Adiponectin, a secretagogue exclusively produced by adipocytes, has been associated with metabolic features, but its role in the development of the metabolic syndrome remains unclear.We investigated the association between serum adiponectin level and metabolic traits, using both observational and genetic epidemiologic approaches in a multiethnic population assembled in Canada.ADIPOQ and GCKR genes.Clinical data and serum adiponectin level were collected in 1,157 participants of the SHARE/SHARE-AP studies. Participants were genotyped for the functional rs266729 and rs1260326 SNPs in GCKR was associated with HOMA-IR (P<0.001), but not with adiponectin level (P = 0.67).Adiponectin level was positively associated with HDL cholesterol and negatively associated with body mass index, waist-to-hip ratio, triglycerides, fasting glucose, fasting insulin, systolic and diastolic pressure . The rs266729 minor G allele was associated with lower adiponectin and higher HOMA-IR . The association between rs266729 SNP and HOMA-IR was no longer significant after adjustment for adiponectin concentration (P = 0.10). The rs266729 SNP was associated with HOMA-IR to an extent that exceeded its effect on adiponectin level . There was no significant interaction between rs266729 SNP and ethnicity on adiponectin or HOMA-IR. In contrast, the SNP rs1260326 in The association of the functional promoter polymorphism rs266729 with lower serum adiponectin and increased insulin resistance in diverse ethnic groups may suggest a causal relationship between adiponectin level and insulin resistance. ADIPOQ) is located on chromosome 3q27 and comprises three exons ADIPOQ locus associated with serum adiponectin level. Even though ADIPOQ is the major genetic determinant of serum adiponectin level, 12 additional contributing loci have been identified through large-scale GWA meta-analyses Adiponectin is a secretagogue exclusively produced by adipocytes and abundantly secreted into the bloodstream where it accounts for 0.01% of total plasma protein ADIPOQ and has functional consequences on ADIPOQ gene expression The common SNP -11377 C>G (rs266729) is located in the promoter region of the gene This study was approved by the Hamilton Health Sciences/Faculty of Health Science Research Ethics Board and written informed consent was obtained from each participant including consent to analyze genetic specimens. The investigation conforms to the principles outlined in the Declaration of Helsinki.The study population was comprised of Canadians of European, South Asian, Chinese, and Aboriginal origin who participated in the Study of Health Assessment and Risk in Ethnic groups (SHARE) and the Study of Health and Research Evaluation in Aboriginal Peoples (SHARE-AP), two cross-sectional studies of cardiovascular disease (CVD) risk factors conducted between 1996 and 1998. Individuals between age 35–75 years were randomly selected from three cities , and from the Six Nations Reservation  as previously described We recorded each participant’s health behaviours and medical history using standardized questionnaires as previously described ADIPOQ was extracted from the gene-centric 50 K single nucleotide polymorphism (SNP) array described elsewhere www.tcag.ca) for SHARE and in the Genome Quebec Innovation Center  for SHARE-AP. In selecting SNP(s) for analysis, we considered functional rather than surrogate SNPs, since the linkage disequilibrium structure in participants may vary according to the ethnic background. The only available functional SNP in ADIPOQ in the gene-centric 50 K array was rs266729. The rs266729 SNP was in Hardy-Weinberg equilibrium within each ethnic group and the call rate for the rs266729 SNP was 98.9%. The polymorphism is common in each ethnic group . The G variant is significantly less frequent in Chinese compared to other ethnic groups (Table S1).The genotypic information of the rs266729 SNP in GCKR as a relevant SNP for this approach. The P446L coding non-synonymous SNP rs1260326 is biologically relevant We also searched for SNPs for use in a reciprocal Mendelian randomization approach The statistical analyses were conducted using SAS version 9.1 , the R package, or PLINK software GCKR is a relevant SNP for a bi-directional Mendelian randomization analysis.To estimate the genetic effect on metabolic traits as a function of genetically lowered adiponectin levels, we performed a Mendelian randomization analysis. First, we used an instrumental variable analysis in which we determined the quantitative trait change per 1 SD of higher genetically predicted adiponectin concentration. We fit the data to a generalized least squares regression model with the rs266729 SNP as a predictor variable, adjusting for covariates , and then we regressed the genetically predicted adiponectin values against the metabolic trait. Second, we calculated a predicted increase in the metabolic parameter based on the effect on adiponectin levels. We converted log adiponectin and log HOMA-IR into z-scores and then used linear regression to model the effect of the rs266729 SNP on log adiponectin z-scores, adjusting for age, sex, and ethnicity. This slope value was then multiplied by the amount that log HOMA-IR z-score changes for every 1 z-score change in log adiopnectin z-score . The resulting value was the predicted increase in the metabolic parameter based on the effect on adiponectin. Third, linear regression was used to assess the effect on log HOMA-IR z-score for the modeled allele. We compared this observed effect with the predicted effect to test whether HOMA-IR may be causally related to rs266729 SNP determined higher adiponectin level. Finally we used a reciprocal Mendelian randomization approach An additive model was used in all the analyses including the rs266729 and the rs1260326 SNP. All of the results, with the exception of the socio-demographic data, are reported based on multivariable analyses. In a sub-analysis, we assessed the association of the metabolic traits with the rs266729 SNP by ethnicity. To further assess the possible heterogeneity in the effects by ethnicity, we conducted a meta-analysis of the parameter estimates obtained for each ethnic group using a random effects model. With a sample size of 1,157, we had 80% power to detect a standard deviation difference in log adiponectin of 0.17  and a standard deviation difference in log HOMA-IR as small as 0.17 (absolute difference in log HOMA-IR of 0.105) for a MAF of 20%.Genotype and phenotype data were available from 1,186 participants from the SHARE and SHARE-AP population. From this sample, 29 individuals who had implausible levels of adiponectin (>100 µg/mL) were excluded, leaving a final sample of 1,157 participants .Table S2. The mean age of the overall population is 50.3 years. Women are less represented among the South Asians compared to Aboriginals (P = 0.02). Significant differences in age, health behaviours including current smoking, measures of adiposity, glycemic index and load, adiponectin and leptin concentration, and insulin resistance are present among ethnic groups (Table S2). For example, Aboriginal people have substantially higher BMI and abdominal obesity relative to other ethnic groups. Conversely people of Chinese origin have the lowest BMI and abdominal obesity. Europeans have a higher BMI, yet less abdominal obesity compared to South Asians. Despite these differences, South Asians and Aboriginal people are more insulin resistant compared to the Europeans . Adiponectin levels are significantly higher in Europeans and Aboriginals compared to the other ethnic groups . (Table S2).The characteristics of participants are displayed in To investigate the association between adiponectin level and metabolic parameters in this multiethnic sample, multivariable linear regression analyses were performed, adjusting for age, sex and ethnic origin. As shown in We assessed the association of the rs266729 polymorphism, under an additive model, with adiponectin level and metabolic parameters found to be associated with adiponectin in our study. The SNP is significantly associated with serum adiponectin level Table 2.The rs266729 polymorphism is also significantly associated with HDL cholesterol and HOMA-IR, after adjusting for age, sex, and ethnic origin Table 2.All other metabolic characteristics which are associated with adiponectin concentration  are not significantly associated with the rs266729 polymorphism.The association between the SNP rs266729 and HOMA-IR In order to examine whether a higher HOMA-IR  may be causally related to the rs266729 SNP determined lower adiponectin level, we performed a Mendelian randomization analysis. If serum adiponectin is causally related to increased insulin resistance, then we would expect that genetically reduced adiponectin  would increase insulin resistance to the same extent as observed in epidemiological studies. We present three main results: i) an estimated causal change in HOMA-IR for a genetically induced change in adiponectin concentration ; ii) a predicted increase in HOMA-IR based on the effect on adiponectin levels (column 6 in GCKR was strongly associated with HOMA-IR (P<0.001), but not with adiponectin level (P = 0.67). Consequently, we did not further explore reciprocal Mendelian randomization analyses using this SNP.Using instrumental variable analyses, we estimated a causal change in log HOMA-IR of –0.61  z-score units for a genetically induced change of one log adiponectin z-score unit. To examine whether the effect of the rs266729 SNP on HOMA-IR is mediated by the variation of adiponectin level, we added log adiponectin to the multivariable model shown in Column 7 of Table S3). To further test if the SNP effects are influenced by ethnicity, we conducted a meta-analysis of the parameter estimates obtained for each ethnic group. The association of adiponectin, HOMA-IR) and the rs266729 SNP were unaltered (Table S4).There is no significant heterogeneity in the gene effect on adiponectin or HOMA-IR variation across ethnic groups   the association of the rs266729 SNP with HOMA-IR is attenuated after adjusting for serum adiponectin level in the regression model, 4) the rs266729 SNP is associated with HOMA-IR to an extent that exceeds its effect on adiponectin level, and 5) the validity of this observation is strengthened because the functional SNP rs1260326 in GCKR while strongly associated with HOMA-IR, is not associated with adiponectin. Thus our findings do not support a reciprocal causal association of HOMA-IR on adiponectin level.By using both observational, genetic epidemiology and Mendelian randomization approaches in the multi-ethnic SHARE/SHARE-AP study we found that the adipocyte-secreted hormone adiponectin may have a causal role in the variation of insulin resistance (as measured by the HOMA-IR index). This study is the first to our knowledge to explore the relationships between adiponectin and metabolic traits using Mendelian randomization. Mendelian randomization is thought to overcome the limitations of classical observational epidemiology and provides useful evidence to support or reject causal hypotheses linking intermediary biomarkers and complex diseases ADIPOQ SNP rs266729, serum adiponectin level and insulin resistance are in line with previous reports A strong body of evidence in the literature supports the view that adiponectin may have a causal role in insulin resistance. Serum adiponectin level has been associated with insulin resistance in cross-sectional and longitudinal studies conducted in European ADIPOQ SNP rs266729 and ethnicity on variation of adiponectin or HOMA-IR. Most of the SNPs identified through GWAS are thought to be proxies in linkage disequilibrium with causal variants and may therefore not been informative in diverse ethnic populations ADIPOQ locus in linkage disequilibrium with the common SNPs. The R221S variant was unique to the Filipino population and was not found in sequences of 12,514 individuals of European ancestry We found no evidence for an interaction between the The data suggest that a consistent association between adiponectin and insulin sensitivity is observed in populations of European, South Asian, East Asian or Native Canadian ancestries. This result is in line with previous reports in SHARE/SHARE-AP and in other populations, since they did not provide consistent evidence of varying associations between adiponectin and metabolic traits like insulin resistance according to the ethnic background ADIPOQ promoter SNP rs266729 and HOMA-IR. One possible explanation is that the association found between adiponectin level and metabolic traits is not causal and explained by confounding factors for all traits but HOMA-IR. However, the modest sample size of the current study  leads us to not totally exclude the presence of subtle genetic effects of the SNP rs266729 on other metabolic traits that we were unable to detect. For instance, the absence of association between the SNP rs266729 and BMI may be unexpected, since insulin resistance is a key feature of obesity Despite the fact that we found a significant association between adiponectin level and HDL cholesterol, HOMA-IR, BMI, WHR, TG, fasting glucose level, fasting insulin level, HOMA-IR, systolic and diastolic blood pressure in our multi-ethnic observational study, we only found a convincing genetic association between the In conclusion, our data suggest a possible causal association between serum adiponectin level and insulin resistance as measured by HOMA-IR, irrespective of the ethnic background. More Mendelian randomization experiments are needed in well-powered multi-ethnic studies to decipher the complex pleiotropic role of the adipocyte-secreted hormone adiponectin on metabolic traits.Table S1Characteristics of the rs266729 SNP genotyped in Aboriginal, South Asian, Chinese and European participants.(DOC)Click here for additional data file.Table S2Characteristics of study participants by ethnicity.(DOC)Click here for additional data file.Table S3Association of metabolic traits with the rs266729 polymorphism. Values are beta-coefficient (95% CI). †(DOC)Click here for additional data file.Table S4Association of metabolic traits with the rs266729 polymorphism, based on meta-analysis of adjusted estimates obtained for each ethnic group. Values are based on fixed effect model. †(DOC)Click here for additional data file.


In 2006 the International Organization for Standardization (ISO) published Standard 15883 for Washer-disinfectors (WD), with Part 3 for human waste containers: what states that manual procedures must be avoided as much as possible. I conducted research to get insight in the awareness of ISO15883 and into bedpan management.Awareness rising for safe bedpan management.Conducting a Global survey (2010) of bedpan management, the awareness of ISO15883 and bedpans causative for HAI. I performed Planned and ‘secret shopper‘ visits in hospital sluice rooms (31) on all continents and had Experience exchange with colleagues (>250).Survey- Response:13% in 54 countriesClostridium difficile, Norovirus, MRSA,- HAI (4-21%) reported with: Pseudomonas aeruginosa, Salmonella species and Acinetobacter baumannii- - Majority never searched for bedpans as a potential source- Manual handling is done (65%)- ISO15883 is known in northwest Europe (76%) in the rest of the world (14-37%)Visits- Diapers and urine catheter as replacement for bedpans is regular done- Nurses, nurse aids, relatives and others do the job without training ‘how to’- Personal Protective Equipment is seldom used- Bedpans without covers are transported to a toilet or slobhopper- Manual empty of the content with (bloody) faeces causing splashes and splatters- Regular only ‘cleaning’ by rinsing or spraying with cold water- Disinfection without attention for the right product, duration and prior cleaning- Not well equipped sluice rooms- Macerators or WD are not maintained or validatedExperience exchangeNurses and physicians seldom look after the process and are glad that it is brought to their attention. Most WD or macerators are often malfunctioning or unused. No available guidelines for bedpan management.There is a risk for HAI caused by poorly performed bedpan management. The risk of handling bedpans can be easily ignored because the chosen practice depends on habits, rituals and budget. Manual handling contributes to the risk of environmental contamination and transmission. In case of using machines maintenance and validation must be regular checked. Hospitals have to recognize their weakness in bedpan management and must set improvements and investments.G. Van Knippenberg-Gordebeke Consultant for For MEIKO Germany


The ability of peripheral nerves to stretch and slide is thought to be of paramount importance to maintain ideal neural function. Excursion in peripheral nerves such as the tibial can be measured by analysis of ultrasound images. The aim of this study was to assess the degree of longitudinal tibial nerve excursion as the ankle moved from plantar flexion to dorsiflexion in a standardised weight-bearing position. The reliability of ultrasound imaging to measure tibial nerve excursion was also quantified.The tibial nerve was imaged over two separate sessions in sixteen asymptomatic participants in a weight-bearing position. Longitudinal nerve excursion was calculated from a three-second video loop captured by ultrasound imaging using frame-by-frame cross-correlation analysis. Intraclass correlation coefficients (ICC) with 95% confidence intervals (CI) were used to assess the intra-rater reliability. Standard error of the measurement (SEM) and smallest real difference (SRD) were calculated to assess measurement error.Mean nerve excursion was 2.99 mm SEM ± 0.22 mm. The SRD was 0.84 mm for session 1 and 0.66 mm for session 2. Intra-rater reliability was excellent with an ICC = 0.93.Assessment of real-time ultrasound images of the tibial nerve via frame-by-frame cross-correlation analysis is a reliable non-invasive technique to assess longitudinal nerve excursion. The relationship between foot posture and nerve excursion can be further investigated. During the gait cycle lower extremity motions such as ankle joint dorsiflexion and pronation of the foot will require the tibial nerve to adapt to positional change imposed by joint motions. To accommodate for positional joint change the tibial nerve possesses mechanical properties which enable it to withstand compression, adapt to repetitive force and stretch and slide in relation to the surrounding tissues . The abiin-vivo longitudinal tibial nerve excursion that can be considered normal in a non-pathological state. In-vitro methodologies have demonstrated that 5% - 10% elongation results in impaired blood flow . The examiner underwent 3 months of training under an experienced ultrasonagrapher prior to commencement of this project.B-mode real time ultrasound and colour imaging was performed using a Philips iU22 ™ ultrasound unit with a linear array transducer (17-5 MHz). Aquasonic 100All participants were instructed regarding measurement protocols and practised foot movements on the weight-bearing platform prior to measurement. Each participant was positioned on the weight-bearing measurement platform as demonstrated in Figure The tibial nerve was located by a transverse scan beginning approximately 1 cm superior to the medial malleolus. The transducer was then moved proximally until the neurovascular bundle was identified. A colour Doppler was used to confirm the location of the tibial artery and assist nerve location, as the tibial artery descends distally with the tibial nerve to enter the tarsal tunnel . The ultFor scanning sessions 1 and 2 three repetitive measurements were taken at one- minute intervals. There was a five- minute interval between scanning session 1 and 2. Between the scanning sessions the transducer was removed, repositioned and the nerve relocated by the examiner.The mean (SD) nerve excursion for each session was obtained by averaging three scans within each session.The video loop of longitudinal nerve excursion captured via ultrasound imaging was converted to digital format (bitmaps). The image size for each of the frames was 800 × 600 pixels. ImageJ  digitial image analysis software was used to calculate the image resolution and also scale conversion from pixels to millimetres. Each video loop was then analysed offline using a method of frame-by-frame cross-correlation analysis software developed in Mat-lab  by Dilley et al. . The sofThe software compares the grey-scale values from the ROIs between adjacent frames of the image sequence by a correlation coefficient calculation for each individual pixel shift. To reduce shift variability and vertical shifting the compared pixel measurements for the nerve were offset against (subtracted from) pixel shifts measurements within the same ultrasound field, from stationary structures such as subcutaneous layers and bone .All continuous data  were screened for normality through calculation of the Kolmogorov-Smirnov statistic. The mean (SD) was obtained for all continuous data. Intra-rater reliability analysis used Intraclass Correlation Coefficients  and 95% confidence intervals (CI) to quantify reproducibility regarding imaging of the nerve. Reliability findings were interpreted by arbitrary benchmarks initially proposed by Fleiss . The strt value of the distribution for a 95% CI (df = 15). The SRD percentage was calculated through dividing the SRD by the mean nerve excursion  or supinated [high-arched] foot type may have an influence on the mechanical functioning of the tibial nerve. We can speculate that foot pronation may have a pre-tensioning effect on the nervous system similar to that of the knee extension and hip flexion, potentially reducing the capacity of the tibial nerve to slide longitudinally. A pronated foot type has been associated with increases in pressure in the tarsal tunnel, creating the potential for a compression neuropathy . SpecifiAs indicated by the SRD there was a relatively high degree of error involved in the measurement of longitudinal nerve excursion. This error maybe explained by the limitations of ultrasound imaging technique employed. The success of ultrasound imaging is operator dependent with the placement of the probe in the exact same position and anatomical plane a potential source of sonographic artefact . The tibIn-vivo measurement of longitudinal tibial nerve excursion with frame-by-frame cross-correlation analysis of ultrasound images is a reliable technique, but this technique demonstrated a relatively large measurement error. Future work will include investigating the effect of foot posture on tibial nerve excursion and the assessment of tibial nerve excursion in lower limb conditions such as tarsal tunnel syndrome and plantar fasciitis, where the tibial nerve has been implicated in symptomology. Clinically the technique may also have applications in the assessment of musculoskeletal structures such as tendons allowing for the quantification of excursion in chronic conditions that affect the foot and lower leg such as rheumatoid arthritis and gout and specific tendon pathologies such as posterior tibialis tendon dysfunction.The authors declare that they have no competing interests.MC and KR designed the study. MC and JY collected and input the data. MC and JY conducted the statistical analysis. MC and KR compiled the data and MC, KR and WH drafted the manuscript. All authors read and approved the final manuscript.


Context integration is an ubiquitious principle in cortical processing underlying many perceptual and cognitive functions. Several neuropsychiatric disorders have been associated with an impairment of integration of context information, particularly schizophrenia . One wayWe investigated orientation-selective center-surround interactions in a network model of the cortical layer 4Cα, the input layer of V1 and the first cortical stage where CSI effects occur. The model is based on a previous one  and consWe found orientation and direction selectivity comparable to findings from neurophysiological studies,4 and thWe have presented a neuronal network model that showed non-specific and orientation-specific center-surround suppression in accordance with experimental and psychophysical data. Our model allows for investigating in detail the mechanisms supporting center-surround suppression and context-integration. This comprises changes of the perceptual input, characteristics of neurons and synapses as well as modulatory influences of GABA, for instance. Finally, our model is suited to systematically model the neural subtsrates of context integration impairments in neuropsychiatric disorders.


Cardiac allograft vasculopathy (CAV) is the principal cause of long-term graft failure following heart transplantation. Early identification of patients at risk of CAV is essential to target invasive follow-up procedures more effectively and to establish appropriate therapies. We evaluated the prognostic value of the first heart biopsy (median: 9 days post-transplant) versus all biopsies obtained within the first three months for the prediction of CAV and graft failure due to CAV.In a prospective cohort study, we developed multivariate regression models evaluating markers of atherothrombosis  and endothelial activation (intercellular adhesion molecule-1) in serial biopsies obtained during the first three months post-transplantation from 172 patients . Presence of fibrin was the dominant predictor in first-biopsy models  and loss of tPA was predominant in three-month models . First-biopsy and three-month models had similar predictive and discriminative accuracy and were comparable in their capacities to correctly classify patient outcomes, with the exception of 10-year graft failure due to CAV in which the three-month model was more predictive. Both models had particularly high negative predictive values .Patients with absence of fibrin in the first biopsy and persistence of normal tPA in subsequent biopsies rarely develop CAV or graft failure during the next 10 years and potentially could be monitored less invasively. Presence of early risk markers in the transplanted heart may be secondary to ischemia/reperfusion injury, a potentially modifiable factor. Modern immunosuppressive regimens have reduced the incidence of acute rejection and extended early survival following heart transplantation but have done little to reduce the incidence of cardiac allograft vasculopathy (CAV), the principal long-term cause of graft failure. CAV, an aggressive form of atherosclerosis that develops within months to a few years after transplantation, accounts for 30% of all deaths first biopsy, obtained 7–12 days post-transplant. Thus, the aim of this study was to determine whether very early data from a single biopsy are sufficient to identify low-risk patients. Our analysis showed that patients with absence of fibrin in the first biopsy rarely develop CAV or graft failure during the next 10 years. Furthermore, the high negative predictive value of the first-biopsy was comparable to that of multiple biopsies obtained over three-months, implying that patients with negative findings in the first biopsy potentially could be monitored less invasively, thereby, avoiding the risk and expense of multiple heart biopsy procedures.The Invasive Monitoring Attenuation through Gene Expression (IMAGE) trial recently showed that patients at low risk of rejection can be monitored safely with noninvasive gene-expression profiling Consecutive adult heart-transplant recipients transplanted from August 1989 to August 2004 and followed prospectively until September 2010, were candidates for study. Patients (n = 172) were included if they survived at least three months post-transplantation, had serial endomyocardial biopsies performed in the first three months, and had their coronary arteries examined angiographically and/or histopathologically for CAV at annual follow-ups. Of 241 candidates, 29 patients were excluded because they had missing three-month biopsy data, either because they died prior to three months (n = 14) or because they were transplanted at another institution (n = 15); 38 survived three-months but were excluded because they had incomplete biopsy data; and two survived but were excluded because of missing follow-up coronary evaluations. The study protocol was approved by the Indiana University local Institutional Review Board and all subjects signed a consent form.All patients received triple-drug immunosuppression Baseline (time-zero) endomyocardial biopsies were performed on all of the 172 donor hearts at the time of transplantation but before reperfusion. Additional biopsies were performed serially during the first three months after transplantation, with the first post-transplant biopsy obtained within a median 9 days of transplantation.Cytomegalovirus disease was defined during follow-up by clinical symptoms and by cytopathologic-tissue culture evidence of invasion. Cytomegalovirus prophylaxis with gancyclovir was used in seronegative recipients of seropositive donors.CAV was evaluated in side-by-side comparisons of identical projections of serial angiograms performed annually (Mean: 5.25±1.0/patient) and diagnosed by evidence of coronary artery narrowing or luminal irregularities either in left main or any primary or branch vessels. CAV was considered severe if left main stenosis was >70%, if two or more primary vessels had stenoses >70%, or if branch stenoses were >70% in all three systems 2 fragments served as secondary antibodies . Arteries were identified with fluorescein-labeled mouse monoclonal antibody to human smooth muscle α-actin .Endomyocardial biopsies were tested immunohistochemically for fibrin ; tissue plasminogen activator ; antithrombin  and intercellular adhesion molecule-1 . Affinity-purified, fluorochrome-labeled or peroxidase-labeled polymer conjugated anti-mouse or fluorochrome-labeled anti-rabbit F(ab')For immunofluorescence studies, tissue samples were embedded in optimum cutting temperature compound , snap-frozen in liquid nitrogen and stored at −75°C. Cryostat sections (4 µm) were air-dried overnight without fixation, and immunostained. Rhodamine-conjugated anti-mouse and Alexa Fluor 488 anti-rabbit or anti-mouse were used as secondary antibodies.For immunoperoxidase studies, slides from paraffin blocks were antigen-retrieved using DAKO Target Retrieval solution (pH 6.0) to expose antigens masked by formalin. Endogenous biotin was blocked with avidin/biotin blocking system (DAKO) and endogenous peroxidase with 3% hydrogen peroxide. Fibrin antibody was applied for 60 minutes at room temperature. Slides were developed using DAKO's EnVision+ Dual Link, HRP kit for mouse primary antibodies in a DAKO Autostainer. Immunohistochemical data were evaluated by two investigators unaware of clinical outcome.proportion of abnormal signs for each marker . Proportions were re-scaled by a factor of 10 so that regression coefficients could be interpreted as a 10% change in the proportion of abnormal biopsies, a change in approximately one biopsy from normal to abnormal for the typical patient. Four predictors were tested representing the presence or absence of abnormality in the four markers .Immunohistochemical data were scored as described by Labarrere et al Univariate logistic regression models were estimated using each biomarker in turn as the sole predictor in the equation. Multivariate stepwise models were then developed in two stages. In stage-one, statistically significant biomarkers were identified by stepwise backward elimination to establish base models. In stage-two, clinical and laboratory covariates shown in Using the method of Austin and Tu The Youden Index calculated from receiver operating characteristic (ROC) curves were used to identify optimum cut-off values for the models Predicted values from ten-year models were stratified into three groupings: (1) LOW RISK (lower 25% of risk distribution); (2) MODERATE RISK (middle 50%) and (3) HIGH RISK (upper 25%) and separate Kaplan-Meier curves were constructed for these risk groups.Demographic and clinical characteristics of the patient population assessed at three months post-transplant are shown in When evaluated in univariate regression models , individIn final multivariate models , 4 and 5The odds ratio (OR) in first-biopsy models represents the multiplicative increase in risk associated with the presence of an abnormal marker. In three-month models the OR is the multiplicative increase in risk associated with a 10% increase in the proportion of abnormal biopsy results . As an example, the three-month model predicting graft failure due to CAV at 10 years, which shows an OR for tPA of 1.31 , indicatPerformance characteristics for all models are summarized in Thus, the earliest information available from a single biopsy is sufficient to identify a subgroup of patients with very low odds of long-term (10-year) graft failure due to CAV.Positive predictive values for both first-biopsy and three-month models tended to be low, with the lowest values associated with one-year predictions. By contrast, negative predictive values were high for both sets of models. For example, in first-biopsy models patients with no evidence of fibrin deposition had a 99% chance of avoiding graft failure due to CAV at one year, and continued to have a 98% and 96% chance of being risk-free at five- and 10-years . Similartime to event.Kaplan-Meier curves using risk-stratified predictive values from 10-year regression models  showed ssubstantially reduced risk of developing long-term CAV and graft failure.Prediction models using information derived from a single endomyocardial biopsy obtained within a median 9 days post-transplant accurately identified heart transplant patients with unnecessary in patients with no evidence of fibrin deposits in the first biopsy. Of course, patients that are not identified as low-risk would still need to be followed with definitive monitoring. A potential strategy would be to follow low-risk patients non-invasively with gene expression profiling, as has been suggested for the monitoring of patients with low risk for rejection The high negative predictive accuracies of our models have important clinical implications. First, models that used only the first biopsy had negative predictive values comparable to models that used all biopsies available at three months, confirming our hypothesis that a single early biopsy is sufficient to identify patients with very low risk of long-term (10-year) graft failure due to CAV. This finding implies that it may be possible to reduce the number of follow-up biopsies and coronary angiographies for low-risk patients, provided there is continued absence of symptoms or signs of rejection. Moreover, multiple biopsies during the first three months may be positive predictive accuracies of our models were not as strong as the negative predictive accuracies, suggesting that it is harder to identify high-risk than low-risk patients. A likely explanation is that patients with evolving disease are eventually identified and treated preventively during the 10-year follow-up interval, thereby reducing the odds initially predicted based on prior three-month data.The independent predictors in most multivariate models, suggesting that the underlying biological process upon which statistical prediction is based evolves over time during the first three months following transplantation. We conclude that early absence of microthrombosis and continued persistence of an intact fibrinolytic system are indicative of a protective phenotype against long-term CAV and allograft failure.Absence of myocardial fibrin in the first biopsy and persistence of microvascular tPA in all biopsies obtained during the first three months emerged as the best severe CAV and graft failure due to CAV. If microvascular fibrin continues to be deposited due to the failure of antithrombin to prevent it, the patient will still retain some capacity to remove it as long as there is a sustained presence of tPA. However, if there is also loss of tPA, fibrinolytic capacity will be diminished, fibrin deposition will continue unabated, and the patient's status will worsen. Thus, it is reasonable that three-month models would pinpoint loss of tPA as the single best independent predictor of long-term CAV and graft failure.Lack of antithrombin typifies a system that is failing to prevent microvascular fibrin deposits. Thus, it is reasonable that loss of antithrombin in the first-biopsy model would turn out to be the best early predictor of CAV, and that the subsequent unchecked accumulation of microvascular fibrin, secondary to the loss of antithrombin, would be the best early predictor of In first-biopsy models it is noteworthy that ICAM-1 expression, a marker of endothelial activation, appears with fibrin as a co-predictor of severe CAV and graft failure at one year, indicating that endothelial activation in the presence of microvascular fibrin further heightens the odds of very early and very serious CAV. This is consistent with observations from a transient cerebral artery occlusion model showing that concomitant reduction of both ICAM-1 expression and microvascular fibrin significantly reduced brain injury and improved post-ischemic blood flow sequence in which these markers emerge during the immediate post-transplant period has not been well described and the prognostic significance of the information contained in the first biopsy has been underappreciated.The importance of early microthrombosis, reduced fibrinolysis and microvascular arterial endothelial activation for CAV and graft failure has been previously demonstrated and before reperfusion further confirms that this phenotype developed after the graft was placed into the recipient. These findings rule out the possibility that our risk predictors are a consequence of brain trauma to the donor or damage to the donor heart occurring during the harvesting procedure. Likewise, they increase the likelihood that abnormal markers detected in time-one biopsies are related to I/R occurring immediately after transplantation. I/R appear to damage endothelium by reducing anticoagulation, increasing thrombogenicity and promoting vascular inflammation and microthrombus formation leading to microinfarctions. Massberg et al 15–42 (FX06) was shown to significantly attenuate I/R injury in a heart transplant model with extended cold ischemia by reducing infiltrating leukocytes The very early appearance of myocardial fibrin suggests that ischemia-reperfusion (I/R) may be a trigger of coagulation activation. The absence of a prothrombotic microvasculature in all time-zero biopsies performed at the time of transplant Considering the emergence of antibody-mediated rejection (AMR) as a model of microcirculation injury and endothelial activation negative predictive accuracy, it would be especially important to know whether the positive predictive accuracy of our models could be significantly improved by adding AMR-related factors.From a prognostic perspective, it is important to ascertain how much predictive information is actually added by incorporating a new marker into the best current model, since new markers may contain little or no additional information not already conveyed by existing factors optimally combined Our data suggest that graft failure may depend upon the extent of very early post-transplant microvascular damage and the capacity of the transplanted heart to remove microthrombi through active fibrinolysis. Thus, therapies designed to de-escalate hypercoagulability may be most effective if applied during the pre- to peri- and early post-operative periods.Our study has strengths and weaknesses. Weaknesses include the utilization of angiography rather than intravascular ultrasound The most important clinical message that emerges from our data is that first-biopsy models are comparable to three-month models as evidenced by their similar capacities to classify patients correctly and to single out patients at low risk of CAV and graft failure. The only exception was the superior performance of the three-month model to correctly classify patients with respect to 10-year graft failure due to CAV. The high negative predictive accuracies associated with both, first-biopsy and three-month models are especially noteworthy. Absence of a prothrombotic microvasculature, even when observed as early as 9 days post-transplant in a single biopsy, identifies patients that rarely develop CAV and graft failure. These very low-risk patients are unlikely to derive benefit from further invasive monitoring. Since repeated heart biopsy procedures are both risky and expensive, our findings have implications for both patients and payers. Of course, patients that do not fall within this low-risk group should continue to be followed under standard protocols using more definitive (invasive) monitoring.Although our findings show that it is possible, using markers that are available within days of the transplant procedure, to identify a subgroup of patients that almost never develops long-term CAV or graft failure, they do not show what the impact on patient outcomes would be if physicians used our prediction models as a clinical tool to manage their transplant patients. Clinical impact can only be demonstrated in a prospective outcome trial in which some patients are randomly assigned to receive usual care and others to a protocol that uses our models to guide treatment decisions.


Attention Deficit/Hyperactivity Disorder (ADHD) is a common neuropsychiatric disorder that has not been well studied in older adults. In this study we examined relationships between ADHD symptoms and cognitive ability and compared them between middle-age  and older-age  adults sampled from the same population. ADHD, mood disorder symptoms and cognitive abilities were assessed in a large population-based sample . We measured current ADHD symptoms using the adult ADHD Self-Report Scale (ASRS), which we found to have the same underlying structure in both cohorts. Older adults reported significantly lower levels of ADHD symptoms and 2.2% of the OA cohort scored equal or above the ASRS cut-off score of 14 (which has been previously associated with ADHD diagnosis) compared with 6.2% of MA adults. Symptom levels were not significantly different between males and females. Using multi-group structural equation modelling we compared ADHD symptom–cognitive performance relationships between the two age groups. Generally higher ADHD symptoms were associated with poorer cognitive performance in the MA cohort. However, higher levels of inattention symptoms were associated with better verbal ability in both cohorts. Surprisingly, greater hyperactivity was associated with better task-switching abilities in older adults. In the OA cohort ADHD symptom–cognition relationships are indirect, mediated largely through the strong association between depression symptoms and cognition. Our results suggest that ADHD symptoms decrease with age and that their relationships with co-occurring mood disorders and cognitive performance also change. Although symptoms of depression are lower in older adults, they are much stronger predictors of cognitive performance and likely mediate the effect of ADHD symptoms on cognition in this age group. These results highlight the need for age-appropriate diagnosis and treatment of comorbid ADHD and mood disorders. Attention Deficit/Hyperactivity Disorder (ADHD) is a neuropsychiatric disorder that develops in childhood and can persist throughout life. It is characterised by age-inappropriate levels of inattention and/or excessive motor activity and impulsivity th ed; DSM-5; ADHD is a common disorder affecting ∼5% of children and 1–7% of adults Although ADHD remains a categorically distinct clinical condition, there is increasing recognition that clinical ADHD lies at the extreme ends of the distribution of continuous ADHD symptom dimensions. Furthermore, symptoms of ADHD are common and can lead to functional impairment in adults who do not meet all the criteria for clinical diagnosis Cognitive deficits have been consistently identified in both children and adults with ADHD In this study we extend our analyses of ADHD symptoms to a cohort of older adults (68–72 years) in the Personality and Total Health (PATH) Through Life Study, a population-based longitudinal study of mental health and ageing The study sample was drawn from the PATH Through Life Project, a large longitudinal study of mental health and ageing in participants across three age groups  with a four-yearly follow-up ‘waves’ of assessment for up to 20 years p>0.05) from responders with respect to gender, total years of education and the levels of anxiety or depression symptoms in either cohort. Participants meeting at least one of the following criteria – history of epilepsy, brain tumour/infection and stroke and with missing data were excluded. Mini Mental State Examination (MMSE) was included in the assessment of the OA cohort to screen for probable dementia. Participants with scores of 23 or less (which is indicative of probable dementia The present study used data from the third wave of assessment (the ASRS was introduced in this wave) of the MA (40–44 years at baseline) and OA (60–64 years at baseline) cohorts. There were 2182 participants in MA cohort aged 48–52 years (mean age 50.7±1.5 years) and 1973 individuals in the OA cohort aged 68–72 years (mean age 70.6±1.5 years). Twenty-six (1%) and fifty-three participants (2.8%) in the MA and OA cohorts respectively did not complete the ASRS questionnaire and were excluded from further analysis. Being older was associated with increased odds of not completing the questionnaire in the OA cohort. However, non-responders did not differ significantly . Variables for panic disorder and other anxiety syndromes were combined to generate a binary categorical variable for anxiety symptoms .Depression and anxiety symptoms were assessed using the Patient Health Questionnaire (PHQ). This is a short version of the patient questionnaire component of the Primary Care Evaluation of Mental Disorders (PRIME-MD) instrument Cognitive data were collected from participants as described previously All statistical analyses were conducted using SPSS 18 and Amos version 20 (Chicago: SPSS Inc.). Missing data for cognitive measures were imputed using Expectation-Maximization method in SPSS. Means and standard deviations were computed for the ASRS-6 and cognitive test scores. As recommended We used multi-group structural equation modelling to test cross-group invariance of the ASRS factor structure (Aim 1) and compare ADHD symptom–cognition relationships in MA and OA groups (Aim 2). First, cross-group invariance of the ASRS factor structure was tested using latent factor SEM in the framework of confirmatory factor analysis Inatt ; Model 5b – FL, FC and variance of Hyperact (VHyperact); and Model 6: FL, FC and residual variance (RV) of the ASRS items. Since the more constrained models were nested within the initial model, the Likelihood Ratio Test (difference in Chi-square statistics relative to the change in degrees of freedom) along with other goodness-of-fit indices (see above) was used to select the more parsimonious model.Analysis of ASRS factor structure: Previous factor analysis has established that ASRS measures two correlated constructs, Inattention and Hyperactivity Inatt, Hyperact, sociodemographic variables and mood-disorder symptoms as predictors. A schematic representation of our theoretical model is shown in Inatt and Hyperact and covariance between the factors were constrained to be equal across both groups. All other parameters were estimated freely. To avoid multi-collinearity we used separate models for cognitive variables  that were highly correlated  removed to improve the overall parsimony of the models. Only the final, most-parsimonious model and the coefficients of significant paths are reported. Results were considered significant at p≤0.05.Analysis of ADHD symptom–cognition relationships: SEMs generated for assessing the effect of ASRS latent factors on cognitive performance included cognitive test scores as dependent variables and We also analysed the factor structure of the cognitive tests used in the study. The factor structure that represents the best fit to both cohorts are shown in http://crahw.anu.edu.au/research/projects/personality-total-health-path-through-life/data). The OA cohort reported significantly lower levels for ADHD, depression and anxiety. For the ASRS, 2.2% of participants in the OA cohort scored 14 or above, which has previously been identified as indicative of ADHD diagnosis Demographic characteristics, symptom measures for ADHD, depression and anxiety, and cognitive test scores for both cohorts are presented in Inatt+Hyperact) ASRS structure in both cohorts individually . Significant paths (p≤0.01) in the final model are shown in Using multi-group SEMs we investigated the effects of ADHD symptoms on cognitive performance, while controlling for sociodemographic variables and mood disorder symptoms . The ASRInatt and Hyperact have limited direct effects on cognitive performance. The most notable was a positive association, observed in both cohorts, between Inatt and Spot-the-Word Test, implying that higher levels of inattention symptoms are associated with greater verbal intelligence. Inatt was also positively associated with Choice Reaction Time, i.e., with slower reaction time in the MA cohort. Hyperact was positively associated with the Delayed Recall test (i.e. poor performance) on the MA cohort and Trail Making Test (Part B) (i.e. better performance) in the OA cohort. Inatt was associated with depression and anxiety symptoms more strongly in the MA than the OA cohort.We also investigated the association between ADHD symptoms and latent variables obtained from factor analysis of the cognitive test scores. Factor analysis identified four latent factors  broadly Inatt only in the MA cohort. It had an effect on the Trail Making Test (Part A) in the OA cohort and had no effect on any of the cognitive variables in the MA cohort. Effects of age on the cognitive variables were similar across both cohorts although the effects were stronger in the OA cohort. A point of contrast was the significant effect of age on Delayed Recall test on OA cohort that was absent in the MA cohort. Age was marginally associated with Hyperact in older adults only. Education had negative effects on mood variables in both cohorts although the effect on anxiety was much stronger in the OA cohort. The effect of education on Inatt was positive in both cohorts and comparatively stronger in the MA cohort, implying that more education is associated with higher levels of inattention. Greater levels of depression symptoms were associated with poorer cognitive performance generally, but the effect is much stronger in the OA cohort. These results did not differ significantly when latent factors underlying the cognition tests were included as dependent variables in the SEM models  older adults reported fewer/less severe ADHD symptoms compared to middle-aged adults; ii) the effect of ADHD symptoms on cognitive performance was weaker in older adults compared to middle-aged adults; iii) in OA adults the ADHD symptom–cognition relationship was mostly indirect and mediated through the strong association between depression symptoms and cognition.We found that 2.2% of the OA cohort met the suggested cut-off for the ASRS-6 score that has been linked to clinical diagnosis in previous studies. This estimate falls within the range of 1–2.5% ADHD prevalence reported in the literature for older adults There may be several reasons for this apparent decline in ADHD symptom levels in old age. The effects of ADHD may become less discernable from other general age-related changes. Thus, even though, as we have demonstrated here, the ASRS appears to measure variation in the same psychological construct in old age, it may do so with lower sensitivity. The relative frequency of people with ADHD in the population will also tend to decline in old age because they have a relatively lower life expectancy We did not find any significant gender difference in ADHD symptoms in older adults. This result is consistent with our previous observation Inatt and Hyperact were more strongly correlated in MA than OA adults, however, this difference was not statistically significant. In a previous study of two samples of different age We replicated the factor structure of the ASRS reported by Hesse et al. (2013) Inatt, in contrast with the MA cohort, suggesting that information processing speed is less affected by symptoms of inattention in older adults.In both MA and OA cohorts the Spot-the-Word Test, which measures verbal ability, was positively associated with symptoms of inattention, an effect that we previously reported for the MA cohort p<0.01. This might suggest that hyperactivity symptoms in this age-group are beneficial for certain cognitive activities. It is possible that hyperactivity symptoms in ageing brains make the system noisier at the neuronal level. While noise is generally considered detrimental, under certain circumstances it may aid in processing of weak stimuli through the phenomenon of stochastic resonance Surprisingly, in the OA cohort, greater hyperactivity was associated with better task-switching abilities as measured by the Trail Making Test (Part B). However, this association did not reach the more stringent significance threshold of Our results suggest that the effects of ADHD symptoms are specific to particular cognitive domains, in contrast to a previous study by Biederman et al (2011) Inatt was strongly associated with depression symptoms in both cohorts, and depression symptoms predict worse performance in all cognitive tests in the OA cohort but not in the MA cohort. We also found evidence of statistical mediation of Inatt on Symbol-Digit Modalities Test by depression in OA adults since the negative association between Inatt and Symbol-Digit Modalities Test became non-significant when depression symptoms were included in the model. Depression symptoms were also significantly associated with Symbol-Digit Modalities Test in OA adults. Thus, our results emphasise the importance of controlling for comorbid mood disorders when investigating ADHD symptom–cognition relationship, especially in older adults.Our results suggest that inattention symptoms affect cognitive performance indirectly through their effect on depression in the OA cohort. The strengths of this study are that it was conducted in a large representative population sample, and hence it is likely to be free of the biases that can be associated with clinical and convenience samples. Furthermore, the narrow age range of the sample removes the possible confounding effects of age within each cohort. Consequently, our results are likely to be generalisable to MA and OA populations. Our study included age groups that have not been well studied with respect to ADHD symptoms and thus is a significant addition to the growing literature in this area. We focus on cognitive performance and ADHD symptoms highlighting differences between middle- and old-age, which is important for understanding age-related cognitive change. Finally, we have modelled both direct ADHD symptom-cognition relationships and indirect effects mediated by symptoms of depression, demonstrating age-group differences in these direct and indirect pathways.Limitations of the study include lack of clinical assessments for ADHD, depression and anxiety. In addition, symptom measures are based on self-reports, which may not be completely accurate  and OA (B) cohorts. Standardised factor loadings are shown. IR: Immediate Recall; DR: Delayed Recall; SRT: Simple Reaction Time; CRT: Choice Reaction Time; TMT-A: Trail Making Test A; TMT-B: Trail Making Test B; SDMT: Symbol-Digit Modalities Test; STW: Spot The Word test; DSB: Digit Span Backwards.(TIF)Click here for additional data file.Figure S2p<0.05 are shown. Arrows reflect direction of relationships between variables. Standardised regression coefficients are shown. gen: gender; educ: education; Inatt: latent factor Inattention; Hyperact: latent factor Hyperactivity; DEP: depression symptom measure; ANX: anxiety symptom measure. Indicators of the latent variables are not shown for the sake of clarity.Final model for ADHD symptom–cognition analyses for MA (A) and OA (B) cohorts. Only paths significant at (TIF)Click here for additional data file.File S1Tables S1–S5.(DOCX)Click here for additional data file.


Glutathione (GSH), a sulfhydryl-containing tripeptide present in most organisms at millimolar levels, plays a crucial role in redox homeostasis.35S-cysteine and subsequent phosphor imaging on the gel obtained from 2D PAGE.A conventional method to detect PSSG relies on the metabolic labeling of endogenous GSH with radioactive E. coli glutathionylspermidine synthetase  and The enzyme GspS catalyzes the amide bond formation between GSH and spermidine to generate glutathionylspermidine , a unique GSH derivative found only in a few Gram-negative bacteria and protozoa.2 group of spermidine is near the surface and freely available for additional chemical derivatization. Computational modeling suggested that the biotin moiety of biotin-spm likely protrudes into the solvent and does not participate in substrate binding . We therefore designed and synthesized biotin-spm as the probe to tag GSH (Scheme 1 in the Supporting Information). The Michaelis–Menten kinetics parameters indicate that biotin-spm  is comparable to the native substrate spermidine .The DNA fragment of the GspS, corresponding to the amino acid sequence of the Gsp synthetase domain, was incorporated to the pCMV2B vector to construct pCMV2B-GspS. The introduction of the vector pCMV2B-GspS into human embryonic kidney (HEK) 293T cells allowed expression of the enzyme GspS and the subsequent conversion of endogenous GSH to Gsp in vivo  for 24 h and then with H2O2 (1 mm) for five minutes. Iodoacetamide  was used to cap free thiols that may otherwise be mistakenly identified in later analysis. Acetone was added to precipitate proteins and to remove unreacted biotin-spm and Gspm-biotin. The precipitate was dissolved with trifluoroethanol and digested with trypsin for 20 h. Gspm-biotin S-thiolated peptides were captured by streptavidin-conjugated resin. After several washes and elution, these target peptides were treated with Gsp amidase  to generate glutathionylated peptides for LTQ-Obitrap LC–MS/MS analysis ,We identified 1409 unique glutathionylated cysteine residues from 913 proteins in 293T cells in each of two independent experiments (Data 1 in the Supporting Information). Among them, 49 proteins were identified in previous proteomics studies (highlighted in Data 1 in the Supporting Information), thus suggesting that those proteins may be sensitive to the redox environment in various cell types. However, for these 49 matches no site information was obtained in earlier studies, owing to the methods used . Some of the proteins, including glutathione S-transferase pi (GSTπ) and sarco/endoplasmic reticulum CaIn addition to the proteins involved in redox regulation, we identified numerous glutathionylated proteins that are involved in various cellular activities, for example signal transduction (cAMP-dependent protein kinase), cell cycle (E2 ubiquitin ligase/E3 ubiquitin hydrolase), energy metabolism , and cytoskeleton assembly (actin). These results support the idea that PSSG likely affects various protein functions.Although glutathionylated proteins have been identified in various cell types, none of the available methods are able to detect the attached GSH to provide definitive evidence of site-specific glutathionylation.Our strategy reported herein is the first to label the endogenous GSH with an engineered tag to allow sensitive detection as well as selective purification of glutathionylated proteins. Since spm-biotin can be independently added to the cell culture expressing the enzyme GspS, our approach requires no external dosing of GSH derivatives that may alter the cellular thiol content and the GSH/GSSG ratio. Moreover, the use of Gspm-biotin also requires no reduction of oxidized cysteines, such as sulfenic acid and nitrosocysteine, otherwise necessary for biotin switches. Therefore, our approach is complementary to the recent approaches to dissect the process of PSSG under physiological condition, as well as to decode the constitutive S-glutathionylation Figure .E. coli GspS into mammalian cells for labeling intracellular GSH. By combining enrichment and MS analysis, our method enables to site-specifically pinpoint S-thiolated residues. The quantitative measurement, currently in progress, will further expand the utility of our methods to better define the functional consequences of PSSG.In summary, we developed an efficient chemo-enzymatic approach for probing glutathionylated proteins by introducing


Cancer is a multifaceted molecular disorder that is modulated by a combination of genetic, metabolic and signal transduction aberrations, which severely impair the normal homeostasis of cell growth and death. Accumulating findings highlight the fact that different genetic alterations, such as mutations in tumor suppressor genes, might be related to distinct and differential sensitivity to targeted therapies. It is becoming increasingly apparent that a multipronged approach that addresses genetic milieu  eventually determines the response of individual tumors to therapy. Cancerous cells often acquire the ability to evade death by attenuating cell death pathways that normally function to eliminate damaged and harmful cells. Therefore impaired cell death nanomachinery and withdrawal of death receptors from cell surface are some of major determinants for the development of chemotherapeutic resistance encountered during treatment. It is therefore essential to emphasize underlying factors which predispose cells to refractoriness against TRAIL mediated cell death pathway and the relevant regulatory components involved. We bring to limelight the strategies to re-sensitize TRAIL resistant cells via vitamins to induce apoptosis. TRAIL plays an imperative role in host immunosurveillance in opposition to tumor progression, as it triggers apoptosis of tumor cells but not normal cells, and therefore has great therapeutic potential for cancer treatment. It is a well established fact that TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy  receptors. Various reviews have been written highlighting role of TRAIL mediated signaling cascade in prostate cancer . EscalatConfluence of information suggests that underpinnings of refractoriness to TRAIL induced apoptosis in malignant cells and normal cells is not entirely understood. However substantial fraction of data has been added into the existing web of knowledge with reference to variety of factors that are present in the tumor microenvironment and can influence the response of malignant cells to TRAIL. An emerging paradigm in cancer chemoprevention is use of natural or biological substances to reverse, repress or counteract either the initial phase of carcinogenesis or the progression of neoplastic cells to cancer. This review provides a summary of the impact of vitamins on mechanisms connected with enhancement of TRAIL mediated signaling together with modulation of the apoptotic  families) as well as cell signal transduction cascades.It is becoming gradually more apparent that all trans retinoic acid (ATRA) restricted phosphorylation of FOXO3A in acute promyelocytic leukemia (APL) cells. The reduction in phosphorylation of FOXO3A resulted in its re-distribution by consequent shuttling into the nucleus to stimulate the expression of TRAIL . DetailePromoter mapping, gel retardation and chromatin immunoprecipitation assays unraveled the detail that retinoids induced the expression of TRAIL mainly through crosstalk with NF-kappaB. However there is a differential mode of action in combination with etoposide, camptothecin or doxorubicin as it antagonized the apoptosis triggered by the chemotherapeutic drugs. Discordantly, apoptosis induced by TRAIL was not antagonized by retinoids .Accumulating evidence suggests that that a combination treatment with all-trans retinoic acid (ATRA) and TGF-beta1 leads to remarkable ATRA-induced suppression of cell proliferation, which is simultaneously accompanied by repression of ATRA-induced apoptosis in human leukemia HL-60 cells. Henceforth TGF signal transduction cascade resulted in dampening apoptosis inducing activity of ATRA by enhancing expression of c-FLIP(L) protein .It is worth mentioning that retinoic acid (RA) has antileukemic activity and up-regulates the expression of TRAIL and its DR4 and DR5 receptors. In a RA-sensitive cell line PLB985, targeted inhibition of TRAIL blocked RA-induced apoptosis [It is intriguing to note that in the leukemic blasts of acute promyelocytic leukemia patients, retinoic-acid-induced expression of TRAIL caused blast apoptosis. Therefore, initiation of TRAIL-mediated death signaling is contributory to the therapeutic value of retinoids ,10. CompThe antioxidant, vitamin C stimulated perifosine -induced expression of DR4 and DR5 in head and neck squamous cell carcinoma cells and inhibited the growth of xenografts . ControvConsiderable verification suggests that pretreatment with 1,25-dihydroxyvitamin D3 repressed apoptosis induced by TRAIL and Fas ligand by up-regulating decoy receptor and death receptor 4 and repressing Fas. However abrogation of death receptor 4 or enforced expression of Fas attenuated the suppressive effect of 1,25-dihydroxyvitamin . On a paIt has recently been explored that TRAIL mediated apoptosis is impaired by IL-1beta release from macrophages. Contrarily, Vitamin D(3) interfered with the release of IL-1beta from macrophages thus re-sensitizing tumor cells to TRAIL-induced apoptosis . Center An enhanced appreciation of the molecular biology of cancer cell growth and survival and the role of the microenvironment in supporting the survival of cancer cells in future will result in identification of additional distinct roles of vitamin D in regulating cellular activity.It is perceptible that α-TOS and hrTRAIL in combination act with remarkable synergy to induce apoptosis in breast cancer cells. Moreover, α-TOS repressed the expression of FLIP and c-IAP1 in erbB2-positive cells .In agreement with the same conception, Vitamin E derivative RRR-α-tocopherol ether-linked acetic acid analog (α-TEA) stimulated the expression of TRAIL and DR5and considerably repressed levels of antiapoptotic factor, c-FLIP L ,19. FurtAnother interesting finding suggests that Tocotrienol (T3), an unsaturated vitamin E but not tocopherol, induced transcriptional up-regulation of the death receptor (DR)-4 and DR5 via enhanced production of reactive oxygen species and augmented activation of ERK1 . AnalogoIt is becoming progressively more appreciable that alpha-tocopheryl succinate  re-sensitizes TRAIL-resistant malignant mesothelioma (MM) cells to TRAIL-induced apoptosis. This transformation of TRAIL refractory (MM) cells to TRAIL sensitive (MM) cells occurs in a p53-dependent manner as p53competent MM cells are responsive to sensitization however, p53deficient counterparts are insensitive . FurtherMounting data suggests that tocotrienol-induced caspase-8 activation and apoptosis in malignant (+)SA mammary epithelial cells is irrespective of the activation of death receptors and is dependent on suppression of the PI3K/PDK/Akt signal transduction cascade and succeeding repression in FLIP expression .It is becoming more and more evident that exposure of the cells to TRAIL results in transient activation of NF-kappaB, a process that is suppressed by cell pretreatment with alpha-TOS. alpha-TOS re-sensitizes cells to TRAIL killing, through repression of NF-kappaB activation .It is worth mentioning that alpha-TOS is nontoxic to normal cells however simultaneously triggers apoptosis in p53 deficient and p21deficient cancer cells .It is contemplating to note that multiple gene variants that have small to moderate individual phenotypic effects contribute to the overall progression of cancer. A particular individual's level of cancer susceptibility and recurrence depends on interactions between environmental factors and a wide range of modifier genes, which systematically impact derailed cellular activities and resistance against a multiple therapeutic interventions especially TRAIL. The latest genome-wide association studies in large cohorts of patients with different cancers provided new insights into the pathophysiology of these molecular disorders and have suggested contribution of previously unsuspected molecular signaling pathways. Additionally, model studies have identified supplementary susceptibility genes for inducing resistance against TRAIL mediated apoptosis. We therefore developed a roadmap of various signaling pathways which were contributory in driving or suppressing carcinogenesis alongwith effect of vitamins at nanoscale level. Next, we discuss the factors governing sequestration of death receptors off plasma membrane.It is praiseworthy that new techniques are deepening our insight into the dynamics of membrane organization. Here, we discuss how the field of internalization and degradation of death receptors has matured and present growing model in which membranes are occupied by fluctuating nanoscale assemblies of sphingolipids, cholesterol and proteins that can be stabilized into platforms that are important in TRAIL mediated signaling and membrane trafficking. TRAIL mediated signaling occurs by various mechanisms, which can be divided into those that are clathrin dependent and those that are clathrin independent Figure Emerging evidence suggests that stable TRAIL-resistant sub-population of the metastatic colon cancer cell line LIM6-TR displays an increased cell-surface expression of galectin-3. Interestingly, protein purification strategies indicated that galectin-3, an endogenous lectin, co-precipitated with death receptors. Targeted inhibition of galectin-3 re-sensitized resistant cells to TRAIL and facilitated TRAIL-mediated endocytosis of TRAIL/death receptors complexes . It is gAdditionally, TRAIL cytotoxicity in hepatocellular carcinoma cells is triggered by lysosomal permeabilization. Death receptors behave in a differential manner in terms of trafficking to lysosomes as DR5 complex under go immediate endocytosis upon ligand stimulation, while DR4 complex is not proficiently internalized. Upon TRAIL treatment, DR5 co-existed with lysosomes following internalization and abrogation of DR5 trafficking to lysosomes in Rab7 deficient cells also repressed TRAIL-mediated lysosomal disruption and apoptosis . EmerginMoreover, in MDA-MB-231 breast cancer cells, endocytosed DR4 is cleaved in a caspase-dependent manner. It is worth mentioning that receptor internalization does not inhibit caspase activation, instead stimulates apoptosis after TRAIL treatment. Consistent with the same concept, it is obvious that TRAIL-induced receptor endocytosis suppresses its pro-apoptotic potential .Besides, impairment of endocytosis or disruption of clathrin-dependent endocytosis signaling components (adaptor protein 2 and clathrin) recapitulates cell surface appearance of the death receptors and sensitize TRAIL-resistant cells to TRAIL-induced apoptosis. Structural overview of DR4 indicates that endocytosis is modulated by its cytoplasmic domain EAQC(337)LL. Therefore unquestionably clathrin-mediated endocytosis pathway could be a prospective target for therapeutics to circumvent tumor resistance to TRAIL receptor-targeted therapies .It is becoming sequentially more apparent that death receptors trigger selective destruction of the clathrin-dependent endocytosis nano- machinery. DR stimulation initiated instantaneous, caspase-mediated cleavage of clathrin-pathway apparatus, impeding retrieval of cargo protein transferrin. Analogously, caspases cleaved the clathrin adaptor subunit AP2alpha between characteristically discrete domains, while effector caspases processed clathrin's heavy chain .Escalating evidence suggests that COX-2 abrogation sensitizes human colon carcinoma cells to TRAIL induced apoptosis by enhancing assembly of the TRAIL receptor DR5 at the cell surface. Simultaneously, it is also associated with reorganization of the death-inducing signaling complex components into cholesterol-rich and ceramide-rich domains known as caveolae. This progression is dependent on accumulation of arachidonic acid and consequent activation of acid sphingomyelinase for the generation of ceramide within the plasma membrane outer leaflet .It is intriguing to note that enforced expression of caveolin results in de-sensitization of cells towards TRAIL as engagement of TRAIL with its respective receptor DR4 reduces the localization of DR4 in caveolae and results in its internalization. Obstruction of caveolae-mediated internalization of DR4 by filipin III outstandingly improved TRAIL-induced apoptosis .Post translational modifications of proteins underpin membrane-protein association and influence protein trafficking, stability or aggregation, thus playing an important role in protein signalling. In accordance with this assumption recent data suggests that DR4 is palmitoylated, whereas DR5 and TNFR1 are not. In addition, DR4 palmitoylation is essential for its raft localization and its ability to undergo oligomerization .Consistently, Ursodeoxycholic acid (UDCA) triggered formation of lipid rafts via ROS production/PKCδ activation pathway that played fundamental roles in UDCA-induced apoptosis. Lipid rafts are essential not only for provision of a platform for DR5 action but also for regulation of DR5 expression . Taken tWhile drawing a parallel between adherent and detachment cells apoptosis assay revealed that detached cells are more sensitive to DR5 antibody-induced apoptosis than adherent cells. As transition from adhesion to detachment of EC9706 cells results in DR5 relocalization, and enhances cytoplasmic translocation of DR5 to cell surfaces via a Golgi-dependent pathway .Cellular organelles in the endocytic pathways have a characteristic spatial distribution and communicate through a highly structured system of vesiculo-tubular transport. TRAPP proteins harmonize consecutive stages of transport, such as vesicle formation, vesicle motility and tethering of vesicles to their target compartment. These protein complexes are well compartmentalized in organelle membranes, making them exceptional mediators for determining transport specificity.A study delineating localization patterns of the receptors in melanoma cells by confocal microscopy suggested that TRAIL-R1 and R2 were located in the trans-Golgi network, but the inhibitory receptors TRAIL-R3 and -R4 were located in the nucleus Figure The signal recognition particle complex (SRP) is a molecular machinery of the “signal hypothesis” that is contributory in protein sorting through the secretory pathway. Similarly, experiments demonstrated that expression of the signal recognition particle (SRP) complex was vital for apoptosis mediated by DR4, but not DR5. Targeted inhibition of SRP subunits by RNA interference resulted in a remarkable decrease in cell surface DR4 receptors that interrelated with inhibition of DR4-dependent cell death. Expression of cell surface DR4 was severely repressed in cells lacking expression of SRP54 or SRP72 . MembranIt is imperative to explore whether DR4 is indeed translocated to the Golgi and the impairment of migratory pathway to properly traffic to the plasma membrane is a secondary effect of SRP depletion. Likewise, it will be essential to find out how DR4 and other proteins are translocated to the Golgi and whether these events are dependent on chaperones or the ER.Keeping in view the trafficking of receptors from endoplasmic reticulum to golgi and later to plasma membrane is a well organized mechanism. We have several hints of protein complexes which might play a role in the translocation. TRAPP proteins are identified to be component of a large multi-protein complex involved in ER-to-Golgi and intra-Golgi trafficking. Details were comprehensively reviewed by . For insRecent molecular genetics study indicated that TRAPPC9 encoded the NIK- and IKK-beta-binding protein (NIBP) . NIBP inIt is indubitable that despite tremendous information added into the developing landscape of death receptor trafficking pathways many protein complexes and assemblies are still lost and a better concept of multi-component, multi-layered trafficking pathway is required. How TRAPP proteins interact with death receptors and facilitate or repress migration from endoplasmic reticulum to golgi and further towards plasma membrane is a missing link. As a consequence, supplementary structure-function studies will be needed to describe the particular sequences necessary for DR4 cell surface localization. In this regard, how DR5 and other TNF family receptors find their way to the plasma membrane remains fascinating, yet an outstanding and under-defined question.Accumulating evidence using confocal microscopy suggested that DR5 were localized in the nucleus in HeLa and HepG2 cells. Nuclear trafficking of DR5 was inhibited in the cells deficient for importin β1 however enforced expression of importin β1 restored redistribution of DR5 in nucleus It is imperative to note that certain viral proteins counteract killing through death receptors by maximizing internalization and degradation. In agreement with this interpretation, human adenovirus type 5 encodes three proteins, named RID suppressed TRAIL-induced apoptosis of infected human cells. RID promoted endocytosis of TRAIL-R1 from the cell surface, as indicated by flow cytometry and indirect immunofluorescence studies for TRAIL-R1. TRAIL-R1 was internalized in discrete vesicles and degradation of TRAIL-R1 was noted .Correspondingly, localization of DR4 but not DR5 in lipid rafts was mediated by TRAIL and fludarabine treatment .It is also necessary to make a note of Nitrosylcobalamin (NO-Cb) that inhibits survival signaling by enhancing drug efficacy by preventing concomitant activation of negative regulators of apoptosis (NF-kappaB or AKT). Additionally, use of NO-Cbl and Apo2L/TRAIL represents a promising anti-cancer combinatorial drug because DR4 (TRAIL R1) is S nitrosylated following NO-Cbl treatment. S-nitrosylation at cysteine 304 facilitates relocalization of Fas to lipid rafts, formation of the death-inducing signalosome, and induction of cell death-55.It is undeniable that nutrigenomics has emerged as an important facet of molecular biology and employs high-throughput genomics technologies to disentangle how nutrients modulate gene and protein expression and eventually influence cellular and organism metabolism. It is vital to use and integrate nutrigenomics in all future nutrition studies portray broader landscape for evidence-based nutrition in near future. Concordant with same viewpoint, whole-genome sequencing is possible, comprehensive strategies for integrating genomic data and counseling of patients need to be developed.Incontrovertibly, vitamins exert pleiotrophic responses in malignant cells leading to cell cycle arrest, differentiation, and apoptosis. Apart from their implications in killing cancer cells via apoptosis, vitamins regulate expression of genes involved in cell proliferation and cell death in a "subapoptotic" manner. For instance, these vitamins mediate the cell cycle machinery, resulting in cell cycle arrest. We have elucidated an overview of cooperative antitumor effect when vitamins are combined with immunological agents. Vitamins and TRAIL synergize to kill cancer cells either by stimulating the expression or appearance of death receptors on cell surface respectively or by amplifying the mitochondrial apoptotic pathway without being toxic to normal cells. Lipophilic vitamins are being explored for their anticancer properties by many researchers . Silica It is significant that a single-agent 'magic' pill for chemoprevention is not highly effective and that using different combinations of phytochemicals and vitamins may be the answer.The authors declare that they have no competing interests.All authors contributed equally to the development of the ideas and the writing of this review. All authors read and approved the final manuscript.


The vascular-stromal elements of lymph nodes can play important roles in regulating the activities of the lymphocytes within. During model immune responses, the vascular-stromal compartment has been shown to undergo proliferative expansion and functional alterations. The state of the vascular-stromal compartment and the potential importance of this compartment in a spontaneous, chronic model of autoimmunity have not been well studied. Here, we characterize the vascular expansion in MRL-lpr/lpr lymph nodes and attempt to ask whether inhibiting this expansion can interfere with autoantibody generation. We show that characteristics of vascular expansion in enlarging MRL-lpr/lpr lymph nodes resemble that of the VEGF-dependent expansion that occurs in wild-type mice after model immunization. Surprisingly, treatment with SU5416, an inhibitor of VEGF and other receptor tyrosine kinases, did not have sustained effects in inhibiting vascular growth, but attenuated the anti-dsDNA response and altered the phenotype of the double negative T cells that are expanded in these mice. In examining for anatomic correlates of these immunologic changes, we found that the double negative T cells are localized within ectopic follicles around a central B cell patch and that these T cell-rich areas lack the T zone stromal protein ER-TR7 as well as other elements of a normal T zone microenvironment. SU5416 treatment disrupted these follicles and normalized the association between T zone microenvironmental elements and T cell-rich areas. Recent studies have shown a regulatory role for T zone stromal elements. Thus, our findings of the association of anti-dsDNA responses, double negative T cell phenotype, and altered lymphocyte microenvironment suggest the possibility that lymphocyte localization in ectopic follicles protects them from regulation by T zone stromal elements and functions to maintain autoimmune responses. Potentially, altering the lymphocyte microenvironment that is set up by the vascular-stromal compartment can be a means by which to control undesired autoimmune responses. Lymph nodes are sites of immune responses, and, within lymph nodes, the activities of and interactions among immune cells are supported and regulated by a highly plastic vascular-stromal compartment that can expand and undergo phenotypic alterations during immune responses. The mechanisms that regulate these vascular-stromal changes and how they contribute to the progression and regulation of the immune response are just beginning to be better understood . In lupuThe anatomic compartmentalization of B cells to polarized follicles at the cortex and T cells and dendritic cells to the “T zone” in the paracortex within lymph nodes is in part is dictated by the unique identity of fibroblastic reticular cells (FRCs) in each compartment. Specialized FRCs within the follicles express B cell-attracting CXCL13, while T zone FRCs express CCR7 ligands that promote the localization of CCR7-expressing T cells and dendritic cells to the T zone hi presumed dendritic cells The blood vessels of lymph nodes bring in cells, nutrients, and oxygen. The high endothelial venules (HEVs) are specialized postcapillary venules that are the portals of entry for circulating lymphocytes and are mostly found in the T zone and medulla In this paper, we characterize the vascular expansion that occurs with progressive lymph node enlargement in the MRL/lpr model of lupus and attempt to inhibit the vascular growth to ask about the subsequent effects on autoantibody generation. The MRL/lpr model is characterized by massive lymph node enlargement in part due to the accumulation of CD4-CD8-B220+ syndecan+ (herein referred to as “double negative”) T cells The Institutional Animal Use and Care Committee of the Hospital for Special Surgery specifically approved this study and all animal procedures were performed in accordance with their regulations.http://jaxmice.jax.org/strain/006825.html). The experiments utilizing 4–5 weeks of SU5416 treatment used JAX strain 000485, which were the rederived MRL-lpr/lpr line that had the more severe phenotype typical of the original MRL-lpr/lpr mice.MRL+/+ and MRL-lpr/lpr mice were either obtained from The Jackson Laboratory  or bred at HSS from Jackson Laboratory breeders. For the initial characterization of the vascular expansion and the 2 and 11.5 week SUGEN compound treatments, the JAX 006825 strain was used. This strain of MRL-lpr/lpr mice demonstrated an attenuated lymphoproliferative phenotype and longer life spans than had been typical for their MRL-lpr/lpr mice  or DMSO vehicle every other day for indicated time periods.For all of our flow cytometric analyses except analyses of plasma cells, we prepared single cell suspensions by digesting with collagenase type II  and stained cells as described previously Lymph nodes were flash-frozen and 7-µm sections were cut on a cryostat. Sections were dried for 1 h, fixed for 10 min in acetone and immunohistochemical staining of fresh-frozen sections was performed as described previously All antibodies used were from BD Biosciences  unless otherwise specified. Abs used were against CD45, CD31, peripheral node addressin (PNAd), gp38 , CD11c (HL3), B220, CD19, IgD, CD21/CD35, CD3, syndecan, BrdU, ER-TR7 , and LYVE-1 . Biotinylated peanut agglutinin (PNA) was from Vector Laboratories .For lymph node VEGF levels, popliteal lymph nodes were solubilized in modified RIPA buffer and subjected to VEGF measurement using a commercial kit  as previously described Anti-dsDNA ELISA was as described Plates were coated with poly-L-lysine and dsDNA as for ELISAs before adding cells suspended in RPMI with 2% FCS  for 4 hours. The rest of the assay was carried out as described negCD31pos cells to delineate the total endothelial cell population . Within this population, endothelial cells can be divided into peripheral node addressin (PNAd) pos and PNAd neg cells. PNAd marks HEV endothelial cells pos endothelial cells here as “HEV endothelial cells”. PNAd neg endothelial cells represent a mixture of mostly blood endothelial cells and less lymphatic endothelial cells . Total lymph node cellularity was also not consistently altered by SU5416 (data not shown). Remarkably, however, SU5416 attenuated the rise in anti-dsDNA titers  is shown for comparison purposes. In contrast to the distinct B cell follicles and T zones in the stimulated MRL+/+ node  and consmed . Some ofmed . Some ofma cells  and the med (double negative) T cells , similar to that of normal B cell follicles , althoug T cells . Further T cells  and can  T cells .neg T cells that normally inhabit the paracortex could be observed in patches between some of the ectopic follicles  along with CD21/CD35med cells (B cells) could still be observed (The relative lack of ER-TR7 staining within the ectopic follicles is reminiscent of the ER-TR7 exclusion from normal B cell follicles. In support of a B cell follicle-type microenvironment within the ectopic follicles, these follicles contained a network of CD21/CD35hi cells  resemblihi cells . Upon SUobserved . Howeverobserved , SU5416 observed . InteresOur results showed that lymph node hypertrophy in the MRL-lpr/lpr model of lupus is accompanied by vascular expansion and also revealed that double negative T cells and B cells are organized within ectopic follicles that were disrupted in association with SU5416-induced attenuation of the anti-dsDNA response. The T cell-rich corona of the ectopic follicles lacked a normal T zone microenvironment, and SU5416-induced follicular disruption normalized the T cell-microenvironment association. The association of the microenvironmental alterations with anti-dsDNA responses and double negative T cell phenotype alterations raises the possibility that these phenomena are related and that microenviromental alterations might contribute to regulating autoimmune responses. T zone FRCs have been shown to have a regulatory capacity hi cell:CD11cmed cell further supports this idea and also raises the possibility that, similar to lymph nodes after model immunization, CD11chi cells may also regulate this phase of quiescence in MRL-lpr/lpr nodes. Short term VEGF receptor blockade with SU5416 reduced HEV numbers, suggesting that, similar to lymph nodes after model immunization Our results showed that the characteristics of vascular expansion accompanying the progressive lymph node enlargement in MRL-lpr/lpr mice are similar to the vascular expansion that occurs after model immunizations. By examining at long intervals in the MRL-lpr/lpr mice, we were likely to have missed any proliferative burst that characterizes the first days after acute immunization of wild-type mice Our results suggest the possibility that localization of double negative T cells with B cells in ectopic follicles is related to the maintenance of the anti-dsDNA response. Whether and how they are related remains to be fully elucidated, but double negative T cells from lupus patients can help B cells to generate an anti-dsDNA response hi presumed dendritic cells were not reduced in number with long term SU5416 treatment, but whether SU5416-mediated blockade affected dendritic cell function was not assessed. Dendritic cells are important for the generation of autoantibodies in MRL-lpr/lpr mice The targets of the SU5416 that are involved in the reduction in anti-dsDNA responses and in lymph node alterations are currently unclear. The lack of effect of SU1498 on these parameters would suggest that VEGF receptor inhibition is not involved. SU5416 also inhibits Flt3, which is important for dendritic cell development Identifying the critical SU5416 targets that are important for the effects in the MRL-lpr/lpr lymph nodes may have implications for potential new therapies in lupus. SU5416 was tested in clinical trials for its anti-angiogenic and anti-tumor properties and was discontinued as a clinical agent because of side effects and suboptimal pharmacokinetics. Sunitinib is a subsequent generation VEGF receptor inhibitor that has a similar profile of activity against multiple receptor tyrosine kinases and that is orally available. It is in clinical use as a first line agent in patients with metastatic clear cell renal cell carcinoma Our results provide new insight into the features of the immune system that are associated with autoantibody generation. Although more work will be needed to fully establish the relationship between autoantibody generation and lymph node microenvironmental alterations, these associations raise the possibility that physical sequestration of autoimmune cells from regulatory microenvironmental elements could potentially be a mechanism for sustaining pathogenic responses and that targeting the association of lymphocytes with the microenvironment may be a strategy by which to control autoimmune responses. Testing this model and understanding the generalizability of our findings in other autoimmune models will be helpful to better understand the potential utility of this strategy and also to provide further insight into mechanisms of disease.