TNF-α are involved in the regulation of its expression. The present study explores TNF-α promoter polymorphisms and correlates them with TNF-α transcript and protein levels in vitiligo patients and controls of Gujarat along with its effect on disease onset and progression. PCR-RFLP technique was used for genotyping of these polymorphisms in 977 vitiligo patients and 990 controls. TNF-α transcript and protein levels were measured by Real time PCR and ELISA respectively. The genotype and allele frequencies for the investigated polymorphisms were significantly associated with vitiligo patients. The study revealed significant increase in TNF-α transcript and protein levels in vitiligo patients compared to controls. In particular, haplotypes: AATCC, AACCT, AGTCT, GATCT, GATCC and AGCCT were found to increase the TNF-α levels in vitiligo patients. Analysis of TNF-α levels based on the gender and disease progression suggests that female patients and patients with active vitiligo had higher levels of TNF-α. Also, the TNF-α levels were high in patients with generalized vitiligo as compared to localized vitiligo. Age of onset analysis of the disease suggests that the haplotypes: AACAT, AACCT, AATCC and AATCT had a profound effect in the early onset of the disease. Moreover, the analysis suggests that female patients had an early onset of vitiligo. Overall, our results suggest that TNF-α promoter polymorphisms may be genetic risk factors for susceptibility and progression of the disease. The up-regulation of TNF-α transcript and protein levels in individuals with susceptible haplotypes advocates the crucial role of TNF-α in autoimmune pathogenesis of vitiligo.Tumor Necrosis Factor (TNF)-α, is a paracrine inhibitor of melanocytes, which plays a critical role in the pathogenesis of several autoimmune diseases including vitiligo, as abnormal immune responses have frequently been observed in vitiligo patients. Moreover, vitiligo patients show higher lesion levels of TNF-α. Genetic polymorphisms in the promoter region of  Vitiligo is an acquired, non-contagious disease in which progressive, patchy, multifocal loss of pigmentation of skin, overlying hair, and often mucous membranes results from loss of melanocytes from the involved areas MHC, ACE, CAT, CTLA-4, COMT, ESR, GCH1, MBL2, PTPN22, and VDRHLA, PTPN22, NALP1, XBP1, FOXP1, IL2RA have been tested for genetic association with vitiligo Vitiligo is a polygenic disease; however, recent genome-wide association studies (GWAS), have identified generalized vitiligo susceptibility genes which almost universally involve immune regulation and immune targeting of melanocytes, that have led to the general consensus that generalized vitiligo is a primary autoimmune disease, though the biological triggers of the autoimmune process remain unknown Cytokines are important mediators of immunity and their response due to imbalance or deficiency in the cytokine network may largely determine autoimmune disease susceptibility and severity. Tumor necrosis factor (TNF)-α is a multifunctional, proinflammatory cytokine which plays an important role in several autoimmune diseases like rheumatoid arthritis, pernicious anemia, diabetes mellitus etc.TNF-α plays important role in apoptosis through activation of the receptor-mediated apoptosis pathway in numerous cell types TNF gene locus is located within the Class III region of the human major histocompatibility complex (MHC) on chromosome 6 (6p21.31) spanning about 3 kb and contains 4 exons. Regulation of TNF-α production occurs at both the transcriptional and post-transcriptional levels, with regulatory sequences within the 5′ end of the gene controlling the rate of transcription TNF-α gene promoter region having the potential to cause structural changes within regulatory sites that could affect the function or regulation of TNF-α production. The location of its gene within major histocompatibility complex and biological activities has raised the possibility that polymorphisms within this locus may contribute to the pathogenesis of wide range of autoimmune and infectious diseases.TNF-α gene transcription whereas −863 leads to decrease in the transcription. These polymorphisms combined could contribute to the autoimmune process making it an ideal candidate for the development of vitiligo.The promoter polymorphisms at positions: −238, −308, −857, and −1031 may lead to a higher rate of TNF-α  are associated with vitiligo susceptibility and modulate TNF-α transcript and protein levels. ii.) to measure and compare TNF-α transcript and protein levels in patients with vitiligo and in unaffected controls. iii.) to correlate TNF-α polymorphisms/levels with onset and progression of the disease.In the present study, we have made an attempt to understand the role of TNF-α in vitiligo pathogenesis. Hence, the aims of this study were: i.) to determine whether the promoter polymorphisms of The study group included 977 vitiligo patients [733 generalized  and 244 localized vitiligo cases] comprised of 451 males and 526 females who referred to S.S.G. Hospital, Vadodara and Civil Hospital, Ahmedabad, Gujarat, India . The diaThe study plan was approved by the Institutional Ethical Committee for Human Research (IECHR), Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India. The importance of the study was explained to all participants and written consent was obtained from all patients and controls.3EDTA coated  tubes. Genomic DNA was extracted from whole blood using ‘whole blood DNA extraction kit’  according to the manufacturer’s instructions. After extraction, concentration and purity of DNA was estimated spectrophotometrically, quality of DNA was also determined on 0.8% agarose gel electrophoresis and DNA was stored at −20°C until further analyses.Five ml. venous blood was collected from the patients and healthy subjects in KTNF-α gene , 0.3 µL of 10 µM corresponding forward and reverse primers , and 0.3 µL (5 U/µL) Taq Polymerase . Amplification was performed using a PTC-100 thermal cycler  according to the protocol: 95°C for 10 min. followed by 30 cycles of 95°C for 15 sec., primer dependent annealing (Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used to genotype all five promoter polymorphisms of F-α gene . The prinnealing  for 30 sBamHI, NcoI, TaiI and BbsI for digesting amplicons of −238 G/A, −308 G/A, −857 C/T, −863 C/A and −1031 T/C of TNF-α gene  used were: F-α gene . 5 µL ofMore than 10% of the samples were randomly selected for confirmation and the results were 100% concordant  and also confirmed by sequencing.Total RNA from whole blood was isolated and purified using Ribopure™- blood Kit  following the manufacturer’s protocol. RNA integrity was verified by 1.5% agarose gel electrophoresis/ethidium bromide staining and O.D. 260/280 absorbance ratio >1.95. RNA was treated with DNase I  before cDNA synthesis to avoid DNA contamination. One microgram of total RNA was used to prepare cDNA. cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit  according to the manufacturer’s instructions in the MJ Research Thermal Cycler .TNF-α and GAPDH transcripts were measured by real-time PCR using gene specific primers  as shown in GAPDH gene was used as a reference. Real-time PCR was performed in duplicates in 20 µl volume using LightCycler®480 SYBR Green I Master  following the manufacturer’s instructions and carried out in the LightCycler®480 Real-Time PCR . The thermal cycling conditions included an initial activation step at 95°C for 10 min., followed by 45 cycles of denaturation, annealing and amplification . The fluorescence data collection was performed during the extension step. At the end of the amplification phase a melting curve analysis was carried out on the product formed. The value of Cp was determined by the first cycle number at which fluorescence was greater than the set threshold value.The expression of -α in patients with vitiligo and controls were measured by enzyme-linked immunosorbent assay (ELISA) using the Immunotech Human TNF-α ELISA kit  as per the manufacturer’s protocol.Serum levels of TNFTNF-α promoter polymorphisms for patients and control subjects were compared using the chi-squared test with 3×2 and 2×2 contingency tables respectively using Prism 4 software . p-values less than 0.01 were considered as statistically significant due to Bonferroni’s correction for multiple testing. Odds ratio (OR) with respective confidence interval (95% CI) for disease susceptibility was also calculated.Evaluation of the Hardy-Weinberg equilibrium (HWE) was performed for both the polymorphisms in patients and controls by comparing the observed and expected frequencies of the genotypes using chi-squared analysis. The distribution of the genotypes and allele frequencies of http://analysis.bio-x.cn/myAnalysis.php2-values for the pair of the most common alleles at each site were estimated using the Haploview programe version 4.1 TNF-α and serum TNF-α levels in patient and control groups was plotted and analyzed by nonparametric unpaired t-test using Prism 4 software . The statistical power of detection of the association with the disease at the 0.05 level of significance was determined by using the G* Power software Haplotype analysis was carried out using TNF-α promoter polymorphisms in 733 generalized vitiligo patients and 990 controls are summarized in p>0·05).The genotype and allele frequencies of the investigated TNF-α were found to be in significant association with generalized vitiligo patients (p<0.0001) when genotypes were compared using chi-squared test-3×2 contingency table with Bonferroni’s correction for multiple testing (p<0.0001)  . Interes<0.0001) . However<0.0001) . This stTNF-α promoter polymorphisms in 244 localized vitiligo patients and 990 controls are summarized in p>0·05) except for −238G/A and −863C/A in patients (p = 0.0001 and p = 0.014 respectively).The genotype and allele frequencies of the investigated TNF-α were found to be in significant association with localized vitiligo patients (p<0.0001) when genotypes were compared using chi-squared test-3×2 contingency table with Bonferroni’s correction for multiple testing (p<0.0001)  . Althougds ratio . This stTNF-α gene were in low to moderate LD association in both generalized as well as localized vitiligo patients . Also, localized vitiligo patients exhibited significantly different frequencies of haplotypes as compared to controls    & 4.The susceptible haplotypes: AACAT, AACCT, AATCC, AATCT and AGCCT were more frequently observed in generalized vitiligo patients as compared to controls and were found to increase the risk of generalized vitiligo as suggested by odds ratio . HoweverFurthermore, susceptible haplotypes: AACAT, AACCT, AATCC, AATCT, AGCCC and AGCCT were more frequently observed in localized vitiligo patients as compared to controls and were found to increase the risk of localized vitiligo as suggested by odds ratio ; howeverTNF-α promoter haplotypes, patients with AACAT, AACCT, AATCC and AATCT haplotypes showed early onset of the disease as compared to GGCAT, GGCCT, GGTCC and GGTCT  (p = 0.04). Moreover, patients with haplotype AATCT showed early onset of the disease as compared to AGTCT and GATCT haplotypes (p = 0.001 and p = 0.025 respectively) (p = 0.045); however, there was no significant difference in age of onset of the disease for haplotype AGCCT as compared to GACCT and GGCCT haplotypes (p = 0.147 and p = 0.481 respectively) (p = 0.248 and p = 0.582 respectively) (p<0.0001) (When age of onset of the disease was correlated with the ctively) . Patientctively) . Also, pctively) . Patientctively) . Interes<0.0001) .TNF-α transcripts in 157 vitiligo patients than in 174 unaffected controls after normalization with GAPDH expression as suggested by mean ΔCp values (p = 0.0005)   . Moreove 0.0295) . The 2−controls .TNF-α for the −238 GG genotype did not differ significantly between vitiligo patients and controls (p = 0.294). However, patients with −238 GA and AA genotypes revealed higher TNF-α transcript levels as compared to controls (p = 0.028 and p = 0.008 respectively) (TNF-α expression differed significantly between patients and controls for −308 GA and AA genotypes (p = 0.042 and p = 0.002 respectively) whereas for GG genotype the expression did not differ (p = 0.064) (TNF-α expression was higher in patients with CT and TT genotypes (p = 0.008 and p = 0.001 respectively) whereas the expression did not differ with CC genotypes (p = 0.173) (TNF-α expression was reduced in patients with −863 CA and AA genotypes (p = 0.032 and p = 0.001 respectively) whereas the expression did not differ with CC genotypes (p = 0.828) (TNF-α expression was higher in patients with TC and CC genotypes (p = 0.036 and p = 0.032 respectively) whereas the expression did not differ with TT genotypes (p = 0.284) (The expression levels of ctively) . Also, T= 0.064) . For −85= 0.173) . However= 0.828) . For −10= 0.284) .TNF-α were analyzed with respect to haplotypes generated from the five investigated promoter polymorphisms of TNF-α ; however, no significant difference was observed in TNF-α expression for the haplotypes: GGTCC, GGCAC, GGCCT and GGTCT .Further, the expression levels of of TNF-α . InteresTNF-α expression based on the progression of the disease i.e. active vitiligo and stable vitiligo (TNF-α transcripts as compared to the patients with stable vitiligo (p<0.0001). To check the susceptibility of the disease based on the gender differences TNF-α expression was analyzed for male and female vitiligo patients. Female patients with vitiligo showed significantly higher TNF-α expression as compared to male patients (p = 0.0073)  .TNF-α promoter polymorphisms with its level in the serum, TNF-α levels were measured in 214 vitiligo patients and 236 unaffected controls. Vitiligo patients showed significant increased serum TNF-α (sTNF-α) levels as compared to controls (p = 0.0003) (p = 0.014)  . Moreove= 0.014) .p = 0.018 and 0.002 respectively). However, for the −238 GG genotype sTNF-α levels did not differ between these groups (p = 0.062) (p = 0.019 and p<0.0001 respectively) whereas the levels were not differed for GG genotypes between patients and controls (p = 0.082) (p = 0.023 and p = 0.020 respectively) whereas the levels did not differ with CC genotypes (p = 0.246) (p = 0.023 and p = 0.035 respectively) whereas the levels did not differ with CC genotypes (p = 0.067) (p = 0.033 and p = 0.001 respectively) whereas the levels did not differ with TT genotypes (p = 0.211)  . Further= 0.082) . For −85= 0.246) . However= 0.067) . For −10= 0.211) .TNF-α haplotypes: GATCC, AGTCT, AGCCT and AACCT were found to increase sTNF-α levels  with susceptible alleles  as compared to controls   .p<0.0001) (p = 0.0066)  . Additio 0.0066) .CTLA-4 and IL-4 gene polymorphisms also supported the autoimmune pathogenesis of vitiligo in Gujarat population MBL-2, ACE, PTPN22 polymorphisms did not show significant association Vitiligo susceptibility is a complex genetic trait that may include genes involved in melanin biosynthesis, response to oxidative stress and regulation of autoimmunity. The importance of genetic factors for vitiligo susceptibility is evident by reports of its significant familial association TNF-α are reported to be involved in modulating expression of TNF-α gene which may be responsible for melanocyte death.Cytokines are important mediators of immunity and there is now convincing evidence that cytokines also have an important role in the pathogenesis of autoimmunity In vitro, direct analysis of skin T cells from margins of vitiliginous skin show that polarized type-1 T cells (CD4+ and particularly CD8+), which predominantly secrete interferon (IFN)-γ and TNF-α are associated with the destruction of melanocytes during active vitiligo TNF-α, is an important multifunctional cytokine secreted by macrophages, T-lymphocytes, fibroblasts and keratinocytes with wide-ranging biological effects of protection from infection, surveillance against tumors and stimulation of inflammatory responses. In the epidermis, the epidermal melanin unit consists of the close interaction of a melanocyte and an associated pool of keratinocytes. Close relationship between these two cell types is important for melanocyte survival and differentiation mainly as keratinocyte-derived cytokines act on melanocytes via specific receptors It has been reported that cytokines such as IFN-γ and TNF-α can initiate apoptosis and thus lead to melanocyte death in the context of autoimmunity TNF-α promoter polymorphisms in adequate number of vitiligo patients and controls to elucidate the role of these polymorphisms in vitiligo susceptibility and to analyze the possible genotype - phenotype correlation. Here, we report that TNF-α −238, −308, −857, −863 and −1031 promoter polymorphisms are significantly associated with Gujarat vitiligo patients. Our results clearly suggest the important role of TNF-α in pathogenesis of vitiligo. Vitiligo patients showed significant increase in TNF-α transcript and protein levels as compared to controls suggesting that melanocyte death in patients could be triggered due to the increased TNF-α levels.Thus, it becomes pertinent to study all TNF-α transcript and protein levels as compared to localized vitiligo patients which indicate involvement of autoimmunity in precipitation of generalized vitiligo. Our results also indicate that active vitiligo patients have significantly higher TNF-α transcript and protein levels as compared to the patients with stable vitiligo which signifies the role of TNF-α in disease progression. Our results also suggest that there are significantly higher transcript and protein levels of TNF-α in female patients as compared to male patients. Moreover, female patients have an early onset as compared to male patients suggestive of the fact that females have increased susceptibility towards vitiligo as compared to males, implicating gender biasness in the development of autoimmunity For the first time we report that generalized vitiligo has significantly higher TNF-α −308 G/A and −238 G/A polymorphisms were found to influence serum TNF-α levels in patients with sarcoidosis of Asian Indian population TNF-α expression wherein TNF-α −238, −308, −857 and −1031 were found to increase whereas −863 was found to decrease the expression. Furthermore, a genotype-phenotype study carried out on SLE patients showed increased TNF-α transcript levels with −238 AA and GA genotypes as compared to GG genotypes The TNF-α −238 G/A & −308 G/A polymorphisms in moderate LD association as compared to the other investigated polymorphisms and are strongly associated with the disease risk in patients as suggested by the odds ratio. Moreover, the haplotype analysis showed the presence of haplotypes involving the susceptible alleles of TNF-α −238 and −308 polymorphisms, having increased levels of TNF-α in patients as compared to controls.LD analysis suggests that TNF-α repressor site TNF-α expression in transfected B cell line Raji and monocytic cell line U937 with a series of mutant constructs within the repressor region, including one where the –238G allele was replaced by a 10 bp linker sequence containing the –238A mutant allele. U937 and Raji cells containing the –238 minor A allele construct showed consistent increase (1.4 to 1.8-fold) in both basal and inducible promoter activity suggesting that the –238 SNP and the region surrounding it could be important in TNF-α regulation and a mutation at position –238 could disrupt its regulation The region between −254 to −230 contains a regulatory sequence that acts as a TNF-α gene expression. Furthermore, Wilson et al. TNF-α transcription using reporter gene assays suggesting that the −308 minor A allele is a much more powerful transcriptional activator than the common allele in a human B cell line.Kroeger et al. TNF-α −308 G/A polymorphism in Iranian population have revealed significant association of −308A allele with vitiligo patients TNF-α −308 G/A polymorphism has no significant influence on vitiligo susceptibility TNF-α promoter polymorphisms on its expression in vitiligo patients and the present study revealed the significant role of these promoter polymorphisms on the levels of TNF-α which might be playing a central role in vitiligo pathogenesis.The study of It has been known that the ROS microenvironment decides the fate of a cell for TNF-α mediated apoptosis TNF-α −308 G/A polymorphism was significantly associated with susceptibility to asthma in patients of South Iran and with susceptibility to inflammatory bowel disease in European population TNF-α −238G/A and −308G/A polymorphisms might be used as biomarkers for psoriasis risk prediction TNF-α −238G/A and −308G/A polymorphisms Disturbances in TNF-α metabolism have been well documented and found to be associated with several other autoimmune and infectious diseases such as rheumatoid arthritis TNF-α was involved in the pathogenesis of vitiligo. Alghamdi et al. Simon and Burgor-Vargas TNF-α gene influence the expression both at transcriptional as well as translational levels in vitiligo. The study also emphasizes the influence of TNF-α on the disease progression, onset of the disease and gender biasness for developing vitiligo. More detailed studies regarding role of TNF-α in precipitation of vitiligo and the development of effective anti-TNF-α agents may prove to be useful as preventive/ameliorative therapies.In conclusion, our findings suggest that the increased TNF-α levels in vitiligo patients could result, at least in part, from variations at the genetic level. For the first time, we show that the promoter polymorphisms of the Figure S1PCR-RFLP analysis of TNF-α promoter polymorphisms. (A) PCR-RFLP analysis of TNF-α −238 G/A polymorphism on 3.5% agarose gel: lanes: 1, 2, 3, 4, 5, 11, 12, 13, 14 & 18 show homozygous (GG) genotypes; lanes: 16, 17 & 19 show heterozygous (GA) genotypes; lanes: 6, 7, 8, 9 10 & 15 show homozygous (AA) genotypes; lane M shows 100 bp DNA ladder. (B) PCR-RFLP analysis of TNF-α −308 G/A polymorphism on 10% polyacrylamide gel: lanes: 1, 2, 4 & 5 show heterozygous (GA) genotypes; lanes: 3 & 6 show homozygous (GG) genotypes; lane M shows 100 bp DNA ladder. (C) PCR-RFLP analysis of TNF-α −857 C/T polymorphism on 10% polyacrylamide gel: lanes: 1, 2, 4, 5 & 6 show heterozygous (CT) genotypes; lane: 3 shows homozygous (GG) genotype; lane M shows 100 bp DNA ladder. (D) PCR-RFLP analysis of TNF-α −863 C/A polymorphism on 10% polyacrylamide gel: lanes: 1, 3, 4, 5 & 6 show homozygous (CC) genotypes; lane: 2 shows heterozygous (CA) genotype; lane M shows 100 bp DNA ladder. (E) PCR-RFLP analysis of TNF-α −1031 T/C polymorphism on 2.0% agarose gel: lanes: 4 & 5 show homozygous (TT) genotypes; lanes: 6 & 7 show heterozygous (TC) genotypes; lanes: 1, 2 & 3 show homozygous (CC) genotype; lane M shows 100 bp DNA ladder.(TIF)Click here for additional data file.Figure S2Linkage disequilibrium analysis of TNF-α promoter polymorphisms. (A) Linkage disequilibrium (D’) among TNF-α promoter SNPs in generalized vitiligo patients and controls from Gujarat population. (B) Linkage disequilibrium (D’) among TNF-α promoter SNPs in localized vitiligo patients and controls from Gujarat population.(TIF)Click here for additional data file.Figure S3Expression fold change of TNF−-α transcript in 157 vitiligo patients against 174 controls showed 0.445 fold change as determined by 2CpΔΔ method.(TIF)Click here for additional data file.Table S1Demographic characteristics of vitiligo patients and unaffected controls of Gujarat.(DOC)Click here for additional data file.Table S2Primers used for TNF-α promoter SNPs genotyping and gene expression analysis.(DOC)Click here for additional data file.Table S3Pairwise linkage disequilibrium (D’) values between TNF-α SNPs with >3% minor allele frequencies within generalized vitiligo patients and controls from Gujarat population.(DOC)Click here for additional data file.Table S4Pairwise linkage disequilibrium (D’) values between TNF-α SNPs with >3% minor allele frequencies within localized vitiligo patients and controls from Gujarat population.(DOC)Click here for additional data file.


Identifying ankylosing spondylitis (AS) patients who are likely to benefit from tumor necrosis factor-alpha (TNF-α) blocking therapy is important, especially in view of the costs and potential side effects of these agents. Recently, the AS Disease Activity Score (ASDAS) has been developed to assess both subjective and objective aspects of AS disease activity. However, data about the predictive value of the ASDAS with respect to clinical response to TNF-α blocking therapy are lacking. The aim of the present study was to identify baseline predictors of response and discontinuation of TNF-α blocking therapy in AS patients in daily clinical practice.AS outpatients who started TNF-α blocking therapy were included in the Groningen Leeuwarden Ankylosing Spondylitis (GLAS) study, an ongoing prospective longitudinal observational cohort study with follow-up visits according to a fixed protocol. For the present analysis, patients were excluded if they had previously received anti-TNF-α treatment. Predictor analyses of response and treatment discontinuation were performed using logistic and Cox regression models, respectively.n = 32), etanercept (n = 137), or adalimumab (n = 51). At three and six months, 68% and 63% of patients were Assessments in Ankylosing Spondylitis (ASAS)20 responders, 49% and 46% ASAS40 responders, and 49% and 50% Bath Ankylosing Spondylitis Disease Activity Index (BASDAI)50 responders, respectively. Baseline predictors of response were younger age, male gender, higher ASDAS score, higher erythrocyte sedimentation rate (ESR) level, higher C-reactive protein (CRP) level, presence of peripheral arthritis, higher patient's global assessment of disease activity, and lower modified Schober test. In August 2010, 64% of patients were still using their TNF-α blocking agent with a median follow-up of 33.1 months (range 2.4 to 68.2). Baseline predictors of discontinuation of TNF-α blocking therapy were female gender, absence of peripheral arthritis, higher BASDAI, lower ESR level, and lower CRP level.Between November 2004 and April 2010, 220 patients started treatment with infliximab (Besides younger age and male gender, objective variables such as higher inflammatory markers or ASDAS score were identified as independent baseline predictors of response and/or continuation of TNF-α blocking therapy. In contrast, higher baseline BASDAI score was independently associated with treatment discontinuation. Based on these results, it seems clinically relevant to include more objective variables in the evaluation of anti-TNF-α treatment. Randomized controlled trials (RCTs) have demonstrated that the tumor necrosis factor alpha (TNF-α) blocking agents infliximab, etanercept, and adalimumab are effective in the treatment of Ankylosing Spondylitis (AS). However, a significant proportion of patients has to withdraw from TNF-α blocking therapy due to inefficacy or adverse events -3. IdentSeveral studies using clinical data from RCTs have focused on the identification of predictors of response to anti-TNF-α treatment in AS -6. HowevDisease activity in AS encompasses a wide range of concepts and is therefore difficult to measure. Recently, the Ankylosing Spondylitis Disease Activity Score (ASDAS) has been developed ,11. ThisSince 2004 AS outpatients with active disease, who started treatment with the TNF-α blocking agents infliximab, etanercept, or adalimumab at the Medical Center Leeuwarden (MCL) and the University Medical Center Groningen (UMCG), were included in the Groningen Leeuwarden Ankylosing Spondylitis (GLAS) study, an ongoing prospective longitudinal observational cohort study with follow-up visits according to a fixed protocol. All patients were over 18 years of age, fulfilled the modified New York criteria for AS or the Assessments in Ankylosing Spondylitis (ASAS) criteria for axial spondyloarthritis including MRI , and staPatients were evaluated at baseline, after three and six months of anti-TNF-α treatment, and then every six months. Disease activity was assessed using the Bath Ankylosing Spondylitis Disease Activity Index  , physiciAt every visit, continuation of treatment was based on a decrease in BASDAI, amounting to at least 50% (BASDAI50 response) or two units compared with baseline, and/or expert opinion in favor of treatment continuation. The ASAS20 and ASAS40 response criteria have been developed for defining treatment response in clinical trials. ASAS20 response was defined as an improvement of at least 20% and absolute improvement of at least one unit  compared with baseline in three or more of the four domains: physical function (BASFI), pain, patient's GDA, and inflammation (mean from BASDAI questions 5 and 6), with no worsening by more than 20% in the remaining domain. ASAS40 response was defined as an improvement of at least 40% and an absolute improvement of at least two units compared with baseline in three or more of the four domains, with no worsening at all in the remaining domain ,16. In t125I-radiolabeled F(ab)'2 fragment of infliximab or adalimumab was added. After overnight incubation, non-bound radiolabel was washed away and Sepharose-bound radioactivity was measured. Test results were converted into arbitrary units per milliliter (AU/ml) by comparison with dilutions of a reference serum. The reference value was set at 12 AU/ml, as derived from 100 healthy donors.Antibodies to TNF-α blocking agents were measured in patients who discontinued infliximab or adalimumab treatment due to inefficacy. Antibodies were detected by radioimmunoassay (RIA) as described in detail previously ,18. The P-value ≤0.3 in the univariate analysis. P-values < 0.05 were considered statistically significant.Statistical analysis was performed with SPSS 16.0 software . Results were expressed as mean ± SD or median (range) for normally and non-normally distributed data, respectively. The Independent Samples T test and Mann-Whitney U test were used to compare differences between groups. The Chi-Square test and Fisher Exact test were used to compare percentages between groups. Predictor analyses of ASAS20, ASAS40, and BASDAI50 response (yes/no) were performed using binary logistic regression. Predictor analysis of time to discontinuation of TNF-α blocking therapy (yes/no) was performed using Cox regression. Multivariate analysis was performed with conditional stepwise forward inclusion of predictors that had a n = 163; UMCG: n = 57) started treatment with a first TNF-α blocking agent; 32 receiving infliximab, 137 etanercept, and 51 adalimumab. Mean age of all patients was 42.9 years (SD ± 11.9), median disease duration was 15 years (range 1 to 53), and 69% were male. The three treatment groups were comparable for age, gender, HLA-B27 status, BASDAI, ASDAS, patient's GDA, CRP, ESR, concomitant medication, and presence of peripheral arthritis at baseline. In the infliximab group, time since diagnosis was significantly longer, the percentage of patients with a history of inflammatory bowel disease (IBD) was significantly higher, and occiput to wall distance was significantly larger compared to the etanercept and adalimumab groups. In the adalimumab group, the percentage of patients with a history of uveitis and physician's GDA were significantly lower and chest expansion was significantly higher compared to the infliximab and/or etanercept group  , male gender (OR: 2.991), and higher ASDAS score (OR: 1.573) or alternatively, presence of peripheral arthritis (OR: 2.518) and higher patient's GDA (OR: 1.173), were independent baseline predictors of ASAS20 response Table .P = 0.216 and P = 0.421, respectively)  was the only independent baseline predictor of ASAS40 response at three months of anti-TNF-α treatment.At six months of anti-TNF-α treatment, younger age , male gender , and higher patient's GDA  or alternatively, higher ASDAS score , were independent baseline predictors of ASAS40 response.P = 0.358 and P = 0.866, respectively)  Table .Multivariate logistic regression analysis showed that younger age , male gender , and higher CRP level  or alternatively, higher ESR level , were independent baseline predictors of BASDAI50 response at three months of anti-TNF-α treatment.At six months of anti-TNF-α treatment, younger age , male gender , presence of peripheral arthritis , and lower modified Schober test  were independent baseline predictors of BASDAI50 response.n = 40, 51%), adverse events ; allergic reaction (n = 4); diarrhea or IBD (n = 5); cardio-vascular disease (n = 2); demyelization problems (n = 1); bladder cancer (n = 1)), both inefficacy and adverse events ; allergic reaction (n = 1); diarrhea or IBD (n = 2); uveitis (n = 1); malaise (n = 1)), or other reasons ; pregnancy wish (n = 5); lost to follow up (n = 2)).In August 2010, 141 (64%) patients were still using their TNF-α blocking agent with a median follow-up of 33.1 months (range 2.4 to 68.2). The remaining 79 (36%) patients discontinued TNF-α blocking therapy after median treatment duration of 7.0 months (range 0.2 to 55.6). Reasons for discontinuation of TNF-α blocking therapy were inefficacy (n = 7) or adalimumab (n = 14) treatment due to inefficacy. Antibody data were missing for one adalimumab patient. Antibodies against infliximab and adalimumab were detected in 5 of 7 (71%) and in 8 of 13 (62%) patients who discontinued treatment due to inefficacy, respectively. In total, 5 of 13 (38%) patients with antibodies to TNF-α blocking agents were primary non-responders and 8 of 13 (62%) patients were secondary non-responders.Antibodies to TNF-α blocking agents were measured in patients who discontinued infliximab   was not significantly associated with achieving response or discontinuation of treatment. However, these findings should be interpreted with caution since there were differences in disease duration, the percentage of patients with extra-articular manifestations, physician's GDA, and spinal mobility measures at baseline and there was an uneven distribution of patients among the different treatment groups.This prospective longitudinal observational cohort study identified higher ASDAS score, higher ESR or CRP level, presence of peripheral arthritis, younger age, male gender, lower modified Schober test, higher patient's GDA, and lower BASDAI as independent baseline predictors of response and/or continuation of TNF-α blocking therapy in AS patients. These findings may help clinicians to identify AS patients who are more likely to benefit from TNF-α blocking therapy in daily clinical practice.AS: Ankylosing Spondylitis; ASAS: Assessments in Ankylosing Spondylitis; ASDAS: Ankylosing Spondylitis Disease Activity Score; BASDAI: Bath Ankylosing Spondylitis Disease Activity Index; BASFI: Bath Ankylosing Spondylitis Functional Index; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; GDA: global disease activity; HR: hazard ratio; IBD: inflammatory bowel disease; MCL: Medical Center Leeuwarden; OR: odds ratio; RA: rheumatoid arthritis; RCTs: randomized controlled trials; RIA: radioimmunoassay; TNF-α: tumor necrosis factor alpha; UMCG: University Medical Center Groningen; VAS: visual analogue scale.EB has received unrestricted research grants from Abbott, Schering-Plough, and Wyeth. AS has received unrestricted research grants from Abbott and Wyeth. The other authors declare that they have no competing interests.SA performed the statistical analysis and interpretation of data and drafted the manuscript; EB and AS participated in the design of the study, performed the acquisition of data, and critically revised the manuscript. EV and HG contributed to the statistical analysis and interpretation of data and critically revised the manuscript. ML, PH and TJ contributed to the acquisition of clinical data and critically revised the manuscript. CK participated in the design of the study and critically revised the manuscript. All authors read and approved the final manuscript.


Intracranial aneurysm (IA) is a socially important disease due to its high incidence in the general public and the severity of resultant subarachnoid hemorrhage that follows rupture. Despite the social importance of IA as a cause of subarachnoid hemorrhage, there is no medical treatment to prevent rupture, except for surgical procedures, because the mechanisms regulating IA formation are poorly understood. Therefore, these mechanisms should be elucidated to identify a therapeutic target for IA treatment. In human IAs, the presence of inflammatory responses, such as an increase of tumor necrosis factor (TNF)-alpha, have been observed, suggesting a role for inflammation in IA formation. Recent investigations using rodent models of IAs have revealed the crucial role of inflammatory responses in IA formation, supporting the results of human studies. Thus, we identified nuclear factor (NF)-kappaB as a critical mediator of inflammation regulating IA formation, by inducing downstream pro-inflammatory genes such as MCP-1, a chemoattractant for macrophages, and COX-2. In this study, we focused on TNF-alpha signaling as a potential cascade that regulates NF-kappaB-mediated IA formation.We first confirmed an increase in TNF-alpha content in IA walls during IA formation, as expected based on human studies. Consistently, the activity of TNF-alpha converting enzyme (TACE), an enzyme responsible for TNF-alpha release, was induced in the arterial walls after aneurysm induction in a rat model. Next, we subjected tumor necrosis factor receptor superfamily member 1a (TNFR1)-deficient mice to the IA model to clarify the contribution of TNF-alpha-TNFR1 signaling to pathogenesis, and confirmed significant suppression of IA formation in TNFR1-deficient mice. Furthermore, in the IA walls of TNFR1-deficient mice, inflammatory responses, including NF-kappaB activation, subsequent expression of MCP-1 and COX-2, and infiltration of macrophages into the IA lesion, were greatly suppressed compared with those in wild-type mice.In this study, using rodent models of IAs, we clarified the crucial role of TNF-alpha-TNFR1 signaling in the pathogenesis of IAs by inducing inflammatory responses, and propose this signaling as a potential therapeutic target for IA treatment. Intracranial aneurysm (IA) is a lesion with a regional bulging of intracranial arteries, usually located at bifurcation sites. IA is a common disease in the general public, with a prevalence of 1–5 percent . SubaracVarious studies using human IA specimens have revealed the involvement of active inflammatory responses in IA lesions, such as the expression of inflammatory cytokines, the infiltration of inflammatory cells into lesions, and the positive linkage of inflammatory-gene polymorphisms with IAs -9. FurthTumor necrosis factor (TNF)-alpha is a cytokine that can strongly activate NF-kappaB, and TNF-alpha signaling has been implicated in the pathogenesis of human IAs. For example, increased TNF-alpha mRNA expression in lesions as determined by reverse transcription PCR (RT-PCR) , increasAll of the following experiments, including animal care and use, complied with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Kyoto University Graduate School of Medicine.Male Sprague–Dawley rats were purchased from Japan SLC . TNFR1-deficient mice were purchased from The Jackson Laboratory . Animals were maintained on a light/dark cycle of 14 h/10 h, and had free access to chow and water. To induce IA, under general anesthesia by intraperitoneal injection of pentobarbital sodium (50 mg/kg), 7-week-old male rats or mice were subjected to ligation of the left carotid artery and systemic hypertension, induced by the combination of salt overloading and ligation of the left renal artery. This procedure is designed to increase hemodynamic stress, the trigger of IA formation -17, on tImmunohistochemical analyses were performed as previously described . BrieflyThe following primary antibodies were used: mouse monoclonal anti-smooth muscle alpha actin antibody , rabbit monoclonal anti-phospho NF-kappaB p65 (ser536) antibody , rat monoclonal anti-TNF-alpha antibody , goat polyclonal anti-MCP-1 antibody , rabbit polyclonal anti-COX-2 antibody , rat monoclonal anti-F4/80 antibody .2 around the dome of induced aneurysms .Macrophages were defined as cells positive for F4/80 staining in immunohistochemistry. The number of infiltrated macrophages was calculated within 10000 umRNA purification from the ring of Willis of mice 5 months after aneurysm induction (n = 4 in wild-type and 3 in TNFR1-deficient mice) and subsequent reverse transcription were performed using a RNeasy Fibrous Tissue Mini Kit  and a High Capacity cDNA Reverse Transcription Kit , respectively, according to the manufacturers’ instructions. For the quantification of gene expression, PCR reactions were performed using the LightCycler 480 system with a SYBR Premix Ex Taq II , using the expression of beta-actin as an internal control.The following primers  were used in experiments: 5′-tctgcagccatttccttctctcc-3′ and 5′-aaaggcctccattgaccagagc-3′ for COX-2, 5′-atgtctggacccattccttcttgg-3′ and 5′-tcccaatgagtaggctggagagc-3′ for MCP-1, 5′-gctcgttgccaatagtgatgacc-3′ and 5′-cgtgcgtgacatcaaagagaagc-3′ for beta-actin.The TNF-alpha protein content of intracranial arteries with aneurysm induction was examined using multiplex suspension array techniques, according to the manufacturer’s instructions. Briefly, intracranial arteries were collected from 6 rats at each indicated time period after aneurysm induction, and total protein was purified by Bio-Plex Cell Lysis Kit . TNF-alpha protein content was then quantified by Bio-Rad DC Protein Assay (Bio-Rad), using the Luminex 2000 System  for detection, and DNASIS Plex version 2.5  for analysis, according to the manufacturer’s instructions.The total protein from intracranial arteries after aneurysm induction was purified by Complete M Lysis kit  according to the manufacturer’s instructions (n = 4 in each group). Each experiment used 25 ug of protein. Total protein and the fluorescence-labeled synthetic substrate for TACE  were incubated in buffer containing 50 mM Tris–HCl (pH 7.4), 25 mM NaCl and 4% glycerol for 1 h at 37°C. Enzyme activity was evaluated based on the increase in fluorescence at 535 nm (excitation 485 nm).Data are shown as the mean ± SEM, and 2 groups were statistically compared using the Mann–Whitney U test. Statistical comparisons among more than 2 groups were conducted using the Kruskal − Wallis test followed by the Steel − Dwass test. The incidence of IAs was analyzed by Fisher’s exact test. A p value smaller than 0.05 was defined as statistically significant.In this study, we used the rodent model of IAs to examine the contribution of TNF-alpha signaling to IA formation. In this model, IA was induced through increased hemodynamic stress, a trigger of IAs in humans -17, loadThe immunoreactivity of TNF-alpha was remarkably increased in IA lesions compared with control intracranial arteries, especially in endothelial cells and the outer membrane, and the difference in relative intensity of positive signals in IA walls was statistically significant  Figure A. ConsisNext, to clarify the involvement of TNF-alpha signaling in IA formation, we used mice deficient in its receptor, TNFR1, based on the finding that TNFR1 was abundantly expressed in intracranial arteries (data not shown). As a result, 15 out of 22 wild-type mice, 1 out of 7 TNFR1-heterozygous, and 1 out of 8 TNFR1-deficient mice, had IAs at right ACA-OA bifurcations. Statistically, the incidence of IA was significantly suppressed in TNFR1-heterozygous and TNFR1-deficient mice compared with wild-type mice  , and confirmed the suppression of NF-kappaB activation under TNFR1 deficiency (Figure 6 residueTaken together, these results suggest a crucial role for TNF-alpha-TNFR1 signaling in IA formation, by regulating inflammatory responses through the induction of NF-kappaB activation and subsequent macrophage infiltration into intracranial arteries.In this study, we first revealed increased TNF-alpha content in IA lesions during IA formation and progression. Second, we clarified a crucial role for TNF-alpha-TNFR1 signaling in IA formation by regulating inflammatory responses via NF-kappaB activation, using TNFR1-deficient mice. Based on these experimental findings, we propose TNF-alpha-TNFR1 signaling as a potential therapeutic target for IAs.A degenerative change in the tunica media is the histological feature of IA walls. Because the degenerative change in the tunica media in IA walls decreases arterial stiffness and, as a natural consequence, raises the probability of rupture, the mechanisms regulating the degeneration of media must be understood. In a previous study, the authors revealed the contribution of TNF-alpha to the phenotypic modulation of vascular smooth muscle cells, a major cell component of media . They alIn this study, as well as previous studies using human specimens ,12-14,23In this study, we used rodent models of IA through increased hemodynamic stress to investigate the contribution of TNF-alpha-TNFR1 signaling to the pathogenesis of IA formation and progression. We confirmed an increase in TNF-alpha content in IA lesions in rat models during IA formation. Furthermore, TNFR1 deficiency suppressed IA formation by inhibiting inflammatory responses in IA walls such as NF-kappaB activation, MCP-1 induction, and macrophage infiltration. These results suggest a crucial role for TNF-alpha-TNFR1 signaling in IA formation and progression.ACA: Anterior cerebral artery; IA: Intracranial aneurysm; MMP: Matrix metalloproteinase; OA: Olfactory artery; TACE: Tumor necrosis factor alpha converting enzyme; TNF-alpha: Tumor necrosis factor alpha; TNFR1: Tumor necrosis factor receptor superfamily, member 1a.The authors declare that they have no competing interests.TA conceived the study, carried out the molecular biological experiments, secured grants, and drafted the manuscript. MF carried out the molecular biological experiments. MN and KN participated in the design and coordination of the study. SN conceived the study, secured grants, and participated in the design and coordination of the study. SN also helped to draft the manuscript. All authors read and approved the final manuscript.


This study explores the short-run spillover effects of popular research papers. We consider the publicity of ‘Male Organ and Economic Growth: Does Size Matter?’ as an exogenous shock to economics discussion paper demand, a natural experiment of a sort. In particular, we analyze how the very substantial visibility influenced the downloads of Helsinki Center of Economic Research discussion papers. Difference in differences and regression discontinuity analysis are conducted to elicit the spillover patterns. This study finds that the spillover effect to average economics paper demand is positive and statistically significant. It seems that hit papers increase the exposure of previously less downloaded papers. We find that part of the spillover effect could be attributable to Internet search engines’ influence on browsing behavior. Conforming to expected patterns, papers residing on the same web page as the hit paper evidence very significant increases in downloads which also supports the spillover thesis.A11, C2197K80The online version of this article (doi:10.1186/2193-1801-2-168) contains supplementary material, which is available to authorized users. In the weeks that followed it amassed some 175000 downloads and a global coverage in print, television and online media. Tim Harford of Financial Times dubbed it the ‘smash-hit economics research paper of the summer’. Arguably the whole incident with all its publicity was completely unanticipated, and came as a surprise to everyone involved. If nothing else, an attractive natural experiment came into being.Economics research papers seldom make headlines. As infrequently do they permeate the online community beyond the academic confines. Something quite unlike was on display in July 2011 following the publication of ‘Male Organ and Economic Growth: Does Size Matter?’ [henceforth MOEG]  specification.Qi, tis the number of monthly downloads, ECONi is a dummy for inclusion in the HECER series, MOEGi, t is the treatment and i ∈ {1,…, N} denotes individual papers. The parameter of interest is γ which identifies the average treatment effect on the paper demand. In order to ensure that paper specific unobservables do not drive our results, we also estimate (1) using paper FEs.fwhere k times. Hence the specifications alleviate the issue that the average downloads can be distorted by handful of papers that receive very considerable attention. These are estimated by a linear probability model of the formDue to skewed distribution of downloads we also estimate the probability that a paper is downloaded during a month at least k = 0, which is used to estimate the spillover effect’s tendency to change the probability that a given paper is downloaded at least once. Subsequently different values of k > 0 are employed to determine the cut-off point at which spillover effects are still observable. The parameter of interest is again γ.where notation is same as above. In the baseline specification g Three different specification are utilized: baseline, baseline with time trend and baseline with time trend and FEsh. The first two are given byIn this section we use log data of daily downloads of economics discussion papers. Estimation of the effect of MOEG on daily download patterns is based on RDD.X includes only a constant and weekend dummies. Our time trend specification supplements the baseline model with a third degree polynomial time trend interacted with the treatment dummy.i Our third alternative specification includes paper fixed effects and weekend dummies.In our baseline model, γ and η, and the former captures the effect of MOEG on the average paper downloads. The latter is the treatment for the 4 papers on the same web page. The treatment MOEG takes place on 15th July, and corresponds to its appearance in Marginal Revolution and Freakonomics. Weekend dummies capture the substantial within-week download volatility.The parameters of interest are Due to the rotational behavior of Earth and Helsinki’s location at the GMT+2 time zone, some [local time] Friday [Monday] downloads from Western [Eastern] Hemisphere are recorded at weekends. However, we postulate that these errors largely cancel each other out, and hence that our spillover estimates are insulated by orbital factors.We first describe the download profile of MOEG through July 2011. Subsequently OLS regression estimates with DID and RDD specifications are presented. Then the role of Internet search engines is briefly discussed.j. During the 15th July MOEG was mentioned in both blogs, the total downloads ratcheted up from 240 to 5346. One week from there the daily downloads peaked on 22th July at 24410 after which they started to fall. By the end of the July the rate had declined to 750. The total number of MOEG downloads on July was some 175000 which amounts to 60% of all article and paper downloads at the University of Helsinki within the month. Were it not for the Internet, visibility on this scale could not take place. The graph vividly illustrates the effect of blogosphere, Facebook, Twitter and traditional online media combined. Furthermore, the patterns corroborate with the figures presented in McKenzie and Özler  they are the two largest groups and 2) similar to the economics working paper series the two working paper series consist of preliminary research papers written in English.e For a text book discussion of DID, see Cameron and Trivedi ( Trivedi  pp. 55–5f We have also experimented using humanities and science working papers as controls separately. This does not affect the estimates.g For a text book discussion of RDD see Cameron and Trivedi ( Trivedi , pp. 879h We also estimated a separate model for the 4 papers on the MOEG web page. However, due to the small sample the parameter estimates were insignificant.i The order of the polynomial is chosen by the Akaike Information Criterion.j In McKenzie and Özler (nd Özler  Freakonok The average treatment effect on monthly downloads is obtained by multiplying the daily figures by 17. This is the number of days after the publication of MOEG in July.l The download statistics are aggregated at calendar months. Due to different submission dates within months, the data can be quite noisy. Hence the first month downloads on average represent only 15 days of downloads. However, DID specification accounts for this. We also dropped observations with submission dates on December 2010 and January 2011 since search engines and/or backup procedures added exactly 20 downloads to all papers on the latter month. This January peak can be observed in all papers irrespective of the field or series.


Recent information has shown the importance of the V1/V2 domain as a target in HIV-1 vaccines. This includes the localization of epitopes to potently neutralizing antibodies to this region, the demonstration that binding between a site in V1/V2 and α4β7 receptors facilitates infection in primary CD4-positive T cells, and the demonstration that the presence of antibodies binding to the native form of the V1/V2 domain of HIV-1 gp120 correlated with protection in the RV144 vaccine trial. These results highlight the need to better understand the structure and immunological properties of this region, in order to optimally express the relevant targets in HIV-1 vaccines.The native V1/V2 domain of a clade B sequence (CaseA2) was expressed by fusion to the C-terminus of a 273 aa fragment of the MuLV gp70 domain. This fusion glycoprotein was characterized by SDS-PAGE under various conditions, by radioimmunoprecipitation experiments with a panel of mAbs directed against V1/V2-specific epitopes, and by MALDI-TOF analysis of immunologically fractionated forms.SDS-PAGE analysis of this protein after deglycosylation under non-reducing conditions revealed the presence of a closely migrating doublet, and mass-spec analysis of the immunologically fractionated forms suggested that these consisted of different disulfide-linked conformers. The two forms reacted differentially with a panel of V1/V2-specific antibodies and possessed variable reactivity with different polyclonal sera.These studies identify a conformational heterogeneity in the V1/V2 domain that is due to alternative disulfide bonding patterns. These conformations profoundly affect the immunoreactivity of this region, and will presumably influence their immunogenicity as well. Due to the importance of this region as a target for neutralizing and otherwise protective antibodies, it is likely that methods for resolving this heterogeneity would improve the efficacy of induction of relevant antibodies to this region by HIV Env vaccines.


The immune correlate analysis of the RV144 trial identified antibodies (Abs) targeted at the V1-V2 domain as potentially protective vaccine responses. Reactivity of vaccinee serum Abs with a glycosylated gp70-V1-V2 fusion protein or with certain V2 loop peptides was significantly associated with a lower risk of viral infection. A V2 loop segment approximately comprising amino acid residues 165-178 of gp120 was also identified by pilot vaccinee studies as a key immunogenic region.To identify whether the segment identified by pilot studies coincides with the reagents reactive in the case control study, I mapped the locations of the reagents associated with lower risk of viral infection onto the V1-V2 domain structure. With a single segment identified as associated both with immunogenicity in the pilot studies and protection in the case control study, I calculated the Dayhoff evolutionary sequence distance between the the sequences of this segment in the immunogens and in a panel of V2 loop based reagents used in the case control study, each with associated odds ratios (OR) for risk. OR was then plotted against distance.The same V2 loop segment from positions 165-178 that was specifically most immunogenic in the pilot studies appears to have been associated with protection in the immune correlate analysis. This segment corresponds to the "C" strand of the V1-V2 domain beta-sheet fold, exactly where the broadly neutralizing antibody PG9 binds. Plotting the evolutionary distance between this segment in the RV144 immunogens and synthetic V2 loop based antigens used in the case control study shows that lower risk (lower OR) correlates with greater evolutionary distance.A V2 loop segment from positions 165-178 of gp120 was highly immunogenic in humans. Abs elicited by the RV144 subtype E immunogen displaying segment may have been protective only if they cross-reacted distantly with subtype B.


Systemic vasculitis constitutes a group of rare diseases, characterized by inflammation of the arterial or venous vessel wall, causing stenosis and thrombosis. Treatment of the different type of vasculitis mainly relies on steroids and immunosuppressive drugs. In case of refractory or relapsing diseases, however, a second line of treatment may be required. Anti-TNF-α drugs have been used in this setting during the last 15 years with inconsistent results. We reviewed herein the use of anti-TNF-α therapy in different kind of vasculitis and concluded that, except for Behcet's disease, this therapeutic option has not demonstrated significant improvement in the treatment of vasculitis.TNF- These molecules have also been tested in other autoimmune and inflammatory systemic diseases such as severe vasculitis refractory to conventional treatment but, to date, vasculitis are not included in the list of therapeutic indications of anti-TNF-α agents.Tumour necrosis factor alpha  which is proteolytically cleaved in a soluble form (17 kda) by a TNF-α converting enzyme (TACE) [κB) and Mitogen Activated Protein (MAP) Kinase pathways, as well as proapoptotic genes transcription by the induction of death signal pathways [α induces leukoendothelial adhesion via increased expression of various adhesion molecules, such as E-selectin, Intercellular Adhesion Molecule 1 (ICAM-1), and Vascular Adhesion Molecule 1 (VCAM-1), and mediates tissue leukocyte infiltration through chemokine synthesis [α induces metalloproteinase production and may also participate in endothelial cell death directly via apoptosis or indirectly via neutrophil activation [Two forms of TNF-e (TACE) . These tble form 7 kda by pathways . TNF-α iynthesis . TNF-α itivation .α may play a role in neutrophil “priming” inducing membrane expression of proteinase-3 or myeloperoxidase, which are subsequently recognized by ANCA in ANCA-associated vasculitis (AAV) [α to membrane-associated TNF-α can have an agonistic action, initiating reverse signaling and processes such as apoptosis, cytokine suppression, and cell activation, which could constitute an interesting target in the treatment of vasculitis [α drugs have been developed and are commercially available, 3 consisting in monoclonal antibodies (infliximab (IFX), adalimumab (ADA), and golimumab). IFX is usually used intravenously at 3 to 5 mg/kg every 8 weeks, and ADA and golimumab are used subcutaneously, 40 mg every 2 weeks for the former and 50 mg once a month for the latter. The fourth available drug is a dimer of a chimeric protein genetically engineered by fusing the extracellular ligand binding domain of human tumour necrosis factor receptor 2 (TNFR2/p75) to the Fc domain of human IgG-1 (etanercept (ETN)) and is used subcutaneously at 25 mg twice a week. The last is a humanised Fab fragment conjugated to polyethylene glycol (certolizumab pegol) but has never been used in vasculitis. Monoclonal antibodies and certolizumab are active on the two molecular forms of TNFα, whereas etanercept neutralizes soluble TNF-α only.In addition, TNF-is (AAV) . This cysculitis , 13. To α in this disease are sparse [α treatment in corticodependent GCA patients have been reported, a comparative double blind study was conducted using IFX but was subsequently stopped due to the lack of efficacy on the prevention of relapse [α therapy is not recommended in GCA  suggested some therapeutic efficacy which was confirmed by other studies (90% rate of remission and 60% for sustained remission) [α may constitute an interesting therapeutic option in TA patients who have been unable to achieve or maintain remission with steroids alone or common immunosuppressive agents.TA is characterized by inflammation of large vessels, primarily the aorta and its main branches, resulting in anevrysm formation, vascular stenosis, or occlusion, affecting mainly young women. TA is a chronic idiopathic granulomatous panarteritis, resulting from infiltration of the three layers of the vessel wall by macrophages, T lymphocytes, and natural killer cells , 24. Firmission) –28 (Tablmission) . In summPAN is a rare necrotizing vasculitis complicating hepatitis B virus chronic carriage affecting medium size vessels, whose incidence has declined since the introduction of hepatitis B vaccination and antiviral treatments . The treα [α have been reported with conflicting results, some patients even experiencing severe flare-up of the disease [MC is a small vessel vasculitis involving skin, joints, peripheral nerves, and the kidney, which is mainly associated with hepatitis C, Sjogren's syndrome, or lymphoma . MC is aα . Before  disease , 35. At  disease .AAV is a group of multisystemic diseases characterized by a pauci-immune small vessel vasculitis which includes three different entities: two recently renamed granulomatosis with polyangeitis (GPA), eosinophilic GPA (EGPA), and microscopic polyangeitis (MPA). AAV pathogenesis is consistent with a primary role for neutrophils, which are both the effector cells responsible for endothelial damage via oxygen radical synthesis and enzyme degranulation and the targets of ANCA . Currentα monoclonal antibodies have also been tested in refractory AAV patients. The efficiency of infliximab has been observed in prospective observational studies [n = 9) or rituximab (n = 8) in association with steroids and immunosuppressive drugs in refractory GPA for a median follow-up of 30.6 months (+/−15.4). Efficacy of infliximab and/or rituximab to obtain remission at one year was observed, with an advantage for rituximab. During long-term follow-up, rituximab was also more effective at obtaining and maintaining remission [α. However, this was a noncontrolled study and groups lacked homogeneity [Etanercept was tested initially in an open label trial including 20 relapsing or incompletely controlled GPA and seemed efficient with a 3-point reduction of the Birmingham Vasculitis activating (BVAS) score at 6 months . These r studies , 43. Oneemission . Recentlogeneity . The lasogeneity .α therapy using infliximab may be useful in complement to standard regimen in some refractory AAV patients, but the recent published data on the beneficial effects of rituximab for obtaining remission and preventing relapses in MPA and GPA appear much more convincing and may significantly limit the use of anti-TNF-α treatment in AAV in the future [In conclusion, it seems that a short course anti-TNF-e future , 48  and triggering extrinsic factors is suspected , 50. Firctive BD , 53.α treatments were first tested for severe eye involvement showing promising results [In 2001, anti-TNF- results . A meta- results . A recen results . Interes results .Five open prospective studies demonstrated a beneficial effect of long-term infliximab treatment on the prevention of relapse, maintenance of visual acuity, and immunosuppressive drugs tapering –61 Tabl. Intraviα treatments were tested in severe refractory cutaneous manifestations, especially etanercept which revealed a significant efficacy compared to placebo on oral ulcers, and nodular lesions [Anti-TNF- lesions .Infliximab in monotherapy was also tested in refractory entero-Behcet during a prospective open trial and showed 100% improvement on clinical, CT-scan, and colonoscopy . Adalimuα failure in refractory BD, a switch of molecule was made in up to 25% of cases with a 70% improvement [In addition, infliximab has been used in BD affecting the central nervous system (CNS) in open prospective studies and was almost always successful , 70. Therovement .In summary, infliximab seems effective in induction treatment and relapse prevention in severe BD refractory to glucocorticoids or immunosuppressive drugs, especially in case of eye involvement. It could also be an alternative therapy to immunosuppressive drugs in case of central nervous system or gastrointestinal manifestations. Nevertheless, the main limitation of the present analysis is that most information originated from limited cases or noncontrolled studies, strongly raising the need for properly randomized controlled clinical trials.α are often prescribed as second/third line treatments in patients already immunocompromised by long-term use of glucocorticoids and immunosuppressive drugs. Despite this fact, side effects have been reported up to 46% and are mostly moderate. The literature review suggests that these drugs are rather safe, in agreement with what is known in RA and spondyloarthropathy [In vasculitis, anti-TNF-hropathy , 74.α are prone to develop soon after initiation bacterial and viral infections mostly affecting respiratory or urinary tracts and cutaneous or soft tissues. Furthermore, reactivation of latent tuberculosis or extrapulmonary forms of this infection is another well-known threat [Patients treated with anti-TNF-n threat –81.α therapy in carcinogenesis and tumor progression remains a matter of controversy. A large study assessed the risk of cancer in a RA cohort treated with anti-TNF-α and showed a relative risk (RR) of 1.00 (95% CI: 0.86–1.15) compared to the biotherapy naïve RA cohort. RR did not increase with longer exposure or with the cumulative duration of active anti-TNF-α therapy during a 6-year follow-up period [α therapy in RA and nonmelanoma skin cancers with a follow-up period of 3 years [n = 6) who developed cancer also received standard therapy associating MTX or CYC, which are known to be involved in cancerogenesis [The role of anti-TNF-p period . Regardip period . One obs 3 years . In vascogenesis . Withoutα treatments can induce acute infusion reaction, which may lead to discontinuation of the treatment. Anti-TNF therapy may also favor antinuclear antibodies appearance which are, however, weakly associated with clinical symptoms [α treatments, such as the occurrence of demyelinating and sarcoid-like granulomatous diseases [Besides infection and malignancy, anti-TNF-symptoms , 86. Othdiseases .α treatments in vasculitis did not demonstrate the same efficacy as in other inflammatory diseases such as RA. Nevertheless, anti-TNF-α should be used in the treatment of BD refractory to immunosuppressive drugs, especially in case of ocular, CNS, or digestive tract involvement. Some interesting data are also available for the use of anti-TNF-α treatments in refractory AAV, but the recent reports on rituximab efficacy in these diseases and the relative innocuity of this drug may limit the use of anti-TNF-α in these diseases, in the future. In both BD and AAV, an anti-TNF monoclonal antibody especially infliximab should be preferred to etanercept. In addition, physicians must be aware of the risk of infection using these drugs, especially in patients already immunocompromised by previous treatments. Regarding other vasculitis, published data are not in favor of efficiency of anti-TNF-α which, therefore, should not be used.Anti-TNF-


Accumulating experimental evidence suggests that TNFR2 plays an important role in renal disorders associated with acute cellular rejection and clear cell renal carcinoma but its exact role in these settings is still not completely understood. This papers reviews the factors that may mediate TNFR2 induction in acute cellular rejection and clear cell renal carcinoma and its contribution to these conditions and discusses its therapeutic implications. A greater understanding of the function of TNFR2 may lead to the development of new anti-TNF drugs.Tumor necrosis factor receptor 2 (TNFR2) is a type I transmembrane glycoprotein and one of the two receptors that orchestrate the complex biological functions of tumor necrosis factor (TNF, also designed TNF- It can transduce signalling both as a membrane-integrated protein and as a soluble cytokine released after proteolytic cleavage; its soluble form is a noncovalently bound trimer of 17 kDa components [Tumor necrosis factor  involves the interplay between mechanisms that maintain tolerance to the graft and factors that promote rejection . Cytokinde novo malignancy is 100-fold increase compared with the healthy nontransplantation population [Renal cell carcinoma (RCC), a TEC malignancy, is the most common type of kidney cancer in adults, responsible for approximately 80% of cases . A clearpulation –30. Up tpulation , 30. Curpulation  and in cpulation , with TNligand passing” [TNFR2 signaling pathways are illustrated in passing” . Howeverpassing” .κB (NFκB) and the activator protein 1 (AP-1) [κB is a critical transcription factor that plays an important role in cell growth, survival, and immune regulation. AP-1 is a heterodimeric transcription factor, the most common form consisting of c-Jun and c-Fos [κB and c-Jun N-terminal kinase (JNK) activation [κB and JNK activation by TNF treatment [κB and JNK activation despite its high TRAF2 binding capability. This may be attributed to a novel carboxyl-terminal TRAF2-binding site (T2bs-C) that prevents the delivery of activation signals from the conventional TRAF2 binding site (T2bs-N) [κB activation [κB involves RIPK1 [rip−/− mice (lacking RIPK1) show no NFκB activation in response to TNF, and TNFR2 was identified as the primary receptor that induced IκBα degradation, the initiating event in NFκB activation [κB activation and induced cyclin D1 instead [κB cannot be exclusively relied on as a marker for signalling via TNFR2 as ligand-bound TNFR2 can activate NFκB in only some cell types. Following ligation of TNFR2, the downstream signaling effects can depend on which of the genes NFκB transcribes. Although the effect is most frequently proliferation, it can include differentiation, cytokine production and apoptosis. TNFR2 activation can induce cell death in T lymphocytes [in situ, where downstream signalling has been shown to mediate reno- and cardioprotection [Transduction of extracellular signals by TNFR2 occurs via recruitment of cytosolic proteins to its intracellular domain as a result of the conformational changes induced by ligand binding . The lig1 (AP-1) . NFκB isnd c-Fos . Howevertivation . Only whreatment . Thus, i(T2bs-N) . Howevertivation . Anotheres RIPK1 . Indeed,tivation . In keep instead . Regardlphocytes ; howeverphocytes , probablphocytes . TNFR2 cphocytes , 18, 47.phocytes , 47 resuphocytes . Thus, potection , 18.κB, AP-1, IRF, and GAS [TNF is a mediator of tissue injury associated with allograft rejection after renal transplantation . TNF gen and GAS . Interac and GAS , 53, 55. and GAS , 18, 56. and GAS . The induction of TNFR2 protein and mRNA in TECs in allograft rejection strongly suggests that it is an important receptor in mediating and/or regulating the rejection process. TNFR2 has been shown to participate in the process of xenograft rejection in pigs  and its γ (IFN-γ), and chemokines which mediate activation and recruitment of leukocytes within the graft tissue as a hallmark of allograft rejection [in situ in tubulitis associated with ACR and may be instrumental in inducing TNFR2 in TECs via production of TNF [Renal TECs, the predominant parenchymal cell type in the kidney and the principal target of renal injury, can be induced to synthesize/secrete inflammatory mediators including TNF, interleukin (IL)-6 and -10, interferon-ejection –64. The n of TNF , 18. in situ by TECs and colocalizes with TNFR2 during ACR [in vivo and in vitro [Another factor that may regulate TNFR2 induction in TECs is TNF-converting enzyme  synthesized ring ACR . TACE acring ACR , allowinring ACR . Indeed,ring ACR  and renaring ACR . The solin vitro . κB-independent pathways [in situ. Another important mediator of TNFR2 induction in TECs is TL1A , the principal ligand for DR3 , 68. TL1pathways . Hence, in vivo and in vitro studies using a TNFR2-specific mutein and TNFR2-deficient cells have confirmed that Etk is a TNFR2-specific kinase involved in cell adhesion, migration, and survival [TNFR2 protein and mRNA can also be induced in organ culture of renal allograft rejection treated with wild type TNF or a TNFR2-selective mutein , resultisurvival . CollectRCC is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene . A mutatκB and promotes cell cycle entry (indicated by expression of nuclear phospho-Histone H3S10 and ki67)  , leadingin vitro data [κB activation which may halt cell death. Indeed, lack of pVHL protein confers RCC cell resistance to the cytotoxic effects of TNF, which is restored upon reconstitution of the VHL protein, with a concordant expression of NFκB target anti-apoptotic genes such as A20, c-FLIP, Survivin, c-IAP-1, and cIAP-2, blocking the activities of caspases-8 and -3 [Signals induced by TNFR2 in malignant TECs are distinct from the signals induced by VEGFR2, as in contrast to TNF, VEGF does not induce Etk activation or cell cycle entry . Moreovetro data  whereby tro data . The fin8 and -3 . 196MET) to arginine (TNFR2196Arg) has been associated with increased risk of chronic inflammatory disorders [196Arg variant in genetically modified cells cause diminished recruitment of TRAF2 to TNFR2196Arg resulting in impairment of TNFR2-mediated NF-κB activation and TNFR1-induced cell death [Single nucleotide polymorphism (SNP) in the TNFR2 gene can predict outcome in other forms of cancer and autoimmune disease –85. A SNisorders , 84, 86.ll death . Collectll death . Moreovell death  and the ll death , 90. A model of the contribution of different cell populations to the induction of TNFR2 in TECs in ACR and ccRCC is presented in Anti-TNF drugs such as infliximab, etanercept, adalimumab, certolizumabpegol, and golimumab have been used for treatment and management of various autoimmune diseases –93; howein vitro systems clearly demonstrate an association of TNFR2 with ACR and ccRCC, suggesting that this receptor may play a crucial role in mediating cellular responses in these disorders. Thus, new therapeutic strategies to either activate TNFR2 signalling in ACR (to facilitate tubular regeneration) or to selectively inhibit TNFR2 signalling in ccRCC  may be a better approach in the treatment of these conditions than global blockade of TNF. Clearly, future clinical studies are warranted to address the prognostic and therapeutic role of TNFR2 in clinical nephrology.Experimental data from human organ culture and in


It has become widely accepted that whenever animals are used in scientific procedures, the 3Rs principle of replacement, reduction and refinement described by William Russell and Rex Burch should be adhered to. Animals should be replaced with non-sentient alternatives if possible, the number of animals used should be reduced and experimental procedures should be refined to minimise pain, suffering and distress. Administration of analgesic agents to animals undergoing surgical procedures is a refinement used to alleviate pain. In this study, a structured literature review was carried out to examine current trends in analgesic administration to rabbits undergoing experimental surgical procedures.P < 0.001). Papers that reported ethical approval were more likely than papers that did not specify approval to report systemic analgesic administration (P < 0.001). When systemic analgesics were administered, buprenorphine was the most frequently used agent and non-steroidal anti-inflammatory drugs were used less frequently than opioids in both time periods.128 papers from 51 peer-reviewed journals were selected for inclusion in this review. Reporting administration of systemic analgesia to rabbits in peer-reviewed scientific papers increased significantly from 16% to 50% between 1995-1997 and 2005-2007  changing the timing of analgesic administration by giving systemic analgesics pre- or perioperatively rather than only postoperatively, 2) using multimodal analgesia when pain is likely to be moderate to severe and 3) increasing the use of non-steroidal anti-inflammatory drugs and use of other techniques such as epidural analgesia particularly for orthopaedic procedures. The 3Rs principle of replacement, reduction and refinement has become widely accepted, and is incorporated into the legislation governing the use of animals in several EU member states and throughout the world. This study was undertaken to gain an indication as to how widely one clear refinement, administration of analgesics to prevent pain in animals undergoing surgery, had been implemented in rabbits.When used in biomedical research, rabbits are most frequently used in toxicity studies , for antThe overall aim of this study was to follow up on a smaller previous study  and exam128 papers, 64 from each time period (1995-1997 and 2005-2007) were selected from 51 journals for inclusion in this review  from 16% to 50% between the two time periods. Buprenorphine was the most commonly reported systemic analgesic administered in both 1995-1997 (60%) and in 2005-2007 (70%)  was reported in one paper which from 1995-1997 where flunixin was administered (e.g. 3% of rabbits undergoing orthopaedic procedures receiving a NSAID). From 2005-2007, 5 (13.5%) of the papers describing orthopaedic procedures reported NSAID administration: carprofen (n = 3), metamizol (n = 1) and paracetamol (n = 1).When procedures were classified according to their potential to be painful, the number of procedures in each category did not differ significantly between 1995-1997 and 2005-2007. In both time periods, rabbits that underwent the most potentially painful procedures were not more likely to receive systemic analgesia than rabbits that underwent less potentially painful procedures. For example, in 1995-1995 22% of papers describing the least painful procedures (craniotomies and skin incisions) compared to 13% of papers describing the most painful procedures (thoracotomies and orthopaedic procedures) reported systemic analgesic administration. Similarly in 2005-2007, 44% of papers describing the least painful procedures compared to 51% describing the most painful procedures reported systemic analgesic administration.When systemic analgesics were reported, all papers from both time periods specified the timing of administration. In 1995-1997 20% of systemic analgesics were administered perioperatively compared to 80% postoperatively. In 2005-2007 9% of systemic analgesics were administered preoperatively, 9% perioperatively, 66% postoperatively, 6% both preoperatively and postoperatively and 9% both perioperatively and postoperatively.P = 0.015) and were commonly combined with the use of a systemic analgesic, whereas use of local anaesthetic agents decreased significantly between time periods (38% to 6%) (P = 0.001)  was similar in the two time periods (83% in 1995-1997 and 87% in 2005-2007). The use of 'agents with analgesic properties' are summarised in Table None of the papers reported the administration of an opioid analgesic in combination with a non-steroidal anti-inflammatory drug and only one paper published between 2005 and 2007 reported the use of two systemic analgesic agents: buprenorphine and fentanyl. When systemic analgesic agents were used, they were always combined with an anaesthetic regimen with analgesic properties in both time periods (Table P < 0.001). 44% of papers from 1995 to 1997 compared to 95% of papers from 2005 to 2007 specified that ethical approval was obtained. Overall, papers that reported ethical approval were also more likely to report the administration of systemic analgesic agents (P < 0.001).There was a significant increase in the number of papers reporting ethical approval between time periods (The majority of the papers (96%) reported the number of animals used. In both time periods the median number of animals used was similar ; 2005-2007: median = 23, range = . Male rabbits were more commonly used than females. From 1995-1997 the gender reported was 34% , 22% , 10%  and 34% (unspecified). From 2005-2007 the gender reported was 43% , 18% , 4%  and 35% (unspecified). Neither the gender of rabbits nor the number of animals used differed significantly between time periods.The reporting of administration of systemic analgesic drugs to laboratory rabbits undergoing surgical procedures is increasing. Unfortunately, even in papers published between 2005 and 2007, not all rabbits undergoing potentially painful surgical procedures received a systemic analgesic agent.Guidelines on refining experimental surgical procedures involving rabbits summarized from several sources are presented in Table When systemic analgesic administration was reported the choice of the agent administered was generally appropriate for rabbits Tables  and 4. OWhen doses of systemic analgesics were reported, they were generally within the recommended range Tables  and 4. OThere was no evidence that rabbits that underwent the potentially most painful procedures were more likely to systemic analgesia than rabbits that underwent less potentially painful procedures in either time period. This is a significant welfare concern as analgesic administration should be matched to the severity of the experimental procedure. Although there was no relationship between probability of receiving a systemic analgesic and the severity of the experimental procedures, there was a relationship between reporting systemic analgesic administration and in reporting ethical review permissions. Papers that reported ethical approval were more likely to report systemic analgesic administration. This may represent a positive influence of local ethical review or differences in animal care regulation.The administration of systemic analgesic agents was rarely reported to occur pre- or perioperatively and systemic analgesics were usually administered postoperatively in both time periods. When analgesics are only administered post-operatively, there is a delay until the agent reaches an effective concentration and therefore a period of time where the animal is likely to experience pain postoperatively . AdminisAlthough the majority of papers in both time periods reported some form of analgesic provision, analgesia was frequently only in the form an anaesthetic agent with analgesic properties Table . This isMultimodal analgesia is recommended in cases when pain is likely to be moderate to severe ,14, as tThe inclusion of animal details within the methods sections of peer-reviewed journals is important as it enables replication of studies, allows readers to judge the scientific quality of the work and because it addresses public concerns about the use of animals in research . The majPeer-reviewed papers were less likely to report the gender of rabbits than the number of animals used in the study. In both time periods included in this review 34% of papers did not report the gender of rabbits used. Similarly 38% of chronic experimental studies published in 2000 in a range of laboratory species did not specify the gender of experimental animals used .Although reporting the administration of analgesic agents to rabbits undergoing potentially painful procedures increased between the two time periods examined, only half of papers published between 2005 and 2007 specified the use of a systemic analgesic agent. The literature review highlights some areas where practices in rabbit analgesia could be further improved including: routine administration of systemic analgesics, administration of systemic analgesic agents pre- or perioperatively, use of multi-modal analgesia and increased administration of non-steroidal anti-inflammatory drugs and use of other techniques such as epidural analgesia, particularly for orthopaedic procedures.http://www.sciencedirect.com was used to identify relevant studies published in English from 1995 to 1997 and from 2005 to 2007. ScienceDirect was accessed between 12-06-2008 and 30-07-2008. The search terms "skin incision"/"surgery" and "rabbit" were used and only papers that were available in electronic format at Newcastle University were selected. The materials and methods section of each paper was screened to identify whether the inclusion criteria were met.The ScienceDirect search engine The inclusion criteria were as previously described . A paperBecause the number of rabbits used in scientific research is decreasing, the number of the papers from 2005 to 2007 that met the inclusion criteria was the limiting factor in determining how many papers to include in the review. All of the papers from 2005 to 2007 that met the inclusion criteria were included in the review and the same search strategy was used to locate papers from 1995 to 1997. Each paper published from 1995 to 1997 was given a number based on the ScienceDirect display and a random number generator was then used to select the number of papers equivalent to the number of papers that met the inclusion criteria ten years earlier.Classification of procedures was as previously described  modified2 agonist (medetomidine or xylazine) were classified as having 'an anaesthetic regimen with an analgesic component' [Anaesthetic agents were also classified according to whether they contained an analgesic component. Rabbits anaesthetised with ketamine and/or an alphamponent' . Similarmponent' . With thmponent' The gender of the rabbits and the total number of animals used in each study were also noted. Specification of ethical review permissions  were also noted.P < 0.05 was considered statistically significant.All statistical analyses were conducted using SPSS software . A two-tailed Mann-Whitney test was used to compare the number of rabbits used in each study between time periods. Chi-squared analyses were used to compare: systemic analgesic administration, use of anaesthetic regimens with analgesic components, local anaesthetic use, use of any analgesic agent , the severity of experimental procedures, number of papers reporting ethical approval and the gender of rabbits used between time periods and to compare whether ethical approval was reported. A value of CAC was involved in the study design, carried out the data collection and assisted with data analysis. PAF was involved in the study design and drafting the paper. MCL was involved in the study design. CAR was involved in the study design, carried out the data analysis and drafted the manuscript. All authors read and approved the final manuscript.Journals included in this study. A list of journals included in this study.Click here for file


TNF-a promoter region, such as 308G/A and 238G/A, could affect the risk of various types of cancer by regulating TNF-a production. In this study, a meta-analysis was performed to investigate the association between common polymorphisms of TNF-a promoter region and colorectal cancer susceptibility.Tumor necrosis factor-alpha (TNF-a) was related to inflammation and involved in the development of colorectal cancer. Polymorphisms located in TNF-a polymorphisms and colorectal cancer. Summary odds ratios (ORs) with their 95% confidence intervals (95% CIs) were calculated using random-effects models. Stratified analyses based on ethnicity and control population source were also conducted.Searching of several databases was performed for all publications on the association between TNF-a 308A polymorphism showed a significant association with increased risk of colorectal cancer in worldwide populations under homozygote comparison  other than heterozygote comparison . TNF-a 238A was not associated with colorectal cancer risk under homozygote or heterozygote comparisons. In stratified analysis, significant association was observed only in Western populations  other than in Eastern populations under homozygote comparison. No significant difference was observed between population-based subgroup and hospital-based subgroup.Overall, TNF-a 308A was moderately associated with an increased risk of colorectal cancer in Western populations, and TNF-a 238A polymorphism was not significantly associated with colorectal cancer risk. Colorectal cancer is the third most commonly diagnosed cancer and the fourth leading cause of cancer-related death worldwide Recently, a number of risk factors for colorectal cancer were identified Biological and epidemiological studies indicated a clear association between chronic inflammation and colorectal cancer TNF-a is regulated by the promoter region of TNF-a gene, many studies have shown that polymorphisms located in TNF-a promoter region  could regulate TNF-a production, thus affecting the risk of cancers Tumor necrosis factor-alpha (TNF-a) is the most important proinflammatory cytokine involved in cell growth, differentiation, and apoptosis TNF-a 308 polymorphism was confirmed as a risk factor for a range of cancers by meta-analysis, such as gastric, breast and hepatocellular cancers TNF-a 308 polymorphism claimed that it was not significantly associated with risk of colorectal cancer TNF-a 308 polymorphism is associated with colorectal cancer remains inconclusive. Therefore another well-conducted, independent review of this issue was required to get a definitive conclusion.Recently, For the association between colorectal cancer risk and other SNPs such as 238, 857, 863 and 1031, no integrated analysis has been made. In light of the heterogeneity of each published study, none of the work could achieve a reliable and consistent conclusion.TNF-α polymorphisms and colorectal cancer, and a meta-analysis containing 3372 cases and 4523 controls was conducted to investigate whether TNF-α 308, TNF-α 238 promoter polymorphisms were associated with the risk of colorectal cancer.In this article, we gathered data from all previous studies of TNF-a polymorphisms and the risk of colorectal cancer was performed, using the following terms in the MEDLINE, PubMed, EMBASE:  AND  AND . All articles were updated on Oct 25, 2013. References of all research and review articles were reviewed for additional references. Searching was conducted by two independent researchers to make sure that no published papers were missed.This meta-analysis was performed according to the PRISMA guideline  usable genotype frequencies in cases and controls provided; and (iii) reporting outcomes and risk estimates and/or presentation of data necessary for calculating ORs with 95% CIs. We excluded studies overlapping with other studies or overlapping with data from the same authors.Included studies met the following criteria: (i) case–control studies on the association between TNF-a 308, TNF-a 238 polymorphisms and the risk of colorectal cancer were 14, 4, respectively. Most of the studies used frequency-matched controls to cases by age and sex.Two researchers extracted the data independently. Items of the author’s last name, year of publication, country of origin, source of the study population, genotypes and numbers of cases and controls were extracted. The numbers of studies on the association between TNF-a polymorphisms and risk of colorectal cancer under homozygote comparison and heterozygote comparison. Random-effects models were used to calculate overall summary ORs and 95% CIs. Study populations were classified as Western (Europe and America) or Eastern .The Hardy–Weinberg equilibrium (HWE) was tested to compare the observed genotype frequencies with expected genotype frequencies in controls of all studies. ORs and 95% CIs were calculated to assess the strength of association between p<0.05 was considered as statistically significant. The Q-statistic was calculated to examine result heterogeneity among studies, and p<0.1 was considered significant. The 2I-statistic was also calculated to efficiently test heterogeneity 2I<25%, 25–75% and >75% considered to represent low, moderate and high degree of inconsistency, respectively. Begg’s funnel plot was plotted to examine the underlying publication bias The significance of the summary ORs was determined by the Z-test, in which two-sided TNF-a polymorphisms and risk of colorectal cancer, and a total number of 3372 cases and 4523 controls were included. Detailed screening process was shown in TNF-a 308 and TNF-a 238, which were reported in 14 and 4 studies, respectively TNF-a 857, TNF-a 863, and TNF-a 1031 were also investigated in this comprehensive searching, but the corresponding sample sizes were too small to perform quantitative data synthesis. Genotype and allele distributions of TNF-a 308 and TNF-a 238 were shown in TNF-a 308A allele were 15.0% in Western populations and 7.1% in Eastern populations. Corresponding frequencies for TNF-a 238A allele were 3.5% and 7.1%, respectively.In this article, 15 studies were identified to evaluate the relationship between TNF-a 308G/A polymorphism, 14 studies with a total number of 2837 cases and 3601 controls were included in this analysis. TNF-a 308 polymorphism under homozygote comparison . TNF-a 308 polymorphism under heterozygote comparison .For TNF-a 308 and colorectal cancer risk was observed under homozygote comparison , but not heterozygote comparison . For Eastern populations, no significant association was found under homozygote or heterozygote comparison. Subgroups characterized by control population source , and neoplasm location (colon or rectum) were also analyzed with the same method as above  = 1.23 (0.24–6.23); TNF-a 308 instead , and no variation for TNF-a 238 .For the overall meta-analysis, the Q-statistic was significant and TNF-a 308, 2I showed moderate variations in both Eastern  and Western populations . For PB and HB subgroups, 2I showed no variation  and a strong variation , respectively, which indicated that hospital-based studies might be important source of heterogeneity. Moreover, variation in the frequency of this TNF-a 308 polymorphism among the populations or some unidentified factors may also be the source of heterogeneity.In the stratified analyses of TNF-a 308, the corresponding pooled ORs were not changed significantly , indicating that our results were statistically robust. Sensitivity analysis was not performed for TNF-a 238 because there were only 4 studies included.To further strengthen our conclusions, the sensitivity analysis was performed. A single relatively small study involved in the meta-analysis was excluded each time. For TNF-a polymorphisms and colorectal cancer. No evidence for bias was detected in TNF-a 308 polymorphism under homozygote comparison  is also a powerful methodology in genetic studies. As for GWAS studies of colorectal cancer Several limitations of this study should also be addressed. Firstly, the sample size was relatively small for stratified analyses, which weakened our conclusions, especially under homozygote comparison. Therefore more studies were needed to be included to obtain a more reliable result. Secondly, detailed information of individual level was lacking in this analysis, for which many stratified analyses were not able to be performed. If individual raw data were available, effect induced by age, gender, drug use and other environmental factors could also be investigated. Thirdly, cytokines, such as interleukins, transform growth factor, tumor necrosis factor, may exert interacting functions with each other, so SNPs of other cytokines should also be taken into account to conclude a true effect if possible. Fourthly, results of GWAS studies were not included because the raw data had not been published. Additionally, gene-gene, gene-phenotype, and gene-environment interactions should be considered.TNF-a polymorphisms and colorectal cancer, and indicated that TNF-a 308 was moderately associated with an increased risk of colorectal cancer in Western populations. TNF-a 238 polymorphism was not significantly associated with colorectal cancer risk. Additionally, large well-designed cohort studies would warrant to confirm this conclusion, and to fully understand the molecular mechanism of colorectal cancer. Further prospective studies in combination with analyses of other cytokines and environmental factors are also required.In summary, this meta-analysis had pooled all the available data related to Table S1PRISMA Checklist of items to include when reporting a systematic review or meta-analysis.(DOC)Click here for additional data file.