W. Stevens

Calcinosis is associated with digital ulcers and osteoporosis in patients with Systemic Sclerosis: A Scleroderma Clinical Trials Consortium Study

OBJECTIVESnWe sought to identify the clinical factors associated with calcinosis in an international multicenter collaborative effort with the Scleroderma Clinical Trials Consortium (SCTC).nnnMETHODSnThis is a retrospective cohort study of 5218 patients with systemic sclerosis (SSc). Logistic regression was used to obtain odds ratios (OR) relating calcinosis to various clinical features in multivariate analyses.nnnRESULTSnA total of 1290 patients (24.7%) had calcinosis. In univariate analyses, patients with calcinosis were older than patients without calcinosis, more likely to be female, and had longer disease duration from the first non-Raynaud phenomenon symptom. Patients with calcinosis were more likely to have digital ulcers, telangiectasias, acro-osteolysis, cardiac disease, pulmonary hypertension, gastrointestinal involvement, arthritis, and osteoporosis, but less likely to have muscle disease. Anti-Scl-70, RNA-polymerase-III, and U1-RNP autoantibodies were significantly less common in patients with calcinosis, while anticentromere (ACA), anti-PM/Scl, and anticardiolipin antibodies were more frequent. In multivariate analysis, the strongest associations with calcinosis were digital ulcers (OR = 3.9; 95% CI: 2.7-5.5; p < 0.0001) and osteoporosis (OR = 4.2; 95% CI: 2.3-7.9; p < 0.0001).nnnCONCLUSIONnOne quarter of patients with SSc have calcinosis at some time during their illness. Our data confirm a strong association of calcinosis with digital ulcers, and support a novel association with osteoporosis.

Quantifying change in pulmonary function as a prognostic marker in systemic sclerosis-related interstitial lung disease

SUPPLEMENTSex Bias In Autoimmune Diseases : Increased Risk Of 47,XXX In Systemic Lupus Erythematosus (SLE) and Sjogrens Syndrome (SS) Supports The Gene Dose HypothesisBackground/Purpose: Human FoxP3+ Th-cells are heterogeneous in function and include not only suppressive cells (TRegs) but also nonsuppressive cells that abundantly secrete proinflammatory cytokines. We have previously shown that FoxP3+ Th-cells were increased in GPA-patients during remission as compared to healthy controls (HCs). In this group of patients, however, we observed a defective suppressor function of TRegs, and an increase in the percentage of Th-17 cells. These observations make it tempting to investigate whether increased FoxP3+ Th-cells in GPA-patients are attributed to an increase in the cytokine-secreting non-suppressive FoxP3+Th-cells. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 46 GPA-patients in remission and from 22 age- and sex-matched HCs. Expression of CD4, CD45RO, and FoxP3 were determined by flow cytometric analysis. The expression levels of FoxP3 and CD45RO were used for distinction between activated suppressor TRegs (FoxP3HighCD45RO+; ASTReg), resting suppressor TRegs (FoxP3LowCD45RO-; RSTReg), and cytokine-secreting non-suppressor TRegs (FoxP3LowCD45RO+; NONTReg) cells. Intracellular expression of IFNg, IL-17, and IL-21 were determined in the various FoxP3+ Th-cell subsets after in vitro activation of PBMCs by PMA and Ca-Ionophore. Results: A significant increase in the frequency of NONTReg cells was observed in GPA-patients as compared with HCs, whereas no differences were detected in RSTReg- and ASTReg cells between GPA-patients and HCs. The distribution of RSTReg- and NONTReg cells did not differ between ANCA-negative and ANCA-positive patients, whereas lower percentages of ASTReg cells were observed in ANCA-positive patients as compared to ANCA-negative patients and HCs. Importantly, a significant increase in the percentage of IL-17+ and IL-21+ cells was seen within the NONTRegcells from ANCA-positive patients (n= 9) when compared to ANCA-negative (n= 10) and HCs (n= 12), whereas no differences were found between ANCA-negative and HCs. Conclusion: Increased FoxP3 expression in Th-cells from GPA-patients is related to an increase in a subset of non-suppressive Th-cells. Increased production of IL-17 and IL-21 cytokines, in NONTReg cells from ANCApositive patients points towards FoxP3+ effector cells and decrease in suppressive TReg cells in relation to ANCA production.Complex Functional Effects Within The HLA Contribute To Sjogrens Syndrome Pathogenesis and May Influence Both Transcriptional Regulation and Peptide Binding

Early mortality in Australian, Canadian and Spanish scleroderma patients: rationale for establishing a multi-national inception cohort of patients with systemic sclerosis

This free journal suppl. entitled: Special Issue: 2014 ACR/ARHP Annual Meeting Abstract Supplement

Asymmetric dimethylarginine levels in the early detection of systemic sclerosis-related pulmonary arterial hypertension

SUPPLEMENTSex Bias In Autoimmune Diseases : Increased Risk Of 47,XXX In Systemic Lupus Erythematosus (SLE) and Sjogrens Syndrome (SS) Supports The Gene Dose HypothesisBackground/Purpose: Human FoxP3+ Th-cells are heterogeneous in function and include not only suppressive cells (TRegs) but also nonsuppressive cells that abundantly secrete proinflammatory cytokines. We have previously shown that FoxP3+ Th-cells were increased in GPA-patients during remission as compared to healthy controls (HCs). In this group of patients, however, we observed a defective suppressor function of TRegs, and an increase in the percentage of Th-17 cells. These observations make it tempting to investigate whether increased FoxP3+ Th-cells in GPA-patients are attributed to an increase in the cytokine-secreting non-suppressive FoxP3+Th-cells. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 46 GPA-patients in remission and from 22 age- and sex-matched HCs. Expression of CD4, CD45RO, and FoxP3 were determined by flow cytometric analysis. The expression levels of FoxP3 and CD45RO were used for distinction between activated suppressor TRegs (FoxP3HighCD45RO+; ASTReg), resting suppressor TRegs (FoxP3LowCD45RO-; RSTReg), and cytokine-secreting non-suppressor TRegs (FoxP3LowCD45RO+; NONTReg) cells. Intracellular expression of IFNg, IL-17, and IL-21 were determined in the various FoxP3+ Th-cell subsets after in vitro activation of PBMCs by PMA and Ca-Ionophore. Results: A significant increase in the frequency of NONTReg cells was observed in GPA-patients as compared with HCs, whereas no differences were detected in RSTReg- and ASTReg cells between GPA-patients and HCs. The distribution of RSTReg- and NONTReg cells did not differ between ANCA-negative and ANCA-positive patients, whereas lower percentages of ASTReg cells were observed in ANCA-positive patients as compared to ANCA-negative patients and HCs. Importantly, a significant increase in the percentage of IL-17+ and IL-21+ cells was seen within the NONTRegcells from ANCA-positive patients (n= 9) when compared to ANCA-negative (n= 10) and HCs (n= 12), whereas no differences were found between ANCA-negative and HCs. Conclusion: Increased FoxP3 expression in Th-cells from GPA-patients is related to an increase in a subset of non-suppressive Th-cells. Increased production of IL-17 and IL-21 cytokines, in NONTReg cells from ANCApositive patients points towards FoxP3+ effector cells and decrease in suppressive TReg cells in relation to ANCA production.Complex Functional Effects Within The HLA Contribute To Sjogrens Syndrome Pathogenesis and May Influence Both Transcriptional Regulation and Peptide Binding

The lectin pathway of complement - a potential role in the pathogenesis and disease manifestations of systemic sclerosis

This free journal suppl. entitled: Special Issue: 2014 ACR/ARHP Annual Meeting Abstract Supplement

Mycophenolate mofetil versus azathioprine in scleroderma-associated interstitial lung disease: results from the Australian Scleroderma Cohort Study

This free journal suppl. entitled: Special Issue: 2014 ACR/ARHP Annual Meeting Abstract Supplement

Autoantibody associations among a well-characterized Australian systemic sclerosis (SSc) cohort

Butterworth P1, Urquhart D2, Cicuttini F2, Menz H1, Strauss B3, Proietto J4, Dixon J5, Jones G6, Wluka A2 1Department of Podiatry, La Trobe University, 2Department of Epidemiology and Preventive Medicine, School of Public Health and Preventive Medicine, Monash University, 3Department of Medicine, Southern Clinical School, Monash University, 4University of Melbourne and Austin Health Melbourne, 5Baker IDI Heart and Diabetes Institute and Alfred Hospital, 6Menzies Research Institute, Hobart

Assymetric dimethylarginine (ADMA) levels in the early detection of systemic sclerosis related pulmonary arterial hypertension (SSc-PAH)

Methods: Patients with RA (as per 1987 ACR) having high disease activity (DAS28-3v 5.1) were included.They were treated with oral methotrexate for 12 weeks, starting at 15 mg/wk, escalating to 25 mg (increased 2.5 mg biweekly). At baseline and 12 weeks, disease activity measured by DAS28-3v (Disease Activity Score 28 joints, 3 variables) and serum stored. In addition, serum stored from 16 controls.Treatment response assessed by DDAS28-3v (0–12 weeks). Serum levels of MPIF1 and MCP2 determined by ELISA (RayBio).

THU0005 An immunochip based interrogation of scleroderma susceptibility variants

Background Understanding the genetic architecture of scleroderma (SSc) susceptibility is vital both in gene discovery and in determining the influence of previous identified susceptibility variants. Objectives To perform an immunochip-based interrogation of scleroderma susceptibility variants using cases from the Australian Scleroderma Cohort Study. Methods We selected 557 cases from the Australian Scleroderma Cohort Study for genotyping with the Immunochip, a custom Illumina Infinium genotyping array containing 196,524 rare and common variants shown to be important in a wide variety of autoimmune disorders. 4,537 controls were taken from the 1958 British Birth cohort. Genotype data were analysed with PLINK. Samples and SNPs with low call rates were excluded, as were SNPs in Hardy-Weinberg disequilibrium or with less than two occurrences of the minor allele. Eigenstrat was used to analyze population structure. The final dataset consisted of 505 cases, 4,491 controls and 146,867 SNPs. Allelic association analyses were conducted using Fisher’s exact test. Genotype clusters were manually examined for all associations of p<10-5 since calling is difficult for some rare variants. Results Significant and suggestive associations were detected at seven loci. Several of these have been previously implicated in scleroderma susceptibility (HLA-DRB1 and STAT4) and several are novel associations, including SNPs near PXK (p=4.4×10-6) and CFDP1 (p=2.6×10-6). The strongest associations were with SNPs in the class II region of the MHC. One of the most strongly associated SNPs (rs4639334; p=1.6’10-8; OR=1.8) is in linkage disequilibrium (r2=0.46) with the class II allele HLA-DRB1*11:01. This allele has been associated with SSc. Another strongly associated SNP is rs2857130 (p=1.6’10-8; OR=0.67), which lies in the promoter region of HLA-DRB1, but is not in LD with any classical MHC alleles. Outside the MHC, there were six regions of association with p<10-5, including the confirmed SSc locus at STAT4. Several SNPs implicate a locus at PXK, which has been previously associated with systemic lupus erythematosus (SLE) but not with SSc. The remaining associations are novel for both SSc and SLE and require replication. Of particular interest is a rare variant located within a non-coding RNA on chromosome 6q21 which was approximately 20 times more frequent in cases than controls. We are currently dissecting the potential biological implications of this locus. Conclusions This pilot study has confirmed previously reported SSc associations, revealed further genetic overlap between SSc and SLE, and identified putative novel SSc susceptibility loci including a rare allele with major effect size. Disclosure of Interest None Declared

Novel biomarkers of dysregulated angiogenesis are not specific to pulmonary arterial hypertension in systemic sclerosis

Aim: The aim of this study was to identify miRNA that target infl ammatory pathways during arthritis. Specifi cally we are interested in production of the key infl ammatory cytokine IL-1β, which is generated by a complex of proteins known as the infl ammasome. Methods: Synovial fl uid monocytes were isolated from patients with rheumatoid or psoriatic arthritis and analysis of miRNA expression was performed. NLRP3, a key component of the infl ammasome, was identifi ed as a potential target of miR-223 and this was validated by several approaches. Results: We have identifi ed the fi rst miRNA that targets an infl ammasome complex. This is miR-223, which has a single, highly conserved binding site in the NLRP3 3′UTR. miR-223 expression decreases as monocytes differentiate into macrophages, and NLRP3 protein increases during this time. However overexpression of miR-223 prevents accumulation of endogenous NLRP3 protein levels, as for monocytic Thp-1 cells differentiated into macrophages with PMA. NLRP3 function was also impacted by miR-223, with decreased IL-1β production after stimulation with nigericin or uric acid crystals, but not poly dAdT (AIM2) or salmonella (NLRC4). Consistent with this, mice lacking miR-223 are reported to have spontaneous infl ammatory disease associated with increased NLRP3 protein expression. miR-223 is known to be decreased in type 2 diabetes, Crohn’s disease, and now we show a specifi c decrease in synovial monocytes from rheumatoid and psoriatic arthritis patients. All of these diseases are associated with increased IL-1β and the effect of miR-223 on NLRP3 could be a mechanism to account for this. Conclusions: In summary we have identifi ed an endogenous miRNA that limits NLRP3 infl ammatory capacity during myeloid differentiation, and is decreased in synovial fl uid monocytes from patients with rheumatoid or psoriatic arthritis.