Molecular markers of systemic autoimmune disorders: the expression of MHC-located HSP70 genes is significantly associated with autoimmunity development.

OBJECTIVES: To analyse the expression regulation of two inducible HSP70 genes - HSPA1A and HSPA1B - located within the major histocompatibility complex (MHC) in patients with various systemic autoimmune diseases and to prove the reliability of MHC-located HSP70 genes as molecular markers reflecting the autoimmune process. METHODS: 94 adult patients with idiopathic inflammatory myopathy (IIM, n=31), systemic lupus erythematosus (SLE, n=31) or systemic sclerosis (SSc, n=32) and 37 healthy individuals were analysed. The mRNA expression level was determined using quantitative real-time PCR method. The expression of intracellular HSP70 was established by flow cytometry, the extracellular HSP70 protein was measured in plasma samples using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The expression of HSPA1A gene was significantly up-regulated in patients with autoimmune diseases (SLE: p<0.01; SSc: p<0.01; IIM: p<0.0001) compared to healthy controls. The expression of HSPA1B gene was increased only in patients with myositis (p<0.05). Furthermore, the HSPA1B gene expression is associated with the HLA-DRB1*03 risk allele in patients with IIM. In addition, we have found a relation between HSPA1A gene expression regulation and the presence of disease specific autoantibodies in patients with SLE and myositis. The level of intracellular HSP70 was not increased; however, the level of extracellular HSP70 protein was increased in patients suffering from SSc and IIM as compared to controls. CONCLUSIONS: The results suggest an involvement of the MHC-linked HSP70 genes in the pathology of studied autoimmune disorders. Therefore, the HSPA1A and HSPA1B genes might serve as an interesting candidate molecule for development of distinct types of autoimmunities.

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+IL1ß/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.

Microarray analysis of circulating micro RNAs in the serum of patients with polymyositis and dermatomyositis reveals a distinct disease expression profile and is associated with disease activity.

OBJECTIVES: The aim of this study was a large scale investigation of myositis-associated circulating miRNA molecules and also determination of expression of these candidate molecules in relation to clinical activity of myositis. METHODS: RNA, containing also miRNAs, was isolated from sera of 28 patients suffering from idiopathic inflammatory myopathies (IIM) and 16 healthy controls. Expression of miRNAs was determined using a miRNA microarray method. Statistical analysis of miRNA expression was carried out using Arraystar software. RESULTS: Our results showed 23 significantly differentially expressed miRNAs. Six miRNAs were differentially expressed in IIM compared to healthy controls. In dermatomyositis (DM) we found 3 and in polymyositis (PM) 6 differentially expressed miRNAs compared to controls. Three miRNAs were up-regulated in patients with highly active disease compared to patients with low disease activity. Furthermore, we found 26 significantly differentially expressed miRNAs in SLE patients compared to IIM, DM and PM patients. CONCLUSIONS: This is the first study that comprehensively describes expression levels of circulating miRNAs in serum of patients suffering from IIM. It can be expected that some of these deregulated miRNA molecules are involved in aetiology of IIM and may potentially serve as molecular markers for IIM development or for monitoring of disease activity.

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 Sjögren's 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.

Increasing incidence of immune-mediated necrotizing myopathy: single-centre experience.

OBJECTIVES: Immune-mediated necrotizing myopathy (IMNM) is characterized by the predominant presence of necrotic muscle fibres in muscle biopsy and variable response to immunosuppressive treatment. The aims of this study were to analyse the temporal trend of IMNM incidence in our centre over the past 10 years and to explore the role of statins as possible causative agents. METHODS: A retrospective evaluation of muscle biopsy results, clinical and laboratory data, including antibody associations of all patients with idiopathic inflammatory myopathy newly diagnosed between 2004 and June 2014, was performed. Available sera were tested for the presence of anti-3-hydroxy-3-methylglutaryl coenzyme A reductase (anti-HMGCR) autoantibodies. RESULTS: Of 357 biopsied patients, 233 fulfilled criteria for inflammatory/immune-mediated myopathy, including 27 (11.6%) classified as IMNM. There were no patients with IMNM diagnosed between 2004 and 2007; subsequently, two to three cases of IMNM per year were seen during the period 2008-11, with a substantial increase to 18 cases (66.6% of all IMNM biopsies) in 2012-14. Thirteen of 27 patients (48%) had a history of statin use, 11 (85%) of whom had positive anti-HMGCR antibodies. There was no IMNM patient without a history of statin use who was anti-HMGCR antibody positive. CONCLUSION: Our data show an increasing incidence of IMNM, which is mainly accounted for by anti-HMGCR-positive IMNM associated with the use of statins.

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.

INTRODUCTION: The aim was to evaluate S100A4 protein as a biomarker of disease activity and potential cancer development in patients with myositis. METHODS: Serum 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. RESULTS: All 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 (ß = 0.552; P = 0.002) in PM patients and with MYOACT (ß = 0.557; P = 0.003) and CRP levels (ß = 0.391; P = 0.029) in DM patients. CONCLUSIONS: Circulating 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.

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.

Arthritis in idiopathic inflammatory myopathy: clinical features and autoantibody associations.

OBJECTIVE: To determine the prevalence, distribution, and clinical manifestations of arthritis in a cohort of patients with idiopathic inflammatory myopathies (IIM). Associations with autoantibody status and HLA genetic background were also explored. METHODS: Consecutive patients with IIM treated in a single center were included in this cross-sectional study (n = 106). History of arthritis, 68-joint and 66-joint tender and swollen joint index, clinical features of IIM, and autoantibody profiles were obtained by clinical examination, personal interview, and review of patient records. High-resolution genotyping in HLA-DRB1 and HLA-DQB1 loci was performed in 71 and 73 patients, respectively. RESULTS: A combination of patients' medical history and cross-sectional physical examination revealed that arthritis at any time during the disease course had occurred in 56 patients (53%). It was present at the beginning of the disease in 39 patients (37%) including 23 cases (22%) with arthritis preceding the onset of muscle weakness. On physical examination, 29% of patients had at least 1 swollen joint. The most frequently affected areas were wrists, and metacarpophalangeal and proximal interphalangeal joints. Twenty-seven out of the 29 anti-Jo1-positive patients had arthritis at any time during the course of their illness; this prevalence was significantly higher compared to patients without the anti-Jo1 autoantibody (p < 0.0001). No association of arthritis with individual HLA alleles was found. CONCLUSION: Our data suggest that arthritis is a common feature of myositis. It is frequently present at the onset of disease and it may even precede muscular manifestations of IIM. The most common presentation is a symmetrical, nonerosive polyarthritis affecting particularly the wrists, shoulders, and small joints of the hands. We have confirmed a strong association of arthritis with the presence of the anti-Jo1 antibody.

Resistin in idiopathic inflammatory myopathies.

INTRODUCTION: The 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. METHODS: Serum 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. RESULTS: In 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)-1ß and IL-6 and monocyte chemoattractant protein (MCP)-1 in mononuclear cells but not in myocytes. CONCLUSIONS: The 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.

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

INTRODUCTION: The 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). METHODS: Serum 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). RESULTS: The 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). CONCLUSIONS: This 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.