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Introduction. Understanding the genetic architecture of scleroderma (SSc) susceptibility is vital both in gene discovery and in determining the influence of previously identified susceptibility variants. It is particularly important in understanding disease mechanism in a disease with few therapies and great morbidity and mortality. Methods. We selected 557 cases from the Australian Scleroderma Cohort Study (ASCS), 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. A total of 4537 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 analyse population structure. The final data set consisted of 505 cases, 4491 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; odds ratio (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 wit

Rheumatoid arthritis (RA) is a chronic autoimmune-disease of unknown origin that primarily affects the joints and ultimately leads to their destruction. The involvement of immune cells is a general hallmark of autoimmune-related disorders. In this regard, macrophages, T cells and their respective cytokines play a pivotal role in RA. However, the notion that RA is a primarily T-cell-dependent disease has been strongly challenged during recent years. Rather, it has been understood that resident, fibroblast-like cells contribute significantly to the perpetuation of disease, and that they may even play a role in its initiation. These rheumatoid arthritis synovial fibroblasts (RASFs) constitute a quite unique cell type that distinguishes RA from other inflammatory conditions of the joints. A number of studies have demonstrated that RASFs show alterations in morphology and behaviour, including molecular changes in signalling cascades, apoptosis responses and in the expression of adhesion molecules as well as matrix-degrading enzymes. These changes appear to reflect a stable activation of RASFs, which occurs independently of continuous exogenous stimulation. As a consequence, RASFs are no longer considered passive bystanders but active players in the complex intercellular network of RA

We investigated the expression of cartilage oligomeric matrix protein (COMP) in normal and rheumatoid arthritis (RA) synovial fibroblasts. In situ hybridization (ISH) was conducted on synovial specimens from five RA patients applying specific probes for COMP or fibroblast collagen type I. ISH was combined with immunohistochemistry, applying antibodies to the macrophage marker CD68. Ribonuclease protection assay (RPA) and rapid amplification of 3'-cDNA ends (3'-RACE) were performed on total RNA from normal and RA synovial fibroblast cultures. Protein extracts from fibroblasts and culture supernatants were compared with synovial fluids and protein extracts from isolated chondrocytes by Western blot utilizing polyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA was detected in fibroblasts of RA synovium by ISH, and in normal and RA synovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homology of chondrocyte and synovial fibroblast COMP along the coding sequence. COMP protein was detected in synovial fibroblasts and culture supernatants by immunoblot. Using polyclonal antibodies, the major portion of COMP from fibroblasts and culture supernatants was present as low-molecular-weight (LMW) bands, corresponding to those found in synovial fluids. These LMW COMP bands, however, were not detected in any of the cells or tissues tested using 18-G3 mAb. In protein extracts from chondrocytes and in COMP purified from cartilage, these LMW bands could not be detected. In conclusion, the data suggest that certain forms of COMP detected in synovial fluid are secreted from synovial fibroblasts and could be distinguished by specific mAbs from COMP secreted by chondrocytes

AimsDysregulation of the bone morphogenetic protein receptor type 2 (BMPR2) is a hallmark feature that has been described in several forms of pulmonary hypertension. We recently identified the microRNA miR-20a within a highly conserved pathway as a regulator of the expression of BMPR2. To address the pathophysiological relevance of this pathway in vivo, we employed antagomiR-20a and investigated whether specific inhibition of miR-20a could restore functional levels of BMPR2 and, in turn, might prevent pulmonary arterial vascular remodelling.Methods and resultsFor specific inhibition of miR-20a, cholesterol-modified RNA oligonucleotides (antagomiR-20a) were synthesized. The experiments in mice were performed by using the hypoxia-induced mouse model for pulmonary hypertension and animal tissues were analysed for right ventricular hypertrophy and pulmonary arterial vascular remodelling. Treatment with antagomiR-20a enhanced the expression levels of BMPR2 in lung tissues; moreover, antagomiR-20a significantly reduced wall thickness and luminal occlusion of small pulmonary arteries and reduced right ventricular hypertrophy. To assess BMPR2 signalling and proliferation, we performed in vitro experiments with human pulmonary arterial smooth muscle cells (HPASMCs). Transfection of HPASMCs with antagomiR-20a resulted in activation of downstream targets of BMPR2 showing increased activation of Id-1 and Id-2. Proliferation of HPASMCs was found to be reduced upon transfection with antagomiR-20a.ConclusionThis is the first report showing that miR-20a can be specifically targeted in an in vivo model for pulmonary hypertension. Our data emphasize that treatment with antagomiR-20a restores functional levels of BMPR2 in pulmonary arteries and prevents the development of vascular remodelling.