Ladislav Šenolt

Resistin in rheumatoid arthritis synovial tissue, synovial fluid and serum

Background: Resistin is a newly identified adipocytokine which has demonstrated links between obesity and insulin resistance in rodents. In humans, proinflammatory properties of resistin are superior to its insulin resistance-inducing effects. Objectives: To assess resistin expression in synovial tissues, serum and synovial fluid from patients with rheumatoid arthritis, osteoarthritis and spondylarthropathies (SpA), and to study its relationship with inflammatory status and rheumatoid arthritis disease activity. Methods: Resistin expression and localisation in synovial tissue was determined by immunohistochemistry and confocal microscopy. Serum and synovial fluid resistin, leptin, interleukin (IL)1β, IL6, IL8, tumour necrosis factor α, and monocyte chemoattractant protein-1 levels were measured. The clinical activity of patients with rheumatoid arthritis was assessed according to the 28 joint count Disease Activity Score (DAS28). Results: Resistin was detected in the synovium in both rheumatoid arthritis and osteoarthritis. Staining in the sublining layer was more intensive in patients with rheumatoid arthritis compared with those with osteoarthritis. In rheumatoid arthritis, macrophages (CD68), B lymphocytes (CD20) and plasma cells (CD138) but not T lymphocytes (CD3) showed colocalisation with resistin. Synovial fluid resistin was higher in patients with rheumatoid arthritis than in those with SpA or osteoarthritis (both p<0.001). In patients with rheumatoid arthritis and SpA, serum resistin levels were higher than those with osteoarthritis (p<0.01). Increased serum resistin in patients with rheumatoid arthritis correlated with both CRP (r = 0.53, p<0.02), and DAS28 (r = 0.44, p<0.05), but not with selected (adipo) cytokines. Conclusion: The upregulated resistin at local sites of inflammation and the link between serum resistin, inflammation and disease activity suggest a role for resistin in the pathogenesis of rheumatoid arthritis.

Prospective new biological therapies for rheumatoid arthritis.

Advances in the current knowledge of pathogenetic mechanisms of rheumatoid arthritis have contributed to the development of biological therapy, and translated research findings into clinical practice. TNF-alpha (infliximab, etanercept, adalimumab), IL-1 (anakinra) and IL-6 (tocilizumab) inhibitors, a B-cell depleting agent (rituximab) and a drug blocking T-cell costimulation (abatacept) have been approved for rheumatoid arthritis. The progress in manufacturing biotechnology has contributed to the development of several other prospective agents that may form the basis for the therapy of rheumatoid arthritis in the near future. New or modified TNF-alpha inhibitors (golimumab, certolizumab pegol), new monoclonal antibodies against other cytokines (e.g. IL-1, IL-6, IL-12, IL-15, IL-17, IL-23), and other agents targeting B-cell depletion (e.g. ocrelizumab, ofatumumab) are in various stages of development. Many pharmaceutical companies have focused on developing small molecule inhibitors with possible peroral administration, which are considered promising drugs for rheumatoid arthritis. In most cases, these small molecules inhibit cellular kinases (e.g. p38, JAK or Syk) that mediate the signaling and transcription of proinflammatory genes. In this review, we describe the cytokine inhibitors and modulators of the immune response currently in ongoing clinical trials, the results of which may further expand the spectrum of efficient therapies for chronic autoimmune diseases.

Increased serum adiponectin levels in female patients with erosive compared with non-erosive osteoarthritis

Adipocytokines including adiponectin and resistin are suggested to be associated with obesity-related complications.1 In general, higher systemic concentrations of resistin and adiponectin compared with paired synovial fluid counterparts were demonstrated in patients with osteoarthritis of the knee.2–4 In contrast, resistin and adiponectin levels were found to be increased at local sites of inflammation in patients with rheumatoid arthritis.3–5 It is suggested that adiponectin in particular actively participates in the process of immune response, inflammation and matrix degradation in destructive arthritides.6 7 Erosive osteoarthritis represents a subtype of generalised osteoarthritis primarily affecting the small joints of the hands, with prominent local inflammation …

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.

Vaspin and omentin: new adipokines differentially regulated at the site of inflammation in rheumatoid arthritis

Scientific interest in adipose tissue-derived peptides has increased dramatically in recent years (1). Several mediators known as adipo(cyto)kines were first associated with the pathophysiology of obesity-related complications; however a significant role for adipokines such as leptin, adiponectin, resistin and visfatin in regulating immune responses and inflammation has recently been discovered (1, 2). Several reports (3-6) have already demonstrated association of these adipokines with the severity of rheumatoid arthritis (RA). Vaspin, a member of the serine protease inhibitor family, and omentin (also known as intelectin) were recently identified in adipose tissue (7, 8). Vaspin is an adipokine with insulin-sensitizing effects that has been suggested to be a compensatory mediator for abrogating obesity and its inflammatory complications (7). Expression of the omentin gene was demonstrated in omental adipose tissue of patients with Crohn’s disease, suggesting that it may be implicated in chronic inflammatory diseases (8). The aim of the present report was to compare local concentrations of vaspin and omentin in synovial fluid of RA patients with those in osteoarthritis (OA) patients and to characterize their potential association with the severity of the disease. Synovial fluid was obtained during therapeutic arthrocentesis from 33 patients with RA and 33 patients with knee OA. The disease activity of RA patients was assessed by DAS28. C-reactive protein (CRP), IgM-rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPA) were routinely analyzed from peripheral blood obtained at the time of arthrocentesis. Characteristics of the patients are given in Table 1. Vaspin (AdipoGen Inc. Korea) and omentin (Apotech Corporation) were analyzed in synovial fluid by ELISA assays. Ethical approval was obtained from the local Ethics Committee and all patients provided informed consent. Statistical analysis was performed using GraphPad Prism 5.0 software. To meet a normal distribution, vaspin and omentin concentrations were naturaly logarithmically (log)-transformed. Differences between two independent parameters were determined by Kruskal-Wallis or T-test. The Spearman test was used for correlation of parameters. As shown in figure 1, the mean (SD) levels of vaspin were significantly higher in the synovial fluid of RA patients than in OA patients (-2.439±1.226 vs. -3.366±1.318 (log) pg/ml; p=0.003), but interestingly the levels of omentin were significantly lower in the synovial fluid of RA patients compared to OA patients (1.491±0.948 vs. 1.964±0.902 (log) ng/ml; p=0.045). After log-transformation, synovial fluid vaspin, but not omentin, had a tendency to correlate with DAS28 (r=0.320, p=0.070) in RA patients. However, neither vaspin nor omentin correlated with serum CRP or leukocyte counts in synovial fluid. In addition, levels of synovial fluid omentin, but not vaspin, significantly correlated with serum ACPA (r=0.398, p=0.029) and IgM-RF (r=0.592, p<0.001). The levels of synovial fluid vaspin and omentin were not affected by body mass index (BMI) or age of the patients. The mean concentration of synovial fluid vaspin, but not omentin, was twice as high in female as in male patients, but possibly due to the low number of male patients in this study, it failed to reach statistical significance. Our data shows different levels of the new adipokines vaspin and omentin at the site of local inflammation. We demonstrate here for the first time elevated levels of vaspin and reduced levels of omentin in synovial fluid of patients with RA compared with those with OA.

S100A4 is expressed at site of invasion in rheumatoid arthritis synovium and modulates production of matrix metalloproteinases

The metastasis-associated protein S100A4 promotes the progression of cancer by regulating the remodelling of the extracellular matrix. The expression of S100A4 in vivo is shown and the functional role of S100A4 in the pathogenesis of osteoarthritis and rheumatoid arthritisis is explored. The expression of S100A4 in rheumatoid arthritis, osteoarthritis and normal synovial tissues was determined by immunohistochemistry. The expression of matrix metalloproteinase (MMP) mRNA was measured in rheumatoid arthritis and osteoarthritis synovial fibroblasts treated and untreated with S100A4 oligomer by real-time polymerase chain reaction. Levels of released MMPs were confirmed by ELISA in cell culture supernatants. S100A4 protein was expressed in rheumatoid arthritis and osteoarthritis synovial tissues, in contrast with normal synovium. S100A4 up regulated MMP-3 mRNA in rheumatoid arthritis synovial fluid, with a peak after 6 h. This resulted in release of MMP-3 protein. MMP-1, MMP-9 and MMP-13 mRNA were also up regulated in synovial fluid, but with different kinetics. MMP-14 mRNA showed no change. Thus, S100A4 protein is expressed in synovial tissues of patients with rheumatoid arthritis and osteoarthritis in contrast with healthy people. It induces the expression and release of MMP-3 and other MMPs from synovial fluid. The data suggest that S100A4-producing cells could be involved in the pathogenesis of osteoarthritis and rheumatoid arthritis, including pannus formation and joint destruction.

Increasing the infliximab dose in rheumatoid arthritis patients: a randomised, double blind study failed to confirm its efficacy

Objective: To evaluate the effect of infliximab dose escalation in incomplete responders in a randomised controlled trial. Methods: 141 rheumatoid arthritis (RA) patients treated with infliximab for 12 months (3 mg/kg; intervals 0, 2, 6 and then 8 weeks) who responded to the drug (disease activity score in 28 joints (DAS28) decrease >1.2) but who were not in remission (DAS28 >2.6) were enrolled into the study. Patients were randomly assigned into arm A, 3 mg/kg, and arm B, 5 mg/kg infliximab every 8 weeks. Outcome measures included the DAS28, its components and C-reactive protein (CRP). Results: There were no significant differences in changes in the DAS28, its components, or CRP in patients in arms A and B during the 12 months of treatment. All patients showed a DAS28 decrease greater than 0.6 after 28 weeks. Eleven patients interrupted therapy in arm A and 14 in arm B. Infusion reactions and non-serious adverse events were observed in 4.2% and 28.2% of arm A patients and in 7.2% and 47.8% of arm B patients. The frequency of serious adverse events was comparable between arms A and B (16.9% and 15.9%, respectively), and the frequency of serious infections was not significantly greater in the higher dose group (5.8%) than in the lower dose group (5.6%). Conclusions: In this setting, increasing the infliximab dose from 3 mg/kg to 5 mg/kg in RA patients with residual disease activity did not improve efficacy but moderately increased toxicity. These data indicate that a switch to another biological treatment would be a more appropriate strategy in incomplete responders.

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.

Monoclonal antibodies to human cartilage oligomeric matrix protein: epitope mapping and characterization of sandwich ELISA.

BACKGROUND Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP 5) is one of the most promising serologic markers with regard to an ability to prognose development of osteoarthritis (OA). Our aim was to map the epitopes of three monoclonal antibodies (mAb) to COMP and to develop and characterize a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring COMP levels in human body fluids. METHODS COMP was digested with trypsin and the NH(2)-terminal sequence of the fragments recognized by each of the mAbs was determined. Steric competition among the mAbs was tested with an antibody capture assay. A sandwich ELISA was developed using unlabeled mAb 16-F12 as a capture antibody, and mAb 17-C10 labeled with biotin as the second antibody. RESULTS Epitopes of the three mAbs were mapped to three different domains within the COMP subunit (16-F12, NH(2)-terminal domain; 17-C10, EGF-like domain; 12-C4, COOH-terminal domain). These epitopes did not overlap. mAbs 17-C10 and 12-C4 yielded similar serum COMP results when used as the secondary antibodies. Serum COMP levels measured with the new sandwich ELISA using mAbs 16-F12 and 17-C10 correlated strongly with results based on an inhibition ELISA with mAb 17-C10 alone (r(2) = 0.836; P < 0.0001). We characterized the new sandwich ELISA with regards to inter- and intra-assay variability, the range of COMP levels that can be expected in human synovial fluids (SF) and sera (controls and OA and rheumatoid arthritis (RA) patients), and the day-to-day and diurnal variability of COMP levels in sera. CONCLUSIONS We have developed and characterized a sandwich ELISA for COMP that is sensitive and yields highly reproducible COMP results upon analysis of human sera and synovial fluids.

The metastasis associated protein S100A4: a potential novel link to inflammation and consequent aggressive behaviour of rheumatoid arthritis synovial fibroblasts

The metastasis-associated protein S100A4 belongs to the large family of S100 calcium-binding proteins that appear to play regulatory roles in diverse biological activities. Moreover, a prognostic role of S100A4 has been suggested for patients with several types of cancer. Cancer promoting properties for S100A4 have been demonstrated, particularly through its regulation of cell motility, proliferation and apoptosis, as well as by stimulation of angiogenesis and remodelling of the extracellular matrix. Increased expression of S100A4 mRNA has been detected in proliferating synovial fibroblasts in rheumatoid arthritis. Furthermore, strong upregulation of the S100A4 protein in rheumatoid arthritis synovial tissue compared with osteoarthritis and control tissues has been demonstrated recently, especially at sites of joint invasion. Several immune and vascular cells were also identified to be producing S100A4 within the synovium. The local upregulation of S100A4 was accompanied by high plasma and synovial fluid concentrations of the S100A4 protein existing in the bioactive oligomeric form in patients with rheumatoid arthritis. Consistent with data from cancer studies, the extracellular S100A4 oligomer appears to be involved in regulation of several matrix-degrading enzymes and modulation of the transcriptional activation function of the tumour suppressor protein p53 in rheumatoid arthritis synovial fibroblasts. Taken together, one can speculate that increased S100A4 protein in circulation and locally at sites of inflammation, particularly at sites of joint destruction, might be linked to the process of aggressive fibroblast behaviour contributing to the pathogenesis of chronic autoinflammatory diseases such as rheumatoid arthritis.