H. Mann

Association of circulating miR-223 and miR-16 with disease activity in patients with early rheumatoid arthritis

Background Identification of parameters for early diagnosis and treatment response would be beneficial for patients with early rheumatoid arthritis (ERA) to prevent ongoing joint damage. miRNAs have features of potential biomarkers, and an altered expression of miRNAs was shown in established rheumatoid arthritis (RA). Objective To analyse RA associated miRNAs in the sera of patients with ERA to find markers of early disease, clinical activity or predictors of disease outcome. Methods Total RNA was isolated from whole sera in ERA patients (prior to and after 3 and 12 months of therapy with disease modifying antirheumatic drugs), in patients with established RA and in healthy controls (HC) using phenol–chloroform extraction. Expression of miR-146a, miR-155, miR-223, miR-16, miR-203, miR-132 and miR-124a was analysed by TaqMan Real Time PCR. Results From all analysed miRNAs, levels of miR-146a, miR-155 and miR-16 were decreased in the sera of ERA patients in comparison with established RA. A change in circulating miR-16 in the first 3 months of therapy was associated with a decrease in DAS28 in long term follow-up in ERA (p=0.002). Levels of circulating miR-223 in treatment naïve ERA correlated with C reactive protein (p=0.008), DAS28 (p=0.031) and change in DAS28 after 3 months (p=0.003) and 12 months (p=0.011) of follow-up. However, neither miR-16 nor miR-223 could distinguish ERA from HC. Conclusions Differential expression of circulating miR-146a, miR-155 and miR-16 in the sera of ERA patients may characterise an early stage of the disease. We suggest miR-223 as a marker of disease activity and miR-16 and miR-223 as possible predictors for disease outcome in ERA.

Decreases in serum levels of S100A8/9 (calprotectin) correlate with improvements in total swollen joint count in patients with recent-onset rheumatoid arthritis

IntroductionThe aim of this study was to examine the serum levels of S100 proteins and to evaluate their role in patients with recent-onset rheumatoid arthritis (RA).MethodsSerum levels of S100A8/9 and S100A12 were analysed in 43 patients with recent-onset RA, both before and three months after the initiation of conventional treatment, as well as in 32 healthy individuals. Disease activity was assessed based on serum levels of C-reactive protein (CRP), the Disease Activity Score for 28 joints (DAS28) and the total number of swollen joints count for 66 joints (SJC).ResultsThe levels of serum S100A8/9 and S100A12 were significantly higher in patients with recent-onset RA compared to the levels in healthy individuals (P < 0.0001) and normalised after three months of treatment. Using age- and sex-adjusted analysis, S100A8/9 levels were correlated with CRP (r = 0.439, P < 0.01), DAS28 (r = 0.501, P = 0.002) and SJC (r = 0.443, P = 0.007), while S100A12 was less significantly correlated with these parameters. Higher levels of S100A8/9 at baseline predicted improvement in the levels of CRP and SJC over time. Moreover, decreases in serum S100A8/9 were associated with decreased serum levels of CRP (r = 0.459, P = 0.005) and improvements in SJC (r = 0.459, P = 0.005). In multiple linear regression analyses, decreases in S100A8/9 but not CRP were significant predictors for improvements in SJC (P = 0.001).ConclusionsThis study is the first to show normalisation of elevated S100 proteins in patients with recent-onset RA after the initiation of conventional treatment. Therefore, S100A8/9 might potentially be a predictive marker for improvement in the total number of swollen joints in patients in the early phase of RA.

Resistin in idiopathic inflammatory myopathies

IntroductionThe purpose of this study was to evaluate and compare the serum levels and local expression of resistin in patients with idiopathic inflammatory myopathies to controls, and to determine the relationship between resistin levels, inflammation and disease activity.MethodsSerum resistin levels were determined in 42 patients with inflammatory myopathies and 27 healthy controls. The association among resistin levels, inflammation, global disease activity and muscle strength was examined. The expression of resistin in muscle tissues from patients with inflammatory myopathies and healthy controls was evaluated. Gene expression and protein release from resistin-stimulated muscle and mononuclear cells were assessed.ResultsIn patients with inflammatory myopathies, the serum levels of resistin were significantly higher than those observed in controls (8.53 ± 6.84 vs. 4.54 ± 1.08 ng/ml, P < 0.0001) and correlated with C-reactive protein (CRP) levels (r = 0.328, P = 0.044) and myositis disease activity assessment visual analogue scales (MYOACT) (r = 0.382, P = 0.026). Stronger association was observed between the levels of serum resistin and CRP levels (r = 0.717, P = 0.037) as well as MYOACT (r = 0.798, P = 0.007), and there was a trend towards correlation between serum resistin and myoglobin levels (r = 0.650, P = 0.067) in anti-Jo-1 positive patients. Furthermore, in patients with dermatomyositis, serum resistin levels significantly correlated with MYOACT (r = 0.667, P = 0.001), creatine kinase (r = 0.739, P = 0.001) and myoglobin levels (r = 0.791, P = 0.0003) and showed a trend towards correlation with CRP levels (r = 0.447, P = 0.067). Resistin expression in muscle tissue was significantly higher in patients with inflammatory myopathies compared to controls, and resistin induced the expression of interleukins (IL)-1β and IL-6 and monocyte chemoattractant protein (MCP)-1 in mononuclear cells but not in myocytes.ConclusionsThe results of this study indicate that higher levels of serum resistin are associated with inflammation, higher global disease activity index and muscle injury in patients with myositis-specific anti-Jo-1 antibody and patients with dermatomyositis. Furthermore, up-regulation of resistin in muscle tissue and resistin-induced synthesis of pro-inflammatory cytokines in mononuclear cells suggest a potential role for resistin in the pathogenesis of inflammatory myopathies.

Interleukin 35 Synovial Fluid Levels Are Associated with Disease Activity of Rheumatoid Arthritis

Objectives To study the association of systemic and local interleukin-35 (IL-35) levels in rheumatoid arthritis. Methods 37 patients with treatment naïve early RA, 49 with established RA and 29 control patients with osteoarthritis (OA) were studied. Serum and paired synovial fluid samples were analysed for IL-35. Disease activity of RA patients was assessed according to the 28-Joint Count Disease Activity Score (DAS28). Results The levels of serum IL-35 were significantly higher in patients with treatment naïve early RA compared to those with established disease and control OA subjects. In addition, serum levels of IL-35 significantly decreased 12 weeks after initiation of glucocorticoids and conventional synthetic disease modifying antirheumatic drugs in patients with treatment naïve early RA. Synovial fluid IL-35 levels were significantly higher in RA compared to OA patients, were significantly elevated compared to serum counterparts and correlated with synovial fluid leukocyte count (r=0.412; p<0.01), serum CRP levels (r=0.362; p<0.05) and DAS28 (r=0.430, p<0.01). Conclusion This is the first study showing elevated circulating levels of IL-35 in treatment naïve early RA, its significant decrease after treatment initiation and positive association between increased synovial fluid IL-35 and disease activity in patients with long-lasting RA.

High levels of metastasis-inducing S100A4 protein and treatment outcome in early rheumatoid arthritis: data from the PERAC cohort

Abstract The aim of this study was to evaluate the role of S100A4 as a biomarker in patients with early rheumatoid arthritis (RA). S100A4 levels were measured in 59 patients with early RA and in 41 healthy controls. The association between the S100A4 levels and the treatment outcome after 12 months was determined using multivariate regression analysis. Serum S100A4 levels were significantly higher in the patients with early RA than in the healthy subjects and significantly decreased after 3 months of treatment. Diseases activity at 12 months was significantly higher in female patients who had initially high levels of S100A4. Persistently high S100A4 levels predicted poor treatment outcome and S100A4 may thus represent promising biomarker for assessing treatment response in patients with RA.

The metastasis promoting protein S100A4 levels associate with disease activity rather than cancer development in patients with idiopathic inflammatory myopathies

IntroductionThe aim was to evaluate S100A4 protein as a biomarker of disease activity and potential cancer development in patients with myositis.MethodsSerum levels of S100A4 were determined in 43 dermatomyositis (DM), 39 polymyositis (PM) and 22 cancer associated myositis (CAM) patients as well as in 77 healthy controls. The associations between S100A4 levels, inflammation, disease activity, muscle strength and cancer development were evaluated.ResultsAll myositis patients had significantly higher serum levels of S100A4 protein compared to healthy controls (median (IQR): 31.5 (17.4 to 59.5) versus 23.8 (14.5 to 33.7) ng/ml, P <0.05). In patients with PM, serum levels of S100A4 protein were significantly higher than in healthy controls (41.6 (24.2 to 123.1) versus 23.8 (14.5 to 33.7) ng/ml; P <0.001) as well as in patients with DM (26.7 (11.3 to 47.5) ng/ml; P <0.05). The levels of S100A4 were comparable between myositis with and without cancer. In all myositis patients, serum S100A4 levels correlated with MYOsitis disease ACTivity assessment (MYOACT) score (r = 0.34; P = 0.001), constitutional (r = 0.30; P = 0.003), pulmonary (r = 0.43; P = 0.0001) and extramuscular disease activity (r = 0.36; P = 0.0001), as well as with creatine phosphokinase (r = 0.27; P = 0.015) and lactate dehydrogenase (r = 0.37; P = 0.002) or c-reactive protein (CRP) levels (r = 0.24; P = 0.038). Multiple regression analysis showed significant association between S100A4 serum levels and extramuscular disease activity (β = 0.552; P = 0.002) in PM patients and with MYOACT (β = 0.557; P = 0.003) and CRP levels (β = 0.391; P = 0.029) in DM patients.ConclusionsCirculating levels of S100A4 are elevated in patients with myositis and associate with several disease activity parameters, particularly with extramuscular components. No relation between S100A4 levels and presence of cancer associated myositis was found.

SAT0436 Abatacept in the Treatment of Adult Dermatomyositis and Polymyositis: Artemis, a Randomized, Treatment Delayed-Start Trial

Background Polymyositis (PM) and dermatomyositis (DM) are chronic inflammatory diseases primarily affecting skeletal muscle leading to muscle weakness. T cells are likely to have a role in the disease process indicated by the predominance of T cells in the inflammatory infiltrates found in affected muscle, and further supported by the associations with certain HLA class II alleles. The role of T cells may be direct as mediators of muscle fiber necrosis and as producers of inflammatory molecules that may have a negative effect on muscle fiber contractility. Objectives To assess the clinical efficacy of Abatacept, an agent blocking T cell co-stimulation, on disease activity in adult DM and PM patients in a trial with randomized treatment delayed-start design. Methods DM and PM patients with persisting signs of inflammatory active disease after treatment with glucocorticoids and ≥1 immunomodulating drug for ≥3 months were randomized to receive either immediate active treatment with Abatacept intravenous (10 mg/kg) infusions or a delayed start after 3 months. The primary endpoint was the number of responders, defined as improved according to the International Myositis Assessment and Clinical Studies (IMACS) Group definition of improvement (DOI), after treatment for 6 months. The secondary endpoint included the number of responders in the delayed onset arm compared to the active treatment arm at 3 months, and the efficacy after 6 months treatment on the individual components of the IMACS core set measures for the disease activity, and health-related quality of life assessed by SF-36. Results Among 20 randomized patients (9 DM, 11 PM; 13 female, 7 male), 17 were included in the analyses and 8 (47%) achieved the DOI after 6 months of active treatment. No differences between DM and PM, or female and male patients could be seen. At 3 months after study start, 5 of the 10 (50%) patients in the active treatment arm were responders achieving the DOI compared to only 1 of the 7 (14%) patients in the delayed onset arm. After active treatment for 6 months (n=17), significant improvement was seen in muscle strength, assessed by the manual muscle test (MMT)-8, from (median) 70 to 73 (p=0.0082), in gastrointestinal disease activity from 3 to 0 (p=0.0156), and in muscle disease activity from 18 to 10 (p=0.0133). SF-36 physical was significantly improved from median 31 at start to 36 at end of treatment (p=0.0054). There were 36 adverse events (AE) reported. Eight AE were judged as related to the drug, of which 4 were mild and 4 were moderate. These included infections, flank pain and dizziness. There were 3 serious AE, none of which was related to the drug. These included hospitalization due to fracture, worsening in muscle weakness and reconstructive surgery. Conclusions In this pilot study, treatment of PM and DM patients with Abatacept resulted in improved muscle performance and health-related quality of life in half of the patients, and warrants further investigation. Acknowledgements This research received funding support from Bristol-Myers Squibb. Disclosure of Interest A. Tjärnlund: None declared, M. Dastmalchi: None declared, H. Mann: None declared, J. Tomasová Studýnková: None declared, R. Chura: None declared, N. Gullick: None declared, R. Salerno: None declared, P. Gordon Paid instructor for: Bristol-Myers Squibb, J. Vencovský Consultant for: Medimmune, Servier, Novartis, I. Lundberg Consultant for: Novartis, Servier, Astra-Zeneca och Bristol-Myers Squibb

Splicing variant of WDFY4 augments MDA5 signalling and the risk of clinically amyopathic dermatomyositis

Objectives Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of rare autoimmune diseases in which both genetic and environmental factors play important roles. To identify genetic factors of IIM including polymyositis, dermatomyositis (DM) and clinically amyopathic DM (CADM), we performed the first genome-wide association study for IIM in an Asian population. Methods We genotyped and tested 496 819 single nucleotide polymorphism for association using 576 patients with IIM and 6270 control subjects. We also examined the causal mechanism of disease-associated variants by in silico analyses using publicly available data sets as well as by in in vitro analyses using reporter assays and apoptosis assays. Results We identified a variant in WDFY4 that was significantly associated with CADM (rs7919656; OR=3.87; P=1.5×10−8). This variant had a cis-splicing quantitative trait locus (QTL) effect for a truncated WDFY4isoform (tr-WDFY4), with higher expression in the risk allele. Transexpression QTL analysis of this variant showed a positive correlation with the expression of NF-κB associated genes. Furthermore, we demonstrated that both WDFY4 and tr-WDFY4 interacted with pattern recognition receptors such as TLR3, TLR4, TLR9 and MDA5 and augmented the NF-κB activation by these receptors. WDFY4 isoforms also enhanced MDA5-induced apoptosis to a greater extent in the tr-WDFY4-transfected cells. Conclusions As CADM is characterised by the appearance of anti-MDA5 autoantibodies and severe lung inflammation, the WDFY4 variant may play a critical role in the pathogenesis of CADM.

THU0065 Expression Profiling of Extracellular Serum Micrornas in Patients with Idiopathic Inflammatory Myopathy

Background Idiopathic inflammatory myopathies (IIMs) are chronic autoimmune disorders characterized by skeletal muscle weakness, inflammatory immune cells in muscle tissues and frequent presence of serum autoantibodies. Besides a genetic risk located within the MHC complex, also epigenetic regulations including changes in miRNAs expression profiles have been implicated recently in many autoimmune diseases. Extracellular miRNAs circulate in the bloodstream and such miRNAs are remarkably stable. It is hypothesized that circulating miRNA may be delivered to recipient cells and regulate translation of target genes. Objectives The aim of this study was to analyze the differences in expression profiles of circulating extracellular miRNAs between patients and controls with possible selection of relevant candidate miRNA molecules involved in disease progression. Methods Expression profile of 1,347 miRNA molecules was analyzed in 47 myositis patients and 16 healthy controls. Circulating miRNAs were prepared from serum using the Trizol® reagent. The expression of miRNAs was measured using the 44K high density microarray from Agilent Company. Results The miRNA expression profiles clearly separate between healthy controls and patients with myositis. In total, 87 miRNA molecules were found to be differentially expressed in myositis patients. Analysis of the associated biological pathways regulated by the detected miRNAs revealed that 44 (50%) differentially expressed miRNAs are predicted to regulate immune response, 38 (43%) miRNAs are related to muscle activity and 40 (46%) miRNAs were predicted to be involved in cell death. Conclusions A number of miRNAs differentially expressed in patients with IIMs were identified. These molecules are potentially involved in the etiopathogenesis of the disease since all are related to important immune mechanisms or muscle function. Acknowledgements This study was supported by the Internal Grant Agency of the Ministry of Health in the Czech Republic [MZČR NT 12452-4], Institutional support of MHCR VZ (00023728) and The Charles University Grant agency (No.621812). Disclosure of Interest None Declared

AB0239 Development of a novel „molecular disease activity“ tool composed of 8 serum biomarkers for monitoring rheumatoid arthritis

Background Accurate disease activity measurement is a key component of rheumatoid arthritis (RA) management. In 2012, multi-biomarker disease activity (MBDA) test using 12 serum proteins was presented1. Objectives To develop a novel test for measuring inflammatory activity in RA. Methods Serum samples were obtained from 82 patients with RA (60 females, median disease duration 4.2 years, median age 55.2 years, RF positivity 60.3%, ACPA positivity 69.7%). A detailed stepwise analysis of 20 candidate biomarkers selected from the literature search (IL1β, IL-6, IL-7, IL-8, IL-12p70, IL-17A, IL-22, IL-33, IL-34, IFNγ, VEGF, YKL-40, CXCL-13, MMP-3, resistin, visfatin, leptin, adiponectin, calprotectin and CRP) was performed. Methods of factor analysis (a statistical method used to describe variability among observed, correlated variables in terms of a potentially lower number of unobserved variables called factors) were used in order to develop a tool composed of different serum markers that would optimally reflect disease activity in RA. Spearman correlation index was used to explore associations between newly designed tool and clinical as well ultrasound parameters (German US7 score) of disease activity. Results We have developed a “Molecular Disease Activity” test covering underlying pathophysiological processes composed of 8 serum markers: calprotectin, CRP, IL-6, MMP-3, VEGF, resistin, IL-22 and IL-7 that optimally reflected inflammatory activity in RA. This model was significantly associated with clinical (DAS28-ESR, DAS29-CRP, CDAI, SDAI) and ultrasound variables (PD syn score, GS syn score) of disease activity in RA, as shown in table 1.Abstract AB0239 – Table 1 Cross-sectional correlations between “Molecular Disease Activity” tool composed of 8 biomarkers and clinical and ultrasound parameters of disease activity Parameters Molecular disease activity(factor score) p (significance) DAS28-ESR 0.664** <0.001 DAS28-CRP 0.666** <0.001 SDAI 0.731** <0.001 CDAI 0.669** <0.001 SJC 0.546** <0.001 GS syn score 0.657** <0.001 PD syn score 0.701** <0.001 ** Correlation is significant at≤0.01 CDAI, Clinical Disease Activity Index; DAS28-CRP, Disease Activity Score with C-reactive protein; DAS28-ESR, Disease Activity Score with erythrocyte sedimentation rate; GS syn, Grey Scale synovitis; PD syn, Power Doppler synovitis; SDAI, Simplified Disease Activity Index; SJC, swollen joint count Conclusions We believe that this newly designed test based on 8 serum markers may contribute to more accurate measurement of inflammatory activity in RA and improvement of patients‘ outcomes. Further evaluation of this tool in a larger cohort is needed. Reference [1] Centola M, Cavet G, Shen Y, et al. Development of a Multi-Biomarker Disease Activity Test for Rheumatoid Arthritis. Plos One2013Apr 9;8(4). Acknowledgements Supported by the project of Ministry of Health, Czech Rep. No. 0 23 728 and SVV No. 260 373. Disclosure of Interest None declared