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INTRODUCTION:Fibromyalgia (FM) is characterized by chronic pain and reduced pain threshold. The pathophysiology involves disturbed neuroendocrine function, including impaired function of the growth hormone/insulin-like growth factor-1 axis. Recently, microRNAs have been shown to be important regulatory factors in a number of diseases. The aim of this study was to try to identify cerebrospinal microRNAs with expression specific for FM and to determine their correlation to pain and fatigue. METHODS:The genome-wide profile of microRNAs in cerebrospinal fluid was assessed in ten women with FM and eight healthy controls using real-time quantitative PCR. Pain thresholds were examined by algometry. Levels of pain (FIQ pain) were rated on a 0-100 mm scale (fibromyalgia impact questionnaire, FIQ). Levels of fatigue (FIQ fatigue) were rated on a 0-100 mm scale using FIQ and by multidimensional fatigue inventory (MFI-20) general fatigue (MFIGF). RESULTS:Expression levels of nine microRNAs were significantly lower in patients with FM patients compared to healthy controls. The microRNAs identified were miR-21-5p, miR-145-5p, miR-29a-3p, miR-99b-5p, miR-125b-5p, miR-23a-3p, 23b-3p, miR-195-5p, miR-223-3p. The identified microRNAs with significantly lower expression in FM were assessed with regard to pain and fatigue. miR-145-5p correlated positively with FIQ pain (r=0.709, p=0.022, n=10) and with FIQ fatigue (r=0.687, p=0.028, n=10). CONCLUSION:To our knowledge, this is the first study to show a disease-specific pattern of cerebrospinal microRNAs in FM. We have identified nine microRNAs in cerebrospinal fluid that differed between FM patients and healthy controls. One of the identified microRNAs, miR-145 was associated with the cardinal symptoms of FM, pain and fatigue.

Background Rheumatoid arthritis (RA) is a joint destructive disease, which leads to physical disability. There is no cure of RA today. Thus, recognition of RA at preclinical stages is essential for successful treatment. Objectives To validate and improve known prediction rules aiming at early recognition of clinically suspect arthralgia and diagnosis of RA. Methods Medical records of 241 first-time patients with clinically suspect joint pain were evaluated for RA diagnosis by the 2010 EULAR/ACR criteria (1) and by the RA prediction rules (13-points, Amsterdam (2)); and 16-points clinical assessment (Leiden (3)) and prospectively followed during 29–47 months. All the patients were a part of the inception cohort of 5455 patients whose samples were tested for RF and/or ACPA during 12 consecutive months at the Laboratory of Clinical Immunology at the University Hospital of Gothenburg. Patients with known diagnosis of RA, other rheumatic disease and chronic pain conditions were excluded. The utility of arthritis-specific autoantibody status (ACPA and/or RF) and the family history for RA prediction were analyzed. Results The RA criteria applied to the records of the first visit identified 24% of patients as RA (EULAR score mean 7.6), 30% as undifferentiated arthritis (UA, EULAR score mean 3.6), while the remaining 45% of the patients were characterized as arthralgia (EULAR score mean 2.3). The 13-point and 16-point prediction rules allocated RA patients to the high-risk group with comparable sensitivity of 78% and 91%. The significant correlation between the two models was present in UA (r=0.398; p=0.0005) and arthralgia (r=0.411; p Conclusions Both prediction rules are shown to be sensitive identify RA patients at assessment. The clinical 16-point assessment has prospective sensitivity for new cases. This is shown independently of autoantibody status and family history of RA. References Cader MZ et al. Performance of the 2010 ACR/EULAR criteria for rheumatoid arthritis: comparison with 1987 ACR criteria in a very early synovitis cohortAnn Rheum Dis. 2011;70:949–55. van de Stadt LA et al. A prediction rule for the development of arthritis in seropositive arthralgia patients. Ann Rheum Dis. 2013;72:1920–6. Krabben A et al. Risk of RA development in patients with unclassified arthritis according to the 2010 ACR/EULAR criteria for RA. Rheumatology (Oxford). 2013;52:1265–70. Disclosure of Interest None declared

Background Recognition of rheumatoid arthritis (RA) at preclinical stages is essential for successful treatment, long-term functional ability and survival. Available serological markers of the disease are restricted to acute-phase reactants and presence of autoantibodies - rheumatoid factor (RF) and ACPA. Although specific, these markers cover less than a half of the patients with RA. Oncoprotein survivin has been recently identified as a marker of severe RA frequently associated with progressive joint damage and poor response to antirheumatic treatment (1). A pilot study on the samples from the Nordic Biobank showed that survivin could predict development of RA several months ahead of clinical symptoms (2). Objectives To validate the utility of survivin measurements for early recognition of clinically suspect arthralgia and diagnosis of RA in the epidemiological setting. Methods The first-visit patients with joint pain from the inception cohort of 5455 subjects tested for RF and/or ACPA during 12 consecutive months at the Laboratory of Clinical Immunology at the University Hospital of Gothenburg, were evaluated for RA diagnosis by the 2010 EULAR/ACR criteria (3). Patients with known diagnosis of RA, other rheumatic disease and chronic pain conditions were excluded. The subjects not fulfilling RA diagnosis at the first visit were prospectively followed during 29–49 months. The utility of survivin measurement and arthritis-specific autoantibody status (ACPA and/or RF) for RA diagnosis were analyzed. Results The total of 385 patients were included in the study. RA criteria applied to the records of the first visit identified 17% of patients as RA. Additional 21% of patients were recognized as undifferentiated arthritis (UA), while the remaining 62% of the patients were characterized as arthralgia. Majority of the RA patients were survivin positive (52%) with an overlap for antibodies (RF and/or ACPA) in 88%. The UA and arthralgia groups had low prevalence of patients positive for survivin (p=0.018) and for autoantibodies (p=0.002). During the follow-up period, 23 cases of new RA and 5 new UA were registered. The incidence of new cases was significantly higher among the survivin-positive patients and corresponded to the increased risk compared to survivin-negative (p=0.008). The combination of survivin and autoantibodies increased further the risk for development of the disease compared to negative patients (RR 4.05[ 1.73–9.20], p=0.002). The presence of isolated survivin was also associated with higher risk of RA (RR 2.55 [1.14–5.71], p=0.028), while no increase in RA risk could be shown for the isolated presence of autoantibodies. Conclusions This prospective study showed in the epidemiological setting that the presence of survivin was associated with an increased risk of RA. Combination of survivin and autoantibodies further increased this risk. References Svensson B et al. Increased expression of proto-oncogene survivin predicts joint destruction and persistent disease activity in early RA. Ann Med. 2010;42:45–54. Bokarewa M et al. Survivin but not Flt3 ligand is up-regulated before the onset of RA: a pilot study. Arthritis Res Ther. 2014;16:R45. Cader MZ et al. Ann Rheum Dis. 2011;70:949–55. Disclosure of Interest None declared

Background Insulin-like growth factor (IGF-1) signaling is essential for development and function of the brain. It helps to improve neuronal plasticity in response to stress, and recently it has been reported to mediate depression [1]. In rheumatoid arthritis (RA), depression is a serious comorbidity with a tight connection to functional disability and early mortality [2]. The serum levels of IGF-1 and the local expression of its receptor (IGF-1R) in inflamed joints are affected in RA [3]. However, the brain IGF-1R signaling in arthritis and its consequences for behavior has not been studied previously. Objectives In this study we analyze how arthritis affects IGF-1R expression and activity in the brain and its relation to behavioral changes. Methods Arthritis was induced in DBA/1 mice by immunization with collagen type II. Development of arthritis was followed clinically. One group was treated with shRNA targeting IGF1R, second group obtained control shRNA, and naive healthy siblings comprised the control group. Behavioral pattern of each mouse was registered by filming at different stages. Protein and mRNA levels of IGF-1R, CD68 and IL-1b were measured in different parts of the brain with IHC and real-time PCR. Serum IGF-1 and IL6 were analyzed with a sandwich ELISA. We validated the findings from experimental arthritis with a cohort of 235 patients with established RA. Results In RA patients, the arbitrary measure of pain (VAS) was mainly associated with depression index (FIQ, r=0.46), fatigue (r=0.44), and disability index (r=0.68) rather than to RA activity (DAS28, r=0.36). In mice, arthritis decreased locomotion and rearing, corresponding to the depressive-like behavioral pattern. Severity of arthritis and decreased mobility correlated with the increased mRNA levels of inflammatory cytokine IL-1, and a marker of activated microglia CD68. We observed a decreased expression of IGF-1R and a remarkable reduction in the number and regularity of the Purkinje cells in the cerebellum accompanied by an accumulation of phosphorylated tau along these cells. Arthritis increased serum levels of IGF-1, which correlated with behavioral pattern in arthritic mice. When the IGF1R signaling was inhibited with shIGF1R-treatment, the arthritic mice showed aggravation of the depressive-like behavior, including increased immobility time, which was no longer correlated to severity of arthritis. Conclusions Depression and functional disability aggravate pain experience in RA patients. Arthritis-induced inhibition of IGF-1R expression and signaling provide molecular mechanism of depression in RA. References Kopczak A, et al. IGF-I in major depression and antidepressant treatment response. Eur Neuropsychopharmacol. 2015 Jun; 25(6):864–872. Ang DC, et al. Comorbid depression is an independent risk factor for mortality in patients with rheumatoid arthritis. J Rheumatol. 2005 Jun; 32(6):1013–1019. Bostrom EA, et al Resistin and insulin/insulin-like growth factor signaling in rheumatoid arthritis. Arthritis Rheum. 2011 Oct; 63(10):2894–2904. Disclosure of Interest None declared

Background Smoking is the most extensively studied environmental risk factor of rheumatoid arthritis (RA). We have recently shown that smoking is associated with high serum levels of survivin (Svensson, Hafstrom et al. 2014). Survivin is a novel biomarker for RA that can predict severe joint destruction as well as treatment outcome for RA patients (Levitski et al, 2015). We hypothesized cytotoxic activity to be the reason for the increased serum levels of survivin observed in RA patients and we wished to further investigate possible molecular mechanisms behind cytotoxicity in RA. Objectives We have studied the effect of nicotine on cytotoxic activity of T lymphocytes during RA paying special attention to the expression of PD-1, a receptor that inhibits activation of T cells. Methods We used female Balb/c mice that received 0.03% nicotine in drinking water or regular tap water as controls, after 18 days mice were immunized with collagen and subsequently developed arthritis (CIA). At the end of experiment bone marrow, lymph nodes and serum were collected and analyzed using flow cytometry (FACS), qPCR and ELISA. To confirm nicotine-dependent nature of experimental findings, we analyzed blood samples of female RA patients and healthy women with known smoking status. Results The proportion of CD8+ cells was 1.9 times larger in nicotine treated mice compared to control (p=0.00020). Meanwhile, the nicotine treated mice had a 2.3 times less PD-1 expressing CD8+ cells in the bone marrow (p=0.032), but PD-1 expression in the lymph nodes was not affected by nicotine. This resulted in an accumulation of the CD8+PD-1- population in the bone marrow of the nicotine treated mice (p=0.0074). The nicotine treated mice had higher levels of serum survivin (0.0 vs 0.29 ng/ml, p=0.017) which also correlated to the size of CD8+PD-1- population in the bone marrow (r=0.52, p=0.024). The decrease in PD-1 expression of CD8+ T cells caused by nicotine was confirmed in smoking women. Smokers had a 1.7 times less PD-1 positive CD8+ T cells compared to non-smokers (p=0.041). In patients, the serum levels of survivin correlated to the expression of the cytotoxic marker CD107 on CD8+ T cells (r=0.52, p=0.047). Conclusions Nicotine exposure supports cytotoxic phenotype of CD8+ T cells characterized by high CD107 and low PD-1 expression in experimental arthritis and RA patients. Increased cytotoxic activity of CD8+ T cells is a plausible mechanism for the enrichment of survivin in serum in RA patients. See graphical abstract. References Svensson B, Hafstrom I, Erlandsson MC, Forslind K, Bokarewa MI. Smoking in combination with antibodies to cyclic citrullinated peptides is associated with persistently high levels of survivin in early rheumatoid arthritis: a prospective cohort study. Arthritis Res Ther. 2014;16(1):R12. doi: 10.1186/ar4438. Levitsky A, Erlandsson MC, van Vollenhoven RF, Bokarewa MI, Serum survivin predicts responses to treatment in active rheumatoid arthritis: a post hoc analysis from the SWEFOT trial. BMC Med. 2015;13:247. doi: 10.1186/s12916-015-0485-2. Disclosure of Interest None declared

Background MicroRNA (miR) represent a part of epigenetic contol of autoimmunity and gain increasing attention in the pathogenesis of RA. Genome-wide and candidate miR surveys indicated deregulation of selected miRs in RA blood and synovial tissue. Smoking belongs to enviromental factors with the ability to reprogram epigenetic control. It is also the most studied risk factor in RA being tightly connected to the production of autoantibodies, high levels of oncoprotein survivin, and resistance to antirheumatic therapy (1,2). Objectives The aim of this study was to determine effect of smoking on the expression of miRs in leukocytes of RA patients. Methods Leukocytes of the peripheral blood of 46 female RA patients (age 21–69 years, disease duration 1–22 y) were used as a source of miR. Information about smoking status was collected via a questionnaire and identified current smokers (CS, n=12), former smokers (FS, n=21) and never smokers (NS, n=13). We used the Toray 3D-Gene microRNAs assay-based approach to compare the levels of 2598 miRs between the RA patients with different smoking status. For the comparison between the groups we used only miRs with abundant (raw values >100) expression in leukocytes. Smoking and RA-associated miRs were retrived from the available comprehensive reviews (3,4). Results The number of detectable miR was highest in the PBMC samples of NS. Former and current smoking was associated with a step-wise decreased global miR expression being most severe in CS. We identified 267 miRs, which had an abundant expression in NS. The expression of 93 of these miRs was significantly affected by smoking being 1.75-times lower in FS and CS (p=0.0002). Analyses of miRs, which had previously been reported sensitive to smoking showed that FS+CS had lower expression of the miRs belonging to the let-7 family compared to NS (2.43 times, p=0.034). Additionally, FS had low expression of the miR-17/92 cluster, where miR-17 family was affected most, reduced 2.05 times (p=0.066), followed by miR-19 and 18 families (1.74 times) and miR-92 family (1.56 times). FS had also 2.1 times lower expression of the miR-16 family. The analysis of miRs described in relation to RA showed that 20 of 30 RA-related miRs were abundantly expressed in the studied samples. The expression of miR-16–5p, miR-126–3p, miR-142–3p, miR-150–5p, miR-22–3p and miR-221–3p showed more then 1.75 times difference between FS and NS. Conclusions Smoking is associated with a global reduction in miR expression in RA patients, where the members of let-7; miR-17/92 and miR-16 families with key roles in regulation of cell cycle progress, tissue development and immune responses clusers are most affected. References Klareskog L et al. A new model for an etiology of rheumatoid arthritis: smoking may trigger HLA-DR (shared epitope)-restricted immune reactions to autoantigens modified by citrullination. Arthritis Rheum. 2006;54(1):38–46 Svensson B et al. Smoking in combination with antibodies to cyclic citrullinated peptides is associated with persistently high levels of survivin in early rheumatoid arthritis: a prospective cohort study. Arthritis Res Ther. 2014;16(1):R12 Churov AV et al. MicroRNAs in rheumatoid arthritis: altered expression and diagnostic potential. Autoimmun Rev. 2015;14(11):1029–37 Vrijens K et al. MicroRNAs as potential signatures of environmental exposure or effect: a systematic review. Environ Health Perspect. 2015;123(5):399–411 Disclosure of Interest None declared

Lubricin (or proteoglycan 4 (PRG4)) is an abundant mucin-like glycoprotein in synovial fluid (SF) and a major component responsible for joint lubrication. In this study, it was shown that O-linked core 2 oligosaccharides (Galβ1–3(GlcNAcβ1–6)GalNAcα1-Thr/Ser) on lubricin isolated from rheumatoid arthritis SF contained both sulfate and fucose residues, and SF lubricin was capable of binding to recombinant L-selectin in a glycosylation-dependent manner. Using resting human polymorphonuclear granulocytes (PMN) from peripheral blood, confocal microscopy showed that lubricin coated circulating PMN and that it partly co-localized with L-selectin expressed by these cells. In agreement with this, activation-induced shedding of L-selectin also mediated decreased lubricin binding to PMN. It was also found that PMN recruited to inflamed synovial area and fluid in rheumatoid arthritis patients kept a coat of lubricin. These observations suggest that lubricin is able to bind to PMN via an L-selectin-dependent and -independent manner and may play a role in PMN-mediated inflammation.

Objective. To assess the potential of metastasin S100A4 as a biological marker in patients with RA.Methods. A total of 87 unselected patients with established RA (disease duration 2-44 years) and treated with MTX and infliximab at a single rheumatology centre were included in a cross-sectional study. Radiographs of hands and feet were taken prior to infliximab treatment and at inclusion (time interval 48 ± 27 months) and scored for the radiographic damage. S100A4 levels were analysed in relation to radiographic damage, clinical disease activity (DAS-28), inflammation (IL-6, CRP, ESR), bone and cartilage markers [MMP-3, COMP, C-telopeptide of type I collagen (CTX-I)] and proto-oncogenes [survivin, insulin-like growth factor 1 (IGF-1), Flt3 ligand].Results. High levels of S100A4 were associated with severe radiographic damage (OR = 3.40, P = 0.025), non-response to infliximab (OR = 4.63, P = 0.003), presence of antibodies to infliximab (OR = 6.24, P = 0.003) and high levels of Flt3 ligand (OR = 2.73, P = 0.04). Regression analysis showed that high S100A4 was predictive for radiographic progression during infliximab treatment [positive predictive value (PPV) 0.68, P = 0.05]. Low levels of S100A4 were associated with response to infliximab (OR = 2.67, P = 0.049), clinical remission (OR = 4.01, P = 0.0047) and negative RF (OR = 9.22, P = 0.0047). S100A4 correlated with survivin (r = 0.71, P > 0.0001).Conclusion. S100A4 levels are increased in proportion to radiographic damage and its further progression in RA patients. High S100A4 levels were associated with a poor clinical response to infliximab and high rate of anti-infliximab antibodies. The finding of a correlation between S100A4 and survivin and Flt3 ligand suggests that these proteins may represent a new cluster of biomarkers predicting radiographic progression and poor treatment response in RA patients.

Survivin is known as an inhibitor of apoptosis and a positive regulator of cell division. We have recently identified survivin as a predictor of joint destruction in patients with rheumatoid arthritis (RA). Flt3 ligand (Flt3L) is expressed in the inflamed joints and has adjuvant properties in arthritis. Studies on 90 RA patients (median age 60.5 years [range, 24–87], disease duration 10.5 years [range, 0–35]) show a strong positive association between the levels of survivin and Flt3L in blood. Here, we present experimental evidence connecting survivin and Flt3L signaling. Treatment of BALB/c mice with Flt3L led to an increase of survivin in the bone marrow and in splenic dendritic cells. Flt3L changed the profile of survivin splice variants, increasing transcription of the short survivin40 in the bone marrow. Treatment with an Flt3 inhibitor reduced total survivin expression in bone marrow and in the dendritic cell population in spleen. Inhibition of survivin transcription in mice, by shRNA lentiviral constructs, reduced the gene expression of Flt3L. We conclude that expression of survivin is a downstream event of Flt3 signaling, which serves as an essential mechanism supporting survival of leukocytes during their differentiation, and maturation of dendritic cells, in RA.

The influence of 38 IgG fractions with either cardiolipin antibodies only (CLa) or both CLa and lupus anticoagulant activity (LA) on the response to activated protein C (APC) and on the snake venom activation of protein C were investigated. Five of eight IgG fractions with LA activity showed a tendency to reduce the effect of APC in the aPTT method and to simulate the APC-resistance phenomenon. This effect was not observed in IgG fractions without LA activity. The addition of IgG fractions did not decrease enzymatic activation of protein C in normal plasma. No correlation was found between sample protein C activity and CLa level or the extent of clotting time prolongation. These observations suggested that acquired resistance to APC in the presence of some types of phospholipid antibodies may be a cause of thrombophilia in such patients.

Objective.To evaluate the safety and effectiveness of rituximab (RTX) in combination with methotrexate in patients with active rheumatoid arthritis (RA) after failure of a single tumor necrosis factor-α (TNF-α) inhibitor. Changes in patient-reported outcomes after primary treatment or retreatment with RTX and factors determining retreatment in clinical practice were also evaluated.Methods.In this phase 3b open-label, multicenter trial, patients received 2 slow infusions of RTX 1000 mg 14 days apart after premedication (primary treatment). Patients with a clinically relevant response could receive retreatment between 24 and 48 weeks. The primary endpoint was evaluation of safety. Secondary outcomes were safety of retreatment, effectiveness of primary treatment and retreatment, and changes in patient-reported outcomes after primary treatment or retreatment.Results.Of 120 patients enrolled at 36 centers and receiving primary RTX treatment, 77 received retreatment, 112 completed the 24-week primary treatment period, and 25 completed the 48-week primary treatment and retreatment period following a single course of RTX. The most common adverse events were mild to moderate nausea, vomiting, nasopharyngitis, and headache. No infections or infusion reactions were considered life-threatening. At 24 weeks, 58%, 27%, and 7% of patients achieved American College of Rheumatology 20, 50, and 70 improvements, respectively, and similar improvements were seen after retreatment.Conclusion.RTX was well tolerated, with a low incidence of infusion reactions and infections. Efficacy results, including enhanced response in rheumatoid factor-positive patients, were comparable to those reported in the literature. Based on its efficacy and safety profile and retreatment schedule, RTX is an attractive treatment option for patients that have not responded to a single TNF-α inhibitor.

TKs are intracellular signaling molecules essential for cell homeostasis. Inhibition of TKs is used in treatment of malignancies and diabetes mellitus. The present study evaluated the role of Flt3 in antigen-induced arthritis. Mice were immunized with mBSA, and arthritis was induced by an i.a. injection of mBSA. Treatment with the Flt3 inhibitor sunitinib was started together with mBSA immunization or together with the induction of arthritis. The mBSA-injected joints were evaluated morphologically for signs of synovitis and bone/cartilage destruction. Markers of bone metabolism and antibody responses were measured by ELISA. Maturation of DCs in the bone marrow and spleen was evaluated by flow cytometry. Sunitinib treatment reduced the intensity of synovitis and the incidence of bone destruction. The reduction in bone destruction was seen when the treatment was started at the time of immunization or at the time of arthritis induction. The antiarthritic effect was achieved by inhibition of DCs, reduction of antibody production, and bone metabolism. Inhibition of Flt3 is a potent antiarthritic mechanism reducing antigen presentation, synovial inflammation, and bone resorption. Down-regulation of TKs may be a useful tool in the treatment of human RA.

Introduction Activated fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA) share many characteristics with tumour cells and are key mediators of synovial tissue transformation and joint destruction. The glycoprotein podoplanin is upregulated in the invasive front of several human cancers and has been associated with epithelial-mesenchymal transition, increased cell migration and tissue invasion. The aim of this study was to investigate whether podoplanin is expressed in areas of synovial transformation in RA and especially in promigratory RA-FLS. Methods Podoplanin expression in human synovial tissue from 18 RA patients and nine osteoarthritis (OA) patients was assessed by immunohistochemistry and confirmed by Western blot analysis. The expression was related to markers of synoviocytes and myofibroblasts detected by using confocal immunofluoresence microscopy. Expression of podoplanin, with or without the addition of proinflammatory cytokines and growth factors, in primary human FLS was evaluated by using flow cytometry. Results Podoplanin was highly expressed in cadherin-11-positive cells throughout the synovial lining layer in RA. The expression was most pronounced in areas with lining layer hyperplasia and high matrix metalloproteinase 9 expression, where it coincided with upregulation of α-smooth muscle actin (α-sma). The synovium in OA was predominantly podoplanin-negative. Podoplanin was expressed in 50% of cultured primary FLSs, and the expression was increased by interleukin 1β, tumour necrosis factor α and transforming growth factor β receptor 1. Conclusions Here we show that podoplanin is highly expressed in FLSs of the invading synovial tissue in RA. The concomitant upregulation of α-sma and podoplanin in a subpopulation of FLSs indicates a myofibroblast phenotype. Proinflammatory mediators increased the podoplanin expression in cultured RA-FLS. We conclude that podoplanin might be involved in the synovial tissue transformation and increased migratory potential of activated FLSs in RA.

Background: Ecs is an ATP-binding cassette (ABC) transporter present in aerobic and facultative anaerobic Gram-positive Firmicutes. Inactivation of Bacillus subtilis Ecs causes pleiotropic changes in the bacterial phenotype including inhibition of intramembrane proteolysis. The molecule(s) transported by Ecs is (are) still unknown.Methodology/Principal Findings: In this study we mutated the ecsAB operon in two Staphylococcus aureus strains, Newman and LS-1. Phenotypic and functional characterization of these Ecs deficient mutants revealed a defect in growth, increased autolysis and lysostaphin sensitivity, altered composition of cell wall proteins including the precursor form of staphylokinase and an altered bacterial surface texture. DNA microarray analysis indicated that the Ecs deficiency changed expression of the virulence factor regulator protein Rot accompanied by differential expression of membrane transport proteins, particularly ABC transporters and phosphate-specific transport systems, protein A, adhesins and capsular polysaccharide biosynthesis proteins. Virulence of the ecs mutants was studied in a mouse model of hematogenous S. aureus infection. Mice inoculated with the ecs mutant strains developed markedly milder infections than those inoculated with the wild-type strains and had consequently lower mortality, less weight loss, milder arthritis and decreased persistence of staphylococci in the kidneys. The ecs mutants had higher susceptibility to ribosomal antibiotics and plant alkaloids chelerythrine and sanguinarine.Conclusions/Significance: Our results show that Ecs is essential for staphylococcal virulence and antimicrobial resistance probably since the transport function of Ecs is essential for the normal structure and function of the cell wall. Thus targeting Ecs may be a new approach in combating staphylococcal infection.

Rheumatoid arthritis (RA) is characterized by an uncontrolled spread of destructive joint inflammation resembling malignancy. Epidemiological studies have established strong correlation between inflammation and predisposition for cancer. Here we assess the predictive role of the circulating proto-oncogene survivin for clinical and radiological outcome of early RA.Serum survivin was measured by sandwich ELISA in 651 patients with early RA (mean duration 6 months). X-rays of hands and feet were prospectively obtained at base-line and after 1, 2, and 5 years and evaluated for the presence of bone destruction by a modified Sharp method. The predictive value of survivin for radiological destruction was calculated using multivariate regression models including antibodies against cyclic citrullinated peptides (aCCP) and rheumatoid factor (RF). Remission was assessed by the EULAR (European League Against Rheumatism) criteria and by criteria proposed by Mäkinen.At base-line, 391 patients (60%) had high levels of survivin. Radiological progression at 5 years was significantly more frequent (P= 0.001) among survivin-positive patients than among survivin-negative. Survivin positivity predicted radiological progression independently of aCCP and RF. The positive predictive value of survivin was proved both in the group of patients with and in the group without erosions at base-line. The combination of positive tests for both survivin and aCCP had the highest prediction for radiological progression (positive predictive value 0.75). Additionally, a positive test for survivin was an independent predictor of not being in remission.Detection of survivin in early RA predicted joint destruction and failure of achieving remission after 5 years in patients with early RA.

Objectives. Viruses may contribute to RA. This prompted us to monitor viral load and response to anti-CD20 therapy in RA patients. Methods. Blood and bone marrow from 35 RA patients were analysed for CMV, EBV, HSV-1, HSV-2, parvovirus B19 and polyomavirus using real-time PCR before and 3 months after rituximab (RTX) treatment and related to the levels of autoantibodies and B-cell depletion. Clinical response to RTX was defined as decrease in the 28-joint disease activity score (DAS-28) >1.3 at 6 months. Results. Before RTX treatment, EBV was identified in 15 out of 35 patients (EBV-positive group), of which 4 expressed parvovirus. Parvovirus was further detected in eight patients (parvo-positive group). Twelve patients were negative for the analysed viruses. Following RTX, EBV was cleared, whereas parvovirus was unaffected. Eighteen patients were responders, of which 12 were EBV positive. The decrease in the DAS-28 was significantly higher in EBV-positive group compared with parvo-positive group (P = 0.002) and virus-negative patients (P = 0.04). Most of EBV-negative patients that responded to RTX (75%) required retreatment within the following 11 months compared with only 8% of responding EBV-positive patients. A decrease of RF, Ig-producing cells and CD19+ B cells was observed following RTX but did not distinguish between viral infections. However, EBV-infected patients had significantly higher levels of Fas-expressing B cells at baseline as compared with EBV-negative groups. Conclusions. EBV and parvovirus genomes are frequently found in bone marrow of RA patients. The presence of EBV genome was associated with a better clinical response to RTX. Thus, presence of EBV genome may predict clinical response to RTX.

Introduction: Rheumatoid arthritis (RA) is frequently complicated with infections. The aim of our study was to evaluate vaccination response in patients with RA after B-cell depletion by using rituximab. Methods: Influenza (Afluria) and pneumococcal polysaccharides (Pneumo23) vaccines were given 6 months after rituximab (post-RTX group, n = 11) or 6 days before rituximab treatment (pre-RTX group; n = 8). RA patients never exposed to RTX composed the control group (n = 10). Vaccine-specific cellular responses were evaluated on day 6 after vaccination, and vaccine-specific humoral responses, on day 21. Results: On day 6 after vaccination, formation of influenza-specific B cells was lower in post-RTX group as compared with the pre-RTX group and controls (P = 0.04). Polysaccharide-specific B cells were found in 27% to 50%, being equally distributed between the groups. On day 21, the impairment of humoral responses was more pronounced with respect to influenza as compared with the pneumococcal vaccine and affected both IgG and light-chain production. Total absence of influenza-specific IgG production was observed in 55% of the post-RTX group. Conclusions: RTX compromises cellular and humoral vaccine responses in RA patients. However, repeated RTX treatment or previous anti-tumor necrosis factor (anti-TNF) treatment did not accentuate these defects.

Toll-like receptors (TLRs) are a family of cellular structures activated by recognition of pathogen associated molecular sequences. The activation of TLRs triggers a variety of intracellular mechanisms aiming to protect the host from the invading microorganisms. Lipopolysaccharide (LPS) is the main ligand for TLR4. Here we show that resistin, a cystein-rich protein believed to regulate carbohydrate metabolism, competes with LPS for binding to TLR4. Binding of recombinant resistin to human myeloid and epithelial cells was assessed by flow cytometry and its co-precipitation with TLR4 was demonstrated. Antibodies against TLR4 abolished resistin binding to human leucocytes and cytokine production by peripheral blood mononuclear cells in response to resistin stimulation. In contrast, isotype-matched murine IgG or TLR2 antibodies were unable to prevent binding of resistin to the cells. Similarly, TLR4-dependent pattern of resistin binding was observed in epithelial cell line HEK293 (human epithelial kidney cell), where TLR4 transfected, but not myeloid differentiation factor 2/CD14-transfected, TLR2 transfected or HEKnull cells, responded functionally to resistin stimulation. Intracellular signalling of resistin was assessed using inhibitors of transcription factors mitogen activated protein kinases, nuclear factor-κB, phosphoinositide 3-kinase and siRNA targeting TLR4 and human myeloid differentiation factor 88. Results demonstrate that TLR4 serves as a receptor for the pro-inflammatory effects of resistin in human cells. This may partly explain the multifunctional role of resistin in chronic inflammation, atherosclerosis and insulin resistance.

Introduction: Bacterial arthritis causes rapidly progressing joint destruction in humans. We have shown that addition of bisphosphonates or corticosteroids to conventional antimicrobial agents decreases the activity of osteoclasts, thereby reducing bone destruction. Here we assess the effect of RANKL-targeted treatments using soluble receptor decoy and osteprotegerin (OPG) on the course and outcome of staphylococcal arthritis. Methods: Treatment was initiated 3 days after Staphylococcus aureus inoculation and included RANK-Fc, OPG-Fc, and OPG-Fc in combination with antibiotics. Control groups were treated with antibiotics, huFc, and PBS. Joints were evaluated for clinical signs of arthritis and histologically for bone and cartilage destruction. Bone mineral density (BMD) was evaluated using a peripheral quantitative computed tomography. Circulating markers of bone metabolism, inflammatory cytokines, and chemokines were analyzed in each group. Results: Mice treated with RANK-Fc or OPG-Fc in combination with antibiotics preserved total BMD and trabecular bone as compared to huFc or antibiotics. Treatment with RANK-Fc or OPG-Fc diminished the levels of bone resorption markers (osteocalcin, CTX-I, and TRACP5b). Neither RANK-Fc nor OPG-Fc influenced significantly the frequency and severity of arthritis. Conclusions: Inhibition of RANKL signalling efficiently prevents bone loss in the mouse model of bacterial arthritis even when started in the overt phase of infection.

Viruses may be part of the pathogenesis of rheumatoid arthritis (RA). Double stranded RNA (dsRNA) is a prototypic viral conformation of nucleic acid that is highly arthritogenic in mice. Therefore, we developed an ELISA to detect dsRNA in sera and synovial fluids (SF) in RA patients and in osteoarthritic controls. The developed ELISA recognizes picogram levels of viral or synthetic dsRNA but shows no reactivity against DNA, synthetic ssRNA, or total RNA prepared from mammalian cells. Before analysis by ELISA, each sample was subjected to RNA precipitation. The RA patients had significantly higher levels of dsRNA than the osteoarthritis patients in SF and in sera. In 7 of 17 RA patients, EBV was present in SF and in all but one of these this was accompanied by the presence of dsRNA. No parvovirus, cytomegalovirus, or polyomavirus was detected. The anti-viral cytokine IFN-alpha was detected in SF in 10 of 21 RA patients, but in none of the osteoarthritis patients. Notably, RA patients with erosive disease course had significantly higher levels of dsRNA in SF than non-erosive patients, but no correlation between dsRNA levels and the presence of RF or levels of C-reactive protein, IL-6, or IFN-alpha was observed.

To assess the role of resistin in primary Sjögren's syndrome (pSS) and its relation to local inflammation.Blood and saliva were collected from 37 patients with pSS (duration of symptoms 12.6+/-1 yrs) and 32 healthy controls. Expression of resistin in salivary glands was visualized immunohistologically, and levels of resistin were detected by ELISA. Levels of resistin were evaluated at baseline and following oral dehydroepiandrosterone (DHEA) treatment (50 mg/day). The effect of DHEA treatment on the secretion of resistin was assessed in vitro in human leukocytes after challenge with insulin and lipopolysaccharide.Levels of resistin in saliva were significantly higher in patients with pSS than in controls, while circulating levels of resistin were similar in both groups. Resistin was expressed in the epithelial cells of striated ducts and in the lymphocytic foci. Resistin levels in saliva were related to the intensity of inflammation in the minor salivary glands of pSS patients. No changes of the levels of resistin in blood or saliva were observed during DHEA treatment. Exposure of naive leukocytes to DHEA in vitro induced significant expression of resistin compared to nonstimulated peripheral blood mononuclear cells (p=0.031).We showed that levels of resistin are upregulated locally in the salivary glands of patients with pSS; and that the levels of resistin correspond to the intensity of lymphocytic inflammation in patients with pSS. We suggest that resistin is expressed in the salivary glands of patients with pSS and may be a driving factor of local inflammation.

The present study assessed the inductory effects of ds- and ssRNA on the leukocyte production of proteins belonging to fibrinolytic and coagulation cascades. Murine splenocytes were stimulated with dsRNA [polyinosinic:polycytidylic acid (polyIC)] and ssRNA sequences [polyinosinic acid (polyI), polycytidylic acid (polyC), and polyuridylic acid (polyU)]. The expression of plasminogen (Plg), tissue factor (TF), IL-6, and IFN-α was assessed. Intracellular tranduction mechanisms activated by oligonucleotides were evaluated using specific inhibitors of signaling pathways and genetically modified mice. polyIC efficiently and dose-dependently induced the expression of Plg, IL-6, and IFN-α, whereas TF was not induced by polyIC. polyI was unable to trigger IFN-α production, and it was efficiently inducing Plg and TF. IFN-αR and dsRNA-dependent protein kinase signaling were not required for the polyI-induced production of Plg or TF. Neither polyU nor polyC induced the expression of Plg or TF. Importantly, the presence of U- and C-nucleotide strands in the dsRNA significantly reduced expression of Plg and TF compared with polyI alone. Exposure of splenocytes to polyI activated the NF-κB pathway followed by the expression of TF and IL-6. In contrast, Plg production did not require NF-κB, was only partly down-regulated by p38 MAPK inhibitor, and was efficiently inhibited by insulin, indicating a different mechanism for its induction. ssRNA exerts its TF-generating properties through NF-κB activation in an IFN-α-independent manner. The expression of fibrinolytic versus coagulation proteins is regulated through distinctly different transduction pathways. As fibrinolytic and coagulation cascades are important components of inflammatory homeostatis, these findings might have importance for developement of new, targeted therapies.

Objectives. The aim of this study was to investigate associations between serum levels of resistin, an adipokine and markers of inflammation, bone metabolism, plasma lipids and kidney function in post-menopausal RA patients and to evaluate if HRT during 2 yrs affected resistin levels. Methods. Eighty-eight women were randomly allocated to receive HRT, vitamin D3 and calcium or vitamin D3 and calcium alone. Serum levels of resistin, IL-1� , IL-1 receptor antagonist (IL-1Ra), IL-6, IL-6 soluble receptor, TNF-� were measured by ELISA, markers of bone metabolism, carboxyterminal cross-linked telopeptide of type I collagen (ICTP) and carboxyterminal propeptide of type I procollagen by RIA, ESR, CRP, Hb, creatinine and lipids by standard laboratory techniques, BMD and total lean mass (TLM) by DXA and joint destruction by Larsen score. Resistin was also measured in 42 healthy control women. Results. There was no difference in resistin concentration between patients and healthy controls. Resistin was significantly correlated with IL-1Ra, CRP, TNF-� , ICTP, glucocorticosteroids and Larsen score and inversely with BMD, hip and with TLM. In multiple regression analysis, IL-1Ra, TLM and use of corticosteroids remained determinants of resistin. Patients treated with HRT displayed significant increase in resistin compared with controls in the first but not the second year. Conclusions. Resistin was associated with increased inflammation, particularly by the acute-phase reactant IL-1Ra antagonizing IL-1� , joint destruction, glucocorticosteroids and with reduced BMD and TLM. These findings suggest resistin being a significant mediator in the inflammatory process in RA. Further studies examining the mechanisms behind the relation between resistin and IL-1Ra are encouraged. HRT does not seem to have important long-term effect on resistin.

To retrospectively evaluate longterm clinical and immunological effects of anti-CD20 treatment in patients with systemic lupus erythematosus (SLE) with active nephritis or autoantibody-mediated cytopenias refractory to conventional immunosuppressive treatment.Anti-CD20 treatment (rituximab) was added to the ongoing immunosuppressive treatment in 31 SLE patients with active nephritis (n = 17), thrombocytopenia (n = 10), and hemolytic anemia (n = 4) refractory to conventional therapy. Disease activity was evaluated by the SLE Disease Activity Index. The median followup time after anti-CD20 treatment was 22 months (range 1-61 mo).Complete B cell depletion was obtained in all patients. In 11 of the 17 lupus nephritis patients complete or partial responses were achieved after 6-12 months. Eight of these patients increased their glomerular filtration rate (GFR) by25%. The responders were characterized by having shorter nephritis duration, a baseline GFR30 ml/min, and detectable circulating CD19+ B lymphocytes before B cell depletion. Anti-CD20 treatment was highly effective in patients with autoimmune thrombocytopenia, inducing a significant increase of platelet counts after 1 month (p0.01). Five of 10 patients achieved complete normalization of their platelet counts within 6 months. The anti-CD20 treatment was followed by a significant reduction of autoantibodies against dsDNA and platelets, in nephritic and in thrombocytopenic patients, respectively.Addition of anti-CD20 treatment to conventional immunosuppressive therapy may be a beneficial strategy in refractory lupus nephritis and autoimmune cytopenias, possibly by reducing the levels of pathogenic autoantibodies.

Hyperfibrinolysis is a consequence of imbalance between fibrinolytic activators and their inhibitors. Increased levels of circulating plasminogen (Plg) activators such as tissue- or urokinase-type plasminogen activators (tPA or uPA respectively) are the most common causes of hyperfibrinolysis, occasionally causing major hemorrhages. We found that staphylokinase (SAK), a well-known Plg activator of bacterial origin, inhibits Plg activation mediated by endogenous tPA and uPA. Furthermore, mixture of SAK with tPA led to a significantly reduced Plg-dependent fibrinolysis. This inhibitory effect was exerted through direct action of SAK on Plg rather than indirectly on tPA or uPA. Inhibition of Plg activation by SAK is readily abrogated by interaction of SAK with human neutrophil peptides (HNPs). Finally, we show that NH2-terminal residues of SAK are important for the inhibitory effect of SAK on tPA- and uPA-mediated Plg activation. In conclusion, SAK reduces tPA/uPA-mediated Plg activation by means of SAK.Plg complex formation, consequently downregulating tPA/uPA-induced fibrinolysis.

Background: One of the hallmarks of rheumatoid arthritis (RA) is hyperplasia and inflammation of the synovial tissue being characterized by in situ occurrence of highly differentiated leukocytes. Fms-like tyrosine kinase 3 (Flt3) has a crucial role in hematopoiesis, regulation of cell proliferation, differentiation and apoptosis. Typically, Flt3 is expressed on early myeloid and lymphoid progenitors and is activated by its soluble ligand (Flt3-L). The highly differentiated cellular pattern in the synovium of the RA patients made us hypothesize that Flt3-L, with its ability to induce proliferation and differentiation, could be of importance in induction and/or progression of arthritis.\ud \ud Methodology/Principal Findings: To investigate occurrence of Flt3-L in RA we have measured its levels in matched serum and synovial fluid samples from 130 patients and 107 controls. To analyse the pro-inflammatory role of Flt3-L, we continuously overexpressed this protein locally in healthy mouse joints using homologous B-cell line transfected with Flt3-L gene. Additionally, recombinant Flt3-L was instillated intra-articularly in combination with peptidoglycans, a Toll Like Receptor 2-ligand with stong arthritogenic properties. Our results show significantly higher levels of Flt3-L in the synovial fluid as compared to serum levels in RA subjects (p = 0.0001). In addition, RA synovial fluid levels of Flt-3-L were significantly higher than these obtained from synovial fluids originating from non-inflammatory joint diseases (p = 0.022). Intra-articular administration of B-cell line transfected with Flt3-L gene resulted in highly erosive arthritis while inoculation of the same B-cell line without hyperexpression of Flt3-L did not induce erosivity and only in a minority of cases caused synovial proliferation! Flt3-ligand potentiated peptidoglycan induced arthritis as compared to mice injected with peptidoglycan alone (p

Rheumatoid arthritis (RA) is characterized by chronic joint inflammation and destruction. B cells play important role in modulating immune responses in RA. In the present study we assessed the impact of the B cell targeting as a third line treatment option. Forty-six patients with established erosive RA non-responding to combination treatment with DMARDs and TNF-alpha inhibitors were treated with anti-CD20 antibodies (rituximab). Rituximab was given intravenously once weekly on four occasions. All patients continued with the previous DMARD. Patients were followed by DAS28, levels of circulating B cells, frequency of immunoglobulin-producing cells, immunoglobulins, and rheumatoid factor levels during the period of 12-58 months. Clinical improvement was achieved in 34 of 46 patients (73%) supported by a significant reduction in DAS28 (from 6.04 to 4.64, P < 0.001). Infusion of rituximab resulted in the elimination of circulating B cells in all but one patient. Within 12 months follow-up, B cells returned to circulation in 86% of patients. Fifty-three percent of the patients were successfully retreated with rituximab or re-started with anti-TNF-alpha treatment. Of the 11 non-responders, five were retreated with anti-CD20 within 2 months, four of them with success, four patients received TNF-alpha inhibitors, the remaining two patients received an additional DMARD. Most of the RA patients resistant to TNF-alpha inhibitors may be effectively treated with anti-CD20 antibodies. The treatment is well tolerated and may be used repeatedly in the same patient and potentially increase sensitivity to previously inefficient treatment modalities.

Rheumatoid arthritis (RA) is an inflammatory joint disease leading to cartilage and bone destruction. Insufficient apoptosis in the inflamed RA synovium along with accumulation of highly differentiated B- and T-lymphocytes as well as invasive growth of macrophages and fibroblasts is among the major mechanisms supporting joint destruction. We have recently shown that circulating survivin, an apoptosis inhibitor tightly bound to tumorigenesis, is an independent predictor of development and progression of joint destruction in RA. In this review we discuss the possible connectivity between viral infection, leading to interferon (IFN)-alpha production, survivin expression, and subsequent joint inflammation. The role of IFN-alpha and the involvement of IFN transcription factors and phosphoinositide-3-kinase signalling as essential modulators of arthritogenic process are discussed in the context of survivin.

To assess the interaction between cystatin C (CysC) and serum amyloid A protein (SAA).Levels of CysC and SAA and antibodies against these proteins were assessed in the paired blood and synovial fluid (SF) samples of 90 patients with rheumatoid arthritis (RA). Age and sex matched individuals having normal iohexol clearance (n = 90) and SF following joint trauma (n = 40) were used as controls. In vitro experiments included assessment of interaction between CysC and SAA by ELISA and the influence of CysC on SAA functions.A pilot screening for cystatins C, E, and F in blood and SF of patients with RA found CysC to be by far the predominant extracellular cystatin. Circulating CysC levels were significantly lower in patients with RA compared to the matched controls (0.81 +/- 0.03 vs 1.01 +/- 0.03 mg/l; p = 0.05). These low CysC levels could not be explained by the presence of anti-CysC antibodies in patients with RA. In contrast, concentrations of CysC that accumulated in the inflamed SF were significantly greater in patients with erosive RA (1.66 +/- 0.08 mg/l) compared to nonerosive RA (1.36 +/- 0.06 mg/l; p = 0.003) and controls (1.18 +/- 0.03 mg/l; p = 0.043). In vitro studies showed direct binding of CysC to SAA. CysC/SAA binding impaired proinflammatory effects of SAA, reducing its ability to trigger expression of proinflammatory cytokines.Our study shows a relative deficiency of circulating CysC during systemic inflammation in RA. Physical interaction between CysC and the acute-phase protein SAA (1) provides an explanation for CysC deficiency; and (2) suggests that CysC is regulating inflammatory responses. We hypothesize that decreased systemic CysC levels predispose to accelerated atherosclerosis and development of amyloidosis in patients with RA.

Background Rheumatoid arthritis (RA) is an established risk factor for premature development of cardiovascular disease. Adipokines are signal molecules originating from adipose tissue and exhibit strong proinflammatory effects. We have previously shown that resistin utilizes IGF-1R (1) and TLR4 (2) pathway to induce inflammation in RA leukocytes and synovia. Objectives In the present study we evaluated the role of adipokines and adipokine-related genes on the cardiovascular risk in female RA patients. Methods The estimated cardiovascular risk was calculated in 187 female RA patients, using the modified Systematic Coronary Risk Evaluation (mSCORE). Serum levels of adipokines and IGF-1 and transcription of genes affected by Resistin and IGF-1 signalling in peripheral blood leukocytes and in subcutaneous fat tissue were analyzed in relation to mSCORE. To assess the predictive value of adipokines and gene transcription for the increased mSCORE, two independent binary logistic regression models were designed where high mSCORE or high resistin were chosen as the dependent variables. Results mSCORE was directly related to the age, disease duration, smoking habits and blood fat profile (cholesterol and triglycerides). These parameters were higher in the group with mSCORE≥1 (n=96) indicative for the increased cardiovascular risk. The patients with mSCORE≥1 had significantly higher levels of leptin (p=0.007), and resistin (p=0.03), while the levels of IGF-1 were significantly lower (p The logistic regression analysis with mSCORE as a dependent variable showed that high serum levels of resistin and leptin were independently associated with the increased cardiovascular risk. Similar associations were observed for adipocyte expression of resistin-triggering genes TLR4 (OR 1.15 [95% CI 1.01-2.15]; p=0.03) and STAT3 (OR 2.39 [95% CI 1.00-5.74]; p=0.05). The logistic regression model with high serum resistin as a dependent variable revealed an association to high transcription of STAT3 (OR 1.12 [95% CI 1.00-3.30]; p=0.02), and low transcription of AKT1 in leukocytes and in adipocytes. Conclusions The results of our study indicate that transcriptional activity of subcutaneous adipose tissue in women with RA is associated with the increased cardiovascular risk. High transcriptional activity of resistin-triggering genes may be a sufficient force driving premature cardiovascular disease in RA patients. References Bostrom EA, Svensson M, Andersson S, Jonsson IM, Ekwall AK, Eisler T, Dahlberg LE, Smith U, Bokarewa MI. Resistin and insulin/insulin-like growth factor signaling in rheumatoid arthritis. Arthritis Rheum. 2011,63:2894-904 Tarkowski A, Bjersing J, Shestakov A, Bokarewa MI. Resistin competes with lipopolysaccharide for binding to toll-like receptor 4. J Cell Mol Med. 2010,14:1419-31. Disclosure of Interest None declared

Background Survivin is a sensitive biomarker predicting development of rheumatoid arthritis (RA) several years ahead of clinical symptoms (1). In early RA, more than 50% of the patients have high serum levels of survivin (2), which recognises the patients with poor treatment responses and progressive joint damage (3). Objectives In this study we evaluated predictive potential and specificity of serum survivin for early recognition of RA. Methods All serum samples of individuals >18 years of age referred during the period of 12 months to the Laboratories of Clinical Immunology at the University Hospitals of Gothenburg and Umea in Sweden were enrolled in the analysis of survivin, rheumatoid factor (RF) and/or anti-citrullinated peptide (ACPA) antibodies. Serum levels of survivin were measured using a sandwich ELISA, and the levels >0.45 ng/ml were considered positive. Medical records of the individuals positive for survivin and RF/ACPA antibodies were reviewed by independent experts to identify cases with newly-diagnosed RA, established RA, and unexplained arthralgia. Results The total of 8868 (5422 Gothenburg, 3446 Umea) adult individuals were analysed and the prevalence of survivin-positive samples was 11.2% and 14.7%, respectively. Among 921 survivin-positive samples, 46% were also positive for RF and/or ACPA vs 10% of survivin-negative, and indicated that survivin-positive individuals had increased estimated risk for being also RF/ACPA positive (RR 4.44[4.01-4.92], p -7 ). Survivin levels were significantly higher in patients with combined positivity of survivin and RF/ACPA (p -7 ). This difference between the cohorts was due to the increasing prevalence of newly-diagnosed RA compared to arthralgia cases in survivin-negative-RF/ACPA-positive cohort (15.3% vs 7.2%, p=0.0004), while this prevalence did not change in surviving-positive cohort (27% vs 21%, respectively). Conclusions In the unselected serum samples, high levels of survivin were associated with RF and/or ACPA antibodies. Cases with arthralgia and newly-diagnosed RA were accumulated within the Survivin-positive samples and suggest a predictive potential of survivin early in the disease course. References Bokarewa M et al., Arthritis Res Ther 2014;16(1):R45 Svensson B et al., Ann Med 2010;42:45-54 Svensson B et al., Arthritis Res Ther 2014;16(1):R12 Disclosure of Interest None declared

Background Survivin is an oncological biomarker. In rheumatoid arthritis (RA), elevated serum survivin is common and has been used to predict disease onset and progressive joint damage. Objectives We investigated the predictive capacity of survivin in clinical disease activity and/or response to various antirheumatic treatments in patients with early RA. Methods Survivin levels from serum were measured using ELISA at baseline in 302 patients enrolled in the Swedish pharmacotherapy (SWEFOT) trial with follow-up at 3, 12 and 24 months. After methotrexate (MTX) monotherapy for 3 months, responders (DAS28≤3.2) remained on MTX, while non-responders were randomized to triple therapy (MTX+sulfasalazine+hydroxychloroquine) or anti-TNF (MTX+infliximab). Survivin levels >0.45 ng/mL were considered positive. Based on survivin status over 24 months, core-set outcomes (i.e. DAS28, HAQ, pain & global VAS) were evaluated at 3, 12, and 24 months. Results Over one-third of all patients (n=114) were survivin -positive at baseline. S urvivin -positive ever-smokers (51/71 vs. 64/112, OR 1.91 [95% CI 1.01-3.62], p=0.045) and survivin -negative patients who converted to positive over 24 months (13/161 vs. 2/100, OR 4.39 [0.97, 19.88], p=0.037) responded seldom to MTX. At 3 months, survivin- positive patients who converted negative (n=11) had greater reductions in DAS28 (p=0.002), HAQ (p=0.011) and global VAS (p=0.025) vs. those who remained positive (n=28), which were maintained over 24 months. At 12 months, survivin -positive MTX-responders who continued monotherapy had a higher risk of disease re-activation on MTX compared to the survivin -negative patients (12/36 vs. 7/52, OR 3.21 [1.12-9.24], p=0.032) and showed no improvement in HAQ over 24 months. Survivin -positive MTX non-responders who converted to negative (n=32) or remained negative (n=83) had greater improvements from 3 to 12 months than those who converted to positive (n=13) (ΔDAS28>1.2, 63% & 60% vs. 31%, p=0.053, p=0.046, respectively). Among survivin -positive patients on triple therapy, converting to negative (n=19) yielded a lower DAS28 at 12 months (2.34 vs. 4.12, p=0.046) and a high frequency of DAS≤3.2 (86% vs. 37%, p=0.056) at 24 months vs. converting to positive (n=7). They also had lower pain (p=0.048) and global VAS (p=0.015) at 24 months compared to the same subgroup, which was not observed among anti-TNF – where no differences in core-set outcomes were observed between the survivin groups. Survivin -positive patients on anti-TNF had a higher risk to have active disease at 24 months compared to those on triple therapy (16/29 vs. 9/32, OR 3.15 [1.09-9.10], p=0.037). Conclusions Survivin -positive patients have worse 24-month outcomes than survivin -negative patients if treated with MTX monotherapy, but conversion to survivin- negative is associated with a good and stable response to MTX monotherapy. For survivin -positive patients with early RA who fail MTX, triple therapy is associated with a better likelihood for response than anti-TNF therapy. Disclosure of Interest A. Levitsky: None declared, M. Erlandsson: None declared, R. van Vollenhoven Grant/research support from: AbbVie, BMS, GSK, Pfizer, Roche, UCB, Consultant for: AbbVie, Biotest, BMS, Crescendo, GSK, Janssen, Lilly, Merck, Pfizer, Roche, UCB, Vertex, M. Bokarewa: None declared

Environmental factors are thought to play a major role in the development of rheumatoid arthritis. Because the use of ethanol is widespread, we assessed the role of ethanol intake on the propensity to develop chronic arthritis. Collagen type II-immunized mice were given water or water containing 10% (vol/vol) ethanol or its metabolite acetaldehyde. Their development of arthritis was assessed, as well as the impact of ethanol on leukocyte migration and activation of intracellular transcription factors. Mice exposed daily to this dose of ethanol did not display any liver toxicity, and the development of erosive arthritis was almost totally abrogated. In contrast, the antibody-mediated effector phase of collagen-induced arthritis was not influenced by ethanol exposure. Also, the major ethanol metabolite, acetaldehyde, prevented the development of arthritis. This antiinflammatory and antidestructive property of ethanol was mediated by ( i ) down-regulation of leukocyte migration and ( ii ) up-regulation of testosterone secretion, with the latter leading to decreased NF-κB activation. We conclude that low but persistent ethanol consumption delays the onset and halts the progression of collagen-induced arthritis by interaction with innate immune responsiveness.

The characteristics of human resistin (RETN) are unclear and controversial despite intensive adipose-focused research. Its transcriptional and functional similarity with the murine myeloid-specific and CCAAT/enhancer binding protein epsilon (Cebpe)-dependent gene, resistin-like gamma (Retnlg), is unexplored. We examined the human CEBPE-regulatory pathway by unbiased reference and custom gene expression assays. Real-time RT-PCR analysis demonstrated lack of both the transcriptional factor CEBPE and RETN expression in adipose and muscle cells. In contrast, primary myelocytic samples revealed a concerted CEBPE-RETN transcription that was significantly elevated in inflammatory synoviocytes relative to intact peripheral blood mononuclear cells (PBMC). Mouse Cebpe and Retnlg were predictably expressed in macrophages, whereas Retn was abundant in adipocytes. Quite the opposite, a low and inconsistent RETN transcription was seen in some human white adipose tissue (WAT) biopsies without any relationship to body mass index, insulin sensitivity, or fat depot. However, in these cases, RETN was co-detected with CEBPE and the leukocyte-specific marker, EMR1, indicating the presence of inflammatory cells and their possible resistin-mediated effect on adipocytes. Indeed, addition of human resistin to WAT in culture induced, like in PBMC, the inflammatory cytokines IL6, IL8 and TNF. Importantly, the expression of the adipose-specific markers CEBPA, FABP4 and SLC2A4 was unchanged, while the expected inhibitory effect was seen with TNF. Both cytokines increased the mRNA level of CCL2 and MMP3, which may further promote inflammation in WAT. Thus, the myeloid-restricted nature of CEBPE precludes the expression of RETN in human adipocytes which, however, are targeted by this innate immune-derived proinflammatory cytokine.

Urokinase (uPA) is a serine protease that not only displays fibrinolytic function but also promotes host leukocytes to home to inflammatory sites. We have recently demonstrated that staphylokinase (SAK), which is a fibrinolytic protein secreted by Staphylococcus aureus, forms complexes with human neutrophil peptides (HNPs), which are members of the defensin family and have anti-microbial properties, thereby inhibiting the bactericidal effects of the HNPs. The aim of this study was to assess whether endogenous uPA, which has fibrinolytic properties similar to those of SAK, binds to HNPs and interferes with SAK/HNPs interaction. To this end, an ELISA was used to analyze the interactions between uPA and HNPs. HMW uPA had the ability to bind to both HNP types. The biological consequences of the formation of this complex were analyzed with respect to its bactericidal properties. HMW uPA killed S. aureus, albeit at relatively high doses (50-100 mug/ml). In contrast, the binding of HMW uPA to HNPs had no impact on the bactericidal functions of the HNPs. Importantly, the addition of HMW uPA to SAK eliminated the ability of SAK to neutralize HNPs. Our results demonstrate that endogenous HMW uPA inhibits S. aureus growth both directly, by cytolysis, and indirectly, by abrogation of the neutralizing effect of SAK on the bactericidal activities of HNPs. These findings indicate novel functions of HMW uPA in the host defense against staphylococcal infections.

Tissue inhibitors of matrix metalloproteinases (TIMPs) regulate the breakdown of extracellular matrix components and play an important role in tissue remodelling and growth, in both physiological and pathological conditions. We studied the autoimmune response to TIMPs in patients with rheumatoid arthritis (RA). Eighty-nine paired blood and synovial fluid samples from patients with RA were assessed for their reactivity with recombinant tissue inhibitors of metalloproteinases (TIMPs) 1 to 4 by an ELISA and were compared with blood from 62 healthy controls and 21 synovial fluid samples from patients with degenerative joint diseases. Presence of antibodies was established as the absorbance of the sample more than 2 standard deviations above the mean of the controls. In addition, immunoglobulin G (IgG) from blood samples of RA patients possessing TIMP antibodies was isolated on protein A–sepharose and tested for the in vitro ability to neutralize TIMP-2-dependent effects on metalloproteinase 9 (MMP9). Anti-TIMP antibodies were found in 56% of RA samples but in only 5% of the controls (P < 0.005). RA patients had high frequencies of antibodies against all TIMPs except TIMP-3. TIMP-2 antibodies were most frequently found (33%), being significantly more prevalent (P = 0.024) in patients with nonerosive than erosive RA. TIMP-1 antibodies were significantly more often found in synovial fluid samples than in the matched blood samples (P < 0.025). Importantly, the IgG fraction containing TIMP antibodies down-regulated the TIMP-2 inhibitory effect, thereby supporting MMP9 activity in vitro. In the present study, we show that RA patients frequently develop autoimmune response to TIMPs that may act as a functionally significant regulator of MMP activity and thereby of joint destruction.

Viral infections often lead to arthralgias and overt arthritic states. The inflammatogenic compound of the viruses giving rise to such an outcome has to date not been identified. Because expression of dsRNA is a common feature of all viruses, we decided to analyze whether this property leads to the induction of arthritis. Histological signs of arthritis were evident already on day 3 following intra-articular administration of dsRNA. Arthritis was characterized by infiltration of macrophages into synovial tissue. It was not dependent on acquired immune responses because SCID mice also raised joint inflammation. NF-κB was activated upon in vitro exposure to dsRNA, indicating its role in the induction/progression of arthritis. Importantly, we found that dsRNA arthritis was triggered through IL-1R signaling because mice being deficient for this molecule were unable to develop joint inflammation. Although dsRNA is typically recognized by Toll-like receptor 3, Toll-like receptor 3 knockout mice developed arthritis, indicating that some other receptors are instrumental in the inducing of inflammation. Our results from in vitro experiments indicate that proinflammatory cytokines and chemokines stimulating monocyte influx were readily triggered in response to stimulation with dsRNA. These findings demonstrate that viral dsRNA is clearly arthritogenic. Importantly, macrophages and their products play an important role in the development of arthritis triggered by dsRNA.

Background: Leptin has been shown to participate in bone remodelling and leptin substitution reported to have a protective effect in experimental septic arthritis. Objective: To assess leptin levels in inflamed joints and plasma of patients with RA. Material and methods: Leptin concentrations were assessed in matched blood and synovial fluid samples from 76 patients with RA. Blood samples from 34 healthy subjects acted as additional controls. Results were analysed and correlated with duration and activity of RA, x ray changes, and treatment at time of sampling. Results: In patients with RA, leptin levels were significantly higher in plasma than in synovial fluid samples obtained simultaneously and higher than in control samples. Plasma and synovial fluid leptin levels correlated strongly. Locally in the joint, leptin levels were related to WBC count. Such a relation was not seen in the bloodstream. Leptin levels were not related to sex, age, or disease duration. Difference between leptin levels in plasma and synovial fluid was greater in non-erosive arthritis (5.1 (SEM 1.2) v 3.7 (0.9) ng/ml, p=0.006), than in patients with erosive joint disease (6.2 (1.0) v 5.4 (0.8) ng/ml, NS). Methotrexate treatment was associated with relatively high plasma leptin levels, while treatment with other DMARDs was associated with lower leptin levels than in patients receiving no DMARD treatment (p=0.0005). Conclusions: Leptin production was significantly increased in patients with RA compared with healthy controls. Synovial fluid leptin levels were significantly lower than in matched plasma samples, suggesting an in situ consumption of this molecule.

Background Rheumatoid arthritis (RA) is an established risk factor for premature development of cardiovascular disease. Adipokines are signal molecules originating from adipose tissue and exhibit strong proinflammatory effects. We have previously shown that resistin utilizes IGF-1R and TLR4 pathway to induce inflammation in RA synovia. Objectives In the present study we evaluated the role of adipokines and adipokine-related genes on the cardiovascular risk in RA. Methods Cariovascular risk was estimated in 146 RA patients, all women (median age 57.5 years, range 21-71, median disease duration 8.0 years, range 1-49 years), using the modified Systematic Coronary Risk Evaluation (mSCORE). Serum levels of adipokines and IGF-1 were analyzed by ELISA. mRNA levels of IGF-1R, TLR4, Akt1, STAT3, RETN, and RELA (NF-kB) were analysed in peripheral blood leukocytes and in subcutaneous adipose tissue, using qPCR. The gene expression analysis was performed in the subgroup of younger RA patients (n=54, mean age 46 years, median mSCORE=1). The adipokine levels and gene expression were analyzed in relation to mSCORE. Results mSCORE was directly related on the age, disease duration, smoking habits and blood fat profile (cholestrol and triglycerides), and not to the DAS28 and BMI of RA patients. mSCORE correlated to the serum levels of leptin, resistin and visfatin. The patients with the increased cardiovascular risk (mSCORE≥1, n=86) had higher levels of adiponectin and visfatin, and low levels of IGF-1 and Akt1 mRNA in the peripheral blood leukocytes compared to the patients where cardiovascular risk was below detection level (mSCORE In the subgroup of younger patients, serum resistin correlated to mSCORE and to the systemic inflammation but not to DAS28. Additionally, mSCORE showed a strong correlation to the adipocyte expression of resistin-triggering receptors (IGF1-R and TLR4) and to the mediators of their transcriptional signalling Akt, STAT3, and RELA. We observed no correlation between mSCORE and the transcriptional activity of the peripheral blood leukocytes. The correlation of mSCORE and transcriptional activity in adipocytes was not dependent on BMI and was stronger in the normal weight patients. Conclusions This pilot study shows a correlation between the cardiovascular risk and the transcriptional activity of the subcutaneous adipose tissue in women with RA. The expression of resistin-related genes in adipose tissue in younger women suggests that resistin may be a sufficient force supporting persistent transcriptional activity of adipocytes and driving premature cardiovascular disease in RA patients. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3893

Background Survivin is recognized as a marker of persistently active and erosive RA. The findings in experimental arthritis suggested that survivin co-ordinates the antigen-dependent leukocyte maturation and function by regulating transcriptional activity of Bcl-6, essential factor of germinal center formation and a factor involved neoplastic transformation to lymphoma. Objectives We studied intracellular expression of survivin and Bcl-6 in the peripheral lymphocytes and its relation to clinical features and treatment of RA patients. Methods The intracellular expression of survivin and Bcl-6 was studied on the effector and memory subsets of CD4+ and CD8+ T-cells and in CD19+ B-cells in 18 RA patients using flow cytometry. Serum and mRNA levels of survivin were analysed in 144 RA patients (age 21-71 years, disease duration 1-49 years) using ELISA and qPCR, respectively. Patients with serum levels of survivin >0.45 ng/mL comprised the serum survivin-positive (sSurv+) group. Results The sSurv+ group (n=76) was characterized by higher frequency of RF+ and aCCP+ patients, as well as by higher levels of aCCP and of differentiation factor Flt3-ligand compared to sSurv – (n=68). The sSurv+ and sSurv- patients were similar with respect to age, disease duration, DAS28 and anti-rheumatic treatment. Intracellular mRNA levels of Bcl-6 were lower in sSurv + compared to sSurv – patients, while RNA of survivin were similar. Flow cytometry showed that survivin was present in >85% of non-stimulated T-cells and B-cells lymphocytes of RA patients. The survivin hi subset comprised 2-14% of T-cells and was enriched in the effector (CD62L neg ) population of CD4+ T-cells compared to central memory and naive CD4+ T-cells. The expression of Bcl-6 was found within 7-38% of survivin + effector CD4+ T-cells and in 21-69% of CD19+ B-cells. The subsets of survivin+ and of survivin+Bcl-6+ T-cells was smaller in the sSurv+ patients compared to sSurv – patients, and was inversely correlated to the levels of serum survivin in these patients. The subset of survivin+Bcl-6+ B-cells was similar in sSurv+ and sSurv- patients. Conclusions In this pilot study we demonstrate that RA patients have co-expression of the two oncoproteins involved in neoplastic transformation to lymphoma, survivin and Bcl-6. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5490

Background A calcium-binding protein S100A4 regulates non-muscle myosin assembly and activity and plays important part in cell motility and differentiation. The binding of S100A4 to calcium drives conformation changes and permits S100A4 to regulate the activity of its multiple intracellular protein partners placing S100A4 at the crossroad of several intracellular transduction mechanisms. In RA, S100A4 is abundantly expressed in synovial fibroblasts, macrophages and vascular endothelial cells of the inflamed joints and may be measured in synovial fluid and in blood. The clinical consequences of the high levels of S100A4 in RA patients are associated with resistant joint inflammation and high skeletal damage. Objectives In the present study we evaluated the role of intracellular functions of S100A4 for T-cell responses in rheumatoid arthritis. Methods A preclinical model of the methylated BSA induced arthritis was induced in S100A4-deficent mice lacking the entire S100A4 protein (S100A4KO) and in wild-type (WT) counterparts where the S100A4 gene was inhibited with a specific shRNA-lentiviral constructs (S100A4-shRNA). The subsets of Th-cell in synovial infiltrates were analysed by immunohistology and in spleens by flow cytometry and qPCR. Phosphorylation of STAT3 (pTy5705), CD5 (pTyr453), Fyn (pTyr530) and ZA70 (pTyr319/Syk, pTyr352) were analysed by flow cytometry in spleen cell cultures. Kinase activity of Fyn and Lck was analysed by phosphorylation of CD5 peptide. Results Histological analysis of the inflamed joints demonstrated that S100A4-deficient mice had significantly alleviated joint inflammation and damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice presented accumulation of Foxp3 + Treg, while the effector T-cell population and the number of RORγt + and pSTAT3 + cells were reduced. T-cells of S100A4-deficient mice were characterized by a limited formation of Th17-cells, which was a result of low mRNA levels of RORγt and STAT3 (pTyr705) activity in spleen and low production of IL17 and IFNg. In vitro experiments provide evidence that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards TCR inhibitor CD5. Nuclear magnetic resonance analysis demonstrated an interaction between CD5 cytoplasmic domain and EF2-binding sites of S100A4. This suggests that S100A4 is able to disrupt intracellular interactions of CD5 and could modify its inhibition of TCR. Consequently, S100A4-deficiency was translated in low expression of CD5, and increased lymphocyte proliferation and phosphorylation of ZAP-70. Conclusions Here we provide experimental evidence that S100A4 directly binds the Src-kinases Lck and Fyn and reciprocally regulates their kinase activity. S100A4-deficiency results in reduced activity of STAT3 suppressing transcription of RORgt and lineage differentiation of Th17-cells. The immunological events controlled by S100A4 are functionally important for the pathogenesis of arthritis, since the deficiency in S100A4 alleviates experimental arthritis. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5414

Background Survivin is a known inhibitor of apoptosis and a regulator of cell division. In rheumatoid arthritis (RA), survivin has been shown to be involved in joint destruction. Here, we investigated survivin in early RA patients from the SWEFOT trial who were administered methotrexate (MTX) monotherapy for 3 months. Non-responders (DAS28 at 3 months >3.2) were randomized to triple therapy (MTX + sulfasalazine + hydroxychloroquine, group A) or anti-TNF (infliximab + MTX, group B). Methods Serum survivin levels were measured using ELISA with samples from 294 patients. Values >0.45 ng/mL were considered positive. Independent Samples t-tests and nonparametric Mann-Whitney U tests were performed where applicable. Results Out of 294 patients, 112 (38%) were positive for survivin at BL. The survivin (+) patients were more often male (30% vs. 22%, p=0.036) and RF (+) (80% vs. 58%, p Out of the 112 survivin (+) patients at BL, 37 (33%) converted to (−) by 3 months. These patients had greater improvements in disease activity compared to (+) and significantly greater reductions in tender joint count (TJC) and ESR (8.4 vs. 3.9, 30.9 vs. 19.0, respectively; p Among MTX responders, the (+/−) converters (n=11) had significantly greater reductions in DAS28 and patient9s global VAS than those who remained (+) (n=28) (3.5 vs. 2.5, 46.6 vs. 23.2, respectively; p Likewise, in the MTX non-responders, disease activity was improved more for (+/−) converters in the randomized group, A, compared to those who remained (+) or (−). Among these patients, those whose survivin status converted from (+) to (−) from 3 to 12 months (n=12) had numerically greater improvements, and a significantly greater reduction in patient9s global VAS than those who remained (+) (n=15) (17.3 vs. −2.9 [an increase], p=0.024). Converters (+/−) among the randomized group, B (n=7), on the other hand, had numerically worse changes for most parameters compared to those who remained (+) (n=16). Conclusions Serum survivin is positive in about one third of patients with early RA and correlates with male gender, RF-positivity, and higher ESR. Survivin seroconversion (+/−) upon MTX is not uncommon and is associated with clinical improvements. Survivin conversion from (+/−) led to greater improvements for patients on triple therapy compared to non-converters. This finding was not observed among patients treated with anti-TNF. Disclosure of Interest : A. Levitsky: None declared, M. Erlandsson: None declared, M. Bokarewa: None declared, R. van Vollenhoven Grant/research support: AbbVie, BMS, GSK, Pfizer, Roche, UCB, Consultant for: AbbVie, Biotest, BMS, Crescendo, GSK, Janssen, Lilly, Merck, Pfizer, Roche, UCB, Vertex DOI 10.1136/annrheumdis-2014-eular.4205

Tissue factor (TF) is a transmembrane glycoprotein and the main triggering element of blood coagulation. TF expression on monocytes and endothelial cells is induced by exposure to endotoxin, tumor necrosis factor, and IL-1 and is considered to appear in consequence of inflammation. In order to assess the proinflammatory capacity of TF itself, the recombinant extracellular domain of TF was injected intra-articularly into healthy mice. To characterize the role of immune cells in the TF-induced arthritis, mice deprived of lymphocytes, neutrophils and monocytes were used. Histomorphological analysis of the joints with respect to inflammatory cell infiltration, pannus formation and erosion formation revealed development of arthritis in 80% of animals injected with TF. In most of the cases synovial proliferation was accompanied by pannus formation and cartilage destruction. Inflammatory cell infiltrate consisted of CD4-Mac1+ macrophages. Depletion of monocytes was, however, not enough to abolish inflammation. Indeed, combined deficiency of monocytes and lymphocytes was required to prevent inflammation following the injection of TF. We observed that TF induced chemokine production (MIP-1α and RANTES), but did not induce a proliferative response nor cytokine release by mouse spleen cells. TF has strong inflammatogenic properties mediated predominantly by monocytes and their release of chemokines. Our study shows that TF can simultaneously trigger the immune and coagulation systems.

The composition of antibodies against phospholipids (PLa) was analysed in 54 PLa-positive thrombophilic women, different in the factor V genotype (Arg506-Gln mutation carriers, n=23; and noncarriers, n=31). The presence of antibodies against prothrombin was also studied. The incidence of cardiolipin antibodies (CLa) and lupus anticoagulant (LA) activity was similar among the carriers and noncarriers, while phosphatidylserine antibodies (PSa) were often the only PLa detected in the carriers of the mutation (7/23 vs. 2/31; chi2=5.47, p

The response to activated protein C (APC) was investigated in 28 healthy women, non-carriers of the Arg506-Gln mutation in factor V, throughout pregnancy (gestation weeks 12, 20, 28, 32 and 37) and after the delivery. A suppression of APC response was observed which reached lowest values by week 28 (nAPC-ratio 0.78 +/- 0.13), sustained low up to the end of pregnancy and rose after delivery (1.11 +/- 0.22; P < 0.05). APC resistance (nAPC ratio < 0.75) was registered in 16 of the 28 women (57%). A reduction of APC ratio was directly related to its value in the non-pregnant state, being most pronounced in the women with the highest APC ratio. Factor VIII increased during pregnancy and correlated inversely to APC ratio (Z coefficient = -0.645, P < 0.0001). The correlation became weaker in the course of pregnancy, losing significance by week 32. This was explained by the differences in profiles of the two variables: the lowest measured APC ratio preceded the peak of factor VIII in most cases. The most pronounced rise of factor VIII was found in the women with minimal levels of APC ratio between 0.8 and 0.7. These results allowed us to speculate that APC response is closely regulated during pregnancy, aiming to maintain a certain relevant level. Transitory reduction of APC response is connected to factor VIII and discussed as a prevalent mechanism of functional APC resistance during pregnancy.

Background Cigarette smoking has been recently connected to the pathogenesis of rheumatoid arthritis (RA). The cellular mechanisms and molecular events initiated by smoking and leading to severe inflammation, low efficacy of anti-rheumatic drugs, and autoantibody production, are still poorly understood. Adipokines are signalling molecules originating from adipose tissue and regulating carbohydrate and lipid metabolism. In RA, adipokines are connected to inflammation, disease activity and radiological signs of joint destruction. Effects of adipokines are mediated through insulin receptor/insulin-like growth factor-1 receptor (IGF-1R) complex. Objectives Present study evaluates a link between cigarette smoking, IGF-1 signalling and adipokine cascade in patients with rheumatoid arthritis. Methods One hundred-eleven patients (76 women, 35 men) with established RA (mean disease duration 9.9 years) participated in the structured telephone interview on their smoking habits. Patients were stratified by smoking habits, age, and levels of IGF-1. Levels of adipokines (visfatin, adiponektin, resistin and leptin) were analyzed in blood samples of these patients using commercial ELISAs. Results Thirty-three of 111 patients (29.7%) reported to be current smokers, while 78 patients were non-smokers. Smokers had slightly shorter disease duration and had a prevalence of men, while disease activity, erosivity and presence of RF were similar between the smokers and non-smokers. Smokers had lower levels of IGF-1 compared to non-smokers (median: 2.64 vs 8.08, p=0.035). Levels of IGF-1 are inversely related to age, this enabled distinct analysis of the younger (mean age 50 y, n=62) and older (mean age 69 y, n=49) patient groups. Smoking was associated with low levels of IGF-1 in the younger (5.82 vs 11.12, p=0.030) and in the older patients (1.05 vs 4.28 ng/ml, p=0.05). Levels of adipokines were similar in the total group of smokers and non-smokers, however IGF-1 was inversely related to visfatin levels (r=-0.35, p=0.003). In the absence of IGF-1 stimulation in patients with low levels of IGF-1, visfatin correlated to resistin (r=0.599) and leptin (r=0.450) levels. No such correlation within adipokine network was found in patients with high levels of IGF-1. Direct inhibitory effect of nicotine on IGF-1 levels was proved in mice. Mice supplemented with nicotine (0.02-0.05%) in drinking water had significantly lower levels of IGF-1 in blood compared to their siblings drinking pure water (p=0.0007). Treatment of mice with recombinant IGF-1 resulted in significantly low circulating levels of leptin, visfatin and resistin. Conclusions Smoking functions as a negative regulator of IGF-1 levels in RA patients and has repressive effects on the cascade of adipokine signaling molecules, contributing to inflammation and joint destruction in RA. Disclosure of Interest None Declared

Survivin but NOT fms-like tyrosine kinase ligand (FLT3L) is up-regulated before onset of rheumatoid arthritis

Background Fibromyalgia (FM) is a chronic condition characterized by widespread musle pain and tenderness. Chronic pain is the major symptom that affects patients physically, mentally and socially. Mechanisms of pain in FM are not completely understood. Relation between smoking and pain is contradictive. In epidemiologic studies smoking is associated to chronic pain, while in acute experiment stimulation of nicotine receptors alleviates pain. Objectives The present study evaluates the possible relation between pain, cigarette smoking, and levels of IGF-1 in patients with FM. Methods Pain was characterised in 63 patients with FM (all women, age 52 years) by Fibromialgia Impact Questionnaire (FIQ), tender points count, and pain threshold was assessed by algometer. All patients participated in the structured telephone interview regarding their smoking habits. Levels of IGF-1, leptin, resistin, and adiponectin were measured in blood. The statistical analysis was performed regarding smoking habits, IGF-1 levels and intensity of pain. Patients were categorized by pain (FIQ and threshold) quartiles, and relative risk (95%CI) was calculated. Results Eighteen patients (28.6%) reported to be current smokers, and 45 were non-smokers. Among the non-smokers, 25 patients smoked previously and ceased before the study (median 13 years, range 1-30). Smokers had higher pain experience compared to non-smokers, characterised by higher FIQ (p=0.036), and lower pain threshold (p=0.014). The difference in pain was largest between the current smokers and patients who had ceased smoking (dFIQ 12%, p=0.005). The RR of pain in the upper quartile in smokers was 6.3 (95%CI: 1.41-36.18) compared to previous smokers. Furthermore, previous smokers have lower pain, fewer tender points and a higher pain threshold compared to smokers. Smokers had significantly lower levels of IGF-1 (ng/ml, median: 2.72 vs 4.82, p=0.027) compared to never-smokers and lower levels of leptin (ng/ml, median: 16.9 vs 34.8, p=0.013) compared to non-smokers. Current and previous smokers had frequently low levels of IGF-1 compared to non-smokers (88% vs 12%, p=0.018). Conclusions Smoking in FM patients was associated with high pain experience. Pain experience improved in the patients who had ceased smoking. Current and previous smoking was associated with low levels of IGF-1 suggesting long-term effects of smoking on regulation of IGF-1 levels in FM patients. Disclosure of Interest None Declared

Seventy women with thrombosis in pregnancy were investigated for the presence of APC resistance and the associated Arg506-Gln mutation in coagulation factor V. The mutation was found in 46% of the investigated women. Carriers of the mutation were more prone to develop thrombosis in the first pregnancy (OR (odds ratio) = 3.41; P < 0.05) and had a higher probability of recurrence (OR = 3.86; P < 0.05) compared to non-carriers. The incidence of miscarriage was not related to the mutation but its probability increased in women with thrombosis in a second or subsequent pregnancy (P < 0.025). Negative dynamics of APC response during pregnancy was observed in 20/22 women; eight of them developed APC resistance de novo. The suppression of APC response was independent of the mutation.