to be more frequent in men aged 65 and over. thyroid cancer is predicted to be the most common cancer in women 35 - 64 years of age, whereas colorectal cancer is expected to be the most prevalent in women aged 65 and over. these projections indicate that the incidences of stomach, lung, liver, colorectal, and prostate cancers will increase gradually with age for men. in women, the age - specific incidence rates of stomach, colorectal, liver, lung, and cervical cancers denote a rising trend in these cancers with age ; however, the incidence of breast and thyroid cancer in women is expected to level off after the late 40s and early 50s, respectively (fig. it is estimated that 76,698 cancer deaths will occur in korea during 2015 (table 1, fig. 1). the projected crs per 100,000 of all sites combined in 2015 for men and women are 187.0 and 114.6, respectively, whereas the projected asrs per 100,000 of all sites combined are 121.7 and 54.5, respectively (table 4). the predicted five leading cancer sites causing mortality in men are lung (cr, 50.1 ; asr, 31.9), liver (cr, 31.5 ; asr, 20.4), colon and rectum (cr, 20.5 ; asr, 13.4), stomach (cr, 18.9 ; asr, 12.2), and pancreas (cr, 11.1 ; asr, 7.2). during the same time period, lung cancer (cr, 19.0 ; asr, 8.5) is projected to be the leading cancer site in women, causing mortality, followed by the colon and rectum (cr, 15.0 ; asr, 6.6), liver (cr, 11.1 ; asr, 5.2), stomach (cr, 10.7 ; asr, 4.7), and pancreas (cr, 10.1 ; asr, 4.5). as a result of a continuous decrease in stomach cancer deaths, stomach was first predicted to be the fourth - common cancer death site in women. the predicted age - specific mortality rates of the selected cancers for males and females in 2015 are shown in further detail in fig. 3. when examined by age, korean men and women aged 60 and over are expected to have the highest mortality rates from lung cancer. a total of 280,556 new cancer cases and 76,698 cancer deaths are expected to occur in korea during 2015. in korean males, it is anticipated that stomach, colorectal, lung, prostate, and liver cancers will be the most commonly occurring, whereas it is expected that lung, liver, colorectal, stomach, and pancreatic cancers will be the most common causes of cancer - related deaths. in korean women, it is anticipated that thyroid, breast, colorectal, stomach, and lung cancers will be the most prevalent, whereas it is projected that lung, colorectal, liver, stomach, and pancreatic cancers will be the most common causes of cancer - related deaths. the country will likely see its cancer burden continue to grow with the aging of its population. the current projections of cancer incidence and mortality for 2015 represent an important resource for planning and evaluation of cancer - control programs. as the estimates in this study are model - based, these results should be interpreted with caution. | purposefor estimation of korea s current cancer burden, this study aimed to report on the projected cancer incidence and mortality rates for the year 2015. materials and methodscancer incidence data from 1999 to 2012 were obtained from the korea national cancer incidence database, and cancer mortality data from 1993 to 2013 were acquired from statistics korea. the cancer incidence in 2015 was projected by fitting a linear regression model to the observed age - specific cancer incidence rates against the observed years and then multiplying the projected age - specific rates by the age - specific population. a similar procedure was used for cancer mortality, except a joinpoint regression model was used to determine at which year the linear trend changed significantly. resultsa total of 280,556 new cancer cases and 76,698 cancer deaths are expected to occur in korea in 2015. the crude incidence rate per 100,000 of all sites combined will likely reach 551.6 and the age - standardized incidence rate, 347.6. the estimated five leading primary cancer incidence sites are the stomach, colorectum, lung, prostate, and liver in men ; and thyroid, breast, colorectum, stomach, and lung in women. the projected crude mortality rate of all sites combined and age - standardized rate is 150.8 and 82.4, respectively. conclusioncancer is currently one of the foremost public health concerns in korea, and as the population ages, the nation s cancer burden will continue to increase. |
it occurs in a wide segment of society, from people who react to life conditions in a well - adjusted way, to people who have severe mental disorders. people who attempt suicide may truly desire to die, or this behavior may just be a response to pain, despair, and hopelessness a cry for help. the world health organization (who) divides suicides into 2 categories : suicide attempts and actual (complete) suicides. suicide attempts are those non - fatal attempts individuals may make to kill, harm, or poison themselves voluntarily. according to the who, 900,000 people worldwide die as a result of suicide each year. suicides are a major public and mental health concern in developed and developing countries today. national suicide rates range from 3 to 45 out of every 100,000 people living in various countries. the rates vary greatly between countries, but the most prevalent methods of suicide hold true for suicide in all parts of the world and include intentional medication overdosing, poisoning, hanging, and shooting one - self with a firearm. suicides are more prevalent in men after the age of 45 years and in women after age 50. non - fatal attempts are higher in women, but fatal attempts are higher in men. self - injurious behavior is more prevalent in women and suicide occurs more in men. self - injurious behavior is linked with suicide in men, but self - injurious behavior is linked with motivations other than suicide in women. rates of attempted suicide are higher among single individuals than among married people, and they are higher in the wealthiest and the poorest segments of society than in the middle class. not surprisingly, suicide rates are higher among the unemployed than they are among those who have jobs [6,1012 ]. the main reasons for suicide attempts are physical problems (2575%) and mental disorders (more than 90% of these being major psychiatric disorders), marital discord, and livelihood difficulties such as economic and social problems. the aim of this study was determine the demographic and sociocultural characteristics of suicide attempts by using data from the turkish statistical institute. it is our intent that the work data may contribute to the national suicide data and the development of suicide prevention policies. we obtained our data, which cover the years 2007 to 2012, from the database accessible at the official website of the turkish statistical institute. the chi - square test was used for statistical analysis, and the percentage distribution was calculated along with the odds ratios. according to data from the turkish statistical institute, the total number of suicide deaths in turkey between 2007 and 2012 changed yearly (=42,03559,209 ; p 15 years and 0.00175% of women died due to reported suicide. in the same year, 0.0056% of men and 0.0023% of women died due to suicide in turkey ; an extremely low rate compared to india. these rates vary from country to country and from culture to culture because religious beliefs and societal traditions may have a preventive effect on suicide. therefore, the perception of suicide varies according to religion, culture, and social system. according to islam, suicide is a great sin. with the effects of these thoughts and beliefs, suicide in our country less than 1% of all the deaths around the world occurred because of suicide. in turkey, as with the rest of the world, 0.9% of the deaths between 2007 and 2012 were due to suicide. suicide accounted for 1.6% of the deaths in america in 2010, and 3% of all deaths in india were because of suicide. in the same year, 0.8% of the deaths in turkey were because of suicide. while the suicide deaths were 0.004% in 2007, this rate increased to 0.0043% in 2013, and this increase was caused mostly by an increase in male suicides. during these same years, the numbers of female suicide deaths decreased. males are more prone to increased suicidal thoughts, aggression, and a tendency toward violence. between 2007 and 2012, the number of suicide attempts has increased, but the numbers of suicide deaths decreased. despite the low rate of suicide death in our country, the increase in suicide rates shows that the subject is a significant social problem. suicide deaths between the ages of 20 and 39 years are 39.6% higher than the all - ages rate, while suicides in those aged 60 years are the lowest by 15.0%. suicide death rates in men and women between the ages of 20 and 39 years are highest, and it is noteworthy that the rates of the 2 genders are close together. the rate of suicide deaths in women aged 19 years is 2.4 times more than the rate of suicide deaths in men of the same age. the rate of suicide deaths in men aged 4059 years is 1.7 times more than the rate of suicide deaths in women of the same age. between 2007 and 2012, male suicide deaths in 2012, compared to 2007, showed an increase of 28.0% and an annual average increase of 4.7%. from 2007 to 2012, this can be connected to the increase in the elderly population. between 2007 and 2012, the number of suicides in women decreased in turkey in all age groups except the 40 to 59 group and the 60 age group. female suicide deaths in 2012, compared to 2007, decreased by 7.6%, with an annual average increase of 1.3%. according to the turkish statistical institute, the number of suicide deaths varies according to the place where people live (=699.5 ; p<0.001). the rate of suicide increases in the district centers but showed a decrease in villages and towns. suicide deaths increased in the spring, reached their highest levels in the summer, and dropped to their lowest levels in the autumn. suicide deaths were most likely to occur in july (n : 1689) and least likely to occur in november (n : 1218), and the deaths in july occurred at a rate about 38.7% higher than the november deaths. suicide deaths in men occur least in september (n : 874) and november (n : 876), but they occur most in june (n : 1151) and july (n : 1152).. suicide deaths in women occur approximately 57.0% more in july (n : 537) than they do in november (n : 342). spring and summer months are especially critical, as the suicides occurring around this time of year are most often associated with failure in school. regarding educational level, more than half of the individuals committing suicide in turkey have no more than a primary or secondary education. illiteracy is 3.75 times more common in women than in men. not receiving any education having a high school or equivalent education is 1.6 times more likely in men than in women. several studies conducted in turkey have shown that suicide attempts in patients with low levels of education are much more likely because the lack of education can cause difficulties in expressing him / herself and can negatively affect the individual s economic situation, which may also lead to suicide. men especially prefer hanging and jumping off buildings, whereas women prefer overdosing on medications, intoxication with poison, and cutting wrists. in the deaths between 2007 and 2012, the most commonly used method was hanging. this method is also commonly used in brazil, argentina, thailand, austria, and germany. according to the turkish statistical institute, about half of the suicide deaths both in men and women were committed by the method of hanging. about half the male suicide deaths in brazil, argentina, thailand, austria, germany, france, portugal, spain, england, and georgia, and the female suicide deaths in slovakia, slovenia, georgia, croatia, and mexico have occurred with a similar method of hanging as in our country. this method is 1.8 times more likely in men (29.3%) than in women (15.9%). in the us in 2007, suicide deaths occurred most often by using a firearm. men prefer this method 1.9 times more than women, and this is similar to what has been observed in our country. this finding is consistent with the information that men prefer more severe methods than women. suicide by using chemicals is 2.5 times more likely in females (13.5%) than in males (5.4%). in many countries, such as canada, america, austria, hungary, denmark, the netherlands, and norway, suicide by using chemicals suicide by jumping from a high place was 1.8 times more likely to occur in females (13.4%) than in males (7.3%). this method is also most common in singapore (72.4%, 20002004) and in hong kong (46%, 2003). this method is second most common in saudi arabia (12%, 10861995) and in japan (10.4%, 1994). it is the third most common method in taiwan (9.4%, 19952004) and in south korea (15%, 2003). some contributing factors are depression, alcohol and drug dependence, personality disorders, schizophrenia, chronic diseases, and unfavorable family conditions, economic and social problems, migration, and urban life. in approximately half of suicide deaths, suicide due to economic difficulties occurs 6 times more often in males than in females. suicide because of commercial failure is 18.5 times more likely in males. in turkey, the rate of employment in males was 71.5%, while this same rate was only 24.9% for females. this data can be evaluated as an indicator as to why suicide is higher in men than in women by reason of subsistence difficulties and commercial failure. the rate of suicide due to unrequited love was seen equally in both males and females. knowing the cause of the suicide is extremely important in terms of approaching and orienting patients. examining this subject from etiological, epidemiological, biological, psychological, sociological, and anthropological perspectives is important in order to improve the prevention of suicides. | backgroundthe aim of this study was to determine the demographic and sociocultural characteristics of suicide attempts by using data from the turkish statistical institute. it is our intent that the work data may contribute to the national suicide data and the development of suicide prevention policies.material/methodswe obtained our data, which cover the years 2007 to 2012, from the database accessible at the official website of the turkish statistical institute, which permits the use of its data for research purposes. the data were evaluated by using the spss 10.0 program. the chi - square test was used for statistical analysis, and the percentage distribution and odds ratios were calculated.resultsaccording to data from the turkish statistical institute, the total number of suicide deaths in turkey between 2007 and 2012 changed yearly (2=42,03559,209 ; p<0.001). while suicide deaths in 2007 made up 0.00396% of the total deaths for that year, that figure increased to 0.00426% in 2013. according to the data from the turkish statistical institute, over 1.9 million people died due to all causes between 2007 and 2012 in turkey. over 17,000 deaths (0.9%) were due to suicide.conclusionssuicide is an important public health problem and is multidimensional in nature. examining this subject from etiological, epidemiological, biological, psychological, sociological, and anthropological perspectives is important to improve the prevention of suicides. |
cystic fibrosis (cf) is a common autosomal recessive genetic disease caused by mutation in the cf transmembrane conductance regulatory (cftr) gene. missing or defective cftr gene products in patients with cf lead to multiorgan dysfunctions such as severe lung disease, pancreas dysfunction and elevated sweat chloride. cystic fibrosis is less common among african, american and asian people than among northern europeans. to date, the most common cftr gene mutation is f508, which accounts for about 30% to 80% of all mutant alleles worldwide. the frequency of this mutation is reportedly between 16% and 23% in different parts of iran. because the type and distribution of these mutations vary widely among populations, this study attempted to identify the most common cftr gene mutations in patients in southwestern iran with cf and search for correlations between certain mutations and the clinical presentation of the disease. this cross - sectional study was conducted over a period of three years from 2009 to 2012 in southwestern iran. the diagnosis of cf was based on the typical clinical features of the disease and two abnormal sweat chloride values (> 60 meq / l). patients with cf were included in this study after written informed consent was obtained from the patients themselves (if 18 years old) or their parents (of a, g542x, w1282x, n1303k, f508, 3849 + 10kbc > t, 394deltt, 621 + 1 g > t, s1251n, g551d, r117h, r1162x, r334w, a455e, 2183aa > g, 3659delc, 1078delt, i507, r347p, r553x, e60x, 3120 + 1 g > a, 2789 + 5 g > a, 1898 + 1 g > a, 711 + 1 g > t, g85e, 2184dela and r560 t) were analyzed with the elucigene cf29 v. direct frequency counts and descriptive statistics were used here to report the clinical and genetic features. forty - five patients with cf (27 males and 18 females) from southwestern iran were included in this study. at diagnosis our patients ranged in age from one month to 19 years ; 82% of them were under 2 years old at the time of diagnosis. their early clinical symptoms are summarized in table 1. as shown, most patients with cf had respiratory symptoms. early clinical symptoms in patients from southwestern iran with cystic fibrosis (n = 45) parental consanguinity was found in 40 patients (89%) and a family history of cf was reported by 15 patients (37%) in this group. the homozygous f508 mutation was observed in eight patients (18%), and three patients (7%) were f508 carriers. this patient was compound heterozygous for the mutant f508 allele (lane a) and the 2183aa > g mutation (lanes c and d). due to 2184dela primer cross - reactivity with the 2183aa > g mutant dna sequence, this sample with the 2183aa > g mutation resulted in diagnostic bands of 425 bp and 169 bp in lanes c and d, respectively. a copy of the mutant f508 allele (160 bp) is seen in lane a, and a copy of the normal f508 allele (also 160 bp) is observed in lane b. the top and bottom bands in each lane are internal positive controls, and lane m shows the 1-kb ladder marker (http://www.gen-probe.com/pdfs/downloads/cfcf029v2_ifugb010.pdf). frequencies of cftr gene mutations in a sample of patients in southwestern iran with cystic fibrosis defects in the cftr gene are the main cause of cf, and molecular analysis of this gene is important for the differential diagnosis. however, the type of cftr gene mutation has little effect on the age of onset and clinical presentation of the disease. the results of this study showed no correlation between the mutations we detected and the clinical presentations in our patients. therefore, clinical signs of cf are still the best guide for diagnosing the disease, and should be taken into account by pediatricians. our results document a 1- to 2-year delay between the first clinical presentation and the diagnosis of cf. delayed diagnosis remains a problem which usually results in progressive disease and even irreversible changes. cystic fibrosis, with a prevalence of 1 in 2500, is considered a common disease among caucasians. the carrier rate of cf is reported to be about 1 in 25 among caucasians with no family history. because consanguinity is known to be the main cause of genetic disorders, a high carrier rate implies greater risk in populations with high levels of consanguineous marriage, including some ethnic groups in iran. the overall rate of consanguineous marriage among iranians is reportedly 38.6%, and ranges from 30% to 85% in different parts of the country. in our sample, 89% of the patients belonged to consanguineous families. unlike european countries and in agreement with other reports from iran, the frequency of f508 was around 24% in our sample of patients with cf. we found no new cftr gene mutations in this study, and all the mutations reported here have been previously reported in different parts of iran. although no accurate data are available regarding the prevalence of cf in different parts of iran, the disease appears not to be rare in this country. to reduce the frequency of new cases of cf, genetic counseling before marriage and prenatal diagnosis for families with one or more affected children are necessary. considering the high number of mutations in the cftr gene and the heterogenous distribution of these mutations among different populations, determining the most common mutations in each area is necessary in order to design effective local diagnostic kits. our results showed that a commercial kit designed to detect 29 different mutations failed to identify any cftr gene mutations in 60% of our patients with cf. to design an effective domestic kit, the entire cftr gene should be sequenced and all cftr gene rearrangements should be identified to detect prevalent and specific local mutations. such a kit would be helpful not only for the reliable diagnosis of cf but also for premarital genetic counseling and prenatal diagnosis. | objectivecystic fibrosis (cf) is a common autosomal recessive genetic disease caused by a mutation in the cf transmembrane conductance regulatory (cftr) gene. this study attempted to identify the most common cftr mutations and any correlations between certain mutations and the clinical presentation of the disease in cf patients in southwestern iran.methodstwenty nine common cftr gene mutations were examined in 45 cf patients.findingschronic cough, intestinal obstruction, dehydration, heat exhaustion and steatorrhea were the most common early clinical symptoms among our patients. the most common mutation was f508, with an allele frequency of 21%. the homozygous f508 mutation was observed in eight patients (18%), and three patients (7%) were f508 carriers. the 2183aa > g mutation was observed in four patients, one of whom was also a f508 carrier. the r1162x mutation was detected in two patients. the g542x, r334w and n1303k mutations were detected each in one patient, the first of whom was also a f508 carrier.conclusionout of 45 patients, 27 (60%) had none of the cftr gene mutations we tested for. the most frequent mutations in southwestern iranian patients with cf should be identified by sequencing the entire cftr gene in order to optimize the design of a diagnostic kit for common regional mutations. |
crystal induced acute kidney injury (c - aki) is a recent cause of acute kidney injury (aki) in intensive care units (icus). indeed, cholesterol embolism, oxalate, uric acid, phosphate, and many medications (sulfadiazine, acyclovir, indinavir, triamterene, methotrexate, orlistat, oral sodium phosphate preparation, and ciprofloxacin) can cause c - aki.1 crystallization will vary for each individual condition as the urinary ph necessary for precipitation is extremely different from one condition to another and therefore urine alkalization may be very detrimental. for instance, for ciproxine crystalisation tends to occur at an alkaline ph in the tubules, therefore urine alkalinisation can surely not be recommended.3 some intoxication, such as ethylene glycol poisoning, can also cause c - aki.2 c - aki can also occur with myeloma.3 sodium phosphate solutions are commonly used to cleanse the bowel in preparation for colonoscopy or surgical procedures and eventually for treatment of severe constipation. though relatively safe, these drugs must be used with caution in the elderly and in patients with renal dysfunction, small intestinal disorders, or poor gut motility and are prohibited in bowel obstruction. acute phosphate nephropathy due to the use of agents containing sodium phosphate is a rare, but potentially life - threatening condition. unfamiliarity with this complication in icus, the time gap between drug administration and onset of aki, and the lack of monitoring of renal function during treatment all explain why this diagnosis is easily overlooked. a 71-year - old man was admitted to the icu after heart surgery. his previous history was unremarkable except for adequately controlled arterial hypertension and dyslipidemia. the surgical procedure was complicated by a left atrium tear necessitating aggressive postoperative fluid loading and dobutamine infusion to maintain perfusion pressure. after an initially uneventful evolution, the patient developed pneumonia, rapidly evolving to septic shock complicated by severe cardiac and respiratory failure. treatment with fluids, catecholamines, and antibiotics was started under invasive hemodynamic monitoring. at that time, renal function was normal. hemodynamic stability was restored and the patient could be progressively weaned from ventilation and supportive medication. since he passed no stools for more than a week, rectal enemas (fleet enema ; cb fleet company, inc, lynchburg, va, usa) were repeatedly administered. they had only minimal effect and an oral laxative (fleet oros ; cb fleet company, inc) was added 8 hours later. less than 12 hours later, the patient suddenly developed an acute rise in creatinine and urea levels and became anuric. the patient was started on a small dose of angiotensin converting enzyme (ace) inhibitors 7 days prior to the acute rise of phosphate. the patient did not receive diuretics, any other antihypertensive drugs, or angiotensin ii (at - ii) receptor blockers during this period before the sudden rise in phosphate. blood analysis revealed phosphorus and calcium levels of 21 mg / dl and 4.6 mg / dl, respectively. initially, the patient was put under high flow hemodialysis in order to quickly reduce the phosphate value and indeed, the phosphate went down from 21 mg / dl to 7.1 mg / dl. because of the hemodynamic instability, the patient was switched over to continuous venovenous hemofiltration at 35 ml / kg / hour in order to avoid a rebound of hyperphosphatemia and ensure hemodynamic stability while continuing extrarenal blood purification of the hyperphosphatemia. after correction of electrolyte imbalance, the patient left the icu in good condition, though still needing intermittent renal replacement therapy for persisting anuria. after a follow - up at 6 months, we should be able to confirm his chronic kidney disease (ckd) status in regards to dialysis. the amount of phosphorus in the blood is determined by oral intake and renal excretion. approximately 60% to 65% of dietary phosphate is absorbed in the upper duodenum, jejunum, and ileum either passively or by active transport mediated by vitamin d. renal elimination of phosphorus depends on the filtered load and the renal threshold. even a slight increase of phosphatemia in response to an increased phosphate load results in a reduction of proximal tubular phosphate reabsorption. excess phosphate induces hypocalcemia by precipitating calcium, decreasing calcium absorption from the gastrointestinal tract and lowering of 1,25-dehydroxycholecalciferol synthesis, and produces sodium phosphate complex formation in the serum. the decline in ionized calcium serum concentration triggers the release of parathyroid hormone (pth) which further reduces phosphate renal reabsorption.4 the cathartic action of sodium phosphate, a small volume laxative, results essentially from its osmotic properties, drawing plasma water into the gastrointestinal tract. sodium phosphate - based enemas are commonly used for bowel cleansing or treatment of stubborn constipation. sodium phosphate mainly induces a marked and transient increase in serum phosphorus, sodium, and chloride levels whilst simultaneously decreasing serum calcium and potassium concentrations. hyperphosphatemia associated with administration of preparations containing phosphate may result from excessive and/or repeated doses, increased intestinal absorption, or impaired renal excretion. intestinal absorption is facilitated by impaired gut peristalsis prolonging retention of these substances in the gut lumen.5 when the urine is oversaturated and buffering factors such as ph, citrate, and pyrophosphate are overwhelmed, renal phosphorus excretion becomes compromised and crystallization will occur. tubular damage produced by the release of reactive oxygen species when calcium phosphate crystals bind to tubular epithelial cells is the presumed main pathway leading to impaired renal excretion.6 risk factors for developing c - aki include female gender, ckd, dehydration, diuretic treatment, colitis, and, to a lesser extent, diabetes mellitus and the use of specific drugs such as nonsteroidal anti - inflammatory agents, ace inhibitors, and at - ii receptor blockers. urine alkalinization may also be a trigger in some specific conditions such as ciproxin induced c - aki.1 elderly patients are at particular risk for phosphate intoxication. they are more sedentary, have lower fluid intake, have intrinsically less bowel movements, often take medications that decrease or influence gut motility, and suffer more underlying systemic and gastrointestinal diseases that incapacitate intestinal function. moreover, an age - related decline in renal function is frequently present.7 many concomitant factors played a role in the development of acute hyperphosphatemia in our patient. first, an already inefficient gut peristalsis in this elderly patient became more impaired during a turbulent postoperative phase characterized by severe hemodynamic, respiratory, and metabolic derangement. elderly age in association with concurrent medication known to diminish gastrointestinal tract motility (sedation, analgesics, and catecholamines), resulted in colonic retention and subsequent increased absorption of phosphorus. second, the pharmacological efforts to combat constipation obviously culminated in a too excessive phosphate load. indeed, normal daily dietary phosphorus intake is approximately 1.5 g whereas one fleet enema contains 8.533 g. such high amounts will produce an almost 100% rise in serum phosphate concentration.8 finally, renal dysfunction that may have been induced or worsened by phosphate intoxication in se, prevented excretion of redundant phosphate. the diagnosis is pending on excluding other causes, but also on the urine microscopy that can identify crystallization. unfortunately, in our case, the patient did not pass more urine in order to do this microscopy. nevertheless, the extreme hyperphosphatemia in the absence of other causes of aki did confirm the diagnosis. regarding further insights concerning patho - physiology,6,911 the kidneys play an important role in the regulation of plasma phosphorus leading to high urinary phosphate excretion. when the urine gets supersaturated and inhibiting factors such as ph, citrate, and pyrophosphate concentrations are low, crystallization will take place. regarding the role of pth,6,11 it is well known that an acute phosphate load (although most of the time small in amount) will uniformly lower both ionized and total calcium and produce a compensatory pth response in the presence of intact parathyroid glands. an increase in pth will lead to an increased load in urinary calcium and will accelerate the precipitation process.6,11 in our case, pth was already elevated at admission without increased phosphate and did not increase further after the extreme hyperphosphatemia, but pth was analyzed too late after the sudden increase in phosphate in order to detect a sudden increase in pth that could have induced an increase in urinary excretion of calcium leading to more potential precipitation (see table 1). as important guidelines for bedside intensivists,10,11 we suggest to consider as contraindications for the use of oral and rectal phosphate containing purgatives, patients under the following conditions. patients under medications contributing to renal hypoperfusion, such as diuretics, ace inhibitors, and at - ii receptor blockers should be seen as patients at risk. other risk factors which have been suggested are advanced age, diabetes, and stage 13 ckd, all of which should be seen as potential contraindications. as stated earlier, our patient was on small doses of ace inhibitors for 7 days prior to the acute rise of phosphate and was not on diuretics, other antihypertensive drugs, or at - ii receptor blockers. therefore, we believe that hyperphosphatemia remains the main mechanism of aki in our patient, even though we do not have a biopsy to definitively confirm this diagnosis. as also stated earlier, septic shock may have played a role as a trigger. in summary, we have described a critically ill patient who developed severe hyperphosphatemia, hypocalcemia, and unexpected c - aki after receiving oral and rectal phosphate containing purgatives. this case highlights the importance of using these drugs with caution in patients at risk and explains why the diagnosis is frequently overlooked.9 the presence of any known risk factor should impose the use of alternative preparations. early detection of this complication could potentially prevent the irreversible slippery slope towards ckd and chronic dialysis. | acute phosphate nephropathy or nephrocalcinosis is a tubulointerstitial nephropathy characterized by tubular calcium phosphate deposition crystal nephropathy and slowly progressive renal insufficiency during or following treatment with preparations containing sodium phosphate. we report a patient who developed nephrocalcinosis (crystal induced acute kidney injury) following the administration of a combination of oral and rectal sodium phosphate for treatment of postoperative constipation. a timely renal replacement therapy procedure may reverse the process of crystallization and the irreversible slope towards chronic dialysis. |
the study population was drawn from the 2002 nsfg, a periodic survey designed and administered by the national center for health statistics (nchs). national estimates of factors affecting pregnancy and birth rates, particularly contraceptive use and pregnancy, with oversampling of african american women, latinas, and teenagers. the 2002 survey was designed to obtain detailed information on factors affecting childbearing, marriage, and parenthood from a national probability sample of 12,571 noninstitutionalized men and women 1544 years of age (n = 7,643 nonpregnant women) (17). trained interviewers asked participants questions and entered the responses into a notebook computer. a detailed description of the 2002 nsfg sample design and sampling weights is provided elsewhere (17). our sample was comprised of nonpregnant female respondents 2044 years of age with recorded information on both diabetes status and bmi (n = 5,955). as the appropriate method to assess weight for height in subjects 12 years), total household income (10% were retained in the final model for all analyses. for this analysis, the full and the final models remained the same. all analyses were performed using sas version 9.1.3 for windows (sas institute, cary, nc). for all analyses, the data were weighted to adjust for the survey design, sampling, coverage, and response rates so that accurate national estimates can be made from the sample. participants were asked to list the types of contraceptive methods that they were using at the time of the interview. participants were classified as contraceptive nonusers if they indicated that they were not using a contraceptive method at the time of interview. contraceptive methods were categorized as hormonal (including birth control pills, injectables, implants, intrauterine devices, and the contraceptive patch), barrier (including the male condom, female condom, diaphragm, cervical cap, and sponge), or sterilization. diabetes status was ascertained based on responses to the following two questions : has a doctor or other medical care provider ever told you that you had diabetes or and were you ever told you had diabetes when you were not pregnant ? (17). the second question was only asked to those who reported ever being pregnant and responded self - report of preexisting (nongestational) diabetes has a good concordance () of 0.8 when compared with medical record reviews, with a sensitivity of 85% and specificity of 97% (18). all participants were asked to report their height and weight at the time of interview. data cleaning was performed by the nchs staff to account for some extremely high and low values reported for height and weight (based on the 5th and 95th percentiles). in particular, height for females was bottom coded at 60 to indicate 60 inches or less and top - coded at 70 to indicate weight for females was bottom and top coded at 108 and 240 lb, respectively (17). for the purposes of this analysis, bmi was recoded as a categorical variable and consisted of the following levels : underweight or normal weight (12 years), total household income (10% were retained in the final model for all analyses. for this analysis, the full and the final models remained the same. all analyses were performed using sas version 9.1.3 for windows (sas institute, cary, nc). for all analyses, the data were weighted to adjust for the survey design, sampling, coverage, and response rates so that accurate national estimates can be made from the sample. the study sample consisted of 135 women with diabetes (2.3%) and 5,820 women without diabetes (97.7%). women with diabetes were more likely to be older, of minority status, and to be separated, divorced, or widowed than women without diabetes (table 1). diabetic women reported higher rates of receiving medical help to become pregnant (13.0%) than women without diabetes (9.7%), although differences were not statistically significant. additionally, diabetic women were slightly less likely to have been sexually active in the past 4 weeks and less likely to desire pregnancy and were more likely to report surgical sterility than nondiabetic women. diabetic women reported a higher percentage of contraceptive nonuse compared with nondiabetic women (38.8 vs. 27.3%, respectively ; p < 0.05). characteristics of the study population (%), stratified by diabetes and bmi, in the 2002 nsfg (n = 5,955) rao - scott test, p < 0.05, for diabetes. overweight and obesity were more common among older women than among women aged 2029 years and among women who had less education, lower income, and higher parity than women with bmi < 25 kg / m. obese and overweight women reported higher rates of treatment for infertility than women with bmi < 25 kg / m, although these differences were not statistically significant. overweight and obese women were slightly less likely to have been sexually active in the past 4 weeks and to desire pregnancy compared with women with bmi < 25 kg / m. the association between bmi and current contraceptive method was statistically significant. among sexually active women who were nonsterile (n = 3,822), the unadjusted odds of contraceptive nonuse were 2.61 times higher in diabetic women than in nondiabetic women (95% ci 1.225.58) (table 2). after adjusting for age, race / ethnicity, education, marital status, income level, receipt of medical assistance to become pregnant, desire to become pregnant, and bmi, the odds of contraception nonuse decreased slightly in diabetic women and were no longer statistically significant compared with women without diabetes (adjusted odds ratio [or ] 1.84 [95% ci 0.814.19 ]). unadjusted and adjusted ors and 95% cis for contraceptive nonuse among women at risk for pregnancy models were adjusted for all variables shown in this table. similarly, the unadjusted odds of contraceptive nonuse were higher among women with class ii or iii obesity, although not for overweight women or women with class i obesity. however, the association between class ii or iii obesity and lack of contraception did not persist after adjustment for confounders. covariates such as higher age, non - hispanic black race, history of medical assistance to become pregnant, and the desire to become pregnant were associated with lack of contraception, while higher educational attainment and a cohabitation living environment were associated with contraception use. in this nationally representative, population - based survey of nonpregnant women, we found that 40% of women with diabetes and up to a third of women with elevated bmis did not use contraception. among women who were sexually active and nonsterile, having diabetes was associated with more than a twofold - greater odds of not using contraception, while class ii or iii obesity (bmi 35.0 kg / m) was associated with a 1.6-fold greater odds of not using contraception. however, after adjustment for confounders such as age, racial / ethnic group, education, history of fertility treatment, and desire for pregnancy, neither having diabetes nor being obese was significantly associated with contraceptive nonuse. history of fertility treatment, desire for pregnancy, and ambivalence about pregnancy were associated with the greatest odds of not using contraception. previous studies of unplanned pregnancies suggest low rates of contraceptive use among women with diabetes, ranging from roughly a quarter to half (1114). we found higher (although still suboptimal) rates of contraception use in this sample. the difference between our results and those from previous studies may be due to the fact that previous studies surveyed women who were pregnant at the time of interview, while the nsfg sample that we used only included women who were not pregnant at the time of the interview. in the behavioral risk factor surveillance study, chuang. (15) found similar rates of contraceptive use among women with diabetes to those that we documented. we also found that the majority of overweight and obese women used contraception, similar to their report (15). in our study, we further examined the relationship between severity of obesity and contraception and found that contraception use was significantly lower among women with severe (class ii or iii) obesity compared with women with lesser degrees of overweight and obesity. in other words, lesser degrees of obesity were not a risk factor for lack of contraception before or after adjustment for potential confounders. diabetes and severe obesity were not risk factors for lack of contraception after adjustment for other confounders including history of infertility treatment. diabetes and obesity are associated with conditions inhibiting ovulation, such as polycystic ovary disease, thus inhibiting pregnancy and increasing the likelihood of infertility (21). in our analysis, treatment for subfecundity or infertility was associated with lack of contraception, presumably due to the assumption that contraception would not be needed (22). however, women may incorrectly perceive that they can not become pregnant ; approximately half of these women who were having regular intercourse reported not using birth control because they thought that they could not get pregnant (22). desire for pregnancy and ambivalence about pregnancy were associated with greater risk of contraceptive nonuse. while the obesity and diabetes literature has focused on unintended pregnancy, the family - planning literature has since documented that desire for pregnancy is a better predictor of family - planning practices than intendedness of pregnancy (23). this distinction may explain the lack of contraception in some women who do not intend to become pregnant immediately and may explain why some women do not engage in recommended preconception practices, such as use of family planning until optimal glycemic control is reached. as women with diabetes were less likely to desire pregnancy than nondiabetic women, adjustment for this variable may have strengthened the association between diabetes and lack of contraception, although the association remained nonsignificant. clinicians caring for women with diabetes and/or elevated bmis may target women who desire pregnancy or who feel ambivalent about pregnancy for more intensive preconception management, even if these women do not intend to get pregnant. such management may be more successful if focused on supportive measures and more intensive glucose control, rather than family planning (11). as with other studies (22), we found that older women were at risk for lack of contraception, even after adjustment for other factors. we found that this was true for women aged 3039 years as well as for women aged 40 years. while it is true that fertility declines with age, women in their 30s and early 40s may still conceive spontaneously. it is possible that women believe that declines in fertility negate the need for contraception. we also found that african american women were less likely to use contraception, even after adjustment for other potential confounders such as age, marital status, and education. previous reports have speculated that these differences may be due to race - specific beliefs about the risks and benefits of different types of contraception, particularly hormonal contraception (24). of note, cohabitation was also associated with greater contraception use, while marital status was not. while explanations are speculative, women who cohabit may have greater frequency of intercourse than their single counterparts but also may have less social support for pregnancy than their married counterparts (22). strengths of this report include the population - based nationally representative sample and the available information on pregnancy desire, fertility treatment, previous pregnancy, and demographic variables, all of which are associated with contraceptive use. limitations include little information on reasons for contraceptive nonuse, particularly with respect to reasons specific to chronic disease, diabetes, and obesity. additionally, we are unable to determine from the nsfg data how long women have had diabetes or whether they have type 1 or type 2 diabetes. information about perceived risks of hormonal contraception for glucose control, intention to lose weight, ineligibility for particular methods, and lack of effectiveness of hormonal methods due to weight would have added to these analyses. finally, the nsfg does not inquire about other factors related to health care delivery, such as usual source of care ; it is possible that a lack of a usual source of care may serve as a barrier to the receipt of family - planning services. we conclude that the use of contraception is not optimal among women with diabetes and elevated bmi. however, our findings suggest that the lack of contraception in women in these high - risk groups is related to sociodemographic factors and other factors related to pregnancy and not these conditions per se. efforts to improve family - planning practices could address factors associated with contraceptive use, particularly women 's beliefs about fecundity, specific to age and history of fertility treatment, as well as ambivalence about their desires for pregnancy. if future pregnancies are desired, preconception management may be more successful if daily folic acid use, weight management, and glycemic control are addressed in these clinical discussions instead of focusing solely on initiation of family planning, even if pregnancy is not immediately intended. future research is needed that focuses on interventions targeting these factors, particularly in populations aged 3039 years and women with diabetes, and interactions with health care delivery. | objectiveto examine contraceptive practices among diabetic women and obese women.research design and methodswe analyzed the responses of 5,955 participants aged 2044 years in the 2002 national survey for family growth. diabetes, bmi, desire for pregnancy, history of infertility treatment, sexual activity, parity, and demographic variables (age, race / ethnicity, education, marital status, income, insurance, and smoking history) were obtained by self - report. lack of contraception was defined as absence of hormonal-, barrier-, or sterilization - based methods. associations among contraception, diabetes, and bmi category were assessed in multivariable logistic regression models in nonsterile, sexually active women.resultsin unadjusted comparisons among sexually active women who were not sterilized, women with diabetes were more likely to lack contraception than women without diabetes (odds ratio [or ] 2.61 [95% ci 1.225.58 ]). women with bmi 35 kg / m2 were more likely to lack contraception than women with bmi < 25 kg / m2(1.63 [1.162.28 ]), but associations between contraception use and lesser degrees of overweight and obesity were not significant. in multivariable models, women who were older (aged 30 vs. 2029 years), were of non - hispanic black race, were cohabitating, had a history of infertility treatment, and desired or were ambivalent about pregnancy were significantly more likely to lack contraception. the associations among diabetes, bmi, and contraception were no longer significant after these adjustments.conclusionsolder women with diabetes and obesity who desire pregnancy, regardless of pregnancy intention, should be targeted for preconceptive management. |
manganeseoxidizing bacteria oxidize mn to form insoluble mn and/or mn oxides, and this far best studied bacterial species have been isolated from manganesecontaining environments such as mnoxide encrusted pipelines, manganesecontaining water pipes and aquifers, as well as from marine sediments (schweisfurth, 1973 ; nealson and ford, 1980 ; emerson and ghiorse, 1992). however, the natural niche of manganeseoxidizing bacteria appears not only to require the presence of manganese, but often also features a marked availability of usually recalcitrant organics in nature. thus, an essential role of these bacteria in oxidation of organic material in organicrich environments has been postulated (sunda and kieber, 1994). moreover, mnoxidizing bacteria have been detected in environmental samples contaminated with recalcitrant xenobiotics, such as polycyclic aromatic hydrocarbons or methyl tertbutyl ether (bruns., 2001 ; bodour., 2003 ; raynal and pruden, 2008). to test the potential use of bacterial manganese oxidation to drive the degradation of recalcitrant polycyclic aromatic organic compounds, 17ethinylestradiol (ee2) was chosen as target compound. ee2 is a persistent synthetic estrogen and is often present as a trace pollutant in waste and drinking water bearing the proven risk of undesirable hormonal effects on animals and humans (thomas., 2001). the persistence of ee2 has prompted a number of attempts to achieve degradation of ee2 by bacteria, e.g. by use of rhodococcus zopfii, rhodococcus equi and sphingobacterium sp. all these bacteria require ee2 to be present at milligram levels. in situ, however, only trace concentrations of the pollutants are normally present in waste and drinking waters, which are unlikely to sustain bacterial growth, thus limiting the use of these bacteria for degradation of e.g. trace amounts of ee2. it has recently been demonstrated that commercially available manganese oxides as powder or as specifically produced nanoparticles can mediate removal of ee2 (de rudder., the main consideration for using biogenic mn oxides produced by mnoxidizing bacteria, instead of chemical mn oxides as used before by de rudder and colleagues (2004), was to explore the possibility of working at significantly lower (micromolar) concentrations of mn. it was shown before that biogenic mn oxides have different structure and surface areas compared with chemical oxides (saratovsky., 2006), and this might bring about their increased activity towards ee2. second, we are aiming at construction of a selfregenerating system allowing cyclic oxidation and reduction of mn oxides linked to continuous concomitant degradation of ee2. in this study, we propose an efficient strategy for the removal of trace levels of xenoestrogens from environmental samples by biogenic generation of highly oxidized mn oxides by manganeseoxidizing bacteria, which in turn drive the indirect oxidation and cleavage of ee2. as degradation of substituted phenols, fluoroquinolone antibiotics, aniline and other aromatic amines by manganese oxides have also been reported (stone, 1987 ; laha and luthy, 1990 ; zhang and huang, 2005), the use of highly oxidized biogenic mn oxides can be expanded to the degradation of other organic pollutants. the use of manganeseoxidizing bacteria can be a starting point for the development of a more general strategy for the purification of waste and drinking water from estrogens and other tracelevel organic contaminants. in order to test if manganese oxides derived from oxidation of mn by manganeseoxidizing bacteria may mediate degradation of ee2, four different microbial strains namely leptothrix discophora, pseudomonas putida strains lmg 2321, 2322 and 2323, which are known to catalyse oxidation of mn, were tested for ee2 degradation. these strains were grown on a complete medium with glucose as a carbon source and with a small amount of ee2 (0.5 m) in presence or absence of different concentrations of mn (25, 50, 75, 100 m). all tested strains have shown the ability to oxidize mn, as observed by the decrease of mn content (table 1) and also by accumulation of mn oxides, visible as insoluble brown or orange particulate matter. the amount of ee2 degraded by l. discophora and lmg 2322 strains was proportional with the oxidized mn. without manganese only a slight decrease of ee2 (around 10%) was detected (table 1), which was independent from the culture growth and may be attributed to adsorption of ee2 to biomass. all the remaining levels of ee2 were found to be very stable in the mnfree bacterial cultures even after 4 weeks of incubation (data not shown). elevated concentrations of mn (higher than 100 m) negatively affected oxidation of mn and the degradation of ee2 (data not shown), which again points at mn oxidation to be a prerequisite to the degradation of ee2. ee2 degradation was measured by hplc analysis, and adsorption of ee2 to biogenic manganese oxides were determined as described in experimental procedures section. the initial concentrations of mn used in this study are known from the earlier studies to induce mn oxidation, but were by far represented in excess compared with 0.5 m ee2. in order to establish a lower limit of mn which starts to catalyse degradation of ee2, lmg 2322 was grown with 0.5 m of ee2 and 1, 10 and 50 m mn. degradation of ee2 is dependent on the amount of mn present in the medium and that multifold excess of mn was required (fig. 1). most of ee2 was degraded in the late stationary phase and thus was not linked to the growth of the bacteria (fig. 1). moreover, the degradation of ee2 occurred in time after mn oxidation : for the culture with 50 m of mn most of ee2 was degraded in the period between 24 and 36 h, whereas most of mn was oxidized earlier, in the period between 18 and 24 h (fig. 1). this delay is likely to be linked to the primary accumulation of significant amounts of biogenic mn oxides, which then start to react with ee2. thus, degradation of ee2 is a slow chemical process that is not dependent on the presence of viable bacterial cells, but rather on the amount of biogenic mn oxides produced. influence of microbial activity on mndependent degradation of ee2 was tested by addition of sodium azide, a known inhibitor of electron transportlinked oxidation. pseudomonas putida was grown in presence of 100 m of mncl2 until orange coloured mn oxides were visible and ee2 and sodium azide at a concentration of 1 mm were added. addition of sodium azide still allowed 80% degradation of ee2, whereas without azide complete degradation of ee2 occured. we suspect that the 20% inhibition caused by azide could be linked to inability of the azidetreated cultures to regenerate mn oxides. a. growth of p. putida in the presence of 0.5 m of ee2 and 1, 10 and 50 m of mn. b. dynamics of mn oxidation. c. dynamics of degradation of 0.5 m of ee2 with different initial concentrations of mn. for each measurement, data represent a mean value obtained from three identical cultures. earlier it was suggested that ee2 may be adsorbed in significant amounts to chemically synthesized manganese oxides (de rudder., 2004). in order to rule out the adsorption of ee2 to insoluble mn oxides generated by the mnoxidizing bacteria and thus to distinguish it from oxidation of ee2, cultures were treated with ascorbic acid, which is known to reduce higher oxidation states of mn back to readily soluble mn ions (stone and morgan, 1984), thereby releasing potentially adsorbed ee2. with few exceptions (e.g. lmg 2321 at 25 mm mn, lmg 2323 at 50 m and 100 m mn), where small amounts of apparently adsorbed ee2 were recovered after ascorbic acid treatment, in all other mnsupplied samples no additional ee2 was released after treatment with ascorbic acid (table 1). ascorbic acid by itself did not affect the concentration of ee2, as shown by ee2 concentrations of mnfree control cultures before and after the treatment with ascorbic acid (data not shown). moreover, upon careful examination of hplc spectra an intermediary product of ee2 was detected at low levels and with a retention time of 9.8. because this product disappeared in a manganesedependent fashion and had no measurable estrogenic effect, its structure was not further examined. therefore, decreased levels of ee2 in the respective mncontaining cultures are accomplished by true degradation, rather than by adsorption to the insoluble mn oxides that are formed. as oxidation of ee2 by mn oxides may result in production of metabolites with undesirable estrogenic activity, we performed yeast estrogen screen (yes) bioassays. the yes assay is based on the use of a recombinant yeast strain that harbours a human estrogen receptor gene and is used to measure the estrogenic activity of environmental samples (pierrat., 1992 ; estrogenic response of the culture of the strain lmg 2322 grown with 0.5 m ee2 and 100 m of mn was comparable with the one of negative control, indicating that no residual estrogenic activity in this mncontaining sample was present where ee2 was found to be eliminated (fig. 2). by contrast, in the corresponding culture grown in the absence of mn where ee2 was still present, strong estrogenic response was found as expected (fig. 2). estrogenic responses of cultures of p. putida 2322 grown with 0.5 m ee2 and with / or without 100 m of mncl2 (black or white squares, correspondingly) (b). estrogenic responses of distilled water (black squares) and a solution of 0.5 m ee2 (white squares) were used as corresponding controls (a). it was shown that degradation of ee2 may be linked to the extracellular enzymatic activity in other organisms, such as to the manganese peroxidase and multicopper oxidase activities of white rot fungi (suzuki., 2003). this is unlikely to be the case for the manganeseoxidizing bacteria tested here for the following reasons : (i) the rate of ee2 degradation is strongly correlated with the amount of mn oxidized and such correlation is normally unlikely to be linked to the enzymatic activity ; (ii) oxidation of ee2 does not coincide in time with the oxidation of mn, when the mnoxidizing enzyme is produced, but it is delayed and rather linked with the period when most of mn has been oxidized and the bulk of mn oxides have been accumulated ; (iii) enzymatic treatment of the mn and ee2containing culture with pronase at two different concentrations of the enzyme (100 m, 1 mm) with the aim to abolish the extracellular enzymatic activity has not resulted in any noticeable inhibition of the ee2 degradation (data not shown). to conclude, manganeseoxidizing bacteria are of particular biotechnological interest, not only with respect to their use in the wellestablished biological treatment of manganesecontaining waters, but also for bioremediation of environments contaminated with trace concentrations of recalcitrant organic pollutants. we presented the indirect oxidation of ee2 by biogenic manganese oxides as a concept of a novel biological process aiming to eliminate trace concentrations of organic pollutants from environmental samples. the initial attack of highly oxidized manganese oxides on ee2 is likely to address chemical features of the phenolic groups also found in other polycyclic aromatic compounds, which could thus likewise be degraded by highly oxidized manganese. the breakdown of other compounds like amines, aromatic amines and xenobiotics with sulphydryl groups were also reported to be chemically degraded by mn oxides, and are thus also likely to be degraded in the presence of manganeseoxidizing bacteria and mn. our future studies will be focused on designing a bioprocess aiming at the degradation of ee2 by mnoxidizing bacteria. a serious step to consider is that the lowest concentration which still allows efficient degradation of ee2 found in this study was 50 m, which is still five times higher than the allowed concentrations of manganese in drinking water. however, designing a bioreactor with alternating conditions would result in both complete degradation of ee2 and complete oxidation of mn. the oxidized insoluble manganese oxides would then precipitate and could be this way removed from the water. generally, these results draw attention to the potential value of microbial extracellular oxidative processes (like bacterial manganese oxidation) for biotechnological processes aiming at degradation of trace pollutants. leptothrix discophora (lmg 8142, belgian coordinated collections of microorganisms) and p. putida strains mnb1 (lmg 2321), mnb6 (lmg 2322) and mnb29 (lmg 2323) were cultivated on a medium described by boogerd and de vrind (1987). to assess degradation of ee2 and to determine manganese oxidation by the manganeseoxidizing bacteria, two types of overnight precultures were set without and with filtersterilized manganese chloride at a concentration of 25 m. on the following day, 50 ml of the medium with ee2 (0.5 m) and mncl2 at different concentrations (0, 25, 50, 75, 100 m) were inoculated, bacterial growth (by measuring od600) was monitored and concentrations of ee2 and mn were measured. mn oxidation was monitored in 2 ml aliquots from the cultures, which were centrifuged at 13 400 g for 30 min. after mn concentrations were determined by using the formaldoxime assay described by bartley and colleagues (1957). briefly, 3 ml of the supernatant was mixed with 1 ml of formaldoxime hydrochloride solution (sigmaaldrich, 0.5 g dissolved in 10 ml of distilled water). culture aliquots were filtered through a 0.22 m filter prior to hplc analysis. in order to test for potential adsorption of ee2 to particulate mn oxides generated by bacterial oxidation of mn, ascorbic acid (0.11 m) was added to 5 ml culture aliquots, and samples were shaken for 15 min at 30c. the samples were analysed for ee2 before and after the treatment with ascorbic acid, which, being a strong reductant, was expected to release adsorbed ee2 by regenerating soluble mn from insoluble mn oxides. ee2 and its potential metabolites were analysed by reversed phase hplc with an asi100 autosampler, p580 pump and sth585 column oven (dionex, sunnyvale, ca, usa) on an agilent zorbax sbc18 column (150 mm 4.6 mm, 5 m). elution was performed at 25c and a flow rate of 1 ml / min with solvent a (100% acetonitrile) and solvent b (milliq + 0.1% formic acid). the injection volume was 50 l and the elution program was as follows : 2 min isocratic at 85% b, linear gradient to 60% b in 5 min, linear to 35% b in 15 min, linear to 0% b (or 100% a) in 3 min follow by 5 min isocratic 100% a, linear gradient to 85% b and isocratic at 85% for 5 min. detection of ee2, e2 and e3 was performed at an excitationemission wavelength of 280310 nm (yoon., 2003) by rf2000 fluorescence detector (dionex) and standards ranging from 2 to 1000 g l for ee2 for calibration were prepared, starting from individual ethanol stock solution containing 1 g l of ee2. the yes assay is based on the use of a recombinant yeast strain that harbours a human estrogen receptor gene (pierrat., 1992) and is used to measure the estrogenic activity of environmental samples (baker, 2001). the bacteria were grown either with manganese and ee2 or with manganese alone (control conditions) for 72 h (until latestationary stage of growth) as described above. one millilitre of each culture was then filtered through a 0.22 mfilter and filtrates were used for the yes assay. the yes assay was performed as described (de boever., 2001) in 96well plates in which 10 l of filtrates diluted twice with dimethyl sulfoxide were incubated with 240 l of the genetically modified saccharomyces cerevisiae at an optical density of 0.25 at 610 nm. additional plates with serial dilutions of either the medium alone (negative control), or of medium supplemented with ee2 in dmso (positive control) were made and equally tested. estrogenic activity responses were recorded as the absorbance at 540 nm divided by the optical density at 630 nm [(a540/a630)net ] (sunrise, plate reader, tecan, grdig, austria). based on the data obtained, the data were fitted by a fourparametric logistic model using the marquardtlevenberg algorithm (sigmaplot 4.0 ; spss inc., chicago, il, usa ; de boever., 2001). leptothrix discophora (lmg 8142, belgian coordinated collections of microorganisms) and p. putida strains mnb1 (lmg 2321), mnb6 (lmg 2322) and mnb29 (lmg 2323) were cultivated on a medium described by boogerd and de vrind (1987). to assess degradation of ee2 and to determine manganese oxidation by the manganeseoxidizing bacteria, two types of overnight precultures were set without and with filtersterilized manganese chloride at a concentration of 25 m. on the following day, 50 ml of the medium with ee2 (0.5 m) and mncl2 at different concentrations (0, 25, 50, 75, 100 m) were inoculated, bacterial growth (by measuring od600) was monitored and concentrations of ee2 and mn were measured. mn oxidation was monitored in 2 ml aliquots from the cultures, which were centrifuged at 13 400 g for 30 min. after mn concentrations were determined by using the formaldoxime assay described by bartley and colleagues (1957). briefly, 3 ml of the supernatant was mixed with 1 ml of formaldoxime hydrochloride solution (sigmaaldrich, 0.5 g dissolved in 10 ml of distilled water). culture aliquots were filtered through a 0.22 m filter prior to hplc analysis. in order to test for potential adsorption of ee2 to particulate mn oxides generated by bacterial oxidation of mn, ascorbic acid (0.11 m) was added to 5 ml culture aliquots, and samples were shaken for 15 min at 30c. the samples were analysed for ee2 before and after the treatment with ascorbic acid, which, being a strong reductant, was expected to release adsorbed ee2 by regenerating soluble mn from insoluble mn oxides. ee2 and its potential metabolites were analysed by reversed phase hplc with an asi100 autosampler, p580 pump and sth585 column oven (dionex, sunnyvale, ca, usa) on an agilent zorbax sbc18 column (150 mm 4.6 mm, 5 m). elution was performed at 25c and a flow rate of 1 ml / min with solvent a (100% acetonitrile) and solvent b (milliq + 0.1% formic acid). the injection volume was 50 l and the elution program was as follows : 2 min isocratic at 85% b, linear gradient to 60% b in 5 min, linear to 35% b in 15 min, linear to 0% b (or 100% a) in 3 min follow by 5 min isocratic 100% a, linear gradient to 85% b and isocratic at 85% for 5 min. detection of ee2, e2 and e3 was performed at an excitationemission wavelength of 280310 nm (yoon., 2003) by rf2000 fluorescence detector (dionex) and standards ranging from 2 to 1000 g l for ee2 for calibration were prepared, starting from individual ethanol stock solution containing 1 g l of ee2. the yes assay is based on the use of a recombinant yeast strain that harbours a human estrogen receptor gene (pierrat., 1992) and is used to measure the estrogenic activity of environmental samples (baker, 2001). the bacteria were grown either with manganese and ee2 or with manganese alone (control conditions) for 72 h (until latestationary stage of growth) as described above. one millilitre of each culture was then filtered through a 0.22 mfilter and filtrates were used for the yes assay. the yes assay was performed as described (de boever., 2001) in 96well plates in which 10 l of filtrates diluted twice with dimethyl sulfoxide were incubated with 240 l of the genetically modified saccharomyces cerevisiae at an optical density of 0.25 at 610 nm. additional plates with serial dilutions of either the medium alone (negative control), or of medium supplemented with ee2 in dmso (positive control) were made and equally tested. estrogenic activity responses were recorded as the absorbance at 540 nm divided by the optical density at 630 nm [(a540/a630)net ] (sunrise, plate reader, tecan, grdig, austria). based on the data obtained, dose response curves for doses versus the data were fitted by a fourparametric logistic model using the marquardtlevenberg algorithm (sigmaplot 4.0 ; spss inc., chicago, il, usa ; de boever., 2001). | summarymanganese (ii) and manganeseoxidizing bacteria were used as an efficient biological system for the degradation of the xenoestrogen 17ethinylestradiol (ee2) at trace concentrations. mn2+derived higher oxidation states of mn (mn3 +, mn4 +) by mn2+oxidizing bacteria mediate the oxidative cleavage of the polycyclic target compound ee2. the presence of manganese (ii) was found to be essential for the degradation of ee2 by leptothrix discophora, pseudomonas putida mb1, p. putida mb6 and p. putida mb29. mn2+dependent degradation of ee2 was found to be a slow process, which requires multifold excess of mn2 + and occurs in the late stationary phase of growth, implying a chemical process taking place. ee2derived degradation products were shown to no longer exhibit undesirable estrogenic activity. |
ankle arthrodesis has long been the traditional operative treatment for posttraumatic ankle arthritis, rheumatoid arthritis (ra), infection, neuromuscular conditions, and salvage of failed total ankle arthroplasty.123 ankle fusion remains the treatment of choice for patients in whom heavy, and prolonged activity is anticipated and ankle replacement can be only recommended for those with more modest requirements.4 ankle fusion provides a painless, plantigrade, and stable foot. several different methods have been described to achieve the ankle fusion ; including external fixators and/or internal fixation, use of fibular strut graft, interposition bone grafting, and intramedullary nailing.5678910 however, it is a salvage procedure that causes persistent alterations in gait and a potential for deterioration exists due to the risk of developing ipsilateral hindfoot arthritis.1112 in our study, the raw surface of the medial side of fibula was compressed using the 4.5 mm cortical screws with lag effect, as a strut graft. we present short term functional outcome of patients who underwent transfibular ankle arthrodesis for ankle arthritis. 29 transfibular ankle arthrodesis in 29 patients performed between april 2009 and april 2014 were included in this study. of the 29 patients, 16 were male (55%) and 13 were female (44%) (male to female ratio = 1.2:1). the mean age at surgery was 50 years (range 22 - 75 years). the mean followup period was 32.52 months (standard deviation 10.34). among the 29 patients who underwent transfibular arthrodesis, 22 (76%) patients had post traumatic arthritis (pta), 5 (17%) patients had tubercular arthritis (ta), and 2 (7%) had rheumatoid arthritis (ra). in our study, 18 procedures were done on right ankles (62.07%) and 11 were on left ankles (37.93%). inclusion criteria were a diagnosis of arthritis of the ankle of grade 3 and above (posttraumatic arthritis [pta ], secondary to tuberculosis, and secondary to ra) with a minimum of 1-year postoperative followup. patients with septic arthritis, failed ankle arthroplasty, and charcot 's arthritis were excluded from this study. data obtained from the case sheets included age, gender, indication for surgery, the side affected, operating surgeon, perioperative antibiotic use, duration of surgery, number of screws used for fixation, source of bone graft, length of stay in hospital, surgical complications, duration of postoperative outpatient followup, comments on postoperative radiographs in the clinic in conjunction with the radiologist reports, and patient satisfaction. under regional anesthesia and tourniquet control with bump under the ipsilateral hip, the fibula was osteotomized approximately 2 cm above the syndesmosis level and reflected down and released of all attachment [figure 1a ]. a good exposure of the ankle joint for adequate preparation of the tibial and talar articular surfaces was available. the articular cartilage of the tibial plafond and the talar dome were removed parallel to one another using an osteotome to provide good contact surface [figure 1b ]. (a) peroperative photograph showing osteotomy of the fibula 2 cms above the ankle joint. (b) peroperative photograph showing complete removal of the distal fibula after the osteotomy gives good exposure of the joint. (c) peroperative photograph showing excision of the medial malleolus through a medial incision gives additional exposure when needed. (d) immediate postoperative radiograph anteroposterior and lateral views showing multiple screws used in arthrodesis in cases where the exposure was not adequate, an additional exposure by osteotomizing the medial malleolus was used and this was done in eight cases [figure 1c ]. two 6.5 mm 16 mm cancellous screws were used to compress the two arthrodesis surface with the ankle in neutral. two 4.5 mm cortical screws were used to place the onlay distal fibula graft in compression : one as distal tibiofibular and the other as talofibular [figure 1d ]. the distal fibular cortex was over drilled with 4.5 mm drill to achieve good compression. following wound closure, compression dressing and above knee slab the initial 6 weeks, patient was mobilized with nonweight bearing. at the end of 6 weeks, we allowed weight bearing in the walking cast and plaster boot. after 3 months, once union was ascertained by radiographs, the walking cast was removed, and patients were free to mobilize to improve the subtalar and midfoot movements with crepe bandage in the day time. the initial postoperative anteroposterior and lateral radiograph was taken for each patient on the immediate postoperative day, and subsequent radiographs were taken at each followup visit in the outpatient clinic and were assessed by a radiologist and the surgeon. followup radiographs were examined for bony union and implant position and incorporation of bone graft. bony union was confirmed by the presence of trabecular lines between the tibia and talus at the point of contact, and disappearance of the radiolucent line that was present in the initial postoperative radiograph. all the patients were assessed with the american orthopaedic foot and ankle society (aofas) ankle - hindfoot scale.13 this was done at final followup on outpatient basis. the main emphasis of this system was on pain, the functional activities and hind foot deformity. a normal person would score 100 points. because of lack of ankle motion, the maximum score that the patient with an ankle fusion could have was 92 since they could not earn the 8 points given for the full range of motion. a score of 8092 was considered as excellent result, 7079 a good result, 6069 a fair result, and score less than 60 was considered as a poor result.11 under regional anesthesia and tourniquet control with bump under the ipsilateral hip, the ankle was prepared and draped. the fibula was osteotomized approximately 2 cm above the syndesmosis level and reflected down and released of all attachment [figure 1a ]. a good exposure of the ankle joint for adequate preparation of the tibial and talar articular surfaces was available. the articular cartilage of the tibial plafond and the talar dome were removed parallel to one another using an osteotome to provide good contact surface [figure 1b ]. (a) peroperative photograph showing osteotomy of the fibula 2 cms above the ankle joint. (b) peroperative photograph showing complete removal of the distal fibula after the osteotomy gives good exposure of the joint. (c) peroperative photograph showing excision of the medial malleolus through a medial incision gives additional exposure when needed. (d) immediate postoperative radiograph anteroposterior and lateral views showing multiple screws used in arthrodesis in cases where the exposure was not adequate, an additional exposure by osteotomizing the medial malleolus was used and this was done in eight cases [figure 1c ]. two 6.5 mm 16 mm cancellous screws were used to compress the two arthrodesis surface with the ankle in neutral. two 4.5 mm cortical screws were used to place the onlay distal fibula graft in compression : one as distal tibiofibular and the other as talofibular [figure 1d ]. the distal fibular cortex was over drilled with 4.5 mm drill to achieve good compression. the initial 6 weeks, patient was mobilized with nonweight bearing. at the end of 6 weeks, we allowed weight bearing in the walking cast and plaster boot. after 3 months, once union was ascertained by radiographs, the walking cast was removed, and patients were free to mobilize to improve the subtalar and midfoot movements with crepe bandage in the day time. the initial postoperative anteroposterior and lateral radiograph was taken for each patient on the immediate postoperative day, and subsequent radiographs were taken at each followup visit in the outpatient clinic and were assessed by a radiologist and the surgeon. followup radiographs were examined for bony union and implant position and incorporation of bone graft. bony union was confirmed by the presence of trabecular lines between the tibia and talus at the point of contact, and disappearance of the radiolucent line that was present in the initial postoperative radiograph. all the patients were assessed with the american orthopaedic foot and ankle society (aofas) ankle - hindfoot scale.13 this was done at final followup on outpatient basis. the main emphasis of this system was on pain, the functional activities and hind foot deformity. a normal person would score 100 points. because of lack of ankle motion, the maximum score that the patient with an ankle fusion could have was 92 since they could not earn the 8 points given for the full range of motion. a score of 8092 was considered as excellent result, 7079 a good result, 6069 a fair result, and score less than 60 was considered as a poor result.11 all cases of ankle fusions (100%) progressed to bony union in a mean postoperative duration of 3.8 months (range 36 months). all the patients were assessed clinically and radiologically after a mean followup of 32 months (range 1452 months). two patients responded satisfactorily to oral antibiotics, and two patients were treated by intravenous antibiotics (2 pta, 1 ta, and 1 ra). two patients had skin maceration and break down that was treated by debridement and secondary suturing (1 ta and 1 ra). the incidence of postoperative complications was limited in pta (n = 2/22) when compared to ta (n = 2/5) (p < 0.01) and ra (n = 2/2) (p < 0.001). in our study, the mean aofas score was 74 (pain score = 32, functional score = 42). we found that twenty patients (68.96%) of the 29 had excellent results, 7 (24.13%) had good, and 2 (6.89%) showed fair results. of the 22 cases of pta patients, 18 patients (81.81%) had excellent results and 4 patients (18.18%) had good results [figures 2 and 3 ]., two patients had excellent results, two patients had good results, and one patient had fair results [figure 4 ]. of the two cases of ra patients, one patient had a good result and one patient had fair result. thus, there were better results among patients whose indication for surgery was pta (n = 22) when compared to ta (n = 5) (p < 0.05) and to ra (n = 2) (p < 0.001) [table 1 ]. x - ray of right ankle joint anteroposterior and lateral views of a 45 years old female showing (a) post traumatic malunited bimalleolar fracture (b) immediate postoperative x - rays with trans fibular ankle arthrodesis (c) six months followup x - rays x - ray of right ankle joint anteroposterior and lateral views in a 28 years old male showing (a) failed open reduction internal fixation and malunited bimalleolar fracture. (c) six months followup x - rays showing arthrodesis x - ray ankle joint anteroposterior and lateral views in a 61 years old male showing (a) tubercular arthritis. (c) six months followup x - ray showing arthrodesis functional outcome of cases as per the aofas scores ankle arthrodesis was first described by albert.10 since then, many surgical approaches have been described in the literature for ankle fusion including the anterior, posterior, and transmalleolar approaches. the transfibular approach to ankle fusion was first described by horwitz.14 adams described transfibular ankle arthrodesis by lateral approach where the distal fibular fragment was removed and prepared for use as an onlay graft.15 the graft was secured by three screws : two of which grip the tibia and one the talus. in this approach, clear exposure of the articular surfaces was readily obtained. the fibular graft gives sound stability and provides scaffolding for formation of a stout bone bridge across the joint. close contact between the tibia and talus was essential, and the packing of cancellous bone chips around the bones was an advantage. it is also generally understood that the chances of union are higher when the fixation is rigid. however, in our study, 4.5 cortical screws were used to avoid graft breakage which is common when using 6.5 mm cancellous screws.1617 the factors that are important in achieving maximal healing and ankle fusion include rigid internal fixation, compression of the fusion surfaces, and a good biological environment.141819 various strategies have been used to reduce the incidence of nonunion including multiple compression screws and larger diameter screws to improve mechanical stability and compression. use of multiple cannulated cancellous screws (about 24) provides maximum contact and compression across the viable cancellous bone surfaces. used transfibular compression arthrodesis of ankle by lateral approach with resection of distal fibula.20 they attained compression using a t plate placed across the tibiotalar joint with the aid of an ao compression apparatus. flckiger and weber used multiple lag screws (2 or 3) by trans fibular approach for tibiotalar arthrodesis compression and used the dissected distal fibula as onlay graft to tibia and talus with cortical screws.21 they achieved solid fusion in all cases with an average aofas score being 78.5. used transfibular technique with sagittal splitting of fibula with internal compression with multiple cancellous screws and achieved 100% solid union arthrodesis and subjective outcome with very satisfied results in 54% and satisfied results in 46%.22 in our study, we used trans fibular approach with osteomized distal fibula being used as an onlay graft over tibia and talus with two cortical screws. two criss - cross 6.5 mm 16 fillet cancellous screws used for tibiotalar arthrodesis for internal compression. our results (100% union) were comparable and in some parameters better than results from other series.1114172122 there were no healing - related problems in our series. this may be attributable to the use of fibula as strut graft and the clearing of cartilage from the distal articular surface of fibula and syndesmosis tissues on the medial side of fibula with decortication. they reported union rates similar to our study of 95% (p < 0.05). they concluded that use of a metal burr and curettage reduced the surgery time and did not significantly alter the bone surfaces.23 the limitation of study is short term followup (mean 32 months). finally, the sample size was relatively small but still larger than some previous studies. a long term assessment and larger series of ankle arthrodesis is thus warranted to gain statistically significant results. we excluded charcot 's neuroarthropathy of the ankle as it independently can influence the overall outcome of the procedure and it is best treated by retrograde intramedullary nailing.24 in more complex trauma around the ankle joint, a humeral locking plate fixation technique described by zhang. may be indicated.25 we do not have experience with this procedure, and it was not indicated in any of our patients as the fibula was used as a lateral stabilizing biological plate. in our series, of the two patients with ra, one patient had a fair result and patients with ra and involvement of ankle may not meet the criteria for an ankle arthrodesis ; this may be because they have involvement not only of the ankle but also of the small joints of the foot so that these joints can not compensate for the fused ankle.1112 however, these patients have a low functional demand. therefore, these patients with ra may be better candidates for the total ankle arthroplasty.26 to be considered as a preferable alternative to arthrodesis, a total ankle replacement (tar) should give better results than those presented here, without other disadvantages. spirt. in their study of total ankle had a 5-year survival rate with reoperation as the end point of 54% only. the 5-year survival rate with failure as the end point was 80% for all patients and 89% for patients who were more than 54 years of age.2627 younger age was found to have a negative effect on the rates of reoperation and failure the functional outcome of this procedure is uncertain. tibiotalar arthrodesis following failed tar is an extremely difficult procedure.4 we did not encounter such patients in our series. described the problems encountered when trying to restore function using tibiotalar arthrodesis following failed tar.3 they found that it could not be achieved in every case despite minimal amount of bone resected at the time of ankle replacement. they found that severe bone loss develops on the talar side as the component loosens, and this may extend to the subtalar joint. they suggest that if there is sufficient talar bone stock, a tibiotalar arthrodesis be done.328 this is often the case in patients with oa, but rarely for those with ra. if there is a significant loss of bone from the talus or a markedly arthritic subtalar joint, they recommend a tibiotalocalcaneal arthrodesis with an intramedullary nail.4 an intramedullary nail gives firm fixation both distally and proximally and is the method of choice when tibiotalocalcaneal arthrodesis is indicated. they also found that most serious outcomes occurred, where the lateral malleolus was totally excised in the belief that it would assist in obtaining good, bony apposition and serve as a bone graft. nonunion occurred in such patients and the absence of a lateral malleolus resulted in the ankle becoming grossly unstable. | background : ankle arthrodesis has long been the traditional operative treatment for posttraumatic arthritis, rheumatoid arthritis, infection, neuromuscular conditions, and salvage of failed ankle arthroplasty. it remains the treatment of choice for patients in whom heavy and prolonged activity is anticipated. we present our short term followup study of functional outcome of patients who underwent transfibular ankle arthrodesis for arthritis of ankle due to various indications.materials and methods:29 transfibular ankle arthrodesis in 29 patients performed between april 2009 and april 2014 were included in this study. the mean age was 50 years (range 22 - 75 years). the outcome analysis with a minimum of 1-year postoperative followup were included. all the patients were assessed with the american orthopaedic foot and ankle society (aofas) hindfoot scale.results:all cases of ankle fusions (100%) progressed to solid union in a mean postoperative duration of 3.8 months (range 36 months). all patients had sound arthrodesis. the mean followup period was 32.52 months (standard deviation 10.34). the mean aofas score was 74 (pain score = 32, functional score = 42). we found that twenty patients (68.96%) out of 29, had excellent results, 7 (24.13%) had good, and 2 (6.89%) showed fair results.conclusion:transfibular ankle arthrodesis is a simple and effective procedure for ankle arthritis. it achieves a high rate of union and good functional outcome on midterm followup. |
melanoacanthoma is a benign and uncommon pigmented mucocutaneous lesion characterized by dendritic melanocytes dispersed throughout the epithelium. published the first retrospective review of 10 cases of oral melanoacanthoma reported in the literature in 1983. oral melanoacanthoma usually occurs on the buccal mucosa (51.4%), with fewer lesions originating on the palate (22.2%), lips (15.2%) and gingiva (5.6%). oral melanoacanthoma is seen almost exclusively in blacks, shows a female predilection and is most common during the third and fourth decade of life. the lesion is smooth, flat or slightly raised, dark brown to black in color. lesions often demonstrate a rapid increase in size and they occasionally reach a diameter of several centimeters within a period of few week, potentially masquerading as a melanoma. to the best of our ability, a comprehensive review of literature on gingival melanoacanthoma revealed 12 cases mostly involving singular lesions [table 1 ]. herein, we present an unusual case report of gingival melanoacanthoma, which presented clinically as pigmented unilateral gingival enlargement. reported cases of gingival melanoacanthomas a 13-year - old male patient attended the department of periodontics, mahatma gandhi post graduate institute of dental sciences, pondicherry, in may 2011 for gingival enlargement and difficulty in mastication. he first noticed enlargement 6 months earlier along the buccal aspect of tooth 26, which increased in size slowly to the present clinical picture. the clinical examination was significant for the presence of pigmented diffuse gingival enlargement along buccal and palatal aspects of 24, 25, 26, 27 and lingual aspects of teeth 34, 35, 36, 37 [figure 1 ]. gingival enlargement was extending up to middle third of the crown of tooth 24, partially covering the occlusal surface of teeth 25, 26 and completely covering the tooth 27 [figure 2 ] and extending up to occlusal surface of teeth 34, 35, 36, 37. the gingival enlargement was firm in consistency, painless, brownish black in color with well - defined borders and there was no associated erythematous background. melanoacanthoma on the maxillary posterior gingiva melanoacanthoma on the occlusal surface of 26, 27 other significant dental findings included root canal treated fractured tooth 21 and congenitally missing tooth 23. based on the clinical findings, provisional diagnosis of idiopathic gingival enlargement was made and gingivectomy was planned for the treatment. patients parents provided oral consent for treatment prior to the initial and additional tests and the dental treatment that followed. gingivectomy was performed under local anesthesia on the buccal aspect of ii quadrant [figure 3 ] and the excised tissue was sent for biopsy [figure 4 ]. one week later, gingivectomy was performed on the palatal aspect of ii quadrant followed by lingual aspect of iii quadrant after 1 week. patient experienced an uneventful post - operative recovery [figure 5 ] at 1 year recall examination there was no recurrence of gingival enlargement at the surgical sites [figure 6 ]. patient will continue to be monitored on a 6 month basis and any new gingival enlargement / lesion will undergo histopathologic investigation. gingivectomy carried out in ii quadrant buccal aspect excised tissue after gingivectomy surgical area 2 weeks post - operatively one year follow - up with no recurrence histopathological examination revealed numerous dendritic melanocytes distributed in basal and suprabasal layers of acanthotic epithelium. there was no spillage of melanin pigment / melanocytes in subepithelial zone [figure 8 ]. microscopical features of melanoacathoma showing dendritic melanocytes distributed throughout acanthotic epithelium (h and e ; original magnification 100) melanoacanthoma showing dendritic melanocytes distributed in basal and suprabasal layers of epithelium with normal connective tissue (original magnification 45) a 13-year - old male patient attended the department of periodontics, mahatma gandhi post graduate institute of dental sciences, pondicherry, in may 2011 for gingival enlargement and difficulty in mastication. he first noticed enlargement 6 months earlier along the buccal aspect of tooth 26, which increased in size slowly to the present clinical picture. the clinical examination was significant for the presence of pigmented diffuse gingival enlargement along buccal and palatal aspects of 24, 25, 26, 27 and lingual aspects of teeth 34, 35, 36, 37 [figure 1 ]. gingival enlargement was extending up to middle third of the crown of tooth 24, partially covering the occlusal surface of teeth 25, 26 and completely covering the tooth 27 [figure 2 ] and extending up to occlusal surface of teeth 34, 35, 36, 37. the gingival enlargement was firm in consistency, painless, brownish black in color with well - defined borders and there was no associated erythematous background. melanoacanthoma on the maxillary posterior gingiva melanoacanthoma on the occlusal surface of 26, 27 other significant dental findings included root canal treated fractured tooth 21 and congenitally missing tooth 23. based on the clinical findings, provisional diagnosis of idiopathic gingival enlargement was made and gingivectomy was planned for the treatment. patients parents provided oral consent for treatment prior to the initial and additional tests and the dental treatment that followed. gingivectomy was performed under local anesthesia on the buccal aspect of ii quadrant [figure 3 ] and the excised tissue was sent for biopsy [figure 4 ]. one week later, gingivectomy was performed on the palatal aspect of ii quadrant followed by lingual aspect of iii quadrant after 1 week. patient experienced an uneventful post - operative recovery [figure 5 ] at 1 year recall examination there was no recurrence of gingival enlargement at the surgical sites [figure 6 ]. patient will continue to be monitored on a 6 month basis and any new gingival enlargement / lesion will undergo histopathologic investigation. gingivectomy carried out in ii quadrant buccal aspect excised tissue after gingivectomy surgical area 2 weeks post - operatively one year follow - up with no recurrence histopathological examination revealed numerous dendritic melanocytes distributed in basal and suprabasal layers of acanthotic epithelium. there was no spillage of melanin pigment / melanocytes in subepithelial zone [figure 8 ]. microscopical features of melanoacathoma showing dendritic melanocytes distributed throughout acanthotic epithelium (h and e ; original magnification 100) melanoacanthoma showing dendritic melanocytes distributed in basal and suprabasal layers of epithelium with normal connective tissue (original magnification 45) oral melanoacanthoma is a benign reactive process and shows a rapid increase in size that reaches several centimeters within a few weeks. the reported age of presentation ranges from 6 to 77 years with a mean age of 29 years. though the lesion is most predominantly observed among black patients, occurrences have also been observed among caucasians, hispanics and asians. the lesion usually occur on the buccal mucosa, but involvement of other sites such as the mucosa of lip, palate, gingiva and alveolar mucosa has also been reported. the lesions are usually solitary and well - circumscribed, though a few authors have reported bilateral or multiple melanoacanthoma. with the addition of featured case 13 patients with gingival melanoacanthoma have been reported [table 1 ]. to the best of our knowledge, this is the first comprehensive case of gingival melanoacanthoma presented clinically as pigmented diffuse gingival enlargement. at 1 year follow - up examination of the pathogenesis of oral melanoacanthomas is not yet clear, although these lesions are considered a reactive phenomenon. there may be an idiosyncratic predisposition toward the formation of oral melanoacanthoma in the patient. histopathological examination did not reveal nuclear pleomorphism, hyperchromatism and nests of melanocytes, suggestive of malignancy. there was normal connective tissue with no spillage of melanin pigment / melanocytes in subepithelial zone. in the light of the history, clinical features and the histopathologic examination, the final diagnosis of oral melanoacanthoma the clinical differential diagnosis of localized brown to black gingival pigmentations include smoker 's melanosis, drug induced pigmentation, physiologic pigmentation, addison 's disease, melanotic macule, pigmented nevi, spitz nevus, post - inflammatory melanosis, hemochromatosis, oral melanoma, mccune - albright syndrome and peutz - jegher 's syndrome. in early stages, therefore, sudden onset of oral pigmentation of unknown etiology needs histopathologic assessment for lesional identity and to rule out the presence of oral melanoma. the lesion may undergo spontaneous regression after incisional biopsy. the present case required surgical excision only as a treatment option since the clinical presentation was diffuse gingival enlargement interfering with normal mastication. to the best of our knowledge, this is the first case of gingival melanoacanthoma in the indian sub - continent. pigmented gingival lesions of unusual clinical presentation should undergo histopathologic examination for timely identification and to rule out malignancy. our case report emphasizes the need for clinicians to include gingival melanoacanthoma in the differential diagnosis of multifocal diffuse pigmented gingival enlargement and surgical excision is only treatment modality for such unusual clinical presentation. | gingival melanoacanthoma is a rare, benign pigmented lesion characterized clinically by sudden onset and rapid growth of a macular brown black lesion and histologically by acanthosis of superficial epithelium and proliferation of dendritic melanocytes. this article reports a previously undescribed case of pigmented unilateral diffuse gingival enlargement, which on histopathological examination proved to be melanoacanthoma. intraoral examination revealed pigmented unilateral diffuse gingival enlargement in relation to second and third quadrants buccally, palatally / lingually. based on these clinical findings, gingivectomy was performed and the excised tissue was sent for biopsy. microscopic examination revealed acanthotic and parakeratotic surface epithelium with dendritic melanocytes distributed in basal and suprabasal layers of the epithelium. 1 year follow - up recall revealed no recurrence of lesion at the surgical sites. our patient exhibits an unusual clinical presentation of melanoacanthoma of gingiva. pigmented gingival overgrowth of recent origin and without any etiologic factors warrants histopathologic examination. |
crystal engineering the rational design of crystalline molecular solids remains an important challenge for chemistry. crystal structure prediction is not yet feasible in all cases, and it is therefore useful to develop motifs which allow families of structures to be generated in a reliable fashion. there is special interest in motifs which lead to microporous (or nanoporous) crystals with voids on the scale of 0.52 nm, sufficient to accommodate molecular guests. such materials offer various functionalities, such as inclusion and storage of gases, and other guest molecules, the enhancement of optical properties of included guests, the use of pores as reaction vessels to promote the formation of desired products, and the separation of mixtures including enantiomers from racemates. at the same time crystallizing species will usually attempt to maximize contact with each other, thus minimizing any void space. to counter this trend is not straightforward and will often require the construction of specially shaped rigid components or units capable of strong and directional interspecies interactions. successful approaches to nanoporous crystal engineering may be divided into two categories. on the one hand (pcps) or metal organic frameworks (mofs), formed by combining metal ions with rigid multivalent ligands. on the other are purely organic systems, which rely on noncovalent bonding to regulate crystal packing. the organic systems may be further divided into intrinsically and extrinsically porous molecular crystals. intrinsically porous crystals are based on molecules with predefined open spaces (macrocycles, cages etc.), whereas extrinsic porosity results simply from crystal packing. of these three approaches (hybrid, intrinsic / extrinsic organic), the latter is probably the most challenging as open frameworks must be maintained without the help of powerful directional coordination bonds or pre - existing cavities. some solutions have emerged through serendipity, such as the classic urea inclusion compounds. there are few motifs which generate families of readily accessible nanoporous crystals, allowing tuning of void dimensions and material properties. the work described in this paper is founded on a serendipitous discovery made a few years ago in the course of our program on anion - binding cholapods. these powerful receptors combine a rigid steroidal scaffold, derived from cholic acid 1 (chart 1) with various combinations of h - bond donor groups. most are reluctant to crystallize but a small subset, represented initially by 24, were found to form needles from methyl acetate - water or acetone - water mixtures. all three were subjected to x - ray crystallography, with interesting results. despite the significant differences between 24, the external similarity of the crystals was reflected in the internal structures ; the three were isomorphous, with almost identical unit cell dimensions and packing arrangements for the invariant steroidal cores. the packing involved the formation of helices with hexagonal symmetry (space group = p61), surrounding solvent - filled channels. the arrangement is illustrated in figure 1, using tris - urea 3 as an example. individual steroid molecules bind to a single molecule of co - crystallized water through 5 h - bonds (figure 1a) and stack to form columns (figure 1b). the columns then pack in a hexagonal arrangement to generate the solvent - filled pores. the orientation of the steroids in the columns is such that the terminal groups (methoxy and nhph) face into the pores and largely determine the nature of the channel surface. effectively, the terminal groups can expand into the channel interior without affecting the packing of the columns which maintain the structure. the channels, moreover, are unusually wide. in the case of trifluoroacetamide 2, the average diameter was found to be 16.4. the average diameter for 3 was only slightly less at 15.7, although the surfaces are more irregular (figure 2). there is thus substantial room, in principle, both for guest molecules and for terminal groups. preliminary experiments on 2 implied that guest exchange was possible, at least for certain solvents (meoac, et2o, toluene). evacuation lead to partial degradation (evidenced by crazing), but the powder x - ray diffraction (xrd) pattern remained largely unchanged. the steroid is solvated by a molecule of water which forms hydrogen bonds to all three urea groups. (b) molecules of 3 stack to form columns, running along the crystallographic c axis. representation as for (a) except that core steroidal atoms are colored blue and green in adjacent molecules, and water molecules are shown with thick bonds. one column of steroids is highlighted using the coloring from (b), with the och3 and nhph groups now in spacefilling mode. (d) a single channel sliced in half along the c axis, viewed in spacefilling mode. terminal och3 and nhph groups retain their coloring, other atoms near or at the internal surface are shown as light blue. (e) 3d schematic representation of a channel, showing helical arrangement of methyl groups and aromatic rings (spheres and hexagons, respectively). interior surfaces for trifluoroacetamide 2 and tris - n - phenylurea 3 viewed along the c - axis. the surfaces were calculated using a 1.4 probe. given the space available within the channels, it seemed likely that a wide range of analogues with a common bis-(n - phenylureido)steroidal core (figure 3) would form crystals isostructural with 24. variation should be feasible not only at the c3 substituent (r in figure 3) but also at the c24 ester group (r in figure 3) (npsus) would be isostructural they should be able to form solid solutions (organic alloys), greatly enlarging the range of systems available. since our original publication we have confirmed both of these possibilities. we have described a series of three npsus with aromatic groups in r, and the interesting feature of water wires in the channels, and also a range of npsu - based organic alloys. herein we provide a more complete description of our work surveying the scope and properties of npsus, drawing on 25 examples which have been characterized by x - ray crystallography. we show how the dimensions and shapes of the channels can be tuned, and how their chemical nature can be altered by the introduction of functional groups (including previously unreported alkene and aldehyde functionality). we also report, for the first time, that npsus can be porous in the strictest sense, stable to evacuation and capable of gas adsorption. moreover we show that they can adsorb a remarkable range of guests, including organic dyes with molecular weights up to 300 and even the c30 hydrocarbon squalene (mw = 410). the core bis-(n - phenylureido)steroidal unit maintains the p61 nanoporous structure, while groups r and r control the size and nature of the pore. the first are esters of 3,7,12-tris-(n - phenylureido)-5-cholanoic acid 6 and include 3 as well as the 14 variants 720 represented in chart 2. ester groups r were chosen for variation in size (and thus pore diameter) and surface characteristics (aliphatic vs aromatic vs fluorocarbon) and also to showcase the potential for placing chromophores (e.g., azobenzenes), fluorophores (e.g., pyrenes), and reactive units (e.g., allyl groups) in the channels. the second group are derivatives of methyl 3-amino-7,12-bis-(n - phenylureido)-5-cholanoate 5, including trifluoroacetamide 2, carbamate 4, tris - ureas 2126, and amides 2729 (chart 3). again the variable group (r) was used to change steric and surface properties and to introduce chromophores and functional groups. in this case aalthough 29 is included here for convenience, it does not adopt the npsu crystal packing. for further details see text. although 29 is included here for convenience, it does not adopt the npsu crystal packing. for further details amine 5 is accessible from cholic acid 1 via a multistep but well - established route in 40% overall yield. tris - urea 3 may be prepared from 5 by treatment with phenyl isocyanate or more directly from 1 via methyl 3,7,12-triaminocholanoate. esters 720 (chart 2) are available from 3 via equilibration with lithium alkoxide or hydrolysis to acid 6 followed by o - alkylation or carbodiimide - induced esterification. the derivatives in chart 3 may be prepared from 5 by treatment with an aryl isocyanate (giving 2126) or an acylating agent (giving 2729). the preparations of 9, 10, 12, 1419, 23, 26, and 29 have been reported previously. procedures for the remaining compounds in charts 2 and 3 are given in the supporting information. the steroids in charts 2 and 3 were crystallized from methyl acetate or acetone, to which small amounts of water had been added, through slow evaporation of the organic solvent. in most cases other polar solvents, such as methanol or ethanol, or nonpolar mixtures, such as chloroform - hexane, yielded oils or amorphous solids. all the compounds could be analyzed by single crystal x - ray diffraction (scxrd), and with the single exception of 29 (see below), all formed crystals with the p61 npsu packing. the structures of 24, 14, 16, and 18 have been reported in communications, the remainder are described for the first time herein. as expected these show only minor variations, the differences between the steroids being accommodated by changes to the shape, diameter, and surface characteristics of the pores. unsurprisingly, given the open nature of the pore region, disorder in terminal groups r and (especially) r was fairly common, being present in 11 of 25 structures. however, in most cases the groups concerned were divided between just two positions, so that a reasonable model of the crystal (for estimating pore volume etc.) this applied to 4, 8, 10, 12, 13, and 24 (disorder in r), and 22 (disorder in r). in two cases, 15 and 19, deleting one of the two possible positions did not give a viable structure. however, these crystals could be modeled successfully by assuming equal occupancy of both positions, on an alternating basis. after editing where relevant, the smoothed solvent accessible surfaces and resulting guest - accessible volumes were calculated using materials studio, employing a probe of radius 1.2. these values are given in table 1, while images of the surfaces are available as supporting information. these were estimated by repeating the calculation using probes of increasing size until the surface was no longer continuous. the resulting value is, effectively, the diameter of the largest sphere which can pass through the channel. images of selected structures viewed down the pores, with terminal groups shown in spacefilling mode, are shown in figure 4 (compounds from chart 2, varying ester group r) and figure 5 (compounds from chart 3, varying c3 terminal group r)., obtained from the materials studio program employing a spherical probe of 1.2 radius. calculations of total solvent accessible surfaces give higher values but include small voids outside the channel region. estimated by calculating the smoothed solvent accessible surface using differing probe radii (increments / decrements of 0.05). the value given is the diameter of the largest probe for which the calculation yields a continuous surface. these values are slightly smaller than those reported in ref (17), due to a change in the method of calculation. could be built using the assumption that r in neighboring molecules occupied alternating positions, and this model was used for the pore volume and diameter calculations. when the probe diameter is reduced to this value, voids are generated outside the channel region while a continuous pore surface has not yet appeared. mes disordered over two positions, one being removed before pore volume and diameter calculations. terminal groups r (= nhph) and or are shown in space - filling mode, with r colored gold. terminal groups r and or (= ome) are shown in space - filling mode, with or colored magenta. table 1 and figures 4 and 5 illustrate the wide variety of structural properties available via the npsu system. for example, starting at nearly 20% (for 2), the volume available in the pores can be tuned downward in small increments essentially to zero (for 15 and 19). indeed, by taking advantage of alloy formation, continuous variation should be possible with these compounds. unsurprisingly, pore volumes and diameters are generally determined by the size of the terminal groups, but more subtle effects are also in play. for example, in the series with varying or (chart 2, figure 4), a 2-carbon spacer between the oxygen and an aromatic group tends to allow efficient packing of the aromatic surface against the side of the channels. thus, for pyrenyl derivative 16, space remains down the center for hydrogen - bonded chains of water molecules. in contrast, a 1-carbon methylene spacer directs the aromatic group toward the center of the channel. in the case of pyrenyl derivative 15, this results effectively in full occupation of the channel ; the calculated guest - accessible volume and minimum diameter are both close to zero. paradoxically, therefore, the larger terminal group (in 16) leaves more space than the smaller group in 15. the shape of the channel wall (smooth vs corrugated) is another feature which can be altered. as mentioned above, compounds for which r = ch2ch2ar (e.g., 14, 16, 18) tend to adopt structures in which the aromatic groups line the surfaces of the channels. the resulting pores are relatively smooth and cylindrical, as illustrated for 14 in figure 6 (top). in other systems from chart 2, the channel surface is presumably corrugated, but with random and/or flexible character due to disorder within the crystal. an example is provided by 13, for which the naphthylmethyl group appears in two orientations, one roughly perpendicular and one more nearly parallel to the channel axis. well - defined corrugated pores may be accessed by placing extended substituents at r (which is less prone to disorder). thus for both 24 (r = azobenzene) and 25 (r = biphenyl) the c3-substituent reaches well toward the c axis creating strongly asymmetric helical pores (figure 6, middle and bottom). this work also shows that the chemical nature of the pore walls can be subject to wide variation. the structures collected in figures 4 and 5 feature an alkenyl group c = c (8), a helical strip of fluorocarbon surface (10), an aldehyde (21), a thioether (22), a boc - protected amine (23), and an iodobenzene (26). as illustrated in figure 7, all are positioned where they can interact with guest molecules and participate in reactions or noncovalent interactions. finally, crystallography of acetamide 29 showed that not every molecule defined by figure 3 adopts the p61 npsu structure. in this case a monoclinic (p21) form obtained from methyl acetate / water was denoted 29, and a tetragonal (p432121) form which crystallized from acetone / water was denoted 29. the molecular units in the two forms are almost identical, and qualitatively different to those in the npsus ; in particular, the c3 substituent is positioned so that the nh group points inward, creating a binding site which accommodates two water molecules (see figures s31 and s32). in both crystals the packing is efficient, leaving no substantial voids (see figures s57 and s58). crystal structures of 14, 24, and 25 viewed perpendicular to the c axis. for the images on the left, the groups which dominate the channel surface (or for 14, r for 24 and 25) are highlighted in spacefilling mode. for the right - hand images, the smoothed solvent accessible surfaces have been added using materials studio, and the structures have then been sliced along the c - axis. space - filling representations of the channel regions in 8, 10, 21, and 22. the structures have been sliced along the c - axis to expose the pore interiors and are viewed roughly perpendicular to c and a (see axes attached to 8). conventional coloring is used for the distinctive groups in each structure (or for 8 and 10, r for 21 and 22), the remaining atoms being shown as silver - blue. implies that the crystal is permeable, allowing exchange of small guest molecules, and that this process does not substantially affect the host framework. ideally, the crystals should also be able to survive the removal of all guest molecules without loss of structure and then show reversible gas adsorption to confirm porosity. as mentioned earlier, we previously demonstrated that trifluoroacetamide 2 satisfies, at least, the guest - exchange criterion. the results from evacuation were less clear - cut ; the powder xrd (pxrd) pattern remained essentially unchanged, but the crystals crazed and became opaque. most npsu crystals, especially those with 3-ureido substituents, showed no change in appearance on evacuation. nonetheless it was clearly desirable to establish the solvation state of a typical npsu, show that the solvent could be removed, and demonstrate that the resulting crystals were unchanged and capable of gas adsorption. we chose tris - n - phenylurea 3 for this study, as this compound is the most accessible npsu and has proved the most convenient for routine use. crystals of 3 were obtained as needles from acetone / water (initial ratio 10:1), after washing with acetone and air - drying. samples were then evacuated at room temperature and 100 c for 24 h. the three samples (air - dried, rt evacuated, 100 c evacuated) were then analyzed by h nmr in dmso, using a procedure which allowed the amount of background water to be measured and taken into account. the composition of the air - dried crystals was found to be 3:water : acetone = 1:3.8:0.2. allowing for the single water molecule per steroid embedded in the channel wall, this implies that the pores are filled with 3 molecules of h2o per molecule of 3, with a small amount of organic solvent also present. after evacuation at rt the composition was 3:water = 1:1, implying that the channels are empty. after evacuation at 100 c for 24 h the ratio 3:water reduced slightly to 1:0.9. this suggests some degradation, although microscopy and pxrd again showed no major changes. samples of 3 were also heated to 150 c and above, and in these cases clear signs of decomposition were observed both by microscopy (loss of transparency) and pxrd (loss of diffraction peaks). having established that the pores could be evacuated without loss of crystallinity, we proceeded to confirm the permanent porosity of 3 using n2 gas adsorption measurements for a sample that had been heated under vacuum at 75 c for 9 h. surprisingly, the n2 adsorption predominately takes place at high relative pressures (p / p > 0.7), and there is significant hysteresis between the adsorption and desorption isotherms giving a type iv isotherm (see e.g., figure 8). this hysteresis differs from that observed in mesoporous materials (pore diameter > 20) that generally closes at lower relative pressures (p / po 0.4) and which is related to pore evacuation involving capillary action. furthermore, the desorption isotherm falls below that of the adsorption isotherm at p / po 0.7. desorption cycle is repeated and presumably reflect slow, nonequilibrium, kinetics of n2 adsorption. such slow kinetics is understandable if access to the pores is restricted to the relatively small number of openings located at the end of the long needle - shaped crystals, which are on average > 2 mm in length. we have previously shown that the pores in 3 are parallel to the long axis of the crystals. similar hysteresis was observed by tosi - pellenq. from the n2 isotherms of long (150 m) microporous crystals of alpo4 - 5, which also contain cylindrical channels (0.76 nm in diameter) along the long axis of the crystals. in this case the fact that the desorption isotherm in figure 8 dips below the adsorption isotherm implies that evaporating nitrogen is lost more rapidly from the channels than gaseous n2 is readsorbed. this may relate to pressure differences between the interior and exterior of the crystals ; it is reasonable to suppose that when the crystals are compressed, inward gas transfer could be relatively slow, while internal pressure could expand the crystals and assist n2 efflux. the possibility that the effects are due to collapse of the crystal structure during n2 analysis was discounted by confirming that the structure remained unchanged, as shown by scxrd of a crystal extracted from the sample of 3 used for n2 analysis. the bet surface area calculated from the n2 adsorption isotherm is very low (29 m / g) and probably represents only the external surface area of the crystals. however the pore volume of 0.17 ml / g calculated from the total n2 uptake (4.9 mmol / g) is highly consistent with the guest - accessible volume (16.1%) calculated from the crystal structure (i.e., 0.17 ml / g equates to 16% of the total volume given a crystal framework density of 0.941 g / ml). the pore volume obtained from n2 uptake is also consistent with the values calculated from the adsorption of liquid guests, as discussed in the following section. n2 adsorption () and desorption () isotherms for crystal 3 at 77 k. see text for discussion. air - dried crystals of 3 were placed in each, left for 1224 h, washed briefly with ether, and subjected to h nmr analysis. all of the substrates were adsorbed in significant amounts, as summarized in table 2. aniline 30 formed a well - defined host guest 1:1 complex which could be characterized by x - ray crystallography. as shown in figure 9, the aniline molecules form a helix within the channel, apparently stabilized by a close interguest chn contact (dc hn = 2.68). the anilines are also held in place by specific favorable interactions with the channel wall, including hydrogen bonds between amino nh and host ester carbonyl (dn ho = 2.46 and n ho = 167.8), nh interactions involving the second amino nh and a host phenyl group (dn h = 2.84), and ch interactions to the aniline -system. guest ratio, although in this case the guest could not be located crystallographically. a calculation of the volume of liquid absorbed per unit mass of host gave a value of 0.12 ml / g, consistent with the pore volume obtained by gas adsorption (see above and table 2). this represents a pore - filling efficiency of 70% using the pore volume calculated from the crystal structure, which is consistent with a strong affinity between the crystal and adsorbate. similar calculations based on the uptake of 3336 suggested that these were absorbed less efficiently. however the value for squalene 36, at 65% of the maximum, is remarkable for such a large (30-carbon) guest. x - ray crystal structure of 3 with adsorbed aniline, viewed along the c - axis (top) and a - axis (bottom). the aniline is shown in space - filling mode. samples of air - dried crystalline 3 were place in aniline and then removed, washed with ether, and analyzed by h nmr after periods ranging from 2 to 180 min. the results showed that the crystals are filled to about half capacity very quickly (within the first 2 min), but that subsequent adsorption is much slower. we were also interested to discover whether the aniline could be oxidized to polyaniline within the channels. indeed, treatment of the complex with peroxyammonium sulfate in 0.1 n hcl caused the crystals to turn dark violet (after 4 h) then green (after 12 h). the diffuse - reflectance uv vis spectrum of the product showed adsorption maxima at 420 and 795 nm consistent with polyaniline formation (see figure s75). pxrd analysis showed that the npsu structure was retained, although the crystals were no longer suitable for single - crystal x - ray structure determination. another set of experiments involved the adsorption of larger guest molecules from solutions in diethyl ether. in these cases colored guests were used for ease of analysis and the potential for interesting or useful optical effects., solutions of dyes 3743 in ether (1020 mm) were added to crystals of 3, and the mixtures left to stand for 3 days. after isolation and washing with ether, all crystals were visibly colored. in the case of 3741 the colors were strong enough to show clearly under a microscope (see figure 10). as shown in figure 10, the colors appeared to permeate the crystals and were not localized at ends or edges. interestingly, the crystals containing nile red 41 were observed to be blue - purple (figure 10e). this dye is strongly solvatochromic, its optical adsorption moving to longer wavelengths with increasing solvent polarity, and a blue or purple color suggests a highly polar environment. soaking the crystals in ether for 24 h resulted in loss of color, showing that the dye adsorption was reversible. a second npsu crystal, trifluoroacetamide 2, was also investigated as host and was found to absorb azo - dyes 37 and 38. the combinations of 3 with disperse red 1 (38) and azulene (43) were investigated further, to establish how much dye was included and how fast. in the case of azulene, only 1 mol % was absorbed, while equilibrium was reached within the first hour. in the case of 38, the first 1 mol % was also absorbed quickly, but a further quantity (nearly 1 mol %) was taken up in a slower process over 24 h. crystals of 3 after exposure to ethereal solutions of (a) 37, (b) 38, (c) 39, (d) 40, and (e) 41. despite the appearance of the crystals there was room for concern that the dyes might not be entering the channels but somehow associated with cracks or defects in the crystals. to test this possibility, we examined the colored crystals under a microscope using plane polarized light. if the dyes were occupying the channels, it seemed likely that some (at least) would show preferential alignments. if the transition dipole moments were to lie roughly along the channel axis (the long axis of the crystal) the crystals should be dichroic, i.e., their colors should be dependent on their orientation with respect to the plane of polarization. figure 11 shows pairs of photomicrographs in which crystals of identical composition, but oriented at roughly 90 to each other, are illuminated with polarized light. each pair of images shows the same crystals, with the plane of polarization differing by 90. the crystals are clearly dichroic, changing from colored to almost colorless as the plane of polarization is rotated. the effect was observed for 237, 238, 337, 338, and 341 but not for 339 or 340. the guests which lead to dichroism (37, 38, and 41) possess extended dipoles due to conjugation of an amino group with an electron acceptor. this feature should encourage the molecules to adopt a head - to - tail arrangement parallel to the channel axis. the images in figure 11 provide strong evidence that the dye molecules are indeed in the channels revealed by crystallography. it should be noted that this phenomenon of dye uptake by organic molecular crystals is rare and may be unprecedented. it is well - known that dyes may be adsorbed by inorganic crystals, such as zeolites, or by organic inorganic hybrids (pcps / mofs). however, the inclusion of dyes in organic molecular crystals is normally achieved by cocrystallization, not by the interaction of substrates with macroscopically sized preformed crystals. this ability of npsus to adsorb such large guest molecules highlights their unusual combination of robust crystal structures with spacious accessible interiors. crystals of npsus with included dyes illuminated with polarized light. for each pair of images the plane of polarization is rotated through 90 between left and right. (a) 237, (b) 238, (c) 337, (d) 341. in principle, the npsu crystal packing represents a powerful tool for the design of functional materials. first, the structure needs to be generalizable, forming in (at least) most of the cases where it might be predicted. second, the crystals need to be truly porous so that the space within may be exploited. we have now examined the crystal structures of 26 molecules with the general structure represented in figure 3, and of these only one (acetamide 29) fails to adopt the p61 npsu arrangement. the range of npsus now includes examples with vanishingly small pores sizes, strongly corrugated pore surfaces, and several cases with potentially reactive functional groups (ch2ch = ch2 in 8, ch = o in 21, sme in 22, and nhboc in 23). it is notable that neither the aldehyde nor nhboc groups, both of which are quite polar, disturbed the npsu packing. we have also shown that the pores can be evacuated without loss of integrity and that subsequent gas adsorption is possible (although given the pressures involved and the low pore volume, applications in gas storage are unrealistic). more importantly, organic molecules are also absorbed, including the large rigid nile red 41 (mw 318), and the even larger but more flexible squalene 36 (mw 411). the ability to orient dye molecules suggests applications in display technology and nonlinear optics. although not all dyes showed this behavior, the tunability of the pores implies that the phenomenon should be extendable (e.g., by tailoring of channel diameter). the fact that small molecules can readily access the pores points to further applications in catalysis, sensing, and separations, especially given the chirality of the crystals and the ability to incorporate effector groups through alloy formation. we hope to explore these and other possibilities in future work. | previous work has shown that certain steroidal bis-(n - phenyl)ureas, derived from cholic acid, form crystals in the p61 space group with unusually wide unidimensional pores. a key feature of the nanoporous steroidal urea (npsu) structure is that groups at either end of the steroid are directed into the channels and may in principle be altered without disturbing the crystal packing. herein we report an expanded study of this system, which increases the structural variety of npsus and also examines their inclusion properties. nineteen new npsu crystal structures are described, to add to the six which were previously reported. the materials show wide variations in channel size, shape, and chemical nature. minimum pore diameters vary from 0 up to 13.1, while some of the interior surfaces are markedly corrugated. several variants possess functional groups positioned in the channels with potential to interact with guest molecules. inclusion studies were performed using a relatively accessible tris-(n - phenyl)urea. solvent removal was possible without crystal degradation, and gas adsorption could be demonstrated. organic molecules ranging from simple aromatics (e.g., aniline and chlorobenzene) to the much larger squalene (mw = 411) could be adsorbed from the liquid state, while several dyes were taken up from solutions in ether. some dyes gave dichroic complexes, implying alignment of the chromophores in the npsu channels. notably, these complexes were formed by direct adsorption rather than cocrystallization, emphasizing the unusually robust nature of these organic molecular hosts. |
surgery is currently the only available cure for primary hyperparathyroidism (php). in selected patients, minimally invasive parathyroidectomy (mip) offers the benefits of decreased morbidity and shorter hospital stay ; hence, it is increasingly replacing the traditional bilateral neck exploration (bne) and four - gland evaluation. this technique depends on successful preoperative imaging and localization of the abnormal gland, using ultrasound (uss) and technetium tc sestamibi scanning with single - photon emission tomography (spect). using a combination of these imaging modalities however, the two imaging methods are concordant in only up to 64% of patients with php, and their accuracy is decreased in multiglandular disease (mgd). identification of a macroscopically abnormal parathyroid gland during mip does not exclude abnormalities of the rest of the glands such as double adenoma, multiglandular disease, or malignant tumours, which may be difficult to distinguish from benign adenomas, especially in light of the above limitations of preoperative imaging. on the other hand, histopathology of excised abnormal parathyroid glands is not predictive of the secretory function of the remaining parathyroid glands left in situ. intraoperative parathyroid hormone (iopth) measurement has recently been introduced as a useful adjunct in confirming the successful excision of a parathyroid adenoma. the method is based on the short half - life of the intact pth (4 - 5 minutes) ; successful removal of diseased parathyroid tissue is predicted by corresponding decrease of serum pth concentrations. we present our initial experience of patients with php treated surgically with minimally invasive parathyroidectomy and iopth measurement, and we evaluate the safety, efficacy, and clinical usefulness of the method in a district general hospital. the study included eleven consecutive patients (two male and nine female) who underwent surgery for php over 6 months period in a district general hospital serving a population of approximately 300,000. ten patients were operated as elective cases because of severe hypercalcaemia complicated with decreased bone density. one patient was operated as an emergency due to extreme hypercalcaemia (adjusted serum calcium level, 6.49 mmol / l) with grossly elevated pth concentration of 1059 pg / ml (reference range 1565 pg / ml), serum phosphate 1.79 mmol / l, creatinine 124 mol / l, and urea 16.0 mmol / l. two weeks prior to admission, she had sustained right scaphoid fracture following a fall and was diagnosed with primary hyperparathyroidism (phpt), awaiting elective parathyroidectomy. all eleven patients had ultrasound scan of the neck preoperatively whereas sestamibi scan was feasible in all but one patient, who was severely kyphotic. three patients underwent planned full neck exploration facilitated by iopth measurements, during the initial period of method validation. in the remaining 8 patients, mip surgery was initiated under general anaesthesia via a 3 cm skin crease incision approximately 1 cm above the clavicle and centred over the anterior border of sternoceidomastoid muscle. biochemical profile was assayed on a cobas 6000 analyser (roche diagnostics, mannheim, germany) using standard methods. serum calcium was assayed by a method according to schwarzenbach with o - cresolphthalein complexone and serum albumin was measured by a colourimetric assay. intraoperative measurement of serum pth was performed at baseline (immediately after the adenoma was resected, t0) and then at every 5 minutes post excision of the macroscopically abnormal parathyroid gland for 15 minutes (t5, t10, and t15). a fall in serum pth level by at least 50% of the baseline value within 15 minutes excision of parathyroid adenoma was followed by the predicted fall in intraoperative serum pth concentration of > 50% in ten patients. in one patient, the pth levels increased and remained high for up to 30 minutes post excision, implying the presence of multiglandular disease ; further neck exploration identified an ipsilateral second adenoma, not localised preoperatively by either ultrasound or sestamibi scan. removal of the second adenoma was followed by the expected fall in serum pth concentration of > 50% confirming successful surgery (figure 1). in another patient, the operation was converted to standard collar incision because the localised parathyroid adenoma macroscopically resembled a thyroid nodule ; therefore, better access was deemed necessary ; histology confirmed the presence of a parathyroid adenoma only. in all cases, histology confirmed the presence of discrete, encapsulated adenomas, containing sheets and islands of eosenophilic and chief cells, with no capsular invasion. there was no evidence of hyperplasia or carcinoma in either of the eleven patients. during the postoperative period, ten patients had normal pth and remained normocalcaemic to a median (range) follow up of 6 (110) months. recurrent elevation of serum pth concentration was noted postoperatively in one patient despite a fall of > 50% of baseline intraoperatively. mmol / l) and during the first postoperative week requiring calcium and vitamin - d supplementation orally. the patient who had parathyroidectomy as an emergency when admitted with hypercalcaemic crisis stayed in the hospital for a total of 7 days in order to be monitored for potential complications of extreme hypercalcaemia. the incidence since the introduction of multichannel analysers is approximately 1 : 1000, with the relatively asymptomatic older female being the typical patient. a prevalence of up to 2.1% in postmenopausal women has been reported in population - based health screening. however, in the minority (less than 10%) of affected patients, php is associated with a number of distinct hereditary syndromes including multiple endocrine neoplasia (men) types 1 and 2a (men 1, men 2a), hyperparathyroidism - jaw tumour (hpt - jt) syndrome, and familial isolated hyperparathyroidism. multinodular parathyroid hyperplasia accounts for 15%20% of cases and only occasionally php is caused by parathyroid carcinoma. this retrospective study was based on a small preliminary series of patients and is not devoid of the relevant methodological weaknesses, such as incomplete data (e.g preoperative levels of 25-oh vitamin d, urine calcium levels), accounted for by local policies and the retrospective data collection method ; these have been addressed in prospective larger series [2022 ]. however, it has demonstrated that minimally invasive parathyroidectomy is a safe and effective method of surgical treatment of php, constituting a simple procedure that can be carried out in a district general hospital as day surgery or with overnight stay ; most of our patients are now discharged within 24 hours with calcium and vitamin - d supplements. although the role of iopth measurement is debatable, in our study, it incurred substantial clinical benefit by identifying the correct pathology in one patient with discordant preoperative imaging and in one patient with double adenoma. it also facilitated successful excision of adenomas without the need for intraoperative frozen section, which can be disconcordant with the definitive histology in up to 10% of cases [23, 24 ]. tc sestamibi scanning, using either single - photon emission computed tomography (spect) or oblique pinhole images, is able to localize over 90% of adenomas, including ectopic glands, but is less accurate in multiglandular disease. when ultrasound scan (uss) and technetium tc sestamibi scanning are concordant their accuracy increases, but this is feasible in only two thirds of patients with php (best concordance rate 64%). in our case series, intraoperative serum pth level quantification had higher sensitivity (100%) in detecting the parathyroid pathology (single or double adenoma) compared to each imaging technique alone. concordance between uss, sestamibi scan and iopth was noted in 9 (82%) patients. the false negative rate for uss alone was 18% (2 in 11 patients) and for sestamibi scan alone 10% (1 in 10 patients). combination of all three methods facilitated optimum selection of patients for minimally invasive surgery. a major benefit yielded by use of iopth measurement in this study was the diagnosis of a double adenoma that had not been detected preoperatively. double adenomas have been reported to occur in 2%15% of patients undergoing operation for php and can be missed in up to 15% of patients diagnosed with a single adenoma by sestamibi scan [29, 30 ]. they have a nonuniform distribution, likely to involve both sides of the neck and they can easily be missed when a focused mip approach is employed. failure to recognise the double pathology can lead to unsuccessful surgical outcome, unnecessary patient distress, and exposure to the risks of a redo operation and anaesthesia, along with further potential cost. failure of serum pth to appropriately decrease after excision of a single adenoma should raise suspicion of additional adenomas and the surgical approach should be modified accordingly, if necessary by converting mip to a standard full neck exploration. although intraoperative serum pth level quantification was first introduced in 1990, it has yet to be universally accepted as a routine adjunct in surgery for php. opponents of the method claim the additional incurring cost of surgery, prolongation of the operating time of mip and the reported false negative rate. we did not perform a cost analysis during this small study, but it is anticipated that the short median hospital stay of our patients was cost effective ; the additional cost of pth assays of 32 per patient (4 intraoperative measurements) has been offset by reducing the hospital stay (cost of 657 per overnight stay per patient) and by eliminating the cost of intraoperative frozen section (150 per patient). the use of iopth measurement in our series added 510 minutes extra to the operating time of minimally invasive procedure. mip in experienced hands has been shown to be associated with shorter operating times comparing to the conventional neck exploration, but studies providing high levels of evidence are lacking in this respect. one patient in our study was found to have elevated serum pth during follow up, despite appropriate intraoperative pth fall after excision of adenoma, but remains normocalcaemic and asymptomatic ; hence, no further imaging has been deemed clinically necessary. this phenomenon is not uncommon and its significance is uncertain ; between 8%40% of patients with normocalcemia after surgery have elevated pth levels at follow up [35, 36 ]. in a recent study, ning. demonstrated that 95% of these patients do not develop recurrent hyperparathyroidism (rhp). proposed potential risk factors include higher preoperative pth concentrations, higher alkaline phosphatase activities, and older age (associated with impaired renal function, bone remineralization, and low vitamin d). they supported the hypothesis that persistently elevated pth may be, in part, secondary to bone hunger and suggested that rhp in their series was more likely if postoperative serum calcium was higher than 9.7 mg / dl (2.42 mmol / l). long follow up with yearly serum pth and calcium levels is necessary to detect potential recurrence at an early stage. in summary, the results of our small case series suggest that minimally invasive parathyroidectomy with intraoperative pth measurement is a feasible, safe, and effective method for treatment of primary hyperparathyroidism in the context of a district general hospital. our findings are in accordance with similar studies appealing for its wider use in the uk. there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. this research did not receive any specific grant from any funding agency in the public, commercial or not - for - profit sector. | background. minimally invasive parathyroidectomy (mip) is increasingly replacing the traditional bilateral neck exploration in the treatment of primary hyperparathyroidism (php). intraoperative pth (iopth) measurement has recently been introduced as a useful adjunct in confirming successful excision of abnormal parathyroid gland. aims. we evaluate the safety, efficacy, and clinical usefulness of iopth measurement during mip in a district general hospital. methods. retrospective review of eleven consecutive patients with php who underwent mip with iopth, following preoperative assessment with ultrasound and sestamibi scans. results. all patients had successful removal of the abnormal parathyroid gland. the concordance rate between ultrasound and sestamibi scan in localising the parathyroid adenoma was 82%. iopth measurement confirmed the removal of adenoma in all cases and, in one case, led to identification of a second adenoma, not localised preoperatively. the median hospital stay was 2 days (range 17 days). all patients remained normocalcaemic after a median of 6 months (range 110 months). conclusions. minimally invasive parathyroidectomy is a feasible, safe, and effective method for treatment of php. the use of iopth monitoring potentially offers increased sensitivity in detecting multiglandular disease, can minimise the need and risk associated with recurrent operations, and may facilitate cost - effective minimally invasive surgery. |
inequities in the health status are global problem, equally affecting reach and poor countries (1) and numerous attempts to quantify economic impact of health inequities have shown significant opportunities for savings if these inequities are reduced (2), such as calculations that lost lives due to inequities in health account to 700.000 deaths annually and 33 million diseased across eu (3, 4). studying self - reported health is expanding over the past 15 years (5) and is considered as an indicator for morbidity and mortality in the population that may be used in primary health care to detect poor health in certain population groups and predicts the health care utilization (6). surveys conducted in eu countries show that individuals systematically rank their health status above all other aspects of their everyday life (7). answers on simple questions on self - assessed health status have shown significant independent association between health status indicators and specific covariates that predict mortality (8), even after adjustment for comorbidity (9), such as cardiovascular diseases (10), and self - rated health is correlated with physical health, functional capacity and psychological well - being (11). self - rated health is also an independent predictor of survival that controls for other related health indicators and it is recommended that these types of research should be conducted not only in western countries (12). prognostic value of self - rated health is particularly important among older adults (13) and literature acknowledge self - rated health as lying at the cross - roads of culture and biology, therefore it needs a collaborative effort between different disciplines to improve understanding of this key measure of health status (14). the goal of the survey is to assess the socioeconomic self - rated health gradient and to describe contribution of behavioral risk factors to this gradient among population in republic of macedonia. the study was conducted in 8 statistical regions in republic of macedonia with population of 2 065 769 inhabitants (state statistical office, 2013) in the period march december, 2013. informed consent was an integral part of the questionnaire and was obtained from each survey participant, explaining the objectives, process and expected outcomes of the research and their right to withdraw at any point of answering the questions, as well as ensuring confidentiality of the information gathered before, during and after finishing the study. by the means of case - control study, households of registered tb cases in the period july, 2012 june, 2013 were recruited as cases and one control household was randomly chosen in cases immediate vicinity and who agreed to participate in the study, to provide for comparison with the general population. study sample has been calculated with statistical program for determining sample size (pepi 4.04x), by using the following parameters : average household size 4.5 (state statistical office, 2012), urban / rural ratio 1.5, poverty line 28.7 (index mundi, 2008), the study power of 80%, 95% confidence interval and maximum acceptable difference 0.05. the calculated sample of 530 households was increased by 10% to allow for non - response. data is collected with selected modules from world health survey questionnaire (15), modified to provide data for survey objectives, in accordance with guidelines for developing countries (16). self - reported health is assessed through the question how will you rate your health status today ? with a five - point scale ranging from 1 very satisfied, 2 satisfied, 3 neither satisfied nor dissatisfied, 4 dissatisfied, 5 very dissatisfied). social determinants are assessed by collected data on gender, place of residence (urban - rural), educational and employment status, ethnicity and the region where the patient lives. face - to - face interview was performed by 20 trained dot nurses who visit tb patients 3 times / week ; all data on variables is based on self - reported information. the instrument has been pre - tested on 10% of the sample, with appropriate corrections following feedback from the pre - test, mainly additional explanations for better understanding of questions. the instrument has been also translated into albanian language, as to allow for interviewing ethnic minorities in rm into their mother tongue. data has been analyzed with ibm spss statistics, version 19.0 (ibm corporation, somers, ny, usa), using descriptive statistics to calculate frequencies and mean values. percentages are used to express values and chi - square test to analyze differences between cases and controls for categorical variables. multiple logistic regression was used to assess the relationship between self - reported health as single categorical response and age, gender, place of residence, region, educational and employment status, as well as smoking and alcohol consumption as categorical explanatory variables (17, 18). the total study population is 562 households with total of 2720 respondents, 53.5% households with tb patient and 46.5% households as controls. characteristics of study population are presented in table 1. means, proportion and disribution of explanatory variables in controls (n=1455) and controls (n=1275) respondents are represented in all 8 regions, as per statistical division of the country, most of them interviewed in the north - west region, 47.6% in skopje region, 14.9% in polog, south - west 8.4%, south - east and north - east with 7.4% and 7.2%, respectively, pelagonia 5.9%, east 5.3% and the lowest number in vardar region (3.2%), which corresponds to distribution of tb patients registered in the period july, 2012 june, 2013, as the main inclusion criterion for cases. due to the study design, percentage of controls in the regions is identical, or negligibly lower or higher compared to cases. distribution of respondents by place of residence is quite proportional with 50.9% living in urban and 49.1% in rural areas, with both cases and controls dominantly living in urban areas. statistically significant difference is observed in gender, with dominance of male tb cases (63.2% vs 52.1% in the control group). members of households with tb cases are slightly younger (mean age of 45.17 years + 15.7 sd, compared to controls with mean age of 47.28 + 14.1sd) and live in statistically significant bigger households of average 4.66 members than hh size of 4.36 in controls. by ethnicity, most of the cases are albanians (50.6%) or macedonians (37.3%), followed by roma (7.5%) and turkish (4.2%) and, given the study design, there is no statistically significant differences between the groups (=3.458, df=5, p=0.63). most of the cases have completed primary school education (41.7%) or high school (39.5%), compared to controls with 30.7% and 43.4%, respectively ; the percentage of controls who have completed university education is more than double (9.3%) compared to cases with 4.5% and the difference among groups is statistically significant. education, in turn, is reflected in employment status with majority of cases reporting unemployment (72.4%), although this percentage is also high in controls (52.2%), with high statistically significant difference among groups. percentage of regular smokers among cases is lower (35.6%) compared to cases who regularly smoke in 43.9% cases, but the percentage is similar if we add those who smoke, but are not doing it every day (46% cases vs 50.8% among controls), with statistically significant differences among groups. percentage of regular alcohol users is also higher in controls, with 55.75% reporting alcohol consumption ever in life, compared to 40.07% cases doing so. half of respondents in our study have assessed their health status (including physical and mental health) as good (49.9%), 24.4% have stated their health status is moderate, 17.9% think their health status is very good and only 7.2% and 0.7% assessed their health status as bad or vary bad, respectively. analyzed by group of respondents, 50.8% cases and 49.1% controls assess their health status as good or moderate (27.6% cases and 21.1% controls), but differences are significant in categories bad with dominant cases with 9.3% vs. 4.9% controls and very bad with 1.3% positive answers among cases vs. no such response in controls with statistically significant difference in answers among groups (=26.410, df=4, p<0.001). assessment of self - reported health status is complemented with six questions on mobility, self - care, pain, cognition, interpersonal activities and affect (table 2). to all six questions, percentage of answers none is higher in controls, 56.9% vs 40.5% cases on the question for mobility, 70.7% vs 50.2% cases for self - care, 42.1% vs 31.8% for pain, 59.2% vs 41.4% for concentration and remembering things, 70.6% vs 48.4% for interactions with community, but high 51.6% in cases vs 27.8% controls in answers to question on feeling sad, low or depressed. percentage of answers moderate, severe and extreme / cannot do is higher in cases along all six dimensions and these differences are statistically significant. distribution of answers on questions on mobility, self - care, pain, cognition, interpersonal activities and affect logistic regression shows that gender was strongly associated with self rated health status, with higher odds of males to report poorer health, compared to women as a reference group. although there was no statistically significant difference in answers on self - rated health in different categories of marital status, it was associated with poorer health only in widowed individuals. statistically significant are also answers according to education category, except for respondents who have completed high school education. by ethnicity, macedonians and roma were twice as likely to report poor health and answers differ according to employment status, unemployed being 3 times more likely to report poor health, compared to other employment categories. place of residence also shows statistically significant differences in self - rated health, rural respondents having 6 times higher odds to report poor health compared to respondents residing in urban areas. as for behavioral variables, odds to report poor health were 8 times higher in regular smokers, 5 times higher for irregular smokers and alcohol was associated with 5 times higher odds for reporting poor health. positive association was found between long - standing illnesses and reporting poor health, such as angina pectoris and mental illness, while no association has been found for comorbidity with arthritis and asthma (table 3). adjusted odds ratios for socio - demografic characteristics and explanatory variables associated with self rated health = reference group 1or = odds ratio 2ci = confidence interval 3ngo = non - governmental oranisation our study shows that in the total study population, self - rated health was reported as excellent or good by only half of the respondents, with slightly less positive answers among cases compared to controls. differences are evident in responses for poor or extreme difficulties in everyday life among cases with very low percentage of such answers in controls. a positive association was found between poor rated health and long - standing diseases such as angina pectoris and mental illness and this finding is consistent with study of self - rated health among women in russia, finland and estonia (4). education was also associated with poor self - rated health, with exception of respondents with university education, similar to surveys in the former soviet union and other european countries (19, 20). adding questions on mobility, self - care, pain, cognition, interpersonal activities and affect has only reaffirmed the findings, with statistically significant differences among study groups along all six dimensions. diagnosis of angina pectoris or other cardiovascular diseases was associated with poorer self - reported health, similar to the swedish study that has established evidence on association of cardiovascular disease and low self - rated health with higher mortality (7). similar are also findings from a danish study that as defined predictors of mortality, such as sociodemographic factors, smoking and obesity (21) implying the importance to include self - rated health when studying risk factors for mortality (22). survey of this type has never been performed in republic of macedonia, thus we can emphasize this fact as a strength of the study, providing baseline information on possible factors that influence health of the population. limitations are those typical of self - reported data and the nested case - control study design, with probability of oversampling respondents with similar characteristics, associated with tb disease as the main selection criteria of cases that can somehow act as confounder of the findings. results can not provide evidence on causality and selection bias as well can not be estimated. survey of this type has never been performed in republic of macedonia, thus we can emphasize this fact as a strength of the study, providing baseline information on possible factors that influence health of the population. limitations are those typical of self - reported data and the nested case - control study design, with probability of oversampling respondents with similar characteristics, associated with tb disease as the main selection criteria of cases that can somehow act as confounder of the findings. results can not provide evidence on causality and selection bias as well can not be estimated. the study is the first step towards establishment of evidence that self - rated health is an important indicator to be followed by both clinicians and health planners in republic of macedonia. further research is needed to determine if self - rated health assessment in routine clinical setting can be used to identify groups at risk for increased mortality and other important health outcomes. however, given the ease of use of simple questions to ask for self - rated health, recommendation for routine collection of these data may offer an extremely beneficial tool in health care planning. | introduction : studying self - reported health is considered an indicator for morbidity and mortality that may be used in primary health care to detect poor health in certain population groups that predicts health care utilization.goal:the goal of the survey is to assess the socioeconomic self - rated health gradient and to describe contribution of behavioral risk factors to this gradient among population in republic of macedonia.material and methods : data is collected through a nested case - control study, conducted in the period march december, 2013. cases are households with tb patient(s) registered in the period july, 2012 june, 2013 and controls are households randomly chosen in cases immediate vicinity.results:the total study population is 562 households with total of 2720 respondents. self - rated health was reported as excellent or good by only half of the respondents, with slightly less positive answers among cases compared to controls and evident differences in responses for poor or extreme difficulties in everyday life. positive association was found between poor rated health and long - standing diseases and education was associated with poor self - rated health. adding questions on mobility, self - care, pain, cognition, interpersonal activities and affect has only reaffirmed the findings, with statistically significant differences among study groups along all six dimensions.conclusion:the ease of use of simple questions to ask for self - rated health makes it an extremely beneficial tool in health care planning. |
they are formed in the bone marrow and continuously enter the blood circulation, where they constitute 4% of the total leukocyte population in mice and 10% in humans. in human peripheral blood, three functionally different subsets of monocytes have been identified and characterized based on their expression of surface markers cd14 and cd16. the major monocyte subset, accounting for approximately 90% of the total monocyte population, expresses high levels of cd14 and no cd16 (cd14cd16), and these cells are referred to as classical monocytes. monocytes expressing cd16 can be further divided into two distinct subpopulations : intermediate monocytes that express relatively high levels of cd14 and some cd16 (cd14cd16) and nonclassical monocytes that express low levels of cd14 and high levels of cd16 (cd14cd16). analogously, mouse monocytes can be separated into two functionally different subsets based on their expression of ly6c, ccr2, and cx3cr1. the ly6cccr2cx3cr1 subset is equivalent to human classical and intermediate monocytes, whereas the ly6cccr2cx3cr1 subset is represented by nonclassical monocytes in humans [4, 5 ] (table 1). considering the strong evidence for comparable systems, murine monocyte subsets will be referred to as their human classical / intermediate or nonclassical counterparts from now on in this review. although the monocyte subsets share several common features, distinct functions have been attributed to the classical, intermediate, and nonclassical monocytes. during injury or inflammation, classical monocytes are rapidly recruited to invade the inflamed tissue and contribute to immunological responses, such as recognizing and removing microorganisms and dying cells. intermediate monocytes are recruited at a later stage of inflammation, and they are mainly associated with antigen presentation, high secretion of proinflammatory cytokines and chemokines, wound healing, and parasite recognition. nonclassical monocytes display a patrolling behavior and constantly survey the endothelium as part of the innate local surveillance. although the monocyte subsets are functionally different, hierarchical clustering and gene - expression profiling have shown that the subsets represent stages in a developmental sequence, with classical monocytes differentiating into intermediate and nonclassical monocytes [9, 10 ]. one of the best known functions of monocytes is, however, as a systemic reservoir of precursor cells for the renewal of several populations of tissue macrophages, dendritic cells (dcs), and osteoclasts [11, 12 ]. in the steady - state, the precursor function is primarily associated with classical monocytes, and whether intermediate and nonclassical monocytes can function as precursors for these immune cells in homeostasis has remained more elusive (figure 1). recent evidence indicates that the renewal of tissue macrophages and dcs in the steady - state hardly relies on the recruitment of monocytes but that these populations are rather maintained by longevity and local proliferation. in contrast, the contribution of monocytes as precursors is well documented during inflammation and, as a result, includes populations of immune cells normally not maintained by monocyte recruitment, such as populations of osteoclast in bone, macrophages in the heart, kidney, and liver, and dcs in the lungs (see figure 2). inflammation favors an expansion of cd16-expressing monocytes, resulting in an increased contribution of intermediate and nonclassical monocytes to populations of tissue - resident immune cells. in this way, the cd16-expressing monocytes contribute to the shaping of these immune cell populations during inflammation. the role of precursor heterogeneity in the generation of immune cells during inflammation has long incited immunological research, and new understandings have put the monocyte heterogeneity into sharp focus. this raises the question of the role of monocyte heterogeneity in the development and function of mature immune cells during inflammation. in this review, we outline and evaluate the discoveries that underlie these advances in our understanding of monocyte heterogeneity and its role in the shaping of monocyte - derived populations of macrophages, dcs, and osteoclasts in homeostasis and inflammation. monocytes are recruited sequentially to sites of inflammation as part of the host - protective immune response. in response to natural killer (nk) cell - produced interferon (ifn-), the monocytes locally differentiate into inflammatory macrophages and dcs and efficiently replace the resident mononuclear phagocytes. trafficking of the monocyte subset is controlled by different mechanisms and at least two sequential phases of monocyte recruitment to sites of inflammation have been identified (figure 3). following a myocardial infarction, classical monocytes are recruited within the first few hours, and their egression from the bone marrow is in principle controlled by the chemokine receptor ccr2 and its ligands ccl2 (or mcp-1) and ccl7 (or mcp-3). the recruited classical monocytes arrive in a highly inflammatory milieu where they exert an immediate and potent immune response by producing high levels of proinflammatory cytokines, such as interleukin il-1 and tnf-. in addition, they locally digest extracellular matrix and dead cells and produce il-18 to activate nk cells, thereby playing an important role in the progression of the immune response. a prolonged immune response from classical monocytes can contribute to tissue damage and initiate inflammatory cascades, as well as drive autoimmunity [26, 27 ]. some days later, when the acute inflammation resolves into a cardiac wound, the presence of classical monocytes diminishes and they are subsequently replaced by intermediate and nonclassical monocytes. in contrast to classical monocytes, cd16-expressing monocytes express low levels of ccr2 and rely on migration signals mediated by chemokine receptor cx3cr1 and its ligand cx3cl1. the nonclassical monocytes accumulate in the damaged tissue and contribute to angiogenesis and fibrosis [29, 30 ]. by secreting anti - inflammatory cytokines, such as il-10 and transforming growth factor tgf-, the cd16-expressing monocytes / macrophages counteract the tissue damage caused by an aggressive immune response from classical monocytes / macrophages. the distinct recruitment of the three monocyte subsets was recently also observed when studying an infected kidney mouse model, where classical monocytes / macrophages were observed to appear rapidly after infection. intermediate monocytes / macrophages arrived later and expressed genes associated with wound healing and released vascular endothelial growth factor and tgf-, supporting angiogenesis and collagen production. the nonclassical monocyte / macrophage population peaked 10 days after the kidney infection and expressed genes associated with fibrosis. similar observations have been made in patients with chronic inflammatory and fibrotic liver diseases, where intermediate monocytes accumulated in the inflamed liver as a consequence of enhanced recruitment of these monocytes from the circulation and local differentiation of classical monocytes in response to inflammatory factors. the same study concluded that these intermediate monocytes expressed both early macrophage and dc markers and were associated with increased phagocytic activity, antigen presentation, and secretion of proinflammatory cytokines (such as tumor necrosis factor tnf-, il-6, and il-1) and different growth factors consistent with a role in wound healing [17, 32 ]. thus, observations in both murine disease models and human patients suggest that the delayed recruitment of intermediate and nonclassical monocyte subsets and their subsequent differentiation into macrophages and dcs is a conserved mechanism that reflects a host - driven response to limit possible tissue damage caused by strong immune responses from classical monocytes / macrophages. it should be noted, however, that the fate of differentiated monocytes after resolution of inflammation remains as a subject of debate, although it has been suggested that they are able to undergo in situ phenotype conversation to become tissue - resident macrophages. however, the fact that monocytes are the immediate upstream precursors of these specialized cell populations is a dogma that only recently was refined with the usage of sophisticated fate - mapping techniques and different in vivo disease models [9, 33 ]. instead of depending on monocyte recruitment, several tissue - resident macrophage and dc populations rather appear to be maintained through longevity and local proliferation of precursors seeded during the embryonic development. yet, depletion of tissue - resident cell populations has demonstrated that circulating precursors in the blood can replenish numerous populations of specialized macrophages and dcs [34, 35 ], supporting the idea of blood monocytes as a circulating precursor reservoir that can be exploited on demand. although classical monocytes are contributing to some populations of tissue - resident dcs, macrophages, and osteoclasts in the steady - state, monocyte recruitment is strongly increased during inflammation and the affected distribution of monocytes, favoring an expansion of cd16-expressing monocytes, has great impact on the formation of monocyte - derived immune cells during inflammation. dendritic cells (dcs) are professional antigen - presenting cells and key regulators of innate and adaptive immune responses. a number of positive dc lineage markers have been identified that separates dcs into either the latter are not derived from circulating monocytes and are therefore not discussed further in this review. monocytes cultured in the presence of granulocyte macrophage - colony stimulating factor (gm - csf) and il-4 generate immature dc that differentiate further into mature dcs by tnf- stimulus [38, 39 ]. within the total population of classical dcs, several distinct subpopulations have been identified, each possessing distinct phenotypical and functional features [4042 ]. although dcs primarily are generated from pre - dcs in the circulation, selected dc populations in the dermis and the intestine are continuously repopulated by recruited classical monocytes [34, 44 ]. evidence supporting a role for cd16-expressing monocytes in the replenishment of dc populations in steady - state is lacking, and it is possible that the patrolling nonclassical monocytes leave the blood vessels and function as dc precursors exclusively in response to inflammatory stimuli. during inflammation, monocyte differentiation into dcs is not restricted to the skin or intestine but includes peripheral tissues normally not maintained by monocyte input (figure 2). the monocyte - derived dcs during inflammation have unique features, distinctly different from tissue - resident dcs generated during steady - state conditions. in vivo transfer experiments have shown that injected monocytes migrate to inflammatory sites and differentiate into dcs in various models of inflammation, including rheumatoid arthritis and dengue virus infection. as part of the innate immune system, monocyte - derived dcs during inflammation secrete high amounts of the anti - inflammatory cytokine il-10 and engulf apoptotic erythroid cells. accordingly, blocking differentiation of monocytes into dcs results in tissue damage due to severe and prolonged inflammation, cytotoxic t cell activity, and shortened host survival expectancy. monocyte - derived dcs have been suggested to contribute to the regulatory control of immune responses, and they produce large amounts of proinflammatory cytokines and enhance th2 cell - mediated immunity in the lungs. the specific contributions of classical, intermediate, and nonclassical monocytes to dc populations during inflammation were recently investigated in patients suffering from end stage renal disease, where chronic inflammation and dramatically increased numbers of circulating nonclassical monocytes were associated with an increased generation of dcs. the specific contribution of the monocyte subsets to populations of dcs during inflammation has long been a topic of debate, and it has been reported that functional differences exist between dcs generated from the different monocyte subsets. these differences include more potent immune responses from dcs derived from classical monocytes and better immune tolerance from dcs generated from nonclassical monocytes. similarly, it has been reported that classical monocytes selectively repopulate populations of cd103 dcs, whereas nonclassical monocytes differentiate into populations of cd11b dcs in the lungs. more recently, these findings were supported by similar observations reported in patients with tuberculosis. patients with tuberculosis have increased numbers of both intermediate and nonclassical monocytes in the circulation, and the cd16-expressing monocytes in these patients differentiate into dcs with poor mycobacterial antigen - presenting capacity. this is explained by the observation that stimulated cd16-expressing monocytes differentiate into alternative dcs with poor antigen - presenting function, expressing no cd1a and low levels of dc - sign on their plasma membrane. after lps stimulation, these inflammatory dcs produce large amounts of il-2 and ifn-, further driving the differentiation of monocytes into inflammatory mature immune cells. classical monocytes, on the other hand, generate functional cd1adc - sign dcs that efficiently stimulate t cell proliferation and secrete high amounts of il-12, il-1, il-10, and tnf- upon mycobacterium tuberculosis infection or lps stimulation. the increased presence of circulating cd16-expressing monocytes during inflammation appears to play a critical role in the development of dcs also in other pathological conditions. for example, in sepsis a systemic inflammatory response syndrome that occurs during infection monocytes derived from sepsis patients preferably differentiate into alternative cd1a dcs (similar to the dcs derived from cd16-expressing monocytes in patients with tuberculosis discussed above). these alternative dcs have an increased capacity to induce regulatory foxp3 + t cells, as compared with monocytes derived from healthy controls with a higher distribution of classical monocytes. thus, a growing body of circumstantial evidence suggests that the monocyte subsets give rise to functionally distinct dcs during inflammation and that the enhanced presence of circulating intermediate and nonclassical monocytes shapes populations of dcs during pathological conditions. macrophages are exquisitely adapted to their local environment and acquire organ - specific functionalities as part of their role in the maintenance of tissue homeostasis. development, differentiation, proliferation, and function of macrophages are regulated by the growth factor colony stimulating factor csf-1 and il-34. macrophages belong to a heterogeneous cell population, with several phenotypically and functionally distinct subsets. most macrophage populations are established prior to birth and maintain themselves by longevity and local proliferation, rather than monocyte recruitment. these macrophage populations include microglia in the central nervous system, kupffer cells in the liver, peritoneal macrophages, and splenic macrophages [9, 13 ]. microglia was early shown to originate from embryonic progenitors, and more recent research has identified the microglia precursors as primitive macrophages in the yolk sac. yet, in other tissues, including the intestine and the dermis, classical monocytes are continuously recruited to maintain the local macrophage populations in homeostasis. in addition, monocyte - derived cardiac macrophages appear to replace macrophages seeded during the embryonic development throughout the life span of an individual. it has been reported that monocyte - derived macrophages, similar to monocyte - derived dcs, are functionally different from their tissue - resident counterparts. monocyte - derived macrophages in the intestine express higher levels of cxc3r1, induce differentiation of foxp3 + t cells from nave cd4 t cells, and are required for induction of th17 cells and antigen - specific responses. whether cd16-expressing monocytes also contribute to macrophage populations in steady - state is unknown, and although early reports indicated that nonclassical monocytes differentiate into alveolar macrophages in homeostasis [67, 68 ], more recent research indicates that alveolar macrophages are in fact derived from fetal monocytes with minimal contribution of circulating blood monocytes. although classical monocytes appear to be the primary precursors of selected populations of macrophage during steady - state, the recruitment of all monocyte subsets during inflammation is strongly increased. inflammatory insults result in recruitment of monocytes to populations of tissue - resident macrophages that normally are maintained independently of monocyte influx, such as macrophages in the heart, in the ischemia brain tissue, and in the inflamed liver tissue (figure 2). the monocyte heterogeneity plays an important role in the generation of functionally distinct macrophages and the monocyte subsets appear to function as macrophage precursors in different pathological conditions. for example, infection with helminth parasites schistosoma mansoni and heligmosomoides polygyrus results in rapid invasion of classical monocytes into the adult murine heart, where they drive inflammation and generate oxidative stress. these classical monocytes subsequently differentiate into macrophages with limited capacity to promote tissue repair. however, in the absence of parasite challenge, such as during cardiac pressure overload, preferential recruitment and accumulation of nonclassical monocytes / macrophages in the cardiac tissue have been observed. similar to the selective recruitment of monocytes discussed above in section 2, the sequential differentiation of the monocyte subsets into macrophages in response to myocardial challenges is likely due to the individual features of the different monocytes / macrophages. macrophages derived from the different monocyte subsets have been shown to maintain some of the properties of their progenitors. for example, macrophages derived from classical monocytes express higher levels of cd14 on their surface compared to macrophages derived from nonclassical monocytes when cultured in vitro. while macrophages from classical monocytes exhibit phagocytic, proteolytic, and inflammatory functions, macrophages derived from cd16-expressing monocytes promote healing of the cardiac tissue by angiogenesis and deposition of collagen. the functional differences between macrophages derived from classical and cd16-expressing monocytes have given rise to the idea that classical monocytes differentiate into cardiac m1 macrophages, whereas cd16-expressing monocytes become m2 macrophages. this, however, still needs to be confirmed. in either way, the selective recruitment of specific monocyte subsets is context dependent and based on the nature of the challenge. thus, the sequentially recruited monocyte subsets during inflammation differentiate locally into macrophages with distinct capacities to drive inflammatory responses or promote tissue repair. osteoclasts comprise a subset of specialized macrophages that arise from fusion of monocytes in the presence of the cytokines macrophage - colony stimulating factor (m - csf) and receptor activator of nf-b ligand (rankl). these cells are uniquely capable of resorbing mineralized tissue, like bone, by binding tightly to the surface and by degrading the different matrix components by secreting acid followed by a cocktail of different proteolytic enzymes. although not considered traditional immune cells, a growing body of evidence suggests that osteoclasts contribute to inflammation and immune responses via the release of cytokines and via antigen presentation. functional differences between subsets of osteoclasts have been reported in homeostasis and include differences in size and proteolytic enzymes used for bone matrix digestion. although it was long assumed that monocytes are an important source of osteoclast precursors, this was not proven in situ until recently when fluorescently labeled monocytes were recruited from the circulation to the bone surface and differentiated locally into osteoclasts. accordingly, depletion of blood monocytes decreases osteoclastic bone degradation by limiting the homing of precursors to the bone surface. bone degradation by osteoclasts is crucial for skeletal maintenance, but increased and uncontrolled bone degradation during inflammation results is a severe pathological phenotype. due to their roles as osteoclast precursors, circulating monocytes form an excellent tool to study the early onset of inflammatory bone loss. in healthy individuals, it is the classical monocytes that harbor the highest propensity to differentiate into osteoclasts. interestingly, however, chiu. in 2010 reported a major shift in osteoclast precursors, from classical monocytes in healthy individuals towards an increased osteoclast formation from intermediate and nonclassical monocytes in patients with psoriatic arthritis (a chronic inflammatory arthritis characterized by severe bone erosion). the influence of an affected distribution of circulating monocytes on osteoclast formation during inflammation has also been observed when osteoclastogenesis of monocytes from patients with inflammatory bone loss has been studied in detail. monocytes isolated from patients with gaucher 's disease form osteoclasts faster than monocytes isolated from healthy controls and the generated osteoclasts display an increased bone - resorptive capacity when compared with osteoclast derived from monocytes isolated from healthy controls. similar observations were reported for patients with rheumatoid arthritis, where osteoclasts with increased bone - resorptive capacity were generated from monocytes derived from patients with rheumatoid arthritis. this indicates that it is in fact intrinsic properties of the isolated monocytes that cause the generation of distinct different osteoclasts and not altered cytokine levels in an inflammatory environment. interestingly, in all above - mentioned conditions (psoriatic arthritis, gaucher 's disease, and rheumatoid arthritis), a selective expansion of the intermediate monocyte subset has been reported, suggesting that in particular this subset is involved in the formation of functionally distinct osteoclasts in these inflammatory conditions [6, 87, 88 ]. accordingly, we recently demonstrated that in particular osteoclasts generated from intermediate monocytes expressed an increased capacity to resorb bone when they are treated with the inflammatory cytokine il-17a. taken together, the increased numbers of cd16-expressing monocytes and in particular intermediate monocytes appear to play a critical role in the generation of osteoclasts during inflammation and can possibly serve as an explanation for the increased osteoclast - associated bone loss observed in several inflammatory disorders. the role of monocytes as precursors for various mature immune cells has been well established. as our understanding of monocyte heterogeneity improves, their intriguing role as precursor cells is becoming increasingly important, and targeting of specific monocyte subsets to control differentiation and function of monocyte - derived immune cells emerges as an appealing therapeutic approach. the putative role of classical, intermediate, and nonclassical monocytes as distinct precursor cells during inflammation is of particular interest for immunological research, but our knowledge is limited and several important aspects are still unknown. this is partly due to the fact that the data collected so far mainly consists of in vitro observations and, unfortunately, few studies have investigated the correlation between an affected precursor population and the development of unconventional downstream immune cells during inflammation. defining the distinct differentiation fates of the monocyte subsets in different inflammatory conditions will enable more precise targeting of immune cells and provide a better understanding of the pathophysiology of inflammation. | blood monocytes are precursors of dendritic cells, macrophages, and osteoclasts. they are a heterogeneous cell population with differences in size, phenotype, and function. although monocytes maintain several tissue - specific populations of immune cells in homeostasis, their contribution to populations of dendritic cells, macrophages, and osteoclasts is significantly increased in inflammation. identification of a growing number of functionally different subsets of cells within populations of monocyte - derived immune cells has recently put monocyte heterogeneity into sharp focus. here, we summarize recent findings in monocyte heterogeneity and their differentiation into dendritic cells, macrophages, and osteoclasts. we also discuss these advances in the context of the formation of functionally different monocyte - derived subsets of dendritic cells, macrophages, and osteoclasts. |
autonomic modulation is the ability of the autonomic nervous system (ans) to respond to stimuli, both internal and external, in order to maintain homeostasis. this system is divided into two branches : the parasympathetic (psns) and sympathetic nervous system (sns). both branches affect heart rate (hr), blood pressure (bp) and the smooth muscle tone of blood vessels. ans activity can be measured non - invasively through variations in both hr and bp. spectral analysis of hr variability (hrv) provides information on the autonomic modulation of hr. specifically, parasympathetic activity is associated with the high frequency band of hrv within the range of 0.15 - 0.4 hz. the low frequency band of 0.04 - 0.15 hz is modulated by both psns and sns activity. spectral analysis of bp variability (bpv), as represented by baroreceptor sensitivity (brs) and the low frequency component of systolic bp (lfsbp), is a non - invasive measure of sns activity. tai chi chuan (tcc) is a martial art characterized by its slow, gentle and rhythmic movement patterns. as a method of exercise training, tcc it has been the subject of many research studies of motor control, psychological anxiety and cardiovascular function.[28 ] with regard to autonomic physiology, several authors have examined the acute effects of tcc after several weeks of training. these studies were cross - sectional and compared the autonomic modulation of individuals who were trained in tai chi with age - matched sedentary controls. during both the studies, measurements were taken before and after tai chi training and meditation. as was hypothesized, results were favorable for those who were trained in tcc and meditation as compared to those who were not. a third study compared tai chi training with low intensity cardiovascular training. none of the above studies controlled for respiratory rate during the measurement of the autonomic variables, which may have affected the validity of their findings. it is well established that respiratory rates modulate autonomic parameters and, as such, should be controlled for.[913 ] respiratory depth and frequency have been shown to affect the depolarization of the sinus node and thereby influence cardiac autonomic control. during inspiration, parasympathetic outflow to the sa node is impeded and results in shortening of the r - r interval (cardioacceleration) whereas, during exhalation, this pattern is reversed (cardiodeceleration). in this investigation, the efficiency of the myocardium to perform work can be represented by the myocardial oxygen consumption (mvo2), which is the most important indicator of the load on the heart.[1416 ] rate pressure product (rpp) is a non - invasive method of estimating mvo2 and can be calculated by multiplying hr by sbp and dividing by 100 (rpp = [(hr x sbp)/100 ]). the hemodynamic components of this product, hr and sbp, are modulated by both branches of the ans. hr is influenced by both the psns and sns, whereas, sbp is only affected by the sns. rpp is the parameter that is targeted by the pharmacological community in an attempt to decrease the incidence of ischemic events leading to infarctions. therefore, the aim of this study was to examine the rpp and autonomic responses of experienced tai chi practitioners and non - practitioners, while controlling for respiratory rates. the hypotheses were that the long - term practitioners of tcc would have an attenuated rpp and autonomic modulation favoring parasympathetic output at rest and during physical stressor phases when compared to sedentary - matched controls. based on statistical calculations from preliminary data of the dependent variables, 80% power at p 0.5. this method was based on the calculation of the modulus of the transfer function between sbp and pulse interval powers in the low - frequency band (0.04 - 0.15 hz). all data acquisitions and post - acquisition frequency domain analyses were carried out in accordance with the standards put forth by the task force on hrv interpretation. all recordings received an anonymous code and all dependent variables were analyzed in a blinded manner. several 2-way (group x stressor) analyses of variance (anova) were performed to determine if differences existed within and between groups with regard to autonomic responses to the experimental conditions. resting data were compared to the isometric and standing stressors in the following manner : rest vs. isometric grip and rest vs. standing. significant differences between group means were assessed, during each individual condition of testing, using a 1-way anova for each dependent variable being measured. based on statistical calculations from preliminary data of the dependent variables, 80% power at p 0.5. this method was based on the calculation of the modulus of the transfer function between sbp and pulse interval powers in the low - frequency band (0.04 - 0.15 hz). all data acquisitions and post - acquisition frequency domain analyses were carried out in accordance with the standards put forth by the task force on hrv interpretation. all recordings received an anonymous code and all dependent variables were analyzed in a blinded manner. several 2-way (group x stressor) analyses of variance (anova) were performed to determine if differences existed within and between groups with regard to autonomic responses to the experimental conditions. resting data were compared to the isometric and standing stressors in the following manner : rest vs. isometric grip and rest vs. standing. significant differences between group means were assessed, during each individual condition of testing, using a 1-way anova for each dependent variable being measured. the autonomic responses for rest, isometric contraction and standing stressors are presented in table 2. the hemodynamic responses for rest, isometric contraction and standing stressors are presented in table 3. vagal modulation, represented by the high - frequency modulation of hrv (hfrr nu), was significantly higher at rest (p<0.001) and during the isometric contraction stressor (p=0.002) for the tcc group compared to the nt group. low frequency to high frequency modulation ratio (lf / hf) of hrv, a non - invasive marker of sympathetic modulation, was significantly lower at rest (p<0.001) and during the isometric stressor (p=0.002) for the tcc group compared to the nt group. low - frequency modulation of systolic bpv, a non - invasive marker of sympathetic vasomotor modulation, was significantly lower at rest (p<0.001), during the isometric contraction (p<0.001) and standing stressors (p < 0.001) for the tcc group compared to the nt group. as shown in table 3, rpp and sbp were lower in the tcc group when compared to the nt group during each phase of testing (p<0.001). no significant differences were found in brs (alpha index) during any of the phases between groups. autonomic parameters at rest, during isometric grip and standing phases hemodynamic parameters at rest, during isometric grip and standing phases the major finding of this study was that long - term practitioners of tai chi demonstrated a significantly lower rpp when compared to sedentary - matched controls. in addition, findings in this study suggest that autonomic mechanisms may be in part responsible for the lowering of the rpp. specifically, tcc practitioners demonstrated significantly more autonomic modulation favoring parasympathetic outflow at rest and during the stressors. the stressors that were used simulated functional activities of daily living, such as grasping and holding (isometric grip) and positional changes (standing). however, the tcc group demonstrated responses that maintained a greater parasympathetic modulation and lesser sympathetic response. increased parasympathetic tone has previously been demonstrated as being cardioprotective. decreased hrv, as a result of increased sympathetic tone, has been shown to be unfavorable and can lead to cardiovascular complications. these adaptations are important in that tcc is a form of low impact exercise that can be easily performed by people of all ages. prior work has shown that aerobic exercise is effective in increasing cardiovascular health by reducing blood pressures, resting hr and increasing hrv. tcc requires the individual to perform movement patterns at a slow tempo for extended periods of time. the intensities of this training have been shown to be approximately 6 mets and comparable to low to moderate aerobic exercise of 50 - 60% of vo2 max. the use of tcc as a model of cardiovascular training, without the use of pharmacological intervention, to improve autonomic modulation has not been extensively or thoroughly studied. this may be very relevant considering the new blood pressure classification and suggestions to lower rpp. the major focus of anti - hypertensive treatment is lowering rpp and the modification of associated autonomic mechanisms. the results in this investigation provide suggestive evidence that tcc might be an effective and perhaps preventative non - pharmacological therapy. specifically, rpp is predicated upon the product of sbp and hr. moreover, blood pressure is the product of cardiac output and peripheral vascular resistance. beat - to - beat bpv, obtained using the finapres, provided insight for sympathetic vasomotor modulation. these values (lfsbp nu) were significantly lower at rest and during each stressor phase in the tai chi group. therefore, since arterioles are heavily innervated by sympathetic fibers, attenuation in sympathetic modulation may have resulted in less of a peripheral resistance. tcc practitioners demonstrated higher parasympathetic modulation during the stressors and these responses may have contributed to a lesser rise in rpp. the consistently lower lf / hf ratio supports the notion that the autonomic balance is preserved better in the tcc practitioners when compared to the non - trained - matched controls. respiration has been shown to affect both the frequency domain and hemodynamic parameters of the ans. previous work has demonstrated that slow breathing of 6 breaths per minute (0.1 hz) may increase the low - frequency component of the hrv power spectrum. this is a concern that is often unresolved due to large inter - individual differences in respiratory rate. controlling respiration rate at 12 breaths per minute may have contributed to the higher hr in the tcc group since this frequency of breathing was faster than they were normally accustomed to. prior studies on the effects of tcc and autonomic modulations failed to control for respiration, which may have weakened the validity of the results. although brs, as represented by the alpha index, was not shown to be significantly different between groups during any of the conditions of testing, sbp and lfsbp nu were significantly lower in the tcc group during each stressor. since these components are sympathetically mediated, the attenuated responses further confirm decreased sympathetic activity. one explanation for brs not being significantly different between groups is that tai chi training does not elicit elevated blood pressure responses as compared to other forms of exercise. more recent work has demonstrated an improvement in brs in patients with coronary artery disease when tcc was used in conjunction with cardiac rehabilitation and pharmacological intervention. although a cause - and - effect relationship can not be made due to the nature of this study, the efficiency of the myocardium, in the tcc group, was demonstrated by suppressed rpp values. this product contains elements of both parasympathetic (hr) and sympathetic (sbp) nervous system activity. although hr was significantly higher for the tcc group, a lower rpp value can only be attributed to the reduced blood pressure responses. therefore, results in this investigation appear to suggest that long - term practice of tcc may be effective in lowering rpp and provides additional evidence on some of the autonomic mechanisms responsible for these results. perhaps, tcc may be an effective adjunct to non - pharmacological anti - hypertensive therapy. | background : spectral analysis of autonomic nervous system activity can provide insight into cardiovascular function. rate pressure product is the parameter often targeted pharmacologically to decrease the incidence of myocardial events.aim:the purpose of this study was to investigate whether or not tai chi chuan practitioners would demonstrate autonomic responses that would be more cardioprotective when compared to non - trained controls.materials and methods : this was a cross - sectional study that measured the autonomic responses and rate pressure product of 2 groups of subjects ; a tai chi chuan trained (n = 13) and non - trained sedentary controls (n = 13) at rest and during 2 stressor phases that simulated functional activities of daily living.results:the tai chi group maintained a greater parasympathetic outflow at rest and during the isometric grip stressor phase (p<0.05). sympathetic outflow, systolic blood pressure and rate pressure product were significantly lower in the tai chi group at rest, during the isometric grip and standing stressor phases (p<0.05).conclusion : although a cause - and - effect relationship can not be concluded in this study, the tai chi group was able to demonstrate efficiency of the myocardium with suppressed rate pressure product values and autonomic responses that favored parasympathetic outflow. this type of training may complement non - pharmacological anti - hypertensive therapy. |
prostate cancer (pc) is one of the most common cancers at present in the male population. the number of diagnosed cases has risen predominantly due to the development of health care and the improvement of prevention and diagnostic methods, and, furthermore, presents an increasing tendency in poland. the number of registered cases in the silesian voivodeship in 2011 (with a decreasing population 9000 inhabitants fewer in 2012 compared to 2011) was 1260 (14.1% of cancers registered among men). in 2012, these numbers were 1482 and 15.7% respectively. these patients can be treated radically with surgery or radiotherapy (rt), i.e. external beam radiation therapy (ebrt) and brachytherapy (bt). brachytherapy is a relatively short treatment, but ebrt is time - consuming in the vast majority of cases the overall treatment time (ott) is approximately 8 weeks (the most common ebrt schedule is based on conventional irradiation specifically the delivery of a fraction dose (fd) of 1.82.0 gy to a total dose varying usually from 76 to 81 gy). taking this into account, along with the increasing incidence of pc, the rationale for the numerous attempts at ott reduction is clear. the choice of such a treatment modality is also supported by some radiobiological data suggesting a low value of pc alfa / beta coefficient 1.5 [4, 5 ]. on the other hand, this fractionation schedule is clearly correlated with a higher risk of late adverse effects in healthy vital organs (rectum, bladder). the key to resolving this issue involves high precision beam delivery and limited, narrow margins around the prostate. this condition can be accomplished using modern rt units such as the cyberknife (ck). the aim of the study was an evaluation of the effectiveness and tolerance of cyberknife - based radioablation in low and intermediate risk prostate cancer patients. the analyzed material comprised 200 prostate cancer patients aged 53 to 83 (mean age 69) treated between 2011 - 2014 with cyberknife - based radioablation. there were 94 low (lr) and 106 intermediate risk (ir) patients. according to our protocol of ck - based pc radioablation, we treated patients from lr and ir (except gleason 4 + 3) groups with a maximal prostate dimension smaller or equal to 50 mm. all patients are referred to radiotherapy by urologists (vast majority), or a patient may come directly to our institution (we do not have a urology ward). each patient and case is analysed and consulted on by our urologists or is sent to a local urologist to discuss the possibility of an operation. in total we have two subgroups of patients treated with ck : patients who can not be operated on due to medical reasons and patients who refuse surgery. 3 patients had gleason 2, 1 gleason 3, 19 gleason 4, 66 gleason 5, 95 gleason 6 and 16 a gleason score of 7. at our center prostate biopsies one core is placed in one container whereas the next is put separately into paraffin. six slices are obtained from one core and examined. as of the beginning of 2013, the minimal gleason score diagnosed on the basis of core biopsy is 3 + 2. in doubtful cases (i.e. as in all cases with a gleason score less than six) immunohistochemical examinations (amcr and p63) are performed. prostate cancer is diagnosed only if the amcr expression is positive and the p63 expression is negative. as we are an oncology center and do not have a urology ward, the vast majority of biopsies are done at other (sometimes remote) centers. in the group of patients analyzed, 38 out of 89 cases with a gleason score less than 6 were diagnosed prior to 2013. of the remaining 51 cases, only three were diagnosed at our institution (in all three cases gleason score 3 + 2). the maximum psa concentration varied from 1.05 to 19.53 ng / ml (mean 8.28, median 7.68). 97 patients presented with t1c, 45 with t2a, 40 with t2b and 18 with t2c stage respectively. the mean prostate dimensions were 42.5 x 37.6 x 40.4 mm in the x, y and z axes respectively. 123 patients suffered from hypertension and/or coronary disease, 25 from diabetes, 12 from asthma or obstructive pulmonary disease, 2 from anemia and 1 from polycythemia, rheumatoid arthritis and hyperthyroidism. 104 had nycturia, 67 polyuria, 41 urination difficulties, 10 dysuria, 3 erectile dysfunction, 2 hematuria, 2 anal bleeding and 1 case diarrhea. on the first day of radiotherapy the initial psa concentration varied from 0.008 to 15.24 ng / ml (mean 4.05, median 3.61). directly before the start of rt, 52.5% of patients were using adt (androgen deprivation therapy) (81 a combination of lhrh analogs and flutamide, 16 lhrh analogs alone, 7 flutamide alone and 1 bicalutamide). the duration of adt varied from 1 to 24 months (mean 5.4). according to our standards, there is no justification for adt in the lr group. however, nearly 100% of our prostate cancer patients are referred for radiotherapy by urologists from other centers (in the analyzed group 47 lr patients started adt before irradiation). in a substantial percentage of cases, we endeavour to discuss this with leading urologists and, in a number of cases, have convinced them to withdraw adt. all patients were irradiated using cyberknife a linear accelerator generating a 6 mv photon beam, installed on a 6-degrees - of - freedom robotic arm, integrated with a 6-degrees - of - freedom robotic couch. for prostate cancer patients, one of the options of the multiplan treatment planning system, the prostate template path, specifically designed for these clinical situations, was used. the tracking of implanted fiducials, ensuring the highest treatment precision, was continuously used during the treatment session. all patients underwent implantation with 3 fiducials 20 mm in length, 0.3 mm in diameter, incised each 2 mm golden wires, forming a stable, compressed form (gold anchors) during implantation (figure 1). compressed form of marker (gold anchor). the implantation was performed transrectally, using an ultrasound head with a specially - designed guide and a 203 mm long needle (diameter 0.71 mm) (figured 2 and 3). markers were implanted in a triangular - like configuration to ensure the possibility of potential prostate movement as well as its rotation, evaluation, and, furthermore, its correction (tracking). as one ck fraction delivery takes quite a long time (40 - 65 min.), the prostate can change its position in the pelvis as a result of bladder and rectum filling. for this reason, the prostate position has to be checked periodically and additionally, the position of the patient and the beam inlet geometry should be corrected. this procedure is fully automatic and can be done every 5 to 150 seconds. at the beginning of the fractional dose delivery, the prostate position is checked frequently ; if it is stable / unaltered the frequency of checking may be decreased. patients were irradiated every other day (overall treatment time [ott ] 9 days) using a fraction dose (fd) of 7.25 gy to the total dose (td) of 36.25 gy. td was delivered to the planning target volume (ptv), comprised of the clinical target volume (ctv prostate with proximal 1 cm of seminal vesicles) and an additional margin (3 mm in the posterior and 5 mm in the other directions respectively). the maximal accepted dose in the target (ptv) was 120% of the planned (36,25 gy) dose. the constraints for healthy tissues respected / observed during treatment planning are presented in table 1. constraints for organ at risk for femoral heads, 25 gy was accepted for 45% of their volume. radiotherapy planning (inverse) was performed on the basis of the ct and mri fusion using the multiplan system. patients were controlled at the radiotherapy end, and subsequently at 1, 4, 8, 14, 20 and 26 months, after treatment completion. the gastrointestinal (gi) and genitourinary (gu) acute adverse effects according to the eortc / rtog scale were monitored at the end of treatment and, moreover, 1 and 4 months thereafter. next, the late gi and gu reactions in the eortc / rtog scale were evaluated. during follow - up (fu) the percentage of evaluated patients without adt, gi and gu adverse effects, and their psa concentrations during fu gi gastrointestinal adverse effect (grade), androgen deprivation therapy, rt radiotherapy graphical illustrations of psa concentration decrease and the course of acute and late gi and gu adverse effects during follow - up are presented in figures 6, 7 and 8. the course of acute gi (gastrointestinal) and gu (genitourinary) adverse effects during follow - up. the course of late gi (gastrointestinal) and gu (genitourinary) adverse effects during follow - up. during fu one patient had a confirmed relapse (18 months after rt) (positive phoenix criterion + biopsy) and was treated with salvage brachytherapy. four patients, during follow - up, developed a second malignancy : one bladder cancer, one squamous cell lung cancer, one colon cancer and one papillary cancer of the thyroid gland. one of the largest patient groups (1100) is presented in a metaanalysis by king.. all patients were treated with a fd of 7.25 gy to a td of 36.25 gy (median fu 36 months). the 5-year biochemical failure - free survival rate (bffs) was 93% for the whole group, 95% for low risk (lr), 83% for intermediate risk (ir) and 78% for high risk (hr) cancer patients. 515 pc patients treated to a td of 35 - 36.25 gy are reported (median fu 72 months). the bffs for lr, ir and hr were 95.8%, 89.3% and 68.5% respectively. concerning the group of 477 lr and ir patients (td of 35 - 36.25 gy) reported by katz, psa decreased from 5.3 to 0.11 ng / ml. in the aforementioned study, late g2 gu and gi reactions were reported in 9.1% and 4% of patients respectively. in another study (a 6-year report), katz also describes late g3 adverse effects 2% and 6% for gu and gi respectively. a rather substantial percentage of gu late adverse effects is also described by chen. on the basis of 204 patients (td of 35 - 36.25 gy) three years after treatment, 5.7% reported leaking more frequently than once a day, 6.4% required pad usage and 10.8% indicated frequent dribbling. another large review of 1472 patients shows a very low percentage of severe acute effects after ck - based treatment of pc patients (g2 542% gu and 027% gi ; g3 0.5% gu and 0% gi). the same authors report 0 - 29% of gu g2 and 1.3% of gu g3 late effects. report 11.5% of moderate and 8.5% of severe acute gi reactions (diarrhea) and only 1.5% of late gi events (2 years after treatment) in the group of 269 patients irradiated to a td of 35.036.25 gy. describe 14.5% of gu late effects in a group of 228 patients with a minimum fu of 24 months. in the group of 100 patients describe 12% of g2 gu, 18% gi acute and 3% of g2 and 1% g3 gu late reactions. similarly, as in metaanalyses and in the aforementioned large single center patients group, the authors also report very good results in the smaller single center series. bernetich., in a group of 142 patients irradiated to a td of 35 - 36.25 gy in 5 fractions, present a 5-year 100% success rate bffs for very low risk (vlr), 91.7% for lr, 90% for ir and 86.7% for hr patients. report a decrease in psa median from 5.4 before treatment (4 x 9.5 gy) to 0.05 ng / ml 5 years later (bffs 96.2%) in the group of 79 lr and ir patients. in spite of a very aggressive radioablation schedule, acceptable percentages of g2 acute and g3 late gu adverse events were observed (10% and 6% respectively). a survey published by oliai. (70 patients) showed, during a 3-year - long observation, 100%, 95% and 77.1% of bffs for lr, ir and hr patients respectively irradiated to a td of 3536.25 gy delivered in five fractions. in the, concerning the irradiation of 57 patients (lr, ir, hr [9 patients ]) with a large prostate (> 50 ccm) to 3536.25 gy (5 fractions), a 2-year observation revealed a decrease in psa median from 6.5 to 0.4 ng / ml and a relatively high percentage of late gu g2 (49.1%) adverse effects. it is very difficult to compare our results (longest observation period 26 months) to those mentioned above with a substantially longer fu. moreover, considering the analysis of percentages of late (lack of g3, the maximum observed percentage of g2 0.7% for gi and 3.4% for gu) and acute (the maximum observed percentage of g3 0.6% for gi and 1% for gu ; 2.1% gi and 8.5% gu of g2) adverse effects observed in the analyzed group, these are even lower than those reported by other publications. we conclude from the above results and discussion that cyberknife - based radioablation in low and intermediate risk prostate cancer patients is an effective and safe radiation modality, enabling the achievement of a very low percentage of biochemical failures (0.5%) and adverse effects during a 26-month - long follow - up. | introductionprostrate cancer (pc) is one of the most common malignancies and is frequently treated with an 8-week course of radiotherapy. cyberknife (ck) based radioablation enables completion of therapy within 5 - 9 days. the aim of this study is an evaluation of the effectiveness and tolerance of cyberknife - based radioablation in prostate cancer patients.material and methods200 pc patients (94 low risk [lr ], 106 intermediate risk [ir ]) underwent ck irradiation every other day (fraction dose [fd ] 7.25 gy, total dose [td ] 36.25 gy, time 9 days). psa varied from 1.1 to 19.5 (median 7.7) and t stage from t1c to t2c. the percentage of patients with androgen deprivation therapy (adt), gi (gastrointestinal) and gu (genitourinary) toxicity (eortc / rtog scale), and psa were checked at 1, 4 and 8 months, and thereafter every 6 months up to a total of 26 months post-treatment.resultsthe percentage of patients without adt increased from 47.5% to 94.1% after 26 months. the maximum percentage of acute g3 adverse effects was 0.6% for gi, 1% for gu and g2 2.1% for gi and 8.5% for gu. no late g3 toxicity was observed. the maximum percentage of late g2 toxicity was 0.7% for gi and 3.4% for gu. median psa decreased from 7.7 to 0.1 ng / ml during fu. one patient relapsed and was treated with salvage brachytherapy.conclusionswe conclude that ck - based radioablation in low and intermediate risk pc patients is an effective treatment modality enabling ott reduction and presents a very low percentage of adverse effects. |
the chemokine proteins are a class of small molecules that play a significant role in leukocyte trafficking during immune response (1). ccr1, as one of the chemokine receptors, is expressed on a number of human cells such as monocytes, macrophages, dendritic cells, and t cells (2).a large number of studies have provided strong evidences for a significant role of the chemokines, rantes (regulated upon activation, normal t cell expressed and presumably secreted), and mip-1a (macrophage inflammatory protein-1) in chronic inflammatory diseases. because mip-1a and rantes are agonists for ccr1, antagonists for this protein may be helpful in treatment of these diseases (3). qsar has become a very well known discipline in the drug discovery researches (46). the basis of such relationships is the assumption that the variation of bioactivity of molecules, as expressed by pic50, can be regressed with changes in molecular descriptors. development of qsar involves selection of most informative independent variables to describe different sets of molecules and the application of various algorithms, such as multiple linear regression or ann to construct the qsar model. the advantage of ann is it inherent power to reveal non - linear relationships between idependent and dependent variables in the derivation of the qsar models. a quantitative structure activity relationship (qsar) model based on artificial neural networks (ann) was developed to study the activities of 29 derivatives of 3-amino-4-(2-(2-(4-benzylpiperazin-1-yl)-2-oxoethoxy) phenylamino) cyclobutenedione as c - c chemokine receptor type 1(ccr1) inhibitors (7). biological and chemical data from 29 derivatives of 4 - 3-amino-4-(2-(2-(4-benzylpiperazin-1-yl)-2-oxoethoxy)phenylamino)cyclobutenedione were used in this study (table 1) (7). all the structures were drawn and optimized using the semiempirical quantum - chemical routine of am1 implemented in hyperchem (8). the structures and biological activities of compounds the structures and biological activities of compounds 913 the structures and biological activities of compounds 1427 the structures and biological activities of compounds 2829 compounds selected as test set in order to model the biological activities of the studied compounds, four classes of descriptors were calculated : constitutional, geometrical, topological, and functional group using dragon (9) on the minimal energy conformations. the data set was devided into the training and the testing sets based on kennard and stone algorithm (10). principal component analysis was used to compress a pool of descriptors into principal components (pcs) as new variables. after that, a model using a nonlinear regression model, artificial neural network, was constructed to make a relationship between pcs and the pic50. a feed - forward ann with error back - propagation learning algorithm was applied for model building. pca was performed on the data set that gives 14 significant pcs (% variance explaind>1). therefore, the next steps of study were restricted to these 14 pcs. plotting of first pc vs. second pc showed none of the compounds is outlier (fig. clarification of the theory of the artificial neural networks in details has been adequately described elsewhere (11) and some relevant remarks is presented. the first two components (pc1, and pc2) from the principal component analysis of the 29 studied molecules. the first layer namely input layer has ni neurons, and function of this layer is reception of information (i.e. inputs) which transfers them to all neurons in the next layer called the hidden layer that their number are indicated by nh. the neurons in the hidden layer calculate a weighted sum of the inputs that is subsequently transformed by a linear or non - linear function. the last layer is the output layer and its neurons handle the output from the network and it calculate response vector. the function of synapses is connection of input layer to hidden layer and hidden layer to output layer. the manner in which each node transforms its input depends on the weights and bias of the node, which are modifiable. ba - ann network was trained with the training set of molecules using a back propagation algorithm followed by conjugate gradient descent in the second stage (47). rmsecv was then employed as tool to select optimum value of various parameters (6). figure 2 shows the effect of the different number of pcs on predictability of developed model. on the based of this figure, the ann has the highest degree of predictability when the number of pcs is 4. the parameters of network which should be optimized are learning rate, number of neuron in hidden layer, momentum.the optimal values for these parameters as it is shown in figures 3a - c are 1.2, 9, and 0.9, respectively. optimization of number of neurons in hidden layer (a), momentum (b), and, learning rate (c). the predicted activity of the ann calculated values of pic50 versus the experimental values are shown in figure 4 and reported in table 1. as it was expected, the calculated values are in good agreement with the experimental values. calculated vs. experimental activity of the investigated compounds in training and test sets various statistical criteria for ann model were calculated and reported in table 3 (58). the external predictability of a proposed model was generally tested using test sets and rcv2. the satisfactory prediction of the values of the inhibitory activity of the test set compounds demonstrates the efficacy of the qsar in predicting the activities of external molecules. various statistical parameter for developed pc- ann model r = square regression coefficient rmse = root mean square error press = predicted error sum of square for training set k=yiyiyi2fk'=yiyiyi2 pca was performed on the data set that gives 14 significant pcs (% variance explaind>1). therefore, the next steps of study were restricted to these 14 pcs. plotting of first pc vs. second pc showed none of the compounds is outlier (fig. clarification of the theory of the artificial neural networks in details has been adequately described elsewhere (11) and some relevant remarks is presented. the first two components (pc1, and pc2) from the principal component analysis of the 29 studied molecules. the first layer namely input layer has ni neurons, and function of this layer is reception of information (i.e. inputs) which transfers them to all neurons in the next layer called the hidden layer that their number are indicated by nh. the neurons in the hidden layer calculate a weighted sum of the inputs that is subsequently transformed by a linear or non - linear function. the last layer is the output layer and its neurons handle the output from the network and it calculate response vector. the function of synapses is connection of input layer to hidden layer and hidden layer to output layer. the manner in which each node transforms its input depends on the weights and bias of the node, which are modifiable. ba - ann network was trained with the training set of molecules using a back propagation algorithm followed by conjugate gradient descent in the second stage (47). rmsecv was then employed as tool to select optimum value of various parameters (6). figure 2 shows the effect of the different number of pcs on predictability of developed model. on the based of this figure, the ann has the highest degree of predictability when the number of pcs is 4. the parameters of network which should be optimized are learning rate, number of neuron in hidden layer, momentum.the optimal values for these parameters as it is shown in figures 3a - c are 1.2, 9, and 0.9, respectively. optimization of number of neurons in hidden layer (a), momentum (b), and, learning rate (c). the predicted activity of the ann calculated values of pic50 versus the experimental values are shown in figure 4 and reported in table 1. as it was expected, the calculated values are in good agreement with the experimental values. calculated vs. experimental activity of the investigated compounds in training and test sets various statistical criteria for ann model were calculated and reported in table 3 (58). the external predictability of a proposed model was generally tested using test sets and rcv2. the satisfactory prediction of the values of the inhibitory activity of the test set compounds demonstrates the efficacy of the qsar in predicting the activities of external molecules. various statistical parameter for developed pc- ann model r = square regression coefficient rmse = root mean square error press = predicted error sum of square for training set k=yiyiyi2fk'=yiyiyi2 pca was performed on the data set that gives 14 significant pcs (% variance explaind>1). therefore, the next steps of study were restricted to these 14 pcs. plotting of first pc vs. second pc showed none of the compounds is outlier (fig. clarification of the theory of the artificial neural networks in details has been adequately described elsewhere (11) and some relevant remarks is presented. the first two components (pc1, and pc2) from the principal component analysis of the 29 studied molecules. the first layer namely input layer has ni neurons, and function of this layer is reception of information (i.e. inputs) which transfers them to all neurons in the next layer called the hidden layer that their number are indicated by nh. the neurons in the hidden layer calculate a weighted sum of the inputs that is subsequently transformed by a linear or non - linear function. the last layer is the output layer and its neurons handle the output from the network and it calculate response vector. the function of synapses is connection of input layer to hidden layer and hidden layer to output layer. the manner in which each node transforms its input depends on the weights and bias of the node, which are modifiable. ba - ann network was trained with the training set of molecules using a back propagation algorithm followed by conjugate gradient descent in the second stage (47). rmsecv was then employed as tool to select optimum value of various parameters (6). figure 2 shows the effect of the different number of pcs on predictability of developed model. on the based of this figure, the ann has the highest degree of predictability when the number of pcs is 4. the parameters of network which should be optimized are learning rate, number of neuron in hidden layer, momentum.the optimal values for these parameters as it is shown in figures 3a - c are 1.2, 9, and 0.9, respectively. optimization of number of neurons in hidden layer (a), momentum (b), and, learning rate (c). the predicted activity of the ann calculated values of pic50 versus the experimental values are shown in figure 4 and reported in table 1. as it was expected, the calculated values are in good agreement with the experimental values. calculated vs. experimental activity of the investigated compounds in training and test sets various statistical criteria for ann model were calculated and reported in table 3 (58). the external predictability of a proposed model was generally tested using test sets and rcv2. the satisfactory prediction of the values of the inhibitory activity of the test set compounds demonstrates the efficacy of the qsar in predicting the activities of external molecules. various statistical parameter for developed pc- ann model r = square regression coefficient rmse = root mean square error press = predicted error sum of square for training set k=yiyiyi2fk'=yiyiyi2 in the developed model, a network including a fully connected three layer, feed - forward ann model trained with a back - propagation learning algorithm was used. the input of the network was the eigenvalue ranked pcs, the number of them which entered neural network varied from 1 to 14, of which 4 pcs of them were selected as input of networks. by using this number of pcs the best results on the basis of lowest rmsecv in the output of network were obtained. since there are no exact theoretical principles for choosing the appropriate network topology, before the training of the network, the adjustable parameters as number of nodes in the hidden layer, transfer function, learning rate and etc. the values resulting from hidden layer are transferred to the last layer, which contains a single neuron representing the predicted activity. various ann architectures were run with the four selected pcs as input. in each run, the neuron architecture and parameters were optimized to reach the lowest rmsecv as the performances of the resulted models. according to the criteria proposed by tropsha and roy (46), for testing the reliability and the robustness of qsar models, as discussed above, the calculated pcs have meaning physicochemically, but they may be employed for building statistical models which help the medicinal chemist limit the number of compounds to be synthesized. for instance, medicinal chemist can propose a training set comprised of molecules which have the characters of two or more chemical classes with the smallest amount of similarity. therefore, the qsar model was used to estimate inhibitory activities of a few suggested compounds. the general structures of four suggested compounds and also their calculated activities are reported in table 4. the relative high predicted activity of the tested compounds suggest further study such as synthesis of other compounds with such chemical structures. the main objective of this study was to define and establish a qsar model to predict bioactivity of a series of 3-amino-4-(2-(2-(4-benzylpiperazin-1-yl)-2-oxoethoxy) phenylamino) cyclobutenedione derivatives as novel ccr1 antagonists without any knowledge of the under study system. calculated pcs were used to make model of the relationship between the molecule structures of the studied compounds and the corresponding bioactivities. the study showed that the calculated pcs as input variable to network can improve the predictive ability of the neural networks. moreover, the suggested qsar model was based on nonlinear ann approach, which can be employed to simulate any kinds of complex correlation or function relationship in a given multivariable system. i.e., ann approach is more appropriate for modeling where no clearly defined mathematical model for a system is available. therefore, accurate, well - organized and intelligent qsar model for the bioactivity will be influential for drug design and development. | background and purpose of the studya quantitative structure activity relationship (qsar) model based on artificial neural networks (ann) was developed to study the activities of 29 derivatives of 3-amino-4-(2-(2-(4-benzylpiperazin-1-yl)-2-oxoethoxy) phenylamino) cyclobutenedione as c - c chemokine receptor type 1(ccr1) inhibitors.methodsa feed - forward ann with error back - propagation learning algorithm was used for model building which was achieved by optimizing initial learning rate, learning momentum, epoch and the number of hidden neurons.resultsgood results were obtained with a root mean square error (rmse) and correlation coefficients (r 2) of 0.189 and 0.906 for the training and 0.103 and 0.932 prediction sets, respectively.conclusionthe results reflect a nonlinear relationship between the principal components obtained from calculated molecular descriptors and the inhibitory activities of the investigated molecules. |
chip was performed using tagged tap - abp1 and tap - cbh1 strains with an anti - calmodulin binding protein antibody (millipore) and a polyclonal serum against the native sap1 protein9. high throughput sequencing was performed on the illumina g2 genome analyzer, and analyzed for polymorphism detection or statistical analysis of enrichment. 2d gel electrophoresis was performed as described11, see supplementary information for construction of the episomal system. | centromere - binding protein b (cenp - b) is a widely conserved dna binding factor associated with heterochromatin and centromeric satellite repeats1. in fission yeast, cenp - b homologs have been shown to silence long terminal repeat (ltr) retrotransposons by recruiting histone deacetylases2. however, cenp - b factors also have unexplained roles in dna replication3, 4. here, we show that a molecular function of cenp - b is to promote replication fork progression through the ltr. mutants have increased genomic instability caused by replication fork blockage that depends on the dna binding factor switch activating protein 1 (sap1), which is directly recruited by the ltr. the loss of sap1-dependent barrier activity allows the unhindered progression of the replication fork, but results in rearrangements deleterious to the retrotransposon. we conclude that retrotransposons influence replication polarity through recruitment of sap1 and transposition near replication fork blocks, while cenp - b counteracts this activity and promotes fork stability. our results may account for the role of ltr in fragile sites, and for the association of cenp - b with pericentromeric heterochromatin and tandem satellite repeats. |
polycystic ovary syndrome (pcos) is a common endocrine disorder in women at reproductive age (1, 2). pcos is diagnosed with oligomenorrhea and/or anovulation as well as clinical and/or biochemical hyperandrogenemia with / without ultrasonographic evidence of polycystic ovaries. the diagnosis is made after exclusion of other disorders including thyroid dysfunction, hyperprolactinemia, nonclassical adrenal hyperplasia, cushing syndrome, and androgen producing tumors (3). according to androgen excess society (aes), pcos phenotypes including phenotype of hyperandrogenism and oligomenorrhea had a higher prevalence of metabolic syndrome when matched with the control subjects (4, 5) ; moreover, recent data support an increased incidence of cardiovascular events in women with pcos, which are related to androgens level (6 - 8). pcos is also associated with an increased risk of insulin resistance (ir), type 2 diabetes mellitus, dyslipidemia, and atherosclerosis (9 - 13). ir is also a risk factor for metabolic abnormalities and increases the risk of cardiovascular events. high mean platelet volume (mpv) might contribute to the endothelial injury by platelet activation and higher plasma concentrations of thromboxane a2 (14). several reports have demonstrated that there is an association between mpv and cardiovascular risk factors (15) such as obesity (16), diabetes mellitus (17), hypertension (18), acute coronary syndrome (19), and stroke (20). according to aes, ir and hyperandrogenemia are risk factors for cardiovascular diseases in patients with pcos (4, 5). regarding the current evidences, it is reasonable to investigate the association of mpv with androgens as well as ir in patients with pcos. to our knowledge, just one study investigated the association of mpv with androgen levels in patients with pcos (20) ; however, the mentioned study included overweight and obese patients. therefore, our study was designed to determine whether there was any association between androgens / ir and mpv in nonobese patients with pcos. a total of 136 patients with newly diagnosed reproductive - age pcos (regarding to criteria of new pcos phenotypes, based on the rotterdam criteria) (3) who were nonobese (body mass index [bmi ], 20 - 25 kg / m) were included. for control group, 59 healthy subjects (bmi, 20 - 25 kg / m) were recruited. all patients completed a standard questionnaire regarding their detailed history of menstrual cycles, acne, hirsutism, and medications. oligomenorrhea was defined as menstrual cycles occurring at intervals > 35 days, with only four to nine periods in a year. bmi (kg / m) was calculated by dividing weight in kilograms by the square of height in meters. the presence of terminal hair growth was scored using ferriman gallwey method (21) ; hirsutism was defined as a score > 8 (22). the presence or absence of acanthosis nigricans and acne were noted. individuals with hyperprolactinemia, congenital adrenal hyperplasia, cushing 's syndrome, and those who had taken hormonal treatment for at least three months prior to their evaluation were excluded. furthermore, patients with hypertension and a history of angina or myocardial infarction, obesity, diabetes mellitus, diagnosed coagulation abnormalities or abnormal results of hematological parameters, any systemic disease, vascular or thyroid disorders, infection or inflammatory diseases, and malignancy were excluded. the control group consisted of healthy women with regular menstrual cycles, normal bmi, and no medication during preceding three months. venous blood samples for assessing fsh and lh (miu / ml), estradiol (pg / ml), prolactin (ng / ml), total testosterone (ng / ml), androstenedione (ng / ml), and dehydroepiandrosterone - sulfate (dheas) (g / dl) were obtained from all participants in the morning between 08:00 a.m. and 09:00 a.m. after an overnight fast. serum fsh, lh, total testosterone, estradiol, progesterone, and dheas levels were measured by the chemiluminescent microparticle immunoassay (paramagnetic particle, chemiluminescent immunoassay) using a unicel dxi 800 system immune - analyzer (beckman coulter ireland inc., radioimmunoassay (ria) with commercial kits was employed to assess serum free testosterone (biosource, nivelles, belgium) and androstenedione levels (radim, roma, italy). insulin (iu / ml) was analyzed by electrochemiluminescence immunoassay (eclia) and via roche htach cobalt 600 device. fasting blood glucose (mg / dl) levels were measured by the roche hitachi t800 device. ir was calculated by the ir index as determined by homeostasis model assessment (homa - ir) with the following formula : fasting blood glucose (mg / dl) fasting insulin (iu / ml)/405 ; values > 2.7 were considered as ir. ovarian volume was calculated by the following formula : v = (/6) length (mm) width (mm) thickness (mm). the presence of polycystic ovaries was diagnosed by the presence of 12 or more follicles in each ovary with 2-mm to 9-mm diameter and/or increased ovarian volume (> 10 cm). data analysis was performed using pasw 18 (ibm, armonk, ny, usa). when comparing the continuous variables student s t test was used for the normally distributed subset and mann - whitney u test was used for the otherwise distributed subset. the approval of the study protocol was obtained from institutional review board and each participant signed an informed consent regarding the principles in the declaration of helsinki. a total of 136 patients with newly diagnosed reproductive - age pcos (regarding to criteria of new pcos phenotypes, based on the rotterdam criteria) (3) who were nonobese (body mass index [bmi ], 20 - 25 kg / m) were included. for control group, 59 healthy subjects (bmi, 20 - 25 kg / m) were recruited. all patients completed a standard questionnaire regarding their detailed history of menstrual cycles, acne, hirsutism, and medications. oligomenorrhea was defined as menstrual cycles occurring at intervals > 35 days, with only four to nine periods in a year. bmi (kg / m) was calculated by dividing weight in kilograms by the square of height in meters. the presence of terminal hair growth was scored using ferriman gallwey method (21) ; hirsutism was defined as a score > 8 (22). the presence or absence of acanthosis nigricans and acne were noted. individuals with hyperprolactinemia, congenital adrenal hyperplasia, cushing 's syndrome, and those who had taken hormonal treatment for at least three months prior to their evaluation were excluded. furthermore, patients with hypertension and a history of angina or myocardial infarction, obesity, diabetes mellitus, diagnosed coagulation abnormalities or abnormal results of hematological parameters, any systemic disease, vascular or thyroid disorders, infection or inflammatory diseases, and malignancy were excluded. the control group consisted of healthy women with regular menstrual cycles, normal bmi, and no medication during preceding three months. venous blood samples for assessing fsh and lh (miu / ml), estradiol (pg / ml), prolactin (ng / ml), total testosterone (ng / ml), androstenedione (ng / ml), and dehydroepiandrosterone - sulfate (dheas) (g / dl) were obtained from all participants in the morning between 08:00 a.m. and 09:00 a.m. after an overnight fast. serum fsh, lh, total testosterone, estradiol, progesterone, and dheas levels were measured by the chemiluminescent microparticle immunoassay (paramagnetic particle, chemiluminescent immunoassay) using a unicel dxi 800 system immune - analyzer (beckman coulter ireland inc., radioimmunoassay (ria) with commercial kits was employed to assess serum free testosterone (biosource, nivelles, belgium) and androstenedione levels (radim, roma, italy). insulin (iu / ml) was analyzed by electrochemiluminescence immunoassay (eclia) and via roche htach cobalt 600 device. fasting blood glucose (mg / dl) levels were measured by the roche hitachi t800 device. ir was calculated by the ir index as determined by homeostasis model assessment (homa - ir) with the following formula : fasting blood glucose (mg / dl) fasting insulin (iu / ml)/405 ; values > 2.7 were considered as ir. ovarian volume was calculated by the following formula : v = (/6) length (mm) width (mm) thickness (mm). the presence of polycystic ovaries was diagnosed by the presence of 12 or more follicles in each ovary with 2-mm to 9-mm diameter and/or increased ovarian volume (> 10 cm). data analysis was performed using pasw 18 (ibm, armonk, ny, usa). when comparing the continuous variables student s t test was used for the normally distributed subset and mann - whitney u test was used for the otherwise distributed subset. the approval of the study protocol was obtained from institutional review board and each participant signed an informed consent regarding the principles in the declaration of helsinki. there were no significant differences between patients and controls with respect to age (p = 0.07) and bmi (p = 0.312) (table 1). abbreviations : bmi, body mass index ; and pcos, polycystic ovary syndrome. total testosterone, free testosterone, androstenedione, dheas, lh, and estradiol levels and lh / fsh ratio were significantly higher in patients than in controls (p 0.05). there was difference in mpv values between patients with high androgen levels (mean, 8.7 fl (7.1 - 11.9) and patients with normal levels (mean, 9.5 fl (6.9 - 13.2), p = 0.012) as well as between patients with high androgen levels and controls (mean, 8.9 fl (7.7 - 9.1), p = 0.04). no difference was observed between patients with normal androgen hormones levels and controls in mvp values (p > 0.05). there was a negative correlation between total testosterone and dheas with mpv (p = 0.016, r = -0.229 ; and p = 0.006, r = -0.261, respectively) (table 3 and figures 1 and 2). r, correlation coefficient ; n, number of the patients with pcos ; and dheas, dehydroepiandrosterone - sulfate. abbreviations : dheas, dehydroepiandrosterone - sulfate. many recent studies have demonstrated the close relationship between atherosclerosis and increased mpv (8 - 12). our study revealed that mpv did not differ between nonobese women with pcos and controls. in contrast, a previous study reported an increased mpv in women with pcos in comparison to non - pcos controls (20). the discrepancy between the findings of two studies could be due the inclusion of obese patients with pcos in the previous study and increased mpv in this population might occur as a consequence of obesity, which is known to causes increased mpv (15). a recent study including nonobese and obese patients with pcos demonstrated that mpv did not correlate with pcos except in patients with obesity (23). according to aes (4, 5) and a review (24, 25), growing evidence links androgens with pathophysiology of pcos and metabolic derangements. moreover, a recent study has announced that hyperandrogenism might be the progenitor of inflammation, which is independent of obesity in the patients with pcos. the authors declared that further studies are needed to evaluate the effect of androgen hormones on metabolic abnormalities in patients with pcos (26). in this regard, a recent study has reported a positive correlation between mpv and dheas levels in pcos (20). in contrast, our study showed that high levels of androgens were associated with the low mpv values in the nonobese patients with pcos. this contrast might be due to enrolling obese patients and a smaller sample size of the former study. thus, we suggest that the studies that aim to investigate the association between mpv and any hormonal / metabolic parameters in patients with pcos must exclude the patients with risk factors of atherosclerotic such as obesity, which may affect mpv. in addition, recent studies support an increased incidence of cardiovascular events related to ir and/or obesity in patients with pcos (27). in our study, ir was higher in nonobese women with pcos than in healthy controls. all these findings suggest that ir might not be an independent risk factor for mpv changes in nonobese patients with pcos. finally, if the result of our study could be confirmed by prospective studies including four groups, namely, obese women with pcos, nonobese women with pcos, nonobese healthy women, and obese healthy women, the effect of obesity on mpv could be better understood. additionally, ir, which is one of the most common features of pcos, was not related to mpv in nonobese patients with pcos. lastly, we showed that elevated androgen levels caused a decrease in mpv in the same population. | background : mean platelet volume (mpv) is generally accepted as a new marker of cardiovascular disease risk in several studies.objectives:this study aimed to determine the association of mpv with androgen hormones and insulin resistance (ir) in nonobese patients with polycystic ovary syndrome (pcos).patients and methods : a total of 136 patients with newly diagnosed reproductive - age pcos (regarding the criteria of new pcos phenotypes, based on the rotterdam criteria) who were nonobese with the mean age of 25 years (25.39 5.51) and mean body mass index (bmi) of 21 kg / m2 (22.07 2.13) were included. in addition, 59 healthy subjects with mean age of 26 years (22.07 2.13) and mean bmi of 22 kg / m2 (21.52 3.84) were recruited as control. total blood count (including mpv), total testosterone, free testosterone, dehydroepiandrosterone - sulfate (dheas), and androstenedione levels were recorded. ir was calculated from blood chemistry measurements of fasting insulin and glucose according to updated homeostasis model assessment.results:no differences were observed in mean mpv values between patients and control group (9.02 fl (8.5 - 10.1) and 8.9 fl (7.7 - 9.1), respectively ; p = 0.777). mpv values were similar among nonobese patients with and without ir and control subjects (p > 0.05). we detected significantly lower values of mpv in patients with hyperandrogenemia in comparison to patients with normal androgen levels (8.7 and 9.5 fl, p = 0.012). there was a negative correlation between total testosterone, dheas, and mpv (p = 0.016, r = -0.229 ; and p = 0.006, r = -0.261, respectively). multiple logistic regression analyses confirmed the independence of these associations.conclusions:our study revealed that nonobese women with and without pcos have similar mpv values. while ir does not have any effect on mpv, elevated androgen levels are associated with a low mpv in nonobese patients with pcos. |
dyskeratosis congenita (dc) is an inherited disorder with progressive multi - system involvement leading to bone marrow failure (bmf), squamous cell carcinoma, pulmonary fibrosis (pf), etc. liver transplantation (lt) for cirrhosis secondary to dc has not been reported previously and we describe here the perioperative management of the same. a patient with diagnosed dc at 25 years of age presented with esld to our institute in his 34 year. past history included haematopoietic stem cell transplant (hsct) for bmf secondary to dc. patient was diagnosed with cirrhosis post - hsct and was kept on medical management for 9 years. at the time of presentation he was not on any immunosuppressant. clinical examination revealed multiple dental caries, reticulate skin pigmentation, nail dystrophy, severe clubbing, epiphora, enlarged liver with irregular margins, splenomegaly and ascites. pre - operative liver function tests showed hypoalbuminemia (2.6 g / dl) with slightly elevated transaminases. on arterial blood gas (abg) analysis, partial pressure of oxygen (pao2) was 51.3 mm hg with widened alveolar arterial oxygen gradient (pao2pao2 = 59.7 mm hg) [table 1 ]. abg with 100% o2 supplementation demonstrated an increased pao2 (158 mm hg) and spo2 of 100% [table 1 ]. arterial blood gas values at various time points high resolution computerized tomography (ct) demonstrated mild bilateral interstitial lung disease (ild) [figure 1 ]. saline contrast echocardiography (sce) suggested severe right to left intrapulmonary shunting and macroaggregated albumin scan (maa) demonstrated a 21% shunt fraction. child - turcotte - pugh score was 10 (child c) and model for end stage liver disease score was 14. patient was diagnosed with cirrhosis secondary to dc and posted for urgent living donor liver transplantation (ldlt). high resolution computerized tomography thorax showing mild bilateral interstitial lung disease apart from standard anaesthesia monitoring, invasive haemodynamic monitoring (central venous pressure, invasive arterial blood pressures and continuous cardiac output monitoring using flowtrac ev1000 edwards life sciences) were used. anaesthesia was induced with propofol (1.5 mg / kg), fentanyl (1.5 gm / kg), and rocuronium (0.9 mg / kg) with modified rapid sequence induction. maintenance of anaesthesia was with isoflurane in air (minimum alveolar concentration 11.2), with infusions of atracurium and fentanyl. baseline abg after induction of anaesthesia showed pao2 of 140 mm hg with fio2 of 0.5. persistent svv > 13% in absence of sustained major vascular occlusion or arrhythmias was treated with 250 ml boluses of 5% albumin. noradrenaline and vasopressin infusion were titrated for a targeted mean blood pressure > 65 mm hg. after reperfusion, fio2 requirements increased from 0.5 to 0.7 to maintain the pao2 above 100 mm hg. estimated blood loss during the surgical procedure was 2100 ml. patient was gradually weaned from mechanical ventilation and vasopressors and extubated on post - operative day 2 in the intensive care unit. post - extubation intermittent non - invasive bilevel positive airway pressure (bipap) support was provided. immunosuppression was provided with steroids, tacrolimus and mycophenolate. on post - operative day 7, patient developed tachypnoea, increased o2 requirement, fever and productive cough. chest auscultation revealed bilateral basal crepitations and basal lobe consolidation on chest x - ray. oxygen requirements were increased and were provided via high concentration o2 mask with reservoir bag to maintain acceptable levels of saturation. on availability of sputum culture reports (culture demonstrated escherichia coli sensitive to carbapenems), patient was shifted from broad spectrum empirical antibiotics to culture specific antibiotics and patient gradually improved with decrease in oxygen requirements. patient was discharged from the hospital on post - operative day 29 with a pao2 of 70 mm of hg on room air, which at 2 months was 78 mm of hg. dyskeratosis congenita is a rare multisystem disorder characterized by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leucoplakia. patients exhibit considerable clinical and genetic heterogeneity with x - linked recessive, autosomal dominant and recessive forms. bone marrow failure and pf are the most common causes of mortality(6070% for bmf/15% for pf) in these patients. hsct is the only curative option for bmf but increases the risk of pulmonary complications. hepatic involvements including hepatomegaly, hemosiderosis, fibrosis, and cirrhosis have been reported in 10% of cases. presence of severe hypoxemia and orthodeoxia and absence of ild led to a probable diagnosis of hepatopulmonary syndrome (hps). imbalance between vasoconstrictors and vasodilators leading to pulmonary vascular dilatation at the pre - capillary and capillary level in patients with liver disease results in hps. subsequent intrapulmonary shunting and v - q mismatch causes progressive hypoxia, dyspnoea, and cyanosis. the patient reported here was fulfilling the criteria for hps : (1) chronic liver disease (cld), (2) alveolar - arterial oxygen gradient > 15 mm of hg and intrapulmonary shunt (demonstrated by sce and maa). severe intrapulmonary shunting ~21% and pao2 of 51.5 mm of hg on room air placed him in almost the very severe category of type i hps. lt being a definitive treatment for hps and severity of hypoxaemia limiting time for medical therapy trial, lt was offered to the patient on an urgent basis. the prevalence of hps in patients with advanced liver disease is approximately 1633%. without lt the 5 years survival rate for cirrhotic patients with hps however, more recently gupta. in their study have reported a mortality rate of 9% in patients with severe hps in peri - transplant period. it has also been demonstrated that severity of pre - operative hypoxaemia correlates with the peri - transplant mortality. rapid progression of pre - operative hypoxaemia in these patients mandates minimal transplant waiting time. ldlt reduces transplant waiting time and prevent functional status deterioration and thus provide good results in patients with severe hps. the low acceptable thresholds of pao2 values were kept keeping in mind pre - operative severe hps and hypoxaemia. restrictive fluid therapy using svv along with lung protective ventilation strategies (low tidal volume) were used to minimize pulmonary complications. other supportive therapies like o2 supplementation, incentive spirometry, intermittent non - invasive ventilatory support, adequate attention to asepsis and antibiotics were provided as per institute 's protocol. hps can be an important cause of hypoxemia in patients of dc presenting with cld. directed pre - operative evaluation to determine the primary cause of hypoxaemia can help in identifying patients who may benefit from lt. | dyskeratosis congenita (dc) is an inherited disorder with progressive multisystem involvement. end stage liver disease (esld) in patients with dc is rare. we describe the perioperative management of a patient with dc induced esld and severe hepatopulmonary syndrome for living donor liver transplantation. |
osteoarthritis (oa) is the most common chronic joint disorder, and it causes detrimental effects on the quality of life and functional status. these are characterized by progressively occurring cartilage destruction, osteophyte formation, and subchondral sclerosis1, 2. the histopathological findings of oa show that homeostasis between the destruction and repair mechanisms of the joint cartilage is disturbed by the increased expression of proinflammatory cytokines (il-1, tnf alpha), matrix metalloproteinases, aggrecanases, nitric oxide, and prostaglandins. this causes degradation of the joints along with the insufficiency in the synthesis of growth factors (gfs), collagens, proteoglycans, and anti - inflammatory cytokines (il-4, il-10)3, 4. conservative treatments have been reported to increase the quality of life of patients particularly in the early phases, when the pathophysiology of the disease does nt change5. the effects of these treatments are short term and their local and systemic side effects cause frequent problems. therefore, recent studies have focused on stimulating cartilage healing processes through administration of growth factors (gf), cytokine inhibitors, matrix metalloproteinase inhibitors, or il-1 receptor antagonists5, 6. platelet - rich plasma (prp) is an autologous concentration of a high number of platelets in a small volume of plasma, and it is prepared by centrifugation of blood. platelets contain significant amounts of cytokines and growth factors which are capable of stimulating cellular growth, vascularization, proliferation, tissue regeneration, and collagen synthesis. delivery of high concentrations of cytokines and gfs to damaged tissues by prp is considered to have a beneficial effect on tendon and cartilage tissue regeneration7, 8. in some in vitro and in vivo studies, anti - inflammatory and reparative effects of prp on cartilage, tendon, and ligament recovery have been shown9,10,11 ; however, there is no consensus on eligible patient selection, the number and frequency of injections, the preparation technique, or the appropriate platelet concentration5. in knee oa, prp injections aim to promote cartilage repair and relieve osteoarthritic symptoms, potentially delaying the need for joint replacement surgery12. some studies have reported a reduction in prp efficacy in moderate and advanced (kellgren lawrence grade 34) knee osteoarthritis, as this group of patients have higher pain and functional impairment, which require more medical attention13, 14. in some studies, it was suggested that in terms of prp activity, oa and chondropenia level is more critical than platelet number and function4. grade 4 oa generally requires surgical treatments such as tibial osteotomy and total knee replacement. in the present study the effects of prp administration to control the disease activity of grade 3 knee oa either with one injection, two injections two weeks apart, or three injections separated by 2-week intervals on the patients pain, quality of life and physical activity levels were investigated. patients in the age range of 4075 years who visited our physical medicine and rehabilitation outpatient clinic between may 2014october 2014 because of single knee pain for a minimum 6 months were recruited for this study. oa was diagnosed according to the american college of rheumatology (acr) criteria15. radiological assessment was conducted by standing anteroposterior and lateral knee radiography according to kellgren- lawrence grading system16. a total of 102 patients identified with grade 3 knee osteoarthritis (with multiple osteophytes, definite joint space narrowing, sclerosis and bony deformity) were studied. the exclusion criteria were bilateral symptomatic knee oa ; age older than 75 years ; receiving physical therapy, intra - articular steroid, hyaluronic acid or prp injections in the last 6 months ; recent history of severe trauma of the affected knee ; active infection, inflammation or tumor existence around the knee ; history of diabetes mellitus, severe cardiovascular diseases, coagulopathies, malignant, immunosuppressive, collagen vascular or autoimmune disorders ; hb values of 30 = impaired mobility20. the mean and the means of age and bmi of the groups were analyzed by one way anova followed by the bonferroni post - hoc test. repeated - measures anova is used to compare the means of three or more matched groups. the term repeated - measures strictly applies to treatments repeatedly administered to each subject, and the term randomized block is used for randomly assigned treatments within a group (block) of matched subjects. a repeated - measures experimental design can be very powerful, as it controls factors that cause variability between subjects. if the matching is effective, the repeated - measures test will yield a smaller p value than ordinary anova. the repeated - measures test is more powerful because it separates between - subject variability from within - subject variability21. data were analyzed using repeated anova and multiple comparisons (the bonferroni test) test. the spss statistical program was used to perform statistical analyses and values of p<0.05 were considered significant. patients were randomly and equally divided into three treatment groups, and 98 (15 male and 83 female) patients completed the follow - up period (group 1 : n= 33, group 2 : n=32, group 3 : n=33) (fig. 1fig. the groups were homogenous in terms of age, gender and bmi ; the results are presented in table 1table 1.the mean age and bmi of the patientsvariabletreatment groupsgroup - i meansdgroup - ii meansdgroup - iii meansdage (years)53.66.754.95.455.25.7bmi (k / m)24.92.325.11.625.51.9bmi : body mass index. vas scores, tug scores and womac total and sub - scores were significantly better than preinjection scores in all of the three treatment groups during the follow - up period (p<0.001). table 2table 2.the mean and standard error of meam (sem) values and the test values according to the repeated values for different four timestreated groupsfollow - upsoutcome measuresvas meansemw.total mean semw.pain mean semw.stiffness mean semw.function mean semtug mean sem1pretreatment7.70.191.42.017.90.56.50.167.01.413.00.21 month5.60.281.72.114.90.55.00.261.81.612.80.23 months6.50.289.35.620.75.05.40.263.11.413.00.26 months7.20.287.61.916.90.46.10.264.61.413.10.22pretreatment7.71.281.63.017.20.56.60.258.02.012.70.21 month3.30.265.92.412.60.43.90.249.42.012.10.23 months4.81.269.62.313.70.44.50.251.32.012.40.26 months6.40.274.52.415.20.55.50.253.72.012.50.23pretreatment8.41.289.91.718.90.37.10.263.81.312.60.11 month2.40.167.41.611.90.33.40.252.01.311.60.13 months3.01.269.81.812.50.33.80.253.51.411.70.16 months4.51.275.11.714.10.24.90.256.01.411.70.3vas : visual analog scale, w : womac, tug : timed up and go test, sd : standard deviation presents the mean and standard deviation values and comparisons of the treated groups during the follow - up period, and includes the results of repeated anova and multiple comparisons. vas : visual analog scale, w : womac, tug : timed up and go test, sd : standard deviation the mean differences, the sem of mean differences, p value and 95% confidence intervals of two groups were compared and the mean differences of group 1-group 2, group 1-group 3, and group 2-group 3 in vas and tug scores were found to be significant (p<0.001). also the mean differences of group 1-group 2 and group 1-group 3 in womac total, womac pain, womac stiffness and womac function scores were also found to be statistically significant (p<0.001, table 2). no significant complications were observed other than transient increases in local pain or swelling during the treatment and follow - up periods. one of the major results of this study was the effectiveness of prp treatment for pain and physical function in grade 3 knee oa. however the effectiveness of a single injection was found to be significantly lower than that of two or three injections. in this study, during the follow up period, significant improvements were observed in the vas, womac and tug values of all of the three groups compared to their pre - injection values, and they showed a tendency of gradual decrease over time. according to the study of kon. which examined prp effectiveness on the knee joint, better results were achieved in patients with a low degree of cartilage degeneration9. chang s meta - analysis showed that prp effectiveness was higher at the degenerative chondropathy stage, and the effect decreased at two or lower doses when degeneration was worse5. in a study administering a single injection of prp, a longer sustained period of pain relief noted in a study of 14 patients cartilage thicknesses measured by ultrasonography, that responses to prp decreased as the level of oa and chondropenia increased4. as joint degeneration increases, factors such as decrease of viable cells, muscle function deficiency, joint instability due to increased ligament laxity, decrease in anabolic response to growth factors, loss of chondrocyte and thinning of cartilage plate may diminish the effectiveness of prp12. despite poorer results, patients with advanced oa still benefit from prp. in a comparitive study of prp and hyaluronic acid (ha) in grade 13 knee oa, the prp group showed significantly better results after 6 months and the worst results were observed in ha - treated subjects with grade 3 knee oa23. speculate that, additional biological mechanisms, not currently known, are responsible for the improvement of oa symptoms after prp treatment6. in the advanced stages of oa, prp might not have a direct effect on the chondrocyte anabolic process, but an anti - inflammatory effect through the regulation of joint homeostasis and the cytokine level6, 24.. showed that prp administered three times at weekly intervals to patients with grade 3 and 4 knee oa reported improvements in their quality of life, and reduced levels of pain, and had increased cartilage thickness as measured by ultrasonography at the 6-month follow up25. there is not enough data regarding the effectiveness of prp in the regeneration of substantial and irreversible bone and cartilage damage26. accordingly, objective studies conducted using magnetic resonance imaging or arthroscopic methods will be valuable in this regard. to our knowledge, there are no studies in the literature which have compared different doses of prp administered to patients with grade 3 knee osteoarthritis. in the present study, 3 prp injections separated by 2-week intervals were found to be more effective for the improvement of pain and mobility than 2 injections in grade 3 oa patients ; however, no significant differences were observed in the womac values. a significant effect was observed in the early period after a single injection of prp, but the effect decreased in a short time. based on the present results, we recommend 2 or 3 injections of prp for patients with moderate knee oa, and physicians decisions should be based on various factors such as the level of pain, level of activity, cost - effectiveness, and bmi. we further speculate that repeating the application after 6 months may further relieve symptoms for a longer period and delay oa progression. in the studies conducted so far, the lack of standardization of prp dosing regimens makes it difficult to compare outcomes of studies for the evaluation of clinical effectiveness27. the amount and effectiveness of platelet concentration and the gfs related to platelets in prp content will vary according to prp preparation techniques. for example, using an activator, the existence of leukocytes in prp content, application frequency, and platelet number range are currently debated issues. in addition, the follow - up period in many studies was short term, and there were no control groups5, 27. prp preparation technique in this study was standardized by our transfusion medicine department and no commercial filters were used. the platelet numbers injected in this study were between 1.1 billion1.4 billion, 46 fold higher than the baseline value, a number similar to that used in many studies, and it was also within the recommended range28. the absence of a control group and the relatively small patient numbers were the limitations of the study. considering the evidence, this minimally invasive injection procedure appears to be safe and effective, and since prp injections biologically change the articular cartilage, they may be a worthwhile treatment option even in moderate knee osteoarthritis. further studies are required with larger sample sizes with longer follow - ups and objective outcome measures. in conclusion, prp is an effective and reliable treatment for functional status and pain for grade 3 oa, and a minimum of two injections appears to be appropriate. | [purpose ] to assess the effects of different numbers of platelet - rich plasma (prp) applications on pain and physical function in grade 3 knee osteoarthritis (oa). [subjects and methods ] a total of 102 patients with grade 3 knee oa were randomly divided into three groups : group 1 received a single injection of prp, group 2 received two injections of prp two weeks apart, group 3 received three injections of prp at 2-weeks intervals. all patients were evaluated with a visual analog scale (vas), the western ontario and mcmaster universities arthritis index (womac), and the timed - up and go test (tug) before the treatment and at 1, 3 and 6 months after the treatment. [results ] ninety - eight patients (15 males, 83 females) completed the study. the mean ages of the patients were 53.56.6, 54.95.3, and 55.15.6 years in group 1, group 2, and group 3, respectively. statistically significant improvements were noted in all of the evaluated measures in all of the groups. the mean differences of group 1-group 2 and group 1-group 3 womac total, womac pain, womac stiffness, and womac function scores were statistically significant. [conclusion ] prp is an effective treatment for functional status and pain in moderate knee osteoarthritis and a minimum of two injections is appropriate. |
samples from 96 birds (41 wild ducks, 29 chickens, 23 ducks, and 3 geese) were collected from the padmachar area of rajshahi district during january 2009. in this area, a lake hosts several thousand wintering wild birds ; that lake also is frequented by poultry from surrounding households. each fecal sample, collected by swirling a cotton swab in a bird s cloaca or droppings, was submerged in a bacterial freeze medium and handled as described (5). each sample was placed on an uriselect 4 agar plate (bio - rad laboratories, marnes - la - coquette, france), and assessed for e. coli by biochemical testing and api 20e biochemical strips (biomrieux sa, marcy - l'etoile, france). one e. coli isolate per positive bird sample was tested by disk diffusion against 15 antimicrobial drugs (table 1) according to the recommendations of the european committee on antimicrobial susceptibility testing (www.eucast.org). each sample was also enriched in brain - heart infusion broth (becton dickinson, franklin lakes, nj, usa) supplemented with vancomycin 16 g / ml (icn biomedicals inc. aurora, oh, usa) for 18 h at 37c. for detection of esbl - producing bacteria and genes (blactx - m, blashv, and blatem) in putative esbl isolates, carbapenem - resistant isolates were screened on mueller - hinton agar plates supplemented with 2 g / ml or 8 g / ml meropenem and incubated overnight at 37c. the genetic profiles of the esbl - producing e. coli isolates were determined by using repetitive element pcr. the reaction mixture contained 1 taq pcr buffer, 0.625 mol / l primer eric1r (5-atgtaagctcctggggattcac-3), 1.9 mmol / l mgcl2, 50 mol / l dntps, 0.6 u taq polymerase, and template in a total volume of 20 l. cycling parameters were 1 min at 94c ; 1 min at 36c and 2 min at 72c for 45 cycles, and a final extension for 5 min at at 72c. isolates that had identical strong band patterns but an addition or a loss of a weak band were assigned subtype numbers. one representative for each repetitive element pcr genotype and subtype (n = 18) was characterized by multilocus sequence typing (mlst) (10). after sequencing, allele profiles and sequence types were determined by using the e. coli mlst database (http://mlst.ucc.ie/mlst/dbs/ecoli/ #). one representative sample for each genotype was tested for transferability of the esbl plasmid by conjugation to recipient e. coli da11782 (mcra, mrr - hsdrms - mcrbc, lacx74, deor, reca1, arad139 [ara - leu ] 7697, galk, rpsl, enda1, nupg, rif). equal amounts of donor and recipient overnight cultures in luria - bertani broth were mixed and incubated, without shaking, overnight at 37c. approximately 10 cfu of conjugation mixture was placed on selective plates containing 10 g / ml cefotaxime, 100 g / ml rifampin, and 50 g / ml nalidixic acid and incubated overnight at 37c. e. coli was isolated from 66 samples, yielding an isolation rate of 73.3% regardless of bird species. thirty - five (53%) of the 66 isolates were resistant to 1 antimicrobial compounds. the 3 next most common resistances were to ampicillin, trimethoprim / sulfamethoxazole, and nalidixic acid (table 1). multidrug resistance was found in 22.7% (15/66) of the isolates, and 13.6% (9/66) of the isolates were resistant to 4 or 5 classes of antimicrobial drugs. the overall prevalence of esbl carriage among birds was 30% (27/90) ; 36 e. coli isolates produced esbl. thirty - four of them belonged to the ctx - m-1 group (2 blactx - m-1 and 32 blactx - m-15) and 2 to the ctx - m-9 group, the latter of which were ctx - m-14like. combinations of blactx - m-15 or blactx - m-1 and blatem-1 were detected in 50% of the isolates, whereas none harbored shv - genes. the genetic fingerprints of the esbl - producing e. coli isolates identified 15 genotypes, of which 19 (53%) of 36 were type a (table 2).this genotype was found in wild and domestic birds. mlst analysis revealed 15 different sequence types (sts) and 1 nontypeable isolate (table 2). four isolates had new allele types or a new combination of allele types and were given novel sts (st2690st2693). conjugation was successful for 9/18 isolates, indicating the transferability of plasmids carrying esbl genes. the carriage rate of esbls was high and the predominating antimicrobial - resistant phenotypes of wild birds and poultry appeared to correlate with antimicrobial prescription patterns in bangladesh (6). most esbl - positive samples originated from poultry, and household poultry was the predominant carrier of the blactx - m-15 genotype and the ctx - m-14like enzymes. the ctx - m-15 gene shows a global distribution in clinical settings but has been reported from poultry in the united kingdom (11) and from wild birds in sweden (5), which indicates that this esbl type also is widely disseminated in the environment. the pcr - based genotyping showed the diversity of the esbl - producing e. coli isolates. wild birds and domestic poultry harbored the same strains, and some of the ducks had the same strains as chickens. this commonality of strains might be caused by a common use of natural water resources, and shows with what ease e. coli can travel between species. mlst analysis identified several human - associated genotypes, including st448, st405, st744, st648, and st131. the epidemic e. coli strain o25bst131 did not carry the more common ctx - m-15 gene but a ctx - m-14like gene, a frequent finding in hospitals in taiwan (12). metallo--lactamases of the new delhi metallo--lactamase type have not been found in the environment of bangladesh (13), but st405 and st648 are associated with new delhi metallo--lactamase-1producing organisms on the subcontinent of india (14). esbl - producing e. coli st744 has been reported previously in humans in laos (15). we showed that e. coli that produces ctx - m-15 is endemic to birds in bangladesh. our findings suggest that wild birds and free - range poultry might be crucial environmental indicators of antimicrobial drug resistance. they also might take a more active part than previously thought as spreaders and as long - term reservoirs of medically threatening pathogens and resistance genes. several factors are likely to contribute to the widespread dispersal of esbls in bangladesh, including dense population, poor sanitation, and close contact with livestock combined with a high selective pressure created by unrestricted use of antimicrobial drugs in human medicine, veterinary medicine, and aquaculture. development of a countrywide antimicrobial resistance surveillance system in livestock, wildlife species, and humans in bangladesh, as well as other measures, are needed immediately to control the situation. | multidrug resistance was found in 22.7% of escherichia coli isolates from bird samples in bangladesh ; 30% produced extended - spectrum -lactamases, including clones of ctx - m genes among wild and domestic birds. unrestricted use of antimicrobial drugs in feed for domestic birds and the spread of resistance genes to the large bird reservoir in bangladesh are growing problems. |
behcet s disease (bd) is a chronic autoimmune / inflammatory disorder characterized by recurrent orogenital ulcers, cutaneous inflammation, and uveitis. in addition to its typical mucocutaneous and ocular manifestations, bd targets the musculoskeletal, vascular, nervous, and gastrointestinal systems.13 the prevalence of bd is geographically influenced, and it is more prevalent in countries along the silk route, particularly in the east asia4,5 and the middle east.611 its prevalence is highest in turkey, followed by egypt, morocco, iraq, saudi arabia, japan, iran, korea, and china.12,13 although the specific etiology of bd remains elusive, extensive studies have suggested that autoimmunity, genetic factors, and environmental factors are involved in its pathogenesis.3,14,15 like many autoimmune disorders, bd has significant genetic associations with particular alleles of the class i and ii human major histocompatibility complex (mhc), and studies of these associations have led to significant insights into the molecular underpinnings of these disorders.1618 the human leukocyte antigen (hla) region on chromosome 6p21.31 contains multiple genes encoding highly variable antigen - presenting proteins and plays a key role in antigen presentation and activation of t cells.19 hla protein, hla - b51, encoded by hla - b is the strongest known genetic risk factor for bd. associations between bd and other factors within the mhc have also been reported, although the strong regional linkage disequilibrium complicates their confident disentanglement from hla - b51. single nucleotide polymorphism mapping with logistic regression of the mhc identified the hla - b / mica region and the region between hla - f and hla - a as independently associated with bd.16,2024 genetic association studies on saudi bd patients are scanty.13,25,26 the saudi population being a closed and isolated society with a high rate of consanguinity (inbreeding) represents a valuable resource for studying such genetic associations, and the present study was aimed at investigating the association of hla - a and b genetic variants with bd in saudi patients. in the present study, we recruited 60 bd saudi patients (aged 2064 years) and an equal number of healthy controls, matched for age (2060 years), sex, and ethnicity (saudi) from prince sultan military medical city, riyadh, saudi arabia, for genetic analysis of hla alleles. the exclusion and inclusion criteria were followed strictly for the selection of patients and controls. a questionnaire was filled for each subject to collect past medical history, drug in use, and relevant life style related questions. this study was approved by the research and ethical committee of prince sultan military medical city, riyadh, saudi arabia, and the written informed consent was obtained from each subject before participation. this research work complied with the principles of the declaration of helsinki. the diagnosis of bd was made based on the criteria of the international study group for bd.27 we evaluated the clinical features such as oral ulcers, genital ulcer, ocular inflammation, musculoskeletal, cutaneous, gastrointestinal lesions, nervous, pulmonary, cardiovascular manifestations, and vascular lesions. the active and nonactive forms of bd were determined at the time of study after the assessment of clinical parameters. a detailed information along with demographic characteristics are mentioned in our recently published article.13 peripheral blood (3 ml) from healthy controls and patients was drawn in edta - containing vials, and genomic dna was extracted using the qiaamp dna mini kit (qiagen) according to the manufacturer s protocol. the purity of dna was determined at 260/280 nm using a nano - drop spectrophotometer (thermo fisher scientific). only dna samples having a 260280 nm absorbance ratio between 1.7 and 2.0 and a final concentration of 2030 ng/l were considered appropriate. hla genotyping was performed by the reverse sequence - specific oligonucleotide polymerase chain reaction (pcr) technique using genotyping kits lab type sso (one lambda) as per the manufacturer s protocol. the regions of dna exons 2 and 3 for the loci a and b were amplified. the allele - specific biotinylated primer accompanying the kits was used for the amplification of dna. the pcr amplification was programmed at 96 c for three minutes followed by five cycles of 96 c for 20 seconds, 60 c for 20 seconds, and 72 c for 20 seconds ; 30 cycles of 96 c for 10 seconds, 60 c for 15 seconds, and 72 c for 20 seconds and extension at 72 c for 10 minutes. the amplified product was also run on 5% agarose gel (pulsed field certified agarose ; bio - rad laboratories) to check the amplification of specific exon of each locus. the remaining pcr product was then hybridized with oligonucleotide probes sequence - specific conjugates with fluorescent microspheres. the hybridized products were analyzed by using flow analyzer running lab scan 100 xponent (one lambda) and fluorescence intensity in each microsphere was identified. frequencies of various alleles of hla polymorphism were compared between bd patients and controls and analyzed by fisher s exact test and p values 0.05 were considered significant. the significance of the differences in distribution of alleles was calculated after bonferroni correction to minimize error due to multiple comparison tests. the binary logistic regression analysis was also performed for each of homozygous (two alleles) and heterozygous (one allele) hla - a or hla - b alleles contributing independently to bd. genetic data were also expressed as an odd ratio interpreted as relative risk (rr) according to the method of woolf as outlined by schallreuter.28 rr indicates the number of times the risk of disease is higher in terms of allele in bd patients than in controls. the rr was calculated for all the subjects using the following formula : rr = adbcwhere a is the number of patients with expression of allele, b the number of patients without expression of allele, c the number of controls with expression of allele, and d the number of controls without expression of allele. ef was calculated for positive association only where rr > 1 using the following formula29 : ef=(rr1)frr, wheref = aa+c preventive fraction (pf) indicates the hypothetical protective effect of one specific allele / genotype against the disease. pf was calculated for negative association only where rr 1 using the following formula29 : ef=(rr1)frr, wheref = aa+c preventive fraction (pf) indicates the hypothetical protective effect of one specific allele / genotype against the disease. pf was calculated for negative association only where rr < 1 using the following formula.29 values < 1.0 indicate the protective effect of the allele against the manifestation of disease. clinically, all bd patients (100%) had oral ulcers, 80.32% genital ulcer, 70.49% ocular, 67.21% musculoskeletal, 60.65% cutaneous, 36.06% gastrointestinal, and 22.95% patients had nervous system involvement. the results of genotyping for hla - a and hla - b in bd patients and controls are summarized in tables 15. the frequency of hla - a02 (38.33%) was the highest, followed by that of hla - a26, hla - a31, hla - a68 (10.83% each), hla - a23, hla - a24 (5.83% each), hla - a01, hla - a30, hla - a32 (3.33% each), hla - a03, hla - a33 (2.5% each), hla - a11, hla - a29, and hla - a69 (0.83% each). comparison of allele frequencies between the bd patients and controls indicated that the frequencies of allele hla - a026 and hla - a31 were significantly higher in bd patients than in controls (p = 0.041, or = 3.523, 95% ci = 1.1111.139, ef = 0.546, and p = 0.005, or = 7.168, 95% ci = 1.58132.498, ef = 0.702, respectively). however, after applying bonferroni correction, the p values are not significant (p = 0.08 and p = 0.656, table 1). an increased frequency of hla - a02 was also found in bd patients as compared to controls (38.33% vs. 29.16%), but the difference was not statistically significant (p = 0.172, table 1). when the data were grouped on the basis of the active and nonactive forms of bd, the frequency of hla - a31 allele was significantly higher in the nonactive form than in the active form of bd (p = 0.015), while the frequency of hla - a26 did not differ significantly in two groups (table 2). the frequency of hla - b51 was significantly higher in bd patients than in controls (p = 0.0001, or = 3.631, 95% ci = 2.0866.322, ef = 0.521). moreover, on applying bonferroni correction, the frequency of hla - b51 was found to be significantly higher in bd patients than in controls (p = 0.0022). increased frequencies of allele hla - b07 and hla - b08 however, the difference was not significant (p = 0.689 and p = 0.823, respectively). on the other hand, hla - b15 was significantly lower in bd patients than in controls (p = 0.03, or = 0.254, 95% ci = 0.0690.935, pf = 0.384, table 3), though after bonferroni correction, the significance was lost (p = 0.66). stratification of genotyping results into the active and nonactive forms of bd revealed no significant difference in the allele frequencies among the two groups (table 4). the frequency distribution of homozygous / heterozygous alleles of hla - a26, hla - a31, and hla - b51 in bd and controls is shown in table 5. the binary logistic regression analysis performed for each of the homozygous and heterozygous hla alleles indicated that hla - b51 allele, both in homozygous (two alleles) and heterozygous (one allele) conditions, is significantly associated with susceptibility to bd in saudi patients (p = 0.0001 and p = 010, respectively). on the other hand, hla - a26 is associated in heterozygous (one allele) conditions with bd, while upon stratification of hla - a31 into heterozygous and homozygous conditions, the association lost significance (p = 0.089). the significantly higher frequency of hla - a26 in bd cases than in controls suggested that hla - a26 is associated with susceptibility to bd in saudi patients. the binary logistic regression analysis (table 5) also indicated that hla - a26 is associated in heterozygous (one allele) conditions with bd in saudi patients. the hla - a gene has been genotyped in bd patients with different ethnicities, and hla - a26 was reported to be associated with bd in taiwan, greece, and japan.3032 hla - a26 has been associated with the ocular manifestation, an outcome of bd indicating its contribution to the risk of bd.31,32 itoh.33 found weak association of hla - a26 with bd and suggested some secondary influence on the onset of bd. in addition, an association between the hla - a26:01 subtype and bd has been reported in japanese and korean.16,22 hla - a26:01 not only has been reported to be a primary susceptibility allele of bd in japan,22 but a recent study also found that the frequency of hla - a26:01 was significantly increased in bd patients with uveitis, particularly in the hla - b51 negative subset.32 our results also suggested that allele hla - a31 is associated (or = 7.168, ef = 0.702) with the risk of bd. after applying bonferroni correction, the p value is not significant (p = 0.08) possibly due to the small sample size. as this is the first study where hla - a31 is found to be associated with bd susceptibility risk, these results remain to be replicated in other cohorts. however, when genotypic data were stratified on the basis of active and nonactive forms of bd, we found that the frequency of hla - a31 was significantly (p = 0.015) higher in the inactive form of bd than in the active form. in general, several earlier reports are consistent with the present study and hla - a gene has been suggested to constitute a second independent susceptibility locus.2022 kang.16 showed that certain hla - a alleles are responsible for the unique clinical features of bd. due to the small sample size, we could not assess any relationship between particular hla - a type and clinical feature of the patient. nevertheless, we believe that the results of our study are unlikely to be affected by systematic errors such as population stratification, because the source of our controls and cases represents the same saudi population. on the contrary, some reports indicated that hla - a alleles are not associated with increased risk of bd in palestine, jordan, iran, ireland, italy, and turkey.3438 the significantly higher frequency of hla - b51 in saudi bd patients than in controls with p = 0.0001, or = 3.631, and ef = 0.521 together with bonferroni corrected p = 0.0022 indicated that hla - b51 is strongly associated with bd susceptibility. earlier, yabuki.26 studied 13 saudi bd patients and reported significantly increased frequency of hla - b51 in bd patients as compared to controls. several studies across the globe in different ethnicities have shown strong evidences for hla - b51 susceptibility to increased risk of bd.17,18,3740 hla - b51 alone increases the risk of bd up to 40%80% in different ethnicities and known as universal risk factor for bd.18,31,41 the present finding of hla - b51 and increased risk of bd in saudis (rr = 3.93) are corroborated with earlier reports from different ethnic populations : rr = 3.51 and p = 0.065 for the japanese population,37 p = 1.35 10 and or = 5.15 for the chinese han population,40 p = 0.0003 and or = 2.39 for the korean population, rr = 3.51 for the iranian population,18 or = 6.24 for the turkish population,38 p = 4.11 10 and or = 4.63 for the sardinia population,39 or = 5.15, p = 1.35 10 for the spanish population,17 and many more. however, hla - b51 alone is neither necessary nor sufficient to determine bd, and several hla - a and -b alleles may independently contribute to the risk of bd.17,24,42 on the other hand, the frequency of hla - b15 was significantly lower in saudi bd patients than in controls, suggesting that hla - b15 may be protective against bd in saudis. contrary to our result, hla - b15 has been associated with bd in some populations.24,42,43 ombrello.24 indicated that hla - b51, -a03, -b15, -b27, -b49, -b57, and -a26 each contributed independently to bd risk in turkish population. piga and mathieu42 in a meta - analysis reported that besides hla - b51 being primarily associated, hla - a26, hla - b15, and hla - b5701 are also independently associated with bd and suggested for further studies to clarify the functional relevance of the different genes found to be associated with disease susceptibility and the potential interactions between genes located within and outside the mhc region. our study supports that besides hla - b51 being primarily associated, hla - a alleles are also independently associated with susceptibility to bd. it is concluded that hla - a26, -a31, and -b51 are associated with bd in saudi patients, while hla - b15 may be protective. however, further studies on population genetics with larger sample size are required to strengthen these findings. | backgroundhla - b51 has been universally associated with behcet s disease (bd) susceptibility, while different alleles of hla - a have also been identified as independent bd susceptibility loci in various ethnic populations. the objective of this study was to investigate associations of hla - a and -b alleles with bd in saudi patients.materials and methodsgenotyping for hla - a and hla - b was performed using hla genotyping kit (lab type(r) sso) in 120 saudi subjects, including 60 bd patients and 60 matched healthy controls.resultsour results revealed that frequencies of hla - a26, -a31, and -b51 were significantly higher in bd patients than in controls, suggesting that hla - a26, -a31, and -b51 are associated with bd. the frequency of hla - b15 was significantly lower in bd patients than in controls. stratification of genotyping results into active and nonactive forms of bd revealed that the frequency of hla - a31 was significantly higher in the nonactive form than in the active form of bd, while there was no significant difference in the distribution of other alleles between the two forms of bd.conclusionthis study suggests that hla - a26, -a31, and -b51 are associated with susceptibility risk to bd, while hla - b15 may be protective in saudi patients. however, larger scale studies are needed to confirm these findings. |
in 2000, there were an estimated 151 million cases of type 2 diabetes worldwide. this figure increased to 285 million in 2010, and it is predicted that 438 million people will suffer from diabetes by 2030.1 type 2 diabetes stems from interactions between genetics, behavior and environment. the genetic basis of the disease is still unclear,2 but there is strong evidence that obesity and inactivity are the main causes of type 2 diabetes.2 - 4 many previous studies that have investigated the relationship between physical fitness and type 2 diabetes have had some limitations. one such limitation is the use of self - reported levels of physical activity.5 - 9 self - reporting tends to be imprecise10 and only correlates moderately with objective measures of cardiorespiratory fitness.11 recent studies have overcome this limitation and report that fitness level and diabetes are inversely related.3,10,12,13 most studies have not acknowledged that risk factors for elevated plasma glucose levels are different in men compared to women.14 sui. found that high physical fitness levels did not eliminate diabetes risk in women,3 but fitness predicted all - cause mortality among women with impaired fasting glucose (ifg) or undiagnosed diabetes.15 the aforementioned studies also did not distinguish between ifg and impaired glucose tolerance (igt) at baseline. both ifg and igt are associated with similar risks for the development of type 2 diabetes,16,17 but the pathogenic backgrounds and etiologies may be different.7,18 the present analysis, as part of a diabetes prevention study,19 aimed to evaluate gender - specific relationships between measured cardiorespiratory fitness and factors that predict the development of diabetes, as described by schwarz.,20 in patients with ifg or igt. furthermore, the study aimed to identify gender - specific risk factors that predict levels of fasting plasma glucose (fpg) and 2-hour plasma glucose levels (2-h pg) to gain insight into the early stages of the disease 's etiology. based on the findings of sui.,3 we hypothesized that cardiorespiratory fitness is related to plasma glucose levels in men, but not in women. we also speculated that other differences in diabetes risk factors exist between men and women. general practitioners from western states in austria were informed about the study and invited to participate. the first ten who agreed to participate were asked to recruit patients (males and females) with ifg and/or igt primarily through member screening for high - risk groups, such as first - degree relatives of patients with type 2 diabetes and overweight individuals between the ages of 40 and 65 years. ifg was defined as fpg levels 100 mg / dl and 240/120 mmhg) was noticed. cardiorespiratory fitness was defined as the highest power achieved during the test divided by body weight (watt / kg). the associations between fpg, 2-h pg levels, chol, hdl, tg, bmi and cardiorespiratory fitness (i.e., relative power, watt / kg) were calculated using pearson correlation analyses. a moderate correlation of r = 0.3 between plasma glucose levels and cardiorespiratory fitness was considered meaningful. accordingly, a sample size of 67 provided a power of 80% (one - tailed). all data, except tg data, were normally distributed (kolmogorov - smirnov test) ; therefore, ln - transformed tg (lntg) values were used in all analyses. a stepwise regression with backward variable selection was performed to construct models that predicted 2-h pg and fpg levels. the regression analysis was performed for men and women separately and was initiated with a model that included the risk factors that predict the development of diabetes as described by schwarz.20 (i.e., age, bmi, hdl, tg, bp, and physical activity). to obtain more accurate information on physical activity, measured cardiorespiratory fitness was used in the analysis instead of self - reported levels of physical activity. the p - value for inclusion in the backward regression model general practitioners from western states in austria were informed about the study and invited to participate. the first ten who agreed to participate were asked to recruit patients (males and females) with ifg and/or igt primarily through member screening for high - risk groups, such as first - degree relatives of patients with type 2 diabetes and overweight individuals between the ages of 40 and 65 years. ifg was defined as fpg levels 100 mg / dl and 240/120 mmhg) was noticed. cardiorespiratory fitness was defined as the highest power achieved during the test divided by body weight (watt / kg). the associations between fpg, 2-h pg levels, chol, hdl, tg, bmi and cardiorespiratory fitness (i.e., relative power, watt / kg) were calculated using pearson correlation analyses. a moderate correlation of r = 0.3 between plasma glucose levels and cardiorespiratory fitness was considered meaningful. accordingly, a sample size of 67 provided a power of 80% (one - tailed). all data, except tg data, were normally distributed (kolmogorov - smirnov test) ; therefore, ln - transformed tg (lntg) values were used in all analyses. a stepwise regression with backward variable selection was performed to construct models that predicted 2-h pg and fpg levels. the regression analysis was performed for men and women separately and was initiated with a model that included the risk factors that predict the development of diabetes as described by schwarz.20 (i.e., age, bmi, hdl, tg, bp, and physical activity). to obtain more accurate information on physical activity, measured cardiorespiratory fitness was used in the analysis instead of self - reported levels of physical activity. the p - value for inclusion in the backward regression model men had higher tg levels (p = 0.002), lower percent body fat (p = 0.005), lower hdl levels (p = 0.045) and lower resting diastolic bp (p = 0.011). men had significantly lower la at a workload of 100 watts (p = 0.009) and significantly lower hr values (given as percent of the peak hr) at 50 and 100 watts (p = 0.001). cardiorespiratory fitness was inversely correlated with 2-h pg for the entire group (r = -0.237) and in men (r = -0.404). was inversely correlated with hdl cholesterol for the entire group (r = -0.372, p<0.05). in women, 2-h pg was inversely correlated with hdl cholesterol (r = -0.377, p<0.05), and fpg was positively correlated with lntg levels (r = 0.458, p<0.05). table 2 shows the results of the backward regression analysis for the 2-h pg and fpg levels. lntg levels predicted fpg levels in women, whereas bmi was the only predictor in men. the symptom - limited incremental exercise test was stopped in 47 patients due to exhaustion, in 10 patients because of a bp greater than 240/120 mm hg, in 4 patients because of arrhythmia and in 11 patients due to dyspnea. the main findings of the present analysis were that cardiorespiratory fitness was related to 2-h pg levels but not fpg levels, and this relationship was present in men only. furthermore, we demonstrated that different risk factors predict 2-h pg and fpg levels in men and women. differences in 2-h pg were explained by cardiorespiratory fitness and age in men, whereas hdl was the only predictor in women. the cardiorespiratory fitness findings are consistent with the inter99 study in which high physical activity, as an indirect measurement of cardiorespiratory fitness, predicted a decline in 2-h pg levels in men only.14 furthermore, similar to the ausdiab study23 and studies in pima indians,24 2-h pg levels were associated with the amount of physical activity, whereas fpg levels were not.. showed that physical activity was associated with a lower progression to diabetes in patients with isolated igt but not in patients with isolated ifg.25 it is suggested that the effect of physical activity on 2-h pg levels is mediated through an improvement in insulin sensitivity,26 which in turn increases the insulin - stimulated glucose uptake after the glucose load.14 different pathogenic backgrounds of ifg and igt16,17 may explain these differences ; stable reduced insulin secretion followed by a decline in primarily hepatic insulin sensitivity characterizes the transition from normal glucose tolerance to ifg. in contrast, low whole - body insulin sensitivity with a secondary lack of beta - cell compensation is associated with the development of igt.18 in studies with self - reported physical activity, which only correlates modestly with objective measures (i.e., exercise testing),13 associations between sedentary habits and risk for type 2 diabetes may have been underestimated.10 in the present investigation, cardiorespiratory fitness was objectively determined using a symptom - limited incremental cycle ergometry test. cardiorespiratory fitness is considered an independent predictor of several cardiovascular diseases, diabetes mellitus and all - cause mortality.10,27 the association between cardiorespiratory fitness and 2-h pg level may therefore explain why igt but not ifg is considered a risk factor for cardiovascular disease.28,29 in women, differences in 2-h pg levels were explained by the differences in hdl levels. this finding is consistent with the findings of njolstad., who showed that hdl cholesterol was inversely related to diabetes in women but not in men.30 the differences in the hdl cholesterol effect based on gender may reflect sex hormone effects.31 furthermore, hdl subfractions and habitual alcohol intake disparities between men and women may play a role.31 in women, the only predictor for fpg was lntg level. accordingly, it has been shown that elevated tg levels are a more important risk factor for metabolic syndrome in women than in men.32 regitz - zagrosek. concluded that risk factors for diabetes should be considered in a gender - specific manner that focuses on elevated tg levels in women and waist circumference in men.32 the results of the present investigation may extend this conclusion by adding cardiorespiratory fitness for men and hdl cholesterol for women. from a practical perspective, increasing cardiovascular fitness is of major importance for men, whereas dietary interventions rather than physical activity to decrease body fat percentage are important measures for preventing type 2 diabetes in women. similarly, sui. showed that high levels of physical fitness did not eliminate the increased risk for diabetes in overweight and obese women.3 however, cardiorespiratory fitness should not be ignored because it has been shown to be a significant predictor of all - cause mortality in women with ifg or undiagnosed diabetes.15 in the present investigation, men had lower submaximal performance parameters at fixed workloads (table 1). furthermore, women had a significantly higher diastolic blood pressure at rest than men because most of the women were more than 50 years old, and therefore have decreased circulating estrogen levels. low estrogen levels independently contribute to an increase in blood pressure due to a direct effect on the arterial wall and the activation of both the renin - angiotensin system and the sympathetic nervous system.33,34 some limitations of the present study must be addressed. this study is a cross - sectional analysis with a relatively small sample size. due to the small sample size, we can not entirely exclude a significant correlation between cardiorespiratory fitness and 2-h pg in women. furthermore, we can not rule out the possibility that variables that were removed by the stepwise algorithm of the regression might have some predictive capability. nonetheless, the results are essentially consistent with large cohort studies and add specific information on gender differences. additional limitations include the following : the maximal cycle ergometry test was symptom limited, and gas analyses for the determination of maximal oxygen uptake values were not performed. premature termination of the tests may have led to a slight underestimation of the peak power output in some cases. although gas analysis is considered to be the gold standard for evaluating maximal oxygen uptake and would thus allow better comparison with other studies, the very close correlation between peak power output and maximal oxygen uptake (r = 0.97)22 justifies the decision to consider peak power output as an appropriate surrogate for maximal oxygen uptake. this study 's findings suggest that high cardiorespiratory fitness is more beneficial in preventing the onset of diabetes in pre - diabetic men than in pre - diabetic women and that different risk factors predict 2-h pg levels and fpg levels in men compared to women. whereas increasing cardiorespiratory fitness should be a key goal for men, reducing body fat to improve the lipid profile is considered more beneficial in preventing type 2 diabetes in women. however, it must be emphasized that the present results do not negate the positive effects of increasing cardiorespiratory fitness in women. we express our gratitude to the following members of the project leadership committee : prof. the study was funded by the austrian national bank (oenb), the university of innsbruck (research department), and the government of vorarlberg. | objectives : to investigate gender - specific relationships between cardiorespiratory fitness and factors that predict the development of diabetes and to identify the risk factors that predict fasting plasma glucose and 2-hour plasma glucose levels.introduction:different risk factors (e.g., low cardiorespiratory fitness) may cause elevated plasma glucose levels in men compared to women. therefore, gender - specific analyses are needed.methods:cardiorespiratory fitness (maximal power output achieved during a standard cycle ergometry test), resting blood pressure, total serum cholesterol, high - density lipoprotein cholesterol and triglyceride levels were measured in 32 pre - diabetic men (mean age : 57.26.8 years ; mean body mass index (bmi) : 28.53.0 kg / m2) and 40 pre - diabetic women (mean age : 55.07.3 years, mean bmi : 30.45.7 kg / m2). a stepwise regression with backward variable selection was performed to construct models that predict 2-hour and fasting plasma glucose levels.results:maximal power output was inversely related to the 2-hour plasma glucose level in the entire group (r = 0.237, p<0.05), but this relationship was significant only for males (r = 0.404, p<0.05). no significant correlation was found between female gender and cardiorespiratory fitness. age and cardiorespiratory fitness were significant predictors of 2-hour plasma glucose levels in men. high - density lipoprotein cholesterol was predictive of 2-hour plasma glucose levels in women. triglycerides in women and bmi in men were the only predictors of fasting plasma glucose levels.conclusions:these findings may have consequences for the development of gender - specific diabetes prevention programs. whereas increasing cardiorespiratory fitness should be a key goal for men, improving the lipid profile seems to be more beneficial for women. however, the present results do not negate the positive effects of increasing cardiorespiratory fitness in women. |
cardiovascular effects of tobacco smoking are primarily attributed to the presence of nicotine in cigarettes. this alkaloid may promote decrease in baroreflex sensitivity, increase in heart rate and blood pressure, atherosclerosis, coronary heart disease, and myocardial infarction. nicotine potentiates sympathetic nervous system leading to increase in plasma and brain catecholamine levels [2, 3 ]. in addition, we have previously demonstrated the acceleration of onset and the exacerbation of hypertension in genetic hypertension predisposed rats after nicotine exposure. the activation of sympathetic neurotransmission by nicotine may be based on its direct effect on the central nervous system, on sympathetic ganglia to increase the efferent nerve activity, and/or on peripheral sympathetic nerve endings and adrenal medulla stimulating catecholamine release. nevertheless, the activated sympathetic system might promote the reflex parasympathetic response composing an elaborated physiological effect after nicotine administration. the alkaloid instantly interacts with the central nervous system binding to nicotinic acetylcholine receptors in the hypothalamus, hippocampus, midbrain, and medulla oblongata [6, 7 ] modulating norepinephrine, dopamine, vasopressin, glutamate, neuropeptide y (npy), and other neurotransmitter systems. nicotine acts also on chemoreceptors afferents, enteric nervous system, and visceral sensory afferents (for a review about nicotinic mechanisms in the autonomic control of organ systems refer to de biasi). peripheral areas involved in cardiovascular control are also under the influence of nicotine, such as the nodose / petrosal ganglia. it is well documented the presence of several neurotransmitters such as npy, substance p (sp), catecholamines, and glutamate in nodose and petrosal ganglia, which are related to baroreflex [13, 14 ]. furthermore, baroreflex afferents to nucleus tractus solitarii (nts) contain glutamate as the main neurotransmitter involved in cardiovascular control, which may be regulated by several other neurotransmitters such as catecholamines, npy. and sp. superior cervical ganglion is part of the sympathetic chain located next to carotid bifurcation that participate in generating the vasomotor tonus of head and neck. the study of superior cervical ganglion is useful to evaluate the function of sympathetic autonomic nervous system. it is well established that the presence of the enzyme tyrosine hydroxylase (th) in this ganglion, which participates in catecholamines synthesis, as well as npy and sp immunoreactive profiles and nicotinic acetylcholine receptors that may influence superior cervical ganglion activity. in view of this, the aim of the present study is to evaluate the modulation of glutamate, th, npy, and sp in nodose / petrosal and superior cervical ganglia and as well as th and npy in nucleus tractus solitarii and hypothalamic paraventricular nucleus of normotensive and spontaneously hypertensive rats after prolonged nicotine exposure delivered through subcutaneously implanted pellets. all the procedures and protocols were performed in accordance with institutional and international guidelines for animal care and use. male normotensive wistar kyoto (wky) and spontaneously hypertensive rats (shr), 1 month old, from the institute of biosciences, university of so paulo (so paulo, brazil), had either nicotine or placebo pellets (innovative research of america, ohio) implanted subcutaneously during 8 weeks. initially, 1-month - old rats had 10 mg/21 days pellets implanted in the lateral side of their neck, which were replaced for pellets of 100 mg/60 days until the end of treatment. pellets released nicotine continuously at the average rate of 8 mgkgday along treatment. it was previously demonstrated that exposure to this concentration of nicotine accelerated and intensified the hypertension in shr although the alkaloid promoted no cardiovascular effects in wky rats. six rats of each strain and treatment were submitted either to transcardiac perfusion for immunohistochemistry, or decapitation for binding analysis and in situ hybridization. brain, nodose / petrosal and cervical superior ganglia were excised to the analysis of glutamate, tyrosine hydroxylase, neuropeptide y, and substance p. rats were deeply anaesthetized and perfused with 50 ml of saline (0.9%) followed by 300 ml of a fixative solution consisted of 4% paraformaldehyde (w / v) and 0.2% picric acid (v / v) in 0.1 mol / l pbs, ph 6.9, at 4c. brain sections (14 m) were obtained in a cryostat (leica, cm 3050, germany) at rostrocaudal levels from 13.68 to 14.08 mm for the analysis of the nts and at 1.80 to 2.12 mm to access the pvn according to the atlas of paxinos and watson. sections were incubated for 24 hours at 4c with rabbit polyclonal antisera against either glutamate (1 : 2000, sigma), th (1 : 400, eugene tech), npy (1 : 2000, sigma), or sp (1 : 1000, peninsula) followed by incubation with a biotinylated antirabbit immunoglobulin (1 : 230, vector, usa) for 90 min. sections were next incubated with an avidin - biotin peroxidase complex (1 : 120, vectastain, vector, usa) for 45 min. immunoreactivity was visualized using 3 - 3-diaminobenzidine tetrahydrochloride (dab) as chromogen and h2o2 (0.05%) after 6 minutes of reaction. immunoreactivities were analyzed by semiquantitative microdensitometry, expressed as spmgv (specific mean gray value), using a ks 400 image analyzer (zeiss, germany) linked to a ccd 72 camera (dage ; mti, michigan city, ind., usa) mounted on a zeiss microscope (40x objective), as previously described by zoli and coworkers. the value for each animal was obtained by taking the mean of two adjacent sections of each analyzed region. brain sections were obtained as specified for immunohistochemistry and thaw - mounted onto poly - l - lysine coated slides that were postfixed with cold 4% parafolmaldehyde in phosphate buffered saline (pbs) solution, dehydrated in ethanol and processed for hybridization procedures. probes complementary to nucleotides 13511398 of th mrna were employed for the detection of this mrna and npy was labeled with a probe corresponding to nucleotides 31463194 of rat npy mrna. probes were synthesized by life technologies, labeled with (35s)datp using terminal deoxynucleotidyl transferase (roche, germany) at the 3-end, and purified according to the previously described protocol. sections were incubated overnight at 42c in a humidified chamber with the labeled probe (500000 dpm/50 l / section) in a hybridization buffer. slides containing the sections were extensively rinsed in saline - sodium - citrate (ssc), dehydrated in ethanol and exposed to a radioactivity sensitive film (kodak biomax mr, kodak, usa) for four weeks. for control experiments, films were analyzed using a computer - assisted image analyzer and software developed by imaging research (brock university, canada). npy1r were evaluated in the nts and pvn through binding assay performed in the same group of rat brains used for in situ hybridization. ci / mmol, amersham biosciences) in a buffer containing hepes (20 mm), nacl (137 mm), kcl (5.4 mm), kh2po4 (0.44 mm), cacl2 (1.26 mm), mgso4 (0.81 mm), bacitracin (0.1%), and bovine serum albumin (bsa, 0.3%). ligand concentration, which is near to kd of npy receptors, was chosen based upon our previous results. after incubating for 60 minutes, sections were rinsed with trizma 50 mm (ph 7.4) followed by distilled water, and exposed to biomax mr film (kodak) during 3 days. binding to npy1r was calculated by displacing the radioactive ligand with 10 mm of npy1r specific agonist (leu31-pro34) npy (peninsula). results were evaluated by 2-way analysis of variance (anova) using graphpad prism (graphpad software inc., version 3.00, ca). a p value < 0.05 was considered to indicate statistically significant differences. nicotine increased th immunoreactivity by 40% only in shr petrosal / nodose ganglia as compared to placebo - treated shr (figure 1). however, npy, glutamate, and sp were increased in these ganglia of both strains after chronic nicotine exposure, as compared to placebo - treated rats (figure 1). moreover, shr presented increased levels of npy and glutamate as compared to wky rats in absence of nicotine (figure 1). the results revealed an association between nicotine exposition and the genetic background to increase th immunoreactivity, suggesting the interaction among gene, environment, and physiology. the interaction of these parameters was already previously demonstrated by our group when the alkaloid exacerbated the hypertension in shr while no alteration in blood pressure was observed in the wky. in contrast, npy and glutamate immunoreactivities depend only on the nicotine or strain, and sp immunoreactivity is influenced by nicotine, without the participation of the genetic predisposition to hypertension. th, npy, and sp immunoreactivities were increased in scg of shr as compared to wky rats (figure 2). alteration in the expression of these immunoreactivities were dependent on the interaction between rat strain and nicotine treatment, since the shr presented elevated levels of these substances and nicotine exacerbated them in scg as can be observed in figure 2. th immunoreactivity and mrna expression in the nts did not differ between wky and shr rats without nicotine exposure. however, the pvn of shr presents 3.5x the amount of th mrna of wky rats (figure 3), without any equivalent increase in its immunoreactivity at basal levels. nicotine increased th immunoreactivity and mrna in the nts of shr (figures 3(a) and 3(b)). the alkaloid also increased th immunoreactivity in the pvn of both strains although th mrna in this nucleus was not altered by nicotine exposure (figures 3(c) and 3(d)). th immunoreactivity and mrna expression in the nts are influenced by the interaction between nicotine exposure and rat strain, since only shr responded to nicotine exposure. this might be of relevance considering the elevation of blood pressure exclusively in this strain. in the pvn, th mrna is influenced by strain while the treatment with nicotine is able to increase th immunoreactivity, without interference of rat strain. shr presented decreased levels of npy immunoreactivity in the nts whereas npy1r binding in this area is increased when compared to wky (figures 4(a) and 4(b)). in the presence of nicotine, npy immunoreactivity increased in the nts of shr only while npy1r binding decreased 50% in this area of both strains (figures 4(a) and 4(b)). npy immunohistochemistry increased in the pvn after chronic nicotine exposure only in the hypertensive strain (figures 4(c) and 4(d)). npy1r binding is found 100% increased in placebo shr as compared to control wky (figure 4), nicotine seems not to modify binding parameters in the pvn. the interaction between rat strain and nicotine exposure influenced npy immunoreactivity in nts and pvn. this immunoreactivity is increased by nicotine only when the genetic background of shr is present. npy1r binding in nts is affected by rat strain and treatment with nicotine, separately, since the amount of npy1r binding is increased in shr and nicotine decreased it in the same magnitude in both strains. npy mrna expression was not altered in shr as compared to wky rats in either nuclei analyzed in the presence or absence of nicotine (table 1). in the present study we demonstrated that prolonged nicotine administration through subcutaneously implanted pellets increased th immunoreactivity in brain and ganglia related to blood pressure control, preferentially in shr. this is in agreement with our previous study that showed the increased responsiveness of shr to nicotine when compared to wky. the alkaloid also increased npy immunoreactivity in ganglia, nts, and pvn of shr, in spite of decreasing npy1r binding in nts of both strains. the effects of nicotine encountered at both central and peripheral levels might be due to its action directly upon the brain as well as on the sympathetic autonomic nervous system and on smooth muscle of arterioles. it is important to point out that a recent revision written by zanutto and collaborators emphasized the long - term blood pressure regulation also by the nts in addition to the renal mechanisms, which validate our study using prolonged nicotine administration to analyze areas of central cardiovascular control. glutamate immunoreactivity was evaluated only in nodose / petrosal ganglia in the present study since the analysis of this neurotransmitter in brain cardiovascular areas was conducted previously by our group and the presence of glutamate as neurotransmitter in the scg was not described yet, although the expression of glutamate receptors is found in scg cells. we have demonstrated an increase in glutamate immunoreactivity in nodose / petrosal ganglia of placebo shr as well as after nicotine exposure in both rat strains as compared to wky control rats. the increased glutamate in the ganglia might be released in the nts by baroreceptor afferents to reduce blood pressure since glutamate is a hypotensive agent in this nucleus. several studies have described the sensitivity of neurons from petrosal and nodose ganglia to nicotine [3033 ] and it seems that a minor part of barosensory neurons are responsive to nicotine. in view of this, the response observed in shr may be due to the raise in blood pressure, since the effects of nicotine related to glutamate immunoreactivity over the shr were more pronounced. moreover, nicotine may act directly upon these ganglia cells to determine integrated system responses. because the specificity of the ganglia neurons was not investigated, the effects of these immunoreactivities may be also related to chemoreception, pain, interaction with glial cells, and other physiological processes [38, 39 ]. nicotine also acts upon th and npy immunoreactivities in nodose / petrosal ganglia of shr compared to wky rat ganglia which may be of relevance to the neural mechanisms of blood pressure regulation. since these molecules are involved in the hypotensive response within the nts it could also be that the increase in th and npy immunoreactivities are secondary in response to the raise in blood pressure induced by nicotine. the possible effects over neurotransmission seem to be more pronounced in shr that are more responsive to the effects of nicotine exposure. on the other hand, increased th and npy immunoreactivities in sympathetic ganglia such as the superior cervical ganglion may be indicative of increased activation of the sympathetic nervous system, which ends up with the release of norepinephrine into blood contributing to the development of hypertension [40, 41 ]. the presence of substance p immunoreactivity in nodose / petrosal ganglia is related to sensory afferents from ventricular myocardium, chemoreception [43, 44 ] and it is released as a cotransmitter by baroreceptor afferents into the nts where it may promote an increase in blood pressure. the present findings demonstrated an increase in substance p expression in nodose / petrosal ganglia of both strains after nicotine exposure which indicates a direct action of nicotine in these ganglia. as previously demonstrated, nicotine did not increase blood pressure in wky rats, revealing the participation of substance p in complex regulatory mechanisms other than only blood pressure control. at the scg level, basal sp immunoreactivity level was increased in shr compared to wky rats such as previously demonstrated by gurusinghe and bell. nicotine treatment induced an additional increase in sp immunoreactivity in the shr, as observed with the other transmitters, suggesting again a relation to the high blood pressure level of this strain. matta and colleagues demonstrated the ability of peripherally administered nicotine to stimulate npy and th expression in the nts and pvn associated with the release of adrenocorticotropic hormone. the present study contributes with the understanding of cardiovascular control associated with nicotine abuse, since the results demonstrated the increased responsiveness of hypertension genetic - susceptible rats to nicotine considering npy and th, although the cause and consequence remains to be further investigated. within the central nervous system, the results also suggest an association between the direct effects of nicotine and a reflex response due to the increased blood pressure of the shr strain, an increased th immunoreactivity only in the nts of shr rats was observed. the analysis of tyrosine hydroxylase possibly indicates the tentative of homeostasis maintenance in response to blood pressure elevation, as previously demonstrated by our group in a model of experimental hypertension. in contrast, we demonstrated that npy1r binding is decreased after nicotine exposure which may lead to increase in blood pressure as described by narvaez and colleagues. th mrna and npy1r binding are about 100% increased in the pvn of shr regardless of nicotine exposure. this is in agreement with previous data that demonstrated the increased sensitivity of shr to stress related to augmented npy expression in brain. moreover, yu and sharp recently demonstrated the association between noradrenergic regulation of the pvn neurons and the sensitization to stress induced by nicotine administration, which is in line with the present results considering the shr strain. in conclusion, findings of the present study suggest that nicotine is able to modulate neurotransmitter systems especially in peripheral ganglia, while its central effects are preferentially encountered in the shr, which may be linked to the elevation of blood pressure encountered in this strain. whether these effects are direct or in response to alteration in blood pressure remains to be determined. | considering that nicotine instantly interacts with central and peripheral nervous systems promoting cardiovascular effects after tobacco smoking, we evaluated the modulation of glutamate, tyrosine hydroxylase (th), neuropeptide y (npy), and substance p (sp) in nodose / petrosal and superior cervical ganglia, as well as th and npy in nucleus tractus solitarii (nts) and hypothalamic paraventricular nucleus (pvn) of normotensive wistar kyoto (wky) and spontaneously hypertensive rats (shr) after 8 weeks of nicotine exposure. immunohistochemical and in situ hybridization data demonstrated increased expression of th in brain and ganglia related to blood pressure control, preferentially in shr, after nicotine exposure. the alkaloid also increased npy immunoreactivity in ganglia, nts, and pvn of shr, in spite of decreasing its receptor (npy1r) binding in nts of both strains. nicotine increased sp and glutamate in ganglia. in summary, nicotine positively modulated the studied variables in ganglia while its central effects were mainly constrained to shr. |
a 167 cm, 65 kg, male patient at the age of 27 was observed in the neurosurgery department for a linear fracture at the temporal bone caused by a motorcycle accident that had taken place 11 days before the operation and a tiny amount of subarachnoid hemorrhage corresponding to grade 1 of the hunt and hess grading system (fig. 1). during the observation, facial nerve paralysis, a complication of the fracture, was found and a facial nerve decompression operation was planned. though he had cerebral hemorrhage in the history from the traffic accident 3 years before, he was provided with no particular treatment and the encephalomalacia by the previous trauma and subarachnoid hemorrhage by the recent accident were found in the neuroimaging. all the findings were normal in all the preoperative tests that were carried out in the laboratory, including the electrocardiography and the chest radiography. thirty minutes before the patient 's arrival in the operation room, an intravenous injection of glycopyrrolate 0.2 mg and midazolam 3.0 mg was performed. after arriving at the operation room, an electrocardiograph, an automated noninvasive blood pressure device, and a pulse oximeter were attached to the patient. before the induction, the blood pressure was 115/67 mmhg, the heart rate was 75/min, and the oxygen saturation was 99%. the induction was performed by an intravenous injection of propofol 120 mg, rocuronium 50 mg, and fentanyl 50 g, and endotracheal intubation was carried out after sufficient muscular relaxation. after checking the continuous positive - end carbon dioxide expiration and the uniform breath sound from both sides of the chest, the tube was fixed at the proper endotracheal location and the mechanical ventilation was initiated with o2 1.5 l / min, n2o 1.5 l / min and sevoflurane 2 vol%. after the initiation, the positive end expiratory pressure was kept at 33 - 37 mmhg. when preparing the operation, the systolic blood pressure was maintained at 100 - 120 mmhg, the heart rate at 80 - 90/min, and the peripheral oxygen saturation at 100%. twenty - five minutes after the induction, the doctor performing the operation injected 2% lidocaine 4 ml including 1 : 200,000 epinephrine at the retroauricular temporal lobe parts for incision after checking the negative blood aspiration. between 20 - 30 seconds after the local infiltration, the heart rate suddenly increased to 160/min and the measured blood pressure was 204/135 mmhg. then, since ventricular arrhythmia was found by the electrocardiograph, injection of all the anesthetics and the operation were stopped, providing 100% oxygen. though an intravenous injection of lidocaine 100 mg was immediately carried out, the ventricular arrhythmia continued. even after the second intravenous injection of lidocaine 100 mg, the arrhythmia continued. following the defibrillation at monophasic 200 - 300 - 360 j, the heart rate was turned to the sinus tachycardia of 110 - 120/min and the measured systolic blood pressure was 100 - 110 mmhg. however, pulseless ventricular arrhythmia was found again after 2 minutes, and defibrillation was performed at monophasic 360 j. after that, the heart rate was turned to the sinus tachycardia of 110 - 120/min again, and the measured systolic blood pressure was 100 - 110 mmhg. again, a supraventricular tachycardia unresponsive to vagal maneuver took place and adenosine 6 mg was injected rapidly through the external jugular vein. though the heart rate was turned to the sinus tachycardia, defibrillation at monophasic 360 j after that, sinus rhythm was recovered and the blood pressure and the heart rate were stabilized at 133/81 mmhg and 98/min, respectively. the arterial blood gas test performed at that time showed ph 7.435, paco2 33.3 mmhg, pao2 369 mmhg, bicarbonate 23.1 mmol / l, and sao2 100%. according to cardiac enzyme test, creatine kinase - mb (ck - mb) was increased to 4.2 ng / ml, and troponin - i to 1.23 ng / ml. after the cardiac rhythm of the patient became normal, the assisted respiration was continuously carried out in the operation room until the spontaneous respiration was recovered. the consciousness and the spontaneous respiration were restored about 2 and 1/2 hours after the induction, and then, the patient was transported to the intensive care unit after extubation of the tracheal tube. the fluid injected in the operation room was crystalloid solution 500 ml and colloid solution 500 ml, and the urination was 300 ml. in the intensive care unit, the measured blood pressure was 123/72 mmhg ; the heart rate was 97/min ; and the peripheral oxygen saturation was 100%. the arterial blood gas test results were ph 7.379, paco2 43.3 mmhg, pao2 111 mmhg, bicarbonate 24.5 mmol / l, and sao2 98%. electrocardiography was performed due to the finding of a heart enzyme level increase, and the left ventricular ejection fraction was 35%, indicating dysfunction at the left ventricular which was regarded as the sequel of the defibrillation and cardiopulmonary resuscitation. there was no abnormal finding in the movement of the myocardium wall. on the day of entering the intensive care unit, the blood pressure was maintained with dopamine 5 g / kg / min, but the dopamine injection was stopped on the second day in the intensive care unit. since the heart enzyme and electrocardiography findings were normal and there was no specific finding in the patient 's symptom or the physical examinations from the sixth day forward in the intensive care unit, the patient was moved to the general ward and was discharged after 2 days without any sequel. for the purpose of improving operative vision, a local infiltration of epinephrine is often carried out by mixing it with physiological saline or local anesthetic, and a side effect rarely takes place with the clinical dose. however, as in this case report, there are a few cases where cardiovascular collapse takes place by a little amount of epinephrine local infiltration. wanamaker. reported the case where severe hypertension and tachycardia took place after the hypodermic injection of 1% lidocaine 3 ml mixed with 1 : 100,000 epinephrine for tympanoplasty, followed by ventricular tachycardia and ventricular fibrillation, and thus cardioversion was required. woldorf and pastore also reported the case where pulmonary edema, as well as severe low blood pressure with the systolic blood pressure lower than 30 mmhg and tachycardia, took place after the infiltration of 1% lidocaine 5 ml mixed with 1 : 200,000 epinephrine to the gingivobuccal fold mucosa. in our case report, a hypodermic injection of 2% lidocaine 4 ml mixed with 1 : 200,000 epinephrine was carried out, dividing the total amount into several times, into the part around the retroauricular mastoid, as in the case of the first example. the total amount of epinephrine used in the quoted cases was 30 g and 25 g, respectively, and the amount in our case was 20 g. the maximum allowable dose for adults is the maximum therapeutic dose of 0.5 - 1 mg and the minimum lethal dose of 4 mg for hypodermic injection, which are a little amount compared to the maximum allowed dose of 7 - 8 mg. in addition, considering that the maximum safe dose of epinephrine can be increased due to the endogenous vasoconstriction and the protective effect of lidocaine that is jointly used, the amount in this case was insufficient to cause cardiovascular collapse, even though it is assumed that all the injected epinephrine was immediately absorbed in the linear fracture at the temporal bone or the vein. a few factors were presumed as the causes of the electrocardiographic disorder by the tiny amount of epinephrine in this case report. first, subarachnoid hemorrhage may be one of the factors, though there was no abnormal finding in the physical examination or other tests except the pain at the fractured part in the temporal region. in the acute phase of subarachnoid hemorrhage, cardiovascular crisis can take place due to an imbalance in neurovegetative control, an increase of catecholamine in the circulatory system, and local myocardial tissue or hypopituitarism. long qt, p - pulmonale, st segment - elevation and st segment - depression, inversion of t wave, prominent u wave, and temporary pathologic q wave are generated, and the generation rate is almost 50 - 90%. such changes are frequently found in the acute phase (0 - 72 hours) and turn to the normal states after a few days. however, clinically significant arrhythmias, such as ventricular tachycardia and atrial tachyarrhythmia, are also found in 1 - 4% of the patients. measured the discharged epinephrine and the metabolites after an epinephrine hypodermic injection and reported that the detected amount was 2 times more than the expected value. however, this explanation might be valid only for the clinical pattern when hundreds of small quantity epinephrine units are injected. on the other hand, the studies that compared the vasoconstriction effect depending on the ratio of local anesthetic revealed that there was no significant clinical difference, although a wide range of concentrations were tested from 1 : 50,000 to 1 : 400,000. if so, the use of a lower concentration of epinephrine for the vasoconstriction effect can be helpful in reducing various complications. inhalation anesthetics can also affect the occurrence of arrhythmia by a little amount of epinephrine injected from outside. however, this can hardly be the cause of the ventricular arrhythmia since sevoflurane, which was used in this case report, shows a low epinephrine - induced arrhythmia incidence rate, although it has a negative myocardial inotropic effect similar to that of isoflurane and desflurane. the patient in this case report undertook the operation on the 11th day after the injury, after the acute phase, and did not show any abnormality in the electrolyte test and electrocardiography in the preoperative tests without any past history of heart disease. even though the patient did not have any specific symptoms like those, a little amount of subcutaneous infiltration caused the arrhythmia 11 days after the injury. in conclusion, it can be assumed that the asymptomatic, but definitely existing, subarachnoid hemorrhage might have caused the increase of catecholamine and hypopituitarism that were not clinically revealed as well. under such conditions, to prevent the epinephrine - induced cardiovascular crisis, a patient 's family and personal history regarding cardiovascular diseases, cryptorrhea, and medication should be thoroughly investigated before the operation and attention should be paid to the ventilation, blood pressure, heart rate, and the heart rhythm during the operation. the therapy for epinephrine - induced cardiovascular crisis is symptomatic and similar to the therapy for pheochromocytoma. for the treatment of severe hypertension, -adrenergic blockers such as phentolamine esmolol, the short acting 1-selective blocker, is preferred because it has a short half - life ; it can be used by volume titration depending on the heart rate ; and it can reduce the risk of hypertension and coronary spasm due to the excessive -stimulation that is found in nonspecific -blockers. calcium - channel blockers, such as verapamil and diltiazem, are also used for hypertension, tachycardia, and arrhythmia. the cardiopulmonary resuscitation algorithm is practiced for the treatment of arrhythmia and cardiac arrest. in this case, since ventricular arrhythmia was found a few seconds after tachycardia and hypertension, lidocaine, which has been used for a long time with a less immediate adverse reaction, was used, but there was no response. since the pulseless ventricular arrhythmia was found, the defibrillator was prepared and cardioversion was tried. for the defibrillation, monophasic 200 - 300 - 360 j, the conventional 3-successive - shock, was used, but intravenous injection of amiodarone and biphasic 1 shock are recommended in the present cardiopulmonary resuscitation guidelines. amiodarone affects the sodium channel, potassium channel, and calcium channel, and it has a blocking effect on and sympathetic nerve receptors. it is injected in ventricular fibrillation or ventricular tachycardia patients who are not responsive to cardiopulmonary resuscitation, electroshock, or vasoconstrictor by an initial intravenous injection of 150 mg for 10 minutes, and the daily maximum allowance is 2.2 g. because the 3 successive shocks can interrupt the thoracic compression and inhibit the perfusion pressure to the coronary artery, biphasic 1 shock 200 j is recommended at present. it should be corrected that we did not follow the treatment guidelines revised in 2005, but applied the drugs and procedures that were recommended before the revision in this case. in conclusion, this case shows that cardiovascular crisis, such as arrhythmia and cardiac arrest, took place after a small amount, clinical volume of epinephrine was injected in a healthy patient without any heart disease. as in this case report, for the patients who have the potential of hypersensitization to externally injected epinephrine due to an intracranial lesion, positive monitoring and preparation are required in case of cardiovascular crisis, even if there is no abnormal finding in the physical examination or the laboratory tests for the operation after the acute phase. | the infiltration of dilute epinephrine solution has been used for many years to provide hemostasis. however, epinephrine has adverse cardiovascular effects, such as arrhythmia, pulmonary edema, and even cardiac arrest. we have experienced epinephrine - induced cardiovascular crisis, with severe hypertension, tachycardia, and cardiac arrest after subcutaneous infiltration of a 2% lidocaine and 1 : 200,000 epinephrine solution in a patient with an asymptomatic subarachnoid hemorrhage. we provided successfully advanced cardiac life support in the operating room and cardioverted the patient back into a sinus rhythm with no untoward effects. the patient recovered without any apparent sequelae after intensive care. |
mr imaging and mr spectroscopy were performed twice in 10 c - peptide negative, nonsmoking patients with type 1 diabetes (five male) using insulin treatment by insulin pump therapy (continuous subcutaneous insulin infusion). one study was done after a 3-day period in which subjects aimed at optimal blood glucose levels measured by a continuous glucose monitoring system (medtronic). the second study was performed after 50% reduction in basal and bolus insulin infusions during 24 h, compared with the first study, in order to maintain hyperglycemia with glucose levels between 15 and 20 mmol / l. for both occasions, patients were instructed to maintain the same caloric intake for 3 days before examination. the sequence between the euglycemic and hyperglycemic occasions was randomly assigned. before mr examination, blood samples (postprandial) were taken. mr spectroscopy measurements (1.5-t ; philips) were obtained using a point - resolved, spatially localized spectroscopic pulse sequence to acquire single voxel (8 ml) spectra. for the heart, lipid resonances of myocardial and hepatic tg were summed and calculated as a percentage of the unsuppressed water signal ([tg / water ] 100). to assess left ventricular (lv) systolic function, the heart was imaged in the short axis orientation. to assess lv diastolic function, a phase contrast sequence with velocity encoding analysis was performed using mass and flow (medis) to quantify lv ejection fraction and flow velocities in early diastole (early filling phase [e ]) and at atrial contraction (atrial filling phase [a ], e / a ratio, and e deceleration). patient characteristics at baseline and during hyperglycemia are shown in table 1. during partial insulin deprivation, hyperglycemic dysregulation was present in all patients (mean plasma 24-h glucose was 8.4 0.6 mmol / l during the control study, which increased to 15.9 0.8 mmol / l during partial insulin deprivation [p < 0.001 ]) and associated with an increase in plasma levels of nonesterified fatty acids from 0.31 0.05 to 0.46 0.07 mmol / l (p = 0.015). myocardial tg content was 0.31 0.04% at baseline and did not change during hyperglycemic dysregulation (0.34 0.06% ; p = 0.587). e / a ratio was unaffected (1.9 0.2 at baseline vs. 1.9 0.3). this is the first study to document the effects of short - term hyperglycemic dysregulation on myocardial tg content and lv function in patients with type 1 diabetes. the present study shows that hyperglycemic dysregulation for 24 h, as frequently observed in patients with type 1 diabetes, does not modulate myocardial tg content or myocardial function, despite considerable metabolic dysregulation. we hypothesized that short - term partial insulin deprivation results in changes in myocardial tg content, possibly associated with changes in myocardial function. stiffness of intermediate - sized arteries is rapidly increased in patients with type 1 diabetes during hyperglycemia, whereas larger arteries seem unaffected (5). moreover, in healthy subjects myocardial function and tg content rapidly adapt to changes in metabolic state (2). interestingly, this adaptation could not be evoked by short - term hyperglycemic dysregulation, suggesting that the heart is protected from these short - term effects. nonetheless, we can not exclude the possibility that prolongation of the duration of partial insulin deprivation might have resulted in changes in myocardial function and tg content. myocardial fatty acid utilization is increased, whereas myocardial glucose uptake is considerably lower in diabetic patients compared with that in control subjects (6). accordingly, the present study interestingly shows that myocardial tg content in diabetic patients was not different from the values we observed in studies in healthy subjects (2,3). however, fatty acid kinetics during hyperglycemia can not be derived from the present data, and we can not exclude changes in myocardial fatty acid oxidation. hepatic tg content was measured to study the effects of insulin deprivation on tissue - specific tg distribution because we previously found discrepant effects of interventions on heart and liver (2). however, in the present study design insulin deprivation did not result in altered hepatic tg content. in conclusion, short - term partial insulin deprivation resulting in hyperglycemic dysregulation, which is frequently observed in patients with type 1 diabetes, does not modulate myocardial or hepatic tg content or lv function, despite considerable metabolic dysregulation. | objective to evaluate the effects of hyperglycemia due to partial insulin deprivation on myocardial triglyceride (tg) content and myocardial function in patients with type 1 diabetes.research design and methods myocardial and hepatic tg content and left ventricular (lv) function were measured by magnetic resonance (mr) spectroscopy and mr imaging during optimal glucoregulation and after 24 h of partial insulin deprivation (n = 10).results mean insulin infusion rate was 45 5 units at baseline, whereas it was 27 5 units during hyperglycemia (per 24 h, p < 0.001). plasma glucose levels increased from 8.4 0.6 to 15.9 0.8 mmol / l (p < 0.001), and plasma levels of nonesterified fatty acids from 0.31 0.05 to 0.46 0.07 mmol / l (p = 0.015). hyperglycemia had no effects on myocardial or hepatic tg content and lv function.conclusionsshort-term hyperglycemic dysregulation does not modulate myocardial or hepatic tg content or myocardial function, despite considerable metabolic adaptations. |
c h functionalization logic is rapidly permeating the way organic chemists approach synthesis and deconstruct target molecules. with methodological advances developing at an increasing pace, new disconnections and strategies once thought impossible are now available for consideration during synthesis planning. while these methods have sporadically been utilized to great effect for decades, only recently have these strategies been formalized and articulated as an efficient and effective means to construct molecules of interest. in comparison to traditional prefunctionalization approaches, there are inherent benefits to using c h bonds as latent functional groups in terms of redox, atom, and step economy. furthermore, many issues of chemoselectivity are frequently mitigated by simply removing the functional groups from the equation altogether. c h functionalization methods are particularly compelling from a strategic standpoint because they can challenge preconceived notions in order to provide solutions to longstanding problems in organic chemistry. stereocontrolled synthesis of complex cyclobutanes is one such problem that was identified while surveying the wide diversity of cyclobutane - containing natural products that have been reported in the literature. common among all of these cyclobutanes, with the exception of tripartilactam (4), is that they are pseudodimeric ; they are composed of two similar, but distinct, olefin precursors. for instance, the piperarborenines (1 and 2) have differing degrees of oxidation on the aryl rings, with one ring containing two methoxy substituents and the other possessing three. the dictazoles (5 and 6), anthocertotonic acid (3), and pipercyclobutanamide a (8), on the other hand, are fully unsymmetrical with four different substituents on the cyclobutane ring. additionally, a wide variety of cyclobutane stereochemistries are observed, furthering the difficulty of general strategies for their construction. complex cyclobutane natural products. with increasing interest apparent in the fields of medicinal chemistry, polymer, and material science, a dearth of methods for the construction of cyclobutanes has been revealed, particularly in comparison to its smaller and larger homologues. the most commonly considered and direct approach to cyclobutane synthesis is through a [2 + 2 ] photocycloaddition. while this strategy has proven useful in many intramolecular contexts and homodimerizations, the successful heterodimerization of two olefins is highly dependent upon the proper steric and electronic properties of the monomers. additionally, the resulting stereochemistry is largely at the mercy of the substrates chosen. for the heterodimerization of two similar monomers, a photochemical approach could be highly inefficient, as illustrated in figure 2. this first issue, presuming a photocycloaddition reaction is viable, is one of statistics. since the two monomers are effectively identical in terms of steric and electronic parameters, there is likely no preference for heterodimerization over homodimerization. the orientation of the olefin monomers during the dimerization is another point of consideration, since both head - to - head and head - to - tail modes of cyclization are possible. when these factors are combined with facile e / z isomerism of the starting materials under photochemical conditions, a potentially very complex mixture of dimeric products could arise that presumably would be very challenging to purify. further supporting this line of reasoning, the homodimerization of methyl cinnamate in the presence of bf3et2o leads to 8 of the 11 possible isomeric cyclobutane products. potential products of a hypothetical photochemical [2 + 2 ] heterodimerzation reaction. partial solutions to this problem have emerged from solid - state photochemistry, template - directed photochemistry, and photoredox catalysis. as shown in figure 3a, seminal studies on topochemistry by schmidt demonstrated that direct irradiation of different crystal polymorphs of cinnamic acid (9) in the solid state leads to different cyclobutane dimers. the polymorph leads to -truxillic acid (10), while the form gives exclusively -truxinic acid (11). this chemistry was the basis for the syntheses of the symmetrical cyclobutane dimers dipiperamide a and incarvillateine. notably, the polymorph of cinnamic acid is photoinert due to improper olefin spacing and alignment in the solid state. this strategy is not well suited for heterodimerizations, however, since a 1:1 cocrystallization and precise packing of the two different olefins in the crystal lattice would be required, a challenging crystal engineering problem that has only been observed in highly biased systems. template - directed photochemistry has also seen success in controlling the stereo- and regiochemistry of [2 + 2 ] reactions by placing two olefins in close proximity through molecular imprinting, supramolecular encapsulation, or other noncovalent interactions (e.g., hydrogen bonding). while this approach has allowed the controlled dimerization of several cinnamic derivatives that are otherwise unreactive, recently, impressive progress has been made using visible light photoredox catalysis for highly efficient and stereoselective dimerization of olefins, including reports of controlled heterodimerization by yoon and co - workers (figure 3b). currently, these methods are limited to aryl enone (e.g., 12) or an electron - rich styrene (e.g., 15) substrate for productive cyclization and only generate head - to - head adducts. direct ring - closing strategies are entropically disfavored and are frequently low yielding, even for simple substrates. ketene cycloaddition is one of the most useful methods for cyclobutane synthesis, due to the high levels of regio- and stereoselectivity frequently observed and a variety of methods for ketene formation, though the product always results in a cyclobutanone. examples of (a) solid - state photochemistry and (b) photoredox - catalyzed [2 + 2 ] cycloadditions. cyclobutane natural products also have proven to be challenging to properly elucidate using standard spectroscopic methods, particularly nmr. numerous stereochemical and constitutional errors have been made in the literature when attempting to determine the structure of cyclobutane - containing natural products. these misinterpretations likely derive from the fluxional nature of the cyclobutane ring system that rapidly undergoes ring flipping, resulting in unpredictable nmr chemical shifts that have been described as rather erratic. proton coupling constants, which are routinely used as a diagnostic stereochemical tool in other cyclic systems, are widely varied for cyclobutanes, with cis and trans vicinal coupling ranging 4.611.5 and 2.010.7 hz, respectively. in combination with the frequently observed long - range jh, h coupling across the ring, compounds of mistaken identity are frequently proposed. from the viewpoint of structural confirmation, a direct dimerization strategy would be at a disadvantage, since the true structure would likely not be challenged if the spectral and physical data matched those which were reported. reassignments are generally reliant upon x - ray crystallography, chemical synthesis, and, more recently, computational methods. since the majority of cyclobutane - containing natural products have not been evaluated by one of these means, it stands to reason that many of the structures suggested in the literature are in fact incorrect. while many terpene - derived cyclobutanes are produced through cationic polyolefin cyclization, the role of enzymes in the production of many cyclobutane dimers is unclear. the marine natural products dictazole a (5), dictazole b (6), and sceptrin (19) are isolated from deep - sea sponges where very little sunlight penetrates, making a purely photochemical [2 + 2 ] pathway improbable. furthermore, sceptrin (19) is isolated as an enantiopure molecule, almost certainly implying enzymatic intervention. a recent report by molinski demonstrated the production of benzosceptrin c from its monomer, oroidin, using a metabiosynthetic approach with cell - free enzyme extracts. this oxidative dimerization, proposed to occur through a series of single - electron - transfer events, suggests that a similar enzymatic pathway is operative for the conversion of hymenidin (18) to sceptrin (19) (figure 4). additional support for this arises from the reluctance of hymenidin (18) and aplysinopsin (21) to undergo photochemical [2 + 2 ] reactions. the piperine cyclobutane natural products (2325), on the other hand, are isolated from pepper plants and are necessarily exposed to light. these molecules are isolated as racemic mixtures and could be produced by unselective photochemical [2 + 2 ] photocycloaddition reactions, as a variety of dimeric products with differing stereochemical patterns have been isolated. curiously, the intermolecular [2 + 2 ] photocycloaddition of these monomers is highly inefficient ; therefore, additional templating or intervention within the plant cell has been proposed. [4 + 2 ] adducts, such as ageliferin (20), dictazoline c (22), and chabamide (25), are also isolated alongside the cyclobutane dimers. hymenidin (18) and aplysinopsin (21) also do not engage in diels piperine (23) can undergo thermal dimerization to chabamide (25), but forcing conditions are required (> 130 c) and the reaction is unselective. an alternate biosynthetic hypothesis for formation of these [4 + 2 ] dimers has been proposed by our group, in which a vinyl cyclobutane rearrangement (vcb) gives the six - membered - ring natural products from the respective cyclobutane dimers. experimental support for this pathway has been provided by the direct conversion of sceptrin (19) into ageliferin (20) and the epimeric nagelamide e in 50% and 28% yields, respectively, after microwave irradiation in water at 200 c. williams also suggested this as a possible pathway for the biogenesis of dictazoline c (22) on the basis of preliminary experiments with naturally isolated dictazole a (5). taking into account the limitations of regio- and stereocontrol of a direct dimerization strategy, an unconventional retrosynthesis of unsymmetrical cyclobutane dimers was considered using c common among many of the cyclobutane natural products shown in figure 1 is a carbonyl group attached directly to the cyclobutane ring. this led us to consider a general cyclobutane strategy in which the carbonyl is viewed as a latent directing group for c this would permit the direct installation of the desired functionality in a facially controlled manner, guided by the preexisting stereocenter. if two c h functionalization reactions could be employed sequentially, the synthetic challenge of pseudosymmetry and stereochemistry would be greatly simplified. while c h functionalization of cyclopropanes had received some attention at the time, examples of direct cyclobutane functionalization were limited to a harsh magnesiation procedure described by eaton and co - workers. h bonds in the literature were generally limited, but a seminal report by daugulis and co - workers in 2005 appeared promising (figure 5a). employing an aminoquinoline directing group, a wide variety of methylene c h bonds could be arylated under palladium (ii / iv) catalysis. furthermore, the only cyclic substrate examined, cyclohexane 26, delivered the bis - arylated product 27 in 61% yield as the all - syn isomer. to test the competence of four - membered rings in this methodology, cyclobutane 28 was prepared and subjected to the reaction conditions with iodobenzene. encouragingly, this substrate outperformed any of the examples described in the original report, giving the bis - phenylated cyclobutane 29 in 97% isolated yield and as a single diastereomer. additionally, the palladium loading could be lowered to 1 mol %, making this one of the most efficient sp c h functionalization reaction reported to date using a pd (ii / iv) manifold. following this initial proof of concept, studies were directed toward two potential problems : sequential cross - coupling reactions and the scope of coupling partners. in order to access the unsymmetrical cyclobutane targets in figure 1, the c h functionalization reactions would need to be performed sequentially in a controlled manner. to test the viability of a monofunctionalization, the phenylation reaction was repeated with 1 equiv of iodobenzene (figure 5b). a statistical mixture (1:1.5:1) of starting material 28, monoarylated cyclobutane 30, and bis - arylated cyclobutane 29 resulted, implying that the rate of the second arylation is nearly identical with that of the first arylation. while this was initially discouraging, we were hopeful that the issue could be overcome through alteration of the reaction conditions or substrate control on a more functionalized system. (b) statistical arylation of 28 with 1 equiv of iodobenzene. to test the generality of the c h cross - coupling reaction, other coupling partners were explored and the scope was found to be broad (scheme 1). electron - rich arenes, such as those found in the piperarborenine natural products (1 and 2), performed excellently to give 31 and 32 in 98% and 96% yield, respectively. two n - tosylated indoles were introduced onto the cyclobutane ring in 92% yield, encouraging potential access to the dictazole natural products (5 and 6). additionally, the c h olefination reaction needed for pipercyclobutanamide a (8) was successful in the daugulis chemistry, with iodostyrene giving 34 in 77% yield. even the bis - dienoate 35 could be prepared using this strategy, introducing a substructure found in tripartilactam (4). finally, alkynylation proved facile according to chatani s protocol to give 36 in 83% yield, which could serve as an alternate entry to the dictazole natural products through a larock indole synthesis. with these preliminary results, efforts were directed toward the total synthesis of the dictazole and piperarborenine families of natural products. reagents and conditions : (a) 5 mol % of pd(oac)2, 80 c, 5 h. reagents and conditions : 10 mol % of pd(oac)2, 80 c, 12 h. reagents and conditions : 5 mol % of pd(oac)2, licl (3 equiv), 100 c, 12 h. the structure of dictazole a (5) offers a number of difficulties for synthesis ; the most notable is the four contiguous stereocenters around the congested cyclobutane core, two of which are quaternary spiroiminoimidazolidinone rings. furthermore, each of the substituents is unique, as only one of the indoles is brominated and a single spiro ring bears methyl groups. to add to this challenge, the spiro stereocenter at c-3 could not be determined by standard spectroscopic means and its relative configuration is unknown. applying the cyclobutane c h functionalization strategy, a retrosynthesis of dictazole a (5) was devised (figure 6). the spiroiminoimidazolidinone rings were first deconstructed ; one could arise through strecker type chemistry (further disconnected to a protected alcohol) and another from an aminoquinoline amide, leading back to intermediate 37. two sequential c h arylation reactions with appropriate 3-iodoindoles would remove two of the stereocenters and lead back to symmetrical cyclobutane 38. notably, the bromide present on one of the indoles in dictazole a (5) should be tolerated in the arylation chemistry, since it proceeds through a palladium (ii / iv) catalytic cycle. finally, the quaternary amino - amide stereocenter at c-1 could arise from an ugi four - component coupling of cyclobutanone 40, 8-isocyanoquinoline 39, methylamine, and a suitable carboxylic acid. retrosynthesis of dictazole a (5) employing c h arylation and an ugi reaction. to test the viability of this approach, (benzyloxy)cyclobutanone 42 was prepared by thermal [2 + 2 ] cycloaddition of benzyl vinyl ether (41) and in situ formed dichloroketene following poisson s one - pot procedure in 50% yield (scheme 2). unfortunately, it was wholly ineffective in the ugi reaction under a variety of reaction conditions explored, despite ample precedent for the use of cyclobutanones in ugi reactions. interestingly, the side reactions were determined to be direct addition reactions of isonitrile 39 with the carboxylic acid or an alcoholic solvent to give dearomatized benzimidazoles (45). while the pivalic acid adduct 45c could be observed by crude h nmr, it was not isolable and hydrolyzed to 45a, which was characterized by x - ray crystallography. these bizarre addition reactions can be rationalized by considering the cyclized zwitterionic isomer 44, wherein a deprotonation / addition mechanism would generate the observed products. during the exploration of an ugi strategy, a model study was also under investigation to examine the effect of quaternary -amino substituents in the daugulis c h arylation reaction. a series of substrates were synthesized from commercially available ethyl 1-amino-1-cyclobutanecarboxylate (see the experimental section for preparations). surprisingly, these proved to have highly deleterious effects on the c h arylation chemistry. azide 46a and cbz - protected amine 46b gave no detectable arylated products on reaction with iodoindole 47, simply decomposing or remaining unreactive after prolonged heating, respectively (table 1). phthalimide - derived 46c required heating to 130 c to initiate the reaction and was accompanied by nonspecific decomposition, yielding only 14% of bis - indolated 48c with full consumption of the starting material. this lowered reactivity was attributed to the coordinating nature of the nitrogen substituents, generating an unreactive chelate with the directing group and preventing cyclometalation. ester - derived cyclobutane 46d was examined next, since it is less coordinating and could be converted to the requisite amine through a curtius rearrangement. while this substrate was also significantly less reactive than the parent cyclobutane 28, it performed the arylation chemistry at much lower temperature (90 c) than phthalimide 46c and the mass balance was largely unreacted starting material. therefore, a 1,1-cyclobutanedicarboxylate derivative was targeted for the second - generation approach to dictazole a (5). a diastereoselective synthesis of the c h activation precursor began following a report from merck for the preparation of cyclobutane hydroxy acids that is scalable and employs inexpensive starting materials. in this reaction, the dianion of 4-methoxyphenylacetic acid (49) was treated with epichlorohydrin in a double - alkylation reaction to deliver hydroxy acid 50 as a single diastereomer (scheme 3). the observed relative stereochemistry can be rationalized by invoking a magnesium chelate that templates the final ring - closing alkylation. fischer esterification and alcohol protection with tbscl generated cyclobutane 51 in 55% yield over the three steps. the electron - rich methoxyarene was selected in anticipation of the ruthenium tetroxide catalyzed arene degradation, which gave acid 52 in 70% yield. notably, performing the reaction in the absence of acetonitrile and at dilute concentrations were necessary to avoid overoxidation of the tbs alcohol to the corresponding cyclobutanone. with the key cyclobutane substrate 52 prepared, studies on the c h functionalization chemistry commenced. two directing groups developed by daugulis and co - workers, 8-aminoquinoline (53a) and o - thioanisidine (53b), were tested in the arylation reaction and were coupled to the carboxylic acid with edc to give 54a and 54b in 75% and 84% yields, respectively (scheme 4). similar to the case for 46d, aminoquinoline 54a was found to be poorly suited for the direct arylation chemistry, delivering bis - indolated cyclobutane 55a in 21% yield (unoptimized) with primarily starting material remaining. the thioanisidine 54b, on the other hand, performed better. under the same reaction conditions, this was especially peculiar, because the thioanisidine - derived directing group was reported to generally be less reactive toward methylene c h bonds in comparison to the aminoquinoline directing group. this observation, combined with the significant effect of substitution, highlights the subtle geometric factors at play in the c h functionalization chemistry. temporarily bypassing the problem of sequential arylation of the two different indoles, attention was directed at removal of the directing groups for the construction of the guanidine - containing spirocycle. removal of the directing group proved to be very challenging, since the inherently strong amide bond is quite sterically hindered after introduction of the indoles. many conditions explored for amide deprotection met with failure, and even hydrolysis of the ester in 55a for a curtius rearrangement resulted in primarily decarboxylation of the generated acid. the difficulty in removal of the amide - based directing groups is consistent with previous studies by chen and co - workers, in which considerable functional group manipulation was required to cleave the aminoquinoline auxiliary. recognizing the need for a new directing group that could be more easily deprotected, we considered an imide - based strategy. since picolinamide was reported to be a competent directing group by daugulis in his 2005 communication, a picolinimide - based directing group seemed logical. imides in general are much more susceptible to hydrolysis than amides, and this would give a second, less hindered carbonyl group for reaction and removal. to test this hypothesis, picolinimide 57 was prepared via the pentafluorophenyl ester according to the andrus protocol in 79% yield over two steps (scheme 5). gratifyingly, this directing group was found to be competent in the c h arylation chemistry, giving the bis - indolated imide 58 along with the corresponding palladium complex pd(58)2 (confirmed by x - ray crystallography). as anticipated, the imide motif was found to be much more easily cleaved than then traditional amide - based systems. treating the mixture of 58 and pd(58)2 with a dcm/2-propanol solution saturated with ammonia in the presence of catalytic scandium while it was possible to separate 58 from its palladium complex, it was more convenient to subject both to the ammonolysis, as they converge to the same product. the acetate derivative of 60 was prepared in an analogous fashion (see the experimental section for details) but strangely proved unsuccessful in the c h arylation chemistry under the same reaction conditions. it is possible that the inductive effect of the acetate influences the efficiency of the reaction or the larger tbs ether locks the ring into a more favorable geometry for c h insertion and cross - coupling. with the successful deprotection of the picolinimide directing group, this ring required regioselective methylation, attempts were made to prepare substrates that would allow for selective alkylation, through either a hydantoin or an appropriately protected spiroguanidine. hydantoin 63 was the expected product from a curtius rearrangement of 59, since the primary amide could intramolecularly collapse onto the intermediate isocyanate (scheme 6). unexpectedly, hydantoin 63 was isolated as the minor product (23% yield) and aminonitrile 62 was isolated as the major product (69% yield) when the carboxylic acid was treated with excess diphenylphosphoryl azide (dppa). this suggests that the hindered primary amide dehydrates competitively with the rearrangement of the intermediate acyl azide under the reaction conditions. interested in moving forward, we alkylated hydantoin 63 with methyl iodide to give 64 in 80% yield, but conversion of the carbonyl to the imino group of 65 through activation with meerwein s salt or lawesson s reagent met with failure. reconsidering the strategy, we turned our attention to the major product of the curtius reaction, aminonitrile 62, as an intermediate to carry forward. bergs hydantoin synthesis was envisioned in which an isocyanate would replace carbon dioxide to directly generate the desired heterocycle. in this reaction, 62 was treated with tosyl isocyanate and heated in ethanol to produce the undesired spirocycle 66. the true identity of the product was initially uncertain because of the ambiguous spectroscopic and mass spectrometry (ms) data (scheme 7). spirocycle 66 could be dimethylated with methyl iodide to give 67, which also appeared to be in agreement with the desired ring system (e.g., 65). during this time, however, crystals were obtained of 66, and the aza - bucherer bergs reaction was demonstrated to be unsuccessful through x - ray crystallographic analysis. instead of the desired oxygen closure, the nitrogen of urea 72 cyclized onto the nitrile to give intermediate 73, which underwent additional sulfonyl migration to produce the observed product 66. still interested in utilizing aminonitrile 62, we were successful in hydrating amide 69 using parkin s platinum catalyst (68), tolerating the free primary amine (scheme 7). unfortunately, this amine was reluctant to react with a number of electrophiles for spirocycle synthesis (isothioureas, cyanogen bromide, bis(methylthio)methylenesulfonamides, etc.) even when combined with a range of bases and salt additives (ag, hg, etc.). recalling the facile reaction of 62 with tosyl isocyanate, amide 69 was also found to react to give urea 71. dehydration of this urea was expected to generate a carbodiimide that would cyclize to the desired product (70), but treatment with burgess reagent gave 66 as the exclusive product in 67% yield for the two steps. again, the hindered primary amide was surprisingly susceptible to dehydration, leading to intermediate 72. given the unforeseen difficulty in constructing the requisite spirocycles, efforts at this time were directed to a separate set of pseudodimeric cyclobutane natural products, the piperarborenines, whose synthesis was being explored concurrently. despite the initial challenges in the synthesis of dictazole a (5), further efforts are aimed at construction of the spirocycles at an earlier stage in the synthesis and application of knowledge gained during the piperarborenine projects for sequential introduction of the differentiated indole substituents. contemporaneous with the dictazole studies, efforts were also being directed toward the synthesis of stereoisomeric piperarborenines b (1) and d (2). the central challenge associated with the piperarborenine natural products is the controlled, sequential installation of the two different aryl rings on the cyclobutane core. piperarborenine b (1) has a cis, trans, cis relative configuration with the two aryl substituents on opposite sides of the cyclobutane ring, whereas the arenes are on the same face of the cyclobutane in the trans, trans, trans piperarborenine d (2) (figure 7). continuing with our general c h functionalization strategy, we viewed the dihydropyridone motif as a latent directing group for c h arylation and devised a divergent strategy from the all - cis cyclobutane 74. from this intermediate, piperarborenine b (1) could be prepared by an epimerization at c-1, directed c h arylation, and further functional group manipulations to install the imide side chains. alternatively, piperarborenine d (2) could be accessed by performing a c h arylation directly on 74, followed by epimerization of both c-1 and c-3 stereocenters. the divergent intermediate 74 was envisioned arising from a desymmetrizing monoarylation reaction of a cyclobutanedicarboxylate derived from 75. while the 1,3-cyclobutanedicarboxylate 75 appears to be quite simple, the shortest synthesis reported in the literature was eight steps in 20% overall yield starting from pentaerythritol (76). viewing this route unsuitable for our needs, we envisioned a new synthesis of 1,3-cyclobutanedicarboxylates starting from methyl coumalate (78). inspired by corey s seminal work on pyrone photochemistry and more recent studies by maulide and co - workers, we selected methyl coumalate (78) as a potential starting material to solve the 1,3-cyclobutanedicarboxylate problem. upon irradiation with ultraviolet light, methyl coumalate (78) was reported to undergo a successful photochemical 4 electrocyclization reaction to generate photopyrone 77. this intermediate was attractive, since only two reductions would be needed to arrive at the desired cyclobutane monocarboxylic acid 75. in practice, it was found that the intermediate photopyrone 77 is quite reactive, rapidly decomposing when treated with acid / base and thermally reverting back to the parent coumalate along with nonspecific decomposition. consistent with earlier reports by corey, hydrogenation of photopyrone 77 with palladium on carbon resulted in varying mixtures of -lactone 79 and the desired acid 75 (scheme 8). resubjection of -lactone 79 to the reaction conditions did not result in further reduction, implying that the c o bond must be reduced first to produce 75. gratifyingly, switching the heterogeneous catalyst to platinum on carbon consistently gave the monoacid 75 as the sole product and diastereomer observed by h nmr. furthermore, both the 4 electrocyclization and the hydrogenation reactions could be performed with dcm as the solvent, allowing the sequence to be further telescoped to an edc coupling with o - thioanisidine (53b), giving 80 in 61% yield in a single operation from methyl coumalate (78). with the cyclobutanedicarboxylate problem resolved, studies commenced toward the development of a desymmetrizing c h monoarylation reaction of cyclobutane 80 with 3,4,5-trimethoxyiodobenzene (81). preliminary results were promising, with the conditions originally reported by daugulis and co - workers (6 equiv of ari, no solvent, 110 c) giving the desired monoarylated cyclobutane 82 in 30% isolated yield (table 2, entry 1). since the carboxylate ligands on the palladium are proposed to be directly involved in the c h cleavage event, it was reasoned that a bulkier carboxylate could hinder the second cyclometalation event and the production of doubly arylated 83. indeed, pivalic acid in combination with tert - butyl alcohol as a solvent proved to be effective (entry 2), though the overall conversion of the reaction was also lowered. further screening of solvents revealed that trifluoroethanol (tfe) improved the reaction, permitting the temperature and catalyst loading to be lowered slightly, but more of the overarylation byproduct 83 was produced (entry 3). switching the solvent to hexafluoro-2-propanol (hfip) maintained the accelerating effects of tfe but almost fully suppressed the second arylation, possibly due to the increased steric bulk. with these optimized conditions, monoarylated cyclobutane 82 was obtained in 65% isolated yield, though the conversion dropped slightly when the reaction was scaled up, leading to a 52% yield on a gram scale. additionally, the beneficial effects of fluorinated alcoholic solvents on c h activation reactions has been reported in other palladium - catalyzed systems since the disclosure of this work. in order to access piperarborenine b (1), a selective inversion of the directing group stereocenter at c-1 was needed, followed by a second c h arylation reaction. while the epimerization of the amide is energetically favorable to create a trans relationship to the aryl ring, the issue is complicated by the presence of the also epimerizable ester moiety at c-3. since inversion of both of the stereocenters is the most thermodynamically favorable result, initial experiments were stopped at incomplete conversion of the starting material to observe the selectivity of the initial epimerization. upon screening various bases, c-3 epimer 85 and double epimer 86 were observed, along with an unexpected transannular cyclization to form imide 87 (table 3). sodium methoxide in meoh / thf showed very little selectivity, resulting in roughly equal quantities of 84 and 85 (entry 1). the hindered amine base dbu showed some selectivity for c-1 epimerization (3/1), though more forceful reaction conditions were required. interestingly, a counterion effect was observed with hindered alkoxide bases (entries 3 and 4). potassium tert - butoxide slightly favored ester epimer 85, while lithium tert - butoxide favored c-1 epimer 84. extending the reaction time of entry 3 to 24 h resulted in nearly full conversion to 86, as anticipated. encouraged by the lithium tert - butoxide result, the solvent was changed to toluene (entry 5). this slowed the reaction rate (15% conversion in 24 h) but only the desired 84 was detectable in the crude h nmr, in addition to starting material. further optimization of temperature, concentration, and reaction time resulted in entry 6, which minimized undesired side reactions while maintaining high conversion of starting material to give 84 in 79% yield. the origins of selectivity in this system are uncertain and are currently under investigation. completion of the piperarborenine b (1) synthesis is shown in scheme 9a. a second, directed c h arylation reaction with 3,4-dimethoxyiodobenzene (88) provided 89 in 46% yield. the reaction conditions developed for c h monoarylation of 80 proved ineffective for this reaction, but performing the reaction in tert - butyl alcohol at high reaction concentrations (1 m) gave acceptable results. attempts to further conversion of the reaction by raising the temperature to 110 c resulted in the production of tris- and tetraarylated cyclobutanes (tentatively assigned by h nmr and lc - ms) in small quantities, along with significant decomposition. with the second c h arylation secured, all that remained to complete piperarborenine b (1) was the conversion of the directing group and ester moieties to dihydropyridone imides. this could also prove problematic, since methods for direct amide bond cleavage are generally very harsh, requiring strong acid or base and heat. this is further complicated by the stereochemical lability of the ester and amide functionalities. while 1,2-trans relationships in cyclobutanes are energetically favored over cis relationships, the 1,3-cis and trans orientations are nearly thermoneutral (0.1 kcal / mol difference for dimethyl 1,3-cyclobutanedicarboxylate). kibayashi and co - workers observed this problem during the synthesis of the natural product dipiperamide a, wherein hydrolysis of 93 with barium hydroxide resulted in equal amounts of the two inseparable epimers 94 and 95 (scheme 9b). fortunately, the two - step deprotection strategy developed by grieco and evans allowed for retention of the carefully constructed stereotetrad. in this reaction, dmap - catalyzed carbamoylation with boc anhydride generated 90 in 90% yield, with x - ray crystallographic analysis confirming the presumed stereochemistry. warming 90 in the presence of lithium hydroperoxide resulted in the hydrolysis of both the amide directing group and the methyl ester in 83% yield. bis - acid 91 was converted to the corresponding bis - acid chloride and heated with dihydropyridone 92 to give piperarborenine b (1) in 77% isolated yield, which matched the spectral and experimental data reported in the isolation paper. the use of 4 molecular sieves as an acid scavenger was uniquely effective for this reaction, with traditional bases resulting in low yields and significant formation of byproducts (possibly resulting from epimerization and ketene generation). initial attempts to synthesize piperarborenine d (2) focused on the resubjection of 82 to the c unfortunately, this consistently resulted in low yields (75 c, decomp) : rf = 0.5 (silica gel, 1/1 hexanes / etoac) [reactive ; spot is from the resulting formamide ] ; hrms (m / z) n / a, unstable ; ir (film) max 3047, 2127, 1682, 1596, 1498, 1389, 826, 762 cm ; h nmr (400 mhz, cdcl3) 9.06 (dd, j = 4.2, 1.7 hz, 1 h), 8.20 (dd, j = 8.4, 1.7 hz, 1 h), 7.87 (dd, j = 8.3, 1.3 hz, 1 h), 7.79 (dd, j = 7.5, 1.4 hz, 1 h), 7.577.47 (m, 2 h) ; c nmr (cdcl3, 101 mhz) 152.0, 142.8, 136.4, 129.5, 128.8, 127.8, 125.9, 122.7, 77.5, 77.2, 76.8. methanol (0.5 ml) was added to a solution of 39 (40 mg, 0.26 mmol) in dcm (0.5 ml) at room temperature. after 4 h, the mixture was concentrated and purified directly by column chromatography (2550% etoac in hexanes) to give methanol adduct 45b (25.3 mg, 52%) as a light yellow oil : rf = 0.2 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c11h10n2o ([m + h ]) 187.0871, found 187.0875 ; ir (film) max br 3373, 2931, 1477, 1340, 1192, 1062, 803, 740 cm ; h nmr (500 mhz, cdcl3) 8.21 (s, 1h), 7.74 (d, j = 8.1 hz, 1h), 7.27 (dd, j = 15.3, 8.0 hz, 1h), 7.19 (d, j = 7.2 hz, 1h), 7.10 (dd, j = 9.8, 1.1 hz, 1h), 6.66 (dd, j = 3.6, 1.1 hz, 1h), 5.91 (dd, j = 9.8, 3.6 hz, 1h), 3.01 (s, 3h) ; c nmr (cdcl3, 126 mhz) 141.6, 140.2, 130.9, 128.1, 123.1, 122.0, 121.0, 120.2, 117.5, 80.4, 50.3. potassium carbonate (960 mg, 6.96 mmol, 2.5 equiv), copper sulfate (7 mg, 0.028 mmol, 0.01 equiv), and the diazo transfer agent s2 (700 mg, 3.34 mmol, 1.2 equiv) were successively added to a solution of commercially available ethyl 1-amino-1-cyclobutanecarboxylate monohydrochloride (s1 ; 500 mg, 2.78 mmol) in methanol (14 ml) at room temperature. after 24 h, the mixture was concentrated, dissolved in etoac (20 ml), washed with 1 n aqueous hcl (10 ml) and brine, and dried over sodium sulfate. after concentration, the crude product was purified by column chromatography (25% et2o in hexanes) to give s3 (324 mg, 78%) as a colorless oil with spectral data which matched those reported (contained 20% of inconsequential methyl ester from concomitant transesterification during the reaction). lithium hydroxide hydrate (131 mg, 3.12 mmol, 2 equiv) was added to a solution of azido ester s3 (265 mg, 1.57 mmol) in thf / h2o (10 ml, 3/1 v / v). the reaction mixture was stirred vigorously for 24 h and quenched with 3 n aqueous hcl (2 ml). the mixture was separated and extracted with etoac (3 5 ml), and the extract was washed with brine (10 ml) and dried over sodium sulfate. after concentration, azido acid s4 (230 mg, 99%) was isolated as a colorless oil : rf = 0.15 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c5h7n3o2 ([m h ]) 140.0465, found 140.0464 ; ir (film) max br 3001, 2100, 1706, 1416, 1248 cm ; h nmr (400 mhz, cdcl3) 2.732.61 (m, 1h), 2.412.26 (m, 1h), 2.161.96 (m, 1h). c nmr (cdcl3, 101 mhz) 178.6, 64.8, 31.2, 14.7. 8-aminoquinoline (260 mg, 1.8 mmol, 1.2 equiv) was added to a solution of s4 (211 mg, 1.5 mmol) in dcm (15 ml) cooled to 0 c, followed by t3p (50 wt % in etoac, 1.34 ml, 2.25 mmol, 1.5 equiv) and triethylamine (0.42 ml, 3 mmol, 2 equiv). the reaction mixture was warmed to room temperature and stirred for 24 h. saturated sodium bicarbonate solution (10 ml) was added, and the biphasic reaction mixture was separated and extracted with dcm (2 10 ml), and the extract was washed with brine (10 ml) and dried over sodium sulfate. after filtration and concentration, the crude product was purified by silica gel chromatography (05% etoac in hexanes) to give 46a (370 mg, 92%) as a colorless oil : rf = 0.6 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c14h13n5o ([m + h ]) 268.1198, found 268.1201 ; ir (film) max br 3328, 2107, 1681, 1523, 1485, 1257, 790 cm ; h nmr (400 mhz, cdcl3) 10.65 (br s, 1 h), 8.84 (dd, j = 4.2, 1.7 hz, 1 h), 8.79 (dd, j = 6.7, 2.3 hz, 1 h), 8.12 (dd, j = 8.3, 1.7 hz, 1 h), 7.557.47 (m, 2 h), 7.42 (dd, j = 8.3, 4.2 hz, 1 h), 2.912.77 (m, 2 h), 2.542.42 (m, 2 h), 2.322.17 (m, 1 h), 2.142.00 (m, 1 h) ; c nmr (cdcl3, 101 mhz) 169.9, 148.6, 138.9, 136.3, 134.0, 128.0, 127.2, 122.1, 121.7, 116.6, 66.6, 31.5, 14.8. triphenylphosphine (367 mg, 1.4 mmol, 1.2 equiv) was added to a solution of 46a (312 mg, 1.17 mmol) in dioxane / h2o (11 ml, 10/1 v / v) at room temperature. a reflux condenser was attached to the flask, and the reaction mixture was placed in an oil bath preheated to 110 c for 36 h. after it was cooled to room temperature, the reaction mixture was acidified with 1 n aqueous hcl (5 ml) and extracted with etoac (3 15 ml). n aqueous naoh (5 ml), saturated with nacl, extracted with etoac (3 25 ml), and dried over sodium sulfate. after filtration and concentration, the crude yellow oil was purified by silica gel chromatography (50100% etoac in hexanes) to give s5 (257 mg, 91%) as a colorless oil : rf = 0.4 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c14h15n3o ([m + h ]) 242.1293, found 242.1294 ; ir (film) max br 3291, 2935, 1671, 1513, 1482, 1324, 790 cm ; h nmr (400 mhz, cdcl3) 8.84 (dd, j = 7.6, 1.5 hz, 1 h), 8.80 (dd, j = 4.2, 1.7 hz, 1 h), 8.06 (dd, j = 8.3, 1.7 hz, 1 h), 7.48 (t, j = 7.9 hz, 1 h), 7.42 (dd, j = 8.2, 1.4 hz, 1 h), 7.36 (dd, j = 8.3, 4.2 hz, 1 h), 2.992.70 (m, 2 h), 2.201.84 (m, 6 h) ; c nmr (cdcl3, 101 mhz) 175.0, 148.5, 139.1, 136.2, 134.7, 128.0, 127.3, 121.5, 121.4, 116.1, 60.0, 35.3, 14.3. cbzcl (88 l, 0.62 mmol, 1.2 equiv) was added dropwise to a vigorously stirred biphasic solution of s5 (124 mg, 0.52 mmol) in dcm / saturated aqueous sodium bicarbonate (7.5 ml, 2/1 v / v) at room temperature. the reaction mixture was stirred for 2.5 h, the phases were separated and extracted with dcm (2 5 ml), and the extract was washed with brine and dried over sodium sulfate. after filtration and concentration, the crude yellow foam was filtered through a plug of silica gel (50% etoac in hexanes) to give 46b (188 mg, 97%) as a white foam : rf = 0.15 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c22h21n3o ([m + h ]) 376.1661, found 376.1663 ; ir (film) max br 3326, 2951, 1688, 1525, 1486, 1256 cm ; h nmr (500 mhz, cdcl3 ; major rotamer) 10.68 (br s, 1 h), 8.83 (br d, j = 7.7 hz, 1 h), 8.66 (s, 1 h), 8.08 (d, j = 8.0 hz, 1 h), 7.50 (t, j = 7.9 hz, 1 h), 7.45 (dd, j = 8.3, 1.5 hz, 1 h), 7.397.29 (m, 3 h), 7.297.21 (m, 2 h), 7.136.79 (br m, 1 h), 6.15 (br s, 1 h), 5.14 (s, 2 h), 2.89 (app s, 2 h), 2.241.91 (m, 4 h) ; c nmr (cdcl3, 126 mhz ; major rotamer) 172.0, 155.1, 148.2, 138.8, 136.1, 134.4, 128.5, 128.0, 127.9, 127.4, 127.3, 126.9, 121.6, 121.5, 116.4, 66.8, 60.4, 31.6, 15.4. triethylamine (212 l, 1.5 mmol, 3 equiv) was added to a solution of s5 (122 mg, 0.51 mmol) in toluene (2.5 ml) at room temperature, followed by phthalic anhydride (150 mg, 1 mmol, 2 equiv). the reaction mixture was placed in an oil bath preheated to 110 c for 20 h. after the mixture was cooled to room temperature, saturated sodium bicarbonate solution (1 ml) was added, the reaction mixture was extracted with etoac (3 3 ml), and the extract was washed with brine and dried over sodium sulfate. after filtration and concentration, the crude product was purified using column chromatography (50% etoac in hexanes) to give 46c (90 mg, 48%) as colorless crystals (188190 c). (note : the low yield likely due to crystallization of the product during chromatographic purification.) : rf = 0.15 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c22h17n3o3 ([m + h ]) 372.1348, found 372.1350 ; ir (film) max 3342, 1774, 1715, 1687, 1530, 1374, 719 cm ; h nmr (400 mhz, cdcl3) 10.66 (br s, 1 h), 8.72 (dd, j = 6.7, 2.3 hz, 1 h), 8.66 (dd, j = 4.3, 1.7 hz, 1 h), 8.08 (dd, j = 8.3, 1.7 hz, 1 h), 7.83 (dd, j = 5.5, 3.1 hz, 2 h), 7.69 (dd, j = 5.5, 3.1 hz, 2 h), 7.527.41 (m, 2 h), 7.36 (dd, j = 8.3, 4.2 hz, 1 h), 3.19 (ddt, j = 13.5, 8.0, 2.5 hz, 2 h), 3.072.94 (m, 2 h), 2.522.35 (m, 1 h), 2.222.09 (m, 1 h) ; c nmr (cdcl3, 101 mhz) 169.7, 168.1, 148.4, 138.7, 136.2, 134.3, 134.1, 132.2, 127.8, 127.2, 123.3, 121.9, 121.6, 116.6, 62.0, 32.0, 18.0. dimethyl cyclobutanedicarboxylate (3.50 g, 20.33 mmol) was dissolved in meoh (150 ml) and cooled to 0 c. an aqueous solution of naoh (813 mg in 150 ml h2o) was then added dropwise over 30 min. the reaction mixture was slowly warmed to room temperature and stirred for 12 h. the meoh was removed in vacuo, and the resulting aqueous solution was washed with et2o (100 ml). the resulting aqueous phase was acidified with 3 n aqueous hcl (10 ml) and extracted with etoac (100 ml, 2 50 ml). the combined organics were washed with brine (100 ml), dried over sodium sulfate, and concentrated to give s6 (3.00 g, 93%) as a colorless oil : rf = 0.1 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c7h10nao4 ([m + h ]) 181.0477, found 181.0478 ; ir (film) max br 3504, 2956, 1705, 1281, 1202, 1138, 688 cm ; h nmr (400 mhz, cdcl3) 3.77 (s, 1h), 2.59 (t, j = 8.1 hz, 1h), 2.081.95 (m, 1h) ; c nmr (cdcl3, 101 mhz) 177.9, 172.2, 100.1, 52.9, 52.6, 29.0, 16.3. 8-aminoquinoline (260 mg, 1.8 mmol, 1.2 equiv) was added to a solution of s6 (237 mg, 1.5 mmol) in dcm (15 ml) cooled to 0 c, followed by t3p (50 wt % in etoac, 1.34 ml, 2.25 mmol, 1.5 equiv) and triethylamine (0.42 ml, 3 mmol). the reaction mixture was warmed to room temperature and stirred for 24 h. saturated sodium bicarbonate solution (10 ml) was added, and the biphasic reaction mixture was separated, extracted with dcm (2 10 ml), washed with brine (10 ml), and dried over sodium sulfate. after filtration and concentration, the crude product was purified by silica gel chromatography (1/1/8 to 1/1/6 dcm / et2o / hexanes) to give 46d (409 mg, 90%) as a pale yellow oil : rf = 0.35 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c16h16n2o3 ([m + h ]) 285.1239, found 285.1244 ; ir (film) max br 3319, 2952, 1735, 1680, 1525, 1484, 1326, 825, 790 cm ; h nmr (400 mhz, cdcl3) 10.42 (br s, 1 h), 8.82 (dd, j = 4.2, 1.7 hz, 1 h), 8.77 (dd, j = 7.2, 1.8 hz, 1 h), 8.13 (dd, j = 8.3, 1.7 hz, 1 h), 7.587.47 (m, 2 h), 7.43 (dd, j = 8.3, 4.2 hz, 1 h), 3.83 (s, 3 h), 2.882.78 (m, 2 h), 2.772.68 (m, 2 h), 2.05 (p, j = 8.0 hz, 2 h) ; c nmr (cdcl3, 101 mhz) 173.7, 168.9, 148.5, 138.8, 136.3, 134.6, 128.0, 127.4, 121.8, 121.7, 116.6, 54.9, 53.0, 29.6, 16.3. 46c (37.1 mg, 0.10 mmol), pd(oac)2 (3.4 mg, 1.5 mol, 0.15 equiv), silver acetate (50 mg, 0.30 mmol, 3 equiv), and n - tosyl-3-iodoindole (47 ; 119 mg, 0.30 mmol, 3 equiv) were placed in a sealed tube, and toluene (200 l, 0.5 m) was added under ambient conditions. the tube was sealed and placed in an oil bath preheated to 130 c for 24 h. the reaction mixture was cooled to room temperature, diluted with dcm (1 ml), filtered through a pad of celite, and concentrated. the resulting dark red oil was purified by silica gel chromatography (1/1/6 to 1/1/3 dcm / et2o / hexanes) to give 48c (12.9 mg, 14%) as colorless crystals (> 175 c, decomp) : rf = 0.4 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c52h39n5o7s2 ([m + h ]) 910.2369, found 910.2355 ; ir (film) max 3334, 1777, 1720, 1682, 1527, 1364, 1170, 906, 719 cm ; h nmr (600 mhz, cdcl3) 10.26 (br s, 1 h), 8.73 (dd, j = 7.5, 1.4 hz, 1 h), 8.02 (dd, j = 8.3, 1.7 hz, 1 h), 7.967.86 (m, 6 h), 7.81 (td, j = 5.3, 2.1 hz, 2 h), 7.67 (dd, j = 4.2, 1.7 hz, 1 h), 7.647.58 (m, 2 h), 7.577.45 (m, 6 h), 7.287.18 (m, 4 h), 7.14 (dd, j = 8.3, 4.2 hz, 1 h), 6.796.71 (m, 4 h), 4.91 (ddd, j = 10.8, 9.6, 1.1 hz, 2 h), 3.21 (td, j = 11.2, 10.4, 6.7 hz, 2 h), 2.09 (s, 6 h) ; c nmr (cdcl3, 151 mhz) : 168.1, 165.4, 147.9, 144.2, 138.3, 135.9, 135.3, 134.7, 134.5, 133.6, 132.0, 131.2, 129.5, 127.7, 127.5, 126.8, 126.4, 124.3, 123.8, 123.0, 121.9, 121.5, 120.3, 120.2, 116.6, 113.6, 73.4, 39.1, 33.8, 21.5. 46d (30 mg, 0.106 mmol), pd(oac)2 (3.6 mg, 1.6 mol, 0.15 equiv), silver carbonate (44 mg, 0.16 mmol, 1.5 equiv), and n - tosyl-3-iodoindole (47 ; 119 mg, 0.30 mmol, 3 equiv) were placed in a sealed tube, and toluene (200 l, 0.5 m) was added under ambient conditions. the tube was sealed and placed in an oil bath preheated to 90 c for 24 h. the reaction mixture was cooled to room temperature, diluted with dcm (1 ml), filtered through a pad of celite, and concentrated. the resulting yellow oil was purified by silica gel chromatography (30% etoac in hexanes) to give 48d (18.3 mg, 21%) as a white crystalline solid, along with recovered 46d (18.7 mg, 62%) : white crystalline solid (150155 c) ; rf = 0.5 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c46h38n4o7s2 ([m + h ]) 823.2260, found 823.2266 ; ir (film) max 3294, 1730, 1673, 1529, 1359, 1170, 904, 726 cm ; h nmr (400 mhz, cdcl3) 10.25 (s, 1 h), 8.46 (d, j = 6.8 hz, 1 h), 8.39 (dd, j = 4.4, 1.7 hz, 1 h), 8.03 (d, j = 8.5 hz, 1 h), 7.887.70 (m, 6 h), 7.48 (d, j = 8.1 hz, 4 h), 7.477.37 (m, 2 h), 7.317.14 (m, 5 h), 6.77 (d, j = 7.9 hz, 4 h), 4.47 (dd, j = 11.7, 8.1 hz, 2 h), 4.05 (s, 3 h), 3.45 (q, j = 11.3 hz, 1 h), 2.90 (q, j = 9.1 hz, 1 h), 2.11 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 173.1, 164.6, 148.5, 144.4, 138.4, 135.9, 135.1, 134.9, 134.0, 130.8, 129.6, 127.8, 127.1, 126.7, 126.0, 124.6, 123.2, 121.7, 121.7, 120.3, 119.9, 116.6, 113.4, 67.2, 53.1, 36.6, 30.1, 21.5. 4-methoxyphenylacetic acid (49 ; 2.00 g, 12.0 mmol) was dissolved in dry thf (3 ml) and added dropwise to a solution of isopropylmagnesium chloride in thf (2 m, 13.2 ml, 26.4 mmol, 2.2 equiv) dropwise, keeping the internal temperature below 50 c. the reaction mixture turned heterogeneous during the addition and was stirred for 30 min at room temperature. epichlorohydrin (1.7 ml, 21.6 mmol, 1.8 equiv) was added dropwise, keeping the internal temperature below 35 c, and the mixture was stirred at room temperature for 45 min. during the addition the solution homogenizes. a solution of isopropylmagnesium chloride (2 m in thf, 12 ml, 24 mmol, 2 equiv) was added to the reaction mixture, which was then warmed to 60 c overnight (14 h). the reaction mixture was carefully quenched with 3 n aqueous hcl (20 ml), keeping the internal temperature below 35 c. the resulting biphasic solution was separated and extracted with etoac (2 50 ml). the combined organics were washed with 1 n aqueous naoh (2 25 ml), and the combined aqueous layer was acidified with 3 n aqueous hcl and extracted with etoac (3 25 ml). the combined organic layers were washed with brine (25 ml), dried over na2so4, and concentrated to give the crude hydroxy acid 50 (2.16 g) as a white solid that was used directly in the next reaction. to a solution of the crude hydroxy acid in meoh (20 ml) was added concentrated sulfuric acid (54 l, 1 mmol), and the mixture was warmed to 60 c for 12 h. the reaction mixture was cooled to room temperature and neutralized with saturated sodium bicarbonate solution (2 ml), and the meoh was removed in vacuo. the resulting mixture was diluted with etoac (50 ml), washed with brine (25 ml), dried over na2so4, and concentrated to give the crude methyl ester (2.16 g), which was used directly in the next step. this material could be further purified using silica gel chromatography (3060% et2o in hexanes) for characterization to give the methyl ester s7 as colorless crystals (mp 6465 c) : rf = 0.4 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c13h16o4 ([m + h ]) 237.1127, found 237.1131 ; ir (film) max br 3419, 2950, 1727, 1511, 1250, 1130, 1031, 832 cm ; h nmr (400 mhz, cdcl3) 7.327.21 (m, 2 h), 6.906.83 (m, 2 h), 4.16 (apparent p, j = 6.9 hz, 1 h), 3.79 (s, 3 h), 3.62 (s, 3 h), 2.972.82 (m, 2 h), 2.722.61 (m, 2 h), 2.56 (br s, 1 h) ; c nmr (cdcl3, 101 mhz) 176.4, 158.5, 133.3, 128.1, 113.9, 62.7, 55.4, 52.6, 44.0, 42.8. tbscl (2.17 g, 14.4 mmol, 1.5 equiv) was added to a solution of crude s7 (2.27 g, ca. 9.6 mmol) in dry dcm (35 ml) at room temperature, followed by imidazole (3.27 g, 48 mmol, 5 equiv). this mixture was stirred at room temperature for 30 min and quenched with meoh (1 ml). the reaction mixture was diluted with dcm (50 ml), washed with 1 n aqueous hcl (50 ml) and brine (50 ml), and dried over na2so4. after filtration and concentration, the crude product was purified by column chromatography (020% et2o in hexanes) to give 51 (2.34 g, 56% for three steps) as colorless crystals (mp 6365 c) : rf = 0.6 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c19h30o4si ([m + h ]) 351.1992, found 351.1987 ; ir (film) max 2952, 1732, 1512, 1251, 1145, 1053, 833 cm ; h nmr (400 mhz, cdcl3) 7.367.29 (m, 2 h), 6.936.86 (m, 2 h), 4.10 (apparent p, j = 7.2 hz, 1 h), 3.80 (s, 3 h), 3.61 (s, 3 h), 2.85 (ddt, j = 8.9, 6.9, 2.4 hz, 2 h), 2.68 (ddt, j = 10.1, 7.5, 2.4 hz, 2 h), 0.88 (s, 9 h), 0.02 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 175.9, 175.8, 158.6, 133.3, 128.2, 113.9, 62.3, 55.3, 52.3, 43.6, 43.2, 25.9, 18.0, 4.7. sodium periodate (31.5 g, 147.3 mmol, 15 equiv) was added to a vigorously stirred biphasic solution of 51 (3.44 g, 9.82 mmol) in etoac / h2o (390 ml/1.15 l) at 4 c. ruthenium oxide hydrate (148 mg, 0.98 mmol, 0.1 equiv) was added in a single portion, and the light yellow mixture was slowly warmed to room temperature and stirred for 14 h. the resulting black mixture was separated and extracted with etoac (2 200 ml). the combined organics were washed with a brine / saturated sodium sulfite solution (200 ml, 10/1 v / v), dried over na2so4 and concentrated. the crude product was filtered through a plug of silica gel (with etoac as eluent) to give 52 (1.97 g, 70%) as a semicrystalline waxy solid. (the yield of this reaction at different scales has varied between 62 and 70% ; larger scales were generally higher yielding.) : rf = 0.5 (silica gel, etoac) ; hrms (m / z) calcd for c13h24o5si ([m h ]) 287.1320, found 287.1328 ; ir (film) max br 3418, 2955, 1712, 1251, 1135, 1048, 835 cm ; h nmr (400 mhz, cdcl3) 4.40 (apparent p, j = 7.3 hz, 1 h), 3.78 (s, 3 h), 2.85 (ddd, j = 9.9, 7.1, 2.8 hz, 2 h), 2.53 (ddd, j = 10.1, 7.5, 2.8 hz, 2 h), 0.87 (s, 9 h), 0.04 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 177.9, 171.4, 62.0, 53.0, 45.8, 41.1, 25.9, 18.0, 4.7. 8-aminoquinoline (180 mg, 1.25 mmol, 1.2 equiv) was added to a solution of 52 (300 mg, 1.04 mmol) in dcm (5.2 ml) cooled to 0 c, followed by edc (210 mg, 1.35 mmol, 1.3 equiv). the reaction mixture was warmed to room temperature and stirred for 24 h. saturated sodium bicarbonate solution (10 ml) was added, and the biphasic reaction mixture was separated, extracted with dcm (2 5 ml), washed with brine (5 ml), and dried over sodium sulfate. after filtration and concentration, the crude product was purified by silica gel chromatography (5% etoac in hexanes) to give 54a (315 mg, 76%) as a pale yellow oil : rf = 0.55 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c22h30n2o4si ([m + h ]) 415.2053, found 415.2052 ; ir (film) max br 3318, 2952, 1740, 1686, 1527, 1145, 1064, 825, 776 cm ; h nmr (400 mhz, cdcl3) 10.63 (br s, 1 h), 8.84 (dd, j = 4.2, 1.7 hz, 1 h), 8.75 (dd, j = 6.5, 2.5 hz, 1 h), 8.15 (dd, j = 8.4, 1.7 hz, 1 h), 7.587.50 (m, 2 h), 7.45 (dd, j = 8.3, 4.2 hz, 1 h), 4.43 (p, j = 7.2 hz, 1 h), 3.84 (s, 3 h), 3.09 (ddt, j = 9.2, 7.1, 2.3 hz, 2 h), 2.64 (ddd, j = 9.9, 7.1, 2.9 hz, 2 h), 0.88 (s, 9 h), 0.05 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 173.1, 168.5, 148.6, 138.8, 136.4, 134.6, 128.1, 127.4, 122.0, 121.8, 116.7, 62.2, 53.1, 47.8, 41.7, 25.9, 18.1, 4.7. 54a (58.8 mg, 0.142 mmol), pd(oac)2 (3.2 mg, 14.2 mol, 0.10 equiv), silver acetate (71 mg, 0.425 mmol, 3 equiv), and n - tosyl-3-iodoindole (169 mg, 0.425 mmol, 3 equiv) were placed in a sealed tube, and toluene (280 l, 0.5 m) was added under ambient conditions. the tube was sealed and placed in a 110 c oil bath for 24 h. the reaction mixture was cooled to room temperature, diluted with dcm (1 ml), filtered through a pad of celite, and concentrated. the resulting orange oil was purified by silica gel chromatography (1/1/8 to 1/1/4 dcm / et2o / hexanes) to give 55a as a yellow foam (29.0 mg, 21% yield) : rf = 0.6 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c52h52n4o8s2si ([m + h ]) 953.3074, found 953.3076 ; ir (film) max br 3314, 2927, 1736, 1676, 1369, 1174, 1126, 747 cm ; h nmr (400 mhz, cdcl3) 10.59 (s, 1h), 8.79 (dd, j = 7.3, 1.7 hz, 1h), 8.56 (dd, j = 4.3, 1.7 hz, 1h), 8.10 (dd, j = 8.3, 1.7 hz, 1h), 7.88 (d, j = 0.8 hz, 2h), 7.887.84 (m, 1h), 7.767.69 (m, 2h), 7.607.47 (m, 7h), 7.35 (dd, j = 8.3, 4.2 hz, 1h), 7.267.20 (m, 5h), 6.756.69 (m, 4h), 5.51 (t, j = 8.2 hz, 1h), 4.20 (dd, j = 8.1, 0.9 hz, 2h), 4.04 (s, 3h), 2.04 (s, 6h), 0.74 (s, 8h), 0.14 (s, 5h) ; c nmr (cdcl3, 126 mhz) 172.7, 164.8, 148.5, 144.4, 138.6, 136.1, 135.2, 134.8, 134.4, 131.4, 129.6, 127.9, 127.5, 126.8, 126.4, 124.6, 123.2, 122.0, 121.7, 119.9, 118.0, 116.8, 113.6, 73.1, 60.4, 53.3, 48.2, 25.8, 21.4, 17.9, 4.4. 2-(methylthio)aniline (40 l, 0.32 mmol, 1.2 equiv) was added to a solution of 52 (77.1 mg, 0.267 mmol) in dcm (1.35 ml) cooled to 0 c, followed by edc (66.5 mg, 0.35 mmol, 1.3 equiv). the reaction mixture was warmed to room temperature and stirred for 24 h. saturated sodium bicarbonate solution (1 ml) was added, and the biphasic reaction mixture was separated, extracted with dcm (2 1 ml), washed with brine (2 ml), and dried over sodium sulfate. after filtration and concentration, the crude product was purified by silica gel chromatography (5% etoac in hexanes) to give 54b (92.1 mg, 84%) as a pale yellow oil : rf = 0.6 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c20h31no4ssi ([m + h ]) 410.1821, found 410.1827 ; ir (film) max br 3315, 2953, 1720, 1692, 1580, 1514, 1434, 1147, 1064, 836 cm ; h nmr (400 mhz, cdcl3) 9.18 (br s, 1 h), 8.30 (d, j = 8.2 hz, 1 h), 7.47 (dd, j = 7.9, 1.6 hz, 1 h), 7.30 (t, j = 7.8 hz, 1 h), 7.08 (t, j = 7.6 hz, 1 h), 4.39 (p, j = 7.2 hz, 1 h), 3.82 (s, 3 h), 3.00 (ddd, j = 9.9, 7.1, 3.0 hz, 2 h), 2.58 (ddd, j = 12.7, 6.0, 2.5 hz, 2 h), 2.37 (s, 3 h), 0.88 (s, 9 h), 0.04 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 173.3, 168.1, 138.2, 132.8, 128.8, 126.1, 124.7, 120.8, 62.1, 53.1, 47.4, 41.7, 25.9, 18.7, 18.0, 4.8. 54b (65 mg, 0.159 mmol), pd(oac)2 (3.6 mg, 1.6 mol, 0.10 equiv), silver carbonate (66 mg, 0.24 mmol, 1.5 equiv), and n - tosyl-3-iodoindole (144 mg, 0.48 mmol, 3 equiv) were placed in a sealed tube, and toluene (320 l, 0.5 m) was added under ambient conditions. the tube was sealed and placed in a 110 c oil bath for 24 h. the reaction mixture was cooled to room temperature, diluted with dcm (1 ml), filtered through a pad of celite, and concentrated. the resulting dark red oil was purified by silica gel chromatography (20/5/75 dcm / et2o / hexanes) to give 55b (77.3 mg, 51% yield) as a yellow foam : rf = 0.6 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c50h53n3o8s3si ([m + h ]) 948.2842, found 948.2841 ; ir (film) max br 3309, 2927, 1716, 1678, 1172, 735 cm ; h nmr (400 mhz, cdcl3) 9.24 (br s, 1 h), 8.10 (dd, j = 8.6, 1.4 hz, 1 h), 7.957.89 (m, 2 h), 7.84 (d, j = 0.8 hz, 2 h), 7.727.59 (m, 6 h), 7.357.21 (m, 6 h), 7.04 (td, j = 7.5, 1.4 hz, 1 h), 6.916.83 (m, 4 h), 5.45 (t, j = 8.1 hz, 1 h), 4.18 (dd, j = 8.1, 0.9 hz, 2 h), 3.99 (s, 3 h), 2.23 (s, 6 h), 1.02 (s, 3 h), 0.75 (s, 9 h), 0.12 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 173.1, 164.1, 144.7, 138.5, 135.2, 134.7, 133.9, 131.1, 129.8, 129.2, 126.9, 126.3, 126.0, 124.7, 124.5, 123.2, 121.2, 119.9, 117.6, 113.5, 72.2, 59.7, 53.5, 48.3, 25.8, 21.6, 17.9, 16.9, 4.3. acid 52 (1.00 g, 3.47 mmol) was dissolved in dry dcm (17.5 ml) and cooled to 0 c in an ice bath. pentafluorophenol (958 mg, 5.2 mmol, 1.5 equiv), triethylamine (1.45 ml, 10.4 mmol, 3 equiv), and hatu (1.58 g, 4.16 mmol, 1.1 equiv) were added sequentially, and the reaction mixture was warmed to room temperature. after 15 h, the reaction mixture was diluted with dcm (15 ml) and quenched with 1 n aqueous hcl (15 ml). the biphasic mixture was separated, washed with brine (15 ml), and dried over na2so4. after filtration and concentration, the crude product was purified by column chromatography (2550% dcm in hexanes) to give s9 (1.30 g, 83%) as a colorless oil : rf = 0.7 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c19h23f5o5si ([m + h ]) 454.1313, found 454.1322 ; ir (film) max 2956, 1789, 1749, 1518, 1244, 1054, 994, 835, 777 cm ; h nmr (400 mhz, cdcl3) 4.38 (apparent p, j = 7.5 hz, 1 h), 3.81 (s, 3 h), 2.96 (m, 2 h), 2.65 (m, 2 h), 0.88 (s, 9 h), 0.05 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 170.0, 168.4, 62.0, 53.2, 45.9, 41.2, 25.8, 18.0, 4.8. s9 (1.27 g, 2.80 mmol) was dissolved in dry thf (14 ml) and cooled to 4 c in a cold room. picolinamide (678 mg, 5.6 mmol, 2 equiv) was added to the cooled reaction mixture, followed by potassium tert - butoxide solution in thf (2.0 m, 3.5 ml, 7 mmol, 2.5 equiv). after 30 min, the reaction was quenched with saturated aqueous ammonium chloride (3 ml). the biphasic mixture was diluted with etoac (30 ml), washed with brine (15 ml), and dried over na2so4. after filtration and concentration, the crude product was purified by column chromatography (1025% etoac in hexanes) to give 57 (1.05 g, 95%) as colorless crystals (mp 8385 c) : rf = 0.25 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c19h28n2o5si ([m + h ]) 393.1846, found 393.1845 ; ir (film) max br 3324, 2952, 1750, 1725, 1698, 1478, 1267, 1062, 837 cm ; h nmr (400 mhz, cdcl3) 10.78 (br s, 1 h), 8.61 (ddd, j = 4.8, 1.7, 0.9 hz, 1 h), 8.18 (dt, j = 7.9, 1.1 hz, 1 h), 7.89 (td, j = 7.7, 1.7 hz, 1 h), 7.53 (ddd, j = 7.6, 4.8, 1.2 hz, 1 h), 4.20 (tt, j = 8.0, 7.1 hz, 1 h), 3.70 (s, 3 h), 2.90 (ddt, j = 9.7, 7.3, 2.6 hz, 2 h), 2.61 (ddt, j = 12.6, 8.2, 2.8 hz, 2 h), 0.83 (s, 9 h), 0.01 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 171.6, 170.9, 162.4, 148.5, 147.7, 138.0, 127.8, 123.3, 62.0, 52.8, 48.3, 40.5, 25.8, 18.0, 4.8. 57 (710 mg, 1.81 mmol), pd(oac)2 (60.9 mg, 0.27 mmol, 0.15 equiv), silver pivalate (1.13 g, 5.41 mmol, 3 equiv), and n - tosyl-3-iodoindole (2.88 g, 7.24 mmol, 4 equiv) were placed in a sealed tube, and toluene (3.6 ml, 0.5 m) was added under ambient conditions. the tube was sealed and placed in a 120 c oil bath for 24 h. the reaction mixture was cooled to room temperature, diluted with dcm (10 ml), filtered through a pad of celite, and concentrated. the resulting dark red oil was purified by silica gel chromatography (1/1/6 to 1/1/2 dcm / et2o / hexanes) to give an orange solid which, upon washing three times with cold et2o (20 ml, 10 ml, 10 ml), gave a white powder (942 mg) containing a 5/1 mixture of 58 and pd(58)2 (55% combined yield), which was used directly in the next reaction. 58 could be separated from its palladium complex for characterization by silica gel chromatography (4/6 etoac / hexanes) : colorless crystals (mp 197200 c) ; rf = 0.5 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c49h50n4o9s2si ([m + h ]) 931.2867, found 931.2850 ; ir (film) max 3301, 2954, 1757, 1738, 1473, 1368, 1173, 1126, 745 cm ; h nmr (400 mhz, cdcl3) 8.26 (ddd, j = 4.8, 1.6, 0.9 hz, 1 h), 7.927.84 (m, 3 h), 7.79 (d, j = 0.9 hz, 2 h), 7.787.74 (m, 4 h), 7.72 (dd, j = 7.7, 1.7 hz, 1 h), 7.677.62 (m, 2 h), 7.34 (ddd, j = 7.6, 4.8, 1.2 hz, 1 h), 7.277.21 (m, 4 h), 7.15 (dd, j = 8.4, 0.9 hz, 4 h), 5.34 (t, j = 8.3 hz, 1 h), 4.21 (dd, j = 8.4, 0.9 hz, 2 h), 3.95 (s, 3h), 2.23 (s, 6h), 0.72 (s, 9 h), 0.15 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 172.0, 165.6, 161.2, 148.2, 147.9, 144.7, 137.5, 135.3, 134.8, 131.0, 130.0, 127.3, 127.1, 126.1, 124.7, 123.2, 122.8, 119.9, 117.2, 113.6, 71.6, 59.9, 53.4, 48.5, 25.7, 21.6, 17.8, 4.4. the mixture of 58 and pd(58)2 from the previous step (942 mg, 1.00 mmol) was added to a saturated ammonia solution of dcm/2-propanol (20 ml, 1/4 v / v) [saturated by bubbling ammonia gas through the solvent mixture for 5 min ]. scandium triflate (24.6 mg, 0.05 mmol, 0.05 equiv) was added, the flask was capped, and the reaction mixture was stirred at room temperature for 24 h. nitrogen was bubbled though the reaction mixture to purge the excess ammonia, and the mixture was concentrated. purification with silica gel chromatography (30% etoac in hexanes) gave 59 (794 mg, 53% for 2 steps) as a white foam : rf = 0.15 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c43h48n3o8s2si ([m + h ]) 826.2652, found 826.2649 ; ir (film) max 3472, 2954, 1733, 1679, 1447, 1366, 1173, 1123, 683 cm ; h nmr (400 mhz, cdcl3) 7.99 (d, j = 8.2 hz, 2 h), 7.80 (d, j = 8.3 hz, 4 h), 7.71 (s, 2 h), 7.60 (d, j = 7.7 hz, 2 h), 7.30 (t, j = 7.3 hz, 2 h), 7.24 (d, j = 7.3 hz, 2 h), 7.20 (d, j = 8.2 hz, 4 h), 6.24 (br s, 1 h), 5.26 (t, j = 8.0 hz, 1 h), 4.99 (br s, 1 h), 4.06 (d, j = 8.2 hz, 2 h), 3.90 (s, 3 h), 2.31 (s, 6 h), 0.72 (s, 9 h), 0.20 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 173.3, 167.8, 144.9, 135.4, 134.9, 131.1, 129.9, 127.1, 126.1, 124.7, 123.2, 119.8, 118.0, 113.8, 72.2, 58.3, 53.3, 47.9, 25.7, 21.6, 17.8, 4.4. triethylamine (450 l, 3.23 mmol, 1.5 equiv) was added to a solution of s7 (505 mg, 2.14 mmol) in dcm (20 ml) cooled to 0 c in an ice bath, followed by acetic anhydride (300 l, 3.23 mmol, 1.5 equiv) and dmap (14 mg, 0.11 mmol, 0.05 equiv). the reaction was mixture was stirred at 0 c for 2 h and then was quenched with saturated aqueous sodium bicarbonate (10 ml). the biphasic reaction mixture was separated, extracted with dcm (2 10 ml), washed with 1 n aqueous hcl (10 ml), washed with brine (20 ml), and dried over sodium sulfate. after filtration and concentration, the crude product was purified by silica gel chromatography (25% et2o in hexanes) to give s7 (552 mg, 93%) as a colorless oil : rf = 0.35 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c15h18o5 ([m + h ]) 279.1232, found 279.1231 ; ir (film) max 2953, 1727, 1512, 1229, 1030, 832 cm ; h nmr (400 mhz, cdcl3) 7.337.27 (m, 2 h), 6.926.84 (m, 2 h), 4.86 (p, j = 7.2 hz, 1 h), 3.80 (s, 3 h), 3.63 (s, 3 h), 3.042.91 (m, 2 h), 2.882.75 (m, 2 h), 2.03 (s, 3 h) ; c nmr (cdcl3, 101 mhz) 175.5, 170.6, 158.8, 132.6, 128.1, 114.0, 64.5, 55.4, 52.6, 45.1, 39.7, 21.1. sodium periodate (3.8 g, 17.7 mmol, 10 equiv) was added to a vigorously stirred biphasic solution of s8 (493 mg, 1.77 mmol) in etoac / mecn / h2o (9 ml/9 ml/30 ml) cooled to 0 c in an ice bath. ruthenium oxide hydrate (13.4 mg, 0.09 mmol, 0.05 equiv) was added in a single portion, and the light yellow mixture was vigorously stirred for 20 h, while being slowly warmed to room temperature. the resulting black mixture was separated and extracted with etoac (2 20 ml). the combined organics were washed with a brine / saturated sodium sulfite solution (200 ml, 10/1 v / v), dried over na2so4, and concentrated to give a crude acid that was used directly in the next reaction without further purification (h nmr (400 mhz, cdcl3) 5.11 (p, j = 7.6 hz, 1h), 3.79 (s, 3h), 3.222.92 (m, 2h), 2.802.60 (m, 2h), 2.04 (s, 3h)). oxalyl chloride (182 l, 2.12 mmol, 1.2 equiv) was added dropwise to a solution of the acid (ca. 1.77 mmol) in dcm (10 ml) containing 1 drop of dmf. after the reaction mixture was stirred at room temperature for 4 h, toluene was added (5 ml) and the solvent concentrated to give the crude acid chloride. this material was dissolved in toluene, and 2-picolinamide (325 mg, 2.66 mmol, 1.5 equiv) was added, followed by 4 molecular sieves (1.7 g). the heterogeneous reaction mixture was heated to 90 c for 16 h, and then the reaction mixture was filtered through celite, concentrated, and purified by column chromatography (2540% etoac in hexanes) to give 60 (251 mg, 44% for two steps) as colorless crystals (mp 138139 c) : rf = 0.25 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c15h16n2o6 ([m + h ]) 321.1087, found 321.1095 ; ir (film) max br 3319, 1735, 1726, 1698, 1481, 1234, 1044, 747 cm ; h nmr (400 mhz, cdcl3) 10.70 (s, 1 h), 8.61 (ddd, j = 4.8, 1.7, 0.9 hz, 1 h), 8.16 (dt, j = 7.9, 1.1 hz, 1 h), 7.89 (td, j = 7.7, 1.7 hz, 1 h), 7.53 (ddd, j = 7.6, 4.8, 1.2 hz, 1 h), 4.91 (p, j = 7.7 hz, 1 h), 3.71 (s, 3 h), 3.06 (ddt, j = 9.6, 7.7, 2.4 hz, 2 h), 2.76 (ddt, j = 10.7, 7.8, 2.6 hz, 2 h), 2.02 (s, 3 h) ; c nmr (cdcl3, 126 mhz) 171.2, 170.4, 170.4, 162.6, 148.6, 147.5, 138.0, 127.9, 123.3, 63.7, 53.0, 49.4, 36.8, 20.9. 59 (400 mg, 0.484 mmol) was dissolved in thf (4.8 ml), and h2o (1.6 ml) was added, followed by lithium hydroxide hydrate (102 mg, 2.43 mmol, 5 equiv). the biphasic reaction mixture was stirred vigorously for 12 h and quenched with 1 n aqueous hcl (3 ml). the layers were separated and extracted with etoac (4 5 ml), and the extract was washed with brine (10 ml), dried over sodium sulfate, filtered, and concentrated to give the carboxylic acid (388 mg, 99%) as a white foam, which was dissolved in dry dcm (4.8 ml) and cooled to 0 c. triethylamine (0.27 ml, 1.94 mmol, 4 equiv) was added, followed by diphenylphosphoryl azide (0.42 ml, 1.94 mmol, 4 equiv). the reaction mixture was slowly warmed to room temperature and stirred for 24 h. the reaction mixture was then heated to 50 c for 6 h and quenched with saturated aqueous sodium bicarbonate (5 ml). the layers were separated and extracted with dcm (2 3 ml). the combined organics were washed with brine (5 ml) and dried over sodium sulfate. after filtration and concentration, the crude product was purified by column chromatography (2550% etoac in hexanes) to give aminonitrile 62 (257 mg, 69%) as a white foam and hydantoin 63 (91 mg, 23%) as a white solid. 62 : white foam ; rf = 0.25 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c41h44n4o5s2sina ([m + na ]) 787.2420, found 787.2421 ; ir (film) max 2928, 1597, 1447, 1368, 1174, 1129, 746 cm ; h nmr (400 mhz, cdcl3) : 8.02 (dt, j = 8.3, 0.9 hz, 2 h), 7.85 (d, j = 8.4 hz, 4 h), 7.807.79 (m, 2 h), 7.63 (ddd, j = 7.9, 1.3, 0.7 hz, 2 h), 7.397.33 (m, 2 h), 7.317.25 (m, 2 h), 7.257.20 (m, 4 h), 4.57 (t, j = 8.3 hz, 1 h), 3.66 (dd, j = 8.4, 0.9 hz, 2 h), 2.33 (s, 6 h), 0.76 (s, 9 h), 0.12 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 145.1, 135.1, 135.1, 130.6, 130.0, 127.2, 125.4, 124.2, 123.6, 119.6, 119.4, 118.0, 114.0, 77.5, 77.2, 76.8, 69.6, 58.2, 52.2, 25.7, 21.7, 17.8, 4.4. 63 : white solid (> 180 c, decomp) ; rf = 0.4 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c42h44n4o7s2sina ([m + na ]) 831.2318, found 831.2332 ; ir (film) max br 3358, 2928, 1727, 1367, 1173, 1127, 745 cm ; h nmr (400 mhz, 1/1 meod / cdcl3) 7.91 (d, j = 8.3 hz, 2 h), 7.74 (d, j = 8.4 hz, 4 h), 7.65 (d, j = 0.9 hz, 2 h), 7.54 (ddd, j = 7.9, 1.2, 0.7 hz, 2 h), 7.28 (ddd, j = 8.4, 7.3, 1.3 hz, 2 h), 7.257.16 (m, 6 h), 5.02 (t, j = 8.1 hz, 1 h), 3.88 (dd, j = 8.1, 1.0 hz, 2 h), 2.30 (s, 6 h), 0.73 (s, 9 h), 0.11 (s, 6 h) ; c nmr (1/1 meod / cdcl3, 101 mhz) 173.5, 157.8, 145.8, 135.4, 135.3, 131.2, 130.4, 127.3, 125.5, 125.3, 124.0, 119.7, 117.7, 114.1, 68.9, 67.3, 50.3, 25.8, 21.6, 18.1, 4.2. 63 (119 mg, 0.147 mmol) was dissolved in dry dmf (1.5 ml), and potassium carbonate (122 mg, 0.88 mmol, 6 equiv) was added, followed by methyl iodide (37 l, 0.59 mmol, 4 equiv). after 2 h, the reaction mixture was quenched with saturated aqueous ammonium chloride (1 ml) and stirred for 30 min. the reaction mixture was concentrated and taken up in etoac (2 ml)/brine (2 ml). the biphasic mixture was separated and extracted with etoac (2 2 ml), and the extract was dried over sodium sulfate. after filtration and concentration, the crude product was purified by column chromatography (25% etoac in hexanes) to give 64 (98 mg, 80%) as a white foam : rf = 0.6 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c44h48n4o7s2si ([m + h ]) 837.2812, found 837.2827 ; ir (film) max 2929, 1768, 1711, 1446, 1368, 1173, 1126, 742 cm ; h nmr (500 mhz, cdcl3) 8.00 (d, j = 8.1 hz, 2 h), 7.79 (d, j = 8.4 hz, 4 h), 7.68 (d, j = 0.8 hz, 2 h), 7.357.28 (m, 4 h), 7.257.19 (m, 6 h), 5.13 (t, j = 7.9 hz, 1 h), 3.89 (d, j = 7.9 hz, 2 h), 3.36 (s, 3 h), 2.42 (s, 3 h), 2.33 (s, 6 h), 0.76 (s, 9 h), 0.10 (s, 6 h) ; c nmr (cdcl3, 126 mhz) 170.3, 155.8, 145.0, 135.3, 134.9, 130.5, 129.9, 127.1, 125.4, 125.1, 123.6, 118.5, 116.3, 114.0, 69.5, 68.9, 25.7, 25.3, 24.3, 21.7, 17.8, 4.4. 62 (120 mg, 0.157 mmol) was dissolved in dioxane (500 l), and h2o (125 l) was added, followed by parkin s catalyst (13.5 mg, 0.031 mmol, 0.2 equiv). the reaction mixture was heated to 80 c for 24 h. the reaction mixture was cooled to room temperature and diluted with etoac (1 ml). the layers were separated and extracted with etoac (3 1 ml), and the extract was washed with brine (2 ml) and dried over sodium sulfate. after filtration and concentration, the crude product was purified by column chromatography (1525% etoac in hexanes) to give 69 (77 mg, 63%) as a pale yellow foam : rf = 0.55 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c41h47n4o6s2si ([m + h ]) 783.2706, found 783.2707 ; ir (film) max br 3453, br 3380, 2928, 1681, 1447, 1366, 1172, 1126 cm ; h nmr (400 mhz, cdcl3) 7.98 (d, j = 8.2 hz, 2 h), 7.78 (d, j = 8.4 hz, 4 h), 7.66 (s, 2 h), 7.62 (d, j = 7.3 hz, 2 h), 7.29 (ddd, j = 8.4, 7.2, 1.3 hz, 2 h), 7.257.17 (m, 6 h), 6.49 (br s, 1 h), 5.01 (t, j = 7.8 hz, 1 h), 4.86 (br s, 1 h), 3.47 (d, j = 8.5 hz, 2 h), 2.31 (s, 6 h), 0.72 (s, 9 h), 0.20 (s, 6 h) ; c nmr (cdcl3, 101 mhz) 173.0, 144.8, 135.3, 134.9, 131.6, 129.8, 127.0, 125.1, 124.6, 123.2, 119.5, 118.5, 113.8, 69.6, 64.3, 54.7, 25.8, 21.6, 17.8, 4.4. 69 (90 mg, 0.115 mmol) was dissolved in thf (2.3 ml), and tosyl isocyanate (21 l, 0.138 mmol, 1.2 equiv) was added at room temperature. after the reaction mixture was stirred for 30 min, the reaction mixture was quenched with aqueous ammonium hydroxide (2 ml). the layers were separated and extracted with etoac (3 2 ml), and the extract was washed with brine (2 ml) and dried over sodium sulfate. after filtration and concentration, the crude 71 (91.1 mg) was obtained as a pale yellow foam and was used directly in the following reaction. burgess reagent (7 mg, 0.03 mmol) was added to a heterogeneous solution of crude 71 (10 mg, 0.010 mmol) in dcm (200 l). the reaction mixture was warmed to 50 c for 2 h (mixture turns homogeneous after 15 min). after concentration, the crude product was purified directly by column chromatography (2% acetone in dcm) to give 66 (8.2 mg, 67%, two steps) as a white solid that is very sparingly soluble when purified, preventing nmr analysis. colorless crystals serendipitously formed from slow evaporation of a dilute tlc sample in wet dcm to further confirm the structure : colorless crystals (> 200 c, decomp) ; rf = 0.5 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c49h51n5o8s3si ([m + h ]) 962.2747, found 962.2734 ; ir (film) max br 3532, br 3395, 2928, 1771, 1636, 1358, 1172, 670 cm ; potassium carbonate (6.9 mg, 0.05 mmol, 6 equiv) was added to a solution of 66 (8.0 mg, 8.3 mol) in dmf (100 l), followed by methyl iodide (2.0 l, 3.2 mol, 4 equiv). the reaction mixture was quenched with saturated aqueous ammonium chloride (200 l) and stirred for 30 min. the reaction mixture was concentrated and taken up in etoac (1 ml)/brine (1 ml). the mixture was separated and extracted with etoac (2 1 ml) and dried over sodium sulfate. after filtration and concentration, the crude product was purified by column chromatography (20% etoac in hexanes) to give 67 (7.8 mg, 95%) as a white foam. alternate procedure : tosyl isocyanate (12 l, 0.08 mmol, 1.25 equiv) was added to a solution of 62 (50 mg, 0.065 mmol) in thf (1.3 ml) at room temperature. the reaction mixture was concentrated after 15 min and dissolved in absolute ethanol (1.3 ml). this reaction mixture was heated to 70 c for 14 h, and then the solvent was evaporated to give crude 66. potassium carbonate (54 mg, 0.39 mmol, 6 equiv) was added to a solution of crude 66 in dmf (650 l), followed by methyl iodide (16.3 l, 0.26 mmol, 4 equiv). after 2 h, the reaction mixture was quenched with saturated aqueous ammonium chloride (500 l) and stirred for 30 min. the reaction mixture was concentrated and taken up in etoac (2 ml)/brine (2 ml). the mixture was separated and extracted with etoac (2 2 ml) and dried over sodium sulfate. after filtration and concentration, the crude product was purified by column chromatography (20% etoac in hexanes) to give 67 (47.2 mg, 73% over 2 steps) as a white foam : rf = 0.6 (silica gel, 1/1 hexanes / etoac) ; hrms (m / z) calcd for c51h55n5o8s3si ([m + h ]) 990.3060, found 990.3071 ; ir (film) max 2927, 1764, 1627, 1447, 1370, 1173, 776, 669 cm ; h nmr (400 mhz, cdcl3) 8.09 (d, j = 8.3 hz, 2 h), 7.95 (d, j = 8.4 hz, 2 h), 7.90 (d, j = 8.4 hz, 4 h), 7.53 (d, j = 8.0 hz, 2 h), 7.49 (s, 2 h), 7.32 (ddd, j = 8.4, 5.5, 2.9 hz, 2 h), 7.257.17 (m, 8 h), 4.74 (t, j = 8.1 hz, 1 h), 3.92 (dd, j = 8.2, 1.0 hz, 2 h), 3.41 (s, 3 h), 3.30 (s, 3 h), 2.52 (s, 3 h), 2.33 (s, 6 h), 0.67 (s, 9 h), 0.36 (s, 6 h) ; c nmr (cdcl3, 126 mhz) 158.9, 154.9, 145.2, 143.5, 139.8, 135.2, 134.7, 130.5, 130.1, 130.1, 127.4, 126.9, 125.2, 125.1, 123.6, 118.4, 115.4, 114.1, 69.5, 68.1, 48.2, 30.8, 26.1, 25.7, 21.7, 21.7, 17.8, 4.9. a flask containing 82 (1.60 g, 3.59 mmol) was evacuated and back - filled with argon. toluene (12 ml) was added, followed by 3.59 ml of a 1.0 m solution of liotbu in hexanes (3.59 mmol, 1.0 equiv). the resulting suspension was warmed to 50 c for 36 h. the reaction mixture was cooled to room temperature and quenched with saturated aqueous nahco3 (15 ml). the mixture was separated, washed with brine (20 ml), dried over sodium sulfate, and concentrated in vacuo. the resulting oil was purified by silica gel chromatography (12.5/12.5/75 to 15/15/70 dcm / et2o / hexanes) to give 87 (110 mg, 7%) as a crystalline solid (mp 200205 c). x - ray - quality crystals were obtained by crystallization from chcl3/et2o : rf = 0.5 (silica gel, 3/1 hexanes : etoac) ; hrms (m / z) calcd for c22h23no5s ([m + h ]) 414.1375, found 414.1380 ; ir (film) max 2936, 1728, 1639, 1586, 1519, 1235, 1124, 814, 747 ; h nmr (600 mhz, cdcl3) 7.31 (ddd, j = 8.4, 7.2, 1.4 hz, 1 h), 7.28 (dd, j = 8.0, 1.7 hz, 1 h), 7.02 (ddd, j = 7.8, 7.1, 1.7 hz, 1 h), 6.38 (d, j = 0.9 hz, 2 h), 5.89 (dd, j = 7.8, 1.3 hz, 1 h), 4.20 (tt, j = 6.0, 1.1 hz, 1 h), 3.85 (s, 3 h), 3.79 (s, 6 h), 3.76 (t, j = 5.8 hz, 2 h), 2.83 (d, j = 9.7 hz, 1 h), 2.68 (dt, j = 9.6, 5.6 hz, 1 h), 2.41 (s, 3 h) ; c nmr (cdcl3, 151 mhz) 173.8, 153.6, 138.1, 137.2, 133.5, 131.2, 129.9, 129.0, 127.0, 126.2, 103.1, 61.1, 56.3, 48.1, 47.1, 30.2, 15.7. 34 (221 mg, 0.513 mmol) was dissolved in dry thf (5 ml), and a toluene solution of potassium tert - butoxide was added (1.7 m, 300 l, 0.51 mmol, 1 equiv) at room temperature. the reaction mixture was warmed to 45 c for 30 min, quenched with saturated aqueous sodium bicarbonate solution (3 ml), and extracted with etoac (2 5 ml), and the extract was washed with brine (5 ml) and dried over sodium sulfate. after filtration, concentration, and purification by a silica plug (dcm), epimer 112 (212 mg, 96%) was obtained as a white solid (mp 136139 c) : rf = 0.7 (silica gel, 3/1 hexanes / etoac) ; hrms (m / z) calcd for c30h26n2o ([m + h ]) 431.2123, found 431.2125 ; ir (film) max br 3349, 2934, 1677, 1519, 1483, 1322, 968, 748, 692 cm ; h nmr (400 mhz, cdcl3) 9.92 (s, 1 h), 8.85 (dd, j = 7.6, 1.4 hz, 1 h), 8.46 (dd, j = 4.2, 1.7 hz, 1 h), 8.10 (dd, j = 8.3, 1.7 hz, 1 h), 7.54 (t, j = 7.9 hz, 1 h), 7.517.43 (m, 5 h), 7.397.31 (m, 5 h), 7.297.22 (m, 2 h), 6.62 (d, j = 15.9 hz, 2 h), 6.46 (dd, j = 15.9, 7.2 hz, 2 h), 3.43 (p, j = 8.5 hz, 2 h), 3.14 (t, j = 9.4 hz, 1 h), 2.53 (dt, j = 10.5, 7.8 hz, 1 h), 2.03 (q, j = 10.2 hz, 1 h) ; c nmr (cdcl3, 101 mhz) 171.4, 148.2, 138.4, 137.3, 136.2, 134.6, 132.1, 132.1, 130.6, 130.6, 128.6, 128.6, 127.9, 127.4, 127.4, 126.4, 126.4, 121.6, 121.5, 116.4, 54.1, 38.7, 38.7, 31.5. expermental data for compounds 1, 2, 8086, 8991, and 99111 can be found in refs (50) and (53). | the application of c h functionalization logic to target - oriented synthesis provides an exciting new venue for the development of new and useful strategies in organic chemistry. in this article, c h functionalization reactions are explored as an alternative approach to access pseudodimeric cyclobutane natural products, such as the dictazole and the piperarborenine families. the use of these strategies in a variety of complex settings highlights the subtle geometric, steric, and electronic effects at play in the auxiliary guided c h functionalization of cyclobutanes. |
regular engagement in social activities is an important component of successful aging. having family and friends to spend time with helps people to find their social identity and purpose in life. social activity starts with the family at home and goes beyond to schools, work, clubs, community, and faith - oriented groups. these opportunities are very important to people 's well - being and quality of life [2, 3 ]. social groups are examples of structured opportunities for people that share similar interests to get together. these interests could range from religion, politics, sports and leisure, and education, among others. although there is no conclusive evidence of causality, a positive association between social group participation and good health is found in the literature. social groups are important to people and they play different roles throughout the life course. older adults engaged in social groups share better physical and mental health than their counterparts who are not engaged [59 ]. studies show that on average, socially engaged older adults have less depression [6, 10 ], live more independently [8, 11 ], have better physical and cognitive functioning, have higher levels of life satisfaction [9, 13 ], and are more likely to be engaged in healthy lifestyles [1417 ]. studies conducted in brazil have reported that only a small proportion of older adults regularly participate in social groups [18, 19 ]. some barriers that prevent older brazilians from engaging in social groups are meetings held in inconvenient locations, lack of time due to family caregiving roles, and lack of company to motivate regular participation. efforts to increase social participation among older adults have been initiated worldwide. in the early 1990s, brazil initiated a program from city halls and faith - based organizations to increase the social engagement of older adults. public efforts were devoted to the creation of community - based social groups known as convivncia groups. this initiative was financially supported by the brazilian national public policy for older adults in 2003 and was consistent with the strategies identified in the 2002 world assembly on ageing held in madrid, spain. this is an effort grounded in public health that aims to promote active aging by encouraging healthy lifestyles, independence, productivity, and participation in civic activities. the structure of convivncia groups involves a daily three - hour period in the afternoon during which a number of different leisure and educational activities are made available to participating older adults aged 60 +. among the most popular activities are folk dance, bingo, seminars about health, exercise classes, choirs, ballroom dance, art craft activities, and watching theater and/or dance performances. meetings are held in church halls and community centers, and have a participation of 30 to 100 people per day [24, 25 ]. convivncia groups are part of a national program for older adults in brazil. there have been some studies to document the benefits of this initiative in different parts of the country. in 2002, a comprehensive survey was conducted in southern brazil which revealed that 20% of older adults were participating regularly in the majority of participants were women of low income and with little education [20, 25, 27 ]. groups were found to be more physically active than their counterparts who were not engaged. findings of this study were disseminated by the local media and through publications. as a result, the municipality of florianpolis launched in 2006 a program called capital do idoso. this ongoing program was developed to improve public health in four target areas of intervention : prevention, promotion, therapy, and rehabilitation. in 2009 - 2010, a follow - up survey was conducted to understand the impact of programs on social engagement among the older adult population. the changes, benefits, and challenges of these public efforts for the older adult population and for the city as a whole are documented in this paper. this paper was based on two surveys conducted in florianpolis, capital of santa catarina state, southern brazil. the first survey was conducted in 2002, and the second in 2010 [28, 29 ]. the first survey interviewed a total of 875 older adults (437 men and 438 women) with average age of 71.6 7.9 years. in 2002, the population of older adults represented 8.4% of the total population. this research was approved by the ethics committee for research on human beings of the universidade federal de santa catarina, brazil (protocol no. the second survey was conducted between 2009 and 2010 with 1,705 older adults (616 men and 1089 women). in 2010, the population of older adults in florianpolis represented 11% of the total population. the research was approved by the ethics committee for research on human beings of the universidade federal de santa catarina, brazil (protocol no. a statement of informed consent was obtained from each participant prior the initiation of data collection. trained the average time taken to conduct each in - home interview was around 60 minutes. a brazilian national database was used to select a representative sample of older persons (instituto brasileiro de geografia e estatstica (ibge)). the research team interviewed one older adult man and one woman per census tract, whereas the second survey selected census tracts and homes at random. the questionnaire in survey one included demographic information, physical health, the use of medical and dental services, activities of daily living and falls, physical activity levels, social resources, socioeconomic status, mental health, and needs and issues that affect older adults lives. survey two also included questions about lifestyle, women 's health, eating habits, mobility, functional capacity, environmental opportunities for physical activity, and elder abuse. for the purpose of this paper we report only demographic information, socioeconomic status, and social resources. the international physical activity questionnaire (ipaq - long form) the ipaq was developed as a relatively simple self - report instrument that would be available in many languages and which would enable researchers to estimate physical activity levels in different countries and compare these data. a key feature of the ipaq questionnaire is its ability to provide, in detail, participation estimates for multiple domains of physical activity, including leisure time physical activity, physical activity for transportation, physical activity in the home, and physical activity at work. although the ipaq explores physical activity levels in four domains, in 2010 we chose to use data only from two domains, transportation and leisure time physical activity. for the purpose of this paper, the middle level proposed in the original ipaq was suppressed, following recommendations from previous surveys using ipaq with older adults in brazil [32, 33 ]. therefore, older adults who carried out moderate or vigorous physical activities within the four domains for 150 minutes per week or over were classified as more active, whereas those who did not reach 150 minutes per week were classified as less active. adjustments for age, sex, education, and income differences were performed using analysis of covariance. chi - squared tests were used for the analyses of categorical variables among the surveys. all statistical analyses were performed using spss 19.0 for windows (spss inc., chicago, il, usa) and statistical significance was set at p < 0.05. table 1 shows the distribution of older adults by gender and socioeconomic status of the two surveys conducted in 2002, and in 2010. the average age of participants was not significantly different between the two surveys. in 2002, it was 71.5 years, and 70.6 years in 2010. the first survey included 50% women, whereas in 2010 they comprised 64% of the sample. in both surveys, the great majority of older adults was married and had less than 8 years of education. table 2 shows participation of the older adults in convivncia groups, between 2002 and 2010. table 3 shows a list of public programs available to all older adults in florianpolis city, between years 2002 and 2010. data from both surveys shows a clear increase in opportunities for social activities among older adults in florianpolis from 2002 to 2010. by 2010, many new programs that were offered to the community and the enrollment of older adults had also increased. for instance, between 2002 and 2010, the total increase in convivncia group members was 6,849 users. this outnumbers the 5,972 new users for the all other non-convivncia physical activity, education, and dance groups offered in the city. convivncia groups are viewed as extremely popular social groups among older adults in florianpolis. adding totals from table 3, there were 14,849 reported users of the convivncia groups in 2010, and 9,388 enrolled in other non-convivncia programs., activities such as pilates, bailes (ballroom dance), and other physical activities had become very popular. in addition, in the 2010 survey, our data suggest that greater attention was paid by health professionals to the promotion of healthy lifestyles and disease management. in order to understand the influence of engagement in social programs, we examined changes in social activity over the time course of the study (figure 1). groups when compared with year 2002 ; reflecting a greater percentage of older adults engaged in social activities with friends. one of the main goals of the convivncia groups is to engage local residents of same cohort in social activities and therefore increase and strengthen their circle of friends. to increase our understanding about lifestyle and healthy behaviors of older adults in florianpolis table 4 shows the average time spent in physical activity in leisure activities, transportation, and total physical activity in 2002, 2010. by 2010, in 2002, about 20% of older adults participated in convivncia groups, whereas by 2010 the participation increased to 42%. by 2010, many more older adults were engaged in social activity with friends and many more were active in physical activity for leisure and transportation when compared to the 2002 survey. this study underscores the potential of convivncia groups to promote socialization as well as healthy behaviors among the older adults population. in brazil, a number of public policy initiatives have facilitated the creation of community - based social groups. in 2003, the brazilian national public policy for older adults was implemented, and the capital do idoso idoso em forma em florianpolis program was started in 2006. the capital do idoso program was awarded a national recognition for health and prevention services offered to the older adult population. some researchers have documented the potential association of these public investments with reduced healthcare costs in the municipality. a study reported that selected hospital costs were reduced about 30% between 2006 and 2010. other health indicators followed similar trends, such as improvements in sleeping quality, increased self - steem, and decreased usage of the public health system. groups visited friends more often in 2010 than they did in 2002. in 2010, they were more engaged in other social groups available in the community. also, in 2010 they spent more time in physical activity during leisure time and for transportation when compared to 2002. groups may have played a role in facilitating positive health behaviors among group members. a study conducted with older women in canada examined the influence that social opportunities have on health and functioning capacity. the results of this study showed that as the older women felt more accepted, they presented with less prevalence of diseases ; in addition, they had enhanced functional capacity to perform household chores and other daily activities. the demographic characteristics of the participants in his study are broadly consistent with profile of older brazilians as a whole. the aging process is often accompanied by declines in opportunities for social interactions and for establishing new relationships. retirement from working activities, children leaving home, and loss of loved ones are associated with increased social isolation among older adults. the initial purpose of the capital do idoso program was not to promote social integration among the underserved older population of the city, but rather to develop educational activities that target disease prevention, health promotion, therapy, and rehabilitation. shortly after its start, it became apparent that social engagement of older adults was one of the program 's most successful outcomes. it was apparent how socially engaged participants became, and how much more frequently they would leave home to visit their friends. older adults are the most vulnerable age group for physical inactivity, and they are the least likely to meet physical activity guidelines. the present study has certain limitations that need to be taken into account when considering the study and its contributions. the most important limitation lies in the fact that we did not include control groups, which would have assisted with the interpretation of findings, such as the participation in convivncia groups and the positive impact of these groups on health behaviors. for example, it is not clear whether participants in convivncia groups differed in their physical activity levels from the general population. in addition, our results may have been influenced by other aspects that were out of our study scope and control, such as increase of regional or national public health campaigns and resources. in summary, our study suggests that the establishment of convivncia groups made a difference to the lives of older adults in florianpolis. most cities in brazil have established some kind of convivncia group. with this in mind, we can build a new culture of healthy and active aging throughout brazil, as proposed by the who in 2002. | in old age, social groups can be a crucial component for health and well - being. in 2009 - 2010, a follow - up survey was carried out in florianpolis, brazil to understand the impact of a variety of programs established since 2002 that were designed to enhance social activities among the older adult population. this study employed two surveys within the population of older adults in florianpolis. the first survey interviewed a total of 875 older adults in 2002, and the second survey involved 1,705 older adults between 2009 and 2010. by 2010, many new programs were offered in the community and the enrollment of older adults in social programs followed similar trends. convivncia groups stood out as extremely popular social groups among this population. this paper discusses some of the potential outcomes associated with participation in convivncia groups. |
for almost six decades, the scientist practitioner (s p) model or accredited programmes in counselling psychology encompass training experience in the science of psychology, theoretical foundations and in the application of practice skills and research. training focuses on personal and interpersonal functioning across the life span and on emotional, social, vocational, educational, health - related, developmental and organisational concerns. p model, whereby research is complemented by practice - based experience and working in clinical settings using case conceptualizations that are upheld by empirical research. counselling psychology distinguishes itself from other counselling and psychotherapy training courses in its endorsement of a clear evidence base for practice. counselling psychology is a branch of psychology practice that is strongly influenced by human science research as well as by the main psychotherapeutic traditions. while aligning itself with the importance of an empirical basis for theory and practice, it also places a high value on the therapist 's use of self, and the inevitable interplay of subjective and intersubjective factors within human interactions, particularly the therapeutic relationship (kirk, 1982). these foundations are the basis for personal therapy being made a part of the core training criteria within counselling psychology. besides requiring personal therapy as a base for intense self - experience, other benefits include the establishment or consolidation of self - other boundaries and the provision of a central space for developing the skill of self - reflexivity (grimmer & tribe, 2001). in practice, counselling psychologists help people experiencing physical, emotional and mental problems to improve well - being, alleviate distress and maladjustment, and resolve crises. in addition, practitioners in this professional specialty provide assessment, diagnosis and treatment of psychopathology. thus, while the profession has developed alongside other applied fields of psychology, counselling psychology is distinctive from a treatment point of view because of its attention to both normal developmental issues and problems associated with physical, emotional and mental disorders. the essence of the profession is that it is embedded in a particular set of values and ethics (e.g. walsh & frankland, 2009 ; woolfe, 1996). goldstein defines it concisely : counselling psychology is unique in that its competencies are founded upon a philosophically oriented and explicit statement of values (2009, p. 36). mick cooper (2009) goes further by examining a range of core counselling psychology texts (british psychological society qualifications office, 2008 ; gillon, 2007 ; orlans & van scoyoc, 2009 ; woolfe, 1996) and identifying six key principles : a prioritisation of the client 's subjective, and intersubjective, experiencing (versus a prioritisation of the therapist 's observations, or objective measures). a focus on facilitating growth and the actualisation of potential (versus a focus on treating pathology). an orientation towards empowering clients (versus viewing empowerment as an adjunct to an absence of mental illness). a commitment to a democratic, non - hierarchical client - therapist relationship (versus a stance of therapist - as - expert). an appreciation of the client as a unique being (versus viewing the client as an instance of universal laws). an understanding of the client as a socially and relationally embedded being, including an awareness that the client may be experiencing discrimination and prejudice (versus a wholly intrapsychic focus). working from a humanistic value base, counselling psychologists strive to engage with their clients first and foremost as agentic human subjectivities that can not be reduced to, or treated as, objects of natural scientific inquiry. the final principle identified by cooper (2009) also points to the rather nuanced and complex area of research. an ongoing challenge for the scientist practitioner is the predominance of medicalised models, which espouse that the most dependable findings are from large, randomized and well - controlled clinical trials (or rcts) with quantitative analysis of the generated data. one difficulty with this is that the findings of rcts may not be easily generalizable ; real world therapy is not manualised, can take varying lengths of time and may include a variety of therapeutic approaches and interventions depending on the client and the current state of therapy, etc. although counselling psychology asserts the relative value of conventional rcts, it recognizes that many of the factors in the process and outcome of therapy simply can not be reliably measured in this way. given the inherent complexity of psychotherapy / psychological therapy, no one theory, methodology or epistemology can provide a comprehensive view of the therapeutic exchange (castonguay, 2011). in accounting for the somewhat competing challenges posed by examining the nature of evidence and the constitution of holistic and person - centred therapeutic work, a pragmatic balance needs to be attained between various veins of evidence (quantitative, qualitative and particularly case study research), recognising the value of each rather than focusing solely on one methodology. significant strides have been made in increasing the rigour of pragmatic psychotherapy research in the past decade (mcleod, 2011 ; timulak, 2008). however, counselling psychologists who conduct research still need to bear in mind that the existence of an at - times critical wider scientific community may mean that it is necessary to balance the conveyance of research findings to that community with maintaining the integrity of individual client work within a methodologically sound framework. the nature of application of training to practice can vary between individual counselling psychologists but, in general terms, a counselling psychologist can consult with a variety of agencies (e.g., schools, government, private organisations), teach at university levels (undergraduate and graduate levels), conduct research, practise therapy (group, individual, couple, family) and hold academic administrative positions. it could be interpreted that the identity of counselling psychologists in ireland is weakened by the diffuse professional identities of counselling psychologists who work in different settings with varied populations, using a range of theories and practices. further, there is in many instances a high degree of similarity between the roles of counselling and clinical psychologists once they enter the workforce. this perhaps suggests that counselling psychologists do not hold a visibly unique identity beyond the distinctions ascribed to them within university training programmes. it could be argued that greater involvement in community programmes fostering diversity and social justice, key components of counselling psychology 's history and identity as a profession, would further highlight its values in an identifiable way. recent social changes, such as the increasing dialogue and recognition of the possibility of same sex marriage in ireland and other countries and changing multicultural policies, allow counselling psychologists the opportunity to develop knowledge and interventions that reflect diversity and uniqueness for those seeking clinical help. in this regard, the profession considers itself to have a unique identity, which can contribute to the development of client - centred psychology services across all areas of service delivery. this may be within statutory services such as the irish health service executive (hse) or across a range of private services. the psychological society of ireland 's (psi) division of counselling psychology (dcop) has had a central role in the development of that identity in an irish context. despite a broad presence across services and populations, a lack of clarity about what defines a counselling psychologist is still frequently present in many psychology services and among the broader professional community. one could partially attribute this phenomenon to the relative recency of the germination of the profession ; globally it stands < 60 years old. yet its qualities merit a reflection on how psychology has expanded to become more inclusive and holistic in the latter half of the twentieth century. in the us, counselling psychology was established soon after the second world war when the reintegration needs of war veterans greatly increased the demand for psychological services. division 17 (entitled the division of counselling and guidance until it was changed to the dcop in 1951), progressed through several name changes during its early years with varying emphases on personnel ', vocational ', today division 17 in the american psychological association (apa) is known as the society of counselling psychology. in the uk, the british psychological society (bps) established the counselling psychology section in 1982 and by 1994 it had become the bps division of counselling psychology with over 1200 members. as it has developed as a profession in the western world, counselling psychology has become recognised as a general practice and health service provider specialty in professional psychology. the beginnings of counselling psychology in ireland can be traced to the formation of the counselling and therapy interest group (ctig) within the psi in 1987. this interest group was set up before the establishment of any professional training in the field to provide a home within the society for psychologists with an interest in the area of counselling and psychotherapy. at this point in time, the awareness and practice of counselling and psychotherapy, as disciplines, were emerging globally from both inside and outside of psychology. the development of professions around these practices was shaped somewhat differently within different countries due to the operation of different historical and social forces (strawbridge & woolfe, 2010). by the time the ctig was being set up in ireland, counselling psychology was already firmly established in the usa and it was steadily emerging elsewhere in the world in places such as australia, canada and the uk (orlans & van scoyoc, 2009). counselling psychology initially emerged as a recognised discipline in the usa at the time of the reorganisation of the apa into the divisional structure it continues to use. alongside this change, the developments in professional training in psychology in the usa during the 1940s and the discussions between the apa - dcop and the veterans association that lead to widespread demand for counselling psychologists in the usa are all regarded as key developments in the american emergence of the specialism (munley, duncan, mcdonnell, & sauer, 2004). the development of the discipline was also encouraged by a number of significant contemporaneous influences, such as the vocational guidance movement, the mental hygiene movement, psychometrics and the study of individual differences and particularly the publication of carl rogers 's seminal work counselling and psychotherapy (1942). in the decades after its establishment, the apa - dcop fostered the development of this new discipline through the hosting of a number of national conferences. this brought into focus many of the core issues it faced in its early development including training and accreditation, public image, professional practice in various settings, research and organisational and political issues in counselling psychology (weissberg., 1988). by the time the ctig was established in ireland, a wide network of accredited professional university programmes, delivering training at doctorate level, along with two dedicated professional journals, the journal of counseling psychology and the counseling psychologist, had been established in the us in the uk, counselling psychology owes its inception to the recommendations of the bps working party on counselling (nelson - jones, 1999). this group was set up in 1979 with the purpose of researching the relationship between counselling and psychology in the uk. by that time the practice of counselling had already become quite established in the uk. there was growing concern from many within the bps who had a professional interest in the field that counselling would develop independently from psychology in the uk. as a result, a significant number of members lobbied for the establishment of a professional interest group in counselling within the bps. in 1982, following the recommendations of the working party, the section of counselling psychology was established. by the end of its first year the development of the discipline in the uk to represent a set of professional competencies with full divisional status is reported to have been a long and challenging road. it has been reported that progress towards this goal was frustrated by significant resistance from within other bps divisions and from some members within the special interest group itself (orlans & van scoyoc, 2009). however, during this time the field continued to develop and significant milestones were achieved. these included the establishment of the bps diploma in counselling psychology and the launch of the counselling psychology section newsletter, which in 1989 became the counselling psychology review. orlans and van scoyoc (2009) note that by 2009 the bps - dcop had become the third largest division of the bps with a membership of 1947. in 1989, the ctig in ireland was starting to lay down important roots for counselling psychology by facilitating professional activities, workshops, peer supervision and networking in counselling and psychotherapy for interested psychologists. in that same year, a one - year diploma in counselling psychology was established in trinity college dublin (tcd), which evolved into a two - year full time master 's degree programme in 1991. the psychology departments of university college cork (ucc) and university college dublin (ucd) also commenced master 's programmes in counselling psychology or psychotherapy meaning that 2530 students were graduating each year. in 1995, the ctig was renamed the counselling psychology interest group and the group actively pursued divisional status. a register was established of its rapidly growing membership and in 1996 the counselling psychology newsletter was launched. the first dcop committee in 1997 included maire dooey (secretary), sheila young (treasurer), eimear burke, joan clinton, sylvia caffrey, anna o'reilly - trace (ordinary members) and john broderick (chairperson). dr eleanor o'leary (ucc) and nuala rothery (tcd) acted as special advisors to the division. margaret quinn, maureen gallagher and many others represented dcop and served on numerous psi committees and task forces. since then, several members of the division have devoted their free time to serve on dcop committees, regional groups and working parties and have represented the division across the many psi committees, including psi council. in recent years, the division committee has been focused on developing and disseminating a deeper understanding of the unique value that counselling psychologists can bring to psychology and multi - disciplinary teams. a major challenge for the profession has been the creation of a distinction, not only between other psychology professions but also between counselling / psychotherapy and counselling psychology. the division has undertaken a number of initiatives to address these issues, including the development and publication of an information leaflet that provides details on the identity and competencies of counselling psychologists, presentations to undergraduate students and negotiations with key stakeholders including important employers. so much energy, it would appear, has gone into such initiatives that counselling psychologists in ireland have perhaps neglected reflecting on the profession as a whole, at least in publication format. while numerous journal articles (e.g. lalande, 2004 ; moller, 2011 ; spinelli, 2001) have been published critically examining the status and nature of the profession in the uk and canada, there has been a paucity of articles published here in ireland. the notable exception has been elaine martin 's contribution of a thought - provoking and powerful piece taking a family systems slant on counselling psychology and its relationship with clinical psychology in the irish psychologist in 2008. it is fair to say that counselling psychology in ireland has developed in many ways over the years since its foundation, first as a special interest group and subsequently as a division. much credit is due to those who promoted the ethos of counselling psychology in its early days. while the division has made significant progress in developing and promoting the counselling psychology identity and creating a niche within counselling services, it has been less successful to date in making its presence felt within more traditional psychological services such as the hse. this lack of progress is not matched by a lack of effort, with records going back at least 15 years documenting efforts of successive division committees to market the unique skillset of counselling psychologists as a valuable addition to both established and developing psychology service. despite these efforts, those within the profession have often found themselves in the position of having to justify their existence by highlighting how counselling psychology is different from other professions and, in particular, from clinical psychology and from counselling and psychotherapy. a lack of representation of counselling psychologists in senior and influential positions has been listed as a significant issue for the profession in ireland in this respect and is something on which the wider counselling psychology community needs to collaborate address (o'brien & timulak, 2012). despite the slow progress, it is important to acknowledge what has been achieved. although to date there have been no changes to hse recruitment policies that excluded counselling psychologists from most positions in the health service, the division committee has made progress on this objective. over the last few years, the committee has called on the hse to undertake recruitment on a competency basis, rather than solely a qualification basis, a position that has recently been supported by the commission for public service appointments. the division has also regularly sought psi council 's support for competency - based recruitment. this support has been realised and firmly led by successive presidents, dr michael drumm, eric brady and dr margaret o'rourke, who have all committed to actively working to seek fair, transparent and competency - based recruitment policies for all psychologists. whereas in the past it has been the experience of irish counselling psychologists that there was similar resistance to their profession to that described above as having occurred within the bps, it is encouraging that counselling psychologists have more firmly felt the support of psi council and presidents in recent years. to support these efforts, the division, in close cooperation with the professional doctoral training programme in tcd (currently the only professional counselling psychology training programme in ireland), defined the competencies of counselling psychologists. these competencies are grounded in the professional and accredited training that students must undertake before achieving a professional designation of counselling psychologist. indeed, the significance of the progress made in terms of the professionalisation of counselling psychology in recent years and particularly in terms of the advances in professional training were identified by a number of senior members within the profession as one of the most important developments in terms of opening up future opportunities for counselling psychology in ireland (o'brien & timulak, 2012). this indicates that the steps being taken are having a positive impact on the development of the profession. credit must also go to dr ladislav timulak and his team at tcd for working hard to secure a doctorate level qualification in counselling psychology. despite principles outlined in documents such as a vision for change (department of health and children, 2006), mental - health service - users still continue to have little information on the options for treatment of mental health difficulties with or without pharmacological intervention. the predominance of the medical model leads to service been delivered according to a number of associated assumptions regarding valid understandings and responses to human distress. in a manner that may have some overlap with the values of other psychological disciplines, counselling psychology views the dominance of the psychiatric discourse as inherently problematic. the profession assumes a humanistic standpoint and emphasises the need for thorough, critical examination of the ethical production and consumption of research. employment settings can, to some degree, determine the level of confrontation with dominant systems of categorisation and associated implicit and explicit ideologies. counselling psychologists working in primary and secondary health service settings, for example, often find that treatment guidelines being used are disorder - focused and drawn from research based on the premise that disorder - based classifications systems are assumed to be valid and useful. the integration of dsm the diagnostic and statistical manual of mental disorders (5th ed. ; dsm-5 ; american psychiatric association, 2013) approaches to mental health problems has been deemed to be incongruent with the development and contextual identity of counselling psychology (eriksen & kress, 2006). however, it has also been argued that simply opposing the dsm is futile due to its political nature (sequeira & van scoyoc, 2002), and by so doing, counselling psychologists are in danger of becoming marginalised professionally within the mental health system. this issue can be seen as crucial by some counselling psychologists who feel that they are confronted with a moral - political choice about where their allegiance should lie. although beyond the scope of this article, the reader is referred to strawbridge and james (2001) who raised similarly compelling issues regarding the questionable nature of diagnostic categories, the power dynamics of labelling, the pathologising of distress, the appropriate use of psychiatric categories within specific contexts, practitioners working within their competence in relation to the categories, lack of informed consent, the potential discouragement of service users and the financial consequences of the use of psychiatric language particularly related to funding and insurance. as a means of justifying expertise, a medicalised framework becomes compelling because the uses of psychiatric categories : are seen as offering an interdisciplinary and international language as well as enhancing a professional image in a highly competitive market (strawbridge & james, 2001, p. 4). the questions of whether the making of diagnoses and the use of diagnostic categories serve the practitioner in securing employment and maintaining status and professional image are worth reflecting on. perhaps as a result the question for practitioners is not about whether diagnostic categories are used, but rather how and in what context. in recent years, there has been a growing movement in global research (e.g. the national institute of mental health [nimh ] and the global alliance for chronic diseases) towards formulating a path of action to attend to those living with or vulnerable to mental health difficulties. recommendations include a shift towards investing in research that uses a life course approach beginning in childhood when risk factors for illnesses later in life, such as family violence in the home, are established (nimh strategic plan, 2008). other notable areas of research include critical analyses of aspects of social exclusion and discrimination, whereby suffering caused by non - user - friendly health systems could be minimised by integrating care for mental health difficulties into chronic disease care (ngo., 2013). such ideas warrant consideration in an irish context, particularly with notable data suggesting that the incidence of mental health difficulties is higher than the number of individuals who successfully seek treatment (kocsis, gelenberg, & rothbaron, 2008). satisfactory awareness and appropriate application of psychological and pharmacological therapies in tandem or separately, where advised or chosen by the service user, are important in the refinement of effective mental health care. into the future, counselling psychologists would do well to continue to examine the nature and application of psychological knowledge derived from research primarily established from a base of psychopathological constructs. promoting a move towards direct engagement with well - being - based research and preventative models could equally contribute to fostering the relational skills often sought by clients who attend therapy. placing value on diversity and equality, projects that aim to tackle the stigma of mental health could also benefit from the knowledge and insight provided by counselling psychologists. following trends in the uk, canada and the usa, it will be important for counselling psychologists to work effectively with a range of other disciplines and practitioners whose knowledge, practice and client base overlap. it is also apparent that there is a growing trend towards increasing the provision of counselling and psychological intervention approaches within other helping professions. greater integration within the professional, academic and multidisciplinary community, as relevant to the psychological care of diverse populations, will offer ongoing opportunities for growth as well as a challenge to maintaining the identity of counselling psychology and its ability to make a distinctive contribution to therapeutic and social issues. today psi hosts a rich diversity of specialisms, although most of its 2300 members are not members of any division. the dcop remains very active and is a few members short of being the largest division in psi. it maintains a strong commitment to supervision, ethical practice and continuing professional development, and 2010 marked the fortieth anniversary of the society. it also marked the second year of tcd 's practitioner doctorate in counselling psychology and the graduation of the last cohort of master 's level students. currently, counselling psychologists are employed in university and college counselling centres, in schools and community services, in the hse, in the irish prison service, and in private practice. while such achievements are significant, there is still much work to be done to expand the presence of counselling psychologists in the irish workplace. | this paper discusses the distinctive nature of the specialism of counselling psychology and outlines the development of the discipline in ireland in the context of international developments and its recognition as a professional branch of applied psychology. today, counselling psychologists are employed in varied clinical and non - clinical settings including health and mental health services (statutory, private and voluntary sector) along with education, forensic, justice, industry and private practices. counselling psychologist is the primary professional identity of many practising psychologists in ireland and the psychological society of ireland 's division of counselling psychology is the main affiliation of at least 179 members. with its focus on facilitating personal and interpersonal functioning across the life span and its emphasis on the therapeutic process, the specialism continues to bridge the disciplines of psychology, counselling and psychotherapy. in this article, some of the challenges still faced by counselling psychology are explored as it navigates its way through the changing landscape of further development and evolution. |
in patients with bladder outlet obstruction (boo) due to benign prostatic hyperplasia (bph), transurethral resection of the prostate (turp) has long been considered a standard treatment regimen. in 1995, gilling first introduced holmium laser prostatectomy for the treatment of bph. since then, holmium laser enucleation of the prostate (holep) has been increasingly performed. holep is advantageous in that it can be performed both effectively and safely with no respect to the size of prostate. as compared with open prostatectomy or turp, holep shows a lower morbidity and a shorter length of hospital stay [3 - 5 ]. it is composed a two - stage procedure : enucleation and morcellation. to date, however, most studies have focused on enucleation and its outcomes. given the above background, we developed an inverse technique for tissue morcellation by modifying the conventional upward technique and then examined its safety and efficiency. the current study was conducted in 389 patients (n=389) with bph who underwent holep between july 2008 and december 2010. for tissue morcellation, we used an upward technique for an initial series of 84 patients during a period from july 2008 to june 2009 and an inverse technique for a consecutive series of the remaining 305 patients between july 2009 and december 2010. in the current study, the inclusion criteria were an international prostate symptom score (ipss) of 8, a peak urinary flow rate (qmax) of 15 ml / sec, and significant postvoid residual (pvr) urine volume. in addition, the exclusion criteria were prostate cancer, neurogenic bladder, and urethral surgery. a prostate biopsy was done, if clinically applicable, to rule out the possibility of prostate cancer., all patients underwent a digital rectal examination, serum prostate - specific antigen (psa) measurement, transrectal ultrasonography, uroflowmetry, and residual urine measurement and ipss and quality of life (qol) scores were determined. holep was done preferably under spinal anesthesia and was composed of two maneuvers : enucleation of prostate adenoma and morcellation of adenoma tissue within the bladder. the mechanical morcellator device is constructed of a handpiece and blades (the outer sheath and inner sheath). the inner sheath aspires the tissue, which is followed by the morcellation of the tissue by a sharp portion between the outer sheath and the inner one. when surgeons notice that the bladder is sufficiently filled and then remote from the bladder mucosa, they place a resectoscope central to the bladder. the morcellator contains a two - phase foot pedal. by sharp pressing of the pedal, this is followed by the morcellation of the captured tissue by the blades by full pressing of the pedal. with the use of the currently commercially available form of the morcellator, the most important detail is the shape of the tip of the morcellator. similarly, the inner sheath also has a semi - tubular shape. to prevent the aspiration of the bladder mucosa from the tip of the morcellator, the morcellator was designed to have a blind - ended tip of the inner sheath. when surgeons discontinue operation of the morcellator, they can adjust the location of the blades. surgeons should be aware that there is a complete lack of aspiration if they halt the tip of the inner sheath before the holes made in the outer sheath. the conventional method of tissue morcellation, that is, the upward technique, is operated when the blades are directed upwards and the tissue is first aspirated from their superior part., we used an inverse technique, a modified form of the conventional upward technique, for 305 patients excluding an initial series of 84 patients. in the inverse technique, the prostate tissue is aspirated and then captured superiorly by inversely placing the blades inferior to the bladder and superior to the prostate tissue (fig. the versapulse power suite (lumenis, yokneam, israel) holmium laser was used for enucleation of prostatic adenoma at a laser power of 80 to 100 w. in addition, a 26-fr resectoscope (karl storz, el segundo, ca, usa) with a laser bridge was used. the tissue morcellation was performed by using a percutaneous nephrolithotomy nephroscope and a versacut morcellator (lumenis). between the upward technique group and the inverse technique group, we compared the mean morcellation time, the morcellation efficiency, the incidence and location of bladder injury occurring during the morcellation, and the catheter time. to rule out the possibility of a learning curve, we excluded an initial series of 20 patients. then, we divided our clinical series of patients into the upward technique group and three inverse technique groups on the basis of the timing of the inverse technique : group 1 (early stage), group 2 (middle stage), and group 3 (late stage). in addition, on the basis of a cutoff value of 50 g, we divided our clinical series of patients into two groups : a group with a prostate size of 50 g and a group with a prostate size of 80 g. furthermore, the morcellation efficiency was significantly poor in patients with a weight of enucleated tissue of > 100 g. in the current study, we compared the morcellation efficiency and the incidence of bladder injury during the morcellation according to prostate size. this comparison showed that both values were higher in the group with a prostate size of 50 g than in the group with a prostate size of < 50 g. however, this was not statistically significant (table 4). there are limitations of the current study as shown below : 1) the current study was conducted under a retrospective cross - sectional design. 2) a smaller number of patients underwent the upward technique as compared with the inverse technique. moreover, the complications occurred at a lower incidence during the morcellation in both groups (the upward technique group and the inverse technique group). theoretically, morcellation is a simple procedure for holep. in practice, however, it is one of the complicated procedures of this surgical modality and requires special attention. urologic surgeons should therefore be careful not to cause complications such as bladder mucosal injury or perforation. as described in the current study, the inverse technique might be a more effective, safer, and more excellent method of morcellation as compared with the conventional upward technique. | purposewe developed an inverse technique for tissue morcellation by modifying the conventional upward technique and then examined its safety and efficiency.materials and methodsfrom july 2008 to december 2010, a total of 389 consecutive patients treated with holmium laser enucleation of the prostate (holep) were enrolled in this study. for tissue morcellation, we used an upward technique for an initial series of 84 patients and an inverse technique for a consecutive series of 305 patients. we compared efficiency and safety between the inverse technique and the upward technique.resultsthere were no significant differences in mean age or prostate volume between the two groups. the mean morcellation efficiency was higher in the inverse technique group. the incidence of severe bladder injury was significantly higher in the upward technique group. regarding the site of bladder injury, 7 and 4 cases of bladder injury occurred in the bladder dome and posterior wall, respectively, in the upward technique group. in the inverse technique group, however, the site of bladder injury was limited to the trigone. we divided our clinical series of patients into the upward technique group and three inverse technique groups on the basis of the timing. the mean morcellation efficiency was significantly higher in all three inverse technique groups than in the upward technique group. however, there was no significant difference in mean morcellation efficiency between the three inverse technique groups.conclusionsin conclusion, the inverse technique might be a more effective, safer, and more excellent method of morcellation than the conventional upward technique. |
you work in the neurological intensive care unit (icu) and you are managing a patient who suffered a subarachnoid bleed and, despite appropriate therapy, is left with significant neurological impairment. you have weaned the patient appropriately on the ventilator and you feel that they are strong enough to tolerate extubation. you worry that, given their severe neurological impairment, they may not be able to protect their airway upon extubation, and as such you consider the merits of tracheostomy. luciana mascia, eleomore corno and pier paok terragni during the early phase after acute brain injury, patients with impaired consciousness may require mechanical ventilation to protect their airway, treatment for intracranial hypertension, and ventilatory support to treat pulmonary complications. after the acute phase, and once satisfactory weaning parameters have been achieved, the patient 's impaired level of consciousness and inability to protect their airway represent strong reasons why extubation should be delayed. these patients might benefit from continued intubation through prevention of aspiration and because of their limited ability to clear secretions, but it has been shown that prolonged intubation in traumatic brain injury is associated with a high incidence of pneumonia. conversely, early tracheostomy after trauma reduces icu length of stay and number of ventilator days, and reduces the incidence of ventilator - associated pneumonia [3 - 5 ]. koh and coworkers confirmed that patients undergoing early elective tracheostomy had shorter icu stays than did patients who were given extubation trials before tracheostomy. kluger and colleagues reported a lower incidence of pneumonia when early tracheostomy was performed in brain - injured patients. nowak and coworkers identified an increased risk for severe tracheal complications in brain - injured patients who had been intubated for more than 14 days. the critical issues in developing effective ventilatory management strategies in acute brain - injured patients remain the identification of those patients who are more likely to require long - term ventilatory support and determination of the optimal timing for tracheostomy. major and coworkers suggested the utility of daily assessment of objective scores such as glasgow coma scale (gcs) and simplified acute physiology score ; scores on these scales of below 7 and greater than 15, respectively, on day 4 had a high positive predictive value for identifying those head - injured patients who required tracheostomy for prolonged airway protection. similarly, namen and coworkers found that a weaning protocol for head - injured patients should always include a neurological assessment using the gcs ; a score greater than 8 on the gcs was most accurate in predicting successful extubation without need for reintubation, and avoiding pneumonia and tracheostomy. qureshi and coworkers reported that, in patients with infratentorial lesions, an aggressive policy regarding tracheostomy is justified because of the low rate of successful extubation, and that a that tracheostomy should be performed on day 8 because of the low probability of subsequent extubation or in - hospital death. selection of this subgroup of patients for tracheostomy is justified because infratentorial lesions located in the cerebellum and brainstem may be associated with damage to the primary neural respiratory centres (which are involved in coordinating respiration), to the lower cranial nerve nuclei (which are responsible for protective airway reflexes), and to reticular activating pathways (which are responsible for impairment in the level of consciousness and consequently for reduced protective airway reflexes). although early tracheostomy may reduce the length of icu stay and pulmonary morbidity, the first 710 days after acute brain injury coincide with the greatest incidence of intracranial hypertension ; the appropriate timing for tracheostomy in these patients must be considered in view of the risk for severe intracranial hypertension. stocchetti and coworkers, in a randomized control trial comparing three tracheostomy techniques, included patients ventilated from 4 days but excluded patients with unstable intracranial pressure requiring active treatment. the patient described in the scenario above appears to meet standard weaning criteria and has stable intracranial pressure but a low gcs score, indicating that he patient has impaired ability to protect his airway. we therefore believe that the patient should receive tracheostomy to reduce the length of icu stay and the likelihood of pulmonary complications. david stather and niall d ferguson the management of a brain - injured patient with satisfactory weaning parameters but a decreased level of consciousness is a common critical care scenario. the role of tracheostomy in this setting, however, has yet to be clearly defined. tracheostomy has been shown to decrease the work of breathing, but this is not the issue in this scenario. aspiration of oropharyngeal contents is common in neurologically impaired patients, but tracheostomy may not protect against aspiration. a retrospective study of traumatic brain - injured patients found a high incidence of pneumonia in those with prolonged intubation, probably because of a loss of normal upper airway defences caused by the presence of the endotracheal tube. unfortunately, tracheostomy does not necessarily reduce the incidence of nosocomial pneumonia ; in fact, the presence of a tracheostomy has been associated with a sixfold increased risk for developing ventilator - associated pneumonia. tracheostomy has been associated with decreased icu and hospital mortality in observational cohort studies of mechanically ventilated patients. this effect, however, is probably related to a selection bias created by the fact that patients needed to survive their first 1020 days of ventilation in order to receive a tracheostomy. when the same observational data were examined in a matched case control design, tracheostomy patients had longer icu and hospital lengths of stay, and a lower icu mortality, but importantly they had no decrease in hospital mortality. brain dysfunction can contribute to extubation failure in a number of ways, such as by decreasing the patient 's ability to protect their airway and clear secretions. namen and coworkers found that a gcs score below 8 was associated with an increased likelihood of extubation failure in neurosurgical patients. coplin and colleagues, however, found no relationship between extubation failure and gcs score. in that prospective observational cohort study, those investigators found that 39 out of 49 patients with gcs score of 8 or less, and 10 out of 11 patients with a gcs score of 4 or less tolerated extubation. in addition, they showed that brain - injured patients who had delayed extubation developed more pneumonias, had longer lengths of stay, and incurred more hospital charges than did similar patients who were extubated promptly after meeting standard weaning criteria. it is possible that tracheostomy could improve the outcome of brain - injured patients in whom upper airway obstruction or problematic secretions could cause extubation failure. its utility in other patients with simple decreased level of consciousness is much less certain. for example, evidence - based guidelines for discontinuing ventilatory support do not include brain - injured patients in a list of populations who may derive particular benefit from early tracheostomy. returning to our scenario, if a plan for ongoing aggressive care has been made after appropriate consideration of prognosis and family discussions, then options from this point would include continuing orotracheal intubation, tracheostomy placement, or primary extubation. there is currently no clear evidence to suggest that performing a tracheostomy in this setting would improve outcome. carefully planned prospective studies, both observational and interventional, that examine the need for and optimal timing of tracheostomy in brain - injured patients are needed to better address this common clinical question. luciana mascia, eleomore corno and pier paok terragni although the need for and optimal timing of tracheostomy for brain - injured patients is a common clinical problem, no large randomized trials have unequivocally clarified whether this intervention improves outcome. while we await definitive answers, we should select subgroups of brain - injured patients who may benefit from this intervention. because of his severe neurological impairment, the patient described in the scenario presented above could be a good candidate for trachestomy to protect his airways. david stather and niall d ferguson the common theme underpinning both the argument presented by mascia and colleagues and our own is the clear lack of rigorous, prospective data regarding the utility and optimal timing of tracheostomy in brain - injured patients. even in the non - neurosurgical critical care population, there is insufficient and conflicting evidence regarding whether the timing of tracheostomy alters the duration of mechanical ventilation or extent of airway injury. this paucity of data should not lead us to make definitive recommendations based on an incomplete understanding of the problem. instead, we must recognize that further scientifically rigorous results are needed before this is important clinical question can be answered. | most clinical trials on the topic of extubation have involved patients outside the neurological intensive care unit. as a result, in this area clinicians are left with little evidence on which to base their decision making. although tracheostomies are increasingly common procedures, they are not without complications and costs, and hence a decision to perform them should not be taken lightly. in this issue of critical care two groups debate the merits of tracheostomy before extubation in a patient with neurological impairment. what becomes very clear is the need for more high quality data for this common clinical problem. |
the choledochal cyst can be defined as a congenital anomaly of the biliary system, characterized by cystic dilatation of the intrahepatic duct and the extrahepatic biliary tree individually, or cystic dilatation of both these parts simultaneously. it has been reported that the incidence of choledochal cyst in western countries ranges from 1:100,000 to 1:150,000 live births, and its incidence in japan has been frequently reported to be 1:1,000 live births. however, the precise incidence of choledochal cyst in korea has not been reported so far. it has been known that choledochal cysts are caused by two main factors, namely, weakening of the bile duct walls and an obstruction distal to it. in addition to the causes of this disease, a close relationship between the choledochal cyst and pancreaticobiliary malunion (pbmu) has also been revealed. also, the probability of occurrence of pbmu related to the position of the opening of the papilla of vater and the probability of the occurrence of choledochal cyst related to such a situation has been considered to be one of the causes, and recently a series of reports have been made available. in this connection, we report our experience of choledochal cyst with pbmu and the papilla of vater positioned in the third portion of the duodenum, in a 30-month - old boy. a 30-month - old boy, who was suffering from vomiting and abdominal pain since the past 20 days, underwent abdominal computed tomography (ct) scans at another hospital and was then transferred to our hospital under the impression of choledochal cyst. there was no particular past medical history or family history, except for the history of hospitalization for treatment of pneumonia one month prior to being admitted to our hospital. laboratory data including complete blood count and urinalysis were within the normal limits, but blood biochemistry revealed the following : aspartate aminotransferase 121 iu / l, alanine aminotransferase 155 iu / l, gamma - glutamyl transpeptidase 168 iu / l, amylase 292 iu / l and lipase 129 u / l, showing elevated levels and t - bilirubin / d - bilirubin were 0.59 mg / dl and 0.16 mg / dl respectively, showing normal levels. abdominal ct demonstrated dilatation of the extrahepatic bile duct and central dilatation of the intrahepatic bile duct (fig. 1). also magnetic resonance cholangiopancreatography demonstrated findings similar to abdominal ct but it could not identify the pbmu (fig., the hepatic uptake appeared to be within the normal range, however, the accumulation of isotope at the common bile duct (cbd) and delayed excretion into the duodenum were confirmed based on the findings. under the diagnosis of choledochal cyst the operation was carried out, and the p - b type of pbmu, in which the pancreatic duct joins the cbd, and the papilla of vater is positioned in the third portion of the duodenum were identified through the operative cholangiography (fig., the diagnosis of a type i choledochal cyst was confirmed, and hence excision of the choledochal cyst and, roux - en - y hepaticojejunostomy were carried out. the patient was discharged on postoperative day 11 without any problems and in good general health condition. choledochal cysts are congenital anomalies of the biliary system and consist of cystic dilatation of the extrahepatic biliary tree, intrahapatic biliary ducts or both. based on the clinical and anatomic findings of choledochal cysts, todani and others argued that choledochal cysts can be divided into 5 major types and these types can be further divided into 3 subcategories. despite their classification into many types, the surgical method of treatment for each type is not far different from the conventional method of treatment for choledochal cysts. many studies on the pathogenesis of various types of choledochal cysts and their relationship with the adjacent structures are in full swing and are being reported. and in cholangiographic techniques, there has been a series of reports which suggest that there is a high possibility of a relationship between the choledochal cyst and pbmu. a hepatic diverticulum appears in the ventral wall of the primitive midgut early in the 4th week of intrauterine life in the development of the human embryo. the extrahepatic bile duct system and the ventral pancreas arise from the hepatic diverticulum during the 4th to 6th week of intrauterine life. the main duct of the ventral pancreatic bud develops near the entry of the cbd into the duodenum. as the duodenum rotates to the right and become c - shaped, the ventral pancreatic bud is carried dorsally with the bile duct. the superior ventral branch of the ventral pancreas joins the distal portion of the dorsal pancreatic duct to form the main pancreatic duct, which merges with the cbd, inserting into the duodenum via the papilla of vater. the papilla of vater is generally situated halfway down the medial wall towards the posterior aspect of the descending or second part of the duodenum. there have been rare cases in which the papilla of vater was found in a position other than its normal position and such cases have been reported sporadically. however, such cases are interesting in the anatomical context. especially, very few cases have been reported in which the papilla of vater opens in the third portion of the duodenum [8 - 10 ]. in a series of recent studies, it has been reported that the frequency of choledochal cyst in patients was higher when the papilla of vater was positioned distal to its normal position. in this context from the anatomical viewpoint, the ectopic distal location of the papilla of vater represents the ectopic distal budding of the hepatic diverticulum during early embryonic life, it is straightforward to postulate that the longer distance between the ventral and dorsal pancreatic buds may result in a delay in communication of the ventral duct with the dorsal duct or failure of communication. and also the formation of pbmu at the early embryonic stage is caused by canalization of ventral pancreatic duct and the anomalous union of pancreaticobiliary duct, which originate from the dislocation of the ventral pancreas, and it is also caused by the stretching of primitive common channel and cbd in accordance with the lengthening, which will result in a long cbd and a long common channel. therefore, if growth of the epithelium in the cbd, the common channel and the pancreatic duct fails keep up with this anomalous elongation during fusion, the ducts may become attenuated, resulting in stenosis and weakness of the duct walls, and subsequent dilatation of the ducts, a phenomenon that has been observed clinically. this suggests that the lengths of the common channel and the cbd in patients with choledochal cyst correlate strongly with the location of the papilla of vater, i.e., the more distal the papilla of vater is, the longer the common channel and the cbd. in conclusion, we have reported a case of a 30-month - old child with choledochal cyst, in whom the form of pbmu occurred simultaneously and the papilla of vater was positioned in the third portion of the duodenum. there is no difference in the conventional surgical method of treatment in such cases despite the variation in the opening position of the papilla of vater. but the likely relationship between the opening position of the papilla of vater and choledochal cyst should be considered when a variation in the opening position of the papilla of vater is detected before surgery. | in cholangiographic techniques, the close relationship between choledochal cyst and anomalous union of pancreaticobiliary duct has attracted medical attention. there have been rare cases in which the papilla of vater was found in a position other than its normal position, and such cases have been reported sporadically. however, such cases are interesting in the anatomical context. in this review, we present our experience of choledochal cyst in a 30-month - old boy in whom the papilla of vater was positioned in the third portion of the duodenum. |
a patient with decreased vision underwent complete ophthalmologic examination, ultrasound biomicroscopy, fluorescein and indocyanine green (icg) angiography. the patient presented a nodule of the iris of the os and of the optic nerves of both eyes. fluorescein and icg angiography are the only objective exams to demonstrate the extent of ocular involvement in a patient with sarcoidosis. a 19-year - old black man noticed decreased vision in his os for approximately 2 months. at presentation best - corrected visual acuity measured with a standard snellen chart was 0.8 os and 1.0 od. slit - lamp examination revealed the presence of a granulomatous nodule of the iris of the os (figure 1a), also detected by ultrasound biomicroscopy (ubm) (figure 1b). fundus examination showed a voluminous granuloma of the optic nerve of os (figure 2) and a granulomatous lesion of the od, more visible on fluorescein angiography (figure 3a, b), which shows optic disc leakage of os and hyperfluorescence of od. multifocal choroidal lesions located in the posterior pole of the os only visible with indocyanine green (icg) were demonstrated (figure 3c, d). systemic medical and laboratory work - up was performed in order to diagnose the granulomatous disease. chest computed tomography showed hilar adenopathy. a tissue biopsy obtained from the nose proved the presence of noncaseating granuloma. sarcoidosis is a chronic multisystemic granulomatous disorder thought to result from an exaggerated cellular immune response to a variety of self antigens or nonself antigens (newman 1997). the disease affects predominantly the lungs and thoracic lymph nodes, skin and eyes (margolis 2007 ; chan 2007). in this case report however, only the icg angiography was capable of demonstrating the widespread extent of choroidal involvement. | purposeto report a case with anterior and posterior nodules associated with systemic sarcoidosis.methodsa patient with decreased vision underwent complete ophthalmologic examination, ultrasound biomicroscopy, fluorescein and indocyanine green (icg) angiography.resultsthe patient presented a nodule of the iris of the os and of the optic nerves of both eyes. chest computed tomography and tissue biopsy established the diagnosis.conclusionsfluorescein and icg angiography are the only objective exams to demonstrate the extent of ocular involvement in a patient with sarcoidosis. |
we suggest that bmp-9 has a regenerative role in the human liver. in favor of its establishment in diss s spaces and supported additionally by presence of bmp-9 s receptors on the surface of human hepatocytes, we have reason to believe that bmp-9 exerts the same effects on hepatocytes themselves. our hypothesis is additionally based on the fact that rhbmp-9 binds to human hepatoma cells and stimulates their dna synthesis. properties that bmp-9 exerts locally and systemically resemble physiological effects of insulin [46 ], especially in protecting peripheral tissues in states of hyperglycemia such as diabetes. bone morphogenetic proteins (bmps) are members of transforming growth factor beta (tgf-) a superfamily of cytokines that regulate cell growth and, differentiation during embryogenesis. some bmps, such are bmp-2/4,-3, -5, -6 and -7, justify their name as they regulate skeletal tissue formation and repair [710 ]. bmp-2 is supplementally involved in formation of vascular calcifications, while bmp-7 deficiency plays a role in progression of chronic renal failure. another tgf- superfamily member, growth differentiation factor 15 (gdf-15), although weak in osteogenic potential, has been implicated in playing an essential role in progression of congestive heart failure. these facts open a new chapter on bmps and their role in pathogenesis of human non - skeletal diseases. the mechanism of action of bone morphogenetic protein-7 (bmp-7) is most likely the one understood the best. accordingly, anti - inflammatory action on intestinal mucosal cells (most likely suppressing interleukin-6 [il-6 ]) is shown. two types of bmp receptors (bmpr) mediate activation of protein kinase and consecutive phosphorylation of amino - acids, phosphatidylinositol as 3-kinase and phospholipase c in insulin. the receptors are commonly composed of 2 transmembrane chains. the various combinations of types of chains can generate a broad range of effects in response to the same ligand. phospholipase c generates inositol 1,4,5-triphosphate (ip3) and diacylglycerol (dag) from phosphatidylinositol 4, 5-biphosphate. ip3 is a universal calcium - mobilized second messenger, while dag activates protein kinase c. both paths aim to increase ca influx and/or uptake. second messengers depicts the action of the bmp / bmpr system. just like other tissue - specific bmps, consequently, data suggest that bmp-9 is a potent alleviative of cartilage advance in vitro, and it has a significant role in forebrain embryogenesis, in cancer biology, in iron metabolism and in glucose homeostasis. the first evidence of bmp-9 was found in the mouse liver, where it was expressed during embryonic development. further investigations revealed that bmp-9 binds on human hepatoma cells and primary rat hepatocytes, inducing a proliferative response in both cell lines. although bmp-9 was not detectable in rat hepatocytes, a high level of bmp-9 messenger rna is expressed in non - parenchymal cells of the liver (ie, endothelial, kupffer, and stellate cells). since cells of the hepatic reticuloendothelial system showed high binding affinity for bmp-9, a possible autocrine - paracrine role is proposed for this morphogen in the liver. this was supported by chen., who found bmp-9 inhibits gluconeogenesis and activated expression of pivotal enzymes of lipid metabolism after a single subcutaneous injection of bmp-9. the hypoglycemic effect of bmp-9 was first established in transcription inhibition in rat hepatoma cells. their effects, however, differ. while bmp-9 as potently as insulin regulates total glucose production, insulin s effect on gluconeogenesis is more potent. bmp-9 has been demonstrated to improve glycemia in diabetic mice, which can be proven by in - situ exposition of hepatocytes to the combination of glucose and insulin and oral glucose in fasted rats, and is proposed as a candidate for the hepatic insulin - sensitizing substance (hiss). the process of hepatic regeneration has evoked wide interest since antiquity. despite many models of liver injury (eg, ccl4), hepatic regeneration has been witnessed in various species, but the exact mechanism and control over liver growth are unclear [2729 ]. it is proposed that regenerative capacities of the hepatocytes are dependent on the supply of oxygen and nutrients. the liver lobule is divided into 3 zones (metabolic heterogeneity) : zone i is the periportal part, which gets the maximum of oxygen and nutrients ; zone ii is the middle part of the liver lobule ; and zone iii surrounds the branches of hepatic vein and gets the minimum of oxygen and nutrients. hepatocytes that are closer to the periportal zone have better regenerative abilities compared to those hepatocytes in the central zone. during liver regeneration, hepatocyte proliferation starts in the areas of the lobules surrounding the portal triads and then proceeds to the pericentral areas by 36 to 48 hours. any explanation of this process has to take into account various blood - stream driven molecules : calcium, hepatocyte growth factor, epidermal growth factor, il-6, transforming growth factor - alpha, and tumor necrosis factor - alpha. insulin and norepinephrine, with limited effect on dna synthesis by themselves, are capable of altering growth factors induced liver regeneration. the kinetics of both cell proliferation and the growth factors produced by proliferating hepatocytes suggest that hepatocytes provide the mitogenic stimuli leading to proliferation of the other cells. based on findings of the bmp-9 s expression in the human liver, we hypothesize effects of bmp-9 to be dependent on blood supply (cvijanovic.). it is crucial to distinguish whether the localization of this protein is zone dependent ? if compared to the central zone, higher levels of the bmp-9 s expression in periportal hepatocytes would indicate its possible hepatoregenerative role. thus, in orthopedics it needs to be applied in vast quantities, and in more adoptable quantities, it is active in soft - tissue locations - including the liver, nervous system and bone marrow. is an extremely powerful tool that should help establishing possible therapeutic potential of bmp-9 in the treatment of type 2 diabetes. the suggested work should expand our current understanding of bmps functions other than those concerning heading morphogenesis towards supporting tissue s formation [713 ]. the effect of bmp-9 suggests its importance other than merely a bone formation inducer, most obviously in metabolism of carbohydrates, but nonetheless, fats. such properties, in supporting tissues organization could reform the clinical management of many musculoskeletal disorders, and its capability in differentiation of many other tissues warrants its popularity and attractiveness. precise determination of protein s expression is needed regarding zonal differences in normal and pathologically altered hepatocytes functions. experimental study needs to be carried out in order to give rise to analysis of direct hepatoregenerative effect of bmp-9. therefore, our hypothesis predicts additional evidence to previously introduced ideas of bmp-9 as a local autocrine / paracrine factor in the liver or systemic protein with a possible effect on glucose sensitive peripheral tissues. | summarybone morphogenetic protein-9 (bmp-9) is a member of the transforming growth factor beta (tgf-) superfamily of cytokines, which regulate cell growth and differentiation during embryogenesis. apart of that, the hypoglycemic potential of bmp-9 is of great interest. it has been confirmed that bmp-9, like insulin, improves glycemia in diabetic mice and regulates directional glucose metabolism in hepatocytes ; therefore it is proposed to be a candidate hepatic insulin - sensitizing substance (hiss). in liver fibrosis, due to the portocaval shunt, insulin bypasses the organ and the liver undergoes atrophy. parenteral administration of insulin reverses atrophy by stimulating mitogenic activity of the hepatocytes. because bmp-9 has a signaling pathway similar to other bmps and insulin, it is to be expected that bmp-9 has a certain regenerative role in the liver, supporting the above - mentioned is evidence of bmp-9 expression in diss s spaces and bmp-7 s mitogenic activity in mucosal cells. however, further studies are needed to confirm the possible regenerative role of bmp-9. |
human noroviruses (novs) are a primary cause of viral gastroenteritis throughout the world (siebenga. 2009), and the principle cause of foodborne illness in europe (kroneman. 2008 ; phillips. 2010) and the united states (mead. 1999 ; scallan. 2011). the number of estimated cases in the united states was recently revised to 5.5 million annually (scallan. 2011), while england has an estimated 2 million cases per year (phillips. norovirus illness is contracted through contaminated food and water and direct person - to - person transmission. contamination often arises during the handling and preparation of foods, although there are many instances where foods, particularly shellfish, are contaminated within their environment. products requiring extensive handling are also prone to contamination. in spite of interventions to eliminate product contamination, like thorough cooking, some products, such as salads and molluscan shellfish are typically eaten raw or only lightly cooked and such products represent the greatest risk to the consumer (richards 2001 ; richards. 2010). the development of commercial processing strategies for foods has been largely based on reductions in bacterial pathogens, and only to a lesser extent on the inactivation of enteric viruses. for some of the enteric viruses, like poliovirus and related picornaviridae, astroviruses, parvoviruses, rotaviruses, and adenoviruses 40 and 41, the levels of infectious virus particles can be determined using cell or tissue culture, since these viruses are propagatable. unfortunately, nov can not be routinely propagated in cell culture or animal models. human exposure and resulting illness scientists and regulators have relied on virus detection in foods based on the physical presence of viral rna, as determined by reverse transcription - pcr, which presently can not distinguish between infectious and inactivated viruses (richards 1999), although methods are being developed toward that goal (li. 2011 ; the lack of a suitable assay for infectious nov and other unculturable viruses, like most wild - type strains of hepatitis a virus (hav), has led the research community to focus on viral surrogates. the selection of a surrogate, at least in regard to novs, has been based on the ability of the surrogate to be propagated in culture, and its genetic, physical, or chemical relatedness to the pathogen. over the years, different surrogates have been selected for use in determining the uptake, persistence, distribution, and inactivation of viruses in foods, water, and environmental samples as well as in chemical disinfection studies on surfaces. studies with surrogates have been used with the intent to develop a better understanding about the pathogens they represent as well as applied applications for pathogen elimination. a respiratory virus known as feline calicivirus (fcv) in the genus vesivirus was first used as a nov surrogate in the late 1990s (doultree. 2006 ; dsouza and su 2010 ; doultree. 1999 ; gehrke. 2004 ; hudson. 2007 ; jimemez and chiang 2006 ; kampf. 2005 ; lages. 2008 ; malik. 2006 ; malik and goyal 2006 ; mori. 2007 ; morino. 2009 ; poschetto. 2007 ; sattar. 2011 ; steinmann 2004 ; urakami. 2007 ; whitehead and mccue 2010) ; and processing interventions, like heating (buckow. 2008 ; butot. 2009 ; cannon. ; fino and kniel 2008 ; nuanualsuwan. 2002), marinating / acidification (cannon. 2006 ; hewitt and greening 2004), and high pressure processing (chen. 2005 ; kingsley. (2006) identified murine norovirus-1 (mnv) as a closer genetic relative of nov. now, mnv has become the more commonly used surrogate for chemical disinfection studies (belliot. 2006 ; dsouza and su 2010 ; lee. 2008 ; lim. 2010 ; magulski. 2009 ; park. 2010 ; sattar. 2011) ; and studies on processing interventions like heating (baert. 2008a ; cannon. ; sow. 2011 ; tanner 2009), freezing (baert. ; park. 2011), gamma irradiation (feng. 2011), electron beam (sanglay. 2011), and high pressure processing (kingsley. 2007 ; lou. 2011 ; tang. 2010). poliovirus and bacteriophage ms2 have also been used as potential surrogates for nov (bae and schwab 2008 ; dsouza and su 2010 ; dawson. another potential surrogate is the recently discovered rhesus monkey calicivirus, known as tulane virus, which can replicate in cell culture, but as indicated by tan and jiang (2010), may not serve as a good model for human nov because it is not in the norovirus genus and has not been shown to cause gastroenteritis like the human novs. unfortunately, there are fundamental differences in the inactivation rates of many closely related viruses differences which may limit their role as surrogates. surrogate viruses are generally expected to mimic the viruses they represent, although studies are proving this concept invalid. in direct comparisons between fcv and mnv, their susceptibility to temperatures, ph, and environmental conditions has been shown to be dramatically different (cannon. 2006). in these studies, mnv was significantly more resistant to both acidic and alkaline phs than fcv ; mnv was more resistant than fcv to chloroform, freon, and vertrel ; fcv was more stable than mnv at 56c (p < 0.05), but differences were not significant at 63 and 72c ; and mnv was more stable in solution at room temperature (cannon. gibson and schwab (2011) reported that fcv was significantly less stable than mnv at 50c, but not significantly different at 60c. in these same studies, hav was significantly more resistant than fcv and mnv to heat treatment at 50 and 60c (gibson and schwab 2011). bae and schwab (2008) evaluated the persistence of fcv, mnv, ms2 and poliovirus in surface water and groundwater and found that fcv was significantly less stable than mnv, ms2, and poliovirus. (2011) compared the effects of ethanol - based hand rubs to eliminate fcv and mnv. they showed that over a short contact time (20 s) that fcv was 100 times more resistant to inactivation than mnv. in another study on hand sanitizers, fcv was more sensitive to some low ph sanitizers than mnv ; however, mnv was more readily inactivated by alcohols, thus the recommendation was made to include both surrogates when testing hand sanitizers (park. 2010). a study on the inactivation of fcv and mnv by uv irradiation (254 nm) showed fcv to be more sensitive than mnv (park. 2011). dsouza and su (2010) evaluated the effects of chemical treatments to inactivate fcv, mnv, and ms2 and reported that a 2% trisodium phosphate treatment for 1 min decreased fcv and ms2 by over 6 logs, but mnv by only 1 log. they also showed that 2% glutaraldehyde reduced fcv and mnv titers by 6 logs, but ms2 by <3 logs. seventy percent ethanol was reported to cause no reduction in titers for any of the viruses (dsouza and su 2010). (2007) compared the persistence of nov and fcv in the digestive tissues of the oyster after depuration for 10 days and concluded that fcv was rapidly depleted, whereas nov persisted. we now know that nov binds to histo - blood group antigens (hbgas) present on gastrointestinal cells of oysters, clams, and mussels (le guyader. 2006 ; tian. 2006, 2007), which may account for differences in virus uptake by shellfish. tulane virus also binds to hbgas (farkas. 2010), while mnv and fcv bind to sialic acid on the host cell surface (stuart and brown 2007 ; taube. these results should lead one to conclude that not all virus surrogates are equal ; some surrogates are more similar while others are quite different from the pathogens they represent. a major limitation in these studies is a lack of correlation in the inactivation rates of the surrogates and of the pathogen. koopmans and duzier (2004) suggested the use of the most resistant enteric virus in developing food safety guidelines. this may over regulate the industry if nov is substantially more susceptible than the surrogate. on the other hand, even the most resilient surrogate may not be as resistant as the pathogen, and may lead to a false sense of security relative to the safety of food or water. unfortunately, there is often no information available to directly correlate the pathogen with its ascribed surrogate, leaving regulators unable to promulgate new food regulations based on surrogate studies. in addition, a surrogate may be representative of a pathogen s response to a particular processing scenario (e.g., pasteurization), but may not necessarily represent a pathogen s response under other processing conditions or in other food matrices. data obtained must be carefully scrutinized and treated as presumptive evidence of how the pathogen may respond to a particular treatment. the use of fcv as a surrogate has diminished as more researchers are utilizing mnv, which is genetically more similar to nov than fcv. consequently, past fcv research is being looked on as unreliable, while mnv is rapidly gaining popularity as a more suitable surrogate, in spite of the fact that fcv seems more resistant to chemical disinfectants than mnv. the reason is that mnv may fail to respond in the same manner as the pathogen it represents. this was demonstrated in a volunteer study involving the inactivation of nov in oysters using high pressure processing (leon., pressures of up to 600 megapascals (mpa) for 5 min at room temperature were required to totally eliminate 4 logs of nov (leon. only 250 mpa was required to inactivate 7 logs of fcv in cell culture media under the same conditions, while 4 logs of mnv required 400 mpa for 5 min at 5c for inactivation (kingsley. one of the problems in comparing such studies is differences in processing conditions or matrix between assays. here, we see different matrices (cell culture media and oysters) and different processing conditions (room temperature and 5c) ; however, no other comparative studies exist for this processing technology. another example of the differences in the inactivation of related viruses may be seen for poliovirus and hav. poliovirus has been used as a surrogate for hav because of its similarity in size, shape, and structure to hav ; however, high pressure processing can inactivate hav at pressures around 400 mpa, but poliovirus persists at 600 mpa (kingsley. another study showed that different strains of cell culture - adapted hav have different sensitivities to heat and high pressure, where heating to 60c for 10 h and pressures of 420 mpa reduced virus infectivity by anywhere from 3 to 5 logs, depending on the strain (shimasaki. this represents a 100-fold difference in the inactivation of different strains of hav and highlights how different strains of the same virus do not accurately portray the inactivation dynamics of all hav strains. much like the differences in inactivation noted among hav strains, different responses to inactivation should be anticipated among the nov strains. different strains of fcv also showed widely differing susceptibilities to inactivation by three alcohol mixtures and a chlorine compound (di martino. if strains of the same virus give discordant inactivation results, then one might expect virus surrogates, which are only slightly related to the pathogens, to be poor indicators of the pathogen s inactivation kinetics. novs include a variety of genetically similar strains (also called genotypes or clusters) within two genogroups (i and ii). the uptake of different nov strains by oysters varied with genogroup i, cluster 1 nov (norwalk virus strain) efficiently bioconcentrated, but genogroup ii, cluster 4 nov poorly bioconcentrated (maalouf. 2011). consequently, the use of a surrogate for viruses that differ widely in their genetic composition and likely in their response to various processing techniques, chemical disinfection, or environmental conditions seems counterproductive. although the most resistant surrogates may be used to evaluate processing effectiveness, the pathogens could be several orders of magnitude more resistant to treatment than the surrogate. predictive models have been proposed for nov and hav inactivation in shellfish, based on the use of surrogates, either fcv or a cell culture - adapted strain of hav (buckow. 2008 ; grove. 2009) ; however, such models are not likely to accurately portray the inactivation of pathogenic viruses on or within foods. with the likelihood that mnv will not accurately reflect the conditions necessary to inactivate nov in foods, one questions the funding of surrogate studies. will mnv become another fcv, with millions of dollars spent on research but little confidence in the results ? considerable research funding is provided globally each year to conduct surrogate studies on nov ; however, there is little benefit derived from these studies in regard to the development of food processing strategies. after years of research with fcv as the surrogate, investigators are considering those results highly presumptuous, particularly in light of mnv which is now generally perceived as a better surrogate. the time, effort, and cost that were devoted to surrogate research with fcv may have been misdirected. at what cost was the fcv research conducted ? no one can be sure, but certainly millions of dollars have been spent on what is now considered by most as an ineffectual surrogate. a similar fate will befall mnv when researchers identify still better surrogates for nov and similar or greater dollar losses can be expected. uncertainty concerning the reliability of surrogate studies has, to the best of this author s knowledge, prevented results from being implemented in regulatory actions or new food processing procedures. if information were available on how much money was spent on surrogate studies and one were to conduct a cost benefit analysis on the fcv or mnv studies performed to date, results would be startling, since there has been much money spent, but with little change in the way we process foods or sanitize the workplace. at the time of this writing (september, 2011), a number of papers had already been published during 2011 on the use of mnv as a nov surrogate, including the use of varying processing technologies, like electron beam (sanglay. 2011) ; gamma irradiation (feng. 2011), and high pressure processing (lou. 2011) to inactivate mnv in produce ; heat inactivation of mnv in clams (sow. 2011) ; chemical disinfectants to eliminate mnv on produce and food contact surfaces (predmore and li 2011) and hands (park. 2011) ; and pulsed uv light inactivation of mnv on food contact surfaces (jean. such studies fail to provide reasonable expectations that the surrogates respond in a similar manner to nov or to different genogroups or strains of nov. in spite of the well intentioned and competent research that has been performed on nov surrogates, results derived from surrogate studies have not answered the important questions regarding nov inactivation in foods, water, or on food contact surfaces. we need to ask ourselves if fcv or mnv could ever be used in establishing food processing regulations when they only provide a glimmer of how the pathogen might respond to certain conditions. with all this uncertainty about surrogates, current practices to evaluate surrogate viruses and to employ molecular assays should be limited, particularly for nov inactivation studies. it is hereby recommended that presumptive information derived from the use of surrogates be subjected to proof - of - principle testing and validated in volunteer studies using nov. human challenge studies are essential to determine which processing techniques are effective in reducing nov in foods. nov is considered the primary cause of gastrointestinal illness worldwide. with such high morbidity rates, strategically designed volunteer studies performed under controlled conditions would seem prudent in order to assess the effects of cooking, freezing, irradiation, disinfectants, and other processing technologies on virus inactivation. the cost for challenge studies would be high ; however, the results would be definitive. a case in point was the recent volunteer study to assess the effectiveness of high pressure processing to inactivate nov in oysters, where it was definitively shown that pressure of up to 600 mpa for 5 min at room temperature would be required to inactivate 4 logs of the genogroup i.1 (norwalk virus) strain of nov (leon. does this mean that the same processing conditions would be required to inactivate the same level of other nov strains ? no, not at all, but is seems likely that differences between nov strains would be less than differences between novs and their so called surrogates. another example of the benefit of clinical trials involved a study performed in australia where shellfish containing nov were purified by depuration (grohmann. depurated oysters were subsequently fed to volunteers who became ill, thus demonstrating that commercial depuration was not effective in eliminating nov in contaminated oysters (grohmann. 1981). in this case, volunteer studies provided definitive answers about the infectiousness of the shellfish. volunteer studies can provide a firm basis for developing food and water processing strategies and for making regulatory decisions. until such time that researchers develop a cell culture propagation system for nov, or other means to discriminate infectious from inactive nov, human challenge studies are our best hope for determining true virus inactivation parameters. this recommendation to limit surrogate studies will be highly controversial, since funding for surrogate research has been plentiful and many researchers (including those in my laboratory) are accustomed to conducting nov research with virus surrogates. however, a shift in research directions is essential if we are to identify realistic and practical processing strategies to improve food safety. former and present research conducted with nov surrogates will likely contribute little to improving the world s food supply, while the costs of such studies could be pooled and redirected to more definitive clinical trials on nov inactivation. future surrogate studies should be considered only when the research is well justified with a clear delineation of why a study with surrogates is appropriate and what definitive information is anticipated. surrogate studies should be linked, to the extent possible, with nov volunteer studies to determine if the inactivation rates of surrogates and novs correlate after various processing interventions. clinical trials will again be necessary to validate some of the new and upcoming assays that are designed to differentiate infectious from inactivated viruses, such as pretreatment of viruses with proteinase k and ribonuclease before rt - pcr (nuanualsuwan and cliver 2002), use of ethidium and propidium monoazide in conjunction with rt - pcr assays (kim. 2010), and various receptor and binding assays, like one developed by li. virus surrogates may play a role in the development of assays for nov infectivity ; however, confirmation that the surrogates are truly representative of nov must be accomplished by volunteer studies. for instance, receptor binding assays which show that mnv only binds when it is infectious, should be linked with volunteer studies designed to evaluate the assay using nov of demonstrated infectivity, as determined by clinical trials. many in vitro surrogate studies suggest that nov can be inactivated by one treatment or another, but fail to carry the work forward to validate the results using similar treatments of nov in volunteer studies. over the years my laboratory just completed a 5-year collaborative study that involved clinical trials at emory university in atlanta (leon., there were seven separate trials involving 190 volunteers at emory university and the university of north carolina at chapel hill (lindesmith. 2003, 2005, 2010) and there were no adverse reactions among any of the volunteers (personal communication, christine moe, emory university). other clinical trials involving novs have been performed at baylor college of medicine (graham. 2002, 2005) and elsewhere (wyatt. 1974 ; dolin. 1982). nov illness is usually a temporary imposition where adverse effects outside of the usual nausea, diarrhea, and vomiting are seldom encountered ; therefore, there is reasonable expectation that trials may be conducted with minimal risk of complications. oversight of clinical trials must be provided by institutional review boards for human subjects or similar entities whose mission is to evaluate potential patient risks, to ensure they are minimized to the extent possible, and to weigh the risks versus benefits of the proposed research. costs for studies vary greatly, but an average volunteer may expect compensation of approximately $ 1000 (us) for their participation and incarceration in the hospital for 3 or 4 days. hospital costs, labor, and overhead for researchers add to the overall expense, thus the cost of a volunteer study may approach $ 500,000 (us) in today s market, depending on the number of volunteers required to provide statistically significant results. if monies that were normally used for surrogate studies were pooled, then multiple volunteer studies could likely be funded each year. the threat of lawsuits, in the event of unforeseen circumstances, prevents some governments, universities, and hospitals from conducting or considering the funding of such studies. in reality, there is far more risk in allowing millions of unsuspecting consumers to contract nov each year, when practical interventions are only a few volunteer studies away. the price to pay for inactivity is great, with lost wages, medical costs, and regulatory and epidemiological expenses involved in tracking and managing outbreaks. costs of supporting clinical trials would be a small price to pay for the considerable savings that would be brought about by even a modest reduction in nov outbreaks. a change in attitudes and a shift in research priorities are essential if we are to win the battle against nov illness. | the inability to propagate human norovirus (nov) or to clearly differentiate infectious from noninfectious virus particles has led to the use of surrogate viruses, like feline calicivirus (fcv) and murine norovirus-1 (mnv), which are propagatable in cell culture. the use of surrogates is predicated on the assumption that they generally mimic the viruses they represent ; however, studies are proving this concept invalid. in direct comparisons between fcv and mnv, their susceptibility to temperatures, environmental and food processing conditions, and disinfectants are dramatically different. differences have also been noted between the inactivation of nov and its surrogates, thus questioning the validity of surrogates. considerable research funding is provided globally each year to conduct surrogate studies on novs ; however, there is little demonstrated benefit derived from these studies in regard to the development of virus inactivation techniques or food processing strategies. human challenge studies are needed to determine which processing techniques are effective in reducing novs in foods. a major obstacle to clinical trials on novs is the perception that such trials are too costly and risky, but in reality, there is far more cost and risk in allowing millions of unsuspecting consumers to contract nov illness each year, when practical interventions are only a few volunteer studies away. a number of clinical trials have been conducted, providing important insights into nov inactivation. a shift in research priorities from surrogate research to volunteer studies is essential if we are to identify realistic, practical, and scientifically valid processing approaches to improve food safety. |
sjgren 's syndrome (ss) is the second most common systemic autoimmune disease (after rheumatoid arthritis, ra), with a prevalence of about 0.5% in the general population. it occurs primarily in perimenopausal women (at a ratio of women to men of 9 : 1) [1, 2 ]. key features of the disease include infiltration of exocrine glands (predominantly salivary and lacrimal) by lymphocytes, production of inflammatory cytokines, and the activation of b lymphocytes and the production of autoantibodies. at the level of exocrine glands, loss of secretory activity other systems or organs such as musculoskeletal system (arthralgias, myalgias, and nonerosive arthritis of small joints), lungs, liver, skin, and kidneys can also be involved. compared to the general population, patients with ss exhibit significantly increased risk of developing b - cell lymphoproliferative disorders (usually b - cell lymphoma involving the mucosa - associated lymphoid tissue (malt)), which affects about 510% of patients leading to significant increased morbidity and mortality rates of these patients. several clinical and laboratory manifestations have so far been proposed as adverse predictors for malignant disease and include features related to deposition of immune complexes (palpable purpura, peripheral neuropathy, low levels of complement c4, and cryoglobulinemia), swelling of the parotid glands, as well as high lymphocytic scores, and the presence of ectopic germinal centers in minor salivary gland biopsies. the causative mechanisms of ss have not been fully elucidated. however, based on the current pathogenetic model, the interaction of both genetic, epigenetic [6, 7 ] and environmental factors seems to contribute to disease development. viruses, especially the epstein - barr virus (ebv) and coxsackievirus, hormones (low estrogen levels seen in perimenopausal women), stress, and occupational exposures have been all considered as the main environmental triggers for disease onset. in the current review article, we will mainly focus on the contributory role of genetic influences in the development of the ss as well as in ss - related lymphoproliferation, a major disease complication. familial clustering and coaggregation with other autoimmune disorders in ss has been long considered [1316 ]. in an italian multicenter case control study, the risk of ss development among first - degree relatives with autoimmune disease was sevenfold higher compared to controls. of note, these first - degree relatives of ss patients had a higher risk of autoimmune disease compared to subjects without first - degree relative affected by ss. a large recent study in taiwanese population confirmed previous observations showing that first - degree relatives of ss patients had an increased risk of ss development as well as of other autoimmune disorders, mainly systemic lupus erythematosus (sle), ra, systemic sclerosis, and type 1 diabetes (t1d), compared to the general population. of interest, siblings of affected individuals demonstrated the highest relative risk for ss development compared to other first - degree relatives (parents and offsprings), implying both genetic influences and shared environmental exposures as contributory factors to disease development. since the 1970s, strong associations between specific alleles of the major histocompatibility complex (mhc) and ss development have been suggested [18, 19 ]. over the last decade, high throughput technologies allowed the confirmation of the dominant role of mhc alleles in the pathogenesis of the disease with novel genetic variants outside the mhc locus emerging as susceptibility factors. the latter seem to be involved in signaling pathways of natural and acquired immunity, inflammatory responses and cellular apoptosis. though initially recognized as major determinants of tissue rejection, mhc genes have been soon after appreciated as critical contributors to the pathogenesis of autoimmune disorders as well. they encode components of the human leukocyte antigen (hla) system including hla class i (a, b, and c) and class ii (dr, dq, and dp), which present endogenous and exogenous antigens to t lymphocytes, respectively. mhc class ii molecules, especially those which encode hla - dr and hla - dq antigens, have been proposed as the most important genes associated with ss susceptibility. the first genetic study on ss revealed an association between ss and hla - dr3 (which was in linkage disequilibrium (ld) with the class i allele hla - b8) in caucasian ss patients. subsequent reports highlighted the association between ss and the hla - d locus [18, 23 ], with a diverse distribution between primary ss (high frequency of the alleles hla - drw3 and hla - b8) and secondary ss (increased frequency of allele hla - drw4), as well as in patients with ss characterized by the presence of raynaud 's phenomenon (increased frequency of alleles hla - drw3 and hla - drw4). subsequent studies in other ethnic populations confirmed hla associations with ss susceptibility [2641 ], with drb104:05-dqb104:01 being a risk allele in japanese populations, drb108:03-dqb106:01 in chinese populations, dr3 and dr11 in spanish populations, and drb111:01, drb111:04, dqb103:01, and dqa105:01 in israeli jews and greeks [43, 44 ] (summarized in table 1). in a subsequent meta - analysis, hla class ii alleles drb103:01, dqa105:01, and qb102:01 were shown to predispose to disease development, while dqb105:01 exhibited a protective role. two recent large genome wide association studies (gwas), in caucasian and han - chinese population, confirmed the strong influence of hla locus on ss. further studies are required on the functional role of the hla polymorphic regions in ss pathogenesis as well as their possible associations with disease diagnosis and/or prognosis. a functional deletion of 6.7 kb in the gene leukocyte immunoglobulin - like receptor subfamily a member 3 (lilra3) has been also associated with autoimmune diseases. lilra3 is a soluble receptor of class i mhc antigens involved in the regulation of immune function. it has been found in ss patients of both caucasian and chinese origin, as well as in other autoimmune diseases including multiple sclerosis, ra, and sle. in the following years, research has been directed to the investigation of single nucleotide polymorphisms (snps) in genes outside the mhc locus (summarized in table 2 [5168 ]), already found to be associated with other autoimmune diseases such as sle. these novel ss - associated genetic variants (outside the mhc locus) can be roughly classified into three main groups depending on the implicated signaling pathway. the first group consists of variants in genes involved in the activation of the interferon (ifn) signaling pathway. specific autoantibodies have been found in approximately two - thirds of ss patients and genetic contribution has been proposed. hla class ii ss - related phenotype has been associated with the presence of autoantibodies in various studies [33, 35, 40 ]. snps in the 5 untranslated region of the baff gene as well as in gtf2i and genes implicated in the nf-b pathway have been also associated with the presence of autoantibodies. finally, the third one contains apoptotic and inflammatory genes, which participate in the nf-b signaling pathway. gene expression studies in ss patients over the last decade revealed upregulation of ifn - inducible genes (the so - called ifn signature) at the level of peripheral blood and affected salivary gland tissues in a substantial proportion of these individuals. moreover, recent data revealed that both type i (ifn/) and type ii (ifn) ifn signatures are upregulated in both peripheral blood and minor salivary gland tissues derived from ss patients while the ifn/ifn mrna ratio in diagnostic salivary gland biopsies could predict the in situ lymphoma development in the setting of ss [72, 73 ]. while the mechanisms leading to this activation remain under investigation, several genetic variants in genes implicated in the ifn pathway have been designated as potential contributors. several polymorphisms of the irf5 gene have been previously shown to either increase or decrease sle susceptibility [74, 76 ]. initial studies on ss revealed the irf5 polymorphism rs2004640 (creates an alternate splice site (exon 1b) in the first exon) as predisposing factor to disease development in both scandinavian and french cohorts [51, 52 ]. another strong signal of association was observed between the insertion / deletion (in / del) of the cgggg sequence in the irf5 gene promoter in both ss and other autoimmune diseases [52, 53, 77 ]. this cgggg in / del is part of a polymorphic repetitive dna region, which includes either 3 or 4 cgggg repeats ; the insertion of an additional cgggg unit (the 4 cgggg allele) is the risk allele associated with increased irf5 transcription in peripheral blood mononuclear cells (pbmcs) and cultured epithelial cells derived from the salivary glands of ss patients, possibly through the addition of sp1 binding site in the gene promoter of the 4r allele [53, 77 ]. moreover, reovirus infection of salivary gland epithelial cells from ss patients carrying the 4r allele further increased irf5 expression at mrna level. taken together, these findings suggest a possible association between the irf5 gene variants and the induction of type i ifns (through the induction of the irf5 gene expression after a viral infection) that could lead to the robust activation of the immune system in salivary glands (target organ) as an early event in ss pathogenesis. recent studies revealed genetic association of transportin-3 (tnpo3), an irf5 neighboring gene that encodes a nuclear receptor involved in the import of splicing factors in the nucleus, with both sle and ss susceptibility with specific variants spanning the irf5-tnpo3 locus being identified [45, 76 ]. the transcription factor stat4 is primarily involved in the signal transduction induced by the cytokines interleukin- (il-) 12 and il-23 leading to differentiation of t helper (th) nave cells towards a th1 phenotype and subsequent production of ifn. stat4 intronic variants, namely, rs7582694 and rs7574865, have been associated with ss development in four candidate gene association studies [52, 55, 56, 79 ]. subsequent gwas studies in ss patients with both european and chinese descent confirmed stat4 locus as an important determinant of ss susceptibility [45, 46 ]. while rs7582694 risk variant has been associated with increased expression of several ifn - inducible genes in ss patients, pbmc derived from lupus patients harboring the risk variant of rs7574865 demonstrated increased sensitivity to ifn effects. the recent gwas in the caucasian but not chinese population revealed an important association of il12a gene polymorphisms with ss. the il12a is a cytokine that forms a heterodimer with the il12b subunit inducing through stat4 the differentiation of nave t - cells in t helper 1 cells which promotes immune response through ifn production by t helper 1 cells. ncr3/nkp30 is a natural killer (nk) specific receptor regulating the cross talk between nk and dendritic cells as well as type ii ifn secretion. the minor allele of the rs11575837 polymorphism within the promoter of ncr3 gene has been found as protective allele for ss development that is associated with reduced ncr3 gene transcription. compared to controls, ss patients who lacked this polymorphism demonstrated higher circulating levels of the ncr3 ligand and demonstrated higher focus scores in salivary gland biopsy. ptpn22 gene encodes the protein lymphocyte tyrosine phosphatase (lyp) previously shown to be implicated in both adaptive (inhibition of t - cell receptor (tcr) and b - cell receptor (bcr) signaling) and innate immune responses (type i interferon (ifn) production by myeloid cells through tlr ligation). a single nucleotide polymorphism (snp) of the ptpn22 gene 1858c > t (rs2476601) leading to substitution of arginine (r) by tryptophan (w) at position 620 has been previously shown to increase susceptibility to several autoimmune diseases including t1d, sle, and ra [8385 ]. although the underlying mechanisms leading to autoimmunity are not clearly delineated, a break in b- and t - cell tolerance through altered bcr and tcr signaling, enhancement of t helper follicular cells, and dampened type i ifn responses leading to a proinflammatory microenvironment have been all shown to contribute to autoimmune pathogenesis reviewed in. in regard to ss, available data so far is rather conflicting. in contrast to a french report in which no association with ss development was detected, studies in both colombian and greek populations identified a strong association with ss susceptibility, particularly in patients characterized by low ifn signatures (manuscript in preparation). on this basis, we postulate that the apparent discrepancies between different studies are related to ifn status of ss patients included. this finding implies an additional shared etiological origin in autoimmune disorders, with a putative role of genetic contributors as determinants of distinct ifn patterns in patients with autoimmune diseases. b - lymphocyte kinase (blk). the kinase blk is a member of the family of the src tyrosine kinase, which seems to be involved in signaling and differentiation of b lymphocytes. common polymorphisms in the blk and in the neighboring family with sequence similarity 167, member a (fam167a) genes have been found to predispose to sle, systemic sclerosis, ra, and recently ss [54, 60, 90 ]. two main snps associated with ss development include rs12677843 (located in intron 1 of the blk gene) and rs12549796 (second intron of fam167a). the functional implication of these snps in ss remains unknown, although previous studies on sle showed an association between the presence of risk alleles with decreased blk mrna levels and increased fam167a mrna levels in transformed b lymphocytes. the association of blk / fam167a polymorphisms with ss was also suggested in a large gwas study in caucasian populations. previously published data support a role for several haplotypes in the 5 regulatory region of baff gene in autoantibody positive ss and increased serum baff levels as well as in distinct (both low and high risk for lymphoma development) ss phenotypes. chemokine (c - x - c motif) receptor 5 (cxcr5). in a gwas caucasian study, the chemokine receptor cxcr5 detected in both circulating b - cells and activated cd4 cells contributes to b- and t - cell migration in peripheral lymphoid as well as in inflamed peripheral organs, upon ligation with the cxcl13 chemokine [91, 92 ]. the latter has been previously found to be upregulated in salivary gland tissues derived from ss patients leading eventually to preferential retention of memory cxcr4cxcr5 b - cells in the ss derived salivary gland infiltrates. early b - cell factor 1 (ebf1). in a large candidate gene association study in ss patients of scandinavian origin, genetic variants of the ebf1 gene (previously shown to be involved in antigen independent changes of b - cell differentiation) have been found to confer increased risk for ss. ox40l (or tnfsf4), a tnf family ligand member, expressed on activated dendritic cells, endothelial cells, and the b - cell surface, has been previously shown to get involved in b - cell activation through interaction with ox40-positive t - cells [95, 96 ]. genetic variants of ox40l have been previously associated with susceptibility to sle (in association with increased transcript and protein levels) and scleroderma, but not with primary biliary cirrhosis or ss after bonferroni corrections in a han - chinese population [63, 97, 98 ]. however, in a scandinavian study, two snps (namely, rs1234315 and rs1234314) located in the 5-untranslated region of the gene ox40l have been found to be significantly associated with ss. an interesting finding from a large study (gwas) in han - chinese but not in european population revealed that a polymorphism in the gtf2i gene (namely, rs117026326) (encodes a transcription factor involved in both t - cell signaling and activation of immunoglobulin heavy - chain transcription upon b - lymphocyte activation) is strongly associated with ss development with overall risk (or) scores higher than other ss - associated identified genes including mhc - ii genes, stat4, and tnfaip3. this finding was also confirmed in another study in chinese population and was linked to the presence of anti - ro / ssa autoantibodies. tnfaip3 gene encodes the a20 protein, an enzyme with ubiquitination activity that appears to play an important role in the regulation of inflammation through the nf-b pathway. a20 protein is expressed at low levels on most of the cells but is rapidly induced after activation of nf-b, acting as a negative feedback regulating both inflammation and apoptosis. experiments on mice revealed that a20 is important for survival and normal development since a20-deficient mice fail to regulate tnf induced nf-b activation and die early due to multiorgan inflammation and cachexia. several genetic variants of the tnfaip3 gene have been associated with autoimmune diseases including ss. the coding tnfaip3 polymorphism, namely, rs2230926, which changes the amino acid sequence from phenylalanine (phe) to cysteine (cys) at position 127 has been previously found to confer increased risk for sle. functional analysis showed that the rs2230926 minor allele, which predisposed to disease, is less effective in inhibiting the activity of nf-b after induction by tnf. the association of tnfaip3 rs2230926 polymorphism with ss has been recently confirmed by two large studies (gwas) in both caucasian and chinese population. tnfaip3-interacting protein 1 (tnip1). of note, polymorphisms of the tnip1 gene, a molecule which interacts with the tnfaip3 gene regulating the nf-b activation, have been recently found to confer increased risk to ss [54, 66 ] and other autoimmune diseases [104, 105 ]. the role of tnip1 polymorphisms in ss development was also confirmed in a large gwas study in caucasian population. lymphotoxin gene a (lta). polymorphisms of the lymphotoxin gene a (lta), located on locus lta / ltb / tnf and related to the activation of the nf-b pathway as well as inflammation, have been found to increase the risk of ss. chemokine (c - c motif) ligand 11 (ccl11). finally, the ccl11 (eotaxin) is a chemokine with important role in ss. the expression of ccl11 has been found to be regulated by the nf-b pathway and specific polymorphisms in the ccl11 gene have been associated with ectopic germinal center - like structures present in salivary gland tissues of a proportion of ss patients who are found to be at risk of lymphoma development. animal models are useful tools for elucidating the etiopathogenetic mechanisms of various autoimmune diseases including ss. over the last decade, various murine models have been proposed in an attempt to explore the early initiating and subsequent events leading to disease development. spontaneous or transgenic murine models which are prone to develop sjgren 's syndrome - like symptoms during lifetime include, among others (as reviewed recently in), (nzb / nzw)f1, mrl, nod, nod - aec1aec2, baff tg, opn tg, and act1. the latter has been recently found to develop a disorder which closely resembles sjgren 's syndrome in association with lupus. the act1-deficient mice are characterized by marginal zone - like b - lymphocyte accumulations, salivary and lachrymal gland inflammation, and production of anti - ro / ssa and anti - la / ssb autoantibodies. act1 is a negative regulator of baff and cd40 molecules (both implicated in b - cell survival and activation) while recent findings proposed it to be a critical component of the il-17 signalling pathway. of interest, several snps around the traf3ip2 gene (which encodes the act1 protein) have been recently found to confer increased risk to lupus and may play an important role in the induction of the interferon pathway (interferon-, interferon inducible genes), which is relevant in the context of autoimmune diseases, like lupus and ss. taken together, these findings indicate the putative role of the snps in the traf3ip2 gene in the development of histological and serological features of ss. lymphocytic infiltration of the exocrine glands and ectopic formation of germinal centers have been considered as the sine qua non of lymphoma development. b - cell hyperactivity, the hallmark of ss, molecular events affecting b - cell function and survival, and the deregulation of the nf-b pathway have been recently proposed as potential factors leading to lymphoma development. chronic antigenic stimulation of autoreactive b - cells and tumorigenic events such as chromosomal translocation and gene mutations / polymorphisms have been suggested as possible mechanisms underlying neoplastic diversion in the setting of ss. regarding oncogenic mechanisms, the presence of the translocation t(14 ; 18) (leading to overexpression of bcl-2, an antiapoptotic gene promoting b - cell survival) has been detected in 5 of 7 salivary gland biopsies of patients with sjgren 's syndrome who developed lymphoma and in none of the 50 corresponding biopsies of patients with the syndrome not associated with lymphoma. furthermore, mutations of tumor suppressor gene p53 are possibly associated with the occurrence of lymphoma in patients with ss. additionally, somatic mutations and polymorphisms in the tnfaip3 gene have been also reported in several types of lymphomas including lymphomas of mucosal marginal zone (malt), which is the major type of ss - related lymphoproliferative disease. in a recent study, the rs2230926 tnfaip3 polymorphism along with other genetic alterations has been found to be associated with ss - related lymphoproliferation, especially of malt type, while functional assays found that this polymorphism is associated with increased activation of the nf-b pathway. another study failed to provide evidence for the presence of myd88 l265p gene mutation (a nonsynonymous change at amino acid position 265 from leucine to proline (l265p)) in patients with ss with and without lymphoma. myd88 is an adaptor protein leading to nf-b activation through tlr, il-1r, and il-18 signaling, which has been previously shown to be implicated in patients with waldenstrm 's macroglobulinemia (wm) and other haematological malignancies [116, 117 ]. the absence of mutation in ss patients with or without lymphoma suggests that probably there are different pathogenetic mechanisms in lymphoproliferation in the setting of ss. given that deregulation of b - cell activation has been postulated as fundamental event in both autoimmunity and b - cell lymphomagenesis, the baff / baff - r axis attracted our research interest. specific haplotypes of the baff gene could discriminate ss patients with lymphoma from ss patients without lymphoma and healthy controls and a functional mutation his159tyr of the baff receptor (baff - r), previously found to confer an increased risk in patients with nhl through activation of the alternative nf-b pathway, has been found to be more prevalent in ss population compared to healthy controls. of interest, more than two - thirds of ss patients complicated by malt type nhl with an age at ss diagnosis between 3rd and 4th decade carried this mutation. the role of known polymorphisms of the methylenetetrahydrofolate reductase (mthfr), gene, an enzyme necessary for the dna synthesis and methylation, which have been previously associated with nhl development [119, 120 ] and autoimmune diseases, has been also investigated. mthfr polymorphisms have been found to be associated with both ss and ss non - malt nhl development in association with methylation alterations, implying genetic and epigenetic abnormalities as common pathogenetic pathways in both benign autoimmunity and malignant transformation (fragioudaki., in preparation). while growing evidence over the last years supports a genetic contribution to ss susceptibility, the majority of genetic variants seem to have weak or moderate effect (except, perhaps, for hla locus), implying an additional role for the environmental insults such as viruses, hormones, and stress in disease pathogenesis. given that the vast majority of these genetic loci have been also detected as susceptibility factors in other autoimmune disorders, shared mechanisms leading to deregulation of the immune system imply a central role in autoimmune pathogenesis. heterogeneity of ss clinical expression from local disease confined to exocrine glands to lymphoma development should be always taken into account when genetic studies are designed, since distinct operating immune pathways underlie distinct clinical phenotypes. further multicenter efforts exploring genetic, epigenetic, and environmental interactions are warranted to further clarify the pathogenesis of the syndrome. | we aimed to summarize the current evidence related to the contributory role of genetic factors in the pathogenesis of sjgren 's syndrome (ss) and ss - related lymphoma. genes within the major histocompatibility complex (mhc) locus previously considered conferring increased susceptibility to ss development have been also revealed as important contributors in recent genome wide association studies. moreover, genetic variations outside the mhc locus involving genes in type i interferon pathway, nf-b signaling, b- and t - cell function and methylation processes have been shown to be associated with both ss and ss - related lymphoma development. appreciating the functional implications of ss - related genetic variants could provide further insights into our understanding of ss heterogeneity, allowing the design of tailored therapeutic interventions. |
approximately 25% of patients with localized breast cancer and without axillary lymph node involvement will suffer from a systemic relapse despite successful treatment of the primary tumor. hence, micrometastatic hematogenous spread appears to be independent from lymphatic involvement and the disease must have the ability to persist in secondary sites of the body, a phenomenon referred to as minimal residual disease (mrd). it was hypothesized that disseminated tumor cells (dtcs), which are found in the bone marrow (bm) of 20%-40% of patients with primary breast cancer (pbc), are progenitors of subsequent metastasis and a promising marker for mrd. however, as dtc detection has the disadvantage of requiring an invasive procedure for sample collection, recent research has focused on the use of circulating tumor cells (ctcs) from the peripheral blood instead of bm aspiration. various assays, including immunocytochemistry, polymerase chain reaction (pcr)based methods or microfluidic systems have been described for ctc detection in pbc patients with positivity rates ranging from 10% to 60% [4 - 8 ]. currently, the most widespread approach is the cellsearch system by veridex (raritan, nj), which was developed to standardize and automate immunomagnetic enrichment, immunofluorescence staining and microscopic enumeration of ctcs. detection of ctcs using cellsearch is of prognostic significance in advanced as well as in pbc. in a recent study, schindlbeck. compared ctc enumeration using cellsearch with dtc detection in patients with primary or metastatic breast cancer. the authors found a significant congruence between dtcs and ctcs, which increased in patients with metastases. however, in a recent study for analysis of the simultaneous detection of dtcs and ctcs in metastatic breast cancer, we were not able to confirm these results. thus, the primary goal of the current study was to compare dtcs and ctcs in patients with pbc. in addition to the enumeration of ctcs using cellsearch we used a secondary reverse transcription (rt)-pcr based assay for ctc detection. patients who underwent primary surgery for pbc (t1 - 4, n0 - 2, m0) at the department of gynecology and obstetrics at tuebingen university, germany, between january 2008 and may 2014 were eligible for inclusion in this retrospective study. exclusion criteria were recurrent or metastatic disease, bilateral breast cancer, neoadjuvant systemic therapy, r1-resection, or a previous history of secondary malignancy. while bm aspiration for dtc detection was performed using a standardized method in all patients, ctc detection was performed using either adnatest breast cancer (from january 2008 until december 2009) or cellsearch (from january 2010 until may 2014). adjuvant therapy (including chemotherapy, endocrine therapy, trastuzumab therapy, and radiation therapy) was based on the current st. those patients have been participating in clinical trials involving bisphosphonates (gain, success a / c, natan, or zol - mrd 001), or were offered treatment with zoledronate at 4 mg every 6 months outside of clinical trials. all specimens were obtained after patients had provided written informed consent and the analysis was approved by the ethics committee of tuebingen university (reference number : 560/2012r). during primary surgery, 10 - 20 ml of bm aspirates were collected. briefly, mononuclear cells were separated via density centrifugation (1.077 g / ml ; ficoll, biochrom, germany), spun down onto a glass slide (hettich cytocentrifuge, hettich, tuttlingen, germany) and fixed in 4% formalin. the presence of dtcs (dtc status) was evaluated by immunostaining using the dako autostainer (dako, glostrup, denmark), the monoclonal mouse a45-b / b3 antibody directed against pancytokeratin (micromet, munich, germany), and the dako - apaa detection kit (dako). for each patient two slides (210 cells) were evaluated, based on consensus recommendations for standardized tumor cell detection and the criteria of the european ishage working group. in addition, with each batch of samples, leukocytes from healthy volunteers served as negative control and the cell lines mcf-7 and skbr-3 were used as positive control. from january 2008 until december 2009, ctcs were evaluated 1 - 3 days prior to surgery using adnatest breastcancer (adnagen ag, langenhagen, germany). in brief, 25 ml of peripheral venous edta blood were collected using adnacollect tubes (adnagen ag). for labeling of tumor cells, a ready - to - use mixture (adnatest breastcancerselect) containing antibodies against ga 73.3 and muc1 was used according to the manufacturer s instructions. subsequently, mrna isolation from lysed, enriched cells was performed according to the manufacturer s instructions using the dynabeads mrna direct micro kit (dynal biotech gmbh, hamburg, germany) included in adnatest breast - cancerdetect. for reverse transcription, sensiscript reverse transcriptase (qiagen gmbh, hilden, germany) was used in combination with oligo(dt) coupled dynabeads. analysis of tumor - associated mrna was performed in a multiplex pcr for human epithelial growth factor receptor 2 (her2), muc1, and ga 733 - 2. the primers generate fragments of the following sizes : ga 733 - 2, 395 base pairs (bp) ; muc1, 293 bp ; her2, 270 bp ; and actin, 114 bp. santa clara, ca) using dna 1000 lab - chips and the expert software package (ver. the test was considered ctc positive if a pcr fragment of at least one tumor - associated transcript (muc-1, ga 733 - 2, or her2) and a fragment of the control gene -actin were clearly detected (peak concentration of > 0.15 ng/l) in both blood samples. from january 2009 until may 2014, enrichment and enumeration of ctcs was performed using the cellsearch technology (cellsearch epithelial cell kit / cellspotter analyzer, veridex llc). briefly, 7.5 ml of peripheral venous blood was collected 1 - 3 days prior to surgery using cellsave tubes (veridex llc, warren, nj). epcam - positive cells were then labeled with the nuclear dye 40,6-diamidino-2-phenylindole (dapi) and monoclonal antibodies specific for the leukocyte common antigen cd45. cd45-negative, cytokeratin - positive cells with an intact nucleus were defined as ctcs and enumerated by trained operators. blood samples containing at least one ctc per 7.5-ml blood were considered ctc - positive. associations between categorical variables were analyzed using chi - square and fisher exact test. to determine survival, times from primary surgery to any recurrence of disease (disease - free survival, dfs) and death of any cause (overall survival, os) patients followed up for less than 6 months were not included in the survival analysis. the influence of the dtc and ctc status, respectively, on survival was determined by univariate analysis and expressed as a hazard ratio (hr) and 95% confidence interval (ci). the initial model included menopausal status, histology, tumor size, nodal status, breast cancer subtype, treatment (chemotherapy, endocrine therapy, trastuzumab therapy, and bisphosphonate therapy), ctc - status (adnatest and cell - search), and dtc - status. the effect of each variable was assessed using the wald test and expressed as an hr and 95% ci. 20 (ibm co., armonk, ny) and reported two - sided with significance levels set to p 0.15 ng/l) in both blood samples. from january 2009 until may 2014, enrichment and enumeration of ctcs was performed using the cellsearch technology (cellsearch epithelial cell kit / cellspotter analyzer, veridex llc). briefly, 7.5 ml of peripheral venous blood was collected 1 - 3 days prior to surgery using cellsave tubes (veridex llc, warren, nj). epcam - positive cells were then labeled with the nuclear dye 40,6-diamidino-2-phenylindole (dapi) and monoclonal antibodies specific for the leukocyte common antigen cd45. cd45-negative, cytokeratin - positive cells with an intact nucleus were defined as ctcs and enumerated by trained operators. blood samples containing at least one ctc per 7.5-ml blood were considered ctc - positive. associations between categorical variables were analyzed using chi - square and fisher exact test. to determine survival, times from primary surgery to any recurrence of disease (disease - free survival, dfs) and death of any cause (overall survival, os) patients followed up for less than 6 months were not included in the survival analysis. the influence of the dtc and ctc status, respectively, on survival was determined by univariate analysis and expressed as a hazard ratio (hr) and 95% confidence interval (ci). the initial model included menopausal status, histology, tumor size, nodal status, breast cancer subtype, treatment (chemotherapy, endocrine therapy, trastuzumab therapy, and bisphosphonate therapy), ctc - status (adnatest and cell - search), and dtc - status. the effect of each variable was assessed using the wald test and expressed as an hr and 95% ci. 20 (ibm co., armonk, ny) and reported two - sided with significance levels set to p < 0.05. the median age was 60 years (range, 29 to 88 years) and most women were postmenopausal (68%). most patients had a tumor grade 1 - 2 (71%) and the tumor size was mainly less than 20 mm (pt1, 69%). the majority were nodal - negative (73%), estrogen receptor (er)positive (82%), progesterone receptor (pr)positive (69%), and her2-negative (84%). the dtc status was determined in all 585 patients and positive in 131 of these (22%) (table 1). dtc detection showed significant association with pr status (p=0.024) and her2 status (p=0.040). adnatest breast cancer was used for ctc detection in 202 patients (table 2) and 19 of these (9%) were ctc - positive. the cellsearch technology was used in 383 patients. the number of ctcs per 7.5-ml blood ranged from 0 to 42 (mean, 0.3). at least one ctc per 7.5-ml blood was detected in 18 of these patients (5%). no significant association between ctc and dtc detection was observed by the use of either adnatest or cellsearch (table 3). the concordance between dtc status as compared to adnatest and cellsearch was 75% and 76%, respectively. median follow - up period (95% ci) was 35 months (range, 31 to 39 months) for all patients. for patients who underwent ctc analysis using adnatest and cellsearch, median follow - up period was 52 months (range, 50 to 54 months) and 22 months (range, 20 to 24 months), respectively. 1, risk of relapse was significantly greater for dtc - positive patients than dtc - negative patients (p=0.046). the hr (95% ci) for relapse and death was 2.13 (1.02 - 4.54) and 2.27 (0.97 - 5.32), respectively. use of the adnatest for detection of ctcs had no impact on dfs (p=0.834) or os (p=0.270). however, in ctc analysis using cellsearch, ctc - positive patients had a significantly greater risk of relapse than ctc - negative patients (p=0.007), whereas the impact of ctc detection on os reached borderline - significance (p=0.051). using cellsearch, the hr (95% ci) for relapse was 6.31 (1.36 - 29.24) and the hr for death was 4.09 (0.88 - 19.01). in multivariate analysis, tumor size, nodal status, and the ctc status, as determined by cellsearch, were significant predictors of reduced dfs, whereas grading and nodal status were significant predictors of reduced os(table 4). the median age was 60 years (range, 29 to 88 years) and most women were postmenopausal (68%). most patients had a tumor grade 1 - 2 (71%) and the tumor size was mainly less than 20 mm (pt1, 69%). the majority were nodal - negative (73%), estrogen receptor (er)positive (82%), progesterone receptor (pr)positive (69%), and her2-negative (84%). the dtc status was determined in all 585 patients and positive in 131 of these (22%) (table 1). dtc detection showed significant association with pr status (p=0.024) and her2 status (p=0.040). adnatest breast cancer was used for ctc detection in 202 patients (table 2) and 19 of these (9%) were ctc - positive. the cellsearch technology was used in 383 patients. the number of ctcs per 7.5-ml blood ranged from 0 to 42 (mean, 0.3). at least one ctc per 7.5-ml blood was detected in 18 of these patients (5%). no significant association between ctc and dtc detection was observed by the use of either adnatest or cellsearch (table 3). the concordance between dtc status as compared to adnatest and cellsearch was 75% and 76%, respectively. median follow - up period (95% ci) was 35 months (range, 31 to 39 months) for all patients. for patients who underwent ctc analysis using adnatest and cellsearch, median follow - up period was 52 months (range, 50 to 54 months) and 22 months (range, 20 to 24 months), respectively. 1, risk of relapse was significantly greater for dtc - positive patients than dtc - negative patients (p=0.046). the hr (95% ci) for relapse and death was 2.13 (1.02 - 4.54) and 2.27 (0.97 - 5.32), respectively. use of the adnatest for detection of ctcs had no impact on dfs (p=0.834) or os (p=0.270). however, in ctc analysis using cellsearch, ctc - positive patients had a significantly greater risk of relapse than ctc - negative patients (p=0.007), whereas the impact of ctc detection on os reached borderline - significance (p=0.051). using cellsearch, the hr (95% ci) for relapse was 6.31 (1.36 - 29.24) and the hr for death was 4.09 (0.88 - 19.01). in multivariate analysis, tumor size, nodal status, and the ctc status, as determined by cellsearch, were significant predictors of reduced dfs, whereas grading and nodal status were significant predictors of reduced os(table 4). in general, prognostic and predictive biomarkers for breast cancer therapy, including er and her2 as well as new molecular tests like the oncotypedx 21-gene recurrence score, characterize tissue from the primary tumor to predict response to treatment. this approach, however, assumes that the primary tumor is representative of the entire tumor burden (including mrd) and that the initial phenotype will not change during the course of disease. there is, however, evidence that tumor cells abandon the primary lesion before acquisition of fully malignant phenotypes to undergo somatic progression at a distant site. this hypothesis of early dissemination and the divergent development of the primary tumor and distant tumor cells makes dtcs and ctcs attractive candidates for monitoring and targeting the systemic component of breast cancer. in univariate analysis, the presence of dtcs in bm significantly affected dfs. for os only borderline significance was detected, which is most likely due to the small number of events. however, in the multivariate analysis, the dtc status had no significant impact on survival, which is in contrast to earlier studies reported by us and others. on the one hand, this is also explained by the small sample size. on the other hand as described previously, bisphosphonate treatment might also contribute to the improved survival of dtc positive patients. the cellsearch technology is used for detection of ctcs in translational research programs of various clinical trials. although other, probably more sensitive methods for detection of ctcs exist, cellsearch provides the advantages of high reproducibility and widespread as well as automated and standardized workflow. to the best of our knowledge, this is the largest study to date comparing dtc detection in pbc with ctc detection using cellsearch. the weak correlation between cellsearch and bm sampling is in line with results from smaller trials. however, we found that only 5% of our patients harbor at least one ctc per 7.5-ml blood, which is considerably lower than the dtc detection rate. detected at least one ctc in 22% of pbc patients ; however, 30 ml blood per patient were analyzed. in addition, patients participating in the success study were about to receive chemotherapy and thus at moderate or high risk for disease recurrence. other studies that determined the prevalence of ctcs in pbc using cellsearch reported detection rates ranging from 9% to 24%. these inhomogeneous results are most likely due to different patient populations, time - points of blood sampling and different sample volumes, reflecting the lack of standardization for ctc detection in pbc. in 2004, food and drug administration as a diagnostic tool for identifying and counting ctcs of epithelial origin in patients with metastatic breast cancer. currently, evidence that ctc detection using cellsearch is also an indicator of poor prognosis in pbc is increasing. we confirmed that ctcs detected using cellsearch were associated with reduced dfs, although we found only borderline significance with respect to os, which is most likely due to the short follow - up period of 22 months and the small sample size. in the success trial,. found that the ctc status is not only predictive of prognosis at the time - point of surgery, but also after completion of adjuvant chemotherapy. likewise, we recently demonstrated that dtc detection during or after adjuvant systemic therapy also impacts prognosis. however, due to the invasiveness of bm sampling, repeated analyses of ctcs from venous blood would be more feasible for monitoring the success of adjuvant treatment. using adnatest for ctc analysis in contrast to cellsearch, we found no impact of the adnatest results on survival. in a recent study, molloy. compared the dtc - status of 733 pbc patients with the presence of ctcs as determined using an rt - pcr - based assay. although the rate of ctc - positive patients was in line with our results, the authors found that ctcs were highly associated with the presence of dtcs and that both dtc- and ctc - detection were predictive of survival. however, molloy. used 50 ml of peripheral blood and an expression score of four marker genes (ck19, p1b, egp, and mmg1) for detection of ctc - positive patients. similarly, daskalaki. found a high concordance between bm and blood for cytokeratin-19-mrna - positive cells in patients with pbc and both the dtc and ctc status were predictive of survival. in contrast to the study by molloy. and to our methodology, daskalaki. used rt - pcr not only for detection of ctcs but also for determination of dtcs. the antibody based detection of dtcs, however, allows for cytomorphological assessment to confirm malignancy and is therefore recommended by an international expert panel. other studies using rt - pcr based assays reported ctc - detection rates between 8% and 55% [6,19 - 21 ]. we found that the percentage of ctc - positive patients was nearly twice as high when adnatest was used as compared to cellsearch. a head to head comparison study between the two assays was recently conducted by fehm. however, the study by fehm. was conducted in metastatic breast cancer patients. because adnatest and cellsearch are two completely different approaches to detection of ctcs, each method was analyzed separately. hence, a main limitation of our study was the small sample size in each subgroup. another limitation is the relatively short follow - up period, particularly in the subgroup analyzed using cellsearch. in addition, improved adjuvant treatment interacts with the survival analysis of this retrospectively designed analysis. we recently found that the dtc status might be predictive of the efficacy of bisphosphonate therapy. a considerable proportion of patients in the current study were treated with bisphosphonates (28% of all dtc - positive women, data not shown), which might explain the small differences observed in dtc - positive versus dtc - negative patients with respect to dfs and os. a current pilot study is evaluating the impact of denosumab on dtcs in patients with pbc (nct01545648). in addition, her2-targeted treatment might improve prognosis of her2-overexpressing dtcs and ctcs, which is currently being evaluated in a phase ii randomized trial for her2-negative pbc patients with her2-positive dtcs (nct01779050). hence, next to their mere quantification, characterization of dtcs and ctcs promises to identify targets for individualized breast cancer treatment. the german detect study group is prospectively evaluating whether characterization of ctcs in patients with metastatic breast cancer is useful in tailoring her2-directed therapy (nct01619111). in patients with her2-negative pbc the ongoing treat - ctc study will evaluate whether ctc - positive patients will benefit from additional trastuzumab treatment (nct0154867). in contrast to ctc analysis in metastatic breast cancer, where ctc detection using the cellsearch technology impacts prognosis at the highest level of evidence, the optimal method and clinical implication for ctc analysis in pbc remains to be elucidated. the inhomogeneous prevalence of ctcs, the discordant results with respect to their correlation with the dtc status and the questionable impact on prognosis (especially when using rt - pcr based assays like adnatest) is most likely due to different methodologies and inhomogeneous patient characteristics. although large prospective trials like the success study highly support the prognostic validity of ctcs in patients with pbc, further studies are needed to standardize ctc detection and to determine their clinical potential for the personalization of breast cancer treatment. ctcs have a very short estimated half - life, and therefore must be continuously replenished. after successful treatment of the primary lesion, although both the dtc and ctc status are predictors of an adverse prognosis, we and others found no association of ctc detection with the dtc status. thus, we do not believe that dtcs and ctcs simply represent two sides of the same coin. to better understand their relationship, their biological role in tumor progression and their clinical role for treatment optimization, in future trials analyses of dtcs and ctcs in breast cancer patients. only molecular characterization of dtcs, ctcs, and tumor tissue from the same patient can ascertain whether these are independent subpopulations of malignant cell clones or in continuous cellular exchange with each other. | purposedisseminated tumor cells (dtcs) from bone marrow (bm) are a surrogate of minimal residual disease (mrd) in primary breast cancer (pbc) patients and associated with an adverse prognosis. however, bm sampling is an invasive procedure. although there is growing evidence that circulating tumor cells (ctcs) from the blood are also suitable for monitoring mrd, data on the simultaneous detection of dtcs and ctcs are limited.materials and methodswe determined the presence of dtcs using immunocytochemistry and the pan - cytokeratin antibody a45-b / b3. ctcs were determined simultaneously using a reverse transcription - polymerase chain reaction based assay (adnatest breast cancer) and cellsearch (at least one ctc per 7.5 ml blood). we compared the detection of dtcs and ctcs and evaluated their impact on disease - free and overall survival.resultsof 585 patients, 131 (22%) were positive for dtcs ; 19 of 202 (9%) and 18 of 383 (5%) patients were positive for ctcs, as shown by adnatest and cellsearch, respectively. no significant association was observed between dtcs and ctcs (p=0.248 and p=0.146 as shown by adnatest and cellsearch, respectively). the presence of dtcs (p=0.046) and the presence of ctcs as shown by cellsearch (p=0.007) were predictive of disease - free survival.conclusionour data confirm the prognostic relevance of dtcs and ctcs in patients with pbc. as we found no significant relationship between dtcs and ctcs, prospective trials should include their simultaneous detection. within those trials, the question of whether or not dtcs and ctcs are independent subpopulations of malignant cell clones should be determined by molecular characterization. |
hyperparathyroidism due to parathyroid abnormalities are classified into three forms : primary, secondary, and tertiary. parathyroid adenoma accounts for about 85% of all cases of hyperparathyroidism, considering all the forms : primary, secondary and tertiary. other less prevalent causes include parathyroid hyperplasia (15%) and carcinoma (3 - 4%). however, acute primary hyperparathyroidism, or parathyroid crisis (pc), is rare. parathyroid crisis is characterized by life - threatening hypercalcemia of a sudden onset in subjects with phpt. we describe a patient with pc secondary to a giant intrathyroidal parathyroid adenoma who presented with progressive neurological symptoms, nausea and vomiting without medical response to treatment. a 19-year - old female patient was admitted to the orthopedics department for supra - acetabular fibrous dysplasia treatment. after orthopedic surgery, during the second postoperative day she began to suffer from lethargy, nausea, vomiting, and abdominal pain. also, the patient reported muscle weakness, fatigue and a worsening of depressive symptoms. a blood analysis was performed which showed elevated ca (14.2 mg / dl, normal laboratory level 8.8 - 10.2 mg / dl). the serum phosphorus was low (3.99 ng / ml, normal laboratory level 4.0 - 19.0 ng / ml) with an increased alkaline phosphatase level (171 ui / l, normal laboratory level 20 - 90 endocrine evaluation showed elevated parathyroid hormone (pth) (1207.0 pg / dl, normal laboratory 7 - 82 pg / dl). an urgent ultrasonography was requested and showed a 47 22 mm nodule in the left thyroid lobe. after two days of intensive medical treatment, the ca levels had not decreased and the clinical situation of the patient remained similar or even worse. because of life - threatening hypercalcaemia and a poor response to medical treatment, she directly underwent surgery after a consensus decision between the endocrinologists and the surgeons. a hemithyroidectomy was performed assuming it was an intrathyroiidal adenoma and without time for performing a fine needle aspiration because of the patient 's situation. during the surgical procedure, a superior parathyroid gland was observed and not resected. at the end of the hemithyroidectomy, the specimen was open and a clear intra - thyroidal adenoma surrounded by thyroid tissue was observed [figure 1 ]. unfortunately, intraoperative pth and anathomopathological analyse were avaible but were not used according to the intraoperative findings. on the final anatomopathological examination, it had a maximum diameter of 3 cm and the whole specimen weighted 70 g. the postoperative course was uneventful. the patient received doses of calcium iv during the first day and oral intake began at day two. oral calcium was then given to the patient in the postoperative period until the fist visit at the outpatient clinic. during follow - up, the risk of developing this disease increases with a peak incidence between 50 and 60 years, whereas phpt in children and adolescents is a rare condition. parathyroid crisis is characterized by life - threatening hypercalcemia of sudden onset in subjects with phpt. it has been reported that changes in mental status are characteristic features of hypercalcemic crisis. moreover, she had muscle weakness. although neuromuscular manifestations have been described previously, the pathophysiology of these manifestations is not yet clear. moreover, in pc, as a potentially lethal condition, emergent parathyroidectomy has been advocated. certainly, it has been emphasized that expeditive parathyroidectomy is the cornerstone treatment of patients with pc. there is no clear relationship between surgery and the beginning of toxic, urgent hypercalcaemia. in surgical series there is an estimated reported prevalence of intrathyroidal parathyroid adenoma that varies from 1.4 - 6%. according to embryology, parathyroid glands originate from the third and fourth branchial pouches. after this, the glands caudally migrate to their final positions. any problems during this migration can lead to the glands settling in an anomalous location, even in the thyroid tissue. in a recent study, localization of most of the adenomas (70.6%) was shown to be on the right side and on the lower third of the thyroid lobes. this study also reported that intrathyroidal parathyroid adenomas were present in 3% of patients submitted for parathyroidectomy. however, reports of a giant intrathyroidal parathyroid adenoma in the literature are exceptional. it has been noted that the weight of the adenoma correlates with the severity of the pc. gasparri., found that in a patient with a serum calcium level higher than 15 mg / dl, the mean weight of the removed glands was 4.9 g, whereas it was 2.1 g in patients with serum calcium levels lower than 15 mg / dl. nevertheless, giant parathyroid adenomas are rare., reported that adenomas heavier than 3.5 g accounted for 7.2% of their series of phpt. differential diagnoses, when very high levels of pth are present, must be established with parathyroid carcinoma. parathyroid carcinoma is a rare malignancy with an incidence rate of less than 1% in all patients surgically treated for primary hyperparathyroidism. to the best of our knowledge the clinical manifestations of hyperparathyroidism in parathyroid carcinoma are usually more severe than in patients with parathyroid adenoma. parathyroid crisis is an emergency because high levels of calcium pose a life - threatening situation for patients.. however, changes in mental status are prevalent, hence, the serum calcium level must be determined in any patient with unexplained central nervous dysfunction. once the patient with pc has been stabilized, expedited parathyroidectomy must be performed. | primary hyperparathyroidism (phpt) is not an uncommon endocrine disorder. however, acute primary hyperparathyroidism, or parathyroid crisis (pc), is a rare clinical entity characterized by life - threatening hypercalcemia of a sudden onset in patients with phpt. we describe a patient with pc who presented with acute worsening of depressive symptoms, nausea and vomiting, and required emergency surgery. serum calcium, alkaline phosphatase, and parathyroid hormone were elevated and serum phosphorus was low. an emergency hemithyroidectomy was performed because of none medical control of hypercalcemia. a giant intrathyroidal parathyroid adenoma was diagnosed. phtp can be a life - threatening situation for patients, requiring immediate surgical treatment. a giant intrathyroidal parathyroid adenoma is an uncommon cause of pc. |
they are also known as " ambu ", which is the trademark of a resuscitator and the term used as a synonym for mr by professionals in brazil and other countries. being used in icus in order optimize oxygenation and facilitate the removal of secretion in patients on invasive mechanical ventilation, mrs are also used for intra - hospital transport and as a source of temporary ventilation in intubated and nonintubated patients. on the basis of their inflation mechanism, mrs can be classified as self - inflating or flow - inflating, the former being the most commonly used type in icus in brazil. self - inflating mrs, which is the type of mr evaluated in the present study, consist of a compressible self - inflating bag, an inflating valve, and an inspiratory / expiratory valve (patient valve ; figure 1). when the bag is compressed by the operator, the patient valve occludes the expiratory port and allows the air within the bag to flow to the patient ; when the pressure within the bag ceases, the patient valve returns to its normal position, occluding the communication of the bag with the patient and allowing the air coming from the patient to be exhaled through the expiratory port. a drop in pressure within the bag during exhalation causes the inflating valve to open, allowing the air to enter the bag. additional components of this type of mr include a device for enriching the oxygen mixture, a positive end - expiratory pressure (peep) valve, and a pressure - limiting valve (plv), the use of which in adult patients is not in accordance with the american society for testing and materials (astm) criteria. figure a illustrates all of the components of the resuscitator, and figure b illustrates only the patient connection port, highlighting the positioning of the patient valve during inhalation and exhalation. a : connection port of the oxygen reservoir with an inflation valve ; b : inflating bag ; c : patient valve ; d : pressure - limiting valve (acts only during inhalation) ; e : expiratory port ; and f : patient connection port. in the literature, there are studies evaluating the performance and safety of mrs available in other countries in accordance with international technical standards. the objective of the present study was to evaluate the characteristics and performance of some of the self - inflating mrs for use in adult patients that are available in the brazilian market, in accordance with international standards. the present study was approved by the research ethics committee of the university of so paulo school of medicine hospital das clnicas and was carried out in the experimental mechanical ventilation laboratory of the department of pulmonology of the same institution in 2006. moriya, so paulo ; oxigel, so paulo ; protec equipamentos mdico - hospitalares, so paulo ; rwr equipamentos hospitalares, so bernardo do campo ; and unitec ind. e com, so paulo), two were manufactured in denmark (ambu silicone resuscitator and ambuspur ; ambu a / s, ballerup, denmark), and one was manufactured in the usa (hudson rci(r) lifesaver ; hudson rci, durham, nc, usa). all of the models are still being marketed, and the updates of the mrs since the present study was carried out to this writing are described in table 1. table 1 characteristics of the manual resuscitators for adults as described by the manufacturers.nd : no data. note : from 2006 to this writing, devices 1, 2, 3, 4, and 7 did not change significantly. in device 5, the current patient connection port is a swivel. in device 6, the color of the silicone was changed from blue to transparent. in device 8, the capacity of the bag was increased from 1,000 to 1,500 ml. note : from 2006 to this writing, devices 1, 2, 3, 4, and 7 did not change significantly. in device 5, the current patient connection port is a swivel. in device 6, the color of the silicone was changed from blue to transparent. in device 8, the capacity of the bag was increased from 1,000 to 1,500 ml. since there are no brazilian standards defining the characteristics for the manufacture and marketing of mrs, the present evaluation used the astm criteria, designation f920 - 93, which are equivalent to the international organization for standardization 8382 criteria, used in other countries. using a respiratory system simulator, the following criteria were evaluated : resistance imposed by the patient valve during the inspiratory and expiratory phases ; functioning of the plv ; and tidal volume (vt) delivered by the mr. in accordance with the astm criteria, the following specifications should be met : a drop < 5 cmh2o in inspiratory and expiratory pressure in the presence of a flow of 50 l / min, leading to resistance < 6 cmh2o. l. s ; the use of a plv is not mandatory for mrs for adults, but, if this valve is available, it should be possible to close it ; a vt 600 ml should be delivered to adult patients (weight 40 kg), with resistance of 20 cmh2o. s and compliance of 0.02 l / cmh2o, using the one - handed technique (maximum hand size, 185 mm). the measurement system consisted of a plastic tube to which pressure and flow sensors were connected (figure 2). the signals from the sensors were amplified and stored on a computer for subsequent analysis with the labview software (national instruments, tx, usa). the signal sampling frequency was 200 hz. figure 2schematic representation of the experimental system. 1 : proximal or distal flow sensor ; 2 : pressure sensor ; 3 : resistor ; and 4 : alveolar pressure sensor. in order to measure inspiratory and expiratory resistance, a mechanical ventilator (bear 5 ; bear medical systems, riverside, ca, usa) was connected to one end of the plastic tube with the sensors (figure 2). the ventilator was set to the controlled volume mode, with a constant inspiratory flow of 60 l / min and a peep of zero cmh2o. in the evaluation of expiratory resistance, the patient connection port of the mr was attached to the other end of the plastic tube, and the air from the ventilator left through the expiratory port into room air (figure 2). in the evaluation of inspiratory resistance, the mr was disassembled and only the proximal portion of the set, without the bag, was used (figure 2). the part of the mr that is connected to the bag was attached to the other end of the plastic tube, and the air left through the patient connection port to the environment. the ambu silicone resuscitator was not tested because it could not be disassembled. the two resistances were calculated as the difference between the proximal and distal pressure to the valve (distal pressure was not measured, because it was equal to zero, since the system was open to the environment), divided by the measured flow. for the testing of vt, the mrs were connected to the plastic tube with the sensors, and this tube was connected to a tracheal cannula of 8.5 mm in diameter (rusch, montevideo, uruguay), the distal portion of which, with the cuff inflated, was fitted into another plastic tube that was connected to a respiratory system simulator (ttl 2600 ; michigan instruments, grands rapids, mi, usa ; figure 2). the simulator allowed the compliance of the bellows to be set with springs ; resistance was set with nonlinear resistors. four volunteer icu physiotherapists in the city of so paulo (two males and two females) operated the mrs. the size of the dominant hand (from the wrist to the tip of the middle finger) was 200 mm for male1, 188 mm for male 2, 175 mm for female 1, and 160 mm for female 2. the physiotherapists were instructed to inflate the model as if they were ventilating a patient, first using the one - handed technique and then using the two - handed technique, with the eight mrs, which were tested in a random sequence, under two conditions of resistance and compliance : 1) " astm " condition - compliance of 0.02 l / cmh2o and resistance of 20 cmh2o. l. s, in accordance with the astm criteria ; and 2) " normal " condition - compliance of 0.05 l / cmh2o and resistance of 5 cmh2o. s, simulating a patient without lung disease. in order to calculate vt, three respiratory cycles were analyzed, all of which were randomly selected among the recorded cycles (a minimum of 10 per test condition). since each volunteer (totaling four) handled eight mrs, using the one - handed and two - handed techniques, in two distinct clinical settings (astm condition and normal condition), 128 tests were performed. of the eight mrs, five were equipped with a plv and were submitted to this test. the ventilator, connected to one end of the measurement system (with a flow sensor and a pressure sensor), was set to the controlled volume mode, with an inspiratory flow of 20 l / min, a vt of 1,800 ml, and a peep of zero cmh2o. a part of the mr was connected to the other end, as were another (distal) flow sensor and the simulator, in sequence (figure 2). we determined the pressure at which the plv opened and allowed air leaks (a time point that could be identified when the flow in the distal sensor decreased in relation to that in the proximal sensor), this pressure being referred to as " opening pressure ", and determined the pressure at which all air sent by the ventilator passed only through the plv (a time point at which the distal flow was equal to zero), this pressure being referred to as " maximal pressure ". the functioning of the valve was considered appropriate if the opening pressure was within the pressure interval suggested by the manufacturers, in general, values greater than 35 - 40 cmh2o (table 1). for each mr, only one inspiratory and expiratory resistance value was shown, because the variation between the cycles was negligible. the vt values were expressed as mean and standard deviation and were evaluated by multivariate analysis using a mixed linear regression model for each of the four conditions studied : vt generated with the one - handed technique under the astm and normal conditions ; and vt generated with the two - handed technique under the same two conditions. post hoc tests with sidak correction were used for comparisons of the vt values among the physiotherapists and among the mrs. for this analysis, we used the statistical package for the social sciences, version 17 for windows (spss inc. the most common type of patient valve was a diaphragm, and the most common bag material was silicone. the capacity of the bag of each mr varied (1,000 - 2,500 ml). the three imported mrs did not have a plv, which was a feature of the five brazilian - made mrs, and, in the mr manufactured by rwr, the plv could be closed without there being any leaks. another difference observed between the imported and the brazilian - made mrs was that the patient connection port was fixed in the five brazilian - made mrs, whereas, in the imported ones, this connection port was a " 360 swivel ". table 2 describes the results of the tests evaluating the resistances of the patient valve during inhalation and exhalation, as well as the functioning of the plv. only in the protec rm did expiratory resistance exceeded the limit set by the astm criteria (< 6 cmh2o. s. in the other mrs tested, the resistances met those criteria, the highest inspiratory resistance being found in the oxigel mr (5.69 cmh2o. l. s. the plv, a feature of five of the mrs, opened at low pressures (< 17 cmh2o), and the maximal pressure was 32.0 - 55.9 cmh2o. table 2 inspiratory and expiratory resistance of the patient valve and evaluation of the pressure - limiting valve. the mean delivered vt values, by mr (table 3) and by physiotherapist (table e1, available online at http://www.jornaldepneumologia.com.br/detalhe_artigo.asp?id=2212), varied. in the situations evaluated (handling of the eight mrs, with the use of the one - handed and two - handed techniques, in two distinct clinical settings - astm condition and normal condition), there were significant differences (p < 0.001) among the mrs and among the physiotherapists. all of the mrs, whenever the one - handed technique was used under the astm condition, delivered a lower vt than that recommended by the astm (600 ml). only the two models of mr manufactured by ambu a / s delivered a vt 600 ml when the one - handed technique was used, but only during the simulation of the normal clinical condition. even when the two - handed technique was used, the mrs manufactured by oxigel and rwr did not deliver the minimal vt under the astm clinical condition, whereas the mrs manufactured by protec and unitec, regardless of the clinical setting simulated, delivered a vt < 600 ml. table 3 tidal volume (vt) generated under the normal condition and under the american society of testing materials (astm) condition.aavalues expressed as mean sd, on the basis of 3 measurements, in ml.bcompliance = 0.02 l / cmh2o ; and resistance = 20 cmh2o. l. s ccompliance = 0.05 l / cmh2o ; and resistance = 5 cmh2o. s. p < 0.05 in relation to the resuscitators that did not have a valveambuambu s ; andhudson / rci values expressed as mean sd, on the basis of 3 measurements, in ml. l. s compliance = 0.05 l / cmh2o ; and resistance = 5 cmh2o. s. p < 0.05 in relation to the resuscitators that did not have a valve in addition, when we observe the results reported in table 3, we can see that the vt values generated under the normal clinical condition were, in general, higher than were those generated under the astm condition, which simulates a pulmonary condition with lower compliance and higher resistance, and that most of the vt values obtained with the mrs that did not have a plv were higher than were those obtained with the mrs that had a plv. the main findings of the present study can be summarized as follows : 1) the (inspiratory and expiratory) resistances of the patient valve met the astm criteria in all but one of the mrs tested ; 2) the eight rms evaluated did not deliver the minimal vt under conditions standardized by those same criteria (vt 600 ml, with the use of the one - handed technique, under the astm clinical condition) ; 3) the use of the two - handed technique did not solve the problem in four mrs (vt remained < 600 ml) ; and 4) the plv present in the brazilian - made mrs usually led to the delivery of a lower vt than that delivered by the mrs that did not have a plv. in brazil, there have been few published studies evaluating mrs for adult patients, commonly used in icus in the country. although those studies investigated important characteristics, such as the oxygen fraction delivered by mrs with and without an oxygen reservoir, the present study evaluated other equally important properties that had not yet been reported in the national literature. regarding the evaluation of resistance of the patient valve during inhalation and exhalation, all of the mrs tested were within the specifications of the astm, except for the protec mr, in which expiratory resistance was 7.91 cmh2o. s. this result is not surprising, given that international studies had described mrs in which the resistances of the patient valve were higher than recommended. it is of note that higher expiratory pressure translates to longer exhalation, which can cause air trapping (auto - peep) and risk of barotrauma, especially when the mr is handled at a high respiratory rate and a high vt. for the evaluation of the vt generated with the one - handed and two - handed techniques, we selected male and female volunteers, because it is known that the vt obtained varies with the gender of the operator (vt is lower for female operators). mean vt varied greatly among the mrs tested, a finding that had been reported in other studies. we observed that some brazilian - made mrs delivered a low vt even with the use of the two - handed technique, which generates a higher vt than does the one - handed technique. other authors had reported, even in normal compliance simulations, vt values lower than 600 ml. however, in the study by mazzolini., which evaluated 16 disposable mrs manufactured abroad, only three mrs did not deliver the minimal vt recommended by the astm (600ml). one factor that seems to have led to increased vt values was the size of the hands of one of the physiotherapists (200 mm, a larger size than that recommended by the criteria, male 1, table e1, available online at http://www.jornaldepneumologia.com.br/detalhe_artigo.asp?id=2212). this association between delivered vt and hand size has been described by other authors. factors that could lead to low volumes are the size and the material of the bag of the resuscitator tested. the volume of air in the bag is not totally delivered to the patient when the bag is compressed, and, in general, the volume of the bag should be twice the volume to be delivered. a bag made of a material that is not very compliant (such as polyvinyl) can lead to a lower vt. in our study, the mr that delivered the lowest vt (unitec) had the bag with the lowest volume, and, in addition, this bag was made of polyvinyl. it is of note that vt was generally lower from the brazilian - made mrs than from the imported ones, except for the mr manufactured by rwr, which generated a higher vt, probably because the capacity of the bag was 2,500 ml. one could question whether the low vt values found for some of the mrs tested resulted from fatigue of the volunteers. however, the study by hess., which evaluated the effect of fatigue caused by ventilation with a mr for 30 min under low compliance (20 ml / cmh2o), found no reduction in vt after 30 minutes for the 14 volunteers evaluated. considering that, in our study, total test time for each of the mrs was approximately one minute, which would total eight minutes for each of the four situations evaluated (two impedance values with the use of the one - handed and two - handed techniques), and that there was a rest break between each measurement, it seems unlikely that the occurrence of fatigue would have influenced the results. as expected, the vt obtained in the simulation of a " normal " patient was higher than that found in the tests using the compliance and resistance settings established by the astm criteria, which simulated a patient with decreased compliance and increased resistance, which are proven to lead to a lower vt. the presence of a plv in the brazilian - made mrs possibly impaired their performance. all of the brazilian - made mrs had a plv, and in only one of them could the valve be closed. the valves of all of them initially opened at low pressures, the values of which were below the specified value, causing leaks during inflation, which can partially explain the lower vt obtained with the rms that had a plv. this result can be considered a severe fault, because it can prevent appropriate ventilation in patients with decreased lung compliance or with increased airway resistance, such as those with pulmonary edema and those with asthma attacks, respectively. according to some authors, the presence of plv does not bring additional safety to adult patients, given that the risk of barotrauma is not associated only with high peak pressures during inhalation. in fact, the astm criteria dispense with the use of a pressure - regulating device for mrs for adults and determine that, if this valve is present, it should be possible to lock it and its closing mechanism should be quite evident to the operator. even in mrs for neonatal / pediatric patients, for which the presence of this valve is recommended in order to limit pressure to values below 40 5 cmh2o, malfunctioning of the plv can also impair ventilation. we consider limitations of our study the fact that all tests were performed using only one unit of each mr (however, all mrs were brand - new) ; that resistance of the patient valve was tested at a flow of 60 l / min, rather than of 50 l / min (value recommended by the astm), which can hinder the comparison with other studies ; and that, since the present study was carried out in 2006, some mr models underwent changes (table 1), which might have altered their performances. this should be probably true for the mr manufactured by unitec, in which the capacity of the bag was significantly increased (table 1). despite the limitations of this study, we believe that important deficiencies of the mrs manufactured in brazil are reported, especially those regarding the functioning of the plv (most could not be locked and opened early), deficiencies that, in some clinical conditions, can impair ventilation in critically ill patients. our results indicate that, in order to deliver an appropriate vt, it is necessary that both hands be used to compress the mr bag and that, when allowed by the mr, the plv be kept closed. we believe that the results obtained in the present study can aid in performing future tests and in improving the brazilian - made mrs. avaliar o desempenho de reanimadores manuais (rms) utilizados no brasil conforme critrios definidos por uma norma internacional. utilizando um simulador do sistema respiratrio, oito rms manuais (cinco produzidos no brasil e trs importados) foram avaliados em relao a resistncia inspiratria e expiratria da vlvula para o paciente ; funcionamento da vlvula limitadora de presso ; e volume corrente (vt) gerado por quatro fisioterapeutas voluntrios, utilizando uma ou duas mos. para a realizao e anlise dos testes, foram utilizados critrios sugeridos pela norma da american society for testing and materials (astm) f920 - 93., presente em cinco rms, abriu em baixas presses (< 17 cmh2o), e a presso mxima variou de 32,0 - 55,9 cmh2o. os valores mdios de vt utilizando uma mo foram inferiores ao sugerido pela astm (600 ml). o vt mdio, nas situaes estudadas, foi geralmente menor nos rms nacionais com vlvula limitadora de presso. as resistncias impostas pela vlvula do paciente esto de acordo com os critrios da astm, com exceo de um rm. as vlvulas limitadoras de presso dos rms nacionais geralmente abrem em baixas presses, determinando o fornecimento de um menor vt nas situaes estudadas, principalmente com o uso de uma mo, o que sugere que a ventilao deva ser feita com as duas mos e a vlvula limitadora de presso deva ser fechada sempre que possvel. so tambm conhecidos como ressuscitadores manuais ou " ambu " que uma marca registrada de reanimador e denominao utilizada como sinnimo de rm por profissionais no brasil e em outros pases. os rms so utilizados em utis para otimizar a oxigenao e facilitar a remoo de secreo em pacientes em ventilao mecnica invasiva, para transporte intra - hospitalar, e como forma de ventilao temporria em pacientes intubados ou no. os rms podem ser classificados, de acordo com sua forma de enchimento, em autoinflveis ou inflveis por fluxo, sendo que o primeiro o tipo mais utilizado nas utis brasileiras. o rm autoinflvel, que o tipo que foi avaliado no presente estudo, composto de um balo compressvel e autoinflvel, uma vlvula para o reenchimento do balo e uma vlvula inspiratria / expiratria (vlvula do paciente ; figura 1). quando o balo comprimido pelo operador, a vlvula do paciente oclui a porta expiratria e permite a passagem do ar no interior do balo para o paciente ; quando cessa a presso no interior do balo a vlvula retorna a sua posio normal, ocluindo a comunicao do balo com o paciente e permitindo a exalao do ar que vem do paciente pela porta expiratria. a queda de presso no interior do balo durante a expirao determina a abertura da vlvula de reenchimento, permitindo a entrada de ar para o balo. componentes adicionais desse tipo de rm incluem um dispositivo para enriquecer a mistura de oxignio, uma vlvula de positive end - expiratory pressure (peep, presso expiratria final positiva) e uma vlvula limitadora de presso (vlp) cujo uso, para pacientes adultos, no recomendado pela american society for testing and materials (astm). a figura a ilustra todos os componentes do reanimador, e a figura b ilustra apenas o conector do paciente, com destaque do posicionamento da vlvula do paciente durante a inspirao e expirao. a : conexo para o reservatrio de oxignio com vlvula de reenchimento ; b : bolsa inflvel ; c : vlvula do paciente ; d : vlvula limitadora de presso (atua somente na inspirao) ; e : porta exalatria ; e f : conexo para o paciente. na literatura, encontram - se estudos que avaliaram o desempenho e a segurana de rms disponveis em outros pases de acordo com quesitos exigidos por normas tcnicas internacionais. a realizao de estudos comparativos permite identificar problemas e aperfeioar esses dispositivos. nosso objetivo, no presente estudo, foi avaliar as caractersticas e o desempenho de alguns dos rms autoinflveis, para uso em pacientes adultos, disponveis no mercado nacional, seguindo alguns quesitos estabelecidos por uma norma internacional. o estudo foi aprovado pelo comit de tica em pesquisa do hospital das clnicas da faculdade de medicina da universidade de so paulo e realizado no laboratrio experimental de ventilao mecnica da disciplina de pneumologia da mesma instituio em 2006. moriya, so paulo ; oxigel, so paulo ; protec equipamentos mdico - hospitalares, so paulo ; rwr equipamentos hospitalares, so bernardo do campo ; e unitec ind. e com. de aparelhos hospitalares ltda., so paulo), dois na dinamarca (ambu silicone resuscitator e ambuspur ; ambu a / s, ballerup, dinamarca) e um nos eua (hudson rcilifesaver ; hudson rci, durham, nc, eua). os rms testados foram emprestados pelos fabricantes ou representantes nacionais. todos os modelos ainda so comercializados, e as atualizaes realizadas nos equipamentos desde a realizao do presente estudo at a sua redao esto descritas na tabela 1. tabela 1 caractersticas dos reanimadores manuais para adultos descritas pelos fabricantes.nd : informao no disponvel. do ano de 2006 at o momento da redao do presente estudo, os aparelhos 1, 2, 3, 4 e 7 no sofreram alteraes significativas. no aparelho 5, a conexo atual para o paciente do tipo swivel. no aparelho 8, a capacidade do balo aumentou de 1.000 para 1.500 ml. nd : informao no disponvel. obs. : do ano de 2006 at o momento da redao do presente estudo, os aparelhos 1, 2, 3, 4 e 7 no sofreram alteraes significativas. no aparelho 5, a conexo atual para o paciente do tipo swivel. no aparelho 8, a capacidade do balo aumentou de 1.000 para 1.500 ml. como no existe uma norma brasileira definindo as caractersticas para fabricao e comercializao de rms, para a presente avaliao, foram usados critrios da astm, designation f920 - 93, equivalente aos da international organization for standardization 8382, utilizada em outros pases. os seguintes critrios foram avaliados : a resistncia imposta pela vlvula do paciente durante as fases inspiratria e expiratria ; o funcionamento da vlvula limitadora de presso (vlp) ; e volume corrente (vt) entregue pelo rm em um simulador do sistema respiratrio. de acordo com os critrios da astm, os itens avaliados devem obedecer as seguintes especificaes : queda de presso inspiratria e expiratria < 5 cmh2o na presena de um fluxo de 50 l / min, determinando uma resistncia < 6 cmh2o. s ; a utilizao de vlp no obrigatria para rm adulto, mas, se disponvel, deve poder ser fechada ; oferta de vt 600 ml para pacientes adultos (peso 40 kg) com resistncia de 20 cmh2o. s e complacncia de 0,02 l / cmh2o com o operador usando somente uma mo (tamanho mximo da mo de 185 mm). o sistema de medidas foi constitudo por um tubo plstico onde foram adaptados os sensores de presso e de fluxo (figura 2). os sensores tiveram seus sinais amplificados e armazenados em um microcomputador para posterior anlise em um aplicativo desenvolvido no software labview (national instruments, tx, eua). a frequncia de amostragem do sinal foi de 200 hz. figura 2 representao esquemtica do sistema experimental. 1 : sensor de fluxo proximal ou distal ; 2 : sensor de presso ; 3 : resistncia ; e 4 : sensor de presso alveolar. para as medidas da resistncia inspiratria e expiratria, foi conectado um ventilador mecnico (bear 5 ; bear medical systems, riverside, ca, eua) a uma das extremidades do tubo plstico com os sensores (figura 2). o ventilador foi ajustado em modo volume controlado, fluxo inspiratrio constante de 60 l / min e peep de zero cmh2o. para a resistncia expiratria, o conector do paciente do rm foi adaptado outra extremidade do tubo plstico, e o ar do ventilador saia pela porta expiratria para o ar ambiente (figura 2). na avaliao da resistncia inspiratria, o rm foi desmontado e apenas a poro proximal do conjunto, sem o balo, foi utilizada (figura 2). a parte do rm que se conecta ao balo foi adaptada outra extremidade do tubo plstico, e o ar saia pelo conector do paciente para o ar ambiente. no foi feito o teste com o rm modelo ambu silicone resuscitator porque o aparelho no pde ser desmontado. as duas resistncias foram calculadas pela diferena entre a presso proximal e distal vlvula (a presso distal no foi medida, pois era igual a zero, j que o sistema estava aberto para o ar ambiente), dividida pelo fluxo medido. para o teste do vt, os rms foram conectados a um tubo plstico com os sensores, e esse tubo a uma cnula traqueal de dimetro 8,5 mm (rusch, montevidu, uruguai), cuja poro distal, com o balonete insuflado, era adaptada no interior de outro tubo plstico que se conectava a um simulador do sistema respiratrio (ttl 2600 ; michigan instruments, grands rapids, mi, eua ; figura 2). o simulador permitia o ajuste da complacncia dos foles atravs de molas ; para o ajuste da resistncia, foram utilizados resistores no lineares. quatro voluntrios, fisioterapeutas de uti da cidade de so paulo, sendo dois homens e duas mulheres, operaram os rms. o tamanho da mo dominante (do punho at a ponta do terceiro dedo) desses profissionais era de 200 mm para o homem 1, 188 mm para o homem 2, 175 mm para a mulher 1 e 160 mm para mulher 2. os fisioterapeutas foram orientados a insuflar o modelo como se estivessem ventilando um paciente, primeiro com uma e depois com as duas mos, com os oito rms, que foram avaliados em sequncia aleatria, em duas situaes de resistncia e complacncia : 1) condio " astm " - complacncia de 0,02 l / cmh2o e resistncia de 20 cmh2o. s, de acordo com a norma da astm ; e 2) condio " normal " - complacncia de 0,05 l / cmh2o e resistncia de 5 cmh2o. para o clculo do vt, foram analisados trs ciclos respiratrios, aleatoriamente escolhidos, entre os ciclos gravados (mnimo de 10 por condio teste). como cada voluntrio (total de quatro) manipulou oito rms, com uma e com duas mos, e em dois cenrios clnicos distintos (condio astm e normal), foram realizadas 128 condies teste. dos oito rms, cinco estavam equipados com vlp e foram submetidos a esse teste. o ventilador, conectado em uma das extremidades do sistema de medidas (com sensor de fluxo e presso), foi ajustado no modo volume controlado, com fluxo inspiratrio constante de 20 l / min, vt de 1.800 ml e peep de zero cmh2o. na outra extremidade, foi conectada uma parte do rm e, na sequncia, outro sensor de fluxo (distal) e o simulador (figura 2). foi avaliada a presso na qual a vlp se abria e permitia o vazamento de ar (momento que podia ser identificado quando o fluxo no sensor distal caia em relao ao proximal), a qual foi denominada " presso de abertura " e em qual valor de presso todo ar enviado pelo ventilador passava apenas pela vlp (momento no qual o fluxo distal era igual zero), que foi denominada " presso mxima ". o funcionamento da vlvula foi considerado adequado se a presso de abertura ocorresse no intervalo de presso sugerido pelos fabricantes, em geral, valores maiores do que 35 - 40 cmh2o (tabela 1). para cada rm, somente um valor de resistncia inspiratria e expiratria foi mostrado, pois a variao era desprezvel entre os ciclos. os valores de vt foram mostrados como mdia e desvio - padro e avaliados atravs de uma anlise multivariada de regresso linear mista (mixed model) para cada uma das quatro condies estudadas : vt com uma mo nas condies astm e normal e vt com duas mos nas mesmas duas condies. foi realizada uma anlise post hoc com correo de sidak para a comparao dos valores de vt entre os fisioterapeutas e entre os rms. para essa anlise, foi usado o pacote estatstico statistical package for the social sciences, verso 17 para windows (spss inc., em relao ao tipo da vlvula do paciente, predominou o tipo diafragma e, em relao ao material do balo, o silicone. a capacidade do balo de cada rm era varivel (1.000 - 2.500 ml). os trs rms importados no dispunham de vlp, presente nos cinco equipamentos nacionais, sendo que, no da marca rwr, a vlp podia ser fechada sem que ocorresse vazamento. outra diferena observada entre os rms importados e nacionais foi o fato de que o conector do paciente era fixo nos cinco reanimadores brasileiros, enquanto, nos importados, esse era do tipo " swivel 360 ". na tabela 2, esto descritos os resultados dos testes que avaliaram as resistncias da vlvula do paciente, durante a inspirao e expirao, e o funcionamento da vlp. apenas a resistncia expiratria do rm da marca protec excedeu o limite estabelecido pelos critrios da astm (< 6 cmh2o. s. as demais resistncias dos rms testados estavam de acordo com as normas, sendo o maior valor de resistncia inspiratria encontrado no rm da oxigel (5,69 cmh2o. s. a presso da abertura da vlp (presente em cinco dos rms) foi baixa (< 17 cmh2o), e o valor de sua presso mxima variou de 32,0 - 55,9 cmh2o. tabela 2 resistncias inspiratria e expiratria da vlvula para o paciente e avaliao da vlvula limitadora de presso. o valor mdio de vt ofertado pelos rms (tabela 3) e pelos fisioterapeutas (tabela e1, disponvel no site do jornal brasileiro de pneumologia, http://www.jornaldepneumologia.com.br/detalhe_artigo.asp?id=2212http://www.jornaldepneumologia.com.br/detalhe_artigo.asp?id=2212) foi varivel. nas situaes avaliadas (manipulao dos oito rms, com uma e duas mos, e em dois cenrios clnicos distintos, condio astm e normal), houve diferenas significantes (p < 0,001) tanto entre os rms, quanto entre os fisioterapeutas. todos os rms, sempre que manipulados com uma mo, ofertaram um vt inferior ao valor preconizado pela norma americana (600 ml, na condio astm). apenas os dois modelos do fabricante ambu a / s ofertaram vt 600 ml, utilizando somente uma mo ; porm, somente durante a simulao da condio clnica normal. mesmo manipulando o rm com as duas mos, os rms dos fabricantes oxigel e rwr, na condio clnica astm, no ofertaram o vt mnimo, enquanto que os dos fabricantes protec e unitec, independentemente do cenrio clnico simulado, ofertaram vt < 600 ml. tabela 3 volume corrente (vt) na situao normal e de acordo com os critrios da american society of testing materials (astm).aavalores expressos em mdia dp, a partir de 3 medidas, em mlbcomplacncia = 0,02 l / cmh2o ; e resistncia = 20 cmh2o. l. s ccomplacncia = 0,05 l / cmh2o ; e resistncia = 5 cmh2o. s. p < 0,05 em relao aos equipamentos sem vlvulaambuambu s ; ehudson / rci valores expressos em mdia dp, a partir de 3 medidas, em ml complacncia = 0,02 l / cmh2o ; e resistncia = 20 cmh2o. l. s complacncia = 0,05 l / cmh2o ; e resistncia = 5 cmh2o. s. p < 0,05 em relao aos equipamentos sem vlvula ainda, observando os resultados expressos na tabela 3, possvel verificar que os valores de vt na situao clnica normal foram, no geral, maiores do que na condio astm, que simula uma condio pulmonar com menor complacncia e maior resistncia, e que a maioria dos vt obtidos com os rms sem vlp foram superiores em relao aos obtidos com os rms com vlp. os principais resultados do presente estudo podem ser resumidos nos seguintes itens : 1) as resistncias (inspiratria e expiratria) da vlvula do paciente obedeceram aos padres estabelecidos pela norma americana, com exceo de um rm ; 2) os oito rms avaliados no ofertaram o vt mnimo em condies padronizadas pela mesma norma (vt 600 ml, com uma mo, na condio clnica astm) ; 3) manipular o rm com as duas mos no resolveu o problema em quatro rms (o vt permaneceu < 600 ml) ; e 4) a vlp presente nos rms nacionais determinou, no geral, o fornecimento de um vt menor em relao aos rms sem vlp. no brasil, existem poucos estudos publicados avaliando rms para pacientes adultos, comumente utilizados em utis brasileiras. embora esses estudos tenham investigado caractersticas importantes, como, por exemplo, a frao de oxignio ofertada pelos rms com e sem o reservatrio de oxignio, o presente estudo avaliou outras propriedades igualmente importantes que ainda no haviam sido relatadas na literatura nacional. em relao avaliao da resistncia da vlvula do paciente na inspirao e na expirao, todos os rms testados estavam dentro das especificaes da astm, com exceo do rm do fabricante protec, que apresentou resistncia expiratria de 7,91 cmh2o. s. esse resultado no surpreende, uma vez que outros estudos internacionais j haviam descrito equipamentos com resistncias da vlvula do paciente superiores ao recomendado. importante ressaltar que quanto maior a resistncia expiratria, mais prolongada se torna a expirao, o que pode provocar aprisionamento de ar (auto - peep) e risco de barotrauma, principalmente quando o rm manipulado com alta frequncia respiratria e alto vt. para o teste do vt com uma e duas mos, foram escolhidos voluntrios de ambos os sexos, pois se sabe que o vt obtido varia com o sexo do operador (vt menor nos operadores do sexo feminino). houve grande variabilidade entre as mdias dos vt dentre os rms testados, o que j havia sido observado em outros estudos. observamos que alguns rms nacionais ofertaram um vt baixo mesmo com uso das duas mos, que determina maior vt do que o uso de somente uma mo. outros autores j haviam relatado, mesmo em simulaes com complacncia normal, valores de vt menores do que 600 ml., que avaliou 16 rms descartveis fabricados no exterior, apenas trs rms no ofertaram o vt mnimo preconizado pela astm (600 ml). possivelmente, vrios fatores influenciaram os resultados referentes ao vt. um fator que parece ter determinado valores elevados de vt foi o tamanho das mos de um dos fisioterapeutas (200 mm, medida superior recomendada pelas normas, homem 1, tabela e1, disponvel no site do jornal brasileiro de pneumologia, http://www.jornaldepneumologia.com.br/detalhe_artigo.asp?id=2212). essa associao entre o vt entregue e o tamanho das mos j foi descrita por outros autores. fatores que poderiam determinar volumes baixos so o tamanho e o material do balo do reanimador testado. o volume de ar no balo no totalmente insuflado para o paciente quando ele comprimido, sendo que, genericamente, o volume do balo deve ser duas vezes maior que o volume que se quer insuflar. um balo de material pouco complacente (como o polivinil) pode determinar um vt menor. no nosso estudo, o rm que ofertou os menores vt (unitec) tinha o balo com o menor volume e que era, ainda, constitudo de polivinil. importante ressaltar que o vt dos rms nacionais foi, no geral, menor do que o dos importados, com exceo do rm do fabricante rwr, que apresentou vt maiores, provavelmente porque a capacidade do balo era de 2.500 ml. poderia ser questionado se o baixo vt encontrado para alguns rms ocorreu devido fadiga dos voluntrios. no entanto, no estudo de hess., que avaliou o efeito da fadiga determinada por uma ventilao com rm por um perodo de 30 minutos em uma situao de baixa complacncia (20 ml / cmh2o), no foi observada uma reduo do vt aps 30 minutos para os 14 voluntrios avaliados. considerando que, em nosso estudo, o tempo total de teste para cada um dos rm foi de cerca de um minuto, o que determinaria um total de oito minutos para cada uma das quatro situaes avaliadas (duas situaes de impedncia com uma ou com duas mos) e que ainda havia um perodo de descanso entre cada medida, parece pouco provvel que a ocorrncia de fadiga tenha influenciado os resultados. como esperado, o vt obtido na simulao de um paciente " normal " foi superior ao encontrado nos testes com os ajustes de complacncia e resistncia padronizados pela norma americana, que simulavam um paciente com baixa complacncia e alta resistncia, que comprovadamente determinam um menor vt. todos rms brasileiros tinham a vlp e, em somente um deles, a vlvula podia ser fechada. a abertura inicial das vlvulas de todos os dispositivos ocorreu em presses reduzidas, com valores abaixo do especificado, determinando vazamento durante a insuflao, o que pode explicar, em parte, o menor vt obtido com os rms com vlp. esse resultado pode ser considerado uma falha grave, pois pode impedir a adequada ventilao de pacientes com complacncia pulmonar baixa ou com resistncia de vias areas elevada, como, por exemplo, no caso de pacientes com edema pulmonar e crise asmtica, respectivamente. segundo alguns autores, a presena da vlp no traz segurana adicional para o paciente adulto, j que o risco de barotrauma no est associado somente s presses de pico elevadas durante a inspirao. inclusive a norma americana dispensa o uso do dispositivo regulador de presso para os rms para adultos e, se presente, determina que a vlvula possa ser travada e que o seu modo de fechamento deva ser bem evidente para o operador. mesmo no caso dos rms para pacientes da faixa neonatal / peditrica, para os quais recomendada a presena da vlvula para limitar a presso em valores inferiores a 40 5 cmh2o, o funcionamento inadequado da vlp tambm pode comprometer a ventilao. so consideradas limitaes do nosso estudo a realizao de todos os testes com apenas uma unidade de cada rm (porm, todos os rms eram novos, sem uso prvio) ; o teste da resistncia da vlvula do paciente foi realizado com fluxo de 60 l / min, em vez de 50 l / min, valor esse preconizado pela astm, o que pode dificultar a comparao com outros estudos ; e, como o presente estudo foi realizado em 2006, alguns modelos de rm sofreram alteraes (tabela 1), o que pode ter modificado seus desempenhos. tal fato provavelmente deve ser verdadeiro para o rm do fabricante unitec, que teve a capacidade do balo aumentada significativamente (tabela 1). apesar das limitaes do estudo, acreditamos que so apontadas deficincias importantes dos rms fabricados no brasil, principalmente referentes ao funcionamento da vlp (a maioria no pde ser travada e apresentou abertura precoce), que, em algumas situaes clnicas, podem comprometer a ventilao de pacientes crticos. os resultados apresentados indicam a necessidade de que, para se ofertar um vt adequado, as duas mos devem comprimir o rm e que, quando o rm permitir, a vlp deve ser mantida fechada. acreditamos que os resultados obtidos no presente estudo podero auxiliar na realizao de futuros testes e no aprimoramento dos rms nacionais. | objective : to evaluate the performance of manual resuscitators (mrs) used in brazil in accordance with international standards. methods : using a respiratory system simulator, four volunteer physiotherapists employed eight mrs (five produced in brazil and three produced abroad), which were tested for inspiratory and expiratory resistance of the patient valve ; functioning of the pressure - limiting valve ; and tidal volume (vt) generated when the one - handed and two - handed techniques were used. the tests were performed and analyzed in accordance with the american society for testing and materials (astm) f920 - 93 criteria. results : expiratory resistance was greater than 6 cmh2o. l1. s1 in only one mr. the pressure - limiting valve, a feature of five of the mrs, opened at low pressures (< 17 cmh2o), and the maximal pressure was 32.0 - 55.9 cmh2o. mean vt varied greatly among the mrs tested. the mean vt values generated with the one - handed technique were lower than the 600 ml recommended by the astm. in the situations studied, mean vt was generally lower from the brazilian - made mrs that had a pressure - limiting valve. conclusions : the resistances imposed by the patient valve met the astm criteria in all but one of the mrs tested. the pressure - limiting valves of the brazilian - made mrs usually opened at low pressures, providing lower vt values in the situations studied, especially when the one - handed technique was used, suggesting that both hands should be used and that the pressure - limiting valve should be closed whenever possible. |
his smoking history was 20 pack - years, and there was no specific past medical history. the patient s review of symptoms was normal except for hoarseness, and there were no abnormal findings on the physical examination, laboratory data, or chest radiography. no abnormal findings were detected on the vocal cords that could cause hoarseness, such as nodules or palsy, on the laryngoscope. the patient underwent a computed tomography (ct) scan, and 3-dimensional (3d) chest ct revealed multiple air - filled lesions in the right paratracheal region (fig. esophagography was performed to confirm whether there was communication between the esophagus and paratracheal air cyst. there were no orifices in the tracheal wall that suggested a connection between the trachea and the paratracheal air cyst on bronchoscopy. we inserted a video - assisted mediastinoscope into the anterior mediastinum following the crease of a 5.08-cms uprasternal incision and dissected along the anterior surface of the trachea. there were two large, fluid - filled cystic masses on the right side of the trachea, and one mass was compressing the right recurrent laryngeal nerve. we dissected these masses with the assistance of the video mediastinoscope and retrieved the upper pole of the cystic masses through the incision. there was one narrow communication to the right posterior side of the trachea at the thoracic inlet level (fig. one of the two masses consisted of a thick - walled multilocular cyst, measuring 2.52 cm in size. the other cystic mass also was same the mass, measuring 2.51.7 cm in size. histologic findings showed cysts lined by respiratory ciliated columnar epithelium, suggesting tracheal diverticulum (fig. the patient s voice had made a full recovery when he visited the outpatient department for his 1 weeks later after discharge. the term paratracheal air cyst is a relatively nonspecific term for a paratracheal air collection. the differential diagnosis of such collections includes tracheal diverticulum, laryngocele, pharyngocele, zenker s diverticulum, apical hernia of the lung, and apical paraseptal blebs / bullae. tracheal diverticulum is considered rare, and there is scant published literature for this entity. one study by goo. examined 65 patients with ct evidence of a paratracheal cyst without pathologic differentiation. nearly all of the cysts were located in the right paratracheal region with only one located in the left paratracheal region. the right - sided nature of the diverticulum may be because the esophagus generally lies to the left of the trachea at this level, leaving the right side unsupported. the transverse diameter of the cysts ranged from 5 to 20 mm on the axial scan, and the vertical length ranged from 5 to 25 mm in the coronal or sagittal view. a communicating channel between the air cyst and trachea was found in 5 patients (8%). a total of 31% of the cysts had irregular wall thickening. on respiratory dynamic ct a change in the size of the paratracheal air cyst during respiration is indicative of communication between the cyst and the airway. although it was not pathologically proven, the authors felt that the cysts likely represented tracheal diverticulum. other causes of paratracheal air collection include laryngoceles, which can be seen on ct as diverticulum of the saccule of the laryngeal ventricle. an apical hernia has continuity with the body of the lung and has lung markings within the herniated lung on ct scans. apical paraseptal blebs or bullae, which are air cysts within the lung, can easily be recognized on ct scans. our patient s paratracheal air cyst had no lung markings, and there was no connection to the lungs or esophagus. specifically, tracheal diverticulum is characterized by single or multiple invaginations of the tracheal wall. they are most frequently found incidentally in postmortem examinations, reported in 1% of patients at autopsy. the congenital variety is thought to represent vestigial supernumerary lungs or aborted abnormally high divisions of the primary lung bud. they may arise 4 to 5 cm below the true vocal cords at the right tracheal side or a few cm above the carina. they are relatively small and narrow - mouthed and may occur separately or accompany other congenital anomalies within the tracheobronchial tree, such as tracheoesophageal fistula. the wall of congenital tracheal diverticulum is similar to the actual tracheal wall, containing smooth muscle fibers, cartilage, and respiratory epithelium. the acquired variety is thought to represent an outbulging at a weak spot in the posterior tracheal wall as a result of increased intraluminal pressure, as with a chronic cough [1,2, 4 ]. in the study by goo., patients with paratracheal air cysts had pulmonary function abnormalities that indicated an obstructive pattern more often than the control group, suggesting a possible association of tracheal diverticuli with emphysema. acquired tracheal diverticulum can arise at any level, and they are typically wide - mouthed and larger than congenital diverticulum. histologically, they are lined by respiratory epithelium ; however, no smooth muscle or cartilage is found in the wall. it is an air - filled tubular structure, most often found posterior and slightly to the right of the trachea and communicating with the trachea. cartilaginous rings within the wall of the diverticulum suggest a congenital form, whereas the absence of cartilaginous rings may suggest an acquired form. ct may also reveal whether the neck of the diverticulum is small or large and whether its wall is thickened as a result of repeated inflammation. when the neck of the diverticulum is very small, communication with the trachea may not be clearly visible on ct. patients with tracheal diverticulum are usually asymptomatic and tend to be incidentally detected ; however, tracheal diverticulum can act as a cavity filled with secretions and so may present clinically with chronic cough, dyspnea, stridor, and repeated episodes of respiratory infection. rarely, tracheal diverticulum can present with symptoms resulting from direct compression of the adjacent tissue or organs. treatment options in tracheal diverticulum include surgical resection in young or symptomatic patients and conservative medical treatment with antibiotics, mucolytics, bronchodilators, and physiotherapy in the elderly and debilitated. in our case, because the patient had hoarseness owing to the mass effect of the paratracheal air cyst and was young, surgical treatment was the appropriate decision. in this case, the patient presented with hoarseness owing to the mass effect of paratracheal diverticulum. during the work - up, there were no significant findings or causes of hoarseness on the laryngoscope. findings on the chest ct scan and 3d reconstruction demonstrated paratracheal air cyst with a channel between the cysts and the trachea. air collection located at the right posterior side of the trachea at the thoracic inlet with fistula was a specific finding that suggested tracheal diverticulum. the disease is rare, but it was not difficult to diagnose. because the patient had a symptom and was young and healthy enough to undergo general anesthesia and surgery, the air cysts were entirely removed via video - assisted mediastinoscopy. reported methods of approach were thoracotomy or video - assisted thoracoscope, but in a small number of cases, resection of the tracheal diverticulum was performed through video - assisted mediastinoscopy in the literature. in our case, most tracheal diverticula are located in the thoracic inlet, and therefore, an approach via mediastinoscopy is possible. the air cysts were confirmed as tracheal diverticula on the histologic examination. immediately after the operation, the hoarseness disappeared. in conclusion, tracheal diverticulum is a rare disease that presents as an outpouching of the tracheal wall. most patients with tracheal diverticulum are asymptomatic but can present with respiratory symptoms, repeated respiratory infection or a mass effect. through chest ct, the physician can obtain a great deal of information such as location, size, air cyst wall thickness, and association with adjacent tissue. depending on the findings, it may be possible to diagnose paratracheal air cyst without histologic examination. if the air cysts do not cause problems, asymptomatic, elderly, or debilitated patients can be treated with conservative management depending on symptoms. however, if symptoms result from direct compression, surgical resection may be an immediate response to the symptoms. | paratracheal air cysts are a rare entity in which cystic formation occurs adjacent to the trachea. most patients with paratracheal air cysts are asymptomatic, and the cysts are detected incidentally on chest radiograph or computed tomography (ct) scan. most symptomatic patients complain of pulmonary symptoms or repeated respiratory infection. rarely, the air cysts can lead to paralysis of the recurrent laryngeal nerve as a result of direct compression. we report a case of a 59-year - old male patient who presented with voice change, and the cause was identified as paratracheal air cysts on a chest ct scan. surgical resection via video - assisted mediastinoscopy was performed, and the voice recovered immediately after the operation. |
its ease of use and patient tolerance has made it popular among gastroenterologists and patients alike. currently, capsule endoscopy is most commonly used to evaluate obscure gastrointestinal bleeding, but its indications are broadening. various reports have described the capsule becoming impacted or retained in the small bowel, requiring retrieval. a 74-year - old female had a history of unidentified gastrointestinal bleeding and underwent a capsule endoscopy. the patient was not able to spontaneously pass the capsule and had intermittent obstructive symptoms. her past medical history was significant for severe copd, and she had undergone hysterectomy and appendectomy in the past. the gastroenterologist performed a colonoscopy, and the capsule was visualized in the terminal ileum. attempts at endoscopic removal of the capsule were not successful. therefore, she was seen in the surgery clinic for removal of the capsule. a decision was made to perform a laparoscopic exploration to evaluate the entire small bowel and colon to identify any associated pathology. just before the procedure, an abdominal x - ray was taken, confirming the presence of the retained capsule in the right lower quadrant (figure 1). after proper informed consent was obtained, the patient was taken to the operating room. laparoscopic instruments were placed through 4 trocars. the patient was found to have adhesions from her previous hysterectomy, which were divided without difficulty. intraoperative examination of the small bowel revealed a stricture in the terminal ileum just proximal to the ileocecal valve with a bulge consistent with the retained capsule (figure 2). the mesenteric vessels were controlled with the ligasure device (us surgical corporation, norwalk, ct) and 2.0-mm vascular loads of the endo - gia stapler (us surgical corporation, norwalk, ct). the colon was transected distal to the cecum, and then delivered through a 4-cm infraumbilical incision. careful inspection revealed the capsule in the terminal ileum at an area that was significantly strictured. the small bowel was transected proximal to the strictured area, and the specimen was sent to pathology. next, a side - to - side anastomosis was formed between the ileum and the colon with a 3.5-mm endo - gia stapler and the incision was closed. she was advanced to sips of clear liquid on the evening of postoperative day 1. she was tolerating oral intake, but on postoperative day 4 she developed abdominal distension consistent with an ileus. on postoperative day 5, she had passage of flatus and was restarted on her clear liquid diet. by postoperative day 7, she was tolerating her diet well with no nausea or vomiting and was discharged home. she was seen several weeks later in the surgery clinic and was doing well without any complications. final pathology demonstrated no evidence of inflammatory bowel disease or malignancy. despite the fact that no pathologic bleeding site was found, the patient has had no further bleeding or obstructive symptoms at 1-year follow - up. after proper informed consent was obtained, the patient was taken to the operating room. laparoscopic instruments were placed through 4 trocars. the patient was found to have adhesions from her previous hysterectomy, which were divided without difficulty. intraoperative examination of the small bowel revealed a stricture in the terminal ileum just proximal to the ileocecal valve with a bulge consistent with the retained capsule (figure 2). the bowel was extensively evaluated, and no other abnormalities were found. an ileocectomy was chosen to remove both the stricture and the retained video capsule. the mesenteric vessels were controlled with the ligasure device (us surgical corporation, norwalk, ct) and 2.0-mm vascular loads of the endo - gia stapler (us surgical corporation, norwalk, ct). the colon was transected distal to the cecum, and then delivered through a 4-cm infraumbilical incision. careful inspection revealed the capsule in the terminal ileum at an area that was significantly strictured. the small bowel was transected proximal to the strictured area, and the specimen was sent to pathology. next, a side - to - side anastomosis was formed between the ileum and the colon with a 3.5-mm endo - gia stapler and the incision was closed. the patient tolerated the procedure well. she was advanced to sips of clear liquid on the evening of postoperative day 1. she was tolerating oral intake, but on postoperative day 4 she developed abdominal distension consistent with an ileus. on postoperative day 5, she had passage of flatus and was restarted on her clear liquid diet. by postoperative day 7, she was tolerating her diet well with no nausea or vomiting and was discharged home. she was seen several weeks later in the surgery clinic and was doing well without any complications. final pathology demonstrated no evidence of inflammatory bowel disease or malignancy. despite the fact that no pathologic bleeding site was found, the patient has had no further bleeding or obstructive symptoms at 1-year follow - up. since its introduction several years ago, capsule endoscopy has rapidly become an important diagnostic modality in the evaluation of several gastrointestinal conditions. the ease of administration and the ability to visualize the entire small bowel are two of the main advantages of capsule endoscopy. as patients request less invasive approaches to their medical care, primary care physicians and surgeons alike will be increasingly exposed to capsule endoscopy. video capsule endoscopy is most commonly used to detect obscure gastrointestinal bleeding not located by endoscopy. the role of video capsule endoscopy has expanded to inflammatory bowel disease, small - bowel neoplasms, malabsorption disorders, iatrogenic disease (nsaid strictures), radiation enteritis, clarification of previous imaging, and chronic abdominal pain. capsule endoscopy has been contraindicated in patients with known small - bowel obstruction, strictures, extensive crohn 's disease, swallowing disorders, pseudo obstruction, motility disorders, cardiac pacemakers, and defibrillators. relative contraindications include pregnancy, chronic nsaid use, extensive diverticular disease, gastroparesis, and previous pelvic or abdominal surgeries. recently, capsule endoscopy has been reported for use in the evaluation for recurrent small - bowel obstruction and strictures. in one study, 19 patients with suspected small - bowel obstruction underwent capsule endoscopy. a definitive diagnosis was made in 32% of the cases. in 3 cases, the capsule was retained and laparotomy was required, but in no instance was the capsule the cause of an acute small - bowel obstruction. although controversial, this provides an example of the potential for expansion of this new technology. the retention rate appears to be between 0.75% to 3% but may be higher in patients with crohn 's disease. if the capsule has not passed after 7 days, then a plain film is obtained. endoscopic retrieval and extraction by push - and - pull enteros - copy with the double - balloon technique have been used with success. as presented in this case, surgical retrieval in fact, it is widely felt that a retained capsule is diagnostic of a clinically relevant stricture or mass. we describe a safe laparoscopic retrieval of a capsule endoscopy retained in the terminal ileum secondary to scar tissue. a review of capsule endoscopy at 2 referral centers revealed 7 of 197 capsules retained. the surgical findings included nonspecific inflammatory strictures (twice), a radiation stricture, a crohn 's stricture, and an umbilical hernia. two cases of open surgical retrieval of an impacted capsule requiring stricturoplasty and a segmental resection have recently been reported in patients with known crohn 's disease. retention or nonpassage of the capsule endoscopy is an uncommon occurrence that is likely to become a more frequent surgical situation as the indications for the technology broaden. in the meantime, for example, many would recommend small bowel follow through or enteroclysis before capsule endoscopy in patients with known crohn 's disease or suspected strictures. in addition, a standard bowel preparation or even a dose of metoclopramide before capsule administration has been shown to increase the rate of complete small - bowel examinations. in the near future, dissolvable radio - opaque patency capsules may prove beneficial for patients with known small - bowel strictures. as in our case, patients with strictures in the small bowel or intraabdominal scar tissue may make passage of the capsule difficult to predict. using the fundamentals of safe laparoscopy and maintaining the principles of surgical management in specific disease processes | video capsule endoscopy is now a first - line tool in evaluating and diagnosing gastrointestinal bleeding, inflammatory bowel disease, and small bowel neoplasms. capsule nonpassage or retention is an uncommon but clinically significant occurrence. how to best retrieve these retained capsules is currently being debated. we report a laparoscopic approach for the retrieval of a retained capsule in the terminal ileum. |
since the 1950s, the global health community has focused on eradication campaign to eliminate malaria and malaria related death all over the world. but it failed globally because of some problems including the lack of innovative research and leaderships, the resistance of mosquitoes to insecticides used to kill them, the resistance of malaria parasites to drugs used to treat them, and implementation difficulties. the priority accorded to reductions of malaria mortality in developing countries is shown by its choice as the fourth millennium development goals (mdgs). much of recent malaria related studies have focused on infant mortality and young children 's and adult 's exposure to the disease and to some extent the impact on pregnant women, without classifying the malaria situation of other subgroups population. scanty studies have looked specifically at the effect of malaria on adult and old - age people and mobile group population. a lot of data have been taken from heath facilities at different levels that may be dominated by patterns of health services utilization rather than clearly representing malaria patterns within communities. some works have taken any forms of data which are perhaps available and built to generalize patterns of malaria burden using proper statistical modeling techniques and others have in - depth network members who maintain population surveillance in health and demographic surveillance system sites across africa and asia. despite these efforts and visibility, there was broad concern that malaria remains a major cause of death in asia and much of infectious morbidity and 90% of malaria mortality are considered to be in many parts of africa. although a lot of tools have been developed for malaria control in asia, it has been argued that the enormous disease burden due to malaria is grossly underestimated. in 2011, about 1.33 billion people or 75% of the region 's total population resided in areas that were at risk of malaria in asia. among them there were 2144849 confirmed malaria cases and 1819 malaria deaths reported by the national malaria control programmes in asia. malaria control and elimination in southeast asia reported that the 11 member countries in the who southeast asia region and among them 10 are endemic to malaria., there have been significant malaria control efforts in recent years but it is unclear what impact they have had and six countries, bhutan, democratic people 's republic of korea, indonesia, nepal, sri lanka, and thailand, are aiming for malaria elimination as a longer - term goal. sri lanka is already in the elimination phase and subnational malaria elimination is progressing well in indonesia and thailand. this paper provides estimated malaria prevalence and mortality by age and country for 2006 to 2011. in addition, it allows us to produce maps of all ages ' group mortality rate adjusted with total population. for the analysis subjects of this study we cover 10 countries out of 50 asian counties. these countries have the highest malaria prevalence rate and close geographical region and fought against malaria for several decades. in this paper, to address the objective, our main aim is to estimate malaria mortality rate and its trend in countries over the said time period. the main sources of the longitudinal data are the world health organization and un database. we utilize two sources (table 1) of data to create trend of falciparum malaria mortality children under 5 years, children greater than 5 years, and all ages group population over the period 20062011 in asian countries. however, since some of this data was missing, we also created a second task to determine malaria mortality in asian countries using the united nation database. if a country was missing a value for malaria mortality in the who data, in that case, we replace that value of malaria mortality with the one from the un data. the total population in million was collected from the world development indicator database. who and un database provide good quality data ; in previous studies many authors used multiple data sources that we also used in this study. the descriptive statistics are prevalence rate per 10,000 populations and death rate per 10,000 populations for all age. prevalence rate is calculated using the number of malaria reported cases divided by total population with multiplying 10,000 populations for each country and year. similarly, we also determined a death rate per 10,000 populations for all ages using the number of malaria attributed death divided by total population with multiplying 10,000 populations for each country and year. in view of the differential patterns of malaria mortality, we analyzed the data into three age groups : aged under five years, aged greater than five years, and all ages using stata software version 12.0 (statacorp lp, lakeway drive college station, texas, usa). to identify summary measures of malaria related death in each country we estimated mean and standard deviation with confidence interval at 95% level of significance. the confidence interval indicates that there is a 95% probability of malaria death that encompasses the true values mean of malaria mortality at different age groups. we also exported the maps to view the geostatistical relationship between death rate for all ages and total population within countries. during 20062011, the prevalence rate per 10,000 people for bangladesh was raised from 2.308 to 3.440, myanmar from 43.573 to 96.261, republic of korea from 2.684 to 3.140, and timor - leste from 373.566 to 167.866. again for bhutan it decreased from 27.687 to 2.628, india from 15.428 to 10.555, indonesia from 15.118 to 10.589, nepal from 1.563 to 0.751, sri lanka from 0.298 to 0.059, and thailand from 4.503 to 3.581 (table 2). compared to these major asian countries, myanmar had one of the highest prevalence rate of increase in malaria related to prevalence rate within this time period (table 2). the death rate per 10,000 people for india increased from 0.068 to 0.069 and for indonesia from 0.100 to 0.123. on the other hand death rate for bangladesh is decreased from 0.120 to 0.055, bhutan from 0.133 to 0.014, myanmar from 0.531 to 0.288, nepal from 0.024 to 0.005, sri lanka from 0.001 to 0.000, thailand from 0.023 to 0.012, and timor - leste from 2.642 to 0.935. the effective death rate change has been conducted in sri lanka and republic of korea within this time period in selected asian countries. the malaria reported cases steadily changed in india and notably changed in sri lanka (figure 1) and the number of malaria deaths was decreasing in both children aged greater than 5 and all ages but increasing in younger children aged less than 5 years (figure 2). the overall rate of malaria prevalence decreased from 486.7 in 2006 to 298.8 in 2011 and death rate also decreased from 3.6 in 2006 to 1.5 in 2011 (table 2) in asia. among ten asian countries over the time period, the prevalence rate per 10,000 populations is still notably present in republic of korea and sri lanka but these countries have zero deaths. figure 3 showed the trend of number of malaria deaths under the age of 5 with zero deaths in sri lanka and republic of korea and consistent decrease in nepal, thailand, and also timor - leste. on the other hand when we considered malaria death age greater than 5, different pictures showed that zero deaths in sri lanka and republic of korea and others countries have a decreased trend over the period (figure 4). between 2006 and 2011, the highest average malaria death rate in younger children aged less than 5 years of 6.867 (95% ci 4.7678.969), children aged greater than 5 years of 0.539 (95% ci 0.2860.793), and all ages of 1.639 (95% ci 0.9872.292) was in timor - leste and the lowest was in korea republic (table 3). similarly, the highest malaria death rate per 10,000 population in timor - leste was 0.935 and in republic of korea was zero (table 2, figure 5) in 2011. the number of children deaths due to malaria in asia has been steadily decreasing since 2009 (figure 2). contrary to this trend, the number of deaths due to malaria in asian individuals aged greater than 5 years accounts for a significant decrease (figure 2). the second highest average malaria death rate in younger children aged less than 5 years of 2.072 (95% ci 1.8852.259) was in myanmar and the third highest of 0.929 (95% ci 0.7331.125) was in indonesia (table 3). but the third highest average malaria death rate in children aged greater than 5 years of 0.048 (95% ci 0.0090.086) was in bhutan (table 3). when we looked at malaria death rate for the all age 's group people in table 3, the patterns that arose for the first, second, and third highest countries average malaria related deaths rate are timor - leste 1.639 (95% ci 0.9872.292), myanmar 0.397 (95% ci 0.3090.486), and indonesia 0.109 (95% ci12 0.0930.126). findings from analysis showed that, in the year 2011, on the basis of data pattern and a refined understanding about malaria mortality, malaria is the underlying cause of death for 13970 individuals, including 12150 children aged younger than 5 years and 1820 individuals aged 5 years or older. since 2006, we noted that substantial improvement has been made in the combat against malaria, with a 6% reduction for all ages of malaria deaths in asia, which was very low with global malaria death reduction rate. however, our findings also show that the substantial acceleration in the decreases of death for ages of 5 years or older is 60% especially in asia and provide hope that rapid reduction will be held in near future. the rate of increase of malaria death in children aged younger than 5 years is 18% since 2006. this rate of change in malaria mortality of children aged younger than 5 in asian countries has become greater with previous estimates and does not support ambitious aspirational goals. malaria mortality rate under 5 aged groups is still high compared to other aged groups in all most selected countries. malaria death of children younger than 5 years is a serious issue in asian countries, although a lot of initiatives have been taken to reduce malaria mortality. as new asian aims for malaria mortality are decreased, it will be necessary to take lessons from these insights but also begin future planning for the evolving and more effective malaria reduction programme. some achievement of random goals established without exact regard to the distribution of rates of change dominating at the time is a political form that confuses knowledge and acclaim for the substantial progress that has been made to reduce malaria mortality in the past decade. moreover, accelerated decreases in malaria mortality will be more likely if we strongly maintain commitment for malaria control (zero death) and increase investment for new strategies, and evidence from policy responses in the asian countries is widely and effectively circulated and implemented. our future aimed to identify which factors have impact to reduce malaria mortality rate in asian countries with included wide range of significant indicators. the malaria mortality rate is steadily decreasing ; moreover malaria is still now a major health hazard in asia. malaria prevalence rate in most of the selected asian countries is not decreasing sharply as we expected and more attention is needed in these countries to control new cases of malaria. our findings provided potential evidence that these countries ' death rate is not increasing compared to increased prevalence rate. our analysis refined that the children aged less than 5 were more affected by the malaria compared to older ones in asian countries. henceforth, children aged less than 5 are more vulnerable than others and scientific community also needs the highest priority to pay attention to this group of population. | background. to control the malaria mortality, the global and national communities have worked together and produced impressive results in the world. some of the asian counties ' malaria mortality rate is more compared to countries with high health facilities around the world. this paper 's main aim is to describe trend of malaria cases and mortality in 10 asian countries using the world health organization data. methods. malaria mortality data was collected systematically from who and un database for the period 20062011. we estimated malaria mortality by age and countries. we also explored the dynamic relationships among malaria death rate, total populations, and geographical region using a map. during 20062011, the average malaria death per 10,000 population of all ages was 0.239 (95% ci 0.104 to 0.373), of children aged less than 5 year 1.143 (0.598 to 1.687), and of age greater than 5 years 0.089 (0.043 to 0.137) in asian countries. malaria prevalence per 10,000 populations steadily decreased from 486.7 in 2006 to 298.9 in 2011. conclusion. the findings show that malaria mortality is higher for children aged less than 5 years compared with with adults selected in asian countries except sri lanka. |
depression has a high prevalence of symptoms related to social, physical, and neurophysiological factors.1 cognition is a function that is altered in depression and has received increasing interest.2 neurophysiological changes that occur in depressive states, such as prefrontal cortex and cingulate activation, are partly responsible for reduced attention, memory, and visuospatial capacities. depression also affects executive processing, causing difficulties in planning strategies and mental flexibility, which, in turn, lead to impaired motivational and decision - making functions.2 impaired cognition is thus intimately associated with the severity of disease and the impairment of daily activities during and after a depressive crisis, including during remission. in addition to the various treatments proposed for depression, physical exercise may have beneficial effects as an add - on therapy.3,4 however, activities that demand divided attention in daily life (e.g., walking) present an increased risk of falls and impaired cognition. combined concurrent tasks (dual - task interventions) have been studied in subjects with neuropsychiatric disorders and in normal subjects, although few data exist on depression in the elderly. studies have produced divergent results regarding other variables such as age and the presence of motor or neuropsychiatric disorders.5 - 8 some hypotheses and theories attempt to explain cognitive and motor performance for the dual - task paradigm. dietrich proposed the transient hypofrontality hypothesis,9 which suggests that the cognitive functions associated with the frontal areas are impaired during physical exercise because the brain prioritizes motor control and the maintenance of vital functions (e.g., blood pressure and temperature control). in contrast, the bottleneck theory assumes that dual tasks are processed by the same neural networks, resulting in impaired execution of the second task.10 indeed, bloem. noted that young adults adopt a posture first strategy, in which they automatically prioritize motor tasks rather than cognitive tasks.11 conversely, elderly subjects and neurological patients (alzheimer 's disease, parkinson 's disease, and stroke patients) focus on the cognitive task when performing dual - tasks, thereby increasing the risk of falls.8 an increasing body of data has shown that moderate physical exercise has preventive and therapeutic effects on several clinical and mental disorders,3,12 especially mild to moderate depression.4,13 although much is already known about the effects of routine exercise on cognitive function, the acute effect of exercise remains to be studied in depth. diverging results might be partly explained by factors such as the following : the time of assessment (during or after exercise) ; the type, intensity, and duration of the exercise ; and the type of cognitive task under study.14,15 studies with young subjects have shown that reaction time, attention, and working memory improve after one session of aerobic exercise.16,17 however, little is known about the effect of acute exercise in the elderly with or without psychiatric disorders. hoffman. observed that routine physical exercise did not improve the cognition of depressed elderly.18 another study examined the effect of acute exercise on the cognition of adults with depression19 and showed that patients improved their attention and inhibitory control immediately after an exercise session. to the best of our knowledge, however, there are no studies on combined cognitive and motor tasks in depressed elderly individuals. the present study aimed to assess cognitive performance during and after one session of physical exercise among elderly persons with major depression. we hypothesized that the most complex cognitive functions would worsen during and improve after moderate physical exercise. this study included a series of elderly subjects (n = 10) diagnosed according to the dsm - iv criteria20 with major depression with mild to moderate severity who had already been participating in a exercise program for at least six months. exclusion criteria consisted of illiteracy, any presentation of degenerative neurological dementia, history of stoke, or any limitation of gait. all patients were weighed and measured for height using a mechanic anthropometric scale (welmy, brazil). depressive mood was assessed using the brazilian validated version of the hamilton depression scale (hamd).21,22 the hamd evaluates the severity of 17 depressive symptoms with a range of zero to 49. patients with scores from seven to 13 are considered mildly depressed, from 14 to 18 moderately depressed, from 19 to 22 severely depressed, and more than 23 very severely depressed. cognition was assessed by a battery of tests including the mini mental state examination (mmse), the digit span test (dst)23 and the stroop color - word test (scw). the mmse brazilian validated version assesses global cognitive functions, with scores ranging from zero to 30.24,25 the dst assesses short - term memory, working memory, and attention. it consists of two subtests, one of which requires the patient to orally repeat a sequence of digits forward and another one that requires that the patient repeat a sequence backward. the scw is a test of selective attention26 in which patients are required to name the ink color in which incongruent color names are printed. the victoria version was used because of its short administration time (the approximate time required is 5 minutes) and its sensitivity to frontal lobe disorders. the victoria version uses three 21.514 cm cards presented in the following order : part d, part w, and part c. each card has six rows of four items. part d contains colored dots (red, green, blue, and yellow), and the task on this card is to name the colors as quickly as possible (congruence). part w has words printed in the following colors : red, green, blue, or yellow. the examinee must name the colored ink in which each word is printed as quickly as possible. in part c, the words red, green, blue and yellow are printed in a color not denoted by the word (e.g., the word red is printed in green ink, etc.), and the examinee must name the colored ink in which the word is printed as quickly as possible (incongruence). for each part, time, and number of errors a ratio index (c / d) of interference was used.27,28 the subjects were tested on two different occasions. in the first session, all cognitive tests were conducted, followed by a 30-minute walk on an electric treadmill (bh fitness - explorer pro action). after a 5-minute warm - up, subjects trained for 25 minutes at 65%-75% of the maximum heart rate estimated for their age (220-age).29 the cognitive tests were conducted again at the 20 minute of the training, immediately after the completion of the training, and again 15 minutes later. one month after this first trial, the cognitive tests were conducted again using the same schedule without submitting the subjects to any physical exercise. this project was approved by the ethics committee of the institute of psychiatry of the federal university of rio de janeiro. all subjects signed informed consent forms before any procedure was undertaken. descriptive data are shown as the mean and standard deviation (sd) or median and confidence intervals (ci). shapiro - wilk and levene tests were used to assess the normality and the homogeneity of variances, respectively. because the data were characteristically non - parametric, two analyses using friedman 's repeat measures were performed for the exercise and control sessions. this analysis compared the cognitive test results among the time points (before, during, immediately after, and 15 minutes after exercising or at the corresponding times during the control session). statistical package for social sciences (spss) version 15.0 all cognitive tests were conducted, followed by a 30-minute walk on an electric treadmill (bh fitness - explorer pro action). after a 5-minute warm - up, subjects trained for 25 minutes at 65%-75% of the maximum heart rate estimated for their age (220-age).29 the cognitive tests were conducted again at the 20 minute of the training, immediately after the completion of the training, and again 15 minutes later. one month after this first trial, the cognitive tests were conducted again using the same schedule without submitting the subjects to any physical exercise. this project was approved by the ethics committee of the institute of psychiatry of the federal university of rio de janeiro. all subjects signed informed consent forms before any procedure was undertaken. descriptive data are shown as the mean and standard deviation (sd) or median and confidence intervals (ci). shapiro - wilk and levene tests were used to assess the normality and the homogeneity of variances, respectively. because the data were characteristically non - parametric, two analyses using friedman 's repeat measures were performed for the exercise and control sessions. this analysis compared the cognitive test results among the time points (before, during, immediately after, and 15 minutes after exercising or at the corresponding times during the control session). statistical package for social sciences (spss) version 15.0 was used, and the level of significance was p<0.05. the patients had a mean age of 71.5 (6.0) years and a mean hamd score of 14.1 (3.8). exercise session : there were no statistically significant differences among the time points with regard to the results of the dst forward and backward (p = 0.692, x = 1.456 ; p = 0.569, x = 2.016, respectively) (figure 1). the scw results were statistically significantly different among the time points for both the congruent and incongruent subtests (p = 0.038, x = 8.400 ; p = 0.011, x = 11,160, respectively). there was improvement in the results for the post-15 minutes time point vs. the before exercise time point (p = 0.009) and for the post-15 minutes time point vs. the during - exercise time point (p = 0.013) for the congruent subitem, whereas in the incongruent trial, there was improvement in the results for the immediately after time point vs. the before exercise time point (0.017), for the post-15 minutes time point vs. the before exercise time point (p = 0.005) and for the post-15 time point vs. the immediately after time point (p = 0.028) (figure 2). furthermore, there was a trend toward improvement for the post-15 time point vs. the during - exercise time point (p = 0.059). control session : there were no statistically significant differences in the dst results for the different time points (forward p = 0.069, x = 7.091 ; backward p = 0.857, x although the congruent subitem of the scw did not present a statistically significant difference (p = 0.062, x = 7.320), the incongruent task showed a difference among the time points (p = 0.015, x = 10.440), with improvement in the immediately after time point vs. the before exercise time point (p = 0.005) and a trend toward improvement for the during exercise time point vs. the before exercise time point (p = 0.059). this study aimed to assess changes in cognitive performance in depressed elderly persons during and after a session of physical exercise. to the best of our knowledge, this is the first study to examine this issue using a dual - task paradigm (motor and cognitive stimuli). we found an improvement in attention and inhibitory control but not in working memory after a 30-minute moderately intense walk on a treadmill. these results only partially confirmed our hypothesis because we found changes only after but not during the exercise. our results corroborate those of other studies of depressive subjects that have found improvements in attention and inhibitory control and no change in working memory.19 the improvements in attention and inhibitory control immediately after and 15 minutes after exercise can be associated with activated brain areas such as the anterior cingulate, which present impaired activation in depressive subjects. moreover, exercise may promote the activation of the reticular formation that is responsible for the modulation of attention and arousal. bartholomew.30 have shown that acute exercise can improve the mood of depressed individuals in addition to yielding cognitive improvement. studies with young and elderly healthy subjects have observed that cognitive function is immediately improved after physical exercise.16,19 this effect appears to occur only when aerobic exercise is applied.16 the improvement in cognition and mood after exercise in depressive and healthy subjects has been attributed to the acute release of neurotransmitters.31 however, recent data have confirmed that acute exercise also increases the levels of neurotrophic factors, such as bdnf (brain - derived neurotrophic factor).32 higher levels of bdnf immediately after exercise may enhance neurogenesis, neuronal plasticity, learning abilities, memory, and mood.33 dual tasks that comprise only cognitive tasks are more difficult to perform than dual tasks that include one physical task, especially for depressive subjects.34 cognitive performance during exercise is still a matter of debate. some studies have shown an impaired performance in complex tasks that depend on frontal functions,7,35 whereas other studies have shown improvement.5,6 contrary to our expectations, we did not observe differences in cognitive function during exercise. however, this study was conducted with physically active patients, which might explain why we observed no impairment during exercise. because the subjects included in this study were trained and adapted to the motor tasks, the processing demand required to perform the task was reduced (task automatization hypothesis), thus minimizing the detrimental effect of the dual task. in the present study, we used a session with no exercise to control for possible responses generated by the learning effect. however, we did not observe significant changes in the test scores during this session except for the scw incongruence subitem for the immediately after time point vs. the before exercise time point. this response may not be explained by the learning effect because the post-15 and during test results were not different. this study has some limitations that should be taken into consideration. as a preliminary report, we assessed a small number of patients. further studies that focus on healthy controls and that use direct markers of exercise intensity such as oxygen consumption are warranted. in conclusion, our study provides evidence that the cognitive function of depressed elderly persons is enhanced immediately after and 15 minutes following a session of physical exercise. moreover, cognitive function is not impaired during the dual - task activity (motor and cognitive tasks). the dual task may be a safe and useful tool to assess cognitive function during and after exercise. this work was supported by the brazilian national council of research and the foundation for research support of rio de janeiro. this project was conducted at the center for alzheimer 's disease and related disorders, institute of psychiatry, federal university of rio de janeiro, brazil. | objective : the goal of this study was to assess the acute effect of physical exercise on the cognitive function of depressed elderly patients in a dual - task experiment.introduction:physical exercise has a positive effect on the brain and may even act as a treatment for major depressive disorder. however, the effects of acute cardiovascular exercise on cognitive function during and after one session of aerobic training in elderly depressive patients are not known.methods:ten elderly subjects diagnosed with major depressive disorder performed neuropsychological tests during and after a moderate physical exercise session (65 - 75%hrmax). a digit span test (forward and backward) and a stroop color - word test were used to assess cognitive function. the elderly participants walked on an electric treadmill for 30 minutes and underwent the same cognitive testing before, during, immediately after, and 15 minutes after the exercise session. in the control session, the same cognitive testing was conducted, but without exercise training.results:the results of the digit span test did not change between the control and the exercise sessions. the results of the stroop color - word test improved after physical exercise, indicating a positive effect of exercise on cognition.conclusions:these data suggest that the cognitive functions of depressed elderly persons, especially attention and inhibitory control, are not impaired during and after an acute session of physical exercise. in contrast, the effect of dual - tasks showed beneficial results for these subjects, mainly after exercise. the dual - task may be a safe and useful tool for assessing cognitive function. |
beauty depends on size as well as symmetry symmetry is an important concept in biology, being related to health, mate selection strategies, and ultimately the survival of the species. while deviations from symmetry are critical perceptual units in detecting the appearance of health, the natural subtle asymmetry of the human face may be relatively unimportant for judging facial attractiveness. a grossly perceptible asymmetry of the face, however, as seen in hemifacial hypertrophy can be highly disconcerting to the patient. the treatment of this condition is unpredictable with an uncertain prognosis and remote chances of patient satisfaction. we have reported a case of true hemifacial hypertrophy in a young female adult and attempted to summarize the possible treatment alternatives to the conventional surgical treatment. a 23-year - old female patient presented to the department of oral and maxillofacial surgery with a chief complaint of asymmetry of her face. she gave a history of an abnormally enlarged left half of her face since birth which became more prominent following menarche. the patient had visited multiple plastic surgery units since childhood and had been diagnosed with hemihypertrophy [figure 1 ]. she was under a regular follow - up regimen by her gp to look for developing cancers. there was no history of such complaints in the family and serum chemistry revealed no abnormalities. the swelling was diffuse, soft, and extended from the frontal bone to the lower border of the mandible on the left side. the zygomatic root was enlarged on palpation [figures 2 and 3 ]. mouth opening and the left half of the hard palate was enlarged with a normal complement of teeth in the maxillary arch. the molars in the mandibular arch had been extracted at an earlier date due to caries. three soft growths were present ; at the lower labial mucosa, the buccal mucosa, and the retromolar area. an excisional biopsy of the growth at the left buccal mucosa was found to be lipomatous in origin. panoramic radiograph showed that the left palate, mandibular body, ramus, condyle, and coronoid were asymmetrically enlarged. the computed tomography showed uniform bony enlargement of the left half of the face including the frontal bone [figure 4 ]. based on the clinical and radiographic features, the patient was provisionally diagnosed with true hemifacial hypertrophy of the left face. axial section computed tomography scan the patient had been informed of the possible treatment options including extensive surgery for hard and soft tissue debulking and more conservative options for only soft tissue recontouring. she had been informed that irrespective of the treatment done, it would not be possible to achieve complete symmetry. the patient, however, was adamant and wished for treatment only if complete symmetry could be achieved. congenital hemihypertrophy or partial gigantism was first described by meckel in 1822 and gained widespread recognition after gesell 's classic description as essentially a developmental deflection of the normal process of birth. hemihypertrophy or hemihyperplasia may be an isolated finding, or it may be associated with other syndromes such as beckwith idiopathic hemihyperplasia is associated with mild cognitive disability, genitourinary anomalies, and a high association with wilm 's tumor. our case showed the presence of only an enlarged half of the face with no other part of the body exhibiting asymmetry. the criteria for the hemifacial type as specified by rowe includes unilateral enlargement of the viscerocranium, limited superiorly to the frontal bone (not including the eye), the lower border of the mandible inferiorly, the mid - line of the face medially, the ear, the pinna being included within the hypertrophic area laterally and enlargement of all tissues - teeth, bone, and soft tissues within this area [table 1 ]. classification of congenital hemihypertrophy by roubeir while khanna and andrade, noted that a relative disproportion is maintained due to the proportional growth of the normal and the abnormal sides, arora. observed that the asymmetry becomes accentuated with age. in this case, the asymmetry was detected at birth but became more apparent as the affected part enlarged at a greater rate than the rest of the body, most noticeably at the onset of menarche. our case, similar to previous case reports revealed lipomatosis as the dominant feature at the abnormal half. the teeth, mandible, and maxilla should be carefully scrutinized for deformities to help one reach the correct diagnosis. true hemihypertrophy involves not only the soft tissues of the body but the hard tissues as well. the professed causes for this condition include inherited chromosomal abnormalities endocrine dysfunctions, vascular or lymphatic malformation, atypical forms of twinning, altered intrauterine environment, and disturbances of the central nervous system pollock. advocated that an enlarged neural tube on one side is responsible for the unilateral increase in size. the growth of the neural tube follows a cephalocaudal gradient of growth and it reasonable to expect that some tube segments are more involved than others. rowe described abnormalities in the dentition of the abnormal side in three respects ; the crown size, the root size and shape, and rate of development. he noted that not all teeth were similarly affected. in the deciduous dentition, the second molar and in the permanent dentition the cuspids were the most commonly affected teeth followed by the first molars and premolars. it was also interesting to note that the premolars were the most enlarged among the teeth ; however, the second molars which develop around the same period are not. early shedding of deciduous teeth, delayed eruption of permanent teeth, prematurely developed teeth with short roots and congenitally missing teeth were often seen on the affected side. rowe described the alveolar bone to be larger and thicker on the affected side with the largest bulk distal to the largest tooth. there was also a propensity for an open bite as both posterior ridges developed exostoses which contacted each other during jaw closure. pollock. described the buccal mucosa as one which hangs in soft pedunculus folds. the tongue enlargement has also been described to be uniform, beginning abruptly in the midline with excrescences resembling large fungiform papillae. in the present case, the buccal mucosa on the affected side showed lipomatous growths, the tongue, lip, and palate were uniformly enlarged ; however, the teeth appeared to be normal in size, and the patient gave no history of abnormalities during shedding. to the unfamiliar clinician, this condition can constitute a baffling diagnostic problem. deformities of the teeth and their related hard tissues in the jaw are key findings for correct diagnosis, particularly in hemihypertrophy limited to the face. the treatment modalities extend from subtle soft tissue contouring to extensive surgeries to correct the underlying bony defect and reshape the overlying soft tissues. the hard tissues can be treated by a combination of condylar recontouring, osteotomies, and ostectomies to achieve the required shape, followed by debulking of soft tissues. extensive surgical treatment had been performed by pollock. and khanna and andrade who used extra- and intra - oral approaches to the deformed skeleton. khanna and andrade performed extensive surgeries in three stages using a hard and soft tissue debulking to achieve an acceptable result, while pollock used osteotomies and recontouring to correct the bony deformities. he advocated the treatment of the soft tissue deformity by excision, rhytidectomy, and cheiloplasty. even though there have been sporadic reports of acceptable results following surgical treatment, dearth of literature, the possibility of aggravating the growth, the difficulties in recontouring an entire half of the face, the uncertainties of how much soft tissue reduction should be done and the possibility of suboptimal postoperative esthetics dissuaded us from approaching the condition in an aggressive surgical manner. the ideal conditions for the use of liposuction are the existence of sufficient elasticity of the skin ; little excess skin and sufficient subcutaneous adipose tissue to be suctioned. liposuction not only allows the flattening of contours but correction of sagging teguments, resulting in some skin retraction. later, a small amount of fluid was introduced into the fat (wet liposuction). klein revolutionized liposuction surgery when he developed the tumescent technique that permits large - volume liposuction under local anesthesia without sedation or narcotic analgesics. tan in his case report described tumescent liposuction in the treatment of partial hemifacial hypertrophy. he performed the procedure using almost 50 ml of modified klein solution before the suction and reported positive results. as described by klein < 1% of the suctioned material in this technique is whole blood. a number of variations of this technique has been utilized for the face and neck regions. ultrasound assisted liposuction consist of repetitive expansion and passive contraction of adipocytes, resulting in rupture of their cellular membrane and liquefaction of fat. there are however chances of contact of the tip of the ultrasonic probe with the under surface of the skin complications such as burns, necrosis and subsequent scarring, nerve injury, and seroma formation. another system used is the powered lipoplasty (vibroliposuction) by rebelo ; this involves a reciprocal to - and - fro motion of the cannula tip using a pneumatic - driven system. this movement mimics action during traditional suction - assisted liposuction due to which, surgeons experience less fatigue during power liposuction with easier penetration and removal of adipose tissue. disadvantages of power liposuction include seroma formation, the creation of larger diameter tunnels (more than the actual cannula size used), swelling and bruising. recently, new laser systems have stimulated lipolysis by the means of small laser fibers introduced through a small cannula directly into the subcutaneous fat. a confluent red diode aiming beam at the tip of the cannula provides visual guidance through transcutaneous - illumination during treatment. large volume liposuction may require higher laser energies, increasing the risk of burns and skin necrosis. in addition, this technique is more time consuming, as the surgeon has to move the laser cannula relatively slowly through then skin to allow for necessary time for the laser - tissue interaction and a second pass with a suction cannula is necessary for fat aspiration. phosphatidylcholine is safe and nontoxic with no significant acute or chronic toxicity, mutagenicity or teratogenicity. although phosphatidylcholine is the main ingredient in most injectable preparations, it must be mixed with sodium deoxycholate (a bile salt) to make it soluble enough for injection. once injected into the subcutaneous tissue, the chain reaction continues over a period of 810 weeks. peckitt demonstrated the destabilization of the adipocyte cell membrane following injection of a critical concentration of phosphatidylcholine. this change in the physical properties of the cell membrane then appears to cause a complex enzymatic cascade involving the release of lipases and other enzymes as well as a rapid apoptotic cascade that causes adipocyte cell death. if the critical concentration is not reached, the unstable cell membrane forms gaps or pores, allowing efflux of some of the cytoplasmic contents. despite the temporary side effects that cause a degree of discomfort, phosphatidylcholine injections can be used successfully in the treatment of localized and small fatty areas of the face, as it requires a few injections and < 1 vial. injection of phosphatidylcholine seems to be a better, safer, and more cost - effective treatment than liposuction in these specific cases. the treatment modalities extend from subtle soft tissue contouring to extensive surgeries to correct the underlying bony defect and reshape the overlying soft tissues. the hard tissues can be treated by a combination of condylar recontouring, osteotomies, and ostectomies to achieve the required shape, followed by debulking of soft tissues. extensive surgical treatment had been performed by pollock. and khanna and andrade who used extra- and intra - oral approaches to the deformed skeleton. khanna and andrade performed extensive surgeries in three stages using a hard and soft tissue debulking to achieve an acceptable result, while pollock used osteotomies and recontouring to correct the bony deformities. he advocated the treatment of the soft tissue deformity by excision, rhytidectomy, and cheiloplasty. even though there have been sporadic reports of acceptable results following surgical treatment, dearth of literature, the possibility of aggravating the growth, the difficulties in recontouring an entire half of the face, the uncertainties of how much soft tissue reduction should be done and the possibility of suboptimal postoperative esthetics dissuaded us from approaching the condition in an aggressive surgical manner. the ideal conditions for the use of liposuction are the existence of sufficient elasticity of the skin ; little excess skin and sufficient subcutaneous adipose tissue to be suctioned. liposuction not only allows the flattening of contours but correction of sagging teguments, resulting in some skin retraction. later, a small amount of fluid was introduced into the fat (wet liposuction). however, the patients frequently required blood transfusions due to blood loss. klein revolutionized liposuction surgery when he developed the tumescent technique that permits large - volume liposuction under local anesthesia without sedation or narcotic analgesics. tan in his case report described tumescent liposuction in the treatment of partial hemifacial hypertrophy. he performed the procedure using almost 50 ml of modified klein solution before the suction and reported positive results. as described by klein < 1% of the suctioned material in this technique is whole blood. a number of variations of this technique has been utilized for the face and neck regions. ultrasound assisted liposuction consist of repetitive expansion and passive contraction of adipocytes, resulting in rupture of their cellular membrane and liquefaction of fat. there are however chances of contact of the tip of the ultrasonic probe with the under surface of the skin complications such as burns, necrosis and subsequent scarring, nerve injury, and seroma formation. another system used is the powered lipoplasty (vibroliposuction) by rebelo ; this involves a reciprocal to - and - fro motion of the cannula tip using a pneumatic - driven system. this movement mimics action during traditional suction - assisted liposuction due to which, surgeons experience less fatigue during power liposuction with easier penetration and removal of adipose tissue. disadvantages of power liposuction include seroma formation, the creation of larger diameter tunnels (more than the actual cannula size used), swelling and bruising. recently, new laser systems have stimulated lipolysis by the means of small laser fibers introduced through a small cannula directly into the subcutaneous fat. a confluent red diode aiming beam at the tip of the cannula provides visual guidance through transcutaneous - illumination during treatment. large volume liposuction may require higher laser energies, increasing the risk of burns and skin necrosis. in addition, this technique is more time consuming, as the surgeon has to move the laser cannula relatively slowly through then skin to allow for necessary time for the laser - tissue interaction and a second pass with a suction cannula is necessary for fat aspiration. phosphatidylcholine is safe and nontoxic with no significant acute or chronic toxicity, mutagenicity or teratogenicity. although phosphatidylcholine is the main ingredient in most injectable preparations, it must be mixed with sodium deoxycholate (a bile salt) to make it soluble enough for injection. once injected into the subcutaneous tissue, the chain reaction continues over a period of 810 weeks. peckitt demonstrated the destabilization of the adipocyte cell membrane following injection of a critical concentration of phosphatidylcholine. this change in the physical properties of the cell membrane then appears to cause a complex enzymatic cascade involving the release of lipases and other enzymes as well as a rapid apoptotic cascade that causes adipocyte cell death. if the critical concentration is not reached, the unstable cell membrane forms gaps or pores, allowing efflux of some of the cytoplasmic contents. despite the temporary side effects that cause a degree of discomfort, phosphatidylcholine injections can be used successfully in the treatment of localized and small fatty areas of the face, as it requires a few injections and < 1 vial. injection of phosphatidylcholine seems to be a better, safer, and more cost - effective treatment than liposuction in these specific cases., rare conditions like hemifacial hypertrophy are ill understood, and the possibility of achieving perfect symmetry in these cases is a herculean task. we feel that safer, conservative and cost effective options such as liposuction and subcutaneous injections of phosphatidylcholine must be done, especially so in cases where the surgical treatment would be deemed too extensive and not without the possibility of unsatisfactory outcomes. 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. | congenital hemihyperplasia is a rare developmental disorder characterized by unilateral overgrowth of one or more body parts resulting in marked asymmetry. we are reporting here, a case of true hemifacial hypertrophy in a young female adult highlighting the clinical features and possible treatment options. |
cranioplasty is a common, but formidable surgical procedure for neurosurgeons, in patients with scalp and/or calvarial defects. there is evidence of cranioplasty having been performed by several early cultures, including pre - columbian incans, using gold or silver plates, and by neolithic celts using bone rondelles. however, the first reported cranioplasty was probably that of a russian nobleman who, after receiving a sword blow to the head, had the resultant defect (and his health) restored with a piece of dog 's cranium (van meekeren, 1668). subsequently, after he had been excommunicated from the russian church (which could not accept the presence of animal bone on a human skull), removal of the graft was impossible, due to bone union. the primary aim of this article is to review the basic principles, to use the split calvarial graft for the reconstruction of a skull defect. the main objectives of cranioplasty are : to achieve primary wound healing, obliterate dead space, and seal off sterile cranial areas from contaminated oronasal cavities, to restore the normal barriers protecting the intracranial structures and obtain a permanent or very durable reconstruction using biologically inert materials, and also to restore the esthetics.[58 ] pathological defects or alterations in the shape of the calvarium may be caused by a number of processes, including traumatic defects, resection of benign or malignant tumors, congenital lesions, and iatrogenic injuries, out of these more common causes of skull defects, including trauma, neurosurgical procedures, and infections.[125915 ] most calvarial reconstructions are performed immediately unless the wound is infected. if infection is present, the reconstruction must be delayed until the infection has been treated. reconstruction of skull defects is technically challenging, but can be achieved with the use of biological tissue, such as the split calvarial bone graft or posterior wall of the sinus or iliac crest, or with artificial materials, such as the 3d titanium mesh. the ideal substitute for undertaking cranioplasty must be biocompatible, strong, and lightweight ; it must be malleable, to precisely fit even complicated cranial defects, nonmagnetic, chemically inert ; radiolucent ; non - ferromagnetic ; readily available ; inexpensive and easily secured, and must have long - term stability. however, no such material currently exists, making natural bone the obvious choice to be used as cranioplasty material. alloplastic implants have the advantage of being readily available, easy to handle and shape, and undergo minimal resorption, however, alloplastic implants are permanent foreign bodies that are susceptible to infection and exposure over time. the advantages of reconstruction with autologous bone include a lower incidence of graft loss than occurs with alloplastic material. also, exposure and infection of the autologous bone can sometimes be managed without complete graft loss, whereas, when alloplastic materials become exposed or infected, often the only choice is removal of the foreign material, (exceptions are porous polyethylene sheet (medpor) allografts, as their infection can be managed by intravenous antibiotics).[2023 ] furthermore, the mechanical, immunological, and technical - grafting properties of autologous bone, together with its superior esthetic, and psychological effects, probably make it the best material for cranioplasty. the split calvarial bone technique was popularized by tessier in 1932, and first applied in nasal bone reconstruction by jackson. the preferred skin incision is generally the coronal one, as it provides a wide exposure of the skull surface and may often proffer simultaneous visualization of the bony defect and the area identified for harvest of the graft. after a bicoronal incision, the scalp flap is raised, a plane is created between the pericranium and skull periosteum, and the region that requires cranioplasty is prepared. any dural dehiscence or loss is first repaired, grafted if required, and can be sealed with fibrin glue or an equivalent. subsequently, the outlines of the cranial defect are traced onto a sheet of transparent plastic and then transferred onto the surface of the skull, chosen as a harvest area. the next step is resection of the graft for which several different techniques can be employed.[122628 ] a bone flap of the same size is removed from the skull and the outer and inner tables can be split apart and used to reconstruct the defect or defects. for this technique a section of the donor skull to be used is split and the outer table is applied to cover the craniotomy defect, leaving the inner table to cover the donor site. larger, split - thickness cranial bone grafts can be used to replace numerous smaller fracture fragments, greatly facilitating the fixation device application and providing much thicker scaffolding, which will better maintain soft tissue contour during remodeling and new bone formation. if possible a single - stage procedure will allow the patient to undergo only one procedure rather than two or more staged operations and avoid several weeks with a substantial bony defect. calvarial bone grafts have the benefit of being harvested from the same operative field as the defect. split outer table grafts are taken from the parietal region of the skull, posterior to the coronal suture, where the skull is the thickest [figures 1 and 2 ]. the graft must not be harvested in the midline because of the risk of injuring the sagittal sinus. treatment of patient with a massive osteoma of the left frontal bone a case of large ewing 's sarcoma involving the right frontal bone although calvarial bone grafts are used today for various scalp defect reconstructions, the complication rates related to the bone graft are surprisingly low. many studies have reported no resorption and no loss of calvarial transplants after repair, on a short - term follow - up, ranging from 1 to 3.7 years.[3135 ] there is almost a unanimous agreement that autogenous calvarium possesses far better characteristics and quality than the most widely used alloplastic materials currently available. there is no type of metal, acrylic resin, or any other type of alloplastic material that fulfills this wide range of requisites, and it is much simpler, less expensive, and safer for the patient to use autologous bone whenever possible. apart from this, fresh autologous bone is the most suitable material for reconstruction of cranial defects in view of its perfect histocompatibility, optimal mechanical properties, and good anatomofunctional fusion of the graf with the adjacent bone, as well as the possibility of partial or total revitalization of the graft itself. live tissue is biologically active and fuses quickly with the adjacent bone, giving excellent results. also, the autologous bone ensures the best possible physiological and cosmetic results, autologous bone grafts usually display bone regeneration processes, do not have a foreign body reaction, and present a low incidence of infection. in addition, calvarial bone grafts in the pediatric age group are fairly mal - leable, allowing reconstructive surgeons to reproduce the precise contour of the calvarium with relative ease. many synthetic substitutions of dura and bone are often used for reconstruction of the skull base ; unfortunately, these methods bear significant disadvantages and can induce chronic inflammation, carry a high risk of infection, and are inferior to the biological sources in terms of strength and sealing quality, [with the probable exception of some materials, such as titanium mashes and cortosstm (orthovita, malvern, usa), which prove to have more strength than the thin split thickness calvarial bone].[4042 ] a disadvantage of calvarial grafts is the limited size of the graft available, particularly when the defect is adjacent to the graft donor site. another disadvantage of calvarial grafts is the risk of violating the inner table or dura during harvest. correction of large calvarial defects with autografts may also be quite time consuming, moreover, both the donor and recipient sites are less biomechanically stable than the adjacent skull. splitting of the bone requires experience, as sometimes the bone cracks into several pieces. other complications specific to the bone relate to its harvest : split calvarial grafts carry the risk of intracerebral hematoma, subarachnoid hemorrhage, dural tears, and csf leaks. in summary, calvarial bone grafts are used today for various scalp defect reconstructions, with incidence of complication rates related to the bone graft being surprisingly low. as no specific cost analysis has been attempted, it is difficult to compare whether the added cost of the synthetic material would be offset by the time saved in the operating room compared to calvarial bone grafting and the subsequent rigid fixation. | cranioplasty is a common, but formidable surgical procedure for neurosurgeons, in patients with scalp and / or calvarial defects. this procedure can be simple or complex. the main objectives of cranioplasty are : to achieve primary wound healing, obliterate dead space, and seal off sterile cranial areas from contaminated oronasal cavities, to restore the normal barriers protecting the intracranial structures (together with a satisfactory cosmetic result) and obtain a permanent or very durable reconstruction, using biologically inert materials, and also to restore the aesthetics. the greatest problem is selecting the optimum material for repair of the cranial defect. many synthetic substitutions of the dura and bone are often used for reconstruction of the skull base ; unfortunately, these methods bear significant disadvantages and can induce chronic inflammation, carry a high risk of infection, and are inferior to biological sources in terms of strength and sealing quality [with the exception of some materials, such as titanium mashes and cortosstm (orthovita, malvern, usa), which are seen to have more strength than the thin split thickness calvarial bone ]. the primary aim of this article is to review the basic principles to use the split calvarial graft for the reconstruction of the skull defect. |
traditionally, the assessment of metabolic acidosis and alkalosis relies on measurement of the base excess, which is the difference between the ' ideal ' buffer base (i.e. the sum of the negatively charged forms of weak acids, [a ] + [hco3 ] + [h2po4 ], at standard conditions (ph 7.4, temperature 37c, partial carbon dioxide tension 40 mmhg) and the ' actual ' buffer base): base excess = buffer baseactual - buffer baseideal (1) during the past few years a novel approach based on assessment of the strong ion difference (sid) has been introduced to evaluate metabolic acidosis and alkalosis. for simplicity, we limit our discussion to these two disturbances. please note that in the following discussion we will refer to the amount of strong ion difference as sid (meq), while we will refer to the strong ion difference concentration as [sid ] (meq / l). by definition, strong ions are always dissociated in a solution. in plasma, as well as in interstitial fluids, the sum of positively charged ions (primarily na, k, caand mg) exceeds the sum of the negatively charged strong ions (primarily cland lactate) of about 42 meq / l. this difference is called the sid, and according to the stewart model its variation is one of the determinants of acid base status. looking at figure 1, the connection between base excess and sid is apparent. in fact, because the ideal sid is equal to 42 meq / l (as is the normal buffer base), it follows that base excess = sidactual - sidideal = buffer baseactual - buffer baseideal (2) because computation of the actual sid is rather complicated, requiring the determination of all of the strong ion concentrations, we believe that the base excess approach may be easier, more rapid and adequate for clinical purposes. indeed, the frequent debate involving the comparison of the ' sid approach ' with the ' base excess approach ' to assessment of metabolic acidosis appears futile because their physiological meanings, as well as their variations, are identical. in other words, the picture is different when one considers the ' understanding ' of acid base and electrolyte equilibria, which everyone has studied in separate chapters of the textbooks. the great merit of the stewart approach is that it considers electrolytes and acid base status in a common framework. here, we would like to propose a comprehensive model that may explain, at least qualitatively, many of the findings observed in clinical practice and in the literature. the sid reflects the difference in electrical charges of the strong ions in the volume of the extracellular compartment (v). at time 0 for example, if at time 0 the sid is normal (i.e. 42 meq / l) then the net amount of electrical charge in the extracellular fluid (15 l) will be 630 meq. during a given period of time there may be an addition of volume to the system (e.g. infusion of a solution) with its own sid (sidinfusion). consequently, a net amount of charge equal to vinfusion [sidinfusion ] will be added to the system. similarly, the urinary system will excrete a volume of urine (vurine) with its own sid (sidurine). the last variable that must be taken into account is endogenous production of sid (sulphates, phosphates, lactate and ketoacids, among other components). it follows that the sid at a given time ' t ' may be derived from a series of equations, which may appear to be complicated in their expression but simple in their meaning. eqn 3 (below) indicates that, in a system, the net amount of electrical charges due to the strong ions is equal to the net electrical charge of the system at time zero plus the net electrical charge added as a result of metabolism plus the net electrical charge added with volume infusion minus the net electrical charge extracted via urine. where epr(t) is the ' endogenous production rate ' of sid (meq / min), ir(t) is the volume infusion rate and upr(t) is the urine production rate. at a given time ' t ', the net fluid volume of the extracellular compartment is equal to the initial volume of the system plus the volume added with infusion minus the volume extracted in the form of urine. because what matters in terms of acid base status is the concentration, rather than the net amount of electrical charge, the sid at a given time ' t ' may be expressed from the above equations as shown in equation 5 at the foot of the page : it is important to remember that an increase in sid will lead the system to become more basic whereas a decrease in sid will lead the system to become more acidic. in general, eqn 5 indicates that metabolic acidosis or alkalosis may occur either by changing the net electrical charge at constant extracellular volume or by changing the extracellular volume at constant electrical charge. looking at eqn 5, we may make several comments. to maintain the metabolic acid base status of a system (i.e. the baseline sid), two conditions must be satisfied : the input quantity of sid should equal the output quantity of sid ; and the distribution volume of sid should remain constant. to the best of our knowledge, the only studies in which the strong ion balance (input and output) was investigated were conducted in cows [6 - 8 ] ; different amounts of sid in the diet caused corresponding changes in urinary sid. as discussed above, sid has been studied in comparison with base excess but without any physiological rationale. the sid approach has been also proposed to explain metabolic acidosis during saline infusion (sid input), but only a few papers have tackled and discussed the problem of urinary sid (sid output) [11 - 13 ]. what we lack is the entire picture of the system ; unfortunately, this requires frequent assessment of urine electrolytes. some clinical findings may be viewed from the perspective of the general framework of eqn 5. this has been attributed to changes in sid due to hyperchloraemia. by looking at eqn 3 because the sid of saline is equal to 0, it follows that, if the urinary output of electrical charge and metabolic production remain constant, the net difference of electrical charges in the system (i.e. the numerator in eqn 5) does not change. what causes the acidosis is the expansion of the extracellular volume (volume input greater than volume output), which leads to decreased concentration of the net amount of electrical charge (i.e. the sid). unfortunately, it is not easy to consider the urinary sid. in fact, although 4042 meq / l of plasmatic negative charge may be derived from the dissociated weak acids ([a ], [hco3 ] and [h2po4 ]), the amount of weak acids is far less in urine and, overall, the range of urinary ph is an order of magnitude greater than that in plasma. in fact, as far as the plasmatic acid base equilibrium is concerned, we must consider only the components of urinary [sid ] that may affect the plasmatic [sid ] (i.e. [k ], [na ] and [cl ]). in fact, in urine [na ] + [k ] + [un ] = [cl ] + [un ] (6) where unand unare the positive and negative unmeasured ions. it follows that [na ] + [k ] - [cl ] = [un ] - [un ] (7) quantitatively, the most important anion in urine is so4, which is derived from the metabolism of sulphur amino acids, whereas the most important cation is nh4. in normal conditions, the sum of urinary [na ] + [k ] - [cl ] amounts to 42 it follows that the concentration of unmeasured anions exceeds the concentration of unmeasured cations of 42 meq / l. when a strong ion such as lactate is added to the plasma, the plasmatic sid will decrease. consequently, the urinary system will react by increasing its excretion of chloride, thereby decreasing the plasma chloride concentration (while [na ] and [k ] must be maintained within normal ranges). therefore, the difference between [un ] and [un ] should decrease (eqn 7). this is accomplished by increasing the excretion rate of nh4, which is a way to augment elimination of clwithout na. indeed, the effects of any volume infusion or other interventions can not be understood if the urinary sid and volume are not taken into account. a merit of the report by moviat and colleagues is that, for the first time in critical care, attention is focused on the urinary system, which is the main regulator of sid. the authors found that the increase in urinary sid (indirectly induced by blocking carbonic anhydrase) was the key driver for correction of metabolic alkalosis. the message is important urinary sid should be a key component of global acid base assessment. we believe that urinary electrolyte monitoring may open a new perspective of research in critical care. acid base equilibrium, one of the oldest research areas in medicine, is still an open field for new discoveries and approaches. meq / l (the strong ion difference [sid ]) and, according to the law of electro - neutrality, is equivalent to the buffer base (bb ; i.e. the sum of [hco3 ], [a ] and [h2po4 ], where aare the weak acids in dissociated form, mainly albumin). during acidosis, | the plasmatic strong ion difference (sid) is the difference between positively and negatively charged strong ions. at ph 7.4, temperature 37c and partial carbon dioxide tension 40 mmhg, the ideal value of sid is 42 meq / l. the buffer base is the sum of negatively charged weak acids ([hco3- ], [a- ], [h2po4- ]) and its normal value is 42 meq / l. according to the law of electroneutrality, the amount of positive and negative charges must be equal, and therefore the sid value is equal to the buffer base value. the easiest assessment of metabolic acidosis / alkalosis relies on the base excess calculation : buffer baseactual - buffer baseideal = sidactual - sidideal. the sid approach allows one to appreciate the relationship between acid base and electrolyte equilibrium from a unique perspective, and here we describe a comprehensive model of this equilibrium. the extracellular volume is characterized by a given sid, which is a function of baseline conditions, endogenous and exogenous input (endogenous production and infusion), and urinary output. of note, volume modifications vary the concentration of charges in the solution. an expansion of extracellular volume leads to acidosis (sid decreases), whereas a contraction of extracellular volume leads to alkalosis (sid increases). a thorough understanding of acid base equilibrium mandates recognition of the importance of urinary sid. |
thermal injury to living tissue is known to begin at 42. lethal exposure times at this temperature range from 3 to 50 hours, depending on the tissue involved (26). at temperatures over 43, lethal exposure times decrease exponentially ; at 46, 8 minutes ' exposure is enough to cause cell death. at temperatures over 60, denaturing of tissue collagen begins and cell death is inevitable (27, 28), and for treatment in cases where injury to normal tissue is not a concern, temperatures of this magnitude are therefore desirable. in clinical practice the tissues around the tip of a needle electrode are heated to 100, the temperature required to induce the desirable degree of injury. it is known, however, that rapid heating to too high a temperature (over 100) dramatically increases tissue impedance, and the deposition of rf energy, heat diffusion, and further coagulation necrosis are limited (29). using either ultrasound or ct guidance, an rf needle electrode is percutaneously introduced directly into a tumor. when the electrode is connected to a generator producing alternating electric current, rf is emitted from its tip, and current within the rf range (460 khz) passes into surrounding tissue. the current agitates ions of tumor tissue around the needle electrode, and the frictional heat produced by ionic agitation induces cellular death via coagulation necrosis. by changing the intensity and duration of the alternating current, and the size of the electrode we currently limit the use of rf ablation to patients who are not considered for surgery, or who decline this option. as yet, there is no consensus as to indication, but investigators generally recommend that hccs smaller than 5 cm in diameter and fewer than four in number are ideal for rf ablation. we are able to treat lesions larger than this by making multiple overlapping ablations with recently developed needle electrodes and powerful generators, but it is known that the larger the tumor, the greater the incidence of incomplete treatment. because rf ablation can cause bleeding, uncorrectable coagulopathy due to severe liver dysfunction is an absolute contraindication. the prothrombin time ratio must be greater than 50% and the platelet count greater than 70,000/mm. patients with large amounts of ascites, obstructive jaudice, evidence of extrahepatic tumors, or extensive portal venous thrombosis are not suitable candidates for the procedure. at the moment three rf devices, all of which consist of rf generators, needle electrodes and dispersive ground pads, are commercially available. the basic principles of each are similar, but the operational techniques are different. generator power varies between 50 and 200 watts ; some are equipped with functions that display output, tissue impedance, and temperatures in the tissue around the tip of a needle electrode. in each device 1). the first device to be introduced in korea, in april 1999, was the rita ' 500 ' model (rita medical systems, mountain view, cal. this consists of a 50-watt electric generator and a 15-gauge needle electrode, insulated except for 0.6 - 1 cm of its tip. its hub is a plunger that advances 4 or 7 prongs from the tip, and when fully deployed, these and the needle electrode resemble an open umbrella 3 cm in diameter. all the prongs and uninsulated tip of the needle are active electrodes, and each curved prong contains a thermocouple at its tip that registers the temperature of surrounding tissue. with the use of recently developed software, the temperature around a needle electrode, power, and impedance can be continuously monitored. the more recent 1500 model, manufactured by the same company, is equipped with a more powerful generator (150 watts) and a larger needle electrode, 5 cm in diameter, which can induce thermal lesions of up to this size with a single ablation. the second device (' rf 2000 ' system) manufactured by radiotherapeutics corporation (mountain view, cal.,) incorporates a 100-watt generator, a leveen 15-gauge, monopolar array needle electrode, and needle electrodes 2, 3, and 3.5 cm in diameter, the largest of which is able to induce lesions with a diameter of up to 4 cm. the 15-gauge electrode consists of a 12- to 15-cm - long insulated cannula that advances ten individual hook - like arms which are deployed after the needle has been introduced into the tumor. the third device, manufactured by radionics corporation (burlington, mass., u.s.a.), includes a needle electrode whose tip is internally cooled with chilled saline, a process thought to increase the size of the thermal lesion. the device is equipped with a 200-watt generator and uses either a single 17-gauge straight electrode or clustered electrodes designed in a triangular shape. with the device rf ablation can be performed percutaneously, laparoscopically, or during laparotomy, and involves conscious sedation or general anesthesia. for the former, we prefer the intramuscular injection of 50 mg pethidine hydrochloride (pethidine ; samsung pharmaceutical, seoul, korea) 10 - 20 minutes before the procedure. whenever patients report intolerable pain during ablation, an additional 50 mg of the same drug is administered intravenously, with continuous monitoring of the cardiovascular and respiratory systems. although lesions can be reliably localized using ct, this part of the procedure has depended mostly on sonography. for grounding, one or two large dispersive pads are attached to the patient 's thigh or back, and after skin cleansing using a sterile technique, local anesthetic (lidocaine ; kwang myung pharmaceutical, seoul, korea) is injected along the expected needle pathway and a small skin nick is made. the electrode is passed through the nick and advanced into the desired portion of the tumor, using real - time sonographic guidance. once the needle tip is appropriately positioned in the tumor, the electrode prongs are deployed and ablation is begun. if a rita device is used, initial ablation involves partial deployment of the curved electrodes, but once the temperature at the tip of these reaches the desired level (usually over 95), they are fully deployed. the temperature is then maintained at 95 - 100 for ten minutes until the entire tumor or the desired portion is ablated. after each ablation, the prongs are retracted and the needle is repositioned. at this point, the needle tract is cauterized and the needle withdrawn from the liver. with an rtc device, the power used for initial ablation after deployment of the multiple arrays is 50 watts. at 1, 2, 3, and 4 minutes, 10-watt increments are applied, to a maximum of 90 watts, until the " roll - off " phenomena is observed (i.e., power output falls precipitously as tissue impedance increases markedly). after a 30-second pause, ablation is restarted, at 75% of the maximum power achieved, until " roll - off " again occurs. each tumor or area of a large tumor is ablated by the application of rf power during these two phases, and at completion the needle is removed with the multiple arrays retracted. with a radionics device either a single electrode with 2 or 3 cm of exposed metallic tip or a triple- cluster electrode, depending on the size and location of the tumor, can be employed. after the needle electrode is introduced into the tumor, rf is usually applied for 12 minutes. to maintain a temperature of 20-25 in the tissue around the needle tip, a peristaltic pump is used to infuse 0 normal saline solution into the lumen of the electrodes. during ablation, generator output, tissue impedance and temperature around the needle tip are continuously monitored. for rf ablation to be successful and complete the ideal goal is to ablate a peripheral margin of 0.5 - 1 cm of normal hepatic tissue surrounding the tumor, as well as the entire tumor itself : without adequate safety margins, untreated viable tumor may remain, leading to treatment failure and local recurrence. in general, a single ablation, or two, may be adequate for tumors less than 3 cm in diameter when a needle electrode at least 3 cm in diameter is used. for larger tumors, however, multiple overlapping ablations should be performed, according to tumor size. with a needle electrode 3 cm in diameter, a single ablation is applied for tumors 3 cm. for tumors located in the mid - zone of the liver rather than its periphery or central zone, this treatment strategy in addition to size, tumor location is one of the important factors influencing the likely therapeutic outcome. if the tumor lies in the subcapsular area, no safety margin along the capsule is possible. in subcapsular tumors, a safety margin of normal hepatic tissue is difficult to obtain, but if the liver capsule overlying the tumor appears to be involved by the tumor, the capsule should be ablated. subcapsular tumors can be effectively treated using either an expandable needle electrode with multiple array or a straight cooled - tip electrode. if a tumor is small and round, the use of an expandable needle electrode is preferred because the lesions it induces tend to be more spherical in shape than those induced by one with a straight tip. for ablating fusiform tumors, a straight - tip needle electrode is better because it frequently makes elliptical lesions. tumors located between the anterior and posterior liver capsules (e.g. in the inferior angle of the right lobe or the lateral segment of the left) are often difficult to ablate completely, especially when they abut on both capsules, and in this particular situation, the choice of needle electrode is important. when an expandable electrode is used in narrow spaces, multiple overlapping ablations involving partial deployment of the hook - like electrodes are unavoidable even if the tumors are small : the full deployment of hooks can penetrate the liver capsule, and this may cause bleeding or unnecessary thermal injury to adjacent organs. because its use involves no risk of penetrating either capsule, and the entire length of active electrode can be employed, the situation requires a straight cooled - tip electrode. additionally, the number of ablations and thus the procedure time are thereby reduced (fig. 3). if the ultrasound - guided percutaneous approach is used, subcapsular tumors exhibiting exophytic growth can also be difficult to ablate completely. the best way to achieve effective necrosis of an exophytic tumor is to ablate the deepest portion first, thus blocking the blood supply to the exophytic portion, which is subsequently ablated (fig. another difficult situation arises when tumors are present in the central zone of the liver, near the hepatic hilum. a tumor in this location is almost always surrounded by large vessels, and the resultant tissue cooling may make it difficult to ablate its periphery and the cuff of normal hepatic tissue. in this situation, the extent of coagulation necrosis is less than usual, and follow - up ct reveals the presence of residual perivascular tumor (fig. 5). on the basis of experimental and clinical studies (30 - 32), several investigators have found that temporary reduction of vascular flow to the liver during rf ablation by mechanical occlusion of the portal vein and/or arterial vessels, or pharmacologic modulation, increased the extent of coagulation necrosis. when rf ablation is performed percutaneously, the use of a balloon for mechanical occlusion of the portal vein is impractical because it is invasive and time consuming. during intraoperative rf ablation after laparotomy, reduction of vascular inflow by clamping the hepatic artery and portal vein at the level of the porta hepatis (pringle maneuver) is very helpful in increasing the extent of coagulation necrosis. pharmacologic modulation of hepatic blood flow has been under investigation and could be the ideal way of creating more extensive coagulation necrosis during percutaneous rf ablation. we are able to suggest a number of ways in which the use of current rf devices for the ablation of tumors surrounded by large vessels may be efficacious. the space between large vessels is usually small and narrow, and safe ultrasound - guided targeting may thus be difficult. it is important not to penetrate large vessels with a needle electrode, since bleeding or pseudoaneurysm may occur. when rf ablation involves the use of an expandable needle electrode and its hooks within the lumen of the vessel are ineffective, the hooks of the multiple array should not be placed beyond the vascular wall. to induce satisfactory coagulation necrosis in this situation, multiple overlapping ablations with partial deployment of the multiple array even if the tumor is less than 3 cm in diameter, the recommendation applies. to avoid penetration of the vessel wall during deployment of the needle electrode, careful monitoring with real - time ultrasound is essential. if the temperature around any tip of the multiple array fails to rise, this indicates that the tip is inside the blood vessel and should be retracted. for ablation of a tumor surrounded by large vessels, a straight - tip internally cooled needle electrode may have advantages over the expandable needle variety. first, because only the straight part of the active electrode is involved, penetration of the vessel wall is not a matter of concern. second, the reduced impedance of the tissues nearest the needle electrode, induced by internal cooling with chilled saline, enables heat to diffuse more easily. this, in turn, allows greater rf energy deposition and can lead to more extensive coagulation necrosis than is possible when an expandable needle electrode is used. for ablation of a tumor surrounded by large vessels, it is also useful to increase the duration of rf application, regardless of the type of needle electrode employed ; one more ablation with the needle tip placed in the same position may help decrease the amount of residual perivascular tumor. this possible outcome is, however, based on our own experience and is somewhat speculative ; firmer conclusions should be based on the findings of further studies. the ablation procedure almost always creates numerous microbubbles in heated tissue, and these increase the echogenicity of the ablated lesion (fig. 6), a condition which is known to persist for between 15 minutes and 6 hours (33, 34). hyperechogenicity may be helpful for monitoring the approximate size of an ablated lesion, but makes further placement of the needle electrode difficult. for large tumors, one good way of treating a large tumor is to deal with the deepest portion first ; in this way increased echogenicity does not obscure its untreated portions. no data concerning the accuracy of ct in depicting residual tumors after rf ablation have been published, and as a standard of reference, the dynamic helical mode suffers certain limitations. nonetheless, ct has been widely used in the evaluation of therapeutic response after rf ablation. complete tumor necrosis (successful treatment) is considered to have occurred when follow - up ct reveals no foci of enhancement within the ablated lesion or at its periphery (figs. 2, 4). any focal enhancement within the tumor or increase in size of the ablated tumor should be considered indicative of residual or recurrent lesion (fig. we treat residual or recurrent tumor with additional rf ablation, using three - phase helical ct to evaluate therapeutic outcome ; because reactive hyperemia in tissue surrounding the ablated lesion makes accurate assessment difficult, such evaluation does not immediately follow treatment, however (fig. 8). whether ablation was successful is usually determined on the basis of ct findings obtained at least one month after the procedure. in our experience, reactive hyperemia has usually resolved by this time. to determine the possible presence of residual or recurrent tumor, follow - up ct dynamic mri with gd - dtpa enhancement is also a good method for evaluating therapeutic response. areas of the tumor in which treatment was successful do not demonstrate the enhancement seen at contrast - enhanced ct. power doppler ultrasonography (pdus) using a microbubble contrast agent has recently been reported to be a promising noninvasive technique for assessing the therapeutic response to rf ablation in hcc (34). nonenhanced pdus was of little help in depicting residual intratumoral flow signals, though contrast - enhanced pdus was very sensitive in the detection of residual tumor vascularity within an ablated lesion (fig. several clinical series using different methods of rf application for the treatment of hcc have been reported (12, 13, 15 - 17, 20). rossi. detailed their seven - year experience with rf ablation in 50 patients with hcc or hepatic metastases (15), reporting very promising survival rates (for hcc patients) of 94%, 86%, 68%, and 40% at 1, 2, 3 and 5 years, respectively. reported complete necrosis rates after rf ablation have varied according to the size and morphology of the hepatic lesions. (16) reported a complete necrosis rate of 90% in 42 patients with 52 small hccs (5.0 cm) (17). in an early experience with 51 hccs (1.0 - 4.0 cm in diameter) at our institution, the complete necrosis rate was 94% (20). during a recent one - year period, we used rf ablation alone for the treatment of 108 patients with 125 nodular hccs. fifty - seven of these 108, with 65 hccs, underwent follow - up helical ct not less than six months after the procedure. forty - five tumors were small (0.7 - 3.0 cm) and 20 were medium (3.1 - 5.0 cm). six - month follow - up ct showed complete necrosis of ablated tumor in 43 (96%) of the 45 small lesions and in 18 (90%) of the 20 that were medium - sized. after rf ablation, we found no major but four minor complications (one small pneumothorax and three cases of minor intraperitoneal bleeding) which disappeared spontaneously with no specific treatment (35). other investigators have also reported low complication rates (12 - 17). on the basis of several clinical studies including ours, we believe that rf ablation is a safe and highly effective technique for the local treatment of hcc, especially for small tumors. rf ablation has several advantages over other existing therapies : it is minimally invasive, and for the treatment of residual or recurrent tumors can be repeated. compared with pei, rf ablation leads to a higher complete necrosis rate and requires fewer treatment sessions (16). the procedure is operator dependent, and for successful ablation, accurate placement of the needle electrode is imperative. in order to obtain good results, medical practicioners should therefore be familiar with sonographically - guided interventional techniques. the other limitation is that abundant portal venous flow in normal liver tissue surrounding the tumor may act as a heat sump, making complete ablation of such tissue difficult. to decrease the effect of portal venous flow, several experimental and clinical studies are in progress. although long - term follow - up evaluation of clinical outcome is needed, rf ablation is safe and effective, and is thus a highly promising technique for the local destruction of hcc. further improvements in the devices and techniques employed may further increase its efficacy and safety. on the basis of several clinical studies including ours, we believe that rf ablation is a safe and highly effective technique for the local treatment of hcc, especially for small tumors. rf ablation has several advantages over other existing therapies : it is minimally invasive, and for the treatment of residual or recurrent tumors can be repeated. compared with pei, rf ablation leads to a higher complete necrosis rate and requires fewer treatment sessions (16). the procedure is operator dependent, and for successful ablation, accurate placement of the needle electrode is imperative. in order to obtain good results, medical practicioners the other limitation is that abundant portal venous flow in normal liver tissue surrounding the tumor may act as a heat sump, making complete ablation of such tissue difficult. to decrease the effect of portal venous flow, several experimental and clinical studies are in progress. although long - term follow - up evaluation of clinical outcome is needed, rf ablation is safe and effective, and is thus a highly promising technique for the local destruction of hcc. further improvements in the devices and techniques employed may further increase its efficacy and safety. | although surgical resection remains the best option as potentially curative therapy for hepatocellular carcinoma, radiofrequency thermal ablation has begun to receive much attention as an effective minimally invasive technique for the local control of unresectable malignant hepatic tumors. most recent radiofrequency devices equipped with a powerful generator and larger needle electrode permit larger thermal lesions, up to 5 cm in diameter, with a single ablation. in this article, the author reviews the technical developments and early clinical results obtained with radiofrequency ablation techniques. |
the best - characterized mutations of egfr conferring sensitivity to egfr - tyrosine kinase inhibitors (tki) in non - small cell lung cancer (nsclc) are deletions in exon 19 (del 19) and point mutations in exon 21 (l858r). conversely, the most common mutation conferring resistance to egfr - tki is the exon 20 point mutation (t790 m). this t790 m mutation is considered the major cause of acquired resistance to egfr - tki. primary t790 m mutation can also occur at baseline (before tki treatment) related to a germinal mutation or somatically mutated subclones. a recent meta - analysis of the predictive role of a pretreatment t790 m mutation in patients with activating egfr mutation receiving egfr - tki treatment shows that the pretreatment t790 m mutation has a negative impact on progression - free survival. here, we present the case of a young patient with a primary lung papillary adenocarcinoma and miliary pulmonary metastases, diagnosed with pretreatment complex egfr mutation combining a heterozygous somatic l858r mutation and t790 m mutation. a 47-year - old man presented with a history of weight loss from 97 to 92 kg within 1 month, cough and fatigue. a chest x - ray (fig. the patient had no fever, and his oxygen saturation was 90% (rest time without oxygen), improving to 94% under oxygenotherapy (1 liter / min). 2) confirmed miliary metastases and a nodule in the right middle lobe and revealed bilateral mediastinal lymph nodes. bronchoscopy was macroscopically normal, but cytology of bronchoalveolar lavage (bal) highlighted an adenocarcinoma, which was confirmed as papillary subtype by mediastinoscopy. molecular analysis of bal, containing 30% of tumor cells, by direct sequencing showed a somatic complex heterozygous egfr mutation (fig. 3) composed of l858r mutation in exon 21 and t790 m mutation in exon 20. t790 m was confirmed to be somatic, as it was not detected in the paired lymphocytes of the patient. after 15 days, the patient returned with worsened dyspnea and an oxygen saturation of 87% with 2 liters / min oxygen. after the first cycle of chemotherapy, there was a dramatic improvement in oxygen saturation (96% without oxygenotherapy) and regression of the miliary metastases. egfr exon 20 insertions account for 4% of egfr mutations and are associated with a lack of sensitivity to egfr - tki in preclinical models and in patients [2, 3, 4 ]. another mutation in exon 20 conferring resistance involves a substitution of methionine for threonine at position 790 (t790 m). this alteration is found as a germ - line variant in 0.5% of never - smokers with lung adenocarcinoma and may confer genetic susceptibility to lung cancer. in fact, egfr germinal mutations are not looked for in routine practice. in the natural history of the disease, t790 m mutation could be present in a small subset of tumor cells and expand selectively under egfr - tki, leading to more than 50% percent of secondary resistance. the frequency of somatic t790 m mutation at baseline (before tki treatment) is controversial, and detection depends on the sensitivity of the molecular technique. studies have suggested that pre - existing resistance may be present at a very low frequency [6, 7, 8 ]. a study found 1 (0.54%) egfr t790 m mutation among 185 nsclc patients without egfr - tki treatment, using mutant - enriched pcr analysis, but not confirmed by direct sequencing. others have found frequencies of up to 35% with very high - sensitive molecular techniques [9, 10, 11 ]. usually, t790 m could not be detected by direct sequencing because of lack of sensitivity. one study reported 2 (0.83%) cases harboring the egfr t790 m mutation among 240 patients with egfr - tki - nave lung adenocarcinoma detected by sequencing but, contrarily to our case, with both germ - line and somatic t790 m mutation and without any other egfr mutation. t790 m mutation impairs the binding of egfr - tki to the egfr adenosine triphosphate - binding pocket, and emerging data suggest that t790 m change itself may potentiate oncogenic activation. patients whose tumors harbor somatic t790 m mutations before treatment had a shorter progression - free survival [9, 10, 11 ]. we underline the clinical importance of pretreatment t790 m mutation detection because the response to egfr - tki is less certain and could lead to a delay in the introduction of classical platinum - based chemotherapy. diffuse, random pulmonary metastases, including miliary metastases, are quite a rare presentation in nsclc and seem to be associated with adenocarcinoma subtype and egfr mutation, suggesting that egfr - tki may be the treatment of choice for such patients especially in the asian population [13, 14, 15 ]. a study reported 5 cases of never - smokers with lung adenocarcinoma with such a pattern of metastases. in the tumor cells of all 5 patients, egfr mutation gene sequencing identified a deletion in exon 19, and all 5 patients had a dramatic response to egfr - tki. in fact, clinical data about patients with lung adenocarcinoma harboring egfr t790 m mutations at diagnosis are not described. this case highlights the link between an uncommon radiological presentation of adenocarcinoma (miliary pattern) and the clinical importance of the detection of a pretreatment (baseline) t790 m mutation in nsclc patients with an activating egfr mutation. | the pretreatment detection of an activating mutation of egfr is now routinely performed in metastatic nonsquamous non - small cell lung cancer (nsclc). the therapeutic impact of such a detection is major, as patients with advanced nsclc exhibiting a mutation of exon 19 or 21 will benefit from egfr - tyrosine kinase inhibitors (tki). the presence of an egfr resistance mutation, such as t790 m in egfr - tki - nave patients, is seldom looked for and is related either to a germinal mutation or to somatically mutated subclones. it has a negative predictive impact. we present the case of a patient with a lung papillary adenocarcinoma and miliary intrapulmonary metastases whose tumor displays a somatic complex heterozygous egfr mutation, combining l858r (exon 21) and a primary resistance mutation t790 m (exon 20), both detected by direct sequencing. |
one of the most serious complications of chronic liver disease is hepatocellular carcinoma. across the world, it is the 4th most common cancer (age standardised rate of 16 per 100,000) and the 3rd most common cause (age standardised rate of 14.6 per 100,000) of deaths from all cancers, accounting for 700,000 deaths per annum. although a global cancer, it is especially prevalent in the asia pacific and sub - saharan africa. the outcome of a patient after the discovery of hepatocellular carcinoma, like many malignancies, very much depends on the stage of the disease at the time of diagnosis. curative treatment can be offered to 30% of cases if diagnosed at bclc stage 0 or a and carries a 5-year survival of 40% to 70%. if diagnosed at stages b or c, the median survival with treatment is 1120 months. at stage d, unfortunately, the lack of symptoms for most part of the natural history of hepatocellular carcinoma is such that many cases are discovered at late stages, limiting the survival of these cases. one of the main strategies for prolonging survival in patients with chronic liver disease therefore lies in the diagnosing hepatocellular carcinoma in the early stages so that effective therapy can be offered. in this regard, screening for hepatocellular carcinoma, that is, detection before the onset of symptoms, forms the backbone of such a strategy. screening refers to the detection of a condition whilst it is still without sign or symptom. the primary aim of screening is to pick up a disease at a stage where treatment is more effective and the outcome, usually measured as survival, is better compared with a later stage of discovery of the condition. the world health organisation in 1968 published the following desirable criteria for a condition to be screened. the total cost of finding the case should be economically balanced in relation to medical expenditure as a whole. case finding should be a continuous process, not just a once and for all project. paraphrasing the above, the ideal screening programme should meet the following criteria : a sufficiently high prevalence of the disease in the population to be screened ; a reliable method for screening, that is, it should have a high true positive rate or low false negative rate (high sensitivity) and a high true negative rate or low false positive rate (high specificity) ; the method of screening should also be easily available, inexpensive, and carries little or no risk of harm to the individual screened ; an effective treatment should be available for the disease to positively impact on the survival of the individual. currently the hepatocellular carcinoma screening programmes advocated by various expert bodies have a high degree of concurrence. the ensuing discussion will review the merits of hepatocellular carcinoma screening vis - - vis the desired characteristics of an ideal screening programme as listed above. the risk of developing hepatocellular carcinoma in this group is over 200 times that of the general population. the risk factors are liver cirrhosis (where the repeated inflammation - necrosis - regeneration cycle increases the risk of carcinogenesis) and chronic hepatitis b virus infection (where the incorporation of hepatitis b virus genome into the hepatocyte dna increases the risk of carcinogenesis). the risk of hepatocellular carcinoma development varies across these conditions and depends on several factors : age, gender, presence of family history of hepatocellular carcinoma, exposure to environmental factors such as aflatoxins, and aetiology of cirrhosis. hepatitis c - related cirrhosis appears to be associated with the highest risk of hepatocellular carcinoma, with a 5-year cumulative risk of 1730%. haemochromatosis - related cirrhosis carries a 5-year cumulative incidence of 21% whereas hepatitis b - related cirrhosis has a 5-year cumulative incidence of 1015% depending on endemicity. the corresponding figures for alcoholic cirrhosis and advanced primary biliary cirrhosis are 8% and 4%, respectively. individuals with chronic hepatitis b virus infections without cirrhosis have a 0.5% annual risk of hepatocellular carcinoma. in this group the risk is higher with advancing age, in men and in those with a family history of hepatocellular carcinoma. women are at lower risk but the risk is increased in women above the age of 50 years old [7, 8 ]. chronic hepatitis b patients with liver cirrhosis have an annual risk of hepatocellular carcinoma of 38%. in patients with liver cirrhosis caused by hepatitis c virus infection or advanced primary biliary cirrhosis (stage 4), cirrhosis caused by other aetiologies such as genetic haemochromatosis and alpha-1-antitrypsin deficiency carry with it a lower annual risk of hepatocellular carcinoma of around 1.5% [913 ]. it is clear that hepatocellular carcinoma can develop from cirrhosis arising from nonalcoholic fatty liver disorder. the effectiveness of a screening strategy can be measured by improvement in survival against the cost incurred to achieve this outcome (cost for each year of life gained). other outcome measures such as quality of life gained, years of economically viable life gained are important but are more difficult to assess. the thresholds for each of these measures will vary according to cultures, individual outlook, and economic status of a country and are arbitrary. utilising the principles of decision analysis and costeffectiveness, the generally accepted threshold for life gain is 3 months, achieved at a cost of less than us $ 50,000 per year of life gained. applying this to decide on the threshold for screening hepatocellular carcinoma, the annual incidence for screening noncirrhotic patients is to be 0.2% and above, and for cirrhotic patients, this translates to an annual risk of 1.5% and above. the difference in the threshold incidences between noncirrhosis and cirrhosis lies in the lower quantum of life gain when hepatocellular carcinoma is diagnosed in an individual with liver cirrhosis. based on the above assumptions and thresholds, there is no justification for population - wide screening of hepatocellular carcinoma. the at - risk groups meeting the above cost - effective criteria for hepatocellular carcinoma screening are patients with liver cirrhosis, male patients with chronic hepatitis b virus infection without cirrhosis who are above the age of 40 years, female patients with chronic hepatitis b virus infection without cirrhosis who are above the age of 50 years, patients with chronic hepatitis b virus infection who have a family history of hepatocellular carcinoma. for younger individuals with chronic hepatitis b virus infection, the annual risk of hepatocellular carcinoma development is lower than the threshold of 0.2%, but the disease is often more aggressive. whether the above thresholds should be used to decide if screening should be recommended for this group should be discussed. whilst not costeffective, the discovery of an early tumour in this age group brings about the greatest potential gain in terms of survival and also economically useful years gained, not to mention the less tangible but no less important benefit of averting the trauma for the patient and family of coming to terms with a fatal cancer in a young individual. the risk of hcc in patients with chronic hbv infection can also be further stratified using prognostic variables age, gender, indices of necroinflammation alanine transaminase level, and hbv dna load. a prognostic scoring system was developed and validated in a large asian population with chronic hbv and gives the risk of hcc development at 3, 5, and 10 years. this scoring system has the potential to be applied to refine the decision - making process with respect to screening in less clear - cut situations, for example, in the group discussed above. currently available methods to diagnose hepatocellular carcinoma comprise blood tests to detect elevation of tumour markers and imaging modalities. blood tests that are elevated with hepatocellular carcinoma include afp (alpha - fetoprotein), the more specific afp - l3 (l3 subfraction of afp), and dcp (descarboxy prothrombin) [1921 ]. afp is released by regenerating hepatocytes, malignant hepatocytes, and also extrahepatic sources such as placental cells and germ cells. among the nonmalignant causes that cause elevated afp are inflammatory liver conditions, pregnancy, and molar pregnancy. the afp - l3 is the glycosylated subfraction of afp and is specific to malignant hepatocytes. it is useful in discriminating between afp elevation arising from a benign condition against that arising from hepatocellular carcinoma. however its sensitivity is low in cases where the afp is not markedly elevated. dcp is also specific to hepatocellular carcinoma ; it is found especially elevated when there is invasion of vascular structures and is a marker of more advanced hepatocellular carcinoma, and therefore may not be a suitable screening tool. afp, taken at a level of 20 ng / ml, has a sensitivity of 66% and a specificity of 82% for hepatocellular carcinoma. whilst specific, it will likely suffer from decreased sensitivity and is not recommended as a general screening tool. more recently the highly sensitive afp - l3 (hs - afp - l3) was evaluated in individuals whose afp was < 20 ng / ml. it was found to increase the sensitivity of detecting hcc from 7% with afp - l3 to 41.5% with hs - afp - l3. in addition, it also predicted poorer outcomes in patients with hcc. at present afp - l3 and hs - afp - l3 remain adjunctive tools in further evaluation in cases of raised afp and is not in a position to supplant the use of afp as a screening tool. liver imaging modalities that have proven effective in detecting hepatocellular carcinoma are ultrasound, ct scan, and mri scan of the liver. in trained operators, it is a test that is generally reproducible, does not carry any adverse effects, and is economical. one limitation of ultrasonography is the difficulty in obtaining a good study in obese patients. ct scan of the liver and mri of the liver provide diagnostic details superior to ultrasound. its cost has decreased with economies of scale. however, the radiation exposure is significant, raising the risk of carcinogenesis if used repeatedly. whilst advocated by some for screening of hepatocellular carcinoma, it is still not yet accepted and therefore not recommended for screening., it is an expensive test, and the acquisition time for one study is considerable, limiting its use as a screening test. at present the use of wither mri or ct scan of the liver lies in the diagnosis of hepatocellular carcinoma where the screening tests (either ultrasound or afp) have flagged up suspicion. at present the interval of screening should be such that the growth of cancer should be picked up between 2 screening. too long an interval, in the other hand, runs the risk of allowing the cancer to grow to a later stage, thereby compromising the effectiveness of the whole screening process. the determinant of this interval is the rate of growth or doubling time of the cancer. studies involving hcv patients suggest that 12-month screening interval brings about survival improvement and is not different from screening at 6-monht intervals [23, 24 ]. in studies of hbv patients, the last 1015 years have witnessed the advent of newer treatment options for hepatocellular carcinoma, and with it, some measure improvement in outcomes. with early diagnosis, cure is possible in 30% of cases, and in the rest, effective control is achievable. surgical resection and local ablation are effective in the treatment of an early, localised hepatocellular carcinoma, and achieving 5-year survival of up to 70%. liver transplant in well - selected patients can bring about a 5-year survival in the order of 80% [26, 27 ]. recent data indicates that rfa is comparable to surgical resection for early hepatocellular carcinoma in terms of survival outcomes and has the advantage of being less invasive [28, 29 ]. transarterial chemoembolisation is an option proven to prolong survival in cases of nonresectable, nontransplantable cases of nonmetastatic hepatocellular carcinoma. hcc screening therefore is an important part of the strategy in improving survival in patients with advanced liver disease. | hepatocellular carcinoma is one of the most serious complications of chronic liver disease and is the third most lethal cancer worldwide. symptoms emerge very late in the course of its natural history with an attendant poor outcome. screening is of paramount importance in a successful strategy to treat hepatocellular carcinoma. a successful screening program rests the availability of an at - risk population, reliable diagnostics tests that are able to diagnose a condition at a stage where effective, and relatively simple and acceptable treatments are available. in hepatocellular carcinoma, all patients with liver cirrhosis or chronic hepatitis b virus infection are at risk. six monthly ultrasound and alpha - foetoprotein determination form the backbone of the screening program. newer modalities and tests show promise but have not supplanted the standard tests. |
striking differences between physics and biology have important implications for interdisciplinary science, technology, engineering, and mathematics (stem) education. the research group in which i work, the center for nonlinear dynamics at the university of texas at austin, is converting into the physics department home for biological physics. many of my collaborations have been with faculty in engineering. for the past 15 years, i have been codirector of the program at the university of texas at austin that prepares secondary science and mathematics teachers (uteach, 2012). the future teachers take a course on scientific research i developed and deliver together with colleagues from biology, astronomy, chemistry, and biochemistry (marder, 2011). this background naturally makes me an enthusiastic advocate of interdisciplinary education at the secondary and undergraduate levels. yet at the same time, i am worried by some features of what may be coming. these worries have to do with what can happen as we are all lumped together under the heading of stem. undergraduate education in biology is confronted with the rapid development of the field and the urge to insert more and more recently discovered facts and ideas into introductory courses. almost all physicists are happy to teach an introductory course whose structure has not changed much since 1960, and for which the content was developed before 1930. indeed physics courses make only occasional excursions past material developed by 1960 up through the second year of graduate school. the reason is not just that physicists are resistant to change, but that the material we view as central simply has not changed for decades or centuries. our educational conservatism has some great advantages. knowing with certainty the topics physics students will study at every level has aided the development of physics education research (per central, 2012), which has developed an impressive body of knowledge on instructional strategies that help students learn the best. but it means that physics will naturally resist presenting its topics in new interdisciplinary combinations. the measured pace at which physics curriculum evolves may finally be sped up by the emergence of stem. this acronym seems a benign and catchy way to market science, technology, engineering, and mathematics in pursuit of improving u.s. competitiveness. it also implies a challenge to physics that first hit me a few months ago as i listened to state officials in texas explaining why undergraduate institutions should shut down inefficient parts of stem, such as physics, so as to get more stem graduates from efficient parts of stem, such as biology (reich, 2011). as we market stem, physics and biology even risk coming into opposition, and physics is already in a weak position, partly due to developments in science education that emerged from the struggle over intelligent design. the debate concerning the teaching of evolution influenced the way that secondary teachers and secondary students understand the nature of science. creation science and intelligent design presented themselves as scientific, so dealing with the attempt to inject them in public schools meant carefully defining science and explaining it well to the public (kitzmiller v. dover, 2005). it reacted to visions of what science was not, particularly the nonscience angling to enter biology textbooks. thus, the nature of science presented to school teachers emphasizes methodological naturalism, the essential role of empirical evidence, science as social construction, and the tendency of science to change over time (evolution and the nature of science institutes, 2012). guarding teachers from intelligent design even evolved into an academic subdiscipline with specialized vocabulary, such as law and theory. for example, understanding the fundamental distinctions and relationships between laws and theories is essential in fully appreciating and evaluating the work of scientists while gaining fluency in the language of science (mccomas, 2003). i look at examples from physics, such as newton 's laws (fundamental theory of motion), curie 's law (an obscure empirical result in magnetism), and the quantum theory (fundamental theory of matter), and see words mainly attached to results through historical accident. passing a vocabulary quiz on law and theory is far from understanding science (wong and hodson, 2008, 2010). given the struggle to maintain the teaching of evolution in schools, i understand why science is defined as it is. but from the vantage point of physics, it creates problems. once students have received a good secondary education in the nature of science, they instinctively reject much of what physicists do as nonscientific. after 10 years teaching undergraduate scientific methods to future science and mathematics teachers, i have learned that, when asked to reason or experiment like physicists, many undergraduates rebel. especially for theoretical physicists, but also for many experimentalists, a great deal of scientific life is occupied with mathematics. indeed, theoretical physics, as well as theoretical chemistry, theoretical biology, parts of mechanical and chemical engineering, and other fields, becomes indistinguishable from applied mathematics. resting in the back of most researchers minds is the idea that at some point their calculations will be compared with experimental work and most papers feature in the end some comparison but the authors time is spent almost exclusively on mathematical calculations and argumentation. much of theoretical high - energy physics in the last 50 years has been carried out in advance of the experiments that may or may not eventually prove it correct. did the theoretical work on the higgs boson only become science once the european organization for nuclear research found the particle ? will calculations of black hole collisions only become science if the collisions are someday observed and the calculations prove correct ? these areas of work have been science from the start, a mode of science focusing on mathematical computations with the possibility of a future comparison with experiment. this mode of science can be carried out at many levels, not just by candidates for the nobel prize. for example, given a prism made of wood and careful measurements of its weight and dimensions, one can compute the location of the water line when one floats it in a bucket. the act of carrying out these computations is a legitimate part of science, one that also uses secondary mathematics. my experience leads me to the undesired conclusion that lots of students do not like it. many mathematics students dislike being pressed to employ ideas as a tool that they were content to know just because they were pretty. for example, i happen right now to be engaged in performing computations of the energy of a certain sort of antiferromagnet as a function of the strength of magnetic fields applied in two directions. colleagues and i will compare these computations with experiments in which neutron beams bounced off magnetic samples under conditions of varying magnetic field (muehlbauer., 2011). searching the entire literature of condensed matter, the largest subfield of physics t tests, p values, and the like (marder, 2010). in much of condensed matter physics, gathering data is cheap once an experiment is operational at all, so the response to random error is to take enough measurements to drive uncertainty down to desired levels. the scientific problems have to do with systematic error, identifying effects that explain functional relations. hypotheses are useful when a complex, noisy system is exposed to a small number of distinct treatments, and one wants to characterize their effects without knowing in detail how they work. hypotheses become irrelevant when a system amenable to detailed mechanistic analysis is exposed to a continuous infinity of treatments. as an example of a very simple case in which one can see these two modes of science in action, consider constructing an experiment with a light bulb and a light intensity sensor. a biological approach to this system might be to say my hypothesis is that when the bulb is farther away the light will be dimmer. this idea would lead to an experiment in which a light bulb is placed at two different locations and intensity is measured multiple times. a low - quality version of the experiment would simply ask which case led to higher mean light intensity, while a higher - quality version would add a t test for significance. i have seen biologists nod contentedly at such a description of student - directed scientific progress, but physicists start to squirm. in fact, with a little geometry, and using the concept of conservation of energy, the student should be able to predict the precise mathematical form of the functional dependence of light intensity upon distance to the sensor. for heaven 's sake, do that, put some error bars on the measurement, and compare it with the expected power law. it has often seemed to me that the difference in the approach to problems of physicists and biologists is great enough that, when given a battery and a bulb, a biologist will design a biology experiment and, when given a frog, a physicist will design a physics experiment (geim, 1998). in the battle for the hearts and minds of secondary school students, many times, while i help college students construct what seems to me a perfectly good physics experiment, i see them turn and say almost in anguish, but i do n't know what my hypothesis is ! students arrive in college with the strong intuition that science requires a hypothesis, an experiment, and minimal mathematics. this singular view of the nature of science is creating several problems that lead back to stem. hidden within the s of science are wildly different modes of thought and action, all of them important. despite the superficial glamor of physics, its mental processes are unfamiliar to most of the public and scarcely recognized as science at all this may be part of the reason that physics enrollments are now at low enough levels that public universities may be increasingly unable to support undergraduate physics programs (hodapp, 2011). few entering undergraduates can imagine what they could learn in physics that would be of any use. the response to this situation is that physicists will have to be more deliberate in teaching and explaining important features of their discipline, just as biologists have long had to do. there is even a threat, although more remote, to mathematics instruction at the secondary level. deprived of contexts such as physics, mathematics at the level of algebra and above threatens to degenerate into a game. the public may not put up with the high stakes attached to this game forever (hacker, 2012). the opportunity is to identify a common core of scientific practices that integrate science, mathematics, engineering, and technology, and make this core a goal for every educated citizen. the common core standards for mathematics and the next generation science standards (common core state standards initiative, 2012 ; achieve, 2013) appear to point in this direction. physics has a contribution to make that is separate from the inviolable list of topics from the introductory physics course. the way that physicists use mathematics in combination with experiment to construct causal models of the world should be part of the common core. we the scientists, mathematicians, and engineers who will be asked to help implement the new standards do not ourselves always possess the full set of skills that stem education will ask of our students. we will struggle to prepare our own undergraduates, as well as current and future secondary teachers, to understand a curriculum broader than what we know. | everyone loves stem, the acronym for science, technology, engineering, and mathematics, that sits in every call to improve u.s. competitiveness. but hidden within science are many different ways of thinking and acting, and unless we protect them separately, the whole scientific enterprise may be at risk. |
atherosclerosis is a serious chronic inflammatory disease of a multifactorial nature, characterized by thickening and loss of elasticity of the arterial walls associated with the presence of atheromas (1 - 7). atheromas are calcified plaques especially composed of lipids and fibrous tissue, which are deposited on the walls of blood vessels (3). when atherosclerosis affects the arteries that supply the brain, i.e., the carotids, strokes can happen (3, 6, 8 - 10). cerebrovascular accident (cva) is the third factor of death in most countries and high costs are annually spent for mental and physical rehabilitation of patients who survive after cva. according to statistics, about 60% of patients who survive, some of these risk factors include hypertension, myocardial infarction (mi), high cholesterol smoking and diabetes (3, 4, 6, 8). the reduced estrogen level and other aging processes associated with menopause in women above 50 years of age are factors that increase the risk of stroke (3, 13). eighty percent of strokes are ischemic and atherosclerotic disease in the region of carotid artery can cause it (6, 8, 11, 12). therefore, if we can trace these lesions, the possibility of preventing strokes will increase considerably. if atherosclerotic plaques are completely or incompletely calcified in the bifurcation of carotid artery, it will be possible to trace them on radiographs. it can be detected on dental panoramic, lateral cephalometry and postero - anterior skull view (13, 14). friedlander and lande described the possibility of diagnosing calcified atheromatous plaque in the carotid artery on dental panoramic images for the first time (19). panoramic radiographs are used routinely in the evaluation of patients with dental problems (10). suggested panoramic radiographs for detecting carotid artery calcifications (cac) to prevent cva (16). described radiopaque nodular lesion (or lesions) as cac, which is separate from the hyoid bone and is adjacent to the cervical vertebrae in the space between c3 and c4 or below it (20). to detect this calcification, we should consider other anatomic and pathologic radiopacities in this area as the differential diagnosis. some of these include the hyoid bone, styloid process, epiglottis, soft palate, tongue, ear lobe, anterior tubercle of the atlas vertebra, stylohyoid and stylomandibular ligaments, thyroid cartilage, triticeal cartilages, and even calcified acnes (10). most of these are readily distinguishable on the basis of location and morphologic features (15). in the differential diagnosis the best way to differentiate atheroma from triticeal cartilage is by considering the fact that atheromas have a more lateral anatomic localization (10). in this study, we are going to study the association of cac on dental panoramic radiographs and two risk factors of cva including hypertension and mi. it should be mentioned that all of these patients needed panoramic radiographs for dental treatment procedures and none of these panoramic images were taken for the purpose of cac detection. therefore, the indication for panoramic request was dental necessity. we provided these radiographs under similar conditions and used conventional panoramic equipment (planmeca proline pm 2002 cc, helsinki, finland), 6 - 8 ma current, 18 seconds, 64 - 66 kvp voltage (based on the size of patient s jaw), agfa gevaert nv ortho cp - g plus (1530 cm) extraoral film (green sensitive universal film) and kodak lanex regular intensifying screens. films were developed and fixed in an automatic film processor (gendex, clarimat 300, london, uk). we evaluated radiographs that had a acceptable diagnostic quality (not overexposed and not underexposed) and cervical vertebrae c3-c5 were observable in them. we diagnosed cac as nodular radiopacity at or below the intervertebral space between c3 and c4. for the differential diagnosis of cacs, other cervical calcifications, as we explained before, were excluded according to carter s study (16). a hypertensive patient was defined as a patient who takes antihypertensive medication or a patient with a systolic blood pressure higher than 140 mmhg and/or a diastolic blood pressure higher than 90 mmhg in two separate evaluations. among 200 studied patients, 52 cases (26%) suffered from hypertension, four (2%) had a history of mi and 22 patients (11%) demonstrated cac on panoramic radiographs (figures 1 and 2). among 52 patients who suffered from hypertension, 11 patients (21.2%) showed cac on dental panoramic radiographs, while among 148 patients who did not have hypertension, only 11 patients (7.4%) demonstrated cac on dental panoramic radiographs (p < 0.001). in other words, the relationship between hypertension and cac in these samples was significant. three patients with a history of mi demonstrated cac on panoramic images (75%), while among people without such a history (196 patients), only 19 (9.7%) had cacs. we observed that the relationship between mi and the occurrence of carotid artery calcification was significant (p = 0.004). the prevalence of cac on panoramic radiographs is detected in different studies and the prevalence has been reported as % 5, % 6.2, % 9.4, % 13, and % 38.8 (1 - 5). a considerable rate of mortalities in the united states refers to strokes that occur in women after the age of 50 (14). atheromas often have been detected on the panoramic radiographs of neurologically asymptomatic male veterans ; however, similar studies have not been conducted among female veterans (14). reported that there is no statistically significant association between the presence of cac on panoramic radiographs and the presence of a significant carotid stenosis or the degree of carotid stenosis (4). in a study conducted by masood. a study carried out by uthman. mentioned that people suffering stroke, had a greater prevalence of cac on panoramic radiographs (8). cohen. demonstrated that 86% of the patients who had cac on dental panoramic images had at least one vascular risk factor (13). friedlander introduced hypertension as the most important risk factor for atherosclerosis in menopausal women, and a significant relationship has been reported between hypertension and the occurrence of carotid artery calcification (15). but ohba from japan in a study on 80-year - old japanese patients found no relation between systolic or diastolic hypertension and the existence or non - existence of cac (11). in our study, this association was completely significant. also, we obtained such a strong association between mi history and cac on panoramic images. cohen. in a study on 73 patients with cac in panoramic radiography observed that 11% had a history of mi, 7% had a history of cva, and 15% died because of vascular accidents. so they concluded that the existence of carotid artery calcification on dental panoramic radiographs could be a strong marker to predict cva (13). thus, the existence of cac on panoramic radiographs along with cva risk factors such as hypertension and a previous mi can be considered as important symptoms for predicting stroke. it should be mentioned that in our studied patients, only four patients had a history of mi, but it was interesting that most of them showed cac on the panoramic radiographs. so nevertheless, we found association between mi and cac on panoramic images, but it is not enough and further studies with more patients reporting a history of mi is recommended. the difference in results among the mentioned studies and also our study can be related to the differences in age, gender and ethnicity of the sample groups. since panoramic radiographs are routinely indicated for dental patients, dentists can evaluate panoramic images to find carotid artery calcification and refer these patients to specialists to prevent all morbidity and mortalities due to strokes and other adverse vascular events that could force high costs for the healthcare system. therefore, training general dentists in order to diagnose this calcification on panoramic radiographs is important. besides dental problems the identification of cac on dental panoramic radiographs must be performed by an experienced evaluator. almog demonstrated that the probability of wrong diagnosis was high when the dentists didnot have enough experience and specialty to detect cac on panoramic radiographs (17). wrong diagnosis can result in enforcing unnecessary considerable costs (for example angiography) to the patient and the society. demonstrated that 50% of the patients who had cac on their panoramic radiographs had a higher than 50% carotid artery stenosis. when panoramic radiographs do not show calcification, the percentage of vessel stenosis will be only 21% (14). panoramic radiographs are not as useful as doppler ultrasonography and ct in detecting atherosclerotic plaques in the carotid arteries and especially stenosis of the vessels, but it is a very cheap and non - invasive method in comparison to other imaging methods and they are commonly used in dental patients. panoramic radiographs may help us to detect cacs in patients with or without other associated risk factors (10). first, identification of mi based on history is not as certain as ecg findings, but it was not possible in our study to detect mi with other methods such as ecg. second, unfortunately, the number of patients with a history of mi in our study was small ; therefore, more investigations in greater sample sizes are necessary. third, some cases of carotid plaques are not calcified so they can not be detected on radiographs. however, the diagnosis of calcified ones can have an important role in preventing cva. in our study, the prevalence of cac was 11% and it shows the importance of panoramic radiograph evaluation in detecting cac. there is a significant relationship between the presence of cac on panoramic radiographs and the two other cva risk factors including hypertension and mi history, so a detailed evaluation of panoramic radiographs is very important. taking panoramic radiographs just to detect cac is not recommended, but patients should be referred for further evaluation when cac is detected on dental panoramic radiographs. dental panoramic radiographs should be carefully examined in the area of the carotid artery in not only patients with systemic disease, but also asymptomatic patients. | background : carotid artery calcification may be related to cerebrovascular accident, which may result in death or physical and mental disabilities in survivors.objectives:our purpose is to study the association of carotid artery calcification (cac) on dental panoramic radiographs and two risk factors of cerebrovascular accident (cva) including hypertension and myocardial infarction (mi).patients and methods : panoramic images of 200 patients that were all women above 50 years of age (a population suffering from vascular diseases) were investigated. all panoramic images were provided under similar conditions in terms of the type of panoramic radiograph equipment, type of applied films and the automatic film processor. then, the patients answered questions about mi history and taking antihypertensive drugs. we also measured the blood pressure of patients in two separate surveys. data analysis was performed by spss statistical program. we used exact fisher test and chi - square test at a significant level of less than 0.05 to study the effect of these variables on the occurrence of carotid artery calcification.results:among 200 studied samples, 22 of the patients (11%) had carotid artery calcification on the dental panoramic radiograph. in total, 52 patients (26%) had hypertension and four people (2%) had a history of mi. eleven individuals among patients suffering from hypertension (21.2%) and three individuals among patients with a history of mi (75%) demonstrated cac on dental panoramic images.conclusions : the relationship between cac found on dental panoramic radiographs and two cva risk factors -- hypertension and mi-- was significant. therefore, it seems that detection of cac on panoramic images of dental patients must be considered by dentists. |
the combined capabilities of both a nonplanar design and nonconventional carrier injection mechanisms are subject to recent scientific investigations to overcome the limitations of silicon metal oxide semiconductor field effect transistors. in this letter, we present a multimode field effect transistors device using silicon nanowires that feature an axial n - type / intrinsic doping junction. a heterostructural device design is achieved by employing a self - aligned nickel - silicide source contact. the polymorph operation of the dual - gate device enabling the configuration of one p- and two n - type transistor modes is demonstrated. not only the type but also the carrier injection mode can be altered by appropriate biasing of the two gate terminals or by inverting the drain bias. with a combined band - to - band and schottky tunneling mechanism, in p - type mode a subthreshold swing as low as 143 mv / dec and an on / off ratio of up to 104 is found. as the device operates in forward bias, a nonconventional tunneling transistor is realized, enabling an effective suppression of ambipolarity. depending on the drain bias, two different n - type modes are distinguishable. the carrier injection is dominated by thermionic emission in forward bias with a maximum on / off ratio of up to 107 whereas in reverse bias a schottky tunneling mechanism dominates the carrier transport. |
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additional information about laboratory methods, study participants, and statistical analysis can be found in the supplementary data. hs - crp level was assessed in plasma from 750 patients : 540 with a confirmed genetic diagnosis of mody (220 hnf1a, 245 gck, 54 hnf4a, and 21 hnf1b), 53 patients with type 1 diabetes, and 157 patients with type 2 diabetes. hs - crp level was assessed in plasma from 750 patients : 540 with a confirmed genetic diagnosis of mody (220 hnf1a, 245 gck, 54 hnf4a, and 21 hnf1b), 53 patients with type 1 diabetes, and 157 patients with type 2 diabetes. subjects (29/750 ; 10.5%) had hs - crp results > 10 mg / l (n = 16 gck - mody, n = 10 type 2 diabetes, and n = 3 hnf4a - mody) and were considered to have an acute inflammatory response and excluded from further analysis. plasma hs - crp was significantly lower in hnf1a - mody than type 1 diabetes and type 2 diabetes (median [iqr ] 0.3 [0.10.6 ] versus 1.1 [0.51.85 ] mg / l [p 1.5 iqr) ;, extreme values (> 3 iqr). the data exclude all subjects with hs - crp levels > 10 mg / l. for discriminating hnf1a - mody from type 2 diabetes, receiver operating characteristic (roc) curve showed good discrimination (area under the curve [auc ] 0.84) and identified a cutoff hs - crp of 0.75 mg / l with 79% sensitivity and 71% specificity (supplementary fig. 1). this equates to a positive predictive value (ppv) of 2.7% and a negative predictive value (npv) of 99.7%, assuming a mody prevalence of 1% (7). hs - crp was less discriminative between hnf1a - mody and type 1 diabetes with a roc auc of 0.78 (79% sensitivity and 67% specificity at a cutoff of 1.5 iqr) ;, extreme values (> 3 iqr). the data exclude all subjects with hs - crp levels > 10 mg / l. for discriminating hnf1a - mody from type 2 diabetes, receiver operating characteristic (roc) curve showed good discrimination (area under the curve [auc ] 0.84) and identified a cutoff hs - crp of 0.75 mg / l with 79% sensitivity and 71% specificity (supplementary fig. 1). this equates to a positive predictive value (ppv) of 2.7% and a negative predictive value (npv) of 99.7%, assuming a mody prevalence of 1% (7). hs - crp was less discriminative between hnf1a - mody and type 1 diabetes with a roc auc of 0.78 (79% sensitivity and 67% specificity at a cutoff of 0.55 mg / l may warrant consideration of the other subtypes of mody. this result may prove to be helpful as an adjunct to identify hnf4a - mody patients, who are phenotypically similar to hnf1a - mody. current crp assays, used to identify inflammation, measure down to levels of 0.20.3 mg / l and, therefore, a specific hs - crp assay may not be necessary. it would be important to determine the comparability between routine crp and hs - crp assays before testing. a limitation of crp is that it will be raised in inflammatory states, reducing its potential use as a discriminative tool. in addition, certain medications reduce crp levels, such as statins (10), aspirin (11), and -blockers (12), which are believed in some cases to reduce crp by 2030%. we did not have drug history available on individual patients, so we were unable to assess the impact of pharmacological agents on our results ; however, owen. (5) showed that removing subjects on statins and/or aspirin in their study did not alter their results. in conclusion, we have confirmed that hs - crp levels are lower in patients with hnf1a - mody than other types of mody, type 1 diabetes, and type 2 diabetes. hs - crp is potentially a cheap, widely available biomarker that might aid in the cost - effective identification of patients with hnf1a - mody. | objectivematurity - onset diabetes of the young (mody) as a result of mutations in hepatocyte nuclear factor 1- (hnf1a) is often misdiagnosed as type 1 diabetes or type 2 diabetes. recent work has shown that high - sensitivity c - reactive protein (hs - crp) levels are lower in hnf1a - mody than type 1 diabetes, type 2 diabetes, or glucokinase (gck)-mody. we aim to replicate these findings in larger numbers and other mody subtypes.research design and methodshs - crp levels were assessed in 750 patients (220 hnf1a, 245 gck, 54 hnf4- [hnf4a ], 21 hnf1- (hnf1b), 53 type 1 diabetes, and 157 type 2 diabetes).resultshs - crp was lower in hnf1a - mody (median [iqr ] 0.3 [0.10.6 ] mg / l) than type 2 diabetes (1.40 [0.603.45 ] mg / l ; p < 0.001) and type 1 diabetes (1.10 [0.501.85 ] mg / l ; p < 0.001), hnf4a - mody (1.45 [0.462.88 ] mg / l ; p < 0.001), gck - mody (0.60 [0.301.80 ] mg / l ; p < 0.001), and hnf1b - mody (0.60 [0.102.8 ] mg / l ; p = 0.07). hs - crp discriminated hnf1a - mody from type 2 diabetes with hs - crp < 0.75 mg / l showing 79% sensitivity and 70% specificity (receiver operating characteristic area under the curve = 0.84).conclusionshs - crp levels are lower in hnf1a - mody than other forms of diabetes and may be used as a biomarker to select patients for diagnostic hnf1a genetic testing. |
tissue homeostasis is characterized by the balance between proliferation and cell growth on one side and cell death on the other side. in response to stressful stimuli, however, in cancer cells activation of pathways that favor cell survival instead of cell death under stressful conditions may contribute to tumorigenesis. in addition, this adaptive stress response promotes the development of acquired resistance, since current treatment approaches such as chemotherapy and irradiation trigger cellular stress pathways, and thus, initiate the activation of survival cascades and anti - apoptotic mechanisms. apoptosis or programmed cell death is the cell 's intrinsic death program that regulates various physiological as well as pathological processes and that is evolutionary highly conserved. hence, further insights into the molecular mechanisms of how cellular stress signals trigger anti - apoptotic mechanisms and how this contributes to tumor resistance to apoptotic cell death are expected to provide the basis for a rational approach for the development of new molecular targeted therapies. there are two major apoptosis signaling pathways, that is, the death receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway. under most circumstances, activation of either pathway eventually leads to proteolytic cleavage and thus activation of caspases, a family of cysteine proteases that act as common death effector molecules. accordingly, caspases are responsible for many of the biochemical and morphological hallmarks of apoptotic cell death by cleaving a range of substrates in the cytoplasm or nucleus. ligation of death receptors of the tumor necrosis factor (tnf) receptor superfamily such as cd95 (apo-1/fas) or trail receptors by their corresponding natural ligands, that is, cd95 ligand or trail, results in the recruitment of caspase-8 into a multimeric complex at the plasma membrane, the death - inducing signaling complex (disc) [6, 7 ]. this in turn leads to caspase-8 activation, which can then directly cleave downstream effector caspases such as caspase-3. alternatively, caspase-8 can promote outer mitochondrial membrane permeabilization by cleaving bid, a bh3-only protein that translocates to mitochondria upon cleavage and causes cytochrome c release. the mitochondrial pathway is initiated by the release of apoptogenic factors such as cytochrome c, apoptosis - inducing factor (aif) second mitochondria - derived activator of caspase (smac)/direct iap binding protein with low pi (diablo) or omi / high temperature requirement protein a (htra2) from the mitochondrial intermembrane space into the cytosol. the release of cytochrome c into the cytosol triggers activation of caspase-3 via the formation of a large cytosolic complex, which is called the apoptosome and consists of cytochrome c, apaf-1, and caspase-9. smac / diablo or omi / htra2 promotes caspase activation by binding to inhibitor of apoptosis (iap) proteins and thereby disrupts the interaction of iaps with caspase-3 or -9 [9, 10 ]. therefore, cancer cells react to cellular stress signals by mounting an anti - apoptotic response, which enables cancer cells to evade apoptotic cell death and ensures cell survival. a wide range of stress signals has been identified, which may evoke a cell survival program in case of sublethal damage, while cell death is usually initiated if the damage is too severe, that is, starvation, hypoxia, dna damaging drugs, irradiation, er stress, and reactive oxygen species just to name a few. the molecular mechanisms that initiate cell death upon cellular stress stimuli have often not exactly been identified and likely depend on the individual stimulus. for example, following exposure to genotoxic substances, damage to dna or to other critical molecules is considered to be a common initial event which is then transmitted by the cellular stress response to the activation of cellular effector systems such as the apoptotic machinery. various stress - inducible molecules, for example, jnk, mapk / erk, nf-b, or ceramide have been implicated in propagating the apoptotic signal [1315 ]. besides caspase - dependent and caspase - independent apoptosis, additional non - apoptotic modes of cell death also exist and have gained increasing attention over the last years, including necrosis, autophagy, mitotic catastrophe, and lysosomal cell death [16, 17 ]. while resistance to these cell death modalities can also contribute to evasion of cell death under stress conditions, the discussion of these alternative modes of cell death is beyond the scope of this review. a characteristic feature of human cancers is the evasion of apoptosis in response to stress stimuli, which contributes to both tumorigenesis and treatment resistance. in principle, cell death pathways can be blocked at different levels of the signaling cascade by upregulation of anti - apoptotic proteins and/or by downregulation or dysfunction of proapoptotic molecules. examples of altered apoptosis signaling pathways that contribute to stress resistance in human cancers will be discussed in the following paragraphs (figure 1). death receptors are part of the tumor necrosis factor (tnf) receptor gene superfamily, which comprises more than 20 proteins, for example, cd95 (apo 1/fas), trail receptors, and tnf receptor 1 (tnfri) [7, 19 ]. death receptors exert many different biological functions, including the regulation of cell death and survival, differentiation, and immune regulation [7, 19 ]. members of the tnf receptor family share a characteristic cytoplasmic domain called the death domain, which is pivotal for transducing the death signal from the cell 's surface to intracellular signaling pathways [7, 19 ]. signaling via death receptor can be impaired in human cancers via downregulation of receptor surface expression as part of an adaptive stress response. for example, in chemotherapy - resistant leukemia or neuroblastoma cells, downregulation of cd95 expression was identified as a mechanism of acquired drug resistance [20, 21 ]. for the apoptosis - inducing trail receptors trail - r1 and trail - r2, abnormal transport from intracellular stores such as the endoplasmatic reticulum to the cell surface rendered colon carcinoma cells resistant to trail - induced cell death. further, membrane expression of death receptors can be reduced by epigenetic changes such as cpg - island hypermethylation of gene promoters in response to stress signals [23, 24 ]. abnormal expression of decoy receptors presents an alternative mechanism of resistance to trail- or cd95-induced apoptosis. to this end, the decoy receptor 3 (dcr3), which counteracts cd95-mediated apoptosis by competitively binding cd95 ligand, was shown to be overexpressed in lung carcinoma or colon carcinoma and in glioblastoma [25, 26 ] and trail - r3 ; a decoy receptor for trail was reported to be expressed at high levels in gastric carcinoma. in addition, anti - apoptotic proteins with a death effector domain (ded) such as cellular flice - inhibitory protein (cflip) and phosphoprotein enriched in diabetes/ phosphoprotein enriched in astrocytes-15 kda (ped / pea-15) can be aberrantly expressed upon cellular stress [28, 29 ]. for example, high oxygen tension (hyperoxia) has been reported to lead to upregulation of cflip, which inhibited apoptosis during hyperoxia by suppressing both extrinsic and intrinsic apoptotic pathways, the latter via inhibition of bax. because of their sequence homology to caspase-8, both cflip and ped can be recruited into the death - inducing signaling complex (disc) upon receptor ligation instead of procaspase-8, thereby preventing caspase-8 activation [28, 29 ]. moreover, the expression of caspase-8 or its function is impaired by genetic or epigenetic mechanisms in various cancers. for example, caspase-8 mutations were identified in some tumors, that is, in colorectal and head and neck carcinomas, although the overall frequency is low [31, 32 ]. alternative splicing of intron 8 of the caspase-8 gene resulting in the generation of caspase-8l, a catalytically inactive splice variant presents another mechanism of caspase-8 inactivation [34, 35 ]. epigenetic silencing secondary to hypermethylation of regulatory sequences of the caspase-8 gene occurs in various tumors, for example, neuroblastoma, malignant brain tumors, ewing tumor, retinoblastoma, rhabdomyosarcoma, or small lung cell carcinoma [33, 3639 ]. furthermore, phosphorylation of caspase-8 on tyrosine 308 by, for example, src has been shown to interfere with its proapoptotic activity. the bcl-2 family of proteins consists of both anti - apoptotic proteins, for example, bcl-2, bcl - xl, and mcl-1, as well as proapoptotic molecules such as bax, bak, and bh3 domain only molecules. there are currently two models to explain the activation of bax and bak by bh3-only proteins. the direct activation model holds that bh3-only proteins, which act as direct activators such as bim and the cleaved form of bid (tbid), bind directly to bax and bak to trigger their activation, while bh3-only proteins that act as sensitizers, for example, bad, bind to the prosurvival bcl-2 proteins. according to the indirect activation model, bh3-only proteins activate bax and bak in an indirect fashion by engaging the multiple anti - apoptotic bcl-2 proteins that inhibit bax and bak, thereby releasing their inhibition on bax and bak [42, 43 ]. regardless of the exact mode of bax and bak activation, the ratio of anti - apoptotic versus proapoptotic bcl-2 proteins rather than the expression levels of one particular molecule of the bcl-2 family regulates apoptosis sensitivity. an increase in the ratio of anti- to proapoptotic bcl-2 proteins has been detected in various cancers and has been correlated to tumor cell survival and apoptosis resistance. more recently, bcl-2 has also been implicated in the regulation of the intracellular redox status. bcl-2 localizes to mitochondrial membranes as well as the endoplasmatic reticulum and the nuclear envelope, which are all sites of ros production. while bcl-2 has initially been described as an anti - oxidant because of its inhibitory effect on h2o2-induced lipid peroxidation, there is also evidence that bcl-2 may promote a prooxidant intracellular milieu. accordingly, ectopic expression of bcl-2 resulted in an elevated constitutive level of superoxide anion and intracellular ph in leukemia cells. conversely, reduction of intracellular superoxide sensitized bcl-2-overexpressing tumor cells to apoptotic stimuli independent of the mitochondria. these findings provide a link between oncogene - mediated alterations in the intracellular redox status and cell survival. besides bcl-2, also cytochrome c has been implicated in the redox regulation of apoptosis. once cytochrome c is released from mitochondria into the cytosol, it triggers formation of the cytochrome c / apaf-1/caspase-9-containing apoptosome, which in turn lead to activation of caspase-9 and downstream effector caspases. there is recent evidence that also the redox state of cytochrome c is involved in the regulation of apoptosis. to this end, the oxidized form of cytochrome c (fe(3 +)) has been reported to induce caspase activation via the apoptosome, while the reduced form of cytochrome c (fe(2 +)) is unable to do so [4951 ]. several mechanisms have been discussed to be responsible for this redox - mediated regulation of cytochrome c activity, including different affinities of the oxidized versus the reduced form of cytochrome c for binding to apaf-1, different abilities of these cytochrome c forms to activate apaf-1, or, alternatively, different affinities for other factors not belonging to the apoptosome. regardless of the exact mechanisms, this regulation of the redox state of cytochrome c opens the possibility of controlling the effector phase of apoptosis at a postmitochondrial level. besides these genetic alterations in bcl-2 family proteins, impairment of mitochondrial apoptosis may also occur at the postmitochondrial level. for example, expression level or activity of apaf-1 may be reduced due to promoter hypermethylation or loss of heterozygosity at chromosome 12q22 - 23, which in turn leads to impaired assembly of a functional apoptosome [5256 ]. moreover, tumor resistance to apoptosis may be caused by aberrant expression or function of inhibitor of apoptosis iap proteins are a family of endogenous caspase inhibitors with eight human members, that is, xiap, ciap1, ciap2, survivin, livin (ml - iap), naip, bruce (apollon), and ilp-2 [10, 57 ]. all iap proteins have at least one baculovirus iap repeat (bir) domain that is required for classification as iap family protein. this domain is also the region of the protein that mediates the interaction with caspases. among the iap family proteins, xiap exhibits the strongest anti - apoptotic properties and inhibits apoptosis signaling by binding to active caspase-3 and -7 and by preventing caspase-9 activation. the expression and function of iap proteins are tightly regulated by several mechanisms, among them is translational regulation. to this end, it is particularly interesting to note that xiap and ciap1 belong to the proteins, which are translated via an internal ribosome entry site (ires). this unique property enables protein translation of these iap proteins even under cellular stress conditions when protein synthesis is usually shut down, for example, because of caspase - dependent breakdown of eukaryotic translation initiation factors coupled with activation of the double - stranded rna - activated protein kinase pkr. however, the mrnas encoding xiap or ciap1 protein contain very long 5 untranslated regions (utrs), which are not amenable to a ribosome - scanning translation initiation mechanism and thus, require a cap - independent translation initiation mechanism, that is, ires - mediated translation. ires - mediated translation allows for the continued translation of xiap and ciap1 even under conditions where cap - dependent translation is inhibited such as cellular stress. in addition, ires - mediated translational regulation of xiap and ciap1 expression enables a rapid response to transient cellular stress conditions in order to delay cell death and ensure survival. of note, cellular stress signals, including low - dose irradiation, anoxia, serum starvation and chemotherapeutic drugs, have been reported to stimulate the ires activity of xiap or ciap1 [6366 ]. this is in line with the concept that such stress signals promote cell survival under stress conditions, at least in part, via ires - mediated upregulation of anti - apoptotic proteins. evasion of apoptosis is one of the hallmarks of human cancers that promote tumor formation and progression as well as treatment resistance. cellular stress signals can contribute to evasion of apoptosis by activating anti - apoptotic and cell survival programs that ultimately block cell death. this interference with proper apoptosis signaling under stress conditions can occur at different points of the apoptosis signaling network, for example, within the death receptor or the mitochondrial pathway or at the postmitochondrial level. whether or not cellular stress eventually engages cell survival or cell death programs also depends on the type and strength of the stress stimulus as well as the cell type. a better understanding of the molecular mechanisms of this interplay between the cellular stress response and anti - apoptotic programs is expected to yield novel molecular targets for therapeutic interventions. the aim is to prevent protective responses in order to maximize the antitumor activity of anticancer treatment approaches. | one of the hallmarks of human cancers is the intrinsic or acquired resistance to apoptosis. evasion of apoptosis can be part of a cellular stress response to ensure the cell 's survival upon exposure to stressful stimuli. apoptosis resistance may contribute to carcinogenesis, tumor progression, and also treatment resistance, since most current anticancer therapies including chemotherapy as well as radio- and immunotherapies primarily act by activating cell death pathways including apoptosis in cancer cells. hence, a better understanding of the molecular mechanisms regarding how cellular stress stimuli trigger antiapoptotic mechanisms and how this contributes to tumor resistance to apoptotic cell death is expected to provide the basis for a rational approach to overcome apoptosis resistance mechanisms in cancers. |
drug hypersensitivity syndrome (dhs), also known as drug rash with eosinophilia and systemic symptoms (dress), is a severe drug reaction. the syndrome can also be caused by other drugs, such as allopurinol, sulfonamides, beta - lactam antimicrobials, antidepressants, and nonsteroidal anti - inflammatory drugs. allopurinol hypersensitivity syndrome (ahs) is characterized by rash, fever, and internal organ involvement. we report a case of an exceptional presentation of agep as a manifestation of ahs. a 67-year - old male was admitted to the dermatology department with history of widespread skin eruption since 10 days. he was started on allopurinol (200 mg / day) for hyperuricemia 8 weeks earlier. clinical examination showed maculopapular eruptions involving trunk, arms and legs, as well as pinhead - sized follicular and nonfollicular pustules on patient 's face and trunk. laboratory investigations revealed a white blood cell count of 13.2 10/l with atypical lymphocytes and hypereosinophilia (eosinophil count 1.9 10/l). iu / l (reference range 340) and alanine aminotransferase 409 iu / l (345). barr virus, hepatitis b, c, hiv, parvovirus b19 and human herpesvirus 6. skin biopsy showed subcorneal spongiform pustules and some single scattered necrolytic keratinocyte [figure 1 ]. the superficial dermis was edematous, with mixed inflammatory infiltration, including numerous neutrophils and rare eosinophils consistent with the diagnosis of agep. subcorneal spongiform pustules and some single scattered necrolytic keratinocytes the patient improved markedly within 72 h on prednisone 30 mg daily (dose of 0.5 mg / kg body weight). full dose corticosteroid therapy was continued for two weeks, and tapered by 2.5 mg every 57 days, with a total duration of 3 months. causality assessment by the naranjo probability scale showed that ahs had probable causal association with the adverse effect. allopurinol hypersensitivity syndrome is a rare adverse reaction characterized by a spectrum of cutaneous reactions and systemic manifestations. ahs is more frequently associated with chronic renal insufficiency and concurrent use of thiazide diuretics. the exact pathogenesis of this syndrome is yet unclear, but different hypothesis are proposed. it seems to be related to the accumulation of allopurinol or oxipurinol a (major metabolite of allopurinol) in patients with renal insufficiency. immunological factors, genetic factors, and human herpes virus - type 6 are also implicated. cutaneous manifestations of hypersensitivity syndrome are heterogeneous, ranging from mild morbilliform exanthema to severe toxic epidermal necrolysis and stevens johnson syndrome. pustules in dhs differ from the typical pattern of agep, in which pustules are more numerous and predominant in main body folds. we scored both the european registry of severe cutaneous adverse reaction (regiscar) criteria for dress and euroscar agep score. the score was 7 for dress indicating a definite case and 5 for agep indicating a probable case. the clinical features which most differentiated dress from agep in our patient were the delayed onset (8 weeks), protracted course and multi - organ involvement. the first case of ahs with generalized nonfollicular pustulosis was described in a 47-year - old man. the second is a 65-year - old man who developed ahs manifested as generalized follicular pustulosis mimicking agep. allopurinol directly inhibits the lymphocytes enzyme purine nucleoside phosphorylase and may affect certain components of the immune system with an alteration of cd4/cd8 rate. the concomitant release of several mediators from eosinophils and/or activation of neutrophils might be important for the clinical evolution of lesions, with appearance of pustules, or aggravation of skin detachment, as well as internal organ involvement in dress. a drug reaction leading to folliculitis and then pustule formation has also been proposed. clinicians should be observant of cutaneous manifestations of hypersensitivity syndrome that may be a nonfollicular and/or follicular pustular eruption as well as the more commonly associated maculopapular rash or erythroderma. | allopurinol hypersensitivity syndrome (ahs) is a severe drug reaction. it is characterized by rash, fever, and internal organ involvement. it may present in different clinical forms. we present a case of acute generalized exanthematous pustulosis occurring as a manifestation of ahs. |
during the last decade much work has been done in the area of thyroid and pregnancy, and significant advances in the understanding of thyroid function modifications have been reached. a general agreement has developed in the potential relationship between different pregnancy pathologies, such as abortion, gestational hypertension, and diabetes, and even subclinical thyroid disorders such as subclinical hypothyroidism and the presence of thyroid antibodies (tai). in particular, a great amount of observations have clearly established that the presence of tai is associated with a significant increased miscarriage risk. furthermore, it has been demonstrated that the prevalence of tai in patients with recurrent miscarriage (rm) is higher with respect to that found in normal fertile control suggesting that tai should be considered as an independent indication of the risk of pregnancy loss [3, 4 ]. nevertheless, the exact mechanism underlying the association between tai and miscarriage remains a matter of debate, and a clear explanation for this phenomenon has not been clearly established. effectively, two possibilities for this association can be considered : immune dysfunction or mild thyroid abnormalities. some authors suggest that the most plausible hypothesis is that women with thyroid autoantibodies have an underlying, more generalized autoimmune activity, which leads to increased fetal losses [5, 6 ]. if this explanation is true, then it would be reasonable to think that the best therapy would require the modulation of the immune function. nevertheless, to date, no studies have demonstrated the efficacy of these therapies in preventing miscarriage in patients with tai. in addition, our previous study has demonstrated that treatment with high - dose immunoglobulin, a therapy which can influence the immune system, does not significantly improve the obstetric prognosis in patients with unexplained rm and tai. in fact, in agreement with other authors, we hypothesise that in women exhibiting tai the thyroid is less able to adapt to the increased requirements of pregnancy leading to an inadequate thyroid hormones release. from this point of view, it is plausible that the increased miscarriage rate in patients with tai could be due to a thyroid dysfunction, rather than a generalized overreaction of the immune system. the aim of the present study was to evaluate the role thyroid autoantibodies in patients with rm focusing on the study of thyroid function throughout a specific test. from january 2001 to january 2010, six hundred and thirty patients with a history of rm attended the outpatient clinic of the university of rome tor vergata. among these patients 46 patients with chronic diseases, with chronic ongoing treatments, or oral contraception were excluded from the study. thyroid function tests included ft4, ft3, and basal and trh - stimulated serum tsh (assessed 20 min after the trh bolus). a standard short trh test was performed in early follicular phase in addition to routine hormonal checks by giving 200 mcgr trh (trh biochem, ferring gmbh, berlin, germany) intravenously and measuring the tsh level at 0 and 20 minutes after bolus. in all studied subjects, trh test was performed at the 7th/8th day of the menstrual cycle after an overnight fasting at 8 am. serum tsh, ft3, and ft4 were measured using a supersensitive electrochemiluminescence immunoassay (roche, mannheim, germany). thyroperoxidase antibody and thyroglobulin antibodies were evaluated by means of an electrochemiluminescence immunoassay (roche, mannheim, germany). thyroperoxidase antibody and thyroglobulin antibodies were considered positive when values exceeded 65 iu / ml for tpo - ab and 115 ui / ml for tg - ab. according to previous studies, impaired response was defined when tsh trh - stimulated level was higher than 15 mui / ml 20 minutes after the bolus. as reported in figure 1, the vast majority of patients showed an abnormal trh - stimulated tsh response. in fact, 30 out of 46 patients (65%) showed a tsh trh - stimulated level higher than 15 mui / ml 20 minutes after the bolus with a mean tsh values of 17.8 8.1 however, as shown in table 1, the mean ft3 and ft4 levels were within the normal range in all cases. mui / l is advisable in women wishing to conceive [10, 11 ]. the present study supports the hypothesis that patients with rm and tai are characterized by a subtle thyroid dysfunction that worsening during early pregnancy can lead to an inadequate thyroid hormones release. in particular, our data demonstrated that in these patients conventional laboratory tests could not reveal this abnormality. in fact, normal ft3, ft4, and tsh levels were found in most of the patients with mean levels within the normal range. interestingly, an abnormal response to short trh stimulation test was found in the vast majority of our patients (65%). from our point of view, this test represents an important tool for the study of thyroid function in patients with rm. in this test basal and trh - stimulated serum tsh (assessed 20 min after the trh bolus) patients with abnormal trh - stimulated tsh response could be considered to have an impaired thyroid function. therefore, we suppose that this exam may represent a sensible test to identify patients in which the thyroid function is less able to adapt to the increased requirement of pregnancy even in presence of normal peripheral thyroid hormones ' concentrations. this hypothesis is supported by a research showing that women with unsuccessful pregnancy, at the time of abortion, displayed high basal and trh - stimulated tsh levels, with normal t4 serum concentrations, higher when compared to those observed among women with normal pregnancy. accordingly, in our previous study, we have shown that, in patients with rm and impaired response to trh test, thyroid replacement therapy can significantly improve reproductive outcome. therefore, the high incidence of an impaired trh test among patients with rm and tai supports the hypothesis that these patients are characterized by a reduced functional thyroid reserve leading to an inadequate concentrations of thyroid hormones necessary for successful pregnancy. as extensively reported, thyroid hormones have a crucial role in all phases of pregnancy. in the early stage of gestation, these hormones can influence the trophoblast endocrine function through direct stimulatory effects on the production and secretion of progesterone, estradiol, hgc (and), and placental lactogen [1315 ]. therefore, an inadequate thyroid hormone availability at the trophoblasts level can lead to an abnormal trophoblast endocrine function [1315 ]. moreover, different studies suggest a possible role of thyroid hormones in regulating apoptosis at throphoblast level. in particular, it has been shown that thyroid hormones suppress apoptosis downregulating the expression of fas and fas ligand in early placental extravillous trophoblast. in addition the importance of an adequate concentration of thyroid hormone for a normal placentation process is further supported by in vitro evidence demonstrating that thyroid can influence the invasiveness and the differentiation of cultured extravillous trophoblast cells hormones upregulating the expression of integrins and metalloproteases. later in pregnancy an impaired thyroid function has been associated with potential repercussions affecting the offspring. recent evidence has suggested that even mild thyroid underfunction may be associated with an impaired fetal brain development. in fact, lower iq scores have been demonstrated in children whose mothers had hypothyroidism during pregnancy in comparison with children with normal maternal thyroid function. moreover, it has been reported that children of pregnant women with normal thyroid function but increased thyroid peroxidase antibodies are at risk for impaired psychomotor development. therefore, our data supporting the hypothesis that the presence of tai identifies women at risk to develop fetal and maternal complications suggesting that, in these patients, a therapy which can reestablish thyroid homeostasis could be essential for a successful pregnancy [7, 8 ]. accordingly, in our previous study we have demonstrated that the use of thyroid replacement therapy, in patients with unexplained rm and tai, was found to be successful in reducing miscarriages. in fact, this therapy resulted in 79% of the pregnancies ending in full - term live births, which represents a significant improvement upon their obstetric prognosis without treatment. in addition, the high rate of successful pregnancy observed in the study could be due to the appropriate time of treatment initiation. in fact, the treatment was started almost one month before pregnancy in order to obtain the optimal thyroid hormone levels necessary to a normal placental development. a more recent study has confirmed the efficacy of thyroid replacement therapy in preventing miscarriage and obstetrics complications in patients with rm and tai. in this study miscarriage rate was compared between thyroid peroxidase antibody positive women who were given levothyroxine beginning in the first trimester of pregnancy to euthyroid thyroid antibody positive women who were not given levothyroxine (the control group). a statistically significant decrease in spontaneous miscarriage was seen in treated women as compared to the controls. in conclusion, our data further support the hypothesis that in patients with rm the presence of tai is associated with a subtle thyroid dysfunction that can be detected by specific test. in fact, the vast majority of our patients with rm and tai showed an abnormal response to trh stimulation tests. therefore, the evaluation of thyroid autoimmunity and the study of thyroid functioning, through trh stimulation tests, could be considered a useful tool for patients with a history of rm. although larger studies are needed, our results suggest that these tests could identify a group of patients in which an appropriate therapy can effectively increase the possibility of a successful pregnancy. | it has been twenty years since the first paper reporting the association between thyroid antibodies (tais) and spontaneous miscarriage was published. following this observation, several studies have clearly demonstrated an increased prevalence of tai in patients with recurrent miscarriage (rm). however, the exact mechanism underlying this association remains a matter of debate. the aim of the present study was to evaluate the thyroid function, throughout a specific test, in patient with rm and tai focusing on the hypothesis that tai should be an indirect sign of a mild thyroid dysfunction. 46 patients with rm and tai were included in the study. all patients underwent short trh stimulation test showing an abnormal response in the vast majority of cases (65%). normal ft4 and ft3 mean values were found whereas tsh values were in the upper normal range (2.64 1.3 mui / l). our data support the hypothesis that in patients with rm the presence of tai is an indirect sign of a subtle thyroid dysfunction detectable by a specific test. this test give the possibility to identify women with rm in which specific therapeutic approaches could effectively improve the possibility for a successful pregnancy. |
since the early 1970s, following the discovery that nutrient fertilizers could enhance the rates of oil biodegradation in marine environments (atlas and bartha, 1972 ; 1973 ; bartha and atlas, 1976), bioremediation has been a potential treatment for mitigating marine oil spills (atlas, 1977 ; 1995 ; swannell., as discussed by swannell and colleagues (1996), the exxon valdez oil spill (evos) has been the most studied case regarding the applicability of bioremediation. bioremediation based on fertilizer addition was used in prince william sound (pws) from 1989 through 1991 with a total of 107000 pounds of nitrogen applied in 2237 separate shoreline applications following physical recovery of oil by shoreline washing and skimming of oil floating on water. figure 1 shows locations and amounts of nitrogen applied 19891990 : amounts applied in 1991 were significantly less. diameter of circles scaled to represent pounds of nitrogen. the application of bioremediation to evos followed extensive laboratory and field testing to demonstrate safety and efficacy. for example, tests conducted for 114 days in large microcosms (30.5 cm diameter, 91.4 cm deep) filled with oiled pws sediments and using rising and falling water to simulate tidal movements showed that 25% of the initial oil mass was lost and that significant degradation of most types of hydrocarbons occurred (bragg., 1992) these experiments showed that the rate of biodegradation slowed down once the more easily degradable components were depleted even when fertilizer was reapplied. it is important to understand that bioremediation does not increase the ultimate extent of hydrocarbon degradation, but only the rate of biodegradation while easily degradable hydrocarbons are present (sugiura., 1997 ; changes in composition of oil on sediments from pws in 1990 laboratory microcosm tests of bioremediation as measured by hplc chromatography (different from more detailed gcms reported for field samples). aromatics (24 + rings) include the 41 target pahs, plus other multiring species. the polars (or nso fraction) include asphaltenes, resins and oxygenated byproducts of biodegradation, and are considered largely inert to biodegradation. field tests jointly conducted in pws by the united states environmental protection agency (usepa), the alaska department of environmental conservation and exxon (prince., 1993) showed, with high statistical significance, that the rate of oil degradation was a function of the ratio of nitrogen / oil, the nonpolar hydrocarbon fraction remaining, and time (bragg., 1994). bioremediation increased the rate of polycyclicaromatichydrocarbon (pah) degradation in the relatively undegraded oil found on the shorelines at this time by a factor of 2, and the degradation rate of total gcdetectable hydrocarbons by a factor of 5 relative to the controls (bragg., 1994). total pah depletion was 44% of that present at the start of the test (fig. depletion of pah groups in subsurface oil residue within fertilized section of kn135b bioremediation field test, pws, 1990. it is important to recognize that biodegradation does not remove all of the hydrocarbons in crude oil some compounds are recalcitrant to microbial attack such as highermolecularweight pahs and the polars (nso molecules containing nitrogen, sulfur and oxygen) (oudot., 1998 ; further, if the biodegradation rate can not be accelerated significantly by a factor of at least 2 it has been suggested that bioremediation is not worth considering (zhu., 2001). as crude oil weathers the rate of biodegradation declines since hydrocarbon biodegradation follows firstorder kinetics (e.g. venosa., 1996 ; wrenn., 2006), which means that as the concentration of degradable hydrocarbons decreases so does the potential applicability of bioremediation. however, some studies indicate that once the easily degraded alkanes and lowermolecularweight aromatics are removed, the rate of biodegradation of the remaining oil residues is no longer limited by nutrients, and the biodegradation rate naturally slows. in bioremediation tests conducted following the prestige spill (diez., 2005 ; gallego., 2006), for example, the rate of biodegradation of all highermolecularweight components slowed dramatically at a point consistent with the disappearance of the lowermolecularweight alkanes (fig. 4). similarly, bioremediation tests conducted by venosa and colleagues (1996) on a simulated spill in delaware bay showed a decline in the rate of biodegradation once the alkanes were removed. wrenn and colleagues (2006), in a series of microcosm tests, also found that the sensitivity of the oil mineralization rate to nutrient input decreased rapidly as the extent of oil degradation increased, and after about 2 weeks the rate of oil mineralization appeared to be independent of nutrient input. they concluded that there may be little value in maintaining a longterm supply of nutrients in contact with oilcontaminated sediments. depletion indices measured at prestige oil spill bioremediation test (moreira site) during a 1year sampling period (gallego., 2006). many of the microorganisms capable of attacking highermolecularweight pahs may operate by cometabolism (cerniglia, 1993 ; kanaly and bartha, 1999 ; van herwijnen., 2003). when alkanes and lowermolecularweight aromatic substrates have been removed from the oil residues, the enzymes needed to attack the higherweight pahs may not be induced, resulting in decreased rates of biodegradation. other ratedecreasing factors that may result from the removal of lowermolecularweight compounds are the increase in viscosity of the remaining oil residue and the decrease in mass fraction of degradable hydrocarbons. these slow the diffusion of degradable hydrocarbons to the oil uraizee and colleagues (1998) systematically altered the concentration of nonbiodegradable heterocyclic polars and asphaltenes in crude oil and analysed its impact on transport of biodegradable hydrocarbons to microorganisms. as the polars content (and oil viscosity) increased, the maximum respiratory oxygen uptake decreased. obviously, the weathering point at which the degradation of pah is no longer nutrient limited will depend on the starting oil composition and the extent of prior weathering, so each case must be evaluated independently. in addition, successful bioremediation in marine environments requires that water containing the applied nutrients must be able to contact the oil. oil that is sequestered (buried under layers of sediment that impede free water flow) may not benefit from applied nutrients. in this case, as well as those where oxygen is limiting, tilling can be considered, but this raises the issue of physical disruption of the environment potentially causing greater environmental harm than benefit. since bioremediation was previously successful in pws and since subsurface oil residues (ssor) remain at some locations, there have been suggestions that bioremediation be applied again (michel., we examine the extent of weathering that had occurred by 2007, over the 18 years since the spill, and we estimate bioremediation potentials by examining the composition of the ssor, its distribution by location and extent of depletion, and the current natural nutrient concentrations in pore water in its vicinity. in 2001 and 2003 the national oceanic and atmospheric administration (noaa) conducted random sampling of 4982 pits dug at 114 sites in pws (short., 2002 ; 2004 ; 2006), and found that 97.8% of the pits had no oil (no) or light oil residues (lor) even though these sites had been heavily oiled in 1989 and previously had significant levels of ssor. separate gridded surveys were also conducted for exxonmobil in 2002 and 2007. the 2007 survey conducted for exxonmobil (boehm., 2008) at 22 sites that were found to be most heavily oiled in noaa 's 20012003 surveys found no ssor in 529 pits (71%). sediments from an additional 162 pits (21.8%) had light levels (lor) or only traces (tr) of ssor. only 34 pits (4.6%) were found with moderate ssor (mor) and 19 pits (2.6%) with heavy ssor (hor). the pits with mor or hor showed a pattern of extreme ssor patchiness, i.e. widely scattered small patches, mostly sequestered under boulder / cobble armour. even where ssor remains it almost no resolvable alkanes remain in samples collected from 2002 through 2007 (fig. most samples also had lost more than 70% of the original pahs through a combination of biodegradation and other weathering processes. we believe 70% depletion of total pah is the approximate threshold above which bioremediation is likely to be ineffective. 4) that showed dramatic reduction in bioremediation effectiveness upon depletion of light alkanes, the almost total depletion of resolvable alkanes in current pws samples (fig. 5), and other studies showing lack of biodegradation rate sensitivity to added nitrogen once lighter alkanes and aromatics were depleted (oudot., 1998 ; we have computed bioremediation indices for all analysed samples for which total pah loss could be quantified. the 70% is meant as a probable threshold, not a specific value above which bioremediation would not work. figure 6 shows the bioremediation index for all samples collected from 2002 through 2007. if the total pah content of a sample was less than 500 ng g sediment, that sample was not included in plots because the total pah concentration was approaching natural background levels in pws and could not be confirmed as evos ssor. however, note that the average percentage of depletion of total pah computed for all samples with total pah content < 500 ng g sediment was 94%. hor residues were found that contained total pah of less than 500 ng g sediment ; however, these comprised 8095% polars they looked like oil but contained only highly degraded evosderived residues. as shown in fig. 6, only about 25% of the total samples from 2002 to 2007 contained ssor that might respond to bioremediation based on the 70% threshold. depletion of total resolvable alkanes in all oiled sediment samples analysed from 2002 to 2007 as function of total pah depletion. figure 7 shows further breakdown of the extent of total pah depletion as a function of tidal elevation [i.e. pit elevation above mllw (mean low low water tide) ]. most samples with less than 70% total pah depletion are located at + 2 and + 3 m (middle to upper intertidal zone). this is not unexpected since ssor at 0 and + 1 m is exposed to water for much longer times. bioremediation indices for samples collected in 2007 as function of pit elevation above mean low tide (mllw). samples least degraded reside in upper intertidal zone (a and b), while few were found that might respond to bioremediation in lower intertidal (c and d). as expected, the trend in individual analyte degradation demonstrates that percentage depletion decreases as the pah alkyl substitution increases (fig. even at 80% total pah depletion about half of the c3 and c4phenanthrenes remain, probably because the analytes become more recalcitrant and less water soluble as alkyl substitution increases. adding nutrients would likely have minimal impact on the rates of biodegradation of remaining highly substituted three and fourring aromatics (oudot., 1998 ; wang., 1998). depletion of c1 to c4alkylated phenanthrenes versus total pah depletion in samples from 2002 to 2007. another factor we evaluated was the concentration of nitrogen naturally available within sediment pore water. the ratio of nitrogen / nonpolar ssor provides a conservative estimate of the ratio of nitrogen / degradable hydrocarbons. it is a convenient ratio for comparing recent nutrient / ssor ratios with values measured during 1990 bioremediation field tests at knight island shoreline segment 135 (kn135), considering measured values of oil loading, polars content and nitrateasnitrogen concentration. figure 9 shows the ratios of nitrogen / nonpolar ssor measured at several sites from 2006 to 2008 normalized to the average value measured over 109 days for the fertilized section of the bioremediation test at kn135b in 1990. horizontal lines indicate the ratios for the fertilized and control sections of kn135b in 1990. clearly, the amount of natural background nitrogen is significantly higher at most sites during 20062008 than at the kn135b control in 1990, and many sites now approach the same value as realized during fertilizer addition in 1990. ratio of (nitrogen concentration / nonpolar hydrocarbons) in 20062008 pore water samples normalized to same ratio measured in fertilized portion of kn135b during 1990 bioremediation test. first, the amount of ssor to be degraded (the ssor loading in mg ssor g sediment) is lower, and the polars content of the remaining ssor is higher, resulting in less nonpolar ssor. second, the average nitrateasnitrogen for 10 segments measured from 2006 through 2008 was 0.36 mg l water (with range by site of from 0.1 to 1.18 mg the concentrations of nitrogen in the pore water of the sediments where ssor remains today are significantly higher than in the incoming seawater. the incoming seawater at sites studied had a concentration of approximately 0.014 mg nitrateasnitrogen l. even in 1990 noaa noted higherthanexpected concentrations of nitrogen in oiled sediments during most of the summer. that raises the question as to the nitrogen source. perhaps it is being generated within the intertidal zone itself by nitrogenfixing cyanobacteria, or it may be coming from groundwater entering the intertidal zone that is possibly enriched in nitrogen by nitrogenfixing frankia species associated with the alder trees that line the upper intertidal zones of many of the shorelines of pws. we believe that the current presence of relatively high levels of natural nutrients shows that the natural biodegradation is no longer nutrient limited ; the rate of biodegradation is lower, and hence demand for nitrogen is lower. third, most of the remaining ssor is sequestered in lowpermeability fine sediments under boulder / cobble armour where it may not have good accessibility to the nutrients that flow with the tides. finally, the small amount of ssor remaining that might respond to bioremediation is scattered in isolated small patches. figure 10 shows the extent of pah degradation remaining in 2007 at sm006b (smith island), which noaa (michel., 2006) identified as the site that in 2001 contained the largest volume of oiled sediment. this site is typical of the way residual ssor is distributed ; few pit locations contain ssor with less than 70% loss of total pah and most have no detectable ssor. at sm006b only two pits near the top of the beach (red crosshatched area) contain ssor that might be amenable to bioremediation. however, ssor remains in these locations because it is sequestered under boulders and cobbles. photo of site sm006b showing the % total pah depletion at pits sampled in 2007. only two small patches of oil remain that might respond to bioremediation. as demonstrated by the extensive depletion of total pah in most samples shown in figs 68, however, effective biodegradation requires that flowing water resupply nutrients to microbes in contact with the oil. the geology (porosity and permeability) of pws shorelines is highly heterogeneous, leading to scattered pockets of sequestered oiled sediment that are not fully in communication with flowing seawater. most of the ssor that is less degraded than 90% resides in such sequestered locations. fertilizer added to shorelines for in situ bioremediation is unlikely to contact this sequestered oil, but would simply be diluted and washed away by water flowing through more permeable zones / paths. thus, the degree of sequestration for individual pockets of ssor probably now is the major factor affecting extent of total pah depletion, not nutrient availability. if microcosm tests were conducted on currently sequestered oiled sediments, results would only indicate the maximum possible increase in biodegradation rate that might be realized if sediments were not sequestered, since contact between oil and added nutrients in microcosms is not impeded by geologic factors. for this reason, microcosm tests would not be indicative of any actual in situ treatment potential on shorelines, but instead would only show the ideal maximum bioremediation rate increase consistent with limitations of the tested oil composition. if in situ bioremediation is unlikely to work because the oiled sediments are sequestered, one might consider tilling of the sediments to increase contact with added nutrients. however, since several studies have shown that pahs from the oil residues in their current sequestered state in pws are not bioavailable to shoreline biota or prey species (boehm., 2004 ; harwell and gentile, 2006 ; neff., 2006), tilling would cause physical disruption to the environment, disturbance of wildlife and, therefore, greater harm than benefit. as shown in figs 68, the highermolecularweight pahs remaining in the oil residue will continue to degrade at rates consistent with adequate natural nutrient content in pore water, but variable extents of sequestration of scattered individual small pockets of ssor. in summary, there is relatively little of the evos oil remaining in pws, it is mostly sequestered under boulders / cobbles, and it will naturally continue to slowly degrade. therefore, we feel that bioremediation would not be appropriate for this ssor, making natural attenuation the preferable treatment. in 2001 and 2003 the national oceanic and atmospheric administration (noaa) conducted random sampling of 4982 pits dug at 114 sites in pws (short., 2002 ; 2004 ; 2006), and found that 97.8% of the pits had no oil (no) or light oil residues (lor) even though these sites had been heavily oiled in 1989 and previously had significant levels of ssor. separate gridded surveys were also conducted for exxonmobil in 2002 and 2007. the 2007 survey conducted for exxonmobil (boehm., 2008) at 22 sites that were found to be most heavily oiled in noaa 's 20012003 surveys found no ssor in 529 pits (71%). sediments from an additional 162 pits (21.8%) had light levels (lor) or only traces (tr) of ssor. only 34 pits (4.6%) were found with moderate ssor (mor) and 19 pits (2.6%) with heavy ssor (hor). the pits with mor or hor showed a pattern of extreme ssor patchiness, i.e. widely scattered small patches, mostly sequestered under boulder / cobble armour. even where ssor remains it almost no resolvable alkanes remain in samples collected from 2002 through 2007 (fig. most samples also had lost more than 70% of the original pahs through a combination of biodegradation and other weathering processes. we believe 70% depletion of total pah is the approximate threshold above which bioremediation is likely to be ineffective. 4) that showed dramatic reduction in bioremediation effectiveness upon depletion of light alkanes, the almost total depletion of resolvable alkanes in current pws samples (fig. 5), and other studies showing lack of biodegradation rate sensitivity to added nitrogen once lighter alkanes and aromatics were depleted (oudot., 1998 ; we have computed bioremediation indices for all analysed samples for which total pah loss could be quantified. the 70% is meant as a probable threshold, not a specific value above which bioremediation would not work. figure 6 shows the bioremediation index for all samples collected from 2002 through 2007. if the total pah content of a sample was less than 500 ng g sediment, that sample was not included in plots because the total pah concentration was approaching natural background levels in pws and could not be confirmed as evos ssor. however, note that the average percentage of depletion of total pah computed for all samples with total pah content < 500 ng g sediment was 94%. hor residues were found that contained total pah of less than 500 ng g sediment ; however, these comprised 8095% polars they looked like oil but contained only highly degraded evosderived residues. as shown in fig. 6, only about 25% of the total samples from 2002 to 2007 contained ssor that might respond to bioremediation based on the 70% threshold. depletion of total resolvable alkanes in all oiled sediment samples analysed from 2002 to 2007 as function of total pah depletion. figure 7 shows further breakdown of the extent of total pah depletion as a function of tidal elevation [i.e. pit elevation above mllw (mean low low water tide) ]. most samples with less than 70% total pah depletion are located at + 2 and + 3 m (middle to upper intertidal zone). this is not unexpected since ssor at 0 and + 1 m is exposed to water for much longer times. bioremediation indices for samples collected in 2007 as function of pit elevation above mean low tide (mllw). samples least degraded reside in upper intertidal zone (a and b), while few were found that might respond to bioremediation in lower intertidal (c and d). as expected, the trend in individual analyte degradation demonstrates that percentage depletion decreases as the pah alkyl substitution increases (fig. even at 80% total pah depletion about half of the c3 and c4phenanthrenes remain, probably because the analytes become more recalcitrant and less water soluble as alkyl substitution increases. adding nutrients would likely have minimal impact on the rates of biodegradation of remaining highly substituted three and fourring aromatics (oudot., 1998 ; wang., 1998). depletion of c1 to c4alkylated phenanthrenes versus total pah depletion in samples from 2002 to 2007. another factor we evaluated was the concentration of nitrogen naturally available within sediment pore water. the ratio of nitrogen / nonpolar ssor provides a conservative estimate of the ratio of nitrogen / degradable hydrocarbons. it is a convenient ratio for comparing recent nutrient / ssor ratios with values measured during 1990 bioremediation field tests at knight island shoreline segment 135 (kn135), considering measured values of oil loading, polars content and nitrateasnitrogen concentration. figure 9 shows the ratios of nitrogen / nonpolar ssor measured at several sites from 2006 to 2008 normalized to the average value measured over 109 days for the fertilized section of the bioremediation test at kn135b in 1990. horizontal lines indicate the ratios for the fertilized and control sections of kn135b in 1990. clearly, the amount of natural background nitrogen is significantly higher at most sites during 20062008 than at the kn135b control in 1990, and many sites now approach the same value as realized during fertilizer addition in 1990. ratio of (nitrogen concentration / nonpolar hydrocarbons) in 20062008 pore water samples normalized to same ratio measured in fertilized portion of kn135b during 1990 bioremediation test. first, the amount of ssor to be degraded (the ssor loading in mg ssor g sediment) is lower, and the polars content of the remaining ssor is higher, resulting in less nonpolar ssor. second, the average nitrateasnitrogen for 10 segments measured from 2006 through 2008 was 0.36 mg l water (with range by site of from 0.1 to 1.18 mg the concentrations of nitrogen in the pore water of the sediments where ssor remains today are significantly higher than in the incoming seawater. the incoming seawater at sites studied had a concentration of approximately 0.014 mg nitrateasnitrogen l. even in 1990 noaa noted higherthanexpected concentrations of nitrogen in oiled sediments during most of the summer. that raises the question as to the nitrogen source. perhaps it is being generated within the intertidal zone itself by nitrogenfixing cyanobacteria, or it may be coming from groundwater entering the intertidal zone that is possibly enriched in nitrogen by nitrogenfixing frankia species associated with the alder trees that line the upper intertidal zones of many of the shorelines of pws. we believe that the current presence of relatively high levels of natural nutrients shows that the natural biodegradation is no longer nutrient limited ; the rate of biodegradation is lower, and hence demand for nitrogen is lower. third, most of the remaining ssor is sequestered in lowpermeability fine sediments under boulder / cobble armour where it may not have good accessibility to the nutrients that flow with the tides. finally, the small amount of ssor remaining that might respond to bioremediation is scattered in isolated small patches. figure 10 shows the extent of pah degradation remaining in 2007 at sm006b (smith island), which noaa (michel., 2006) identified as the site that in 2001 contained the largest volume of oiled sediment. this site is typical of the way residual ssor is distributed ; few pit locations contain ssor with less than 70% loss of total pah and most have no detectable ssor. at sm006b only two pits near the top of the beach (red crosshatched area) contain ssor that might be amenable to bioremediation. however, ssor remains in these locations because it is sequestered under boulders and cobbles. photo of site sm006b showing the % total pah depletion at pits sampled in 2007. only two small patches of oil remain that might respond to bioremediation. as demonstrated by the extensive depletion of total pah in most samples shown in figs 68, however, effective biodegradation requires that flowing water resupply nutrients to microbes in contact with the oil. the geology (porosity and permeability) of pws shorelines is highly heterogeneous, leading to scattered pockets of sequestered oiled sediment that are not fully in communication with flowing seawater. most of the ssor that is less degraded than 90% resides in such sequestered locations. fertilizer added to shorelines for in situ bioremediation is unlikely to contact this sequestered oil, but would simply be diluted and washed away by water flowing through more permeable zones / paths. thus, the degree of sequestration for individual pockets of ssor probably now is the major factor affecting extent of total pah depletion, not nutrient availability. if microcosm tests were conducted on currently sequestered oiled sediments, results would only indicate the maximum possible increase in biodegradation rate that might be realized if sediments were not sequestered, since contact between oil and added nutrients in microcosms is not impeded by geologic factors. for this reason, microcosm tests would not be indicative of any actual in situ treatment potential on shorelines, but instead would only show the ideal maximum bioremediation rate increase consistent with limitations of the tested oil composition. if in situ bioremediation is unlikely to work because the oiled sediments are sequestered, one might consider tilling of the sediments to increase contact with added nutrients. however, since several studies have shown that pahs from the oil residues in their current sequestered state in pws are not bioavailable to shoreline biota or prey species (boehm., 2004 ; harwell and gentile, 2006 ; neff., 2006), tilling would cause physical disruption to the environment, disturbance of wildlife and, therefore, greater harm than benefit. as shown in figs 68, the highermolecularweight pahs remaining in the oil residue will continue to degrade at rates consistent with adequate natural nutrient content in pore water, but variable extents of sequestration of scattered individual small pockets of ssor. in summary, there is relatively little of the evos oil remaining in pws, it is mostly sequestered under boulders / cobbles, and it will naturally continue to slowly degrade. therefore, we feel that bioremediation would not be appropriate for this ssor, making natural attenuation the preferable treatment. in 2007, we sampled 22 pws shoreline sites that had also been sampled in 2002 and have been shown to be among the most persistently oiled in earlier surveys (short., 2002 ; 2004 ; 2006 ; michel., 2006 ; boehm., 2008 ; page., these 22 study sites included all 10 of the 2001 and 2003 noaa hor sites and 18 of the 23 2001 and 2003 mor or hor sites. in addition, two sites were included that were both heavily oiled in 1989 and identified as segments having extensive otter foraging. at each shoreline, 100mlong transects were surveyed either at four elevations (0, + 1, + 2 and + 3 m) or just at two elevations (+ 2 and + 3 m) above mllw. pits were dug 10 m apart along each transect. using established surveying methods (owens and sergy, 2000 ; boehm., 2008), the visual ssor level (heavy, moderate, light, trace or oil film) was noted for each pit, and samples of sediment were collected for analysis. to access sediment showing any visible ssor, it was necessary to remove surface boulders and cobbles and then dig to a depth of 3050 cm. all sediment samples were solvent extracted to determine the total extractable hydrocarbon content (teh). approximately 25% of all samples showing ssor were then selected for detailed gcms chemistry. however, for sites specifically selected for evaluating detailed bioremediation potential, all grid pits containing a teh of greater than 1 mg ssor g sediment were analysed by gcms using established protocols (page., 1995 ; douglas., 2004). concentrations of 41 target pah were measured, including parent and alkyl substituted naphthalene, fluorene, phenanthrene, dibenzothiophene, chrysene and sixring components. also measured were saturated hydrocarbons including resolvable alkanes (c9 to c40normal alkanes plus pristine and phytane), total gcdetectable hydrocarbons, saturated cyclic hydrocarbon biomarkers and seven c20c28 triaromatic steroids. the polars (nso) fraction was estimated by difference of gravimetric mass of total extract less f1 and f2 fractions (atlas and bragg, 2007). the analytical and quality assurance and quality control methods for teh, stearane / triterpane biomarkers, and pah analyses of sediment samples have been published in detail elsewhere (page., 1995). all analyte concentrations were calculated on a dry sample weight basis (ng g sediment) to assess the sediment concentration and on an extract weight basis (g g extract) to calculate analyte losses relative to the original cargo crude oil (atlas and bragg, 2007). we evaluated bioremediation potential by computing the extent of depletion of key components relative to their concentrations in the evos cargo crude and comparing that with depletion results from prior bioremediation studies (atlas and bragg, 2007). the bioremediation potential index was based on the mass ratio method using c29stigmastane as the most stable (conserved) species in evos cargo oil 18 years after the spill (atlas and bragg, 2007). the total loss by any oil component, x, from weathering and biodegradation relative to the evos cargo oil was computed by the following equation : where : cx = mass concentration of component xcc29r = mass concentration of c29rstigmastanes = sampleevos = evos cargo oil stabilized at atmospheric pressure. where : cx = mass concentration of component x cc29r = mass concentration of c29rstigmastane evos = evos cargo oil stabilized at atmospheric pressure. a bioremediation index was calculated from the mass loss data as follows : computing this index, ordering the results in increasing rank and graphing permits a rapid visual determination as to whether the aggregate ssor compositions would respond to nutrient addition. seventy per cent total pah degradation is used as the normalizing factor above which bioremediation effectiveness is unlikely based on results of prior bioremediation tests where nutrient addition was no longer effective after disappearance of light alkanes. to evaluate the concentration of nitrogen present within the pore water at the surveyed grid locations, stainless steel piezotubes were driven into subsurface sediment to a depth where ssor layers exist (3050 cm below boulder / cobble armour), and in situ measurements were taken of dissolved oxygen, water salinity and temperature using a multimeter. then 100 ml of samples of pore water were withdrawn by peristaltic pump and stored frozen until analysed for inorganic nitrate / nitrite as nitrogen. this sampling was completed prior to any pits being dug at the site to prevent contamination of pore water and was usually done 1 day prior to pit sampling. in 2007, we sampled 22 pws shoreline sites that had also been sampled in 2002 and have been shown to be among the most persistently oiled in earlier surveys (short., 2002 ; 2004 ; 2006 ; michel., 2006 ; boehm., 2008 ; page., these 22 study sites included all 10 of the 2001 and 2003 noaa hor sites and 18 of the 23 2001 and 2003 mor or hor sites. in addition, two sites were included that were both heavily oiled in 1989 and identified as segments having extensive otter foraging. at each shoreline, 100mlong transects were surveyed either at four elevations (0, + 1, + 2 and + 3 m) or just at two elevations (+ 2 and + 3 m) above mllw. pits were dug 10 m apart along each transect. using established surveying methods (owens and sergy, 2000 ; boehm., 2008), the visual ssor level (heavy, moderate, light, trace or oil film) was noted for each pit, and samples of sediment were collected for analysis. to access sediment showing any visible ssor, it was necessary to remove surface boulders and cobbles and then dig to a depth of 3050 cm. all sediment samples were solvent extracted to determine the total extractable hydrocarbon content (teh). approximately 25% of all samples showing ssor were then selected for detailed gcms chemistry. however, for sites specifically selected for evaluating detailed bioremediation potential, all grid pits containing a teh of greater than 1 mg ssor g sediment were analysed by gcms using established protocols (page., 1995 ; douglas., 2004). concentrations of 41 target pah were measured, including parent and alkyl substituted naphthalene, fluorene, phenanthrene, dibenzothiophene, chrysene and sixring components. also measured were saturated hydrocarbons including resolvable alkanes (c9 to c40normal alkanes plus pristine and phytane), total gcdetectable hydrocarbons, saturated cyclic hydrocarbon biomarkers and seven c20c28 triaromatic steroids. the polars (nso) fraction was estimated by difference of gravimetric mass of total extract less f1 and f2 fractions (atlas and bragg, 2007). the analytical and quality assurance and quality control methods for teh, stearane / triterpane biomarkers, and pah analyses of sediment samples have been published in detail elsewhere (page., 1995). all analyte concentrations were calculated on a dry sample weight basis (ng g sediment) to assess the sediment concentration and on an extract weight basis (g g extract) to calculate analyte losses relative to the original cargo crude oil (atlas and bragg, 2007). we evaluated bioremediation potential by computing the extent of depletion of key components relative to their concentrations in the evos cargo crude and comparing that with depletion results from prior bioremediation studies (atlas and bragg, 2007). the bioremediation potential index was based on the mass ratio method using c29stigmastane as the most stable (conserved) species in evos cargo oil 18 years after the spill (atlas and bragg, 2007). the total loss by any oil component, x, from weathering and biodegradation relative to the evos cargo oil was computed by the following equation : where : cx = mass concentration of component xcc29r = mass concentration of c29rstigmastanes = sampleevos = evos cargo oil stabilized at atmospheric pressure. where : cx = mass concentration of component x cc29r = mass concentration of c29rstigmastane evos = evos cargo oil stabilized at atmospheric pressure. a bioremediation index was calculated from the mass loss data as follows : computing this index, ordering the results in increasing rank and graphing permits a rapid visual determination as to whether the aggregate ssor compositions would respond to nutrient addition. seventy per cent total pah degradation is used as the normalizing factor above which bioremediation effectiveness is unlikely based on results of prior bioremediation tests where nutrient addition was no longer effective after disappearance of light alkanes. to evaluate the concentration of nitrogen present within the pore water at the surveyed grid locations, stainless steel piezotubes were driven into subsurface sediment to a depth where ssor layers exist (3050 cm below boulder / cobble armour), and in situ measurements were taken of dissolved oxygen, water salinity and temperature using a multimeter. then 100 ml of samples of pore water were withdrawn by peristaltic pump and stored frozen until analysed for inorganic nitrate / nitrite as nitrogen. this sampling was completed prior to any pits being dug at the site to prevent contamination of pore water and was usually done 1 day prior to pit sampling. | summaryin this article we consider what we have learned from the exxon valdez oil spill (evos) in terms of when bioremediation should be considered and what it can accomplish. we present data on the state of oiling of prince william sound shorelines 18 years after the spill, including the concentration and composition of subsurface oil residues (ssor) sampled by systematic shoreline surveys conducted between 2002 and 2007. over this period, 346 sediment samples were analysed by gcms and extents of hydrocarbon depletion were quantified. in 2007 alone, 744 sediment samples were collected and extracted, and 222 were analysed. most sediment samples from sites that were heavily oiled by the spill and physically cleaned and bioremediated between 1989 and 1991 show no remaining ssor. where ssor does remain, it is for the most part highly weathered, with 82% of 2007 samples indicating depletion of total polycyclic aromatic hydrocarbon (total pah) of > 70% relative to evos oil. this ssor is sequestered in patchy deposits under boulder / cobble armour, generally in the midtoupper intertidal zone. the relatively high nutrient concentrations measured at these sites, the patchy distribution and the weathering state of the ssor suggest that it is in a form and location where bioremediation likely would be ineffective at increasing the rate of hydrocarbon removal. |
5-lipoxygenase (5-lox ; ec 1.13.11.34 ; encoded by the alox5 gene) is the key enzyme in the biosynthesis of lipid signaling molecules, that is, the inflammatory leukotrienes and the anti - inflammatory lipoxins [1, 2 ]. the 5-lox system is considered one of the key players in inflammation, an important pathway in the cardiovascular system (particularly in atherosclerosis) [4, 5 ], and a putative modulator of central nervous system (cns) functioning and pathobiology [68 ]. moreover, the 5-lox pathway has been considered in mechanisms of neural plasticity ranging from neurogenesis and neural differentiation to regulation of synaptic plasticity [10, 11 ]. for example, metabolites derived from 5-lox activity have been implicated in the expression of long - term depression in hippocampal slices. it is believed that a nonleukocyte synthesis of leukotrienes does not require 5-lox and that in the cns and in the heart, leukotrienes can be produced via a transcellular biosynthetic pathway [12, 13 ]. nevertheless, there is evidence for 5-lox expression in these organs / systems [6, 1416 ]. thus, regulation of the brain expression of 5-lox participates in mechanisms of neuroprotection. in the cns of rats and mice, 5-lox expression increases during aging [14, 17 ] and in brain ischemia is elevated in alzheimer 's disease [7, 20 ] and in response to ischemia. in the heart, 5-lox content appears to remain stable during cardiac remodeling but can be altered by dietary modifications. furthermore, in the heart, that is, myocardial cells, drugs such as statins and thiazolidinediones trigger anti - inflammatory processes and increase the production of anti - inflammatory lipoxins depending on 5-lox, particularly the status of 5-lox phosphorylation [15, 23 ]. 5-lox gene expression is regulated by epigenetic mechanisms including modifications of dna methylation [1, 2426 ]. epigenetics encompasses the modification of chromatin structure that leads to regulation of the gene expression and phenotype. epigenetic modifications are further influenced by the environment (life experience) and are typically long lasting and sometimes heritable. thus, dna methylation at the sites of cpg dinucleotides has been implicated in gene regulation in virtually all tissues, including the brain. siegmund. studied human cerebral cortex samples in aging subjects and found a progressive aging - associated rise in the dna methylation of 5 cpg islands of certain cns genes, typically in conjunction with declining levels of their corresponding mrnas. these authors concluded that dna methylation is dynamically regulated in the human cerebral cortex throughout the lifespan, involves differentiated neurons, and affects a substantial portion of genes predominantly by an age - related increase. in addition, data are available on epigenetic mechanisms and cardiovascular gene regulation. for example, it was observed that the genomic dna methylation in patients with coronary artery disease is significantly higher than in controls. it has been hypothesized that the 5-lox pathway may be involved in the comorbidity of certain heart and brain disorders [30, 31 ]. furthermore, it was proposed that comparative studies of heart and brain tissues from the same subjects would improve efforts in this area of research. here, we investigated whether aging affects brain and heart 5-lox mrna levels and dna methylation. because mice are used in many 5-lox - related experimental models, including 5-lox - deficient mice, we investigated mouse tissue samples. however, a conundrum of epigenetic research of cns- and heart - expressed genes lies not only in the cell - specificity of epigenetic mechanisms, but also in their species - specificity. thus, the human 5-lox gene is on chromosome 10, whereas in the mouse it is on chromosome 6. comparing the dna regions of the 5-lox promoter and exon 1 (1844 nt, human ; 1937 nt, mouse), we found 132 cpg dinucleotides in the human and only 59 in the mouse gene promoter regions. to study the methylation status of this region, we employed our recently developed methylation - sensitive restriction endonuclease assay. since dna methylation depends on dna - methyltransferases (dnmts), we investigate the tissue content of mrnas for dnmt1 and dnmt3a, the maintenance, and the de novo dnmts, respectively. two - month - old (young) and 22-month - old (old) c57bl/6j male mice were obtained from the national institute on aging (bethesda, md). they were housed in groups of 46 in a temperature controlled room and had free access to laboratory chow and water. cerebellum, frontal cortex, hippocampus, and heart (the apex portion was selected as a source of uniform ventricular myocytes) tissues were obtained following lethal anesthesia (160 mg / kg ketamine ; sigma, st. louis, mo) and transcardial perfusion with 0.9% ice - cold saline to remove the circulating blood cells (i.e., until the outflow from the right atrium was clear). the total rna was extracted using the chemical isolation method with trizol reagent (invitrogen, carlsbad, ca) following the manufacturer 's instructions. to eliminate possible dna contamination, rna samples were treated with a dnase reagent, dna - free (ambion, inc., austin, tx). total rna (3 g) was reverse transcribed with 200 u of cloned moloney murine leukemia virus (m - mlv) reverse transcriptase (gibco brl, carlsbad, ca). the qpcr was performed in a stratagene mx3005p qpcr system (stratagene, la jolla, ca) using maxima sybr green qpcr master mix (fermentas, glen burnie, md) in a two - step cycling protocol as described by the manufacturer. there was an initial 5 minutes denaturing followed by 40 cycles of denaturing at 95c for 15 seconds and annealing / elongation at 60c for 1 minute. table 1 shows primer sequences used to detect 5-lox, dnmts, and cyclophilin mrnas (primers were purchased from integrated dna technologies, inc., the amplification products of all above - noted genes were run on agarose gels. for each product, we obtained one single band of the expected size (data not shown). reactions were performed using three different concentrations (12.5, 25, 50 ng) of reverse transcribed material (in duplicate for each sample). they were homogenized in homogenizing buffer (100 mm nacl, 10 mm tris - hcl ph 8.0, 25 mm edta, 0.5% sds, 0.1 mg / ml proteinase k), 100 l per 10 mg of tissue, and incubated 4 - 5 hours at 37c. the dna was purified with phenol, phenol - chloroform (ph 8.0), precipitated with isopropanol, and dissolved in h2o. the dna methylation assay was designed as follows : the promoter - exon - intron structure of the mus musculus 5-lox gene was reconstructed by aligning the 5-lox (alox5) mrna (nm_009662), the promoter, the 5 untranslated region (utr), and partial coding region sequences (af393814) with the mouse genome sequence. although the mouse 5-lox gene promoter does not contain typical cpg islands, the mouse 5-lox promoter-5utr - first exon, the adjacent intron portion, and the 3 end of the gene contain the highest c and g densities. the 5utr - first exon and the adjacent intron region (comprising 519 nucleotides) contain 66% of all c and g nucleotides. furthermore, this region contains 41 cpg dinucleotides ; 14 are located upstream from the atg translation start codon and 27 are downstream (figure 1). multiple methylation - sensitive endonuclease recognition sites were located in that portion. thus, we investigated their methylation rate (i.e., upstream and downstream from the atg translation start codon) with the aid of four methylation - sensitive endonucleases acii (c cgc), bstui (cg cg), hinp1i (g cgc), and hpaii (c cgg) (their respective recognition sequences cutting sites are shown in parentheses). to measure the 5-lox dna methylation levels, we used the restriction digest - quantitative pcr (sybr green rd - qpcr). the selected approach utilizes the ability of methylation - sensitive endonucleases to digest only unmethylated recognition sites, and their inability to act on sites with methylated cytosine. thus, if the targeted cpg is methylated, the site is blocked for the enzyme 's endonuclease activity and as a result, greater amounts of templates are available for the action of the taq dna polymerase. the assay was performed as follows : 1 g of genomic dna was used in a restriction digest reaction for each of the four endonucleases. digested dna samples were diluted with water and an aliquot (100 ng dna) was used for qpcr (stratagene) with the maxima sybr green qpcr master mix (fermentas) according to the manufacturer 's protocol. the following primers were used : forward 5-agagaaggatgcgttggaaggt-3 and reverse 5-gactccgggcaagtgagtgct -3. these primers amplify the 238 nt region upstream of the first atg translation start codon. this region contains two recognition sites for each of 4 endonucleases selected for the assay. primers for the exon - intron part were as follows : forward 5-agtcatgccctcctacacggtca-3, reverse 5-agtcatgccctcctacacggtca-3. they amplify a 344 bp fragment. for the input control, we used the 394 nt region in the first intron because this region does not contain recognition sites for the selected methylation - sensitive endonucleases. this region was amplified with the following primers : forward 5-tgatgtggctggcctcttatgtga-3, reverse 5-actgggactgagtgcaggaaatgt-3. the qpcr reactions were run with 2 different primer sets (target and input) in separate tubes and the coefficient of variation for the relative amount of a target sequence was calculated. data were analyzed by either one - way anova followed by dunnett 's multiple comparison test, or by an independent sample t - test. results are expressed as the mean s.e.m. from 5 to 10 animals. figure 3 shows that the expression levels of 5-lox mrna in heart tissue are about fifty times greater than in brain tissue. similar to previously published data, we found that during aging, 5-lox content in the brain (e.g., cerebellum and frontal cortex) increases. however, in the heart, aging resulted in significantly decreased 5-lox mrna levels (figure 3). using the 5-lox dna methylation assay, which is based on the ability of methylation - sensitive endonucleases to digest only unmethylated recognition sites, we found that the four endonucleases acii, bstui, hinp1i, and hpaii revealed a differential tissue- and brain - region - specific methylation status of 5-lox dna (figure 4). the dna region targeted by acii was more methylated in the heart compared to the brain ; whereas the dna regions targeted by bstui, hinp1i, and hpaii were more methylated in the brain (frontal cortex and hippocampus but not the cerebellum) than in the heart (figure 4). aging generally increased 5-lox dna methylation but this effect was differential with respect to the dna region studied, the type of the tissue / organ (e.g., heart versus brain), and the brain region we investigated. focusing on the promoter-5utr region (figure 4), we found the effect of aging in the areas targeted by acii, bstui, and hpaii but not hinp1l. thus, the methylation levels assayed by acii and bstui were increased by aging in all tissues studied, whereas the methylation levels studied by hpaii were increased only in the hippocampus. focusing on the exon - intron region, we found that aging increased methylation only in the brain and only in the dna region sensitive to bstui (units as in figure 4 (two - tail t - test) ; frontal cortex : young = 110 14, old = 163 14, p <.05, n = 10 ; hippocampus : young = 86 8, old = 136 17, p <.05, n = 5 ; cerebellum : young = 36 6, old = 58 10, n.s., n = 5 ; heart : young = 42 20, old = 54 10 ; ns, n = 5). the content of mrna for dnmt1 and dnmt3a (maintenance and the de novo dnmts, resp.) was generally greater in the heart compared to the brain, particularly the content of dnmt3a mrna (figure 5). the only aging - associated difference we observed was the decreased dnmt3a mrna content in the heart of old versus young mice (figure 5(b)). in line with previous reports, we confirmed that 5-lox mrna is expressed in the mouse brain, albeit at levels significantly lower than 5-lox mrna levels measured in heart tissue. we also confirmed that aging increases 5-lox mrna content in the brain. in our previous work with rat brain tissue, we found an aging - associated 5-lox increase in the cerebellum and the hippocampus. in this study with mice, we found an aging - associated 5-lox increase in the cerebellum and the frontal cortex but not in the hippocampus. these differences do not appear to be species - related (e.g., rat versus mouse) because others found that aging increases 5-lox expression in the mouse hippocampus but not in the cerebellum and cortex. the reason for these discrepancies is not apparent, but considering the putative epigenetic regulation of 5-lox expression, it is likely that individual life experiences of various colonies of old rodents could result in a colony - specific aging - modified epigenetic regulation of 5-lox expression. regardless of the observed regional differences, all previous studies [14, 17, 37 ] and our present results are consistent as to the direction (i.e., increase) of aging - associated changes in brain 5-lox expression. in contrast to the observed effects of aging in brain tissue, we found that in heart tissue aging was associated with a decrease in 5-lox mrna content. to our knowledge, this is the first time that heart 5-lox expression has been investigated in the context of aging. previous studies reported that 5-lox content in the heart remains stable during cardiac remodeling but can be altered by dietary modifications. on the other hand, 5-lox protein content in the aorta was significantly increased in old (24 months) compared to adult (6 months) rats. dna methylation is an epigenetic mechanism involved in the regulation of gene expression including the effects of aging and tissue - specific expression. we observed that in the heart, which expresses about 50 times greater 5-lox mrna levels than the brain, the dna regions targeted by bstui, hinp1i, and hpaii were less methylated than those in the brain. typically, methylation of proximal promoters containing a high density of cpg dinucleotides (cpg islands) leads to long - term gene silencing.. the methylation status of the nonisland cpg loci appears to be equally relevant for gene regulation. for example, sakamoto. reported that the tissue - dependently and differentially methylated regions were disproportionately distributed in the nonisland cpg loci and that these loci were located not only in 5 regions of genes but also in intronic and nongenic regions. the mouse 5-lox promoter-5 utr sequence has an equal number (i.e., 2) of recognition sites for each of the four endonucleases used in our assay. for example, aging did not affect the methylation status assayed at the hinp1i sites, it only affected the hpaii sites in the hippocampus and produced a similar effect on acii and bstui sites in all tissue samples studied. on the other hand, the exon - intron sequence has 4 bstui, 6 hinp1i, 1 hpaii, and 1 acii site. however, the only effect of aging in this 5-lox dna sequence was observed in brain tissues and only in bstui sites. the pattern of dna methylation depends on enzymes such as dnmts, in particular dnmt1 which is involved in the maintenance of dna methylation patterns and dnmt3a which predominantly contributes to de - novo dna methylation. although the expression of dnmt1 and dnmt3a showed some tissue and regional differences, we did not observe major aging - associated alterations in the mrna levels of these two enzymes, with the only exception that there was lower dnmt3a mrna content in the heart of old mice. however, this finding does not exclude the possibility of aging - altered dnmt activity in the brain. furthermore, multiple enzymatic mechanisms are involved in determining the pattern of dna methylation, including dna - demethylases. for example, recent findings indicate that neuronal activity is capable of triggering epigenetic dna demethylation, and it remains to be investigated whether this process is affected by aging and whether it could alter 5-lox dna methylation in the absence of significant dnmt changes. further research is needed to elucidate if the observed changes in 5-lox dna methylation could play a role in aging - associated modifications of tissue 5-lox mrna levels. for such a functional role, dna methylation status would have to influence the binding of various methylation - sensitive regulatory factors (e.g., transcription activators and repressors). we subjected the 5-lox dna sequences used in our assay (figure 1) to an analysis with the software for searching transcription factor binding sites (tfbind), which uses the weight matrix in transcription factor database transfac r.3.4.. this analysis, along with the survey of available literature, suggests that in the region upstream of the mouse 5-lox atg start codon, the likely candidates are the following transcription factors : ahr / arnt, nmyc, e2f, ap4, ap2, usf, and sp1. in the coding region, putative regulatory factors include ahr / arnt, usf, elk1, hsf1, hsf2, arp1, rfx1, gata1, ap4, e2f, and arp1. nevertheless, one must take into account that the tissue samples we used in this study contained a mixture of cell types and that the link between 5-lox mrna and 5-lox dna methylation may depend on the cell type. inherent in studies with tissue samples as opposed to research in cell cultures is that 5-lox dna extracted from tissue samples derives from both 5-lox mrna - expressing and mrna - nonexpressing cells. this technical limitation negatively impacts the ability to directly relate mrna content and dna methylation status. similarly, cell type may be decisive in determining the type of 5-lox metabolites produced (e.g., leukotrienes versus lipoxins). although leukotriene synthesis in the cns and in the heart can occur via a transcellular biosynthetic pathway that is believed not to require 5-lox expression [12, 13 ], aging - increased leukotriene synthesis in the hippocampus and aorta appears to be associated with increased 5-lox expression. it was proposed that in the cns, metabolites derived from 5-lox activity could influence neural plasticity, such as the expression of hippocampal long - term depression. furthermore, it has been established that the biological effects of leukotrienes in the cns and heart can be counteracted by noninflammatory 5-lox metabolites lipoxins. no data are available on the effects of aging on cns and heart production of lipoxins. this information appears necessary for a full understanding of the biological implications of aging - altered expression of 5-lox in the brain and heart. in conclusion, we found that in addition to altering 5-lox mrna content in heart and brain tissues, aging increased 5-lox dna methylation, and also that this effect was dna - site- and tissue - specific. it has to be stressed that our results were obtained in tissue samples containing a mixture of various cell types and that further insight into functional implications of dna methylation for 5-lox mrna expression would require cell - type directed studies. for example, one possibility is to focus on neuronal dna in brain tissue samples by applying methods for neuronal nuclei isolation. | the expression of 5-lipoxygenase (5-lox) is affected by aging and regulated by epigenetic mechanisms including dna methylation. we used methylation - sensitive restriction endonucleases (acii, bstui, hpaii, and hinp1i) to assess 5-lox dna methylation in brain and heart tissue samples from young (2 months) and old (22 months) mice. we also measured mrna content for 5-lox and the dna methyltransferases dnmt1 and dnmt3a. in young mice, the 5-lox mrna content was significantly greater in the heart compared to the brain ; 5-lox dna methylation was lower, except in the acii assay in which it was higher in the heart. aging decreased 5-lox mrna content in the heart and increased it in the brain. aging also increased 5-lox dna methylation and this effect was site- (i.e., enzyme) and tissue - specific. generally, dnmt1 and dnmt3a mrna content was lower in the brain regions compared to the heart ; the only effect of aging was observed in the mrna content of dnmt3a, which was decreased in the heart of old mice. these results indicate a complex tissue - specific and aging - dependent interplay between the dna methylation system and 5-lox mrna content. interpretation of this data must take into account that the tissue samples contained a mixture of various cell types. |
a 50-year - old man with multiple caf - au - lait spots on his body was transferred to the emergency department of kyungpook national university hospital after the sudden onset of severe dyspnea. we were aware that he had type 1 neurofibromatosis (nf-1) from a previous visit to our hospital 27 years previously, but he had not undergone any treatment. on arrival, his mental status was alert and he had a blood pressure of 119/87 mmhg, but he also exhibited tachycardia, with a heart rate of 130 beats per minute. his initial hemoglobin level was 8.2 g / dl, his hematocrit value was 25.3%, and his platelet count was 6710/l. a chest x - ray showed a massive opacification in the right lung field with a tracheal deviation to the left side (fig. a chest computed tomography (ct) scan with medium contrast confirmed a large right hemothorax and extravasation of the contrast medium from an internal mammary artery (i m a) aneurysm (fig after closed thoracostomy with a chest tube insertion, we removed approximately 1,000 ml of blood. we planned two - stage management for the patient, using endovascular embolization for the proximal portion of the i m a, followed by surgery to evacuate the hemothorax and clip the distal portion of the i m a. immediately after starting the angiogram, the patient suffered cardiac arrest from hypovolemic shock. during cardiopulmonary cerebral resuscitation with volume replacement, we planned to perform emergency embolization through the right common femoral artery, and emergency angiography revealed a ruptured aneurysm in the right i m a. the interventional radiology team occluded the proximal portion of the i m a with two 5-mm tornado embolization microcoils (cook medical inc., subsequently, superselection of the distal i m a was attempted. however, since the i m a was made discontinuous by an aneurysmal segment, the microcatheter could not reach the distal i m a, making distal embolization impossible. nonetheless, the patient s hemodynamic status recovered, although his mental state could not be determined at that time. after confirming complete occlusion of the proximal portion of the i m a, the patient was moved to the intensive care unit (icu) and hypothermic therapy was performed there. in the icu, the patient was semicomatose and a small amount of bleeding through the chest tube persisted. serial chest radiographs revealed an increasing pleural effusion that was suspicious for blood in the right lung field. three days later, the patient was taken to the operating room to drain the pleural effusion and to clip the distal portion of the i m a. through a video - assisted thoracic surgery (vats) a ruptured mediastinal pleura was observed in the thoracic inlet and a small amount of blood was found to be leaking through the distal portion of the i m a. we clamped the distal portion of the i m a with two hemostatic clips (fig. after six months of follow - up, the patient has remained clinically asymptomatic without recurrent hemothorax. spontaneous hemothorax caused by a ruptured aneurysm in nf-1 patients is very rare, but can be fatal. vascular lesions can occur in any vessels, but in the thoracic cavity, ruptures of aneurysms in the intercostal arteries, subclavian artery, and internal mammary artery have been reported in patients with nf-1. the incidence of vascular lesions in patients with nf-1 has been reported to be 3.6%. reported that the most common location of artery rupture was the subclavian and intercostal arteries. spontaneous hemothorax caused by aneurysmal rupture in nf-1 is very rare, but may be critical, especially if it occurs in a large artery. chest pain, dyspnea, and syncope are the presenting symptoms of arterial aneurysmal rupture in the thoracic cavity. in our case, the diagnosis should be confirmed by selective angiography in order to plan an optimal therapeutic intervention. an aneurysmal rupture in a patient with neurofibromatosis should not be corrected by vessel reconstruction. surgical management is more aggressive and complex, and artery reconstruction is limited by arterial fragility [25 ]. in the case of the rupture of an i m a aneurysm, the treatment depends on the patient s hemodynamic condition. in unstable conditions, surgical exploration includes removing the hematoma, surgical ligation for bleeding, and packing to achieve hemostasis. however, this surgery is aggressive and problematic due to difficulties in finding the source of bleeding and performing surgical ligation associated with the fragile nature of vascular tissue. miura. reported that they failed to identify the cause of bleeding during surgery, so they terminated the procedure in a patient with an intercostal aneurysmal rupture. in patients who are hemodynamically stable, endovascular treatment is the best choice for arterial rupture in nf-1 patients. coil embolization or stent grafts are often used in these cases because these treatments are safer and less complicated than surgery. however, fatal rebleeding from the aneurysm or due to backflow from collateral branches has been identified as a possible life - threatening complication after an endovascular procedure. a subsequent vats procedure may be indicated for cases of aneurysmal rupture in the thoracic cavity to prevent rebleeding and to improve the patient s symptoms. in our case, regardless of whether the patient had been hemodynamically unstable on arrival, we planned a two - stage management strategy that combined endovascular treatment and a subsequent vats procedure. the interventional radiology team was already prepared when the patient was transferred to our hospital, and we were concerned about possible difficulties in finding the bleeding site through surgery. first, coil embolization of the proximal rupture of the i m a was undertaken, while the distal portion of the i m a was later clipped using two hemostatic clips via vats. a large arterial rupture in a patient with nf-1 is a life - threatening condition that can result in hypovolemic shock and severe dyspnea. therefore, when encountering an nf-1 patient with a massive hemothorax, early recognition and treatment are necessary. in conclusion, we planned a two - stage management strategy to treat an i m a aneurysmal rupture associated with nf-1. although the patient required cardiopulmonary cerebral resuscitation, successful results were obtained by coil embolization and a subsequent vats procedure. | the rupture of an internal mammary artery (i m a) aneurysm in a patient with type 1 neurofibromatosis (nf-1) is a rare but life - threatening complication requiring emergency management. a 50-year - old man with nf-1 was transferred to the emergency department of kyungpook national university hospital, where an i m a aneurysmal rupture and hemothorax were diagnosed and drained. the i m a aneurysmal rupture and hemothorax were successfully repaired by staged management combining endovascular treatment and subsequent video - assisted thoracoscopic surgery (vats). the patient required cardiopulmonary cerebral resuscitation, the staged management of coil embolization, and a subsequent vats procedure. this staged approach may be an effective therapeutic strategy in cases of i m a aneurysmal rupture. |
all experimental neural responding procedures were approved by the seoul national university animal care and use committee, and all in vivo experimental procedures were performed according to the standards of the association for research in vision and ophthalmology. all neural recordings were made in cortical areas 17 and 18 of two adult cats, weighing 3.1 and 3.4 kg (hanlym lab. atropine (0.05 mg) and dexamethasone (40 mg) were injected subcutaneously into each cat to reduce tracheal secretions and to minimize stress - based responding. the cats were initially anesthetized with ketamine and xylazine (15 mg / kg and 1.5 mg / kg, intramuscular injection). endotracheal tubes were inserted into the trachea of each cat to allow for artificial respiration. the concentration of isofluorane was reduced to 0.5% in o2 during the recording in each cat. the heads of the animals were secured using a stereotaxic device (boardtech, incheon, korea) using ear and mouth bars as well as clamps to the orbital rim. stainless steel pegs were implanted with screws in the frontal bones of both cats. a craniotomy over the lateral gyrus of left hemispheres of both cats exposed the dura, which was surgically removed. after stabilization of anesthesia, the cats were immobilized with gallamine triethiodide (10 mg). the respiratory rates and tidal volumes of both cats were controlled with a respiratory pump (clare ventilator, victoria, australia) to maintain end - tidal co2 levels at 3.8% to 4.2%, and the body temperatures of both cats were maintained at 37.5. a continuous infusion of ringer 's solution, containing gallamine triethiodide (10 mg / kg / hr) and glucose (80 mg / kg / hr), was administered to each cat. the eyes (conjunctival sacs) of both cats were irrigated with a 10% phenylephrine (neosynephrine - pos, ursa pharm., the retinal coordinates were obtained with light to monitor the fixation location of both cats. artificial pupils (5 mm diameter) trial lenses (usually between -2 and -3 diopters) were placed in front of the right eyes to correct for distance, and the left eyes were occluded. stimuli were generated using visionworks for electrophysiology (vision research graphics, durham, nh, usa) on a 19-inch monitor (950nf ; samsung, suwon, korea), placed 57 cm in front of the cats ' right eyes, using a 1024 768 pixel resolution and a 85-hz refresh rate. when action potentials were observed in one or more neurons, preliminary tests were performed. the position was conducted manually by varying the x - y position of an aperture drifting sinusoidal grating display on the monitor. once the receptive field was isolated, the stimulus (15 deg 15 deg) was fixed in a position restricting the number of cortical neurons (to between three and five) which were responding to the stimulus. to measure the effects of various contrasts and velocities, directional and spatial frequency tuning selectivity was measured using drifting grating displays (described below). drifting sinusoidal grating displays were placed to the right eye of each cat within a square aperture (15 deg 15 deg). the spatial frequency and directional tuning properties of the neurons were examined using drifting sinusoidal grating displays with varying spatial frequencies, velocities, and directions [16 - 18 ]. the experimental protocol utilized a series of 10 spatial frequencies and 16 directions at seven velocities (i.e., a total number of stimuli, 1,120). at each spatial frequency (e.g., 0.05 cycle / deg) and orientation (67.5 deg), the stimulus was shown for 1 second at varying velocities (+ 60, + 50.8, + 41.5, + 32.3, + 23.1, + 13.9, + 4.6, -4.6, -13.9, -23.1, -32.3, -41.5, -50.8, and -60.0 deg / sec). this set of velocities was repeated for seven orientations (-22.5 deg intervals). between each orientation set, after all orientations and velocities were presented, the sequence was repeated using nine spatial frequencies (0.27, 0.48, 0.70, 0.92, 1.13, 1.35, 1.57, 1.78, and 2.0 cycle / deg). in total, the stimulus set resulted in 1,120 responses and 80 resting discharges for each animal. the stimulus set was repeated two or four times and resulted in seven directional and spatial frequency tunings (fig. 1). to investigate the effect of the contrast displays on the directional and spatial frequency tuning, the tuning test was conducted at three contrast levels (0.4, 0.7, and 1.0). single and/or multiple units were recorded extracellularly from areas 17 and 18, using a utah multielectrode array (uea ; bionic technologies, salt lake city, ut, usa). the uea was implanted at a depth of 1.5 or 1.0 mm using a high - velocity inserter. the device was implanted at the junction of the lateral and posterior lateral gyri as described by tusa.. neuronal activity was amplified (5,000), filtered (250 - 7, 500 hz), and digitized (30 khz sample rate, with 8 bits / sample, at a selectable, resolution of between 0.5 - 8 v per bit), using a 100-channel data acquisition system (bionic technologies). at the end of the recording session, both cats were euthanized with a lethal dose of pentobarbital in excess of 100 mg / kg body weight intravenous injection. the responses of each neuron to the 160 stimuli (10 spatial frequencies 16 directions 1 velocity) per second are shown in a raster plot of the directional versus spatial frequency domains (fig. 1a). the center (marked with a star) of the inner - most contour represents the optimal response, which is located at the point of optimal directional (along the x - axis) and spatial frequency (along the y - axis). the contour at 50% of the optimal response is indicated by an asterisk. the directional tuning bandwidth and spatial frequency bandwidth a directionality index was computed as 1 minus the ratio of the response in the null direction to the response in the preferred direction. as a result of these calculations, each value (optimal response, optimal direction, optimal spatial frequency, tuning width, and band width) was plotted along the contrast (0.4, 0.7, and 1.0). 2), and the distribution of the slopes for these ' best fit ' lines is shown for each parameter in fig. all experimental neural responding procedures were approved by the seoul national university animal care and use committee, and all in vivo experimental procedures were performed according to the standards of the association for research in vision and ophthalmology. all neural recordings were made in cortical areas 17 and 18 of two adult cats, weighing 3.1 and 3.4 kg (hanlym lab. atropine (0.05 mg) and dexamethasone (40 mg) were injected subcutaneously into each cat to reduce tracheal secretions and to minimize stress - based responding. the cats were initially anesthetized with ketamine and xylazine (15 mg / kg and 1.5 mg / kg, intramuscular injection). endotracheal tubes were inserted into the trachea of each cat to allow for artificial respiration. the concentration of isofluorane was reduced to 0.5% in o2 during the recording in each cat. the heads of the animals were secured using a stereotaxic device (boardtech, incheon, korea) using ear and mouth bars as well as clamps to the orbital rim. stainless steel pegs were implanted with screws in the frontal bones of both cats. a craniotomy over the lateral gyrus of left hemispheres of both cats exposed the dura, which was surgically removed. after stabilization of anesthesia, the cats were immobilized with gallamine triethiodide (10 mg). the respiratory rates and tidal volumes of both cats were controlled with a respiratory pump (clare ventilator, victoria, australia) to maintain end - tidal co2 levels at 3.8% to 4.2%, and the body temperatures of both cats were maintained at 37.5. a continuous infusion of ringer 's solution, containing gallamine triethiodide (10 mg / kg / hr) and glucose (80 mg / kg / hr), was administered to each cat. the eyes (conjunctival sacs) of both cats were irrigated with a 10% phenylephrine (neosynephrine - pos, ursa pharm., the retinal coordinates were obtained with light to monitor the fixation location of both cats. artificial pupils (5 mm diameter) trial lenses (usually between -2 and -3 diopters) were placed in front of the right eyes to correct for distance, and the left eyes were occluded. stimuli were generated using visionworks for electrophysiology (vision research graphics, durham, nh, usa) on a 19-inch monitor (950nf ; samsung, suwon, korea), placed 57 cm in front of the cats ' right eyes, using a 1024 768 pixel resolution and a 85-hz refresh rate. when action potentials were observed in one or more neurons, the position was conducted manually by varying the x - y position of an aperture drifting sinusoidal grating display on the monitor. once the receptive field was isolated, the stimulus (15 deg 15 deg) was fixed in a position restricting the number of cortical neurons (to between three and five) which were responding to the stimulus. to measure the effects of various contrasts and velocities, directional and spatial frequency tuning selectivity was measured using drifting grating displays (described below). drifting sinusoidal grating displays were placed to the right eye of each cat within a square aperture (15 deg 15 deg). the spatial frequency and directional tuning properties of the neurons were examined using drifting sinusoidal grating displays with varying spatial frequencies, velocities, and directions [16 - 18 ]. the experimental protocol utilized a series of 10 spatial frequencies and 16 directions at seven velocities (i.e., a total number of stimuli, 1,120). at each spatial frequency (e.g., 0.05 cycle / deg) and orientation (67.5 deg), the stimulus was shown for 1 second at varying velocities (+ 60, + 50.8, + 41.5, + 32.3, + 23.1, + 13.9, + 4.6, -4.6, -13.9, -23.1, -32.3, -41.5, -50.8, and -60.0 deg / sec). this set of velocities was repeated for seven orientations (-22.5 deg intervals). between each orientation set, after all orientations and velocities were presented, the sequence was repeated using nine spatial frequencies (0.27, 0.48, 0.70, 0.92, 1.13, 1.35, 1.57, 1.78, and 2.0 cycle / deg). in total, the stimulus set resulted in 1,120 responses and 80 resting discharges for each animal. the stimulus set was repeated two or four times and resulted in seven directional and spatial frequency tunings (fig. 1). to investigate the effect of the contrast displays on the directional and spatial frequency tuning, the tuning test was conducted at three contrast levels (0.4, 0.7, and 1.0). single and/or multiple units were recorded extracellularly from areas 17 and 18, using a utah multielectrode array (uea ; bionic technologies, salt lake city, ut, usa). the uea was implanted at a depth of 1.5 or 1.0 mm using a high - velocity inserter. the device was implanted at the junction of the lateral and posterior lateral gyri as described by tusa.. neuronal activity was amplified (5,000), filtered (250 - 7, 500 hz), and digitized (30 khz sample rate, with 8 bits / sample, at a selectable, resolution of between 0.5 - 8 v per bit), using a 100-channel data acquisition system (bionic technologies). at the end of the recording session, both cats were euthanized with a lethal dose of pentobarbital in excess of 100 mg / kg body weight intravenous injection. single unit responses were classified off - line using matlab ver. 2.0 beta (bionic technologies). the responses of each neuron to the 160 stimuli (10 spatial frequencies 16 directions 1 velocity) per second are shown in a raster plot of the directional versus spatial frequency domains (fig. 1a). the center (marked with a star) of the inner - most contour represents the optimal response, which is located at the point of optimal directional (along the x - axis) and spatial frequency (along the y - axis). the contour at 50% of the optimal response is indicated by an asterisk. the directional tuning bandwidth and spatial frequency bandwidth a directionality index was computed as 1 minus the ratio of the response in the null direction to the response in the preferred direction. as a result of these calculations, each value (optimal response, optimal direction, optimal spatial frequency, tuning width, and band width) was plotted along the contrast (0.4, 0.7, and 1.0). 2), and the distribution of the slopes for these ' best fit ' lines is shown for each parameter in fig. recordings were made from a total of 153 cells. of these, 94 cells (61%) were selected for further analysis. the contour plots of these cells showed either one clear peak (direction selective) or two clear peaks (non - direction selective). each of five parameters (optimal response, optimal direction, tuning width, optimal spatial frequency, and bandwidth) was tested to determine whether that parameter was affected by the contrast display. the slope of the relationship between the parameters was extracted by fitting a linear function (fig. the responses of most of the cells increased linearly with increasing contrast (mean, 27.86 ; sd, 36.69) (fig. the optimal direction did not change for most cells (mean, -0.21 ; sd, 79.72) (fig. the tuning bandwidth was also stable (mean, 14.18 ; sd, 72.55) (fig. 3c) across various contrast displays, as were the optimal spatial response frequencies (mean, -0.13 ; sd, 0.50) (fig. 3d) and bandwidths (mean, 0.02 ; sd, 0.13) (fig. the goal of this study was to examine the effects of various contrasts on the directional / spatial frequency tuning properties of neurons in the cat visual cortex. previous research on the selectivity of striate cortical neurons has shown that neurons in this region respond to bars or edges. light and dark bars divide striate cortical neurons into two main groups : simple and complex cells (depending on the discreteness or overlap of these sub - regions). complex cells can be further divided into standard and special complex cells, depending on their length summation. the main objective of this experiment was to examine the effects of contrast displays on directional and spatial frequency tuning of neurons in the straital cortex. additionally, length summation was not able to be tested using a multi - channel recording system. orientation / directional tuning curves have been obtained by comparing the responses to stimuli of different orientations and directions. gratings establish the cells ' tuning to specific spatial frequency [1,5 - 9 ], as well as to orientation and the direction of movement. previously, studies have recorded and analyzed the responses of a single neuron under a particular set of conditions. however, these methods lead to understanding of neuronal behavior under a single, specific condition (e.g., at the optimal direction or optimal spatial frequency). recently, multi - electrodes have become available, including the utah arrays used in this study. the major benefits of using multi - electrodes, include : 1) allowing the placement of multiple electrodes at once, rather than individually ; and 2) the ability to receive data from multiple sites at the same time. when multi - channel recording methods are used, stimulus parameters can not be limited as each cell prefers different values. by using additional non - optimal stimuli, other neurons which prefer such stimuli it may take twice the time, but because more than 50 cells can be recorded simultaneously with the multi - channel recording system, it is more time effective. to get obtain and appropriate isolation of spikes from each channel, the results of this study show that neurons in visual cortex areas 17 and 18 of the cat exhibit contrast - invariant directional and spatial frequency tuning. the stability of both the tuning width and the bandwidth in response to varying contrasts has been established. however, previous work has suggested some level of variability associated with spatial frequency selectivity. previous work demonstrating the stability of these tuning properties has used experiments of different animals, and it was considered necessary to confirm these results in the same animal. additionally, interactions between direction tuning selectivity and spatial frequency selectivity or velocity were also considered to be likely. additionally, the effects of various contrasts on tuning properties were investigated using a drifting stimulus rather than a stationary stimulus. previous work has shown that humans recognize either an object 's direction of movement or its spatial frequency with no effect of contrast. the results of this study show a pattern of responses which is consistent with this ; however, the relationship between the activity of these cells and the perceptions of the animal remain unclear. the saturation of responses in some cells observed in this study (minus and zero slopes in fig. this was not observed in this study, and it may only occur at low levels of contrast (lower than 30%). in summary, these results are consistent with the proposal that both the directional tuning and spatial frequency tuning characteristics of cortical cells are stable when the contrast of the stimulus placed in the visual field is greater than 40%. | purposethe purpose of this study was to investigate the effects of contrast display exposure on neuronal directional and spatial frequency tuning. neuronal responses were recorded from ninety - four neurons in cortical areas 17 and 18 in two adult cats.methodsa multi - channel microelectrode was implanted in cortical areas 17 and 18 of two paralyzed and anaesthetized cats. various drifting sinusoidal grating contrast displays were presented to one of the cats ' eyes in the visual field. contour plots based on the neuronal responses to the drifting sinusoidal grating displays using various contrasts (i.e., 0.4, 0.7, and 1.0) and velocities (i.e., 4.6, 13.9, 23.1, 32.3, 41.5, 50.8, and 60.0 deg / sec) were plotted as a function of the spatial frequency and the direction associated with each velocity and contrast used.resultsfive parameters were extracted from these contour plots : 1) optimum response, 2) preferred direction, 3) optimum spatial frequency, 4) directional tuning width, and 5) spatial frequency bandwidth. to determine the optimal velocity, each parameter was plotted against each of the specific display contrasts used, and a ' best fit ' line was established. response amplitudes were dependent on the type of contrast utilized ; however, the spatial frequency and directional tuning properties were stable for the cortical neurons assessed.conclusionsthe results of the presentation of different contrasts on neuronal directional and spatial frequency tuning are consistent with behavioral results when medium and high contrast displays are used. |
the need for conservative spinal interventions is increasing, corresponding to the increasing number of spinal disorders. the literature describes multimodal therapy programs as successful therapy options in the treatment of acute and chronic low back pain (lbp).13 the multimodal outpatient program at the university of ulm department of orthopaedics has provided an intense multimodal treatment on an outpatient basis for several years. it combines group therapy with individual approaches, pain management, medication, spinal injection procedures, physiotherapy, medical training therapy, spa, massage, transcutaneous electrical nerve stimulation (tens), ergotherapy, traction, and back school. such a constellation of experts provides an intense level of therapy for the patient. despite many reports in the literature, this study does not use the term nonspecific low back pain, but, rather, aims to determine a specific diagnosis based on clinical examination, radiography, and magnetic resonance tomography (mrt). the purpose of this study is to compare the short - term outcomes between patients suffering from sciatica due to a discus intervertebralis herniation and those suffering from low back pain caused by facet joint disease after 3 weeks of intense multimodal outpatient pain management program. the duration of the program is 3 weeks, occurring daily between 8 am and 5 pm. it combines a broad spectrum of active and passive therapy elements, performed in groups as well as on an individual basis. the core elements of the therapy regime are medical, physiotherapy, and psychological, using a collaborative, interdisciplinary, and nonhierarchical team approach. an experienced orthopedic surgeon and an anesthesiologist are responsible for the medical treatment, indication, modification in medication or treatment regime, further diagnosis, and interventional pain management. between five and seven experienced physical therapists provide group and individual exercise, back school, manipulation, massage, spa therapy, tens, superficial heat application, traction, ultrasonography, and physical relaxation techniques. a separate ergotherapy group is included in the treatment regime to improve patients occupational and private functionality. an experienced psychologist is responsible for psychological therapy (psychosomatic treatment and psychotherapy) in a group setting. individual psychological therapy is optional, but is utilized by an increasing number of patients. the team discusses each patient s history, therapy approach, and progress in 23 meetings per week. table 1 describes the separate components of the treatment regime and the frequency of each component s application. the duration of the program is 3 weeks, occurring daily between 8 am and 5 pm. it combines a broad spectrum of active and passive therapy elements, performed in groups as well as on an individual basis. the core elements of the therapy regime are medical, physiotherapy, and psychological, using a collaborative, interdisciplinary, and nonhierarchical team approach. an experienced orthopedic surgeon and an anesthesiologist are responsible for the medical treatment, indication, modification in medication or treatment regime, further diagnosis, and interventional pain management. the orthopedic surgeon supervises each patient s progress two times per day. between five and seven experienced physical therapists provide group and individual exercise, back school, manipulation, massage, spa therapy, tens, superficial heat application, traction, ultrasonography, and physical relaxation techniques. a separate ergotherapy group is included in the treatment regime to improve patients occupational and private functionality. an experienced psychologist is responsible for psychological therapy (psychosomatic treatment and psychotherapy) in a group setting. individual psychological therapy is optional, but is utilized by an increasing number of patients. the team discusses each patient s history, therapy approach, and progress in 23 meetings per week. table 1 describes the separate components of the treatment regime and the frequency of each component s application. one hundred fifty - six (out of 261) patients who underwent the intense interdisciplinary treatment between 2009 and 2011 were included in this retrospective study. the inclusion criteria were as follows : a definitive diagnosis of lumbar disc herniation with sciatica (n = 107) or facet joint arthritis with low back pain (n = 49), based on clinical and radiographic (plain radiograms and mrt) examination. the radiographic criteria proposed by lane were used to analyze the plain radiographs. the criteria outlined by pfirrmann were used to analyze the t2-weighted sagittal magnetic resonance imaging (mri) scans for lumbar disc degeneration, in combination with disc extrusion in the mri and clinical findings of persistent or intermittent nerve root entrapment. finally, the criteria proposed by weishaupt were used to analyze the mri imaging for lumbar facet joint degeneration in combination with changes in plain radiographs (sclerosis, hypertrophy) and clinical findings of local or pseudoradicular lumbar pain.6 no other spinal diseases or systemic disorders contributed to the symptoms. patients with overlapping symptoms and multilocular pathology (spondylarthritis and discus hernia) who could not be characterized precisely after spinal injections into one of the two subgroups were excluded from the retrospective analysis. the duration of symptoms was at least 12 weeks and every patient received standard therapy before entering the program. every patient enrolled in the study was treated for 3 weeks in the program and signed an informed consent about the use of the datasets (history, numerical rating scale [nrs ], and oswestry disability questionnaire [odq ]) acquired at the baseline and at the end of the therapy regime. the exclusion criteria were infection, severe instability, pregnancy, age under 18 years, fracture, anticoagulation, myelopathy, somatoform disease, substance abuse, severe cardiopulmonary alteration, weak overall fitness, paresis, and incompliance. the mean pain intensities of sciatica and lbp (low back pain) were documented using the 10-point nrs. in cases of coexistence of lbp and sciatica, both tools are validated and frequently used to assess the outcome of surgical and nonsurgical treatments for low back pain.7 the mean nrs reduction and odi (oswestry disability index) improvement were analyzed with statistical software (spss statistics 17.0 ; ibm corporation, armonk, ny). first, the normal (gaussian) distribution of variables was excluded using a histogram, the kolmogorov - smirnov test with a lilliefors significance correction, and the shapiro wilk test. whitney test (u - test) and the kolmogorov- smirnov test (z - test) to identify unpaired variables and a significant difference between the discus hernia cohort and the facet joint disease group.8,9 the sample power size was calculated with a t - test analysis (post hoc one - tailed analysis for means). the results of the z - test were reproduced with minitab 16 (minitab inc, state college, pa). one hundred fifty - six (out of 261) nrs and odq outcomes were analyzed (sciatica in lumbar discus hernia group, n = 107 ; low back pain in spondylarthrosis, n = 49). a sample size power of 0.9985 was calculated using a t - test analysis (post hoc one - tailed analysis for means, effect size d = 0.8, = 0.05, critical t = 1.6548, delta = 4.6379). the mean reduction of nrs was calculated to be 4.31 points in the discus hernia group (mean = 4.31, 95% ci : 4.66 to 3.96, sd = 1.82) and 4.43 in the facet joint disease group (mean = 4.43, 95% ci : 5.00 to 3.85, sd = 2.00) (see figure 1a). the mean disability reduction (measured by odi) was calculated to be 10.47% in the discus hernia group (mean = 10.47%, 95% ci : 12.58% to 8.36%, sd = 10.94), and 10.88% for the facet joint disease group (mean = 10.88%, 95% ci : = 13.84% to 7.92%, sd = 10.31) (see figure 1b). neither of the groups presented a gaussian distribution of variables in their outcomes (graphical analysis histogram, kolmogorov - smirnov test with a lilliefors significance correction = 0.000, 0.004 ; shapiro whitney u - test and the z - test were used to calculate the significance of the difference between the results in both groups (h0-hypothesis : the results in the improvement of pain self - assessment and functionality in both groups were equal). the hypothesis of equality (h0) was accepted after calculating a two - tailed significance of 0.757 for pain reduction, 0.702 for odi reduction using the mann whitney u - test, a two - tailed significance of 1.000 for pain reduction, and 0.997 for odi reduction using the kolmogorov smirnov z - test. there were no complications or dropouts during the therapy regime in either of the groups. most unimodal treatments fail to restore functionality and adequately reduce pain.10 one reason for this failure is the biopsychosocial aspect of chronic pain, which is not addressed in unimodal therapy. with its focus on functional restoration, multimodal treatment is a promising option for patients with chronic lbp.11 the results of this comparative study demonstrate equal short - term results in the intense interdisciplinary treatment of patients with lumbar discus herniation with sciatica and patients with lbp due to facet joint disease. despite the efficacy of intense interdisciplinary treatment regimes, there is a lack of research on how this method affects specific spinal disorders. according to the results of this study, we conclude that patients suffering from sciatica due to intervertebral disc herniation and patients with chronic back pain due to facet joint disease have similar short - term results in response to intense interdisciplinary treatment. compared to the results for chronic lbp treatment regimes presented in the literature, both groups presented good clinical outcomes.11 despite many misleading reports in the literature, this study does not use the term nonspecific lbp, but, rather, aims to determine a specific diagnosis based on clinical examination, radiography, and mrt.1214 although there was no significant difference in the outcomes from both groups, the results do not justify the use of nonspecific diagnoses. the results of this study must be interpreted carefully, because imaging and examination findings do not always correlate with symptoms in spinal disorders, nor do they correspond to subjective pain intensity.15,16 therefore, forcing a specific spinal - disorder diagnosis is problematic. wrong diagnoses may lead to overdiagnosis, labeling effects experienced by the patient, and, ultimately, further chronification of pain.17 on the other hand, specific anatomical diagnoses have direct therapeutic implications in orthopedics, especially for the use of spinal injection procedures (epidural, periradicular or facet joint injections).18 in addition to the therapeutic benefit of symptom control, image - guided injections also contribute to higher diagnostic accuracy (eg, verification of facetogenic versus radicular pain).19,20 reports in the literature conclude that 67%75% of lbp cases can be accurately diagnosed as discogenic, facetogenic, or sacroiliac pain using image - guided injections.2123 | the literature describes multimodal pain - management programs as successful therapy options in the conservative treatment of chronic low back pain. yet, the intensity and inclusion criteria of such programs remain debatable. in many studies, the pain originating from spinal structures is described as nonspecific low back pain a diffuse diagnosis without serious implications. the purpose of this study is to compare the short - term outcomes between patients suffering from sciatica due to a discus intervertebralis herniation and those suffering from low back pain caused by facet joint disease after 3 weeks of treatment in an intense multimodal outpatient program in the department of orthopaedic surgery at the university hospital. |
in a preliminary study, 12 male and 12 female weanling sherman strain rats were given a single dose of 1000 mg polybrominated biphenyls (pbbs) firemaster ff1 lot 7042 kg / body weight as a 5% solution in corn oil. three male and three female weanling rats were given corn oil. one day after dosing pbb blood levels ranged from 78 to 162 ppm and 42 days later they ranged from 1.1 to 2.99 ppm. the liver was the only organ with pathological changes. in a long - term recovery study groups of 20 male and female rats, 2 months old, were given 0 or 1000 mg pbbs / kg body weight as a single dose in peanut oil. five rats per group killed 2, 6, 10, and 14 months after dosing had pronounced liver pathology, including hepatic porphyria in the female rats and neoplastic nodules also mainly in female rats. chemical analyses of blood, liver, and adipose tissue for ppbs 10 and 14 months after dosing gave the following mean results. blood levels in females were 2.9 and 2.92 ppm, respectively, and males 0.94 and 1.34 ppm, respectively. adipose tissue levels in females were 1202 and 783 ppm and in males 713 and 866 ppm, respectively. the liver levels in females were 37 and 22 ppm and in males 60 and 63 ppm, respectively.imagesfigure 4.figure 5.figure 6.figure 7.figure 8. |
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understanding of vitamin d role in optimal health has expanded greatly in the past few years. grant indicated that increasing western europeans serum 25(oh)d levels to at least 100 nmol / l all year could significantly reduce rates and economic burdens of several types of diseases whereas the reduction in healthcare expenditures was estimated to be 187,000 million euro per year. the most important benefits would come for cancer, cardiovascular disease, diabetes mellitus, respiratory infections, dental / periodontal diseases and other. for these reasons the polish consensus on vitamin d supplementation and standards were published on the beginning of 2010 and implemented into practice. it negatively affects effectiveness of expensive pharmacotherapies in infectious diseases (chronic hepatitis c, tuberculosis), osteoporosis, multiple sclerosis, epilepsy, chronic kidney diseases and atopic dermatitis. it is estimated that more than 1 billion people of all ages worldwide have serum 25(oh)d values 80 nmol / l, in addition to peg - ifn / rbv combination therapy, had higher rates of rapid virologic response (rvr ; 44% vs. 17%, p 15 ng / ml, or 2.2,) as an independent predictor of viral response indicating that adding vitamin d3 to conventional peg / rbv therapy significantly improved viral response. lange reported that cyp27b11260 rs10877012 was an independent predictor of sustained virologic response (svr) in patients with poor - response il28b genotypes, but not in patients with favorable il28b genotype. patients with chronic hepatitis c showed a high prevalence of vitamin d defficiency [25(oh)d 24 mo of bisphosphonates therapy ; (2) decrease of > 3.0% in bmd at the lumbar spine, bilateral femoral neck, total hip, or trochanter between the baseline and follow - up dexa scans ; (3) incident low - trauma fracture despite > 12 mo of bisphosphonate therapy. patients with a mean 25(oh)d 33 ng / ml had a 4.5-fold greater odds of a favorable response. patients with a mean 25(oh)d 33 ng / ml had a substantially greater likelihood of maintaining bisphosphonate response. the probability of inadequate response to bisphosphonate therapy measured as bmd increase was 4-fold higher in patients with 25(oh)d 80 nmol / l, in addition to peg - ifn / rbv combination therapy, had higher rates of rapid virologic response (rvr ; 44% vs. 17%, p 15 ng / ml, or 2.2,) as an independent predictor of viral response indicating that adding vitamin d3 to conventional peg / rbv therapy significantly improved viral response. lange reported that cyp27b11260 rs10877012 was an independent predictor of sustained virologic response (svr) in patients with poor - response il28b genotypes, but not in patients with favorable il28b genotype. patients with chronic hepatitis c showed a high prevalence of vitamin d defficiency [25(oh)d 100 nmol / l were required to achieve normal levels of 1,25(oh)2 d. therefore ergocalciferol was an effective treatment that delayed the development of secondary hyperparathyroidism in children with ckd23. alvarez documented that vitamin d3 (cholecalciferol) treatment reduced parathyroid hormone levels in 46 adult patients with early chronic kidney disease in 1 y rct study. subjects with early ckd (stages 23) were supplemented with oral vitamin d3 (vitamin d group ; 50,000 iu / wk for 12 wk followed by 50,000 iu every other week for 40 wk). serum 25(oh)d increased in the vitamin d group only [26.7 6.8 to 42.8 16.9 ng / ml ; ] and remained elevated at 1 y. pth decreased from baseline only in the vitamin d group (baseline : 89.1 49.3 to 70.1 24.8 pg / ml ; p = 0.01) at 12 weeks, but values were not significantly different from baseline at 1 y. blood pressure and fgf23 did not change in either group. thus serum pth levels improved after vitamin d3 treatment in adult ckd patients who had secondary hyperparathyroidism. vitamin d supplementation as adjunctive therapy in osteoporosis why only some osteoporotic patients maintain a positive response to prolonged bisphosphonate therapy is unknown. carmel examined bisphosphonate response and its association with serum 25(oh)d level in a real world setting. serum 25(oh)d level was strongly associated with maintaining bisphosphonate response arguing that vitamin d may be involved in optimizing prolonged bisphosphonate therapy. postmenopausal women with low bone mineral density (bmd) treated with bisphosphonates were categorized. the definition of non - response to bisphosphonate therapy was based on the eurofors study, which identified patients for teriparatide therapy after failing anti - resorptive agents (median duration of bisphosphonate treatment was 36 mo). non - response included any of the following : (1) t - score of 24 mo of bisphosphonates therapy ; (2) decrease of > 3.0% in bmd at the lumbar spine, bilateral femoral neck, total hip, or trochanter between the baseline and follow - up dexa scans ; (3) incident low - trauma fracture despite > 12 mo of bisphosphonate therapy. patients with a mean 25(oh)d 33 ng / ml had a 4.5-fold greater odds of a favorable response. patients with a mean 25(oh)d 33 ng / ml had a substantially greater likelihood of maintaining bisphosphonate response. the probability of inadequate response to bisphosphonate therapy measured as bmd increase was 4-fold higher in patients with 25(oh)d < 30 (or, 4.42 ; 95% ci, 1.2215.97, p = 0.02). in patients with vitamin d deficiency response to bisphosphonate therapy measured by bone turnover markers indicated that ctx decreased only by 48% in patient treated with alendronate in comparison to 61% in patient supplemented with cholecalciferol (0.266 mg / wk for 3 mo ; final vitamin d status - serum 25(oh)d 82 ng / ml). bertoldo reported that serum 25(oh)d levels modulate the acutephase response associated with the first nitrogencontaining bisphosphonate infusion. body temperature and (40 individuals with severe chronic periodontitis) bashutski evidenced that placebo patients with baseline vitamin d deficiency [serum 25(oh)d, 1619 ng / ml ] had significantly less clinical attachment loss (cal) gain (-0.43 mm vs. 0.92 mm, p < 0.01) and probing depth (ppd) reduction (0.43 mm vs. 1.83 mm, p < 0.01) than vitamin - d - sufficient individuals. vitamin d deficiency at the time of periodontal surgery negatively affects treatment outcomes for up to 1 y and indicated that vitamin d status may be critical for post - surgical healing. these data are supported by work of hokugo who described increased prevalence of bisphosphonate - related osteonecrosis of the jaw with vitamin d deficiency in rats. necrotic bone in the oral cavity has recently been reported in patients treated with nitrogen - containing bisphosphonates as part of their therapeutic regimen for multiple myeloma or metastatic cancers to bone. similar to human patients, rat onj lesions prolonged the oral exposure of necrotic bone sequestra and were uniquely associated with pseudoepitheliomatous hyperplasia. it was suggested that the pathophysiologic mechanism(s) underpinning onj may involve the interaction between bisphosphonates and compromised vitamin d functions in the realm of skeletal homeostasis and innate immunity. bischoff - ferrari evaluated the effect of 8002000 iu daily cholecalciferol and extended physiotherapy (pt) on complications after hip fracture. at baseline, 50.9% of participants had 25(oh)d levels of less than 12 ng / ml and 97.7% of less than 30 ng / ml. extended pt was successful in reducing falls but not hospital readmissions, whereas cholecalciferol treatment, 2000 thus, the 2 strategies may be useful together because they address 2 different and important complications after hip fracture. hgberg evidenced amelioration of depression by vitamin d supplementation in swedish adolescents. in 54 swedish depressed adolescents increasing mean serum 25(oh)d from 41 nmol / l at baseline to 91 nmol / l after supplementation led to decreased symptoms of depression : well - being increased (p < 0.001) and there was a significant improvement in eight of the nine items : depressed feeling (p < 0.001), irritability (p < 0.05), tiredness (p < 0.001), mood swings (p < 0.01), sleep difficulties (p < 0.01), weakness (p < 0.01), ability to concentrate (p < 0.05) and pain (p < 0.05).vitamin d supplementation alone has therapeutic effect in vitamin d deficient depressed patients. huang described patients with multiple areas of chronic pain and supplemented with vitamin d3 1200 iu daily (if serum 25(oh)d was 20 to 29 ng / ml) or 50,000 iu weekly (if serum 25(oh)d was < 20 ng / ml) improved their pain levels, sleep, and various aspects of qol. khoraminya reported therapeutic effects of vitamin d3 (1500 iu / day) as adjunctive therapy to fluoxetine (20 mg) in 42 patients with major depressive disorder. in the 8-weeks, double - blind, randomized, placebo - controlled trial depression severity based on hamilton depression rating scale hdrs and beck depression inventory bdi decreased significantly after intervention, with a significant difference between the two groups - the vitamin d + fluoxetine combination was significantly better than fluoxetine alone from the 4th week of treatment. the exact mechanism by which vitamin d exerts its beneficial effect in epilepsy is still to be explored. vitamin d receptors as well as the 1-hydroxylase, the enzyme that produces 1,25(oh)2d, are distributed widely in the brain. vitamin d deficiency is known to be highly prevalent among epilepsy patients, but only one study, published nearly 40 y ago, documented that administration of vitamin d2 resulted in a seizure reduction of 30% on average. in small study of hollo vitamin d3 supplementation (oral dose of 40,000200,000 iu bolus in order to normalize vitamin d deficiency, and then a daily maintenance dose of 20002600 iu) results in improved seizure control in patients with pharmacoresistant epilepsy. ten (of 13) patients showed decreased seizure numbers during the 90 d following treatment onset as compared with the baseline period. among all patients, the median seizure reduction was 40%, and there was a non - significant tendency for patients with larger proportional elevation of 25(oh)d levels exhibiting a larger proportional reduction in seizure numbers. median serum 25(oh)d level at baseline was 11.8 ng / ml (range : 4.034.2 ng / ml) and rose close to the normal range in all patients (median : 38.0 ng / ml, range : 23.345.0 ng / ml). there is a substantial and growing body of evidence indicating that the lower end of the adequate range is at least 80 nmol / l and, by some criteria, 100 nmol / l. it was evidenced that transfering patients from vitamin d deficiency to proper vitamin d status (75100 nmol / l serum 25(oh)d) increase effectiveness of therapies in infectious diseases (chronic hepatitis c, tuberculosis), osteoporosis, multiple sclerosis, epilepsy, chronic kidney diseases and atopic dermatitis. for these reasons doctors should take special attension to vitamin d status in patients suffering for these diseases properly implementing recent vitamin d recommendation. | at least 80% of the whole polish population, including prepubertal children and adolescents, adults and seniors, are vitamin d deficient, defined as 25(oh)d < 50 nmol / l. 83% of polish newborns start their lives at the state of vitamin d deficiency because 78% of their mothers are also deficient. it was observed that treating patient vitamin d deficiency to vitamin d status serum 25(oh)d) 75100 nmol / l increased effectiveness of therapies in infectious diseases (chronic hepatitis c, tuberculosis), osteoporosis, multiple sclerosis, epilepsy, chronic kidney diseases and atopic dermatitis.. for these reasons doctors should take special attension to vitamin d status in patients suffering for these diseases properly implementing recent vitamin d recommendation. |
he was taking warfarin for deep vein thrombosis and had a hemoglobin level of 4.5 g / dl. he had undergone low anterior resection and partial bladder resection with adjuvant chemotherapy to treat sigmoid colon cancer 3 years earlier. the anemia and the prolongation of the prothrombin time were easily corrected ; thus, continuous bleeding was not suspected. nevertheless, in order to rule out gastrointestinal bleeding as a cause of the severe anemia, abdominal computed tomography (ct) was performed. however, there was no evidence of intra - abdominal bleeding. instead, a 55-mm abdominal aortic aneurysm with eccentric bulging combined with a right common iliac artery aneurysm (fig. the aneurysm size increased by 10 mm compared with the size 2 years previously. because severe intra - abdominal adhesion and wound problems after laparotomy were expected, endovascular aortic aneurysm repair (evar) was recommended instead of open surgery. evar was started with the deployment of a bifurcated main body using an aortic stent graft of 303040 mm and an inner bare stent 323250 mm in size (s&g biotech inc., subsequent extension of the right graft limb was performed uneventfully using two covered stents (121280 mm and 121260 mm) to exclude the right common iliac artery aneurysm. dreadful complications occurred during the last procedure of the left graft limb extension. after the selection of the left graft limb and the guide wire exchange to a lunderquist stiff wire (cook inc., bloomington, in, usa), a 16f introducer sheath was inserted for the delivery of a limb extension graft, but the tip of the sheath perforated the left proximal external iliac artery during the advancement of the sheath (fig. sheath - pushing against the tortuous iliac artery caused the guide wire to slip back into the sheath, and the dilator tip punctured the arterial wall. although the extension of the left graft limb was scheduled to end at the distal common iliac artery, the extension was promptly determined to be lengthened to the external iliac artery to cover the perforated site. the left graft limb of the main body was reselected, and a stent graft of 1212100 mm was deployed. however, subsequent aortography showed an occlusion of the left graft limb, and acute thrombosis was suspected (fig. a prompt balloon thrombectomy was attempted after the left superficial and deep femoral arteries were clamped to prevent distal embolization. a 6f balloon catheter was advanced through the occlusion and withdrawn carefully not to cause disjunction of the overlapped stent grafts. fresh thrombus was successfully removed, and the graft flows became excellent without any endoleak upon the completion of angiography (fig. 3). at the 3-month follow - up, the patient was doing well without any symptoms like buttock claudication. although the surgery reduces the risk of rupture of the aortic aneurysm, the mortality of surgery has been reported to be approximately 2% to 8%. in fact, in the high - risk group for surgery, the morbidity is as high as 30%. since the first successful endovascular treatment of an abdominal aortic aneurysm in 1991, this procedure has been performed with increasing frequency for high - risk patients. recently, with the development of smaller and more precise devices, evar has been accepted as a useful treatment method for patients at high risk for postoperative complications and patients refusing surgery. however, evar also has many complications related to the procedures, such as ruptures, thrombi within stent grafts, endoleaks, dislocation of the stent grafts, and infections. we experienced combined iliac artery rupture and graft limb thrombosis, which would cause a fatal outcome unless addressed immediately. although the main cause of the iliac artery perforation in this case was the fact that the push of the introducer sheath was careless and jerky, one of the keys for successful endovascular repair of aortic aneurysm is adequate vascular access through the femoral and iliac arteries. calcification, diminished diameter, and severe tortuosity of the iliac arteries have been associated with an increased incidence of iliac injury during evar. however, even after careful preoperative assessment, inadvertent iliac rupture can occur and be a source of morbidity and mortality. this case calls attention to the importance of a careful manipulation of wires and catheters during the endovascular procedure. with respect to the management of acute iliac artery rupture, endovascular repair with a stent graft intraoperative invasive arterial blood pressure monitoring, maintenance of stiff wire access, readily available intra - aortic occlusion balloons, and an inventory of iliac stent grafts are the prerequisites for the prompt endovascular management of a ruptured iliac artery during evar. almost all the previous literature reported delayed or late stent graft thrombosis rather than acute or intraoperative thrombosis as in this case. the incidence rates have been reported to be 2.7% to 23.8%, and underlying stenosis or kinks have been pointed out as the main causes. however, the main cause of the acute graft limb thrombosis in this case was thought to be inadequate anticoagulation during the management of the ruptured iliac artery. the goal of the management of the stent graft thrombosis is the restoration of appropriate blood flow to the lower extremities. therefore, the available treatment methods are thrombectomy, catheter thrombolysis, and extra - anatomical bypass, such as axillo - femoral or cross - femoral bypass. according to the eurostar (european collaborators on stent / graft techniques for aortic aneurysm repair) registry, as a secondary procedure after evar, 11% of the patients required femoro - femoral bypass grafting. in this case, thrombectomy was performed rather than thrombolysis or extra - anatomical bypass because thrombolysis right after the event of iliac artery perforation was inappropriate, and extra - anatomical bypass should be a second - line treatment option considering that the thrombus was fresh. the limitations of thrombectomy are mainly associated with technical difficulty in the introduction of a catheter into the lumen of the stent graft, particularly in the case of late thrombosis and with the possibility of stent migration or disconnection of graft elements, which can lead to type i or type iii endoleaks. to prevent thrombosis in the stent graft, adequate anticoagulation during and after the procedure is necessary. in summary, we report a rare case of combined iliac artery rupture and graft limb occlusion complicating evar, which were successfully managed without additional open surgery. careful manipulation of catheters and guide wires, as well as maintenance of optimal anticoagulation throughout the procedure, is fundamental but the most important requirement for an endovascular procedure. to decide on the best treatment option among the various surgical and endovascular modalities | for high - risk patients, endovascular aortic aneurysm repair (evar) is a good option but may lead to serious complications, which should be addressed immediately. a 75-year - old man with a history of abdominal surgery underwent evar for an aneurysm of the abdominal aorta and iliac arteries. during evar, iliac artery rupture and graft limb occlusion occurred, and they were successfully managed by the additional deployment of an iliac stent graft and balloon thrombectomy, respectively. we, herein, report a rare case of the simultaneous development of the two fatal complications treated by the endovascular technique. |
matrix - assisted chondrocyte implantation (maci) is a commercially available third - generation autologous chondrocyte implantation (aci) technique for the treatment of articular cartilage defects. established treatment algorithms generally agree on the use of microfracture or osteochondral autograft transfer (oat) as first - line treatment for smaller defects (3 - 4 cm) of the knee, mostly due to the expense of these procedures. several studies have demonstrated superiority of aci in larger defects, confirming recommendations for the use of aci in this population ; alternatively, osteochondral allograft transplantation (oca) can be considered but is not readily available in many countries. despite general agreement on these algorithms, they have never been validated nor has surgical adherence to their suggestions been demonstrated. the 4 original studies by brittberg and peterson described periosteum - covered autologous chondrocyte implantation (aci - p). after these initial studies, many surgeons replaced the periosteum with a collagen type i / iii membrane (aci - c) to reduce surgical time, patient morbidity, and the risk of hypertrophy. the current third generation of aci utilizing matrix - seeded chondrocytes (aci - m) the indications for aci treatments have remained consistent with the original suggestions. because the treatment is very costly, much efforts have been put into ensuring that the treatment is only offered to the patients where superiority over microfracture can be expected, for example, larger defect sizes. while evidence for using aci and related treatment continues to expand, it has also become evident that the strict inclusion criteria in prospective randomized clinical trials leads to patient selection that differs from that of patients actually undergoing cartilage repair treatments in clinical practice. the aim of the present study was to investigate these demographic parameters in patients receiving maci and to compare them to the inclusion criteria for current clinical trials, essentially comparing the reality of cartilage repair with the idealized situation in a restricted trial environment. we hypothesized that patients scheduled for maci treatment are different from those enrolled in clinical trials of chondrocyte implantation in terms of age, cartilage defect size, and number of defects, and that there are differences between countries. secondary, we hypothesized that patients included in prospective randomized trials had smaller defects than those included in cohorts of patients treated in clinical practice, rather than a controlled trial. anonymized data were obtained from the genzyme / sanofi database on patients scheduled for autologous chondrocyte implants with maci between 2008 and 2013. only data from countries with more than 10 patients treated patient demographics (age, gender) and cartilage defect characteristics (size, number of defects) were evaluated. age and defect size were reported at the time of biopsy, rather than implantation. the database includes a large number of patients, which in the present study is used as an indicator of the characteristics of patients actually receiving aci treatment. medline and google scholar were reviewed for clinical cohort and comparative studies in english with unique patient data on patients treated with aci techniques for treatment of focal cartilage defects in the knee (on april 8, 2015). arthroscopic treatments could not be discriminated in the database, but the number of procedures is expected to be negligible. arthroscopic approaches were excluded in the study search, due to the possible confounding of treatment indication in the comparison. the studies were evaluated based on patient age, gender, number of defects, and lesion size. maci, autologous chondrocyte implantation, autologous chondrocyte transplantation, matrix - assisted chondrocyte implantation ; combined with knee. average patient age, average defect size, number of defects, and male - female ratio in the included studies were noted and compared with the database. studies not mentioning average age and average defect size, and where these parameters could not be calculated using information in the respective articles, were excluded. weighted average age and lesion sizes were calculated with respect to the enrolled patients in each study. the included studies were grouped by country and compared by country to the database data when possible. hence, t test for independent samples with unequal variances was used to test our hypotheses. variables compared between cohort studies and comparative trials were investigated using weighted 2-sample t test. register data and data in studies (age and defect size) was investigated using t test with weighting of studies based on patient number. a significance level of p 10 cm), and the majority of defects (70%) were medium in size (3 - 10 cm) (fig. a total of 71 studies fulfilled the inclusion criteria with a total of 5,449 patients (tables 1 and 2). four randomized trials were retrospective and were assigned to the cohort group of studies, giving a total of 57 studies in the cohort group. the weighted mean age of all studies was 34.2 years (range = 8 - 65 years), and the combined weighted mean defect size was 4.95 cm (range = 0.5 - 36 cm). the defect size of the patients in the database was significantly larger than that of patients included in the studies (p = 0.001). there were no difference in age between the database and the studies (p = 0.68). aci - p = periosteum - covered autologous chondrocyte implantation ; aci - m = matrix - seeded autologous chondrocyte implantation ; aci - c = collagen type i / iii membrane autologous chondrocyte implantation. age and defect size aci - p = periosteum - covered autologous chondrocyte implantation ; aci - m = matrix - seeded autologous chondrocyte implantation ; aci - c = collagen type i / iii membrane autologous chondrocyte implantation. the weighted mean ages in cohort studies and randomized trials were 34.3 years and 32.9 years, respectively, and this difference of 1.6 years was not statistically significant (p = 0.91). weighted mean defect size was equal for the 2 groups of studies (4.94 cm vs. 4.37 cm ; p = 0.93). male - female ratios in the 2 groups of studies were also similar (60%), which was lower than that in the database (67%). prospective comparative studies included only patients with single lesions while 19% of the treated patients in the register had multifocal lesions. significant differences in patient age and cartilage defects size in the database were observed between countries (fig. defect sizes are presented in descending order : turkey 7.4 cm [6.3 ; 8.5 ] ; spain 6.8 cm [6.0 ; 7.6 ] ; greece 6.5 cm [5.9 ; 7.2 ] ; italy 6.3 cm [5.8 ; 6.8 ] ; singapore 5.9 cm [5.1 ; 6.8 ] ; netherlands 5.7 cm [3.4 ; 8.1 ] ; australia 5.5 cm [5.1 ; 5.8 ] ; denmark 5.2 cm [4.3 ; 6.1 ] ; germany 5.1 cm [4.8 ; 5.5 ] ; england 5.0 cm [4.7 ; 5.2 ] ; israel 3.6 cm [2.2 ; 4.9 ]. stratification by country of the included studies showed that defect size in patients receiving chondrocyte transplantation was up to 1.5 to 2.5 times larger than that of the patients enrolled in their clinical trials. in swedish studies, n = 154), whereas patients in the united stated had the largest average defect size of 6.5 cm (n = 1,591). notably, most studies included operated patients that were younger and had larger defects than their respective trials (table 3). defect sizes and age of patients included in studies compared with patients in the database scheduled for chondrocyte transplantation treatment stratified by country. in the genzyme / sanofi database of 2,690 patients the average age was 33.7 years (range = 11 - 65), and male - female ratio was 67:33. single defects accounted for 81% whereas 19% were multifocal. there were no correlations between mean defect size and number of defects or patient age. on average, 18.9% of cartilage defects were small (10 cm), and the majority of defects (70%) were medium in size (3 - 10 cm) (fig. a total of 71 studies fulfilled the inclusion criteria with a total of 5,449 patients (tables 1 and 2). four randomized trials were retrospective and were assigned to the cohort group of studies, giving a total of 57 studies in the cohort group. the weighted mean age of all studies was 34.2 years (range = 8 - 65 years), and the combined weighted mean defect size was 4.95 cm (range = 0.5 - 36 cm). the defect size of the patients in the database was significantly larger than that of patients included in the studies (p = 0.001). there were no difference in age between the database and the studies (p = 0.68). aci - p = periosteum - covered autologous chondrocyte implantation ; aci - m = matrix - seeded autologous chondrocyte implantation ; aci - c = collagen type i / iii membrane autologous chondrocyte implantation. aci - p = periosteum - covered autologous chondrocyte implantation ; aci - m = matrix - seeded autologous chondrocyte implantation ; aci - c = collagen type i / iii membrane autologous chondrocyte implantation. the weighted mean ages in cohort studies and randomized trials were 34.3 years and 32.9 years, respectively, and this difference of 1.6 years was not statistically significant (p = 0.91). weighted mean defect size was equal for the 2 groups of studies (4.94 cm vs. 4.37 cm ; p = 0.93). male - female ratios in the 2 groups of studies were also similar (60%), which was lower than that in the database (67%). prospective comparative studies included only patients with single lesions while 19% of the treated patients in the register had multifocal lesions. significant differences in patient age and cartilage defects size in the database were observed between countries (fig. defect sizes are presented in descending order : turkey 7.4 cm [6.3 ; 8.5 ] ; spain 6.8 cm [6.0 ; 7.6 ] ; greece 6.5 cm [5.9 ; 7.2 ] ; italy 6.3 cm [5.8 ; 6.8 ] ; singapore 5.9 cm [5.1 ; 6.8 ] ; netherlands 5.7 cm [3.4 ; 8.1 ] ; australia 5.5 cm [5.1 ; 5.8 ] ; denmark 5.2 cm [4.3 ; 6.1 ] ; germany 5.1 cm [4.8 ; 5.5 ] ; england 5.0 cm [4.7 ; 5.2 ] ; israel 3.6 cm [2.2 ; 4.9 ]. stratification by country of the included studies showed that defect size in patients receiving chondrocyte transplantation was up to 1.5 to 2.5 times larger than that of the patients enrolled in their clinical trials. in swedish studies, average defect size was 4.5 cm, in austria 3.2 cm (n = 154), whereas patients in the united stated had the largest average defect size of 6.5 cm (n = 1,591). notably, most studies included operated patients that were younger and had larger defects than their respective trials (table 3). defect sizes and age of patients included in studies compared with patients in the database scheduled for chondrocyte transplantation treatment stratified by country. in the present study, we compared 2,690 patients assigned for treatment with maci for cartilage defects in the knee with 5,449 patients enrolled in cohort studies and prospective clinical trials. the majority of the patients scheduled for maci treatment were comparable in terms of age and defect size, to those included in cohort and prospective comparative studies instructing current treatment guidelines. comparing average defect sizes, however, the defects were larger in the database than the average defect size in the cohort studies. we further discovered that the size of the cartilage defects in patients assigned for maci varied significantly between countries. the differences in cartilage defect sizes between patients enrolled in trials and those scheduled for aci with maci may be multifactorial. obviously, strict inclusion criteria for studies in terms of limiting population sizes for sufficient power may be partly responsible for this difference. however, due to the significant cost of the treatment compared to other modalities, public health care systems and private insurance companies may be reluctant to offer this treatment to patients with defect size in the lower end of the recommended interval. engen. previously addressed the issue of differences between patients enrolled in cartilage repair trials and those seen in their clinic with respect to all different surgical cartilage repair modalities. they found that of 137 patients referred to their clinic with cartilage defects only 4.4% were eligible for inclusion in all randomized controlled trials ranging between 7% and 80% for the individual studies. the main contributor in their review was defect size, while age and additional joint injuries such as meniscal tears were also important. treatment selection for focal articular cartilage lesions requires several patient - specific considerations as well as attention to additional joint pathologies. out of the various factors predicting outcome of aci procedures for cartilage repair, age and defect size are often addressed. while some authors find age to be a factor influencing outcomes, convincing evidence of the role of defect size is still absent. ebert. reviewed patients from 2 of their trials for factors predicting 5-year outcome after maci treatment and found that while preoperative physical and mental scores in the sf-36 questionnaire contributed significantly to the 5-year koos value, cartilage defect size and preoperative duration of symptoms were only predictors of outcome on magnetic resonance imaging evaluation. recently reviewed the evidence of different patient - specific parameters and their effect on outcome after cartilage repair in 13 studies. they found that neither patient age nor defect size were independent factors related to the clinical outcome.., investigating risk factors for revision surgery after aci. other factors for consideration in patient selection include alignment, ligamentous and meniscal injuries, and amount of degenerative changes. in the present article unfortunately, the database was inconsistent in the reporting of anatomical location of the defect and these data therefore were not included in our study. females are less likely to receive aci treatment as seen in the database compared with the studies. the role of gender in focal cartilage damage and outcome after aci has been investigated previously and some controversy exists. while jungmann. found the female gender to be negatively related to outcome, filardo. showed in a match - pair analysis that while females generally had more complex cartilage injuries, all other factors equal, the female gender did not predict worse outcome after aci - m. there are no clear explanation for the international differences observed in patient inclusion for maci treatment. cultural aspects may play a role but different health care and reimbursement systems may also be important. the database does not provide any information on whether patients were treated in private or public hospitals. notably, studies carried out in the united states had the highest average defect size of the study population, but it still remains unclear how this compares to the patient population receiving chondrocyte implantation in that country. the present study used nonstandardized surgeon assessment of defect size, which could potentially confound the data. if all surgeons overestimated or underestimated the defect size during arthroscopy for the database compared with a postdebridement measurement in the studies, this may be a potential source of bias. it is, however, a measurement method similar to that most commonly used in clinical studies. for example, the largest combined defect size in the register is estimated to be 47 cm, which likely represents an outlier. the high number of included patients and surgeons performing the evaluations limit the role of this potential confounder as well as the influence of the very few statistical outliers. as the database contained age at the time of biopsy, the actual age of the patients at the time of surgery will be higher, but the actual age at the time of surgery is unknown. however, it could be argued that since the biopsy is taken at the time of indication for chondrocyte transplantation, this may be the more correct measure to use, as we do not look at the outcome in relation to age. the genzyme / sanofi database did not contain information on body mass index and the reproducible information on anatomical location of the defect was insufficient to allow for analysis. there were also no data on additional knee injury such as meniscal and ligament tears. all these factors are however also important in considering the correct patient selection for treatment of cartilage injuries with autologous chondrocyte implantation. we compared patients scheduled for a commercially available third - generation aci treatment maci. this was compared with patients scheduled for many different types of aci - related treatments. in the comparison made in the present study, emphasis is put on indications, removing potential confounding since the indications are similar for all types of aci treatments regardless of generation or commercialization. in our comparison there is overlap in patient data between the database and the studies, since patients receiving maci in the studies are also present in the database. this could impair the validity in terms of potential bias in the country - stratification comparison if countries with no or little difference between defect size in the database and reported studies (e.g., germany and england) were only using maci in the reported studies. this is however not the case and the risk of bias is presumed to be of small in this comparison. this study shows that the vast majority of patients scheduled for aci with maci have articular cartilage defect sizes that are within the range of what is generally recommend for this procedure, although patients enrolled in clinical trials have significantly smaller defects than those scheduled for treatment outside a trial environment. this study also shows that patients receiving maci treatment in 9 european countries, australia, and singapore have different cartilage defect sizes, and in some countries the difference between patients enrolled in trials and generally assigned for surgery differs significantly. | objectiveto compare characteristics for patients scheduled for autologous chondrocyte implantation with matrix - assisted chondrocyte implantation (maci) with those enrolled in clinical trials and to describe differences in patient selection between countries.designanonymized data from patients scheduled for maci treatment in the knee in europe and australia / asia were obtained from the genzyme / sanofi database. average age, defect size, and male - female ratio were analyzed and compared by country. clinical cohort studies and prospective comparative trials using autologous chondrocyte implantation and related treatments were identified and weighted average age, weighted defect size, and male - female ratio were analyzed and compared with data from the database.resultsfrom the database 2,690 patients were included with mean age 33.7 years and male - female ratio of 67:33. mean defect size was 5.64 cm2 and 70% of the defects were 3 to 10 cm2. there were significant differences between patients mean defect sizes between countries. sixty - nine studies (57 cohorts and 12 prospective comparative trials) with a total of 5,449 patients were identified. the combined weighted mean age was 34.2 years, and the combined weighted mean defect size was 4.89 cm2. patients scheduled for maci had significantly larger defects that those included in clinical trials. there was no significant difference in age. no differences were found between cohorts and prospective comparative trials.conclusionthe vast majority of patients scheduled for autologous chondrocyte implantation with maci have chondral defect comparable to that generally recommended, but differences exist between countries. patients enrolled in clinical trials have significantly smaller defects than those undergoing treatment outside controlled trials. |
sry (sex determining region y)-box (sox) genes encode a group of transcription factors that bind to the minor groove of target dna elements and regulate transcriptional initiation of nearby promoters (1). typically numbering in 20 in diverse vertebrate species, the members of this family can be further divided into subgroups based on the level of amino acid conservation within the hmg box and the presence of other motifs (1). sox5, sox6, and sox13 comprise the soxd subgroup. aside from the highly conserved hmg box, they feature the leucine zipper motif which allows homo- and hetero - dimerization and consequently endows added flexibility in binding site selection (2). soxd genes are expressed widely during embryogenesis and play critical roles in various aspects of development including cell fate specification (2). during development, sox5 is expressed in subsets of cells in the central nervous system (cns), cranial ganglia, neural crest and skeletal / cartilage tissues as well as in melanocytes and th17 cells (26). like sox 5, sox6 is expressed in cns and skeletal / cartilage tissues but in addition is also seen in skeletal and cardiac myocytes and erythroid cells (2, 7) sox13 also is seen in cells in developing cns and in cartilage progenitors (2, 8). sox13 expression is detected in addition in the developing artery, inner ear, hair follicle and a subset of t cells (2, 810). the best known functions of soxd genes have to do with cell fate determination and differentiation. sox5 and sox6 are particularly well characterized for their redundant and synergistic roles in chondrogenic differentiation (1114). loss of either sox5 or sox6 has limited effects but that of both leads to severe chondrodysplasia leading to defects in skeleton formation and embryonic lethality (11, 12). sox5 is also cell - autonomously required for normal th17 cell differentiation in mouse (6). independent of sox5, sox6 functions in terminal differentiation of cardiac and skeletal muscle cells and erythroid cells (7, 1517). sox13 is expressed in t cells and promotes development of this subpopulation while opposing differentiation of t cells (10). consisting a minority subset of t cells, t cells express and receptors rather than and receptors and show distinct preferred tissues of localization and partly overlapping immune functions with t cells (10). sox13-ablated mice show a severe growth retardation compared to wild type mice possibly due to the loss of wnt signaling modulation for which sox13 is responsible (10). the best characterized role of soxd genes in neural development has to do with the regulation of neuronal subtype determination. most of the progress here also is in large part based on analyses of gene - targeted mice during embryogenesis. sox5 is expressed in deep cortical layers (layers v, vi, and subplate) from which distinct neuronal populations are born in succession (18). the neurons in the deepest layer, the subplate are born in embryonic day (e) 11.5 while those in layers vi and v show the peaks of generation around e12.5 and e13.5 respectively (18, 19). neurons in these deep cortical layers, mostly projecting subcortically to spinal cord, pons, midbrain, and thalamus, are born ahead of those in layers i through iv (18). their layer specification is marked and controlled by presence and/or absence of various transcription factors including cux1, cux2, ferzt2, and ctip2 (18). lai and coworkers showed that deletion of sox5 results in an abnormal sequence in generation of layer specific neurons and dys - regulated gene expression leading to abrogation of layer specific characteristics (19). an independent study by kwan and coworkers in addition demonstrated that layer v subcerebral axons are missing and that sp and layer vi axons are misrouted (20). the results indicate that sox5 is critically involved in multiple aspects of neuronal differentiation for subsets of cortical neurons. sox5 and sox6 show largely mutually exclusive expression patterns during corticogenesis (21). in an elegant study by azim and coworkers, sox6 was shown to be expressed in the ventricular zone of pallium (eventually the upper surface of cerebrum) and postmitotic neurons of subpallium (i.e. ganglionic eminence portions of telencephalon) while sox5 expression was detected in subsets of postmitotic neurons in pallium and ventricular zone of subpallium (21). the complementary expression apparently reflects the mutually repressive function of the two transcription factors at least for the progenitor cells in the ventricular zone (21). specifically, loss of one gene leads to expansion of the zone expressing the other. given however that ectopic expression of one transcription factor does not repress the expression of the other, it appears that sox5 and sox6 are necessary but not sufficient for the cross repression. gene deletion of sox6 also revealed that sox6 controls diversity, positioning and maturation of subpallial postmitotic neurons which ultimately constitute cortical interneurons (21, 22). panman and coworkers reported that sox6 is expressed in a medially located subset of mda neurons and its ablation leads to a decrease of substantia nigra pars compacta mda neurons and an increase in ventral tegmental area mda neurons (23). a critical counterpart player appears to be otx2 which is highly expressed in ventral tegmental area mda neurons and deletion of which leads to expansion of the midbrain region occupied by sox6-positive neurons (23). far less is known about the function of sox13 although a brain - wide expression in the forebrain, midbrain, and hindbrain in post mitotic neurons has been reported (8). more recently, expression of sox13 has also been shown in oligodendrocyte lineage cells (24). this limited knowledge on sox13 function likely is due to the redundant role among soxd genes and a lack of distinct neural phenotypes in sox13-null mouse model. therefore, although sox13 deletion on its own may result in subtle to no visible effect insofar as neuronal fate determination is concerned, combinatorial gene deletion will likely lead to better functional assignments. a careful and detailed comparative analysis of expression pattern for the three genes should be carried out from which testable predictions on function can be made. soxd transcription factors are not just expressed in postmitotic neurons but also in proliferating progenitors in the ventricular and subventricular zones (vz and svz respectively) of the developing nervous system (21, 2527). alternatively, they may be involved in determining regional identity as their expression is seen in more restricted parts of the vz and svz compared to members of the soxb1 gene subfamily consisting of sox1, sox2, and sox3 (28). these transcription factors show broad and overlapping expression in the vz and svz of the developing nervous system (28). stemness of neural progenitors during embryogenesis and into adulthood (29). in at least two different developmental contexts, soxd genes using gene targeted models, stolt and coworkers showed that soxd proteins control multiple aspects of oligodendrocyte development (27). specifically, they showed that deletion of either sox5 or sox6 induces premature appearance of cells positive for sox10 which is a definitive oligodendrocytic lineage marker in the central nervous system. furthermore, terminal markers of oligodendrocytes such as proteolipid protein 1 and myelin basic protein were also precociously expressed. importantly, they showed that the effect is more pronounced in compound knockout embryos demonstrating the redundant function of the two transcription factors. recently, by examining sox6 and sox13 compound mutation embryo, the baroti and coworkers demonstrated that sox13 also contributes to inhibition of premature oligodendrocytic differentiation (24). it should be also noted that migration pattern as well showed anomaly in the gene - targeted embryos consistent with the proposed function of sox5 and sox6 in the multiple stages of oligodendrocyte development (27). in another study by lee and coworkers, function of sox6 in a broader population of neural progenitors the study was based on the finding that sox6 is a direct regulatory target of sox2, a critical determinant of consistent with that sox6 was a downstream target of sox2, sox6 expression was shown to occur among sox2 positive cells during neural development. using the chicken in ovo system which had been extensively used to examine spinal cord development, the investigators first demonstrated that sox2 induces expression of sox6 in vivo and subsequently carried out gain- and loss - of - function of sox6 in the neural progenitors. remarkably, inhibition of sox6 expression via rna interference led to a severe depletion of sox2 positive neural progenitors and induction of isl1 positive motor neurons in a spatiotemporally precocious manner. taken together with that ectopic expression of sox6 resulted in enhanced sox2 expression and inhibition of motor neuron differentiation, it was proposed that sox6 forms a positive feedback network with sox2 and that together they maintain a pool of neural progenitor or stem cell population (fig. 1). sox5 shows an expression pattern partially overlapping with that of sox6 in the vz and svz of developing spinal cord (27). like sox6, sox5 has also been proposed to be a direct regulatory target of sox2 (25). it is thus possible that sox5 plays a similar role for the neural precursors in the developing spinal cord. rather, a study by quiroga and coworkers described cell fate specifying function of sox5 in dorsal neural progenitors ultimately controlling the specification of dorsal interneurons (26). as described above, sox5 and sox6 are also expressed in the vz s of subpallium and pallium respectively (21). whether they are directly or indirectly targeted by sox2 or other soxb1 proteins in the cortex and whether they contribute to maintenance of it has been reported that sox5 inhibits premature differentiation of cortical neurons (19). this however is in reference to the proper sequence and timing in the generation of distinct types of cortical neurons and may have little to do with maintenance of stemness. azim and coworkers, who described the mutually exclusive pattern of expression of sox5 and sox6 also noted that a similar pattern is shown by ngn2 and mash1 expressed respectively in pallium and subpallium (21). in addition, other subpallium - restricted genes including olig2, dlx1, dlx2, gxh2, ils1 and meis1 all showed expression in pallium. loss of sox5 however did not show a reciprocal expansion of the zone expressing pallium - specfic genes indicating that regulatory network is not built around sox5 and sox6 as the principal determinants of cell fate. this view is further enhanced by the observation that sox6-deleted pallial progenitor cells co - express ngn2 and mash1 rather than losing ngn2 expression altogether. still, it is clear that the two transcription factors contribute to demarcation of distinct zones of differentially fated cells. it has been long noted that soxd transcription factors lack domains associated with transcriptional activation or repression (2). another issue is how they play apparently distinct roles during multiple developmental stages of given cell lineages. for example, how do their roles switch from maintaining stemness in neural progenitors to regulating timing of differentiation and cell type specific gene expression in developing cortical neurons and oligodendrocytes ? while little is known, interactions with other transcription factors and co - factors, both cooperative and inhibitory, are likely key parts of the answer (2). sox5 and sox6 bind to sites within super - enhancers for the genes involved in chondrocyte development including transcription factors and extracellular matrix (14). importantly, they promote binding of sox9 to a distinct but often closely located sites thereby bringing the key activator of chondrogenesis to relevant target genes (13, 14). stunningly, sox5 and sox6 inhibit the activity of the same sox9 as well as sox10 during oligodendrocyte development (27). the mechanisms thus may be cell type and target gene specific and include competing for binding sites and for other partner transcription factors such as olig2 (27). sox5 has also been reported to inhibit expression of sox10 target genes in the melanocyte lineage by recruiting the co - repressors ctbp2 and hdac1 to their regulatory regions (5). this is in contrast to physical interaction with bmp r - smads and activation of bmp target genes during early ectoderm patterning and neural crest formation (30). sox6 also has been reported to interact with hdac1 and can inhibit -catenin dependent transcriptional activation via direct interaction (31). during cortical neuronal differentiation, sox5 has been shown to inhibit expression of fezf2, a gene required for corticospinal neuron identity and connectivity, by competing with sox4 and sox11 for binding sites in an enhancer (32). finally, sox13 can block canonical wnt signaling by directly interacting with tcf1 and promote t cell development (10). in sum, soxd genes can function as either activators or repressors of transcription depending on the developmental and cellular contexts and the nature of cis - regulatory elements of given target genes as well as the presence and interaction with other transcription factors or cofactors. this represents overall a very complex situation that defies generalization, and direct target genes of soxd transcription factors need to be examined in a gene - by - gene manner. the three soxd genes are expressed in diverse cell types throughout development in partially overlapping manners. recent years have seen significant advances made in understanding of their roles in neural development. in particular, animal models including knockout mice have made major contributions to demonstrating their functions in maintaining stemness of progenitors, in generation of diverse subtypes of cortical neurons and in properly timed differentiation of oligodendrocytes. still, our knowledge is extremely limited in terms of specific molecular mechanisms of transcriptional regulation. this is in large part due to the diversity of molecular functions which soxd transcription factors are capable of performing and the lack of knowledge on direct regulatory target genes in most of the cell types expressing them. this implies that what lies ahead is not a generalization but a gene - by - gene analyses. specifically, we need to define transcriptional profiles regulated by these genes and determine direct target genes. modern techniques and tools of molecular biology including rna - seq, chip - seq, and genomic sequences of multiple vertebrate species should enable the first necessary steps to this end. | soxd transcription factor subfamily includes three members, sox5, sox6, and sox13. like other sox genes, they contain the high - mobility - group (hmg) box as the dna binding domain but in addition feature the subgroup - specific leucine zipper motif. soxd genes are expressed in diverse cell types in multiple organs during embryogenesis and in adulthood. among the cells expressing them are those present in the developing nervous system including neural stem (or progenitor) cells as well as differentiating neurons and oligodendrocytes. soxd transcription factors do not contain distinct activator or repressor domain, and they are believed to function in modulation of other transcription factors in promoter - specific manners. this brief review article will attempt to summarize the latest studies on the function of soxd genes in embryogenesis with a particular emphasis on the regulation of neural development. |
in order to receive an accurate diagnosis and treatment planning, many researchers have used different criteria to determine the anterior - posterior relationship of the jaws. although molar relationship has been used in angel classification, it is clear that canine and molar relationship indicates the anterior - posterior relationship of the jaws when teeth are placed in the accurate position on the dental arch. using plaster models for classification of malocclusion or determination of the jaws relationship, without lateral cephalometric radiograph is not reliable, particularly for patients who have defective dental structures. currently, several types of intracranial plans have been used as reference lines to determine discrepancy of the jaws. the most popular measurement of discrepancy of the jaws in anterior - posterior dimension is a point, nasion, b point (anb) angle, which indicates the relationship of apical base of maxilla to mandible. based on the geometric studies (studies conducted geometrically rather than statistically), following factors are able to change anb angle, without any effect on the anterior - posterior relationship of apical bases : rotation of the jaws around anterior cranial base.rotation of the occlusal plane around anterior cranial base, with or without jaws rotation.position of nasion in the sagittal and vertical dimension and the length of anterior cranial base (s - n).facial height.rotation of s - n plan. rotation of the occlusal plane around anterior cranial base, with or without jaws rotation. position of nasion in the sagittal and vertical dimension and the length of anterior cranial base (s - n). thus, regarding the geometric studies, anb angle can not always indicate anterior - posterior relationship of the jaws accurately. this index is a linear measurement, which is the distance between two perpendicular lines from points a to b to functional occlusal plane. geometric studies have shown that the value of wits appraisal index may be influenced by alteration of functional occlusal plane and vertical distance of points a and b as well as sagittal alteration of points a and b. some researchers have offered linear and angular measurements based on palatal plane. although this plane is constant, with increasing of age, it has variable gradient, so it is difficult to determine normal average. as these indexes are influenced by anatomic landmarks and dental occlusion and regarding to probability of effect of these landmarks on accuracy of evaluations, it is necessary to access an independent index. recently, and angles have been introduced, which are not dependent on cranial landmarks or dental occlusion. therefore, they are reliable indexes in cases that previous indexes such as anb angle and wits appraisal may not be used. angle is a novel method of measurement of skeletal discrepancy between the maxilla and mandible in sagittal plan. this angle uses three anatomic landmarks : point a, point b and mandibular plan : point a : is the deepest point on the midline of the maxilla, which moves from base to alveolar process. point b : is the most anterior part of base of the mandible and the most posterior point of the outer contour of mandibular alveolar process in midline. angle is the angle between ab line and perpendicular line from a to mandibular plan [figure 1 ]. angle also uses three anatomic landmarks : point a, point b and condylar axis : point c (condylion) : is the most posterior - superior point on condylar head of the mandible (in the present study, c is considered as the center of the condyle). angle is the angle between ab line and perpendicular line from point a to cb line [figure 2 ]. considering ethnical, sexual and growth differences, it is necessary to evaluate normal cephalometric average of every population. the aim of this study was to evaluate and to compare the range of and angles of 6 - 17-year - old children of isfahan who have normal occlusion. this is a descriptive analytical study. in order to conduct this study, archive documents of orthodontic department of isfahan dental school a total of 235 cephalometric radiographs of children, who had no history of orthodontic treatment and also based on 13 following criteria categorized as normal occlusion, were selected : symmetric face.having the whole teeth regarding to age (lack of primary tooth was acceptable if there was adequate space).normal tooth form, position and size.class i molar relationship for permanent tooth (class i molar relationship or end to end relationship during mixed dentition or flush terminal plane relationship of second primary teeth [e ] in cases of incomplete growth of permanent first molar).class i canine relationship.proper overbite up to 3 mm and over jet up to 2 mm.slight or no space between teeth.slight or no dental rotation.slight or no dental crowding.no posterior cross bite.no history of previous orthodontic treatment.no history of orthognathic surgery.no congenital disorder. having the whole teeth regarding to age (lack of primary tooth was acceptable if there was adequate space). class i molar relationship for permanent tooth (class i molar relationship or end to end relationship during mixed dentition or flush terminal plane relationship of second primary teeth [e ] in cases of incomplete growth of permanent first molar). acetate tracing paper (dentaurum, germany) was used for tracing and cephalometric protractor (3 m unitek, corporation, monrovia, california, usa) with accuracy of 1 was used for measurement. anb angle, wits appraisal index, and angles were measured. during measurement, 16 subjects were excluded due to clear inconsistency between anb angle and wits appraisal index. furthermore, 17 subjects were excluded due to failure of determination of condylar center position in order to measure angle. three subjects were excluded due to superimposition of right and left mandibular plan and consequently failure of determination of angle. finally, 199 subjects (112 females and 87 males) participated in this study. correlations of and angles with anb angle and wits appraisal index were determined using pearson correlation coefficient. t - test was used to determine the relationship between gender and and angles and anova was used to evaluate the relationship between age groups and and angles (p < 0.05). frequency of 6 - 17-year - old children of isfahan with normal occlusion based on age group mean and standard deviation of and angles are illustrated in table 2. mean and standard deviation of anb angle, wits appraisal, and angles in 6 - 17-year - old children of isfahan with normal occlusion according to t - test, there was a significant difference between the mean of angle and gender. however, there was no significant difference between other angles and gender [table 3 ]. there was significant differences between the mean of angle and 14 - 17-year - old age group (p = 0.01) while the mean of angle showed a significant difference in age groups of 8 - 10-year - old (p = 0.02) and 14 - 17-year - old (p = 0.02). comparison mean and standard deviation of anb angle, wits appraisal, and angles in 6 - 17-year - old children of isfahan with normal occlusion based on sex according to spearmen correlation coefficient there was no significant relationship between angle and age (p = 0.054) (r = 0.138), while there was a direct, significant relationship between angle and age (r = 0.435). the relationship between values of and angles and anb angle are presented on diagrams 1 and 2 and the relationship between these angles with wits appraisal index is presented on diagrams 3 and 4. relationship of angle with a point, nasion, b point angle relationship of angle with a point, nasion, b point angle relationship of angle with wits appraisal index relationship of angle with wits appraisal index accurate measurement of anterior - posterior relationship of the jaws has a major importance in orthodontic treatment plan. the well - known criterion to investigate the anterior - posterior relationship of the jaws is anb angle. a common alternative is wits appraisal index, which is not dependent on cranial landmarks or jaws rotation ; however, accurate diagnosis of functional occlusal plane is difficult and it may occur after orthodontic treatments due to alteration of functional occlusal plane rather than alteration of sagittal relationship of the jaws. in order to compensate the deficiency of anb angle and wits appraisal index, baik and ververidou suggested angle. the researchers claimed that angle not only is independent of cranial landmarks and functional occlusal plane, but also it seems that clockwise or counter - clockwise rotation of the jaws has slight influence on this angle. another advantage of angle is that it can be applied in serial evaluation of orthodontics treatment plan and shows changes in sagittal relationship of the jaws related to growth and orthodontic or orthognathic interference. in addition, it can be used as accurate criteria in orthognathic surgery for patients with anterior - posterior and vertical skeletal disorder. however, major disadvantage of this angle is determination of condylar center, which is not always easy. this issue has been similarly experienced in the present study, which 17 subjects were excluded due to lack of clarity of condylar area. in order to obtain accurate measurement of angle, cephalometric radiographs must have high quality, so that the clinician can easily trace posterior border of ramus and determine condylar head position. the advantage of using the center of condyle rather than condylion point is that accurate tracing of condylar contour is not usually easy. as it is illustrate in figure 2, if real condylar center is point c, but clinician locates it in a circle with a radius of 2 mm, angle is affected less than 1, which makes its application more acceptable. in the present study, baik and ververidou showed the range of angle 27.5 - 43.5 and 27 - 35 respectively. the difference between these studies is probably due to different sample size, inclusion criteria and demographic features of patients. the mean of angle was 31.7 3.3 in the current study, which is consistent with the results of fattahi. suggested angle, which is superior to angle because the determination of mandibular plane is easier and the quality of radiograph does not influence this plane. however, mandibular plane may be defective in some lateral cephalograms due to weak radiographic technique. one of the advantages of angle is that the rotation of the lower jaw from temporomandibular joint area or the rotation of mandibular body does not really influence the angle as a and b points change their position. however, if mandibular plane changes without displacement of a and b points, the angle will be failed. in the present study, the range of angle was 8 - 27, while fattahi. showed 11 - 29. the mean value of angle was 17.34 3.74, which is consistent with the results of fattahi. according to spearman correlation coefficient there was direct significant, but weak relationship between angle and age (r = 0.435). as angle increases with the increase of age, it can be suggested that angle is consistent with cephalocaudal gradient of the growth curve. pearson correlation coefficient revealed that there was reverse significant relationship between anb angle and angle (r = 0.520) and also angle (r = 0.329). as an increase in angle and angle indicated a tendency to class iii and a decrease in anb angle indicates the same tendency, these angles reinforce each other in the classification of the malocclusion. there was also weak and reverse significant relationship between wits appraisal index and angle (r = 0.412) and angle (r = 0.427). as increase of these angles and a decrease of wits appraisal index (more negative) indicate tendency to c1ass iii, both of them reinforce each other in the classification of the malocclusion. biak and ververidou suggested that there is a significant relationship between anb angle and wits appraisal index and angle. according to the present study, there was a direct significant relationship between angle and angle (r = 0.621). therefore, it can be said that these two angles have consistent validity and can be well used in the determination of anterior - posterior malocclusion. in fact, all linear and angular measurement in lateral radiographies has specific advantages and disadvantages, thus none of the methods can be used merely as an accurate method for diagnosis and treatment plan in orthodontics. although angle and angle are able to diagnosis the anterior - posterior relationship of the jaws and bone disorder, previous cephalometric measurements should not be ignored. based on the findings of the current study, further studies on the sensitivity and specificity of angle and angle in three types of malocclusion and also comparison of these angles with other method of measuring jaw relationship such as the angle of convexity and facial angle are recommended. considering that angle and angle do not have the disadvantages of previous measurements such as anb angle and wits appraisal index for evaluation of anterior - posterior relationship of the jaws, they may be an accurate method for diagnosis of jaw disorders and treatment plan. | background : linear and angular measurements such as a point, nasion, b point (anb) angle and wits appraisal index are not accurate enough to evaluate sagittal relationship of the jaws. the aim of this study was to evaluate and compare the range of and angles in 6 - 17-year - old children of isfahan, having normal occlusion.materials and methods : this was an analytical descriptive study. for this study, 235 cephalometric radiographs of patients who did nt receive orthodontics treatments and based on 13 indexes had normal occlusion, were selected. after tracing of cephalograms, anb angle, wits appraisal index, angle (resulted from the intersection of ab line and perpendicular line from point a to mandibular plane) and angle (resulted from the intersection of ab line and perpendicular line from point a on cb line) were measured. data was analyzed by t - test, anova and pierson - spearman correlation coefficient (p < 0.05).results : mean value of and angles were 17.34 3.47 and 31.7 3.31 and ranged from 8 - 27 to 21.5 - 39 respectively. according to t - test, there was a significant difference between two sex groups for angle (p = 0.02) ; however, it was not significant for angle. according to spearman correlation coefficient, there was no significant difference between age and angle ; however, angle was directly and significantly related to age (r = 0.435). there was significant and reverse relationship between and angles with anb angle and wits appraisal index.conclusion: and angles are reliable and can be used to evaluate the anterior - posterior relationship of the jaws. |
docosahexaenoic acid (dha), a type of 3 polyunsaturated fatty acid (pufa), has attracted considerable attention due to its structural and physiological functions.1 dha is an essential compound in the cellular membrane of nervous tissues, such as brain and retina, and can improve development of these tissues. moreover, dha has beneficial effects in preventing cardiovascular diseases, cancer, schizophrenia, and alzheimer 's disease.2 among many microorganisms, aurantiochytrium sp., which is a marine funguslike heterotrophic labyrinthulomycetes, is an effective fermentative microorganism for dha production because it grows fast and accumulates high levels of dha as a main fatty acid contained in microbial lipids.3 the microbial oil from aurantiochytrium sp., containing high levels of dha, was considered one of the most feasible dha sources and could alleviate the problems caused by the traditional fish oil source.3 cultivation of aurantiochytrium sp. for dha production was the most fundamental step because it affects cost and quality of dha. apart from essential macronutrients (carbon, nitrogen sources, and sea salt), some micronutrients, such as vitamins and trace elements, are required for growth of aurantiochytrium sp. and are added to the medium to improve the biomass and dha yield of aurantiochytrium sp.. 4, 5 these micronutrients could reduce cost and increase quality of dha significantly at low concentrations. however, the effects of phytohormone on biomass and dha yields in aurantiochytrium sp. and its mechanisms have never been reported. plant hormones (phytohormones) are natural or synthetic chemical messengers, which regulate growth and development of plants and are effective at very low concentrations. plant hormones have been divided into many groups and they stimulate a variety of biological processes, such as cell growth and metabolism of fatty acids and lipids.6, 7 apart from plants, some nonplant microorganisms, including fungi and microalgae, have also been reported to be affected by plant hormones. chatterjee. reported that 0.1 mg l gibberellic acid could enhance mycelial growth and chitosan yield in rhizopus oryzae.8 in plant fungi symbiosis systems, some plant hormones, as signal molecules, may affect the symbiosis positively or adversely.9 microalgae are primitive eukaryotic plant cells and have a close evolutional relationship with plants. thus, there are various reports on effects of different plant hormones on algal growth and production of metabolites. uses of plant substrates, such as auxins, cytokinins, abscisic acid (aba), polyamines, brassinosteroids, jasmonides, and salicylic acid, were able to stimulate growth of chlorella sp., whether for the production of lipids for biofuels, the production of bioproducts, the treatment of wastewater, or a variety of other reasons.10 salama. found that phytohormones, such as indole3acetic acid (iaa) and diethyl aminoethyl hexanoate (dah), could accelerate growth of scenedesmus obliquus and induce the quality and quantity of fatty acid content for biodiesel production.11 as a plant hormone, 6benzylaminopurine (6bap) is a cytokinin that stimulates plant growth and development, setting blossoms and stimulating fruit maturity by accelerating cell division.12 it is also an inhibitor of respiratory kinase in plants, and increases postharvest life of green vegetables.13, 14 however, few studies on the effects of 6bap on the growth of microorganisms have been reported. thus, 6bap should be considered a growth stimulator in fermentation media for improvements of biomass and dha yield in aurantiochytrium sp. moreover, labyrinthulomycetes are often prevalent in marine ecology and inhabit marine plants as commensals or mutualists.15 the effect of plant hormone on aurantiochytrium sp. and its mechanism can deepen our understanding of the relationship between plants and aurantiochytrium sp. metabolomics has provided new insights into understanding intracellular metabolites and discovering potential biomarkers.16 this technology has been applied in microalgae to improve lipid productivity.17 moreover, metabolomics was also used to reveal the global response of biochemical network reactions to environmental, genetic, or developmental signals.18 gas chromatography mass spectrometry (gcms) was applied to investigate the metabolic profile of aurantiochytrium sp. under different oxygen conditions.18 this metabolomics method was also used to analyze the metabolomics profile of the final fermentative cells of schizochytrium sp.19 in this study, effects of 6bap on growth, lipid and dha yield in aurantiochytrium sp. comparative metabolomics analysis of aurantiochytrium sp. under 6baptreated and control groups (without 6bap) at different time points was performed through a gcmsbased metabolomics method and multivariate analysis. this study could provide a better understanding of the mechanism underlying 6bap enhancement of lipid and dha biosynthesis in aurantiochytrium sp. the nonadecanoic acid, docosahexaenoic acid methyl esters, 6benzylaminopurine, adonitol, methoxyamine hydrochloride, nmethylntrimethylsilyltrifluoroacetamide and trimethychlorosilane were purchased from sigmaaldrich (st. louis, usa). ylh70 (cctcc no : m2014215), which was isolated from the mangrove ecosystem in yueqing bay and was collected in the china centre for type culture collection (cctcc), was used in the present study. the strain was preserved in 20% glycerol at 80 c and was maintained in gpy medium, containing 2% glucose, 1% polypeptone, 0.5% yeast extract and 20% sea salt. the strain was inoculated into a 500 ml flask containing 300 ml of gpy medium and cultured at 28 c for 2 days as seed culture. the seed culture was then inoculated into 5 l fermentation reactors containing 3 l of fermentation medium, composed of 6% glucose, 1% yeast extract, 0.5% peptone and 20 g l sea salt, and cultured at 28 c for 5 days. for 6baptreated cells, different concentrations of 6bap (0, 1, 3, 5, 7, and 9 mg ml) were added to the fermentation medium before cultivation. samples of the control and 6baptreated groups were collected every 24 h until the total fermentation time reached 120 h, and the biomass, lipid content, fatty acid profile and metabolic profile were analyzed as described below. an aliquot of 50 ml of cell suspension was centrifuged at 4 c and 7000 g for 5 min and then washed twice with distilled water. the cell pellets were lyophilized to a constant weight at 50 c for approximately 48 h. the lipid extraction and fatty acid analysis were performed according to the method reported by gao. with moderate modifications.20 the lyophilized cell was ground by mortar and pestle into a fine powder under liquid nitrogen. the cell powder was extracted into 100 ml of chloroform / methanol (2:1, v / v) at room temperature. the lipid extract was dried by evaporation and then weighed. for fatty acid analysis, 0.1 g cell powder was mixed with 5 ml of 0.4 mol l methanolic koh and incubated at 60 c for 1 h, followed by an esterification procedure in 5 ml of bf3methanol (14%, w / w) reagent at 60 c for another 1 h. the fatty acid methyl esters (fames) were extracted with 5 ml hexane and analyzed by agilent 6890 n gc using an hpinnowax column (30 m 0.25 mm, 0.25 m film thickness, agilent technologies) with he as carrier gas. the temperaturecontrol procedure was as follows : from 100 to 240 c at 15 c per min and maintained at 240 c for another 10 min ; the temperature of the injection port and flame ionization port was 250 c. the collected cell was quenched and extracted according to the methods reported by li. the sample was quickly mixed with cold 60% methanol (40 c, v / v) to capture the metabolism of cells instantaneously and then centrifuged at 5000 g and 4 c for 3 min. the cell pellet was washed twice with cold physiological saline (0.9% sodium chloride solution) and ground by mortar and pestle into a fine powder under liquid nitrogen. 1 ml of prechill methanol (40 c) was added to the cell powder (100 mg) and mixed thoroughly for 30 s. the mixture was centrifuged at 5000 g and 4 c for 5 min and the supernatant collected. an aliquot of 0.75 ml of prechill methanol (40 c) was added to the cell pellet and shaken violently for 30 s followed by centrifugation at 8000 g and 4 c for 5 min. six independent repeats were prepared for the control and 6baptreated groups at each time point. for gcms analysis, derivatization of samples was performed according to the method of hu. with moderate modifications.21 the 0.5 ml sample obtained above and 50 l internal standard (adonitol in water, 1 mg ml) were dried in a vacuum centrifuge dryer. 200 l of methoxyamine hydrochloride in pyridine (20 mg ml) was added to the dried sample and incubated at 30 c for 2 h. subsequently, 200 l of mstfa (nmethylntrimethylsilyltrifluoroacetamide) containing trimethychlorosilane (1%, v / v) was added to the sample, and incubated at 37 c for another 1 h. the resulting samples were preserved at 40 c and adjusted to room temperature before injection. the gcms analysis was performed on an aglilent 78905975c gcms solution system (agilent, usa) equipped with a db5 capillary column (30 m 0.25 mm, 0.25 m film thickness ; agilent j&w scientific, folsom, ca). under a constant flow rate of 1 ml min, helium was used as the carrier gas. 1 l of samples were injected into the db5 capillary column at a split radio of 1:3. the gc oven temperature was kept at 70 c for 2 min, raised to 290 c at a rate of 8 c min, and then kept for 3 min. the temperatures of injection, transfer line and ion source were 300 c, 280 c and 250 c, respectively. a mass scan range (50600 m / z) at a rate of 20 scans s was used. the real time analysis software in the gcms system (agilent, usa) was used to obtain mass spectrometric data. all detected metabolites were identified by alignments of mass spectra in nist 05 (national institute of standards and technology, gaithersburg, md) installed in the gcms system. the data set was normalized using division of the intensity of the peaks in each sample by the intensity of the internal standard peak, and then by the quality of each sample on the same chromatograph. the generated normalized metabolite contents (variables) were imported into the simca package (version 10.0, umetrics, umea, sweden) for multivariate analysis. principal component analysis (pca), a nonsupervised method was initially performed to obtain an overview of the gcms data from the control groups from the 6baptreated groups. differences between the samples were detected in the pca score plots, in which each point represents a linear combination of all the metabolites from every sample. distance between the groups of samples shows a measure of the overall difference among the metabolic profiles under different conditions. a supervised plsda was subsequently performed to identify the metabolites contributing to the differences between the control and 6baptreated groups. a coefficient loading plot of the plsda models was used to determine the differential metabolites that could separate the sample groups. based on the loading plots and variable importance in the projection (vip) value threshold (vip > 1), metabolites that were contributing to differing the control and the 6baptreated groups were identified through the plsda model.22 the altered metabolites, which were elicited by 6bap and selected from the plsda model, were further subjected to hierarchical cluster analysis (hca). hca was performed by euclidian distance and complete linkage grouping with the help of gene cluster software, and visualized through treeview software. then, two cycles of median center genes and arrays were performed, which repeated five times. finally, the data sets were applied to three cycles of normalize genes and arrays, which repeated five times. oneway analysis of variance (anova) t tests were performed using spss 12.0 software to analyze the distribution of the concentrations and logarithmically transformed concentrations for metabolites in this study. the nonadecanoic acid, docosahexaenoic acid methyl esters, 6benzylaminopurine, adonitol, methoxyamine hydrochloride, nmethylntrimethylsilyltrifluoroacetamide and trimethychlorosilane were purchased from sigmaaldrich (st. louis, usa). ylh70 (cctcc no : m2014215), which was isolated from the mangrove ecosystem in yueqing bay and was collected in the china centre for type culture collection (cctcc), was used in the present study. the strain was preserved in 20% glycerol at 80 c and was maintained in gpy medium, containing 2% glucose, 1% polypeptone, 0.5% yeast extract and 20% sea salt. the strain was inoculated into a 500 ml flask containing 300 ml of gpy medium and cultured at 28 c for 2 days as seed culture. the seed culture was then inoculated into 5 l fermentation reactors containing 3 l of fermentation medium, composed of 6% glucose, 1% yeast extract, 0.5% peptone and 20 g l sea salt, and cultured at 28 c for 5 days. for 6baptreated cells, different concentrations of 6bap (0, 1, 3, 5, 7, and 9 mg ml) were added to the fermentation medium before cultivation. samples of the control and 6baptreated groups were collected every 24 h until the total fermentation time reached 120 h, and the biomass, lipid content, fatty acid profile and metabolic profile were analyzed as described below. an aliquot of 50 ml of cell suspension was centrifuged at 4 c and 7000 g for 5 min and then washed twice with distilled water. the cell pellets were lyophilized to a constant weight at 50 c for approximately 48 h. the lipid extraction and fatty acid analysis were performed according to the method reported by gao. with moderate modifications.20 the lyophilized cell was ground by mortar and pestle into a fine powder under liquid nitrogen. the cell powder was extracted into 100 ml of chloroform / methanol (2:1, v / v) at room temperature. the lipid extract was dried by evaporation and then weighed. for fatty acid analysis, 0.1 g cell powder was mixed with 5 ml of 0.4 mol l methanolic koh and incubated at 60 c for 1 h, followed by an esterification procedure in 5 ml of bf3methanol (14%, w / w) reagent at 60 c for another 1 h. the fatty acid methyl esters (fames) were extracted with 5 ml hexane and analyzed by agilent 6890 n gc using an hpinnowax column (30 m 0.25 mm, 0.25 m film thickness, agilent technologies) with he as carrier gas. the temperaturecontrol procedure was as follows : from 100 to 240 c at 15 c per min and maintained at 240 c for another 10 min ; the temperature of the injection port and flame ionization port was 250 c. the collected cell was quenched and extracted according to the methods reported by li. the sample was quickly mixed with cold 60% methanol (40 c, v / v) to capture the metabolism of cells instantaneously and then centrifuged at 5000 g and 4 c for 3 min. the cell pellet was washed twice with cold physiological saline (0.9% sodium chloride solution) and ground by mortar and pestle into a fine powder under liquid nitrogen. 1 ml of prechill methanol (40 c) was added to the cell powder (100 mg) and mixed thoroughly for 30 s. the mixture was centrifuged at 5000 g and 4 c for 5 min and the supernatant collected. an aliquot of 0.75 ml of prechill methanol (40 c) was added to the cell pellet and shaken violently for 30 s followed by centrifugation at 8000 g and 4 c for 5 min. both supernatants were pooled together and preserved at 80 c for further analysis. six independent repeats were prepared for the control and 6baptreated groups at each time point. for gcms analysis, derivatization of samples was performed according to the method of hu. with moderate modifications.21 the 0.5 ml sample obtained above and 50 l internal standard (adonitol in water, 1 mg ml) were dried in a vacuum centrifuge dryer. 200 l of methoxyamine hydrochloride in pyridine (20 mg ml) was added to the dried sample and incubated at 30 c for 2 h. subsequently, 200 l of mstfa (nmethylntrimethylsilyltrifluoroacetamide) containing trimethychlorosilane (1%, v / v) was added to the sample, and incubated at 37 c for another 1 h. the resulting samples were preserved at 40 c and adjusted to room temperature before injection. the gcms analysis was performed on an aglilent 78905975c gcms solution system (agilent, usa) equipped with a db5 capillary column (30 m 0.25 mm, 0.25 m film thickness ; agilent j&w scientific, folsom, ca). under a constant flow rate of 1 ml min, helium was used as the carrier gas. 1 l of samples were injected into the db5 capillary column at a split radio of 1:3. the gc oven temperature was kept at 70 c for 2 min, raised to 290 c at a rate of 8 c min, and then kept for 3 min. the temperatures of injection, transfer line and ion source were 300 c, 280 c and 250 c, respectively. a mass scan range (50600 m / z) at a rate of 20 scans s was used. the real time analysis software in the gcms system (agilent, usa) was used to obtain mass spectrometric data. all detected metabolites were identified by alignments of mass spectra in nist 05 (national institute of standards and technology, gaithersburg, md) installed in the gcms system. the data set was normalized using division of the intensity of the peaks in each sample by the intensity of the internal standard peak, and then by the quality of each sample on the same chromatograph. the generated normalized metabolite contents (variables) were imported into the simca package (version 10.0, umetrics, umea, sweden) for multivariate analysis. principal component analysis (pca), a nonsupervised method was initially performed to obtain an overview of the gcms data from the control groups from the 6baptreated groups. differences between the samples were detected in the pca score plots, in which each point represents a linear combination of all the metabolites from every sample. distance between the groups of samples shows a measure of the overall difference among the metabolic profiles under different conditions. a supervised plsda was subsequently performed to identify the metabolites contributing to the differences between the control and 6baptreated groups. a coefficient loading plot of the plsda models was used to determine the differential metabolites that could separate the sample groups. based on the loading plots and variable importance in the projection (vip) value threshold (vip > 1), metabolites that were contributing to differing the control and the 6baptreated groups were identified through the plsda model.22 the altered metabolites, which were elicited by 6bap and selected from the plsda model, were further subjected to hierarchical cluster analysis (hca). hca was performed by euclidian distance and complete linkage grouping with the help of gene cluster software, and visualized through treeview software. then, two cycles of median center genes and arrays were performed, which repeated five times. finally, the data sets were applied to three cycles of normalize genes and arrays, which repeated five times. oneway analysis of variance (anova) t tests were performed using spss 12.0 software to analyze the distribution of the concentrations and logarithmically transformed concentrations for metabolites in this study. to investigate the effects of 6bap on biomass, lipid and dha yields for aurantiochytrium sp. ylh70, different concentrations of 6bap (0, 1, 3, 5, 7 and 9 mg ml) were added to the medium before fermentation and the 6bapfree group (0 mg ml) was used as the control group. as shown in fig. 1, the optimal biomass, lipid and dha production (20 g l, 52.8% biomass and 4.6 g l)1 were obtained when 3 mg ml 6bap was used. there was almost no difference in biomass for aurantiochytrium sp. ylh70 with addition of 6bap, however, compared with the control group, addition of 6bap caused up to 25.9% and 52.8% increases in lipid and dha production in aurantiochytrium sp. combining the results from the biomass, lipid and dha productions, we concluded that 3 mg ml 6bap added to medium was optimal. for this dose, we examined the time courses of lipid and dha yields by aurantiochytrium sp. effects of different concentrations of 6benzylaminopurine on biomass, lipid content and dha yield in aurantiochytrium sp. all data are means of three replicates ; vertical bars represent error bars with the value equal to the standard error of the mean. time course of lipid content (a) and dha yield (b) in aurantiochytrium sp. all data are means of three replicates ; vertical bars represent error bars with the value equal to the standard error of the mean. the metabolic profiles of aurantiochytrium sp. ylh70 from all samples were analyzed by a pca scores plot (fig. ms analysis, mass spectral deconvolution and removal of the internal standard and pseudopositive peaks, such as those caused by noise, the number of peaks were 104 and 156 in the control and 6baptreatment groups, respectively. from these peaks the pca analysis showed that all samples from the control and 6bap treatment groups were well separated, suggesting different metabolic profiles among these samples. in all cases, 76.9% of the total variance in data was described as rx = 0.769 by the 13 principle components. this indicates that 76.9% of variation in the data was related to the 6bap treatment and 6bap was likely to be responsible for the metabolic variation. (a) control group at 24 h ; (b) 6bap treatment group at 24 h ; (c) control group at 48 h ; (d) 6bap treatment group at 48 h ; (e) control group at 72 h ; (f) 6bap treatment group at 72 h ; (g) control group at 96 h ; (h) 6bap treatment group at 96 h ; (i) control group at 120 h ; (j) 6bap treatment group at 120 h. through plsda pairwise comparison, a significant separation of metabolic profiles between the control and 6bap treatment groups at each time point was observed (fig. 4(a), (b), (c), (d) and (f)). as shown in fig. s1 (supporting material), vip plots showed that some of the identified metabolites were responsible for the group separation. the metabolites with a vip value more than 1 were considered the most effective metabolites which could explain the difference.22 thus, based on the vip value (> 1) and pvalue (1) and pvalue (< 0.05), a total of 46 intracellular metabolites were selected and 38 of them were identified with the aid of the nist 05 database. these metabolites were mainly composed of fatty acids, amino acids, organic acids, carbohydrates, alcohols, squalene, cholesterol, and so on, most of which were related with the fatty acids metabolism and growth in aurantiochytrium sp.. plsda derived plots for the control and 6bap treatment groups at each time point. (a) control versus 6baptreated group at 24 h ; (b) control versus 6baptreated group at 48 h ; (c) control versus 6baptreated group at 72 h ; (d) control versus 6baptreated group at 96 h ; (e) control versus 6baptreated group at 120 h. hca was performed to confirm the results from the pca model and plsda model analyses, and to reflect clustering patterns among metabolites. as shown in fig. 5, a clear separation between the first twodays and the last threeday groups was observed. these results verified the reliability of the pca and plsda model and indicated that the effect of 6bap on aurantiochytrium sp.ylh70 was also related to time. (a) control group at 24 h ; (b) 6bap treatment group at 24 h ; (c) control group at 48 h ; (d) 6bap treatment group at 48 h ; (e) control group at 72 h ; (f) 6bap treatment group at 72 h ; (g) control group at 96 h ; (h) 6bap treatment group at 96 h ; (i) control group at 120 h ; (j) 6bap treatment group at 120 h. in this study, addition of phytohormone 6bap had almost no effect on the growth of aurantiochytrium sp. ylh70, however, it caused a significant increase in lipid and dha accumulation. since the lipid and dha productivity was the most important characteristic for aurantiochytrium sp. in the fermentation process, 6bap could be a regulator which enhanced fermentation performance. a gcms method combined with multivariate analyses was used to investigate changes of metabolites under exposure to 6bap at the optimal concentration (3 mg ml). these metabolites responsible for responding to 6bap belonged to different categories of components, including fatty acids, amino acids, organic acids, carbohydrates, alcohols, squalene, cholesterol, and so on. addition of 6bap increased yields of lipid and dha (figs 1 and 2). meanwhile, other fatty acids, such as tetradecanoic acid (c14:0), heptadecanoic acid (c17:0), octadecanoic acid (c18:0), oleic acid (c18:1) and arachidonic acid (c20:4), were also induced by 6bap treatment. these results suggested that the metabolic flux to fatty acids was promoted by 6bap treatment for lipid accumulation (fig. research in jatropha curcas showed that 6bap could modify the fatty acid profile and induce linoleic and linolenic acids.22 moreover, other phytohormones, such as indole3acetic acid, gibberellic acid, kinetin, 1triacontanol, and abscisic acid were often used as regulator for improvement of biodiesel production by microalgae.10, 11, 23 thus, this study is the first report that 6bap promotes the fatty acids and lipid accumulation in microorganism. fatty acids were the main components of the cell membrane and affect the fluidity and permeability of cell membrane.24 some fatty acids, especially unsaturated fatty acids, were induced by phytohormones to increase the fluidity and permeability of cell membrane as a response to phytohormone treatment of the microorganism. as a result, uptake of the substrate in medium into the cell might be accelerated and metabolism might be promoted. zhu. found that oddnumbered fatty acids c15:0 and c17:0 were increased for responses to environmental factors, such as temperature and salinity in schizochytrium limacinum ouc88.25 these results indicated that changes in fatty acid composition induced by 6bap might be correlated with the antistress mechanism in aurantiochytrium sp. metabolic changes induced by 6benzylaminopurine in aurantiochytrium sp. ylh70. at each time point, the content of glucose was decreased by 6bap, indicating that the glycolysis was induced by 6bap and the rate of utilization of glucose was accelerated for production of other metabolites (table 1 and fig. the decrease of the content of glycerol under 6bap treatment indicated that the glycerol pathway was suppressed and more carbon flux was directed to pyruvate. pyruvate was the precursor for many metabolites, such as valine and ethanol, which were decreased in response to 6bap, and more metabolic flux was directed to acetylcoa (table 1 and fig. ylh70 at five different time points the vip scores of all metabolites are higher than 1. the data represents the contents of metabolites and are showed as the mean sd. all data are means of six replicates. acetylcoa is a very important precursor for many metabolic pathways, including mevalonate biosynthesis, tricarboxylic acid cycle (tca) and fatty acids synthesis (fig. squalene and cholesterol, which are the active components in the mevalonate biosynthesis pathway of aurantiochytrium sp. ylh70, were both decreased by 6bap treatment. squalene is a triterpene hydrocarbon and an important ingredient used in cosmetics, pharmaceutical and medical industries. aurantiochytrium sp. has been used for production of squalene.26 a gene encoding squalene synthase, which condensed two molecules of farnesyl pyrophosphate into a squalene, was cloned in schizochytrium limacinum, and several cisacting elements in the promoter were also predicted, suggesting that synthesis of squalene was regulated by some factors at transcriptional level.27 thus, it will be necessary to study the regulation mechanism of 6bap on squalene synthesis in future research. was synthesized by further catalysis of squalene.28 however, we found that unsaponifiable matter, such as squalene and cholesterol, competed acetylcoa with fatty acids synthesis pathway. thus, the mevalonate pathway in aurantiochytrium sp. could be inhibited to improve fatty acids synthesis by metabolic engineering. besides, a portion of acetylcoa flow to the tricarboxylic acid cycle (tca) in aurantiochytrium sp., producing metabolites associated with this pathway. a 6bap treatment caused decreases in fumaric acid, which was an intermediate of the tca cycle (fig. moreover, some amino acids, such as proline, alanine and lysine, which could be converted from ketoglutaric acid, were all reduced by 6bap treatment. these results indicated that the metabolic flux from glycolysis, tca cycle and mevalonate pathway to the fatty acids biosynthesis was promoted by 6bap addition. some polylols, such as myoinositol and quercitol, were also found to make a great contribution to responses to environmental factors, such as oxygen and osmotic pressure in schizochytrium sp. strains.18, 29 further investigation will be necessary to determine the relationship between 6bap addition and myoinositol metabolism. addition of 6bap could cause a significant decrease in phosphoric acid (table 1). ren. found that a phosphatelimitation condition could maintain higher activities of malic enzyme and glucose6phosphate dehydrogenase, which favored accumulations of lipid and dha.30 in the present study, increases in lipid and dha could be explained by a decrease in phosphate induced by 6bap treatment in aurantiochytrium sp., which resulted in changes in enzyme activities. in the current research, a gcmsbased metabolomics method was applied to investigate the metabolic response to 6bap of aurantiochytrium sp. for metabolites profile, group classification and pairwise discrimination between the control and the 6bap groups were obviously observed. some metabolite, such as fatty acids, amino acids, carbohydrates and polyols, were responsible for the response to 6bap in aurantiochytrium sp.ylh70. our findings indicated that with addition of 6bap, fatty acids biosynthesis in aurantiochytrium sp.ylh70 was promoted and the metabolic flux to tca, mevalonate pathway and glycerol metabolism were diminished. this study showed that the metabolomics method based on gcms analysis was an appropriate tool to investigate the response to environmental or fermentative regulators in the molecular mechanism of microorganism, and 6bap had potential as a stimulator for improving lipid and dha production in aurantiochytrium sp.ylh70. (a) control versus 6baptreated group at 24 h using plsda model, (b) control versus 6baptreated group at 48 h using plsda model, (c) control versus 6baptreated group at 72 h using plsda model, (d) control versus 6baptreated group at 96 h using plsda model, (e) control versus 6baptreated group at 120 h using plsda model. | abstractbackgroundphytohormones are chemical messengers that have a positive effect on biodiesel production of microalgae at low concentrations. however, the effect of phytohormone 6benzylaminopurine on lipid and docosahexaenoic acid (dha) production in marine dhaproducer aurantiochytrium has never been reported. in this study, a gcmsbased metabolomics method combined with a multivariate analysis is applied to reveal the metabolic mechanism of 6benzylaminopurine enhancing production of lipid and dha in aurantiochytrium sp.ylh70.resultsin total, 71 metabolites were identified by gcms. the pca model revealed that 76.9% of metabolite variation was related to 6benzylaminopurine treatment, and overall metabolomics profiles between the 6benzylaminopurine and control groups were clearly discriminated. fortysix metabolites identified by the plsda model were responsible for responding to 6benzylaminopurine. metabolic analysis showed that 6benzylaminopurine could accelerate the rate of utilization of glucose in aurantiochytrium sp. ylh70, and the metabolic flux from glycolysis, tca cycle and mevalonate pathway to fatty acids biosynthesis was promoted. moreover, the antistress mechanism in aurantiochytrium sp.ylh70 might be induced by 6benzylaminopurine.conclusionmetabolomics is a suitable tool to discover the metabolic mechanism for improving lipid and dha accumulation in a microorganism. 6benzylaminopurine has the potential to stimulate lipid and dha production of aurantiochytrium sp.ylh70 for industrial purposes. 2015 the authors. journal of chemical technology & biotechnology published by john wiley & sons ltd on behalf of society of chemical industry. |
noncommunicable diseases (ncds) are steadily rising, affecting both developing and developed countries. this is a consequence not only of population aging, but also of the nutrition transition towards westernized diets and sedentary lifestyles. the nutrition transition is fueled by socioeconomic and technological development as well as globalization and accelerated urbanization. among the nutrition - related ncds, diabetes is a major concern because its prevalence is rapidly increasing worldwide and particularly so in developing countries. this number is expected to reach 592 million by 2035, with the middle east, south east asia, and africa showing the fastest increase in the number of cases. according to the international diabetes federation, 80% of people suffering from diabetes live in low- and middle - income countries. diabetes is associated with several complications, leading to morbidity, disability, and premature mortality [2, 3 ]. type 2 diabetes (t2d) is by far the most common form of the disease. diabetes also entails a heavy economic burden for patients, households, and healthcare systems [4, 5 ]. t2d is a lifestyle disease, which can and should be prevented by intensive lifestyle interventions, characterized by changes in dietary habits and increased physical activity. indeed, lifestyle interventions at the prediabetes stage have proved successful at reducing the incidence of t2d by 28.5% to 58%, in china (da qing), india (indian diabetes prevention program : idpp-1), finland (diabetes prevention program, dpp), and the united states (diabetes prevention program and outcomes study, dppos) [68 ]. weight control is key to the prevention and management of diabetes independent of dietary composition. as obesity is a major risk factor for t2d, lifestyle interventions aimed at weight loss or control are also critical to prevent t2d. except for india and china, few studies have been conducted to date on diabetes prevention programs in low- and middle - income countries. in developed nations and even more so in low - resource countries, healthcare spending is a critical economic and political issue. a recent world health organization report recommended addressing common lifestyle risk factors for ncds, considering their cost - effectiveness, and their relative ease, and speed of implementation. in resource - limited settings in particular, decision makers require information on the economic burden of ncds, particularly t2d, and of the potential added value of lifestyle interventions for health and development. the economic evaluation of various preventative interventions is important in view of the urgent need for developing countries to set these ncds as a public health priority, of the rapid increase in diabetes prevalence and of substantial variations in lifestyle intervention components and delivery. there are limited systematic reviews on this topic and the most recent ones covered the period of 19852008 [12, 13 ]. most economic evaluations of t2d prevention programmes pertained to developed countries partly owing to lack of relevant data in developing countries, while cost - effectiveness tends to be context - specific. our objective was to review economic evaluation studies of lifestyle interventions for the primary prevention of t2d and also for the control of obesity as key risk factor, based on data published since 2009. this review was intended to update knowledge on the cost - effectiveness of t2d prevention. in order to identify all relevant studies performing an economic evaluation of lifestyle interventions to prevent t2d and for obesity control, we searched the following databases : pubmed, medline, the british national health services economic evaluation (nhs ees), cinhal, econ lit, web of sciences, embase, and latin american and caribbean health sciences literature (lilacs). we restricted our search to studies published in french, english, or spanish between january 2009 and december 2014 as previous systematic reviews included studies published between 1995 and 2008. we used medical subject headings (mesh) and other relevant terms to the topic as major constructs to build our search strategy. the mesh or other relevant terms are related to economic, diabetes, and intervention constructs. to combine these, we used boolean operators and and or as appropriate. in addition, the reference lists of all included studies were scanned to identify any additional potentially relevant reports. for example, the pubmed search combined (i) cost - benefit - analysis (mesh) or costs and cost - analysis (mesh) or cost - benefit (title) or cost - effectiveness (title) or cost - utility (title) or economic evaluation (title) and (ii) type 2 diabetes (mesh) or non insulin dependent diabetes (mesh) or gestational diabetes (mesh) or obesity (mesh) or impaired glucose tolerance (title) or prediabetes (title) and (iii) diet (mesh) or physical activity (mesh) or diet therapy (mesh) or lifestyle (mesh) or risk reduction behaviour (mesh) or prevention (title) or lifestyle modification (title) or lifestyle advice programme (title) or non pharmacological prevention (title). appendix shows the majors constructs used in our search strategy. in order to select the relevant studies for this review, we screened titles and abstracts using a three - stage process. at the first stage, two authors (koffi alouki and clara bermdez - tamayo) independently selected studies based on abstracts and titles., the same coauthors resolved the disagreements. on the basis of full assessment and discussion of each study the study selection was guided by the following inclusion criteria : original research articles published in peer - reviewed journals were candidates for inclusion.type of economic evaluation : the selected studies conducted a full economic evaluation as defined by drummond.. full economic evaluations are studies in which a comparison of two or more treatments or care alternatives is undertaken and in which both the costs and outcomes of the alternatives are examined in terms of cost - effectiveness, cost - utility, or cost - benefit analyses.the participants : the population groups targeted for the primary prevention of t2d were adult subjects (over 18 years old) who were at high risk of developing the disease because of obesity, impaired glucose tolerance, impaired fasting glycaemia, or gestational diabetes.the interventions : we considered dietary modifications or physical activity or both to prevent t2d or control obesity.the comparison : any comparison arm or group used against the lifestyle intervention was accepted for this review.outcomes : these were cost per qaly (quality adjusted life years) gained, cost per life year gained, cost per daly (disability adjusted life years) averted, cost per diabetes case averted, and other relevant outcomes.studies published between january 2009 and december 2014.studies that were published in english, french, or spanish. type of economic evaluation : the selected studies conducted a full economic evaluation as defined by drummond.. full economic evaluations are studies in which a comparison of two or more treatments or care alternatives is undertaken and in which both the costs and outcomes of the alternatives are examined in terms of cost - effectiveness, cost - utility, or cost - benefit analyses. the participants : the population groups targeted for the primary prevention of t2d were adult subjects (over 18 years old) who were at high risk of developing the disease because of obesity, impaired glucose tolerance, impaired fasting glycaemia, or gestational diabetes. the interventions : we considered dietary modifications or physical activity or both to prevent t2d or control obesity. the comparison : any comparison arm or group used against the lifestyle intervention was accepted for this review. outcomes : these were cost per qaly (quality adjusted life years) gained, cost per life year gained, cost per daly (disability adjusted life years) averted, cost per diabetes case averted, and other relevant outcomes. studies that were published in english, french, or spanish. the first reviewer (koffi alouki) extracted data using a standardized data extraction form built according to the consolidated health economic evaluation statement (cheers). the second reviewer (clara bermdez - tamayo) checked that the extracts and discrepancies were resolved through discussion. data extracted included the type of economic evaluation, subjects ' characteristics (e.g., age, biological and anthropometric characteristics), intervention details (e.g., duration, location, intensity, and mode of delivery of the intervention), comparator, analytical model used, effectiveness data, sensitivity analysis, and reported outcomes relevant to the review. there are some trial - based type studies while others relied on model - based studies or previous trial results to extrapolate by using modeling techniques, something which was previously highlighted in the literature. the interventions compared, the population groups targeted, and the outcomes reported also varied across studies. for these reasons we chose a narrative approach for this systematic review, as is usually done for systematic reviews of economic evaluations. we used the british medical journal (bmj) quality assessment checklist, a 36-item scale, to assess the quality of the studies. this checklist was developed with the aim of standardizing the presentation of study data, thereby contributing to the quality of economic evaluations. we also assessed any risk of bias due to conflicts of interest or sponsorship of studies. each item was answered by no or yes or not applicable. we gave a score of 0 if the answer was yes and 1 if it was no. then we summed up the number of no responses to obtain a global score in which a higher score represented poorer quality. two reviewers (koffi alouki, clara bermdez - tamayo) conducted this operation independently and disagreements were resolved through discussion. quality rating was used to interpret the results but no study was excluded on this basis. the search yielded 176 abstracts. after reviewing the abstracts, subsequent reference tracking, and excluding duplicate articles, we narrowed the focus to 56 candidate studies having performed an original economic evaluation. further review of the full text resulted in 20 studies that met our inclusion criteria. the studies were conducted in the uk, usa, canada, australia, germany, finland, the netherlands, singapore, sweden, and china. three studies performed only a cost - effectiveness analysis while 13 studies assessed only cost - utility. the four remaining studies combined cost - effectiveness and cost - utility analysis. there were 10 studies using a markov - type model or a decision tree, or both, to make projections of the evolution of t2d [2224, 2629, 31, 33, 35 ]. the period for which the potential benefit of the intervention was simulated ranged from three years to lifetime. the two studies targeting women with gestational diabetes were conducted throughout pregnancy [20, 31 ]. in eight studies [16, 17, 22, 23, 25, 2729 ], data on efficacy and effectiveness used in simulations were drawn from the major randomized controlled trials on t2d prevention : dppos, dpp, and da qing. in a further six studies, effectiveness data was based on synthesis of multiple studies [24, 3135 ]. six of the 20 studies used effectiveness data from a specific intervention conducted in the same country where the economic evaluation was carried out [1821, 26, 30 ]. the cost to the healthcare system only or to the whole society was generally considered. the costs and the effectiveness were discounted using rates varying from 3% to 5% according to the countries. to test the robustness of the results, it was not possible to identify the source of funding for only one study. among the other 19 studies, the interventions included in this review varied from the simple provision of information to active behaviour change schemes. the lifestyle changes described across studies pertained to diet, physical activity, or both. in some cases, the screening of subjects at high risk preceded the interventions without explicitly taking account of the screening in the cost calculations [17, 18, 24 ]. of 10 model - based studies, seven simulated interventions based on the dpp [2325, 28, 29 ] or the china da qing study [22, 27 ]. the dpp intervention goal was to achieve and maintain a weight reduction of at least 7% of initial body weight through diet and physical activity of moderate intensity, such as brisk walking for at least 150 minutes per week. the dpp program included a lifestyle curriculum, taught by case managers on a one - to - one basis during the first 24 weeks after enrollment. subsequent individual sessions (usually monthly) and group sessions with the case managers were designed to reinforce the behavioral changes. the aim of nutritional counselling was to help the participants achieve a diet containing 10% of total energy intake as saturated fats, 510% as polyunsaturated fats, 2530% as total fat (saturated, monounsaturated, polyunsaturated, and trans fatty acids), and 25 to 35 grams of fibre per day. one study described a commercial program consisting of a low - calorie diet and physical activity advice. one study carried out the economic evaluation of the dash diet (dietary approaches to stop hypertension) or a low fat diet to reduce the disease burden related to excess body weight. diet emphasizes reduced consumption of fat, red meat, sweets, and sugar - containing beverages, and the program recommends 180 minutes per week of moderate intensity physical activity. one study performed the economic analysis of lifestyle changes achieved through e - learning devices. another economic evaluation from the united kingdom was conducted on a program focusing on very low - calorie diets. two studies reported on the economic evaluation of interventions consisting solely of physical activity [21, 35 ]. it took the form of a structured programme of exercise in a fitness centre and incorporating monitoring of individual performance. the second study pertained to sessions of physical activity that included 60 minutes of walking in addition to exercise. the exercise sessions consisted of aerobic and strength exercises. depending on the studies, the comparison (or control) interventions were common standard care, placebo, metformin (850 mg twice daily), or simple advice in writing for physical activity and nutrition. reported costs depended on the chosen perspective, the nature of the intervention, the target population, and the time horizon. there is no consistency across studies in the items included to estimate the costs of interventions. all studies considered direct costs including medical costs [16, 17, 2023, 25, 30 ]. medical records were the most common source for data on diabetes and complications. generally, the direct medical costs were copayment fees for treatment, diagnostic testing, prescription drugs, and medical supplies. the direct costs also included the costs of visits to healthcare providers and exercise physiologist and metformin cost for controls. direct nonmedical costs pertained to services such as transportation of subjects and family members to clinics, as well as special food in some instances. lost income for the patients and their families and the costs of hiring nurses or care providers were recorded as indirect costs. four studies [18, 20, 21, 23 ] reported on the estimated cost of productivity loss. in four of the 20 studies [22, 24, 25, 29 ], cost estimates were only presented as category totals, without breakdown into individual items and without a separate presentation of the resources needed for the interventions. two studies described the unit costs of various resources, using the ingredient approach [27, 28 ]. physical quantities of necessary inputs were counted and multiplied by unit prices to obtain total costs. price data came from healthcare facilities, from patient records, or else from estimates based on published data. seven studies recorded costs alongside the trials [1719, 21 ]. except for four studies [1821 ], all studies discounted the costs by 3% to 5%. the effectiveness data were usually derived from major randomized controlled trials on t2d prevention [7, 17, 19, 38, 39 ]. nearly one - third of studies estimated effectiveness based on meta - analyses or literature reviews [24, 3135 ]. three out of the 20 studies reported effectiveness as dalys averted [24, 31, 33 ]. diabetes cases averted by the interventions were reported in two studies [19, 24 ]. one study reported change in utility as an outcome measure, based on the 15-dimension questionnaire tool (15d) to assess quality of life but without conversion to quality adjusted life years. nine studies (9/20) used the eq5d (euroqol 5-dimension tool), three studies used the qwb - sa (quality of well being - self - administered), two studies used the hrqol (health related quality of life) system, one study used the vsa (visual scale analog), and one study used the 15d or sf-36 (36-item short form survey instrument). in some cases, the key assumption was that the expected effects of the intervention were for the short term with a linear decrease in effectiveness following the intervention. another study projected the same effectiveness during the whole duration of the time horizon projected. the probability of progressing from prediabetes to t2d in all studies using modeling techniques was always lower in the intervention arm consisting of lifestyle intervention than in the comparison arm, whether placebo, usual care, or metformin treatment. assumptions regarding the effectiveness of screening were needed when screening was required before implementing interventions. the studies reported sensitivity and specificity of screening ranging from 75% to 100% [22, 25 ]. compared with usual care, placebo, or metformin, interventions based on lifestyle modifications were reported as cost - effective in 15 of 20 studies [16, 17, 19, 2230, 3335 ]. the conclusions are usually based on the incremental cost - effectiveness ratio as applied in the study countries. one study considered the who threshold to decide on the cost - effectiveness, according to which intervention is considered highly cost - effective when the icer is below the gdp (gross domestic product) per capita and cost - effective when the icer ranges from 1 to 3 times gdp per capita. when a strategy improves health outcomes at lower cost, it is considered to be dominant and it is obviously preferred as cost - effective. those interventions that are less effective and more costly (considered as dominated interventions) or more costly and more effective but with a resulting more expensive icer would unlikely be cost - effective ; these studies were considered inconclusive. three studies revealed that the interventions were dominant [17, 22, 27 ]. oostdam. reported that a lifestyle intervention implemented during 32 weeks and targeting at - risk pregnant women in germany was not cost - effective. similar results were also reported in another intensive lifestyle intervention targeting at - risk pregnant women. one study reported that a lifestyle intervention offered by nurses was not more cost - effective in reducing t2d risk than the control intervention consisting in the provision of a general health brochure. johansson. examined the cost - effectiveness of lifestyle by sex and concluded that intervention was only cost - effective among women. sensitivity analysis, which allows assessing the reliability and the generalizability of the results, was performed in all studies except one. over half the studies performed univariate sensitivity analysis and eight studies performed bivariate sensitivity analysis. probabilistic sensitivity analysis was used in ten studies [18, 21, 24, 2629, 31, 32, 35 ]. the input parameters that were analyzed through a range of assumed values were discounted cost and outcomes (qalys gained), variations of intervention cost, variation of probability of transition between disease states considered, the duration of interventions, the size of the population at risk, and performance and frequency of screening test. although the icer was sensitive to changes in the above parameters, it remained acceptable for most studies, except for the three studies reporting that the intervention was not cost - effective [20, 27, 31 ]. based on the bmj checklist, the studies included in this review showed quality limitations in reporting as shown in figure 2. out of the remaining 20 studies, 15 presented at least two methodological limitations and five studies showed only one. more than half (12/20) of studies did not address the issue of generalizability. the justification of the parameters used for the sensitivity analysis was presented by only few studies. the models of five studies were not fully described or their choice was not justified. eight studies did not report separately the resources for the interventions and their unit cost. the purpose of this systematic review was to describe economic evaluations of lifestyle interventions to prevent t2d or control obesity in high - risk population groups and which were carried out since 2009. lifestyle interventions in communities and schools for the primordial prevention of t2d were excluded as a recent review reported on their cost - effectiveness. our results confirmed those of former reviews [12, 13 ], which concluded that lifestyle interventions through physical activity or diet or combining both were generally cost - effective, with a few exceptions. in our review, five out of the 20 studies were inconclusive. there was a trend for a higher proportion of interventions targeting the prevention of t2d compared to those focusing on obesity to be cost - effective (11/14 versus 4/6, resp.). the inconclusive studies [18, 20, 21, 31, 32 ] included one on the prevention of t2d, two on the prevention of gestational diabetes among pregnant women, and two on obesity control. these studies were more costly with less effectiveness or more costly with higher effectiveness but resulting in a more expensive incremental cost - effectiveness ratio compared with standard care (or control treatment). three of the seven studies conducted within trial were nonconclusive compared with three of the 13 studies based on models. the short duration of some studies may also explain the absence of significant changes in the outcomes. another inconclusive study pertained to an e - based intervention, which may not induce enough motivation for change. overall, there was considerable heterogeneity in the nature of lifestyle interventions across studies, which hampered comparisons and possibly contributed to the inconsistencies in outcome findings. study results could be influenced by intervention components, selection of participants, and methodological and modeling choices. few studies evaluated effectiveness in terms of qaly although it is known that diabetes is associated with deterioration of the quality of life. qaly is a relevant parameter that allows comparisons of the burden of disease in terms of quality and quantity. preference - based health state classification systems were preferred in most studies to objective methods of evaluation of health states. mcdonough and tosteson showed that, among studies that compared alternative preference - based systems, the eq-5d tended to provide larger change scores and therefore more favourable results than the health utility index (hui), while the sf-6d provided smaller change scores and therefore less favourable ratios than the other systems. hence, the choice of outcome as a measure of effectiveness has an impact on reported results. cost variations were due primarily to the chosen perspective (societal / health system or third party), to the various inputs for the interventions, and to differences of care unit costs across countries. the lifestyle interventions involved several health professionals, which explains the higher costs of these interventions compared to controls. in some but not all simulation studies, the costs of health infrastructure and training of personnel were also provided. such inclusive costs would be particularly useful for low - resource countries where health systems are ill - prepared to tackle a chronic disease like t2d. few studies complied with the recommendation to present quantity and unit cost of inputs separately from total cost as advocated in the guidelines, resulting in lack of the information required for the replication of the intervention in other settings. additionally, the reported costs were often incomplete. for instance, if a societal perspective is adopted, the costs related to lost productivity, premature mortality, and permanent and temporary disability should be computed which is not always the case. as stated in the guidelines [47, 48 ], the future costs beyond one - year time should be discounted, which was done in most studies and using different discount rates. although the selected rates were not justified in most studies, they probably reflect the fact that there are no universally accepted discount rates for economic evaluations studies. the time horizon should be long enough to capture any significant difference between the intervention and the comparison groups in terms of costs and outcomes. yet, it appeared too short in some studies to appraise and capture long - term impact of interventions. particularly, in the management of obesity or gestational diabetes, the inconclusive results may be partly ascribed to too short interventions to capture the long - term benefits. the costs for treating t2d complications are onerous, and the temporal horizon of some studies based on model assumptions can not guarantee that the complications will not appear later on. however, without sustained compliance of subjects with advocated lifestyle changes, weight regain over the long - term can alter the quality of life and impinge on the estimated cost - effectiveness of interventions. considering exclusively the short - term effects of obesity control interventions can indeed be misleading. for this reason, it is not impossible that the cost - effectiveness of some interventions reviewed here and focusing on obesity was overestimated. trial - based studies limited the results of interventions to health gains over the course of the intervention. in contrast, studies supported by models were based on assumptions on a longer temporal horizon. this approach, however, would omit taking into account other relevant events that may occur later on that will have an impact on the costs and on the quality of life or other outcomes. additionally, the nature of the model inevitably has a bearing on the estimated cost - effectiveness of the interventions. modeling diabetes evolution showed variations among studies with regard to the shift from normal glycaemia to prediabetes and then to diabetes and finally to the complications. with the exception of one study, the models however seemed to be consistent with the natural evolution of the disease as described in the literature. the accuracy of the models is one of the criteria for sound economic evaluations, and it reflects the ability to capture the evolution of the pathology in a real situation [51, 52 ]. yet, in some studies, the models showed complication states without disclosing the nature of the complications. another limitation of most models is that complications are taken separately into account while subjects living with t2d can have several complications. some studies [23, 25 ] did not consider the likelihood of glycaemia returning to normal in subjects with prediabetes. the simplification of analytical models may not help to consider these subtleties during the course of evolution of the disease in a real situation. most simulated interventions were based on epidemiological studies of t2d prevention that provide the reference evidence for lifestyle interventions as relevant strategy to prevent diabetes. the randomized controlled trials that measured the long - term effects in real life situations after cessation of the active intervention [37, 39 ] therefore provide important new data for the simulation studies to better estimate the long - term effectiveness of interventions to prevent diabetes. however, the efficacy observed in controlled experimental conditions is different from the expected effectiveness in the real world because of subject selection, recruitment, and follow - up and other factors that have a bearing on economic outcomes [10, 53 ]. moreover, in some model - based studies, the at - risk subjects may not accurately match the at - risk subjects of the original studies, so that the assumed effectiveness in these studies is uncertain. the effectiveness of interventions in the real world often falls a long way short of the maximum efficacy shown in trials. for instance, a retrospective observational analysis of overweight and obese subjects demonstrated that, compared with the 58% reduction in risk of progression to diabetes seen in the dpp trial, risk reduction for incident diabetes in subjects who participated in the study in an intense and sustained way was lower. most articles lacked one or several reporting items of the bmj checklist for quality of economic evaluation studies although this checklist was issued before these studies were conducted. another limitation observed is the lack of a clear description of the models, while the guidelines recommend transparency in their description. in some cases, the models did not adequately capture the natural history of the disease, leading to questionable conclusions. conversely, in another study, the model did not include the complication state, which would have been relevant in the natural history of diabetes. however, although we performed an evaluation of the quality of studies, we deliberately chose not to exclude any study on that basis. in any case, excluding the poorer quality studies would not have altered the conclusions. we used a comprehensive electronic search strategy using preestablished criteria in common medical literature databases. as all studies are not referenced in electronic databases, we revisited the bibliography of each selected study to ensure that our search was exhaustive. two reviewers cross - checked the database to identify the relevant studies. at variance with previous reviews on the economic evaluation of t2d prevention studies, ours also included intervention studies targeting obesity as main risk factor for t2d and not only the interventions aiming directly at t2d prevention. ours also encompassed the assessment of study quality based on the bmj guidelines designed for the critical appraisal of economic evaluation studies. we adopted a dichotomous scale for each item, however, which does not reflect the level of completeness of the information reported for each item considered. it is also recognized that limiting the candidate studies to those published in french, english, and spanish is a potential source of publication bias. due to the heterogeneity in the methods and results of the different studies, we were not able to perform a meta - analysis, which is usually considered the gold standard but which is not often feasible with economic evaluation studies. the present review on the cost - effectiveness of lifestyle modification interventions showed, with only a few exceptions, that these interventions targeting adult subjects at high risk for diabetes were cost - effective despite different assumptions regarding disease progression and variations in the delivery of these interventions. the results are consistent with conclusions of former reviews, confirming the importance of lifestyle interventions combining diet and physical activity to prevent diabetes in at - risk population groups. this review also broke new ground by assessing the methodological limitations of the economic evaluations and the quality of reporting, to aid in interpretation of results. lifestyle interventions should be further stressed as an effective strategy to prevent or delay diabetes. unfortunately, few studies have been conducted in resource - poor countries in spite of a dire need for such data, and the findings from developed countries are not entirely relevant. future research should address the effectiveness and cost - effectiveness of such interventions in low - income country settings, where the prevalence of t2d is soaring. meanwhile, the data of the present review provide compelling arguments for policy makers to implement measures to prevent t2d. | objective. to summarize key findings of economic evaluations of lifestyle interventions for the primary prevention of type 2 diabetes (t2d) in high - risk subjects. methods. we conducted a systematic review of peer - reviewed original studies published since january 2009 in english, french, and spanish. eligible studies were identified through relevant databases including pubmed, medline, national health services economic evaluation, cinhal, econlit, web of sciences, embase, and the latin american and caribbean health sciences literature. studies targeting obesity were also included. data were extracted using a standardized method. the bmj checklist was used to assess study quality. the heterogeneity of lifestyle interventions precluded a meta - analysis. results. overall, 20 studies were retained, including six focusing on obesity control. seven were conducted within trials and 13 using modeling techniques. t2d prevention by physical activity or diet or both proved cost - effective according to accepted thresholds, except for five inconclusive studies, three on diabetes prevention and two on obesity control. most studies exhibited limitations in reporting results, primarily with regard to generalizability and justification of selected sensitivity parameters. conclusion. this confirms that lifestyle interventions for the primary prevention of diabetes are cost - effective. such interventions should be further promoted as sound investment in the fight against diabetes. |
while psychostimulants have acute effects on brain dopamine systems, there is also evidence from several lines of research for the involvement of the opioid system in psychostimulant dependence. for instance, positron emission tomography (pet) studies have revealed functionally important changes in the brain opioid systems of cocaine users as compared to controls (gorelick. there is also pharmacological evidence from animal studies showing that the opioid antagonist naltrexone attenuates amphetamine - induced locomotor sensitization and reinstatement of drug - seeking in rats (hggkvist., naltrexone has been shown to significantly attenuate the subjective effects of amphetamine in amphetamine - dependent subjects and healthy controls, as well as reducing craving and risk of relapse to amphetamine use (jayaram - lindstrm. this is in line with studies linking the opioid system to the hedonic component of reward (berridge. a possible explanation of naltrexone 's effect could be that amphetamine causes an activation of the opioid system and that this activation is inhibited by opioid antagonists, which thereby attenuate the rewarding effects of amphetamine. indeed, several studies have shown evidence of such stimulant - induced opioid release in the ventral striatum of rodents (olive. 2001 ; roth - deri. 2003 ; soderman & unterwald, 2009). recently, a dose of oral amphetamine was shown to induce decreases in [c]carfentanil binding in healthy control subjects. however, in this study there was no within - subject control for expectation effects and the measurements were performed 3 h after dosing (colasanti. administration of amphetamine on the opioid system have not yet been investigated in humans. in this placebo controlled pet study of healthy, drug - naive subjects amphetamine induces an acute opioid release in the brain, specifically in the ventral striatum, measurable as a reduction in binding of the -opioid receptor radioligand [c]carfentanil. the study was approved by the swedish medical products agency, the regional ethics review board and the radiation safety committee and performed in accordance with ich guidelines for good clinical practice. ten healthy young men, aged 26.72.5 yr (means.d.), were recruited by advertising within the karolinska institutet. screening procedures, including the alcohol use disorders identification test, drug use disorders identification test and mini international neuropsychiatric interview, were performed by a study physician in order to exclude subjects with any somatic or psychiatric disease or a history of any substance use disorders either themselves or in a first - degree relative. at screening and on the test days, subjects were tested for alcohol in exhaled air and with urine toxicology to exclude use of any illicit drugs. a t1 weighted magnetic resonance (mr) scan (1.5 t) was performed for every subject prior to pet procedures to exclude intracranial pathology and obtain anatomical references for definition of regions of interest (rois). applying a double - blind, cross - over randomized design, three pet examinations were performed for each subject. the test days were approximately 1 wk apart, although three examinations were delayed for technical reasons (performed after an interval of 2040 d). the first examination served as a baseline measure, while the second and third measurements were done after administration of an i.v. the order of amphetamine or placebo was randomized and double - blind to avoid confounding with expectation effects. after the amphetamine and placebo sessions, subjects were asked to subjectively rate, on a scale from 0 to 100, how strongly they felt any effect of the drug and how strongly they experienced any arousal or they were also asked to rate how strongly they would like to have more of the drug. we used the pet radioligand [c]carfentanil, a selective -opioid receptor agonist (frost. [c]carfentanil has been shown to have excellent test retest reliability, making it a suitable ligand for cross - over experiments (hirvonen. [c]carfentanil is considered sensitive to acute changes in opioid neurotransmission, based on previous findings of reduced [c]carfentanil binding potential (bpnd) in response to behavioural or pharmacological challenges that have been interpreted as increased endogenous opioid release (hirvonen. the injected radioactivity was 306.815.1 mbq (means.d.) and injected mass 0.300 0.181 g (mean s.d.). there were no significant differences in these variables between the baseline, placebo and amphetamine conditions [repeated measures analysis of variance (anova) for injected dose : f2,18=0.41, p=0.67 and for injected mass : f2,18=0.14, p=0.87 ]. pet examinations were performed using the high resolution research tomograph (siemens molecular imaging ; germany). for each subject, an individual helmet was made and attached to a holder on the coach to minimize head movement. after a 6 min transmission scan using a single cs source, the study drug (amphetamine / placebo) was injected into an i.v. two minutes later, the radioligand was administered as a rapid bolus and flushed with saline. list - mode data were acquired for 69 min, starting at the time of ligand injection. pet images were reconstructed from a series of 16 time frames (31, 43 and 93 min), including modelling of the point spread function, after correction for attenuation, randoms and scatter. this reconstruction procedure yields a spatial resolution of 1.5 mm (varrone. pet images were corrected for head movement using frame - by - frame realignment (montgomery. rois were delineated on the mr images for each subject individually, using the human brain atlas software (fig. ventral striatum was selected as the primary roi, while a secondary, exploratory analysis included other brain regions involved in drug abuse and reward, i.e. associative and sensorimotor striatum, prefrontal cortex (divided into orbitofrontal, dorsolateral and medial), anterior cingulate cortex, hippocampus and amygdala. the definitions of rois were based on previously published guidelines (abi - dargham. 1.t1 weighted magnetic resonance image, coronal slice through the striatum with superimposed regions of interest (a). high - resolution research tomograph positron emission tomography image with [c]carfentanil, corresponding slice (b). t1 weighted magnetic resonance image, coronal slice through the striatum with superimposed regions of interest (a). high - resolution research tomograph positron emission tomography image with [c]carfentanil, corresponding slice (b). pet images were co - registered to the mr image using spm 5 and the parameters derived were used to apply rois to the pet images in order to extract time quantitative analysis was performed using the simplified reference tissue model with the occipital lobe as reference region, as has been validated for [c]carfentanil (endres. the bpnd was the parameter of interest, representing the ratio at equilibrium of specifically bound to that of non - displaceable radioligand (innis. bpnd data from the baseline, placebo and amphetamine conditions were analysed with repeated measures anova for effect of treatment. ratings of how much the subjects felt the drug effect ranged from 50 to 100 (mean 8816.9), feeling of (mean 6622.2) and how much they wanted more of the drug from 0 to 100 (mean 6432.6). when analysing the pet data with repeated measures anova, we found no significant effects of amphetamine compared to placebo on the [c]carfentanil bpnd for ventral striatum, which was the primary roi (fig. 2). expressed in terms of difference between the placebo and amphetamine conditions, mean bpnd was 0.037 in our sample, with s.d.=0.240, giving us a 95% confidence interval of 0.186 to 0.111 (or 6.4 to 3.8%). in other words, we are confident that amphetamine does not reduce bpnd in the ventral striatum by > 6.4%, as compared to placebo. the same results were found when extending the analysis to other regions of the striatum (table 1). 2.individual measures of [c]carfentanil binding potential (bpnd) for the ventral striatum. mean bpnd change from baseline for each condition in different regions of the striatumregionbaselineplacebochange from baseline (%) amphetaminechange from baseline (%) f 2,18 p ventral striatum2.900.432.910.42 + 0.22.840.432.20.2920.750associative striatum1.840.341.940.21 + 5.11.860.29 + 0.60.2980.746sensorimotor striatum1.280.231.330.13 + 4.51.250.172.20.7930.468d.f., f and p values from repeated measures analysis of variance of treatment effect, uncorrected for multiple comparisons. individual measures of [c]carfentanil binding potential (bpnd) for the ventral striatum. mean [c]carfentanil binding potential (bpnd)s.d. and mean bpnd change from baseline for each condition in different regions of the striatum d.f., f and p values from repeated measures analysis of variance of treatment effect, uncorrected for multiple comparisons. an exploratory analysis of other brain regions, including the prefrontal cortex, amygdala and hippocampus, did not reveal any effects of amphetamine on [c]carfentanil bpnd (table 2). table 2.exploratory analysis of mean [c]carfentanil binding potential in regions of the prefrontal cortex and medial temporal loberegionbaselineplaceboamphetamine f 2,18 p dorsolateral prefrontal cortex1.1361.2521.2371.410.270medial prefrontal cortex1.1201.1761.1800.3230.728orbitofrontal cortex1.1851.2361.2120.4050.673anterior cingulate cortex1.1691.2701.2770.6260.626amygdala2.4752.3742.5670.1130.113hippocampus0.4530.5060.4551.3410.287d.f., f and p values from repeated measures analysis of variance of treatment effect, uncorrected for multiple comparisons. exploratory analysis of mean [c]carfentanil binding potential in regions of the prefrontal cortex and medial temporal lobe d.f., f and p values from repeated measures analysis of variance of treatment effect, uncorrected for multiple comparisons. measures of baseline [c]carfentanil bpnd were found to be quite similar to the values obtained in a previously published test the inter - individual variation in change between different conditions as indicated by the standard deviation was higher than the corresponding inter - individual variation in test we therefore proceeded in analysing associations between individual bpnd changes and subjective ratings of amphetamine effects. however, we found no evidence of a correlation between changes in [c]carfentanil bpnd and the subjective effects of amphetamine (p>0.1, data not shown). in the present cross - over, randomized, placebo - controlled pet study, we found that an acute i.v. dose of amphetamine did not change [c]carfentanil bpnd in the striatum, nor in the prefrontal cortex, amygdala or hippocampus. the dose of amphetamine administered in this study (0.3 mg / kg bodyweight) is the one most often used in human laboratory studies, causing strong, immediate subjective effects in drug - naive individuals. this dose has been shown to induce a significant dopamine release in the ventral striatum, measurable in human pet studies as a decrease of the dopamine d2-receptor radioligand [c]raclopride bpnd by about 15% (drevets. importantly, a dose of 0.3 mg / kg bodyweight was also used previously in studies showing that naltrexone blunts the subjective effects of amphetamine (jayaram - lindstrm. amphetamine - dependent patients often inject several hundred mg and such a dose naturally gives rise to stronger pharmacological effects, possibly including an activation of the opioid system. however, due to the risk of adverse reactions, such doses can not be administered to humans in an experimental setting. animal studies are therefore needed to investigate the opioid effects of amphetamine across a wider dose range. in this study, we started the pet measurement 2 min after the amphetamine / placebo injection and continued for 69 min. a possibility is that amphetamine does cause an endogenous opioid release that either evaporates too fast or commences too late for us to capture it in this time frame. the 69 min time frame was chosen based on our previous studies, where naltrexone 's effects were already evident after a couple of minutes and stayed significant over several hours (jayaram - lindstrm. amphetamine administration came somewhat later, but was clearly evident during the first hour (olive. evidence from a recent animal study suggests that opioid activation following cocaine administration i.p. peaks at 20 min (soderman & unterwald, 2009). to test for signs of such an early opioid activation, we did a post - hoc kinetic analysis using only the first 39 min of the pet scan, a time frame that has previously been shown to have good reliability with [c]carfentanil (hirvonen. this analysis did not reveal any trend of early opioid release, suggesting that our original time frame was adequate. several rodent studies have shown evidence of psychostimulant - induced opioid activation, a finding that could not be translated to humans in this study. dose of 2 mg amphetamine / kg bodyweight gives rise to an increase of dialysate endorphin levels in the accumbens, peaking at 300% (olive. 2001), and a dopamine increase peaking at 350% (butcher. doses in humans, the dose used in this study is probably less potent, even though it did cause strong behavioural effects. other species differences, particularly in the anatomy of the dopamine system, might therefore be more important in explaining these results (berger. 1991). in human subjects, as opposed to other animals, it is important to acknowledge expectation effects since they may involve opioid mechanisms (scott. 2008). in this study, the subjects were aware that they would not be administered any study drug during the first, baseline pet examination, but in the second and third they would get either amphetamine or placebo intravenously. by randomizing the order of placebo and amphetamine, we could control for expectation effects and anova showed no effect of order on [c]carfentanil bpnd. neither was there any significant difference between the baseline (i.e. without study drug) and medication conditions, which means that the expectation of an amphetamine injection did not give rise to any measurable opioid activation. this study was performed with healthy, drug - naive subjects, but the effects of amphetamine may be quite different in amphetamine - dependent patients. for example, after three doses of the same strength as in this study, a significant sensitization to amphetamine occurs with an increased dopamine release in the striatum (boileau. 2006). such mechanisms might also cause a downstream recruitment of other neurotransmitter systems with repeated drug intake. although we saw no evidence of opioid activation in healthy subjects from this first dose of amphetamine, it is possible that the opioid system becomes involved later in the development of stimulant dependence, forming part of the neuro - adaptations in brain reward systems that create a state of hedonic allostasis in the addicted brain (koob & le moal, 2001). indeed, recent pet studies have shown significant alterations of [c]carfentanil bpnd in chronic cocaine patients as compared to healthy controls, changes that predict treatment outcome and are correlated with drug craving and risk of relapse to cocaine use (ghitza. another recent study of alcohol - dependent patients found that opiate - induced dopamine release in the ventral striatum was correlated with the severity of alcohol dependence (spreckelmeyer. 2011), providing further evidence of the important interactions between these two neurotransmitter systems in substance use disorders. we have previously shown that naltrexone pretreatment attenuates the subjective response to an acute dose of amphetamine to a greater extent in amphetamine - dependent patients than in healthy subjects (jayaram - lindstrm. it is therefore possible that amphetamine does cause a significant opioid release in the amphetamine - dependent, but not in the healthy, human brain. genetic variability between samples, particularly in the prevalence of the asp40 single nucleotide polymorphism in the opioid receptor gene that has been linked to naltrexone response in alcohol dependence, might also have contributed to differences between studies ; although we do not have genetic data to prove this (dlugos. 2012) published a study reporting a decrease in [c]carfentanil bpnd 3 h after an oral amphetamine dose of 0.5 mg / kg bodyweight. there are several differences between our studies that may help explain the differences in results. administration of amphetamine in terms of pharmacokinetics and also in the resulting subjective effects. indeed, in colasanti 's study, the high - dose group did not report any significant euphoria, while the subjects in our study consistently reported strong effects of amphetamine as compared to placebo. while a part of this interval is needed due to the slower absorption of an oral amphetamine dose, it remains a significant difference between our studies. our choice of an immediate measurement was based on several lines of evidence, not least previous studies showing a very fast reduction of (3)h - damgo binding after cocaine administration in rodents (soderman & unterwald, 2009). also, the euphoric effects of an i.v. amphetamine dose are immediate and start to wear off gradually after 12 h. of course, if opioid release plays any important role in these acute rewarding effects, it has to be present within this time interval rather than 3 or 4 h later. there are also some technical differences between the studies that might have influenced the results. the increased resolution of the high - resolution research tomograph system provides a higher signal recovery, which should provide additional sensitivity to changes in binding (schain. 2012). also, we had no bias in terms of differences in injected mass or radioactivity between the different conditions. finally, a particular strength of our study is the cross - over randomization, which allowed us to do within - subject comparisons while still controlling for expectation effects. the results of these two pet studies suggest another possible explanation for the fact that naltrexone attenuates the subjective effects of amphetamine, namely, that weak unspecific adverse effects of naltrexone (e.g. nausea) might somewhat blunt the immediate effects of amphetamine, while its specific action as an opioid antagonist blocks the prolonged euphoric effects possibly caused by opioid release 2 or 3 h later. to conclude, in this placebo - controlled pet study, an acute i.v. dose of amphetamine did not cause any opioid release in the healthy human brain reward system. furthermore, possible expectation effects were not mediated by opioid activation, since order of placebo / amphetamine did not change [c]carfentanil bpnd. these results are in contrast with earlier findings of stimulant - induced opioid activation in rodents as well as recent data showing later effects of oral amphetamine using a slightly different study design. further studies are needed to more fully investigate this issue from a translational perspective. with the recent development of small animal pet systems, the effects of amphetamine on the opioid system could be studied across a wider dose range and timescale than what is possible in humans. studies of stimulant - dependent patients are also needed to investigate the pathophysiological importance of the opioid system and its potential as a therapeutic pharmacological target. lars farde also holds a position as chief scientist, imed cns / pain, astrazeneca, sweden. | studies in rodents have shown that psychostimulant drugs such as cocaine and amphetamine cause endorphin release in the brain reward system. there is also evidence for the involvement of the opioid system in human psychostimulant dependence. the acute effects of an i.v. psychostimulant drug on the brain opioid system, however, have not yet been investigated in humans. we hypothesized that an i.v. dose of amphetamine as compared to placebo would cause an opioid release in the human brain reward system, measurable as a reduction of the binding potential of the -opioid receptor radioligand [11c]carfentanil. ten healthy young men were examined using positron emission tomography (pet) and [11c]carfentanil in three sessions : at baseline ; after placebo ; after an i.v. amphetamine dose of 0.3 mg / kg bodyweight. the order of amphetamine and placebo was double - blinded and randomized. pet examinations were performed with a siemens high resolution research tomograph. data were analysed with the simplified reference tissue model, applying manually drawn regions of interest for every subject. using repeated measures analysis of variance, we found no significant differences in [11c]carfentanil binding potential between amphetamine and placebo conditions in any of the investigated brain regions. in contrast to data from rodent studies and a recent study of oral amphetamine administration in humans, an i.v. dose of amphetamine does not cause any acute opioid release in healthy human subjects. the postulated role of the opioid system in mediating the effects of amphetamine needs to be further investigated in animal models of the disease as well as in patient populations. |
the fabp6 mice used in this study were produced in - house and have been backcrossed to the c57bl/6 background for 8 generations. the mice were housed in a controlled environment with a 12 h light / dark cycle and were given free access to water and standard diet (2020x teklad global rodent diet). immature (2325 days old) female mice were superovulated by administration of equine chorionic gonadotropin (ecg ; 5 iu, i.p.) followed 48 h later by human chorionic gonadotropin (hcg ; 5 iu, i.p.). mice were euthanized at defined time - points during the gonadotropin - stimulated follicular and luteal development. briefly, ovaries were placed in phosphate - buffered saline and large antral follicles were punctured using sterile 27.5 g needle. ovaries were gently squeezed with a spatula to flush out remaining granulosa cells from punctured follicles. the cell suspension was passed through a cell strainer (40 m, bd falcon) to remove cumulus - oocyte complexes. granulosa cells from both ovaries of each mouse were pooled for quantitative real - time pcr (qpcr) and immunoblot analyses. to determine ovulation rate, ovulated oocytes were collected from both oviducts and counted as a measure of number of ovulations for each mouse. total rna was purified from ovaries using the trizol reagent (life technologies, burlington, on) and then converted to cdna for qpcr analysis (bio - rad cfx384 system, bio - rad, mississauga, on). the pre - validated primers used were : fabp6 ; aromatase (cyp19a1), 5-tggagaacaattcgccctttc-3 and 5-ccgaggtgtcggtgacttc-3 ; prostaglandin synthase 2 (ptgs2), 5- tgagcaactattccaaaccagc-3 and 5-gcacgtagtcttcgatcactatc-3 ; succinate dehydrogenase complex a (sdha), 5-ggaacactccaaaaacagacct-3 and 5-ccaccactgggtattgagtagaa-3 ; -2-microglobulin (b2 m), 5-ttctggtgcttggtctcactga-3 and 5-cagtatgttcggcttcccattc-3 ; glyceraldehyde-3-phosphate dehydrogenase (gapdh), 5-aggtcggtgtgaacggatttg-3 and 5-tgtagaccatgtagttgaggtca-3 ; and ribosomal protein l19 (rpl19), 5-atgagtatgctcaggctacaga-3 and 5-gcattggcgatttcattggtc-3. the average combined abundance of sdha, b2 m, gapdh and rpl19 mrnas was used to normalize the mrna abundance of other genes. the thermal cycler profile used in the analyses is as follows : initial denaturation step of 5 min at 95 c, followed by 35 cycles of 15 sec at 95 c denaturation step and 30 sec at 58 c annealing / extension step. for immunoblot analyses, protein extracts were resolved by sds - polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. after blocking with 5% milk in tbs - t (50 mm tris - hcl, ph 7.5 ; 150 mm nacl ; 0.05% tween 20), the membranes were incubated overnight at 4 c with primary antibodies followed by washing with tbs - t (3 times, 10 min each) and incubation with secondary antibody (1:10000) for 1 h at room temperature. the immunoblotted proteins were detected using immun - star kit and chemidoc analyzer (bio - rad). the mouse fabp6 antiserum (used at 1:40,000 dilution) was generated in rabbits using recombinant mouse fabp6 as antigen. other antibodies (diluted 1:1000 as recommended by the suppliers) were purchased from commercial sources : anti - star (sc-25806, santa cruz biotechnology, dallas, tx), anti - actb (ab8227, abcam, cambridge, ma). the secondary antibody, goat anti - rabbit igg (hrp - conjugated) (ab6721, abcam), was used at 1:10,000 dilution. ovaries collected from superovulated mice were fixed in 10% neutral buffered formalin, embedded in paraffin and cut into 4 m sections (leica rm2125rt). following initial steps of deparaffinization, dehydration, antigen retrieval and blocking (blocking solution consisted of 3% bovine serum albumin, 0.4% triton x-100 and 10% normal goat serum), sections were incubated with or without murine fabp6 anti - serum (1:500) for 1 h. the slides were then washed with pbs and incubated with anti - rabbit secondary antibody (1:200) dilutions for 1 h. immunoreactivity to fabp6 antiserum (brown staining) was visualized using the substrate dab chromogen system (k3467, dako, burlington, on). differences between mrna abundance at different time points were evaluated by one - way analysis of variance followed by tukey s multiple comparisons post - hoc test. the fabp6 mice used in this study were produced in - house and have been backcrossed to the c57bl/6 background for 8 generations. the mice were housed in a controlled environment with a 12 h light / dark cycle and were given free access to water and standard diet (2020x teklad global rodent diet). immature (2325 days old) female mice were superovulated by administration of equine chorionic gonadotropin (ecg ; 5 iu, i.p.) followed 48 h later by human chorionic gonadotropin (hcg ; 5 iu, i.p.). mice were euthanized at defined time - points during the gonadotropin - stimulated follicular and luteal development. briefly, ovaries were placed in phosphate - buffered saline and large antral follicles were punctured using sterile 27.5 g needle. ovaries were gently squeezed with a spatula to flush out remaining granulosa cells from punctured follicles. the cell suspension was passed through a cell strainer (40 m, bd falcon) to remove cumulus - oocyte complexes. granulosa cells from both ovaries of each mouse were pooled for quantitative real - time pcr (qpcr) and immunoblot analyses. to determine ovulation rate, ovulated oocytes were collected from both oviducts and counted as a measure of number of ovulations for each mouse. total rna was purified from ovaries using the trizol reagent (life technologies, burlington, on) and then converted to cdna for qpcr analysis (bio - rad cfx384 system, bio - rad, mississauga, on). the pre - validated primers used were : fabp6 ; aromatase (cyp19a1), 5-tggagaacaattcgccctttc-3 and 5-ccgaggtgtcggtgacttc-3 ; prostaglandin synthase 2 (ptgs2), 5- tgagcaactattccaaaccagc-3 and 5-gcacgtagtcttcgatcactatc-3 ; succinate dehydrogenase complex a (sdha), 5-ggaacactccaaaaacagacct-3 and 5-ccaccactgggtattgagtagaa-3 ; -2-microglobulin (b2 m), 5-ttctggtgcttggtctcactga-3 and 5-cagtatgttcggcttcccattc-3 ; glyceraldehyde-3-phosphate dehydrogenase (gapdh), 5-aggtcggtgtgaacggatttg-3 and 5-tgtagaccatgtagttgaggtca-3 ; and ribosomal protein l19 (rpl19), 5-atgagtatgctcaggctacaga-3 and 5-gcattggcgatttcattggtc-3. the average combined abundance of sdha, b2 m, gapdh and rpl19 mrnas was used to normalize the mrna abundance of other genes. the thermal cycler profile used in the analyses is as follows : initial denaturation step of 5 min at 95 c, followed by 35 cycles of 15 sec at 95 c denaturation step and 30 sec at 58 c annealing / extension step. for immunoblot analyses, protein extracts were resolved by sds - polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. after blocking with 5% milk in tbs - t (50 mm tris - hcl, ph 7.5 ; 150 mm nacl ; 0.05% tween 20), the membranes were incubated overnight at 4 c with primary antibodies followed by washing with tbs - t (3 times, 10 min each) and incubation with secondary antibody (1:10000) for 1 h at room temperature. the immunoblotted proteins were detected using immun - star kit and chemidoc analyzer (bio - rad). the mouse fabp6 antiserum (used at 1:40,000 dilution) was generated in rabbits using recombinant mouse fabp6 as antigen. other antibodies (diluted 1:1000 as recommended by the suppliers) were purchased from commercial sources : anti - star (sc-25806, santa cruz biotechnology, dallas, tx), anti - actb (ab8227, abcam, cambridge, ma). the secondary antibody, goat anti - rabbit igg (hrp - conjugated) (ab6721, abcam), was used at 1:10,000 dilution. ovaries collected from superovulated mice were fixed in 10% neutral buffered formalin, embedded in paraffin and cut into 4 m sections (leica rm2125rt). following initial steps of deparaffinization, dehydration, antigen retrieval and blocking (blocking solution consisted of 3% bovine serum albumin, 0.4% triton x-100 and 10% normal goat serum), sections were incubated with or without murine fabp6 anti - serum (1:500) for 1 h. the slides were then washed with pbs and incubated with anti - rabbit secondary antibody (1:200) dilutions for 1 h. immunoreactivity to fabp6 antiserum (brown staining) was visualized using the substrate dab chromogen system (k3467, dako, burlington, on). differences between mrna abundance at different time points were evaluated by one - way analysis of variance followed by tukey s multiple comparisons post - hoc test. detection and semi - quantitative estimation by conventional pcr using primers specific for the entire open reading frame of the murine fabp6 mrna sequence demonstrated its presence in ovary of wild - type (c57bl/6) mouse, but at a lower abundance compared to ileum (fig. 1. detection of fabp6 mrna in the wild - type (c57bl/6) murine ovary. (a) the fabp6 open reading frame was amplified by conventional pcr to detect the fabp6 mrna in murine ovary and ileum. primers specific for the murine 18s ribosomal rna sequence were used to indicate the presence of templates in the reaction. (b) relative mrna abundance (mean sem ; n = 4) of fabp6, cyp19a1 and ptgs2 in pure populations of granulosa and luteal cells isolated from ovaries at specific time points during superovulation protocol as described in the materials and methods section. relative mrna abundance was determined by normalization to the average abundance of sdha, b2 m, gapdh and rpl19 mrnas. therefore, we used total rna from granulosa and luteal cells collected at specific time - points during gonadotropin - induced follicular development and ovulation. analysis of cyp19a1 and ptgs2 mrna abundance by qpcr, in ecg and hcg dependent manner, confirmed that our cell populations represented precise stages of granulosa cell differentiation through follicular and luteal development. (a) the fabp6 open reading frame was amplified by conventional pcr to detect the fabp6 mrna in murine ovary and ileum. primers specific for the murine 18s ribosomal rna sequence were used to indicate the presence of templates in the reaction. (b) relative mrna abundance (mean sem ; n = 4) of fabp6, cyp19a1 and ptgs2 in pure populations of granulosa and luteal cells isolated from ovaries at specific time points during superovulation protocol as described in the materials and methods section. relative mrna abundance was determined by normalization to the average abundance of sdha, b2 m, gapdh and rpl19 mrnas. the pattern of fabp6 gene expression in response to gonadotropin (ecg and hcg) treatment indicated that maximal transcript abundance (p 0.05 ; fig. (a) mean body weight of mice on the first day of superovulation treatment protocol. (b) mean number of ovulations in response to superovulation treatment in c57bl/6 (n = 4) and fabp6 (n = 7) mice as described in the materials and methods section. the number of oocytes in oviducts of each mouse was determined at 1820 h after hcg stimulation. values shown are mean sem. p < 0.03.) on the day of ecg treatment suggesting that overall growth and development of fabp6 mice was normal. the number of oocytes ovulated in fabp6 mice was significantly lower (p < 0.03) than c57bl/6 mice (fig. 3b). nonetheless, the ovulated cumulus - oocyte - complexes of both genotypes appeared normal with expanded cumulus cell layers and mature oocytes. these data suggest that fabp6 plays a significant role in granulosa cell differentiation required for follicular rupture, but is dispensable for meiotic maturation of the oocyte. (a) mean body weight of mice on the first day of superovulation treatment protocol. (b) mean number of ovulations in response to superovulation treatment in c57bl/6 (n = 4) and fabp6 (n = 7) mice as described in the materials and methods section. the number of oocytes in oviducts of each mouse was determined at 1820 h after hcg stimulation. it is generally accepted that fabp6 (also known as ileal lipid binding protein ; ileal bile acid binding protein) is involved in intracellular transport of bile acids in enterocytes [10, 16 ]. however, the fabp6 mrna and protein have also been detected in the ovaries of several species [3, 14, 15, 17,18,19 ], albeit the precise tissue localization and expression pattern in dynamic structures of the ovary were not established. further, the importance of this protein in ovarian function remains unknown since the ovary is not a major site for bile acid metabolism. in the present study, we surveyed murine ovaries for fabp6 distribution and explored its potential importance in ovarian function. fabp6 is known to preferentially bind bile acids but the ovaries are not regarded as an active site for bile acid metabolism, although a recent study reported that some genes involved in bile acid synthesis are expressed in human granulosa cells. we reported recently that bile acids are found in the follicular fluid of the dominant follicle of lactating cows and that granulosa cells of the dominant follicles express slc10a2 and gpbar1 (bile acid transporter and receptor, respectively), suggesting the novel idea that bile acid signaling might be occurring in ovarian cells. on the other hand, fabp6 has been shown to be capable of binding other ligands, including fatty acids [8, 9 ] and progesterone, and therefore may be involved in metabolic pathways beyond bile acid metabolism. it is well established that the progesterone receptor is induced in granulosa cells of ovulating follicles by hcg and mice lacking this receptor do not ovulate. along with these observations, our data showing high mrna abundance of fabp6 at ecg-48h and hcg-1h are indicative of its role in progesterone signaling, which is needed for efficient ovulation. interestingly, despite the attenuated ovulation response displayed by fabp6 mice to superovulatory stimulation, they do not exhibit overt defects in fertility or fecundity. the loss of fabp6 function may not seriously impact the overall fecundity of polytocous animals (such as mice), but may be important for species that release only one egg per cycle (such as humans). detailed understanding of mechanisms by which fapb6 influences the ovulatory process requires further research. in summary, we demonstrated here that fabp6 is present in distinct cell populations within the murine ovaries and loss of fabp6 results in marked reduction in ovulatory response to superstimulation. these findings suggest that fabp6 may have important roles in ovarian physiology that are distinct from its function as an intracellular bile acid transporter in the ileum. | the fatty acid binding protein 6 (fabp6) is commonly regarded as a bile acid binding protein found in the distal portion of the small intestine and has been shown to be important in maintaining bile acid homeostasis. previous studies have also reported the presence of fabp6 in human, rat and fish ovaries, but the significance of fabp6 in this organ is largely unknown. therefore, we surveyed murine ovaries for fabp6 gene expression and evaluated its role in ovarian function using mice with whole body fabp6 deficiency. here we show that the fabp6 gene is expressed in granulosa and luteal cells of the mouse ovary. treatment with gonadotropins stimulated fabp6 gene expression in large antral follicles. the ovulation rate in response to superovulatory treatment in fabp6-deficient mice was markedly decreased compared to wildtype (c57bl/6) mice. the results of this study suggest that expression of fabp6 gene in granulosa cells serves an important and previously unrecognized function in fertility. |
the significance of low - level laser in clinical medicine began with the important works of endre mester. the basic science underlying low - level laser therapy (lllt) is that it involves the treatment of various medical diseases with photomedicine and the use of low - level lasers or light - emitting diodes to alter cellular function, or, in short, the employment of photo - biomodulation effects to help normalize cellular functions, including the energy usage of body cells. in recent years, the various clinical applications of lllt have mainly been based on previous scientific works concerning the effect of low - level lasers, such as increasing adenosine triphosphate production,1 while, at the same time, the exertion of a positive influence on fibroblast2 and collagen synthesis3 at the cellular - molecular level. clinical protocols exist for lllt in the management of common painful conditions such as rheumatoid arthritis, osteoarthritis, acute and chronic neck pain, tendinopathy, and chronic joint disorders. as far as adhesive capsulitis of the shoulder in the elderly is concerned, most clinicians use a combination of nonsteroidal anti - inflammatory medications and conventional physical therapy consisting of ultrasonic therapy, transcutaneous electrical therapy, and short - wave therapy, for management. these forms of conservative treatment represent symptomatic treatment only, without the biomodulation effects offered by low - level lasers ; for instance, ultrasound treatment affords neither anti - inflammatory nor biomodulation effects at the cellular - molecular level. the objective of this paper is to report the clinical result of a study on the efficacy of lllt in the management of the early phase of symptomatic adhesive capsulitis of the shoulder in elderly who failed to respond to the combined use of conventional physical therapy and nonsteroidal anti - inflammatory medications for not fewer than 4 weeks. in this study, we investigated the clinical effect of the combined irradiation of strategic anatomic points of pathology in adhesive capsulitis as well as acupuncture points, which, to the best of our knowledge, has not been reported in the literature to date. we stress that patients presenting late, after 6 weeks of disease onset, were excluded to guard against the possibility that whatever positive clinical result obtained from the use of lllt could have been due to spontaneous disease resolution. published medical literature exists concerning clinical studies of the effect of lllt on frozen shoulder that were positive, albeit being short - lived.46 prior to the start of the present study, we researched the irradiation of two acupuncture points namely, bingfeng and tianzong in addition to the laser irradiation, at a wavelength of 810 nm, of six preselected points that are commonly abnormal on magnetic resonance imaging studies in patients with adhesive capsulitis. we found long - lasting clinical results, particularly with regard to improvement in range of motion ; much longer, in fact, than that previously reported in the literature. this forms the rationale of this clinical study and forms the rationale as to why we followed up the patients for a mean of 2 years. the study population consisted of a prospective cohort of consecutive unselected 35 elderly patients with mean age of 65 (range : 6077) years being referred to our tertiary referral pain center within 6 weeks of onset of symptoms and previously having failed response to a combination of nonsteroidal anti - inflammatory medications and not fewer than 4 weeks of conventional physical therapy. contrast - enhanced magnetic resonance imaging was performed in each patient prior to entry to the study to confirm the typical appearances in the axillary pouch, rotator interval, biceps anchor, and anteroposterior capsule7 to confirm the diagnosis and ensure there was no other concomitant pathology such as rotator - cuff tear. exclusion criteria included patients presenting late after 6 weeks of disease onset ; patients who had concomitant other shoulder pathologies on magnetic resonance imaging ; patients with prior shoulder operations ; and patients who had potential contraindications for the use of laser treatment, such as previous history of tumor or ongoing sepsis. we also excluded patients with previous neuromuscular conditions of the affected upper extremity, such as a previous cerebrovascular accident. all patients signed informed consent detailing that they would be treated by lllt and that only us food and drug administration - approved devices would be used. each subject was clinically examined and followed up by the same orthopedic surgeon to minimize interobserver error. all the low - level laser pain treatments were also performed by the same orthopedic surgeon, and all the serial shoulder scoring was done by the patient with the assistance of the same orthopedic surgeon. during the initial visit, extra care was taken to document the shoulder range of motion, presence of neurological deficit, sites of tenderness, and overall upper extremity function. murley score,8 a 100-point scale composed of a number of individual parameters, to assess the patient s level of pain and ability to carry out normal daily activities. the test is divided into four subscales : pain (15 points) ; activities of daily living (20 points) ; strength (25 points) ; and range of motion : forward elevation, external rotation, abduction, and internal rotation of the shoulder (40 points) the higher the score, the higher the quality of the function. lllt at a wavelength of 810 nm emitted from a gaaias semiconductor laser device (figure 1) with 5.4 j per point, 32.4 j per session, spot area of 0.5 cm, peak power of 50 mw, and power density of 20 mw / cm, and continuous output was employed. the treatment regimen consisted of three sessions of treatment per week for 8 consecutive weeks. each treatment session lasted 180 seconds : 20 seconds each for the six chosen spots such as, the subacromial space, biceps anchor, axillary pouch, anterior shoulder capsule, posterior shoulder capsule, and rotator interval. in addition, we irradiated two acupuncture points9,10 near the shoulder for 30 seconds each the tianzong and bingfeng acupoints (figure 2), otherwise known as si 11 and si 12, respectively, of the small intestine meridian in the field of chinese medicine. we did not add on any other conventional therapy during each treatment session, as all the subjects had had these therapeutic interventions before but had failed to respond. further, no medications were prescribed to the subjects under study, as none of the patients had responded to oral anti - inflammatory agents provided by other clinicians before receiving treatment at our tertiary referral pain center. the only rehabilitation exercises were the home exercises already taught to patients by other physiotherapy centers prior to patients entry to the study. therefore, essentially all subjects in the current study received only lllt as really the sole therapy. in this study of 35 elderly patients and 50 painful and stiff shoulders affected by painful adhesive capsulitis, the male to female ratio was 1.0:1.3 and the right shoulder was affected in 60% of cases. the study period lasted from 2011 to the end of 2014. all 35 subjects in the study population completed the treatment regimen with good compliance ; there were no defaults. only four painful shoulders among the 50 shoulders failed to positively respond to laser treatment, with no improvement in constant table 1 details the relevant demographics of the patient population together with the serial pre- and posttreatment shoulder scores. figure 4 shows the relative breakdown of the mean value of the constant subscores pre- and posttreatment as well as at 2-year follow - up, including the range of motion subscale. in the graph, one can notice that lllt treatment had a positive clinical effect on range of motion, level of pain, and activities of daily living but had no effect on the strength of the elderly patients. apart from the four shoulders of two patients who did not show clinical response to lllt and required surgery, there was a statistically significant improvement in the constant score in the remaining 46 shoulders, from a mean score of 59 at the start of the study (range : 5762), with standard deviation of 1.1 to a mean score of 71 at the end of treatment (range : 6873), with standard deviation of 1.0. statistical testing with student s t - test rejected the null hypothesis that the consistent improvement in constant the mean clinical follow - up of this patient cohort was 2 years (range : 1.52.5 years). a reasonably long follow - up was required to reveal whether the lllt modality alone could provide any lasting benefits in patients, as well as any incipient side effects. in this study, no side effect was observed at the 2-year mark, and all patients tolerated the cold laser therapy very well. over the past years, more than 100 double - blind placebo - controlled studies have been published on the effects of lllt. these articles also showed the favorable anti - inflammatory effect of lllt.11 low - level lasers may reduce pain related to inflammation by lowering, in a dose - dependent manner, levels of prostaglandin e2, prostaglandin - endoperoxide synthase 2, interleukin 1-beta, tumor necrosis factor - alpha, the cellular influx of neutrophil granulocytes, oxidative stress, and tissue edema. the appropriate dose appears to be between 0.3 and 19.0 j / cm.11 unlike traditional laser devices, innumerous clinical studies have shown that low - level lasers are safe and lack side effects, unlike, for example, the lasers used for cosmetic surgery, in which thermal injury to the skin is not an uncommon side effect. the current study employed an 810 nm wavelength laser and we irradiated six important anatomic points around the shoulder region relevant to the shoulder pathology at hand. additionally, we irradiated the bingfeng and tianzong acupuncture points to produce additive effects, especially range of motion improvement, since previous studies of lllt on adhesive capsulitis did not consistently achieve an improved range of motion despite achieving pain relief.2 the total application time of all irradiation, 180 seconds, was well tolerated, even by patients of advanced age. there was absolutely no need for any anesthesia of any kind, since the laser application was noninvasive and painless. the majority of previous clinical studies of lllt concentrated more on shoulder impingement syndrome12 than on frozen shoulder, but the reported positive effects appeared only short - lived,13 as claimed by a recent cochrane systematic review, and there is a complete lack of medium - term clinical follow - up studies on the subject in the medical literature. to add to this, studies that have investigated shoulder pain in general, without pinpointing a particular diagnosis, have, in fact, looked at an admixture of different shoulder disorders, therefore it is precarious to draw any conclusions from them. for instance, in many clinical studies, the researchers relied solely on a clinical diagnosis without confirmation by magnetic resonance imaging. magnetic resonance imaging is imperative to rule out other possible shoulder pathologies, besides confirming the clinical diagnosis beyond an element of doubt. in summary, we chose lllt owing to its lack of side effects, while being noninvasive and well tolerated by the elderly. by contrast, traditional methods of treatment for painful shoulders of the elderly caused by adhesive capsulitis involve nonsteroidal anti - inflammatory agents as well as conventional physiotherapy, such as ultrasound therapy and electrical stimulation therapy. none of these therapies has the biomodulation effects offered by lllt, such as improvement in microcirculation or the upregulation of several genes involved in energy metabolism and oxidative phosphorylation, which stimulates an increase in adenosine triphosphate production, which in turn regulates other cellular processes, leading to the normalization of biological functions at the cellular level. for instance, therapeutic ultrasound devices do not even have anti - inflammatory effects any biomodulation effects.14 in general, for any conservative treatment of adhesive capsulitis to be meaningful, the treatment modality must have lasting effects and benefits for the patient, rather than providing only transient pain relief, such as the administration of painkillers does. we are not aware of any clinical study reporting on the medium - term efficacy of lllt in the management of shoulder adhesive capsulitis. in the study reported here, lllt showed evidence of lasting benefit and represents a viable option to choose rather than surgery, particularly in the elderly population, in whom concomitant medical comorbidities may well increase the risk of surgery. this study lacked a control group, which represents a weakness and is acknowledged as such. a control group was not included, as the majority of patients do not give consent to the idea of a sham laser light source. the fact that the patient population included subjects who failed to respond to a combination of conventional physical therapy and nonsteroidal anti - inflammatory medications but responded positively to lllt warrants further large - scale double - blind placebo - controlled studies to further confirm the clinical results of this study, provided lasers of similar wavelength, power density, and application time, and the six points of application are the same, together with the two additional acupuncture points of bingfeng and tianzong to make any clinical results comparable. the current prospective review of a patient cohort of 35 elderly patients with documented symptomatic adhesive capsulitis of 50 painful shoulders showed a significant positive clinical response in patients, as evidenced by the marked improvement in their constant murley scores, not only in the short - term but also in the medium - term, as the mean clinical follow - up in this prospective study was 2 years. further large - scale studies are worthwhile to further explore the use of lllt, not only in the treatment of adhesive capsulitis but also in other painful shoulder conditions, such as partial rotator - cuff tears. | introductionthis paper reports on the medium - term mean 2-year prospective follow - up of a patient cohort of 35 unselected elderly patients with mean age of 65 years who visited our tertiary referral pain center for painful adhesive capsulitis of the shoulder managed with low - level laser therapy (lllt).materials and methodsall patients in this prospective cohort study had documentation of the diagnosis by contrast - enhanced magnetic resonance imaging before study entry and all had failed to respond to a combination of conventional physical therapy and nonsteroidal anti - inflammatory medications for not fewer than 4 weeks. lllt, at a wavelength of 810 nm emitted from a gaaias semiconductor laser device with 5.4 j per point and a power density of 20 mw / cm2, was employed to irradiate six predetermined anatomic points and two acupuncture points. the treatment regimen consisted of three sessions of treatment per week for 8 consecutive weeks. each treatment session lasted 180 seconds. serial clinical assessment was undertaken using the constant murley shoulder score.resultsa total of 50 painful shoulder joints were treated, as a number of elderly presented with bilateral symptoms. all but four painful shoulders showed significant improvement in constant murley shoulder score at the end of 8-weeks lllt treatment and, surprisingly, the improvement was found maintained at follow - up assessments at 1 year and 2 years.conclusionwe conclude that lllt is a viable option in the conservative treatment of shoulder pain arising from adhesive capsulitis of the shoulder in the elderly, with a positive clinical result of more than 90% and with clinical efficacy both in the short - term and the medium - term. |
the inflammatory and immune reactions induced by the bacterial plaque represent the main characteristics of periodontitis, and this disease represents a particularly cogent example of problem arising from the phenomenon. studies have demonstrated that periodontal disease affects between 10% and 15% of the world 's population, representing the greatest cause of tooth loss. there is strong evidence that this disease affects a specific, predisposed group of the population that presents an exacerbated inflammatory / immune response to the periodontopathogenic bacteria that accumulate on the teeth and around the gingival tissue, which in turn may lead to tissue damage [1, 3 ]. the exact mechanism of periodontitis development, including the prior agents or mediators involved, is not clear. periodontitis manifests itself as a multifactor phenomenon, including the generation of reactive oxygen species (ros). the strong evidence linking ros to the pathological destruction of the connective tissue during periodontal disease rests on the presence of neutrophils infiltration as the main event in the host 's response to bacterial invasion [1, 5, 6 ]. furthermore, hydroxyl radical (oh) is most active in damaging important molecules such as dna proteins and lipids, while hydrogen peroxide (h2o2), even not being considered a potent ros, is capable of crossing the nuclear membrane and also damaging the dna. quantitatively, the main source of superoxide anion (o2) and other ros responsible for initiation reactions is the respiratory chain. however, its presence in the periodontal tissue results first and foremost from the activation of phagocytes (neutrophils and macrophages), such as antibacterial agents [1, 5, 8 ]. it has been suggested that superoxide anion is involved in bone reabsorption which has been corroborated by studies that have demonstrated the presence of this anion in reabsorption zones adjacent to the osteoclasts. the hydroxyl radical is able to initiate a classical chain reaction known as lipid peroxidation leading to the vasodilation production and rat bone reabsorption. an example of the damage caused by hydrogen peroxide is that it can stimulate the phosphorylation of the nf - kb - ikb complex activating the nk - kb and facilitating nuclear translocation and downstream of proinflammatory cytokines, including interleukin-2 (il-2), interleukin-6 (il-6), interleukin-8 (il-8), -interferon, and tumor necrosis factor- (tnf-) that are very important in the pathogenesis of periodontal disease [1012 ]. ros production is inevitable in all aerobic organisms including humans, who necessarily posses a complex system of antioxidant defense [8, 13 ]. if the homeostasis is interrupted in favor of ros, an oxidative stress situation is created. the aim of this study was to evaluate the involvement of proinflammatory and oxidative stress markers in individuals with chronic periodontal disease. september, 2003 in a single centre (peridontologic centre, florianopolis, santa catarina, brazil). eighteen subjects were divided into 2 groups : experimental group (e), comprising individuals with chronic periodontitis, age 52.95.0 (4 men and 5 women), with the following inclusion criteria : presence of chronic inflammation (pain, redness, heat, swelling), diagnosed according to bleeding on probing, at least 5 or 6 sites with probing depth 5 mm, attachment loss 3 mm, and extensive radiographic bone loss. control group (c), was composed of healthy individuals, age 51.19.6 (4 men and 5 women) with no prior history of periodontal disease. for both groups, the following items were considered as exclusion criteria : infection, cardiovascular and/or neurological illness, renal insufficiency and/or diabetes ; pregnancy ; smoking ; use of antibiotics and/or hormonal or nonhormonal anti - inflammatory drugs 6 months prior to tissue collection. in the day of the surgical procedures, this study was in agreement with the ethical principles of the world medical association declaration of helsinki (1964). permission for this study was obtained from the ethical committee for human research of the federal university of santa catarina (project no. 210/2002) and the study included only individuals that agreed to participate after reading and signing a free and informed consent form, except those with difficulty in understanding and communicating, with physical handicap, or both, which could have compromised the sample collection. all of the surgical procedures were assessed and performed by a periodontist, according to the necessity for each treatment. the patients with chronic periodontitis (experimental group) were submitted a pocket depth reduction technique from palatal / lingual, buccal, and interproximal sites. the biopsies were obtained from inflammatory granulation tissues, connective and epithelium tissues. the samples collected from the control group were obtained from quarantined mucosa during the surgical procedures of impacted third molars removed following orthodontic recommendation or after the reopening of dental implants. all samples from the experimental and control groups were removed during the surgical and were immediately frozen in liquid nitrogen (170c) subsequently latter laboratory analysis. on the day of the experiments, the samples were deathward at room temperature to determine the different parameters : cat, gpx, gst, gr, mpo activities, and the contents of tg, gsh, gssg, and tbars. the method described by aebi was employed to measure the catalase activity (cat) by measuring the decay of a freshly prepared 10 mm hydrogen peroxide solution at 240 nm. glutathione peroxidase (gpx) was measured at 340 nm through the glutathione / nadph / glutathione reductase system by the dismutation of tert - butyl hydroperoxide. glutathione s - transferase (gst) activity was determined at 340 nm using cdnb (1-chloro-2, 4-dinitrobenzene) as substrate and a 0.15 m gsh concentration. finally, glutathione reductase (gr) activity was assayed at 340 nm by measuring the rate of nadph oxidation. results were expressed as mmolg(cat) and mol ming (gpx, gst, and gr). nonprotein thiols, mostly present as the reduced form of glutathione (gsh), were measured at 412 nm according to beutler using elmann 's reagent (dtnb : 2-dithionitrobenzoic acid). immediately after thawing, acid extracts were obtained by adding tissue portions to 12% trichloroacetic acid (1 : 4 w / v), which were then centrifuged at 15 000 g for 5 minutes at 5c. supernatants from the acid extracts were added to a buffer containing 0.25 mm dtnb in 0.1 m na2po4, ph 8.0, and the formation of the thiolate anion was immediately determined. total glutathione (tg) was also measured at 412 nm in acid extracts according to the enzymatic method of tietze. oxidized glutathione (gssg) was also determined by calculating the difference (in equivalents of gsh) between total glutathione and reduced glutathione contents. results were expressed as mol g. myeloperoxidase activity was measured according to the method developed by rao., and was estimated by colorimetric measurement at 450 nm on an elisa plate reader. thiobarbituric acid - reactive substance (tbars) contents were determined to assess endogenous lipid oxidation in gingival tissue according to ohkawa. and bird and draper. after thawing, gingival portions were immediately added to 12% trichloroacetic acid (1 : 4 v / v) and were then centrifuged at 15 000 g for 5 minutes at 5c. supernatants were added to 50 mm tris - hcl ph 7.0, vortexed for 20 seconds, added to 0.67% (w / v) 2-thiobarbituric acid, maintained in boiling water for 60 minutes, cooled at 5c for 30 minutes, and then analyzed spectrophotometrically at 535 nm. concentrations were expressed as nmol tbars / g wet tissue using 535=153 mmcm. all the biochemical parameters described above were measured in duplicate, except for the tbars determinations, which were measured in triplicate. the results were expressed as mean sem. statistical differences between groups were determined by independent student t test analysis. for all analyses, p.05) (table 1). however, the values obtained for oxidized glutathione (gssg) showed a significant increase in the experimental group when compared to the control group (p=.019) (see table 1). lipoperoxidation was measured through tbars contents, which were significantly a higher increase in the experimental group (p=.015) (see table 1). few studies have considered the effect of the imbalance between oxidants and antioxidants in patients with periodontitis, which in turn predisposes such individuals to the damaging effects of ros in the periodontium. ellis and collaborators analyzed gingival tissues from patients with severe periodontal disease and showed that the activity of catalase was decreased. in the present study, the activity of catalase was not different when the experimental and control groups were compared. one possible explanation for these different responses is that the patients with periodontal disease were in distinct stages of the disease. in this regard, it is well known that the antioxidant responses found in different pathologies depend on the severity or extension suffered by the patients, and long - term chronic conditions may have jeopardized the antioxidant defenses. the analysis of the enzyme glutathione peroxidase revealed a significant increase in the experimental group. a gpx increase in gingival samples from dogs and humans with periodontal disease the gpx increase may represent possible antioxidant compensation in detoxification reactions of organic peroxides produced during oxidative stress in gingival tissue. furthermore, glutathione s - transferase (gst) also revealed a significant increase in its activities in the experimental group. since gst has a direct role in the neutralization of hydroperoxides derived from the lipoperoxidation processes, increases in gst activities are probably related to the oxidative stress caused by the periodontal inflammatory process [8, 29 ]. gst comprises a group of enzymes that are also able to detoxify a variety of compounds including xenobiotics derived from pathogenic microorganisms, catalyzing their conjugation with gsh. hence, increases in gst activities are excellent indicators of endogenous detoxification from exogenous sources. the enzyme glutathione reductase (gr) has an important accessory antioxidant function related to glutathione peroxidase and glutathione s - transferase. gr intervention continuously regenerates gsh from gssg in the presence of nadph, therefore preventing cellular loss of gsh. however, in the current study, no differences in gr activities were detected in gingival tissue between the two groups. the ubiquitous tripeptide glutathione (gsh) acts directly as a generic ros scavenger or as a cofactor of gpx and gst, either by catalyzing the reduction of hydrogen peroxide and lipid hydroperoxides or by the conjugation / excretion processes of the so - called phase ii reactions. total and reduced glutathione revealed a tendency to increase, but the values were not significantly different in patients with periodontitis compared to the controls. despite gsh, these results suggest a de novo synthesis of glutathione, which is extremely necessary for the homeostasis of cells. some periodontopathogenic bacteria deplete gsh, and this may explain the amount of this antioxidant was not elevated in the gingival tissue of patients with periodontitis combined with an increase of the gpx activity in the affected tissue [33, 34 ]. a similar result was obtained in gingival tissue and blood, but lower levels of gsh were detected in the crevicular gingival fluid of patients with chronic periodontitis, when compared to normal subjects [27, 35 ]. on the other hand, a significant increase in gssg concentrations was detected in the experimental group, which is a clear biomarker of oxidative stress detected in inflammatory processes linked to periodontitis. 2002) found less gssg in gingival cervical fluid of patients with chronic periodontitis. consistent with the results for gssg, tissue lipoperoxidation, measured as tbars contents in the gingival tissue, also displayed a significant increase (p=.015) in individuals affected by periodontitis, and oxidative stress, in the gingival tissue associated with periodontal disease. the systemic depletion of antioxidants clearly indicates that in chronic periodontitis the antioxidant system is affected by a relatively strong oxidation insult, which can also deplete nutritional antioxidants such as vitamin e and c in plasma and also vitamin e in red cell membrane. moreover, myeloperoxidase activity in gingival tissue showed a significant increase in patients with periodontal disease when compared to the control group, an indicative of a chronic inflammatory process also reflected at a systemic level. these results were similar to the measurements obtained from the analysis of crevicular gingival fluid in humans with periodontal disease. oxidative stress processes and alterations in the immune system are closely related and have been described in different diseases, thus both the aspects also seem to be linked to the pathogenesis of periodontal disease, and can also be detected in the plasma of patients with periodontitis [8, 27, 35 ]. however, the extent to which ros overgeneration influences the initiation and progression of periodontal diseases is still unknown. in conclusion, in spite of the limited number of samples examined in the present study, the results indicate a relationship between proinflammatory and oxidative stress biomarkers and periodontal disease. | objective. to evaluate the involvement of proinflammatory and oxidative stress markers in gingival tissue in individuals with chronic periodontitis. subject and methods. eighteen subjects were divided in two groups : experimental (age 52.95.0) and control (age 51.19.6). the activities of enzymatic antioxidants such as catalase, glutathione peroxidase (gpx), glutathione s - transferase (gst), glutathione reductase, nonenzymatic antioxidants : total glutathione and reduced glutathione, oxidized glutathione (gssg), thiobarbituric acid reactive substances (tbars), and myeloperoxidase activity (mpo) were evaluated in gingival tissues from interproximal sites. statistical differences between groups were determined by independent student t test and p<.05. results. individuals with periodontal disease exhibited a significant increase in the activities of mpo, gpx, gst, and also in tbars and gssg levels in gingival tissue compared to the control group (p<.05). conclusion. the results of the present work showed an important correlation between oxidative stress biomarkers and periodontal disease. |
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