Lenka Pleštilová

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.

Disease specificity of autoantibodies to cytosolic 5'-nucleotidase 1A in sporadic inclusion body myositis versus known autoimmune diseases

Objectives The diagnosis of inclusion body myositis (IBM) can be challenging as it can be difficult to clinically distinguish from other forms of myositis, particularly polymyositis (PM). Recent studies have shown frequent presence of autoantibodies directed against cytosolic 5′-nucleotidase 1A (cN-1A) in patients with IBM. We therefore, examined the autoantigenicity and disease specificity of major epitopes of cN-1A in patients with sporadic IBM compared with healthy and disease controls. Methods Serum samples obtained from patients with IBM (n=238), PM and dermatomyositis (DM) (n=185), other autoimmune diseases (n=246), other neuromuscular diseases (n=93) and healthy controls (n=35) were analysed for the presence of autoantibodies using immunodominant cN-1A peptide ELISAs. Results Autoantibodies directed against major epitopes of cN-1A were frequent in patients with IBM (37%) but not in PM, DM or non-autoimmune neuromuscular diseases (<5%). Anti-cN-1A reactivity was also observed in some other autoimmune diseases, particularly Sjögrens syndrome (SjS; 36%) and systemic lupus erythematosus (SLE; 20%). Conclusions In summary, we found frequent anti-cN-1A autoantibodies in sera from patients with IBM. Heterogeneity in reactivity with the three immunodominant epitopes indicates that serological assays should not be limited to a distinct epitope region. The similar reactivities observed for SjS and SLE demonstrate the need to further investigate whether distinct IBM-specific epitopes exist.

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.

Decreased circulating visfatin is associated with improved disease activity in early rheumatoid arthritis: data from the PERAC cohort.

Objective To evaluate circulating visfatin and its relationship with disease activity and serum lipids in patients with early, treatment-naïve rheumatoid arthritis (RA). Methods Serum visfatin was measured in 40 patients with early RA before and after three months of treatment and in 30 age- and sex-matched healthy individuals. Disease activity was assessed using the Disease Activity Score for 28 joints (DAS28) at baseline and at three and 12 months. Multivariate linear regression analysis was performed to evaluate whether improved disease activity is related to serum visfatin or a change in visfatin level. Results Serum visfatin was significantly elevated in early RA patients compared to healthy controls (1.92±1.17 vs. 1.36±0.93 ng/ml; p = 0.034) and significantly decreased after three months of treatment (to 0.99±0.67 ng/ml; p<0.001). Circulating visfatin and a change in visfatin level correlated with disease activity and improved disease activity over time, respectively. A decrease in visfatin after three months predicted a DAS28 improvement after 12 months. In addition, decreased serum visfatin was not associated with an improved atherogenic index but was associated with an increase in total cholesterol level. Conclusion A short-term decrease in circulating visfatin may represent an independent predictor of long-term disease activity improvement in patients with early RA.

Autoantibody Specificities and Type I Interferon Pathway Activation in Idiopathic Inflammatory Myopathies.

Myositis is a heterogeneous group of autoimmune diseases, with different pathogenic mechanisms contributing to the different subsets of disease. The aim of this study was to test whether the autoantibody profile in patients with myositis is associated with a type I interferon (IFN) signature, as in patients with systemic lupus erythematous (SLE). Patients with myositis were prospectively enrolled in the study and compared to healthy controls and to patients with SLE. Autoantibody status was analysed using an immunoassay system and immunoprecipitation. Type I IFN activity in whole blood was determined using direct gene expression analysis. Serum IFN‐inducing activity was tested using peripheral blood cells from healthy donors. Blocking experiments were performed by neutralizing anti‐IFNAR or anti‐IFN‐α antibodies. Patients were categorized into IFN high and IFN low based on an IFN score. Patients with autoantibodies against RNA‐binding proteins had a higher IFN score compared to patients without these antibodies, and the IFN score was related to autoantibody multispecificity. Patients with dermatomyositis (DM) and inclusion body myositis (IBM) had a higher IFN score compared to the other subgroups. Serum type I IFN bioactivity was blocked by neutralizing anti‐IFNAR or anti‐IFN‐α antibodies. To conclude, a high IFN score was not only associated with DM, as previously reported, and IBM, but also with autoantibody monospecificity against several RNA‐binding proteins and with autoantibody multispecificity. These studies identify IFN‐α in sera as a trigger for activation of the type I IFN pathway in peripheral blood and support IFN‐α as a possible target for therapy in these patients.

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.

Expression and Regulation of PIWIL-Proteins and PIWI-Interacting RNAs in Rheumatoid Arthritis

Objective The PIWIL (P-element induced wimpy testis like protein) subfamily of argonaute proteins is essential for Piwi-interacting RNA (piRNA) biogenesis and their function to silence transposons during germ-line development. Here we explored their presence and regulation in rheumatoid arthritis (RA). Methods The expression of PIWIL genes in RA and osteoarthritis (OA) synovial tissues and synovial fibroblasts (SF) was analysed by Real-time PCR, immunofluorescence and Western blot. The expression of piRNAs was quantified by next generation small RNA sequencing (NGS). The regulation of PIWI/piRNAs, proliferation and methylation of LINE-1 after silencing of PIWIL genes were studied. Results PIWIL2 and 4 mRNA were similarly expressed in synovial tissues and SF from RA and OA patients. However, on the protein level only PIWIL4 was strongly expressed in SF. Using NGS up to 300 piRNAs were identified in all SF without significant differences in expression levels between RA and OASF. Of interest, the analysis of the co-expression of the detected piRNAs revealed a less tightly regulated pattern of piRNA-823, -4153 and -16659 expression in RASF. In RASF and OASF, stimulation with TNFα+IL1β/TLR-ligands further significantly increased the expression levels of PIWIL2 and 4 mRNA and piRNA-16659 was significantly (4-fold) induced upon Poly(I:C) stimulation. Silencing of PIWIL2/4 neither affect LINE-1 methylation/expression nor proliferation of RASF. Conclusion We detected a new class of small regulatory RNAs (piRNAs) and their specific binding partners (PIWIL2/4) in synovial fibroblasts. The differential regulation of co-expression of piRNAs in RASF and the induction of piRNA/Piwi-proteins by innate immune stimulators suggest a role in inflammatory processes.

A3.30 Plasma level of HSP70 protein is increased in czech patients with idiopathic inflammatory myopathy

Background and Objectives Heat shock protein 70 (HSP70) plays an essential role as molecular chaperone by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and preventing their aggregation. These proteins have a dual function depending on their intracellular or extracellular location. Intracellular HSP70 proteins have a protective, antiapoptotic role. Under normal conditions they act as molecular chaperones. Under a stress conditions, the expression of HSP70 genes is induced, which helps the cell to survive otherwise lethal condition. This is due to its important role in apoptosis. The extracellular HSP70 proteins, on the other hand, can act as mediators of immune response. They can elicit an immune response modulated either by the adaptive or innate immune system. Mechanisms of extracellular and intracellular action of Hsp70 is expected to be involved also in autoimmunity development. The aim of this study was to establish the plasma level of Hsp70 protein in patients suffering from IIM and find its possible relation to the clinical features of our patients. Materials and Methods We quantified plasma Hsp70 levels in a healthy control group (n = 37) and a group of 34 patient with IIM. Plasma Hsp70 protein was analysed in duplicates using a commercially available high-sensitivity sandwich enzyme-linked immunosorbent assay (ELISA), suitable for detection of Hsp70 protein in serum or plasma samples (Assay Designs EKS-715, USA). Statistical differences between the patients and control groups were established using Mann-Whitney U test. All the expression data are presented as medians with range. Results We have found that plasma levels of Hsp70 were significantly higher in patients with myositis than in healthy controls (0.173 [0.000 – 7.647] vs. 0.000 [0.000 – 1.008] ng/ml; p<0.01). However, plasma Hsp70 levels did not correlate with age, CRP, disease activity index (MYOACT), or other clinical features (muscle activity) of our patients. Conclusions In this study, we have found that the amount of Hsp70 protein in plasma is significantly higher in IIM patients when compared with healthy controls. Our results correlate with our former findings showing that mRNA levels of HSP70 genes were increased in myositis patients. Finally we can assume that the Hsp70 protein is possibly involved in the pathological mechanism of the myositis. Acknowledgement This study was supported by the Internal Grant Agency of the Ministry of Health in the Czech Republic [MZČR NT 13699].

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