Distinct Secondary Structures of the Leucine-Rich Repeat Proteoglycans Decorin and Biglycan: Glycosylation-Dependent Conformational Stability

Biglycan and decorin, closely related small leucine-rich repeat proteoglycans, have been overexpressed in eukaryotic cers and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans. A comparative biophysical study of these glycoforms has revealed that the overall secondary structures of biglycan and decorin are different. Far-UV Circular Dichroism (CD) spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly Beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to. the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the fmal form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that at least in this specific domain, the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provided further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1-2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1-6 M urea, probably indicating the presence of stable unfolding intermediates

Association of CD40 with rheumatoid arthritis confirmed in a large UK case-control study

OBJECTIVE: A recent meta-analysis of published genome-wide association studies (GWAS) in populations of European descent reported novel associations of markers mapping to the CD40, CCL21 and CDK6 genes with rheumatoid arthritis (RA) susceptibility while a large-scale, case-control association study in a Japanese population identified association with multiple single nucleotide polymorphisms (SNPs) in the CD244 gene. The aim of the current study was to validate these potential RA susceptibility markers in a UK population. METHODS: A total of 4 SNPs (rs4810485 in CD40, rs2812378 in CCL21, rs42041 in CDK6 and rs6682654 in CD244) were genotyped in a UK cohort comprising 3962 UK patients with RA and 3531 healthy controls using the Sequenom iPlex platform. Genotype counts in patients and controls were analysed with the chi(2) test using Stata. RESULTS: Association to the CD40 gene was robustly replicated (p=2 x 10(-4), OR 0.86, 95% CI 0.79 to 0.93) and modest evidence was found for association with the CCL21 locus (p=0.04, OR 1.08, 95% CI 1.01 to 1.16). However, there was no evidence for association of rs42041 (CDK6) and rs6682654 (CD244) with RA susceptibility in this UK population. Following a meta-analysis including the original data, association to CD40 was confirmed (p=7.8 x 10(-8), OR 0.87 (95% CI 0.83 to 0.92). CONCLUSION: In this large UK cohort, strong association of the CD40 gene with susceptibility to RA was found, and weaker evidence for association with RA in the CCL21 locus

Corrigendum: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis

B cells are implicated in rheumatoid arthritis (RA) based on the presence of autoantibodies and the therapeutic response to B cell depletion. IL-21 has a significant role in B cell development and function. Here we assess B cell responses to IL-21 and the mechanisms responsible for altered IL-21R expression in RA. Flow cytometry of PBMC and cultured B cells was used to quantify protein and mRNA levels of IL-21R, IL-21 signaling through pSTAT3, specificity protein 1 (SP1) and to determine cytokine production (IL-6) and maturation status of B cells in RA and healthy control subjects. SP1 binding to the IL21R promoter region in B cells was assessed with ChIP-qPCR. We demonstrate an increase in IL-21R expression in total and memory B cells from RA subjects, which correlated with responsiveness to IL-21 stimulation. Stimulation of naïve RA B cells with IL-21 and CD40L resulted in an increase in differentiation into plasmablasts and an increase in IL-6 production in comparison to healthy controls, which was dose dependent on IL-21 stimulation. IL-21R expression on memory B cells in RA synovial fluid was comparable to peripheral blood making our study pertinent to understanding B cell responses in the joint and site of inflammation. We identified an increase in SP1 protein and mRNA in RA B cells and demonstrate an increase in binding of SP1 to the IL21R promoter region, which suggests a mechanism by which IL-21R expression is enhanced on B cells in RA. Taken together, our results indicate a mechanism by which IL-21 enhances B cell development and function in RA through an SP1 mediated increase in IL-21R expression on B cells

Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis

B cells are implicated in rheumatoid arthritis (RA) based on the presence of autoantibodies and the therapeutic response to B cell depletion. IL-21 has a significant role in B cell development and function. Here we assess B cell responses to IL-21 and the mechanisms responsible for altered IL-21R expression in RA. Flow cytometry of PBMC and cultured B cells was used to quantify protein and mRNA levels of IL-21R, IL-21 signaling through pSTAT3, specificity protein 1 (SP1) and to determine cytokine production (IL-6) and maturation status of B cells in RA and healthy control subjects. SP1 binding to the IL21R promoter region in B cells was assessed with ChIP-qPCR. We demonstrate an increase in IL-21R expression in total and memory B cells from RA subjects, which correlated with responsiveness to IL-21 stimulation. Stimulation of naïve RA B cells with IL-21 and CD40L resulted in an increase in differentiation into plasmablasts and an increase in IL-6 production in comparison to healthy controls, which was dose dependent on IL-21 stimulation. IL-21R expression on memory B cells in RA synovial fluid was comparable to peripheral blood making our study pertinent to understanding B cell responses in the joint and site of inflammation. We identified an increase in SP1 protein and mRNA in RA B cells and demonstrate an increase in binding of SP1 to the IL21R promoter region, which suggests a mechanism by which IL-21R expression is enhanced on B cells in RA. Taken together, our results indicate a mechanism by which IL-21 enhances B cell development and function in RA through an SP1 mediated increase in IL-21R expression on B cells

PADI4 genotype is not associated with rheumatoid arthritis in a large UK Caucasian population

BACKGROUND: Polymorphisms of the peptidylarginine deiminase type 4 (PADI4) gene confer susceptibility to rheumatoid arthritis (RA) in East Asian people. However, studies in European populations have produced conflicting results. This study explored the association of the PADI4 genotype with RA in a large UK Caucasian population. METHODS: The PADI4_94 (rs2240340) single nucleotide polymorphism (SNP) was directly genotyped in a cohort of unrelated UK Caucasian patients with RA (n=3732) and population controls (n=3039). Imputed data from the Wellcome Trust Case Control Consortium (WTCCC) was used to investigate the association of PADI4_94 with RA in an independent group of RA cases (n=1859) and controls (n=10 599). A further 56 SNPs spanning the PADI4 gene were investigated for association with RA using data from the WTCCC study. RESULTS: The PADI4_94 genotype was not associated with RA in either the present cohort or the WTCCC cohort. Combined analysis of all the cases of RA (n=5591) and controls (n=13 638) gave an overall OR of 1.01 (95% CI 0.96 to 1.05, p=0.72). No association with anti-CCP antibodies and no interaction with either shared epitope or PTPN22 was detected. No evidence for association with RA was identified for any of the PADI4 SNPs investigated. Meta-analysis of previously published studies and our data confirmed no significant association between the PADI4_94 genotype and RA in people of European descent (OR 1.06, 95% CI 0.99 to 1.13, p=0.12). CONCLUSION: In the largest study performed to date, the PADI4 genotype was not a significant risk factor for RA in people of European ancestry, in contrast to Asian populations

The SPORTSMART study: a pilot randomised controlled trial of sexually transmitted infection screening interventions targeting men in football club settings

Background: Uptake of chlamydia screening by men in England has been substantially lower than by women. Non-traditional settings such as sports clubs offer opportunities to widen access. Involving people who are not medically trained to promote screening could optimise acceptability. Methods: We developed two interventions to explore the acceptability and feasibility of urine-based sexually transmitted infection (STI) screening interventions targeting men in football clubs. We tested these interventions in a pilot cluster randomised control trial. Six clubs were randomly allocated, two to each of three trial arms: team captain-led and poster STI screening promotion; sexual health adviser-led and poster STI screening promotion; and poster-only STI screening promotion (control/comparator). Primary outcome was test uptake. Results: Across the three arms, 153 men participated in the trial and 90 accepted the offer of screening (59%, 95% CI 35% to 79%). Acceptance rates were broadly comparable across the arms: captain-led: 28/56 (50%); health professional-led: 31/46 (67%); and control: 31/51 (61%). However, rates varied appreciably by club, precluding formal comparison of arms. No infections were identified. Process evaluation confirmed that interventions were delivered in a standardised way but the control arm was unintentionally ‘enhanced’ by some team captains actively publicising screening events. Conclusions: Compared with other UK-based community screening models, uptake was high but gaining access to clubs was not always easy. Use of sexual health advisers and team captains to promote screening did not appear to confer additional benefit over a poster-promoted approach. Although the interventions show potential, the broader implications of this strategy for UK male STI screening policy require further investigation

Overlapping genetic susceptibility variants between three autoimmune disorders: rheumatoid arthritis, type 1 diabetes and coeliac disease

INTRODUCTION: Genome wide association studies, replicated by numerous well powered validation studies, have revealed a large number of loci likely to play a role in susceptibility to many multifactorial diseases. It is now well established that some of these loci are shared between diseases with similar aetiology. For example, a number of autoimmune diseases have been associated with variants in the PTPN22, TNFAIP3 and CTLA4 genes. Here we have attempted to define overlapping genetic variants between rheumatoid arthritis (RA), type 1 diabetes (T1D) and coeliac disease (CeD). METHODS: We selected eight SNPs previously identified as being associated with CeD and six T1D-associated SNPs for validation in a sample of 3,962 RA patients and 3,531 controls. Genotyping was performed using the Sequenom MassArray platform and comparison of genotype and allele frequencies between cases and controls was undertaken. A trend test P-value < 0.004 was regarded as significant. RESULTS: We found statistically significant evidence for association of the TAGAP locus with RA (P = 5.0 × 10-4). A marker at one other locus, C1QTNF6, previously associated with T1D, showed nominal association with RA in the current study but did not remain statistically significant at the corrected threshold. CONCLUSIONS: In exploring the overlap between T1D, CeD and RA, there is strong evidence that variation within the TAGAP gene is associated with all three autoimmune diseases. Interestingly a number of loci appear to be specific to one of the three diseases currently studied suggesting that they may play a role in determining the particular autoimmune phenotype at presentation

Study of the common genetic background for rheumatoid arthritis and systemic lupus erythematosus

BACKGROUND: Evidence is beginning to emerge that there may be susceptibility loci for rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) that are common to both diseases. OBJECTIVE: To investigate single nucleotide polymorphisms that have been reported to be associated with SLE in a UK cohort of patients with RA and controls. METHODS: 3962 patients with RA and 9275 controls were included in the study. Eleven SNPs mapping to confirmed SLE loci were investigated. These mapped to the TNFSF4, BANK1, TNIP1, PTTG1, UHRF1BP1, ATG5, JAZF1, BLK, KIAA1542, ITGAM and UBE2L3 loci. Genotype frequencies were compared between patients with RA and controls using the trend test. RESULTS: The SNPs mapping to the BLK and UBE2L3 loci showed significant evidence for association with RA. Two other SNPs, mapping to ATG5 and KIAA1542, showed nominal evidence for association with RA (p=0.02 and p=0.02, respectively) but these were not significant after applying a Bonferroni correction. Additionally, a significant global enrichment in carriage of SLE alleles in patients with RA compared with controls (p=9.1×10(-7)) was found. Meta-analysis of this and previous studies confirmed the association of the BLK and UBE2L3 gene with RA at genome-wide significance levels (p<5×10(-8)). Together, the authors estimate that the SLE and RA overlapping loci, excluding HLA-DRB1 alleles, identified so far explain ∼5.8% of the genetic susceptibility to RA as a whole. CONCLUSION: The findings confirm the association of the BLK and UBE2L3 loci with RA, thus adding to the list of loci showing overlap between RA and SLE

Brief Report: Identification of BACH2 and RAD51B as Rheumatoid Arthritis Susceptibility Loci in a Meta-Analysis of Genome-Wide Data

Objective: A recent high-density fine-mapping (ImmunoChip) study of genetic associations in rheumatoid arthritis (RA) identified 14 risk loci with validated genome-wide significance, as well as a number of loci showing associations suggestive of significance (P = 5 × 10−5 < 5 × 10−8), but these have yet to be replicated. The aim of this study was to determine whether these potentially significant loci are involved in the pathogenesis of RA, and to explore whether any of the loci are associated with a specific RA serotype. Methods: A total of 16 single-nucleotide polymorphisms (SNPs) were selected for genotyping and association analyses in 2 independent validation cohorts, comprising 6,106 RA cases and 4,290 controls. A meta-analysis of the data from the original ImmunoChip discovery cohort and from both validation cohorts was carried out, for a combined total of 17,581 RA cases and 20,160 controls. In addition, stratified analysis of patient subsets, defined according to their anti–cyclic citrullinated peptide (anti-CCP) antibody status, was performed. Results: A significant association with RA risk (P < 0.05) was replicated for 6 of the SNPs assessed in the validation cohorts. All SNPs in the validation study had odds ratios (ORs) for RA susceptibility in the same direction as those in the ImmunoChip discovery study. One SNP, rs72928038, mapping to an intron of BACH2, achieved genome-wide significance in the meta-analysis (P = 1.2 × 10−8, OR 1.12), and a second SNP, rs911263, mapping to an intron of RAD51B, was significantly associated in the anti-CCP–positive RA subgroup (P = 4 × 10−8, OR 0.89), confirming that both are RA susceptibility loci. Conclusion: This study provides robust evidence for an association of RA susceptibility with genes involved in B cell differentiation (BACH2) and DNA repair (RAD51B). The finding that the RAD51B gene exhibited different associations based on serologic subtype adds to the expanding knowledge base in defining subgroups of RA