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Rheumatoid arthritis is an autoimmune syndrome presenting with chronic inflammation of the joints. Patients with the same diagnosis can present with different phenotypes. In some patients severe joint inflammation and early joint destruction are observed, whereas a milder phenotype can be seen in others. Conversely, patients with the same signs and symptoms may exhibit different immunological and molecular abnormalities. Since the introduction of early treatment in clinical practice, the treat to target principle, and new medicines such as biologic disease-modifying antirheumatic drugs, clinical remission can be achieved early in the disease course, albeit not in all patients. The clinical response and efficacy of biologic disease-modifying antirheumatic drugs vary among different individuals. Therefore, there is a need to develop a more personalized approach toward treatment to achieve rapid remission in every patient to prevent disability and restore and maintain quality of life, without unnecessary adverse effects, in a cost-effective manner. The latest data from explorative studies of predictive markers of response are discussed here, together with a preliminary treatment algorithm based on currently available knowledge.

Background Rituximab (RTX) induces more than 98% depletion of the CD20 +B cells in blood after a single injection, yet 35% to 50% of RA treated patients show a poor response to the therapy1. Despite the identification of many different biomarkers, mostly in the B cell compartment2, adequate prediction of response to RTX treatment is still quite challenging. Objectives To test the hypothesis that non-response to rituximab can be predicted by analysing B-cell receptor (BCR) repertoire characteristics before and shortly after rituximab therapy. Methods Paired peripheral blood (PB) samples and synovial tissue (ST) samples were available from a total of 21 patients before therapy with RTX, and at 4 and 16/24 weeks after treatment. Next-generation sequencing was used to analyse the BCR repertoire, and asses the frequency of high expanded clones (HECs:>0.5% of the sequenced reads)3 and load of somatic hypermutation (SHM). Clinical response was evaluated at 6 month following EULAR response criteria. Results In spite of the complete depletion of B cells (measured using CD19) with conventional flow cytometry, we detect a complete BCR repertoire at week 4 and 16/24 after RTX treatment. The post-treatment PB BCR repertoire is composed of fewer, but more expanded and more mutated clones compared to baseline (figure 1). Non-response associates with a higher number of HECs at week 4 (p Conclusions Incomplete depletion of the baseline BCR clonal repertoire in peripheral blood within the first month of treatment predicts poor clinical response at 6 months, revealing the persistence of “rituximab-resistant” BCR clonal signatures associated with treatment failure. In all patients the PB BCR repertoire at 4 weeks after rituximab is dominated by few but highly expanded and highly mutated BCR clones, most likely CD20-negative plasmablasts, while less pronounced and delayed effects are observed in the ST BCR repertoire. References [1] Cohen SB, et al. Arthritis Rheum2006;54:2793–806. [2] Benucci M, et al. Autoimmun Rev2010;9:801–3. [3] Doorenspleet ME, et al. Ann Rheum Dis2014;73:756–628. Disclosure of Interest None declared

Background Several studies have shown that angiopoietin signalling to Tie2 may play a role in the initiation and perpetuation of disease rheumatoid arthritis (RA) and psoriatic arthritis (PsA). However, the cell type(s) involved in this process, as well the specific role of Tie2 signalling in the synovium of arthritis patients, remain unclear. Objectives To examine the role Tie2 signalling in macrophages and fibroblast-like synoviocytes (FLS) within the context of the synovial microenvironment of arthritic patients. Methods Peripheral blood (PB) monocytes from healthy donors (HD) were differentiated with synovial fluid (SF) of RA and PsA patients. PB and SF monocytes from RA and PsA patients were differentiated into pro-inflammatory macrophages with IFN-γ. Tie2 expression was analysed by flow cytometry and quantitative PCR. Macrophages, RA FLS and synovial tissue explants were stimulated with Ang-1 or Ang-2 (200 ng/ml) alone or in combination with TNF (10 ng/ml) for 4 hour or 24 hour. mRNA and protein expression of inflammatory mediators was analysed by quantitative PCR and ELISA and luminex, respectively. Arthritis was induced in wild type (WT) and Tie2 over-expressing (Tie2-TG) mice by intraperitoneal injection of 100 ml of K/BxN serum on day 0 and day 2. Mice were sacrificed on day 14 after serum transfer. Results Tie2 expression was observed IFN-γ-differentiated macrophages, from RA and PsA patients, as well as HD macrophages differentiated with RA and PsA SF. In all cases, both Ang-1 and Ang-2 stimulation significantly enhanced TNF-induced expression of pro-inflammatory cytokines (IL-6, IL-12B) and chemokines (IL-8, CCL-3 and CXCL-6). Tie2 activation also enhanced TNF-mediated production of these inflammatory mediators in RA FLS. The clinical severity of serum-induced arthritis was significantly higher in Tie2-TG mice compared to WT mice, associated with enhanced synovial expression of IL-6, IL12B, iNOS, CCL-2 and CXCL-10. Finally, we found that Ang-2, and to a lower extent Ang-1, induced the production of IL-6, IL-12B, IL-8, and CCL-3 in the synovial tissue explants of RA and PsA patients. Importantly, Ang-2 blockade with a specific neutralising anti-Ang2 antibody suppressed the production of IL-6 and IL-8 in synovial tissue of RA patients. Conclusions These results suggest that Tie2 signalling, even within the complex microenvironment of affected synovial tissue, has an important pro-inflammatory role in the pathology of RA and PsA. As this effect can be reversed by Ang-2 neutralisation, interfering with Tie2 activity may be a promising therapeutic target in arthritic diseases. Disclosure of Interest S. Garcia Perez: None declared, P. A. Kabala: None declared, B. Malvar Fernandez: None declared, M. W. Tang: None declared, S. A. Hartgring: None declared, C. Conde: None declared, M. Sleeman Employee of: Formerly an employee at MedImmune for the duration of this collaboration, D. L. Baeten: None declared, P. P. Tak: None declared, J. Connor Employee of: MedImmune LCC, K. A. Reedquist: None declared, T. R. Radstake: None declared

Background Previous work has suggested subtle infiltration of synovial T cells1 in the absence of overt synovial inflammation2 in individuals at risk of developing rheumatoid arthritis (RA). Objectives To study the molecular changes in synovium preceding arthritis development in preclinical RA. Methods We included sixty-seven individuals who were IgM rheumatoid factor (RF) and/or anti-citrullinated protein antibody (ACPA) positive and without any evidence of arthritis. All individuals underwent mini-arthroscopic synovial biopsy sampling of a knee joint at inclusion and were prospectively followed. First, an explorative genome-wide transcriptional profiling study was performed on synovial biopsies obtained from 13 individuals using Agilent arrays (test cohort). Survival analysis was used to identify transcripts with a significant association with arthritis development. The expression level of differentially expressed genes was validated using quantitative real-time PCR in the total cohort. Immunohistochemistry was used to study gene candidates at protein level in situ. Results Six of the 13 individuals in the explorative study developed RA after a median follow up time of 20 months (IQR 2–44). The 7 individuals who did not develop RA had a median follow up time of 85 months (IQR 69–86). Using a False Discovery Rate of Immunohistochemistry analyses (n=54) showed an abundant expression of CXCL12 and CXCR4 already in most RA-risk individuals. Synovial biopsies from RA-risk individuals who developed arthritis were more likely to show a positive gp38 staining and lower lipid staining. Conclusions This study clearly shows molecular changes appearing in synovial tissue before onset of arthritis in the absence of overt synovitis. Preclinical synovial alterations in immune response genes and lipid metabolism were associated with development of arthritis. References [1] de Hair MJ, et al. Arthritis Rheumatol2014. [2] van de Sande MG, et al. Ann Rheum Dis2011. Disclosure of Interest L. Van Baarsen: None declared, M. de Hair: None declared, J. Semmelink: None declared, I. Choi: None declared, D. Gerlag Employee of: GlaxoSmithKline, UK. GlaxoSmithKline was not involved in this study., P. Tak Employee of: GlaxoSmithKline, UK. GlaxoSmithKline was not involved in this study.

Introduction Rheumatoid arthritis (RA) is an autoimmune disease with unknown etiopathogenesis where systemic autoimmunity precedes clinical disease onset. Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSC) play a crucial role in shaping the immune response and maintaining peripheral tolerance. Objectives We developed an experimental model for studying the functional capacities of human LNSC during the earliest phases of RA and compared their cellular and molecular characteristics to LNSC from healthy volunteers. Methods ACPA +RA patients (n=24), ACPA +RA risk individuals (n=23) and seronegative healthy controls (n=14;HC) underwent ultrasound-guided inguinal lymph node biopsy. Human LNSCs were isolated and expanded in vitro for cellular (flow cytometry), molecular (methylome, transcriptome and microRNA) and functional analyses. Results Key LN chemokines CCL19, CCL21 and CXCL13 were induced in LNSCs upon stimulation with TNFα and lymphotoxin α1β2, but to a lesser extent in LNSCs from RA patients. RNA sequencing was performed on LNSC of HC (n=5), ACPA +RA risk individuals (n=6) and ACPA +RA patients (n=4). Of interest, LNSC from ACPA +RA risk individuals and ACPA +RA patients revealed a common significantly differential expressed gene signature compared with HC LNSC. Pathway analysis of this common signature showed, among others, significant enrichment of pathways affecting actin cytoskeleton, focal adhesion and cell junction. Accordingly, in a gel contraction assay LNSC from ACPA +RA risk individuals and RA patients showed impaired collagen contraction compared to healthy LNSC. In RA LNSC a significant enrichment was observed for genes involved in TGFb signalling while in RA-risk LNSC cell cycle genes were differentially expressed compared with HC. DNA methylation analyses revealed common differentially methylated CpG sites (DMS) in LNSC from ACPA +RA patients (n=5) and ACPA +RA risk individuals (n=3) compared with HC (n=4). These DMS were significantly hypomethylated and associated with antigen processing and presentation (HLA-DRB1). Conclusions This data point towards alterations in the cytoskeleton and antigen-processing and presentation in LNSC from ACPA+RA risk individuals and RA patients. Further studies are required to investigate the influence of this LNSC abnormality on immune responses. Disclosure of interest C. Ospelt: None declared, E. Karouzakis: None declared, J. Hahnlein: None declared, H. Semmelink: None declared, R. Gay: None declared, P.-P. Tak Employee of: Currently: Senior Vice President R and D Pipeline, Global Development Leader and Chief Immunology Officer, GSK, D. Gerlag Employee of: Currently: Head Clinical Unit Cambridge at GSK, S. Gay: None declared, L. Van Baarsen: None declared

Background Sirukumab, a selective, high-affinity human monoclonal antibody to the interleukin-6 (IL-6) cytokine, is under development for rheumatoid arthritis (RA) and other diseases. Objectives To evaluate long-term efficacy and safety of sirukumab in patients (pts) with RA refractory or intolerant to anti-tumor necrosis factor (TNF) agents. Methods This phase 3 study included pts ≥18 years with moderate to severe active RA, and a lack of benefit to ≥1 anti-TNF or intolerance to ≥2 anti-TNFs. Eligible pts were initially randomized 1:1:1 to sirukumab subcutaneous (SC) 50mg q4w, sirukumab SC 100mg q2w, or placebo SC q2w for 24 wks. Placebo-treated pts with Results 878 pts were initially randomized to placebo (n=294), sirukumab 50 mg q4w (n=292), or sirukumab 100 mg q2w (n=292). Of placebo-treated pts, 94 met EE criteria at Wk 18 and 158 crossed over at Wk 24 and were re-randomized to sirukumab. 60% of pts had received ≥2 prior biologics, including non-TNF–targeted biologics. RA signs and symptoms and patient-reported outcomes (PROs [SF-36 scores]) improved significantly with sirukumab versus placebo through Wk 24. Improvements were maintained through Wk 52 with no dose response (Table 1). Through Wk 52 in the combined sirukumab 50mg q4w and 100mg q2w groups, respectively, an adverse event (AE) was reported for 79.6% and 81.3% of pts and a serious AE was reported for 14.2% and 13.2% of pts; injection-site reactions and alanine aminotransferase increases were the most commonly reported AEs. Conclusions In this population intolerant or refractory to anti-TNFs/other biologics, sirukumab SC 50mg q4w and 100mg q2w were well tolerated and reduced signs and symptoms of RA and improved PROs through 52 wks of treatment, also among pts who switched from placebo to sirukumab. Disclosure of Interest Y. Tanaka Grant/research support from: Mitsubishi-Tanabe, Takeda, Daiichi-Sankyo, Chugai, Bristol-Myers, MSD, Astellas, Abbvie, and Eisai, Speakers bureau: Abbvie, Chugai, Daiichi-Sankyo, Bristol-Myers, Mitsubishi-Tanabe, Astellas, Takeda, Pfizer, Teijin, Asahi-kasei, YL Biologics, Sanofi, Janssen, Eli Lilly, and GlaxoSmithKline, D. Aletaha Grant/research support from: AbbVie, Pfizer, Grunenthal, Merck Medac, UCB, Mitsubishi/Tanabe, Janssen, and Roche, Consultant for: AbbVie, Pfizer, Grunenthal, Merck Medac, UCB, Mitsubishi/Tanabe, Janssen, and Roche, P. Agarwal Shareholder of: Janssen Research & Development, LLC, Employee of: Janssen Research & Development, LLC, R. Kurrasch Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, P. Tak Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, S. Popik Shareholder of: Janssen Research & Development, LLC, Employee of: Janssen Research & Development, LLC

Background T-cells are thought to be key players in the initiation and progression of rheumatoid arthritis (RA). Earlier we showed that already at the seropositive “at risk” stage uninflamed synovial tissue contains T-cell infiltrates1. In another study we showed that inflamed synovium selectively harbours expanded T-cell clones that are hardly present in paired blood samples2. Objectives Following up on these observations, we longitudinally investigated whether the same expanded T-cell clones found in the inflamed synovial tissue at onset of RA are already present in the synovium in the seropositive “at risk” stage. Methods Fifty-five individuals without arthritis but seropositive for IgM rheumatoid factor and/or anti-citrullinated protein antibody (ACPA) were prospectively followed. In five aCCP+ individuals synovial biopsies and paired blood samples at inclusion (“at risk” stage) and after development of RA (ACR2010 criteria; mean time to arthritis 27 months (range 11.7–47.3)) were available for analysis. T-cell clones were identified by their unique TCRβ sequence using RNA-based next generation sequencing3. For each sample, 3570 TCRβ sequences were analysed. Clones with a frequency of ≥0.5% were arbitrarily considered as highly expanded clones (HECs). ANOVA and t-test were used for statistical analysis. Results T-cell repertoires in “at risk” and RA synovium were similar (mean (± SD) number of clones 488±70 vs 567±204 respectively, p=0.46), number of HECs (37±7 vs 31±18, p=0.41) and the impact of HECs collectively on the TCR repertoire (mean 48% ± 13% vs 50% ± 20%, p=0.84). Interestingly, of the HECs present in the synovium at onset of arthritis 23% (±9%) were already present as HECs in the synovium at the seropositive “at risk” stage. This overlap was significantly higher than that with paired blood samples taken at the arthritis (3% ± 3%; p=0.01) or at the seropositive “at risk” stage (5% ± 7%; p=0.01; Figure 1a-c patient example; Figure 1d summary of results). Further characterization of the synovial CDR3 sequences (length, total charge, polar, aromatic and aliphatic side chains) showed no significant differences between RA HECs that were and those that were not expanded in the seropositive “at risk” stage. Conclusions Many T-cell clones found in early RA synovial tissue are already present in the pre-clinical “at risk” phase. The resemblance in TCR repertoires indicates that the process leading to disease – at least at the T-cell level – constitutes a smooth development. These clones, being already present in the very early stage of this disease and persisting as dominant clones during contraction of active arthritis, form attractive candidates for further characterization. References de Hair MJ et al. Arthritis Rheum. 2014. Klarenbeek PL et al. Ann Rheum Dis. 2012. Klarenbeek PL et al. Immunol Lett. 2010. Disclosure of Interest None declared

Background: The synovial tissue is a primary target of many inflammatory arthropathies, including psoriatic arthritis (PsA). Identification of proinflammatory molecules in the synovium may help to identify potentially therapeutic targets. Objective: To investigate extensively the features of cell infiltration and expression of mediators of inflammation and joint destruction in the synovium of patients with PsA compared with patients with rheumatoid arthritis matched for disease duration and use of drugs. Methods: Multiple synovial tissue biopsy specimens were obtained by arthroscopy from an inflamed joint in 19 patients with PsA (eight oligoarthritis, 11 polyarthritis) and 24 patients with rheumatoid arthritis. Biopsy specimens were analysed by immunohistochemistry to detect T cells, plasma cells, fibroblast-like synoviocytes, macrophages, proinflammatory cytokines, matrix metalloproteinases and tissue inhibitor metalloproteinase-1, adhesion molecules and vascular markers. Stained sections were evaluated by digital image analysis. Results: The synovial infiltrate of patients with PsA and rheumatoid arthritis was comparable with regard to numbers of fibroblast-like synoviocytes and macrophages. T cell numbers were considerably lower in the synovium of patients with PsA. The number of plasma cells also tended to be lower in PsA. The expression of tumour necrosis factor alpha (TNFα), interleukin (IL) 1β, IL6 and IL18 was as high in PsA as in rheumatoid arthritis. The expression of matrix metalloproteinases, adhesion molecules and vascular markers was comparable for PsA and rheumatoid arthritis. Conclusion: These data show increased proinflammatory cytokine expression in PsA synovium, comparable to results obtained for rheumatoid arthritis, and support the notion that, in addition to TNFα blockade, there may be a rationale for treatments directed at IL1β, IL6 and IL18.

Background: Synovial tissue (ST) from end stage destructive rheumatoid arthritis (RA) and arthroscopic biopsies obtained during active inflammation might exhibit different characteristics. Objective: To define the cell infiltrate and the expression of proinflammatory cytokines, angiogenic factors, and matrix metalloproteinases (MMPs) in ST selected at arthroscopy compared with that from end stage RA. Methods: Synovial biopsy specimens were obtained from the actively inflamed knee joints of 13 patients with chronic RA by arthroscopy and compared with ST from 10 patients with end stage, destructive RA. Immunohistological analysis was performed to detect T cells, plasma cells, macrophages, fibroblast-like synoviocytes (FLS), and the expression of interleukin (IL)1beta, IL6, tumour necrosis factor a (TNFalpha), MMP-1, MMP-3, MMP-13, TIMP-1, and VEGF. Results: The expression of CD68+ macrophages was significantly higher in ST selected at arthroscopy than in samples obtained at surgery, both in the intimal lining layer and in the synovial sublining. The expression of CD3+ T cells also tended to be higher in arthroscopic samples. The expression of TNFalpha, IL6, MMP-1, MMP-3, MMP-13, TIMP-1, and VEGF was on average higher in ST obtained at arthroscopy. In contrast, the expression of IL1beta was on average higher in surgical samples. Conclusion: Active arthritis activity is associated with increased cell infiltration, expression of proinflammatory cytokines, MMPs, and angiogenic growth factors in synovial biopsy samples selected at arthroscopy. Increased expression of IL1beta in the synovium of patients with destructive RA requiring joint replacement may well reflect the important role of IL1beta in cartilage and bone destruction

Objectives: To compare receptor activator of NF-κB ligand (RANKL) production in the synovial tissue from patients with active rheumatoid arthritis (RA), inactive RA, spondyloarthropathies (SpA), osteoarthritis, and from normal subjects. In addition, to establish the cell lineages expressing RANKL in these tissues. Methods: Immunohistological analysis of frozen synovial tissue biopsy specimens was performed using a monoclonal antibody (mAb) to detect RANKL. Sections were evaluated by computer assisted image analysis and semiquantitative analysis to compare RANKL expression between groups. Dual and sequential labelling with mAb RANKL and cell lineage specific monoclonal antibodies were used to determine the types of cells expressing RANKL. Results: Higher levels of RANKL were expressed in tissues from patients with active RA and SpA than in tissues from patients with inactive RA, osteoarthritis, and from normal subjects. RANKL protein was associated with CD3 antigen-positive lymphocytes and some macrophages. RANKL was predominantly associated with activated, memory T cells (CD45Ro positive cells) in patients with active RA and spondyloarthropathy (SpA). Conclusions: The highest levels of RANKL were detected in patients with RA with active synovitis and in some patients with SpA. An increase in RANKL in the inflamed joint of patients with RA, produced by infiltrating activated T cells and macrophages, is likely to be an important cause of joint erosions in RA.

OBJECTIVES—Examination of synovial tissue (ST) obtained at surgery because of end stage destructive rheumatoid arthritis (RA) showed that macrophages and fibroblasts are the major cell types at the cartilage-pannus junction (CPJ). This study aimed at defining the cell infiltrate and mediators of joint destruction in ST selected at arthroscopy from the CPJ in patients with RA who did not require joint surgery. METHODS—Paired synovial biopsy specimens were obtained at arthroscopy from ST adjacent to the CPJ and the suprapatellar pouch from the knee joints of 17 patients with RA. Immunohistological analysis was performed using monoclonal antibodies to detect T cells, B cells, plasma cells, macrophages, fibroblast-like synoviocytes, mast cells, and granzyme B+ cytotoxic cells as well as the expression of metalloproteinase (MMP)-1, MMP-3, and MMP-13. The sections were evaluated by computer assisted image analysis and semiquantitative analysis. RESULTS—The cell infiltrate comprised mainly T cells, macrophages, and plasma cells. The ST was also infiltrated by the other cell types, but at lower numbers. Expression of MMPs was abundant, especially MMP-3. The features of ST at the CPJ were generally similar to those at the suprapatellar pouch. CONCLUSIONS—The synovium at the CPJ in patients with RA who did not require joint surgery exhibits, in general, the same type of cell infiltrate and expression of MMPs and granzymes as ST from the suprapatellar pouch. The pathological changes that have been described at the CPJ in patients with RA with end stage, destructive disease may well reflect the transition to a process in which macrophages, fibroblast-like synoviocytes, and other cell types become increasingly important.

Background: IFN beta may have immunomodulatory effects in rheumatoid arthritis (RA) and its increased production in RA synovium may be a reactive attempt to inhibit inflammation. Objective: To determine the expression of IFN beta in the synovial tissue of patients with RA, osteoarthritis, and reactive arthritis. Methods: Synovial biopsy specimens were obtained by arthroscopy from patients with RA and disease controls for immunohistological analysis using a monoclonal antibody specific for IFN beta. Bound antibody was detected by an immunoperoxidase method. Stained sections were evaluated by computer assisted image analysis. Double stainings were performed with antibodies to detect CD55 positive fibroblast-like synoviocytes (FLS), CD68 positive macrophages, and CD83 positive dendritic cells (DCs) coexpressing IFN beta. Results: IFN beta protein was abundantly expressed in the synovium of patients with RA. Digital image analysis showed a significant increase in the mean integrated optical density for IFN beta expression in RA synovial tissue compared with disease controls. Specific up regulation of IFN beta expression was also seen when the results were controlled for cell numbers. Phenotypic analysis showed that FLS, especially, but also macrophages and DCs may express IFN beta in RA synovial tissue. Conclusions: The increased expression of IFN beta in RA synovium suggests activation of an immunomodulatory mechanism that could inhibit synovial inflammation

Background Systemic autoimmunity may precede the development of clinical signs and symptoms of seropositive rheumatoid arthritis (RA), which offers a window of opportunity to delay or prevent clinically manifest arthritis by targeted intervention. This could represent a paradigm shift from treatment to prevention 1 . Objectives To explore if a single infusion of rituximab (anti-CD20 antibody) can prevent or delay the onset of clinically manifest arthritis in individuals at risk of developing autoantibody positive RA. Methods Eighty-two subjects with arthralgia who had never had clinically manifest arthritis and never used disease-modifying antirheumatic drugs were included in a multicentre, randomised, double-blind, placebo-controlled clinical trial. They were positive for both anti-citrullinated protein antibodies (ACPA) and rheumatoid factor and they had CRP levels ≥3 mg/l and/or subclinical synovitis on ultrasound or MRI of the hands. Subjects were randomized to receive a single iv infusion of either 1000 mg rituximab or placebo after 100 mg methylprednisolone premedication in each group. Subjects were prospectively followed to assess development of clinically manifest arthritis. We performed Kaplan-Meier survival analysis, Cox regression analysis and determined Treatment*Time Cox proportional hazards. Results Eighty-one individuals (52 females; mean age 53 (IQR 13.5) years) received treatment, which was generally well tolerated. One patient withdrew before treatment. The median follow up was 27.0 months (IQR 25.0), during which 30 subjects developed arthritis: 16/40 (40%) in the placebo group and 14/41 (34%) in the rituximab group, after a median period of 11.5 (interquartile range [IQR] 12.5) months in the placebo group versus 16.5 (IQR 19.0) months in the rituximab group. Whereas the risk for development of arthritis in the placebo group was 40%, we found a reduction of 53% of this risk in the rituximab group at 18 months follow up (HR (95%CI)=0.475 (0.190–1.191)). At the 25% quartile (75% free of arthritis) of the cumulative arthritis-free survival, there was a delay in the development of arthritis of 12.0 months (12 months in the placebo group versus 24 months in the rituximab group. As expected, this effect attenuated over time. Treatment*Time Cox proportional hazard analysis showed that the beneficial effect of rituximab was statistically significant (P Conclusions A single infusion of 1000 mg rituximab significantly delays the development of arthritis in subjects at risk of developing RA. This is the first study evaluating the effects of a biopharmaceutical in this population, and the results strongly support the rationale for future clinical trials aimed at prevention of RA by a targeted intervention. References Gerlag DM, Norris JM, Tak PP. RA: from risk factors and pathogenesis to prevention: Towards prevention of autoantibody-positive rheumatoid arthritis: from lifestyle modification to preventive treatment. Rheumatology (Oxford). 2015 Sep 15. Review Acknowledgement N de Vries, DJ van Schaardenburg, E Brouwer, T Huizinga Disclosure of Interest D. Gerlag Shareholder of: GlaxoSmithKline, Grant/research support from: Dutch Arthritis Foundation, Netherlands Organisation for Health Research and Development, Employee of: GlaxoSmithKline, M. Safy: None declared, K. Maijer: None declared, M. de Hair: None declared, S. Tas: None declared, M. Starmans-Kool: None declared, A. van Tubergen: None declared, M. Janssen: None declared, P.-P. Tak Shareholder of: GlaxoSmithKline, Grant/research support from: Dutch Arthritis Foundation, Netherlands Organisation for Health Research and Development, Employee of: GlaxoSmithKline

Background A Treat-to-Target approach (T2T), treating patients with RA towards a target, either remission or low disease activity (T2T-REM or T2T-LDA) is nowadays recommended. However it has never been assessed whether such a strategy in daily clinical practice really leads to more patients meeting that target. Methods BIODAM is a 2-year prospective cohort including patients in daily practice with RA from 10 countries, who were started or changed on DMARD and/or anti-TNF treatment and were followed-up every 3 months. Participating physicians were required to practice T2T per protocol. Per visit was decided whether a patient was treated according to T2T or not. T2T-REM was considered met: i) if a patient had already a disease activity score below the target (DAS28-CRP 6 months). The relationship between T2T and ACR/EULAR Boolean remission 3 months later (or sustained remission) was investigated using generalized estimating equations with auto-regression. Results In total 3084 visits of 539 patients (mean (SD) age: 56 (13) years, 76% female, disease duration 6 (8) years). In 68% of the visits, T2T-REM was applied (in 79% of the visits T2T-LDA was applied). ACR/EULAR-boolean remission was reached in 15% of the visits. Appropriate application of T2T-REM led to a 50% higher likelihood of ACR/EULAR-boolean remission 3 months later than not applying T2T-REM. Both T2T-REM and T2T-LDA strategies led to lower disease activity (with an exception of DAS28 remission or DAS28-LDA). Only 9% of the treatment intensifications followed upon a DAS28 between 2.6 and 3.2, and 79% of the intensifications were applied upon a DAS>3.2. Following T2T strategies led to higher achievement of sustained remission (table). Conclusions A treat-to-target approach, even with a modest benchmark (DAS28 = 3.2), works and leads to a higher achievement of (sustained) ACR/EULAR-remission. Our study illustrates the importance of acting upon the data that is routinely collected in a clinical encounter. Rheumatologists should be encouraged to follow a treat-to-target approach in order to improve the outcome of their patients. Disclosure of Interest None declared

Objective Assess whether CD70 + B cells contribute to EAE. Materials and methods MOG-specific TCR transgenic mice (2D2) were crossed with mice with constitutive CD70 expression on B cells. The development of EAE and the phenotype of B-T lymphocytes were studied in 2D2xCD70 animals. Results Spontaneous EAE developed in 20% of 2D2xCD70 and 3% of 2D2 mice. EAE was also more severe in 2D2xCD70 versus 2D2 animals upon MOG immunization. The susceptibility of 2D2xCD70 to EAE was associated with fewer FoxP3 + T cells. Conclusions Expression of CD70 by B cells aggravates EAE possibly by reducing the number of regulatory T cells.

Recently, synoviolin, a novel E3 ubiquitin ligase, was identified in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and synovial tissue, where it may contribute to the dysregulated proliferation and apoptosis seen in RA.1 2 Synoviolin expression is also associated with arthritis development in mice.1 The exact mechanism of synoviolin regulation and expression remains to be elucidated, but recently a role for tumour necrosis factor α (TNFα) and interleukin 1b was suggested as these cytokines increased the expression of synoviolin in RA FLS.3 Another interesting observation is that high level sustained expression of synoviolin in whole peripheral blood was related to decreased clinical response in RA patients treated with TNF blockade.4 Therefore, we investigated synoviolin expression in synovium and its relationship to later response to TNF blockade in patients with RA compared with psoriatic arthritis (PsA) and osteoarthritis (OA). Synovial tissue samples were obtained …

Reumatoide artritis (RA) is de meest voorkomende chronische ontstekingsziekte van de gewrichten, waarbij vooral het weefsel dat het gewricht aan de binnenkant bekleedt, de zogenoemde synoviale membraan, is aangedaan (synoviitis: figuur 4.1). De verschijnselen zijn pijn, stijfheid, zwelling en bewegingsbeperking (artritis). RA komt voor bij circa 1% van de bevolking (drie keer meer bij vrouwen dan bij mannen) en openbaart zich meestal tussen het 40e en 60e levensjaar.

Background Current rheumatoid arthritis (RA) treatment guidelines recommend treating patients early according to the treat to target principle aiming for remission with a “step-up” treatment paradigm to biologics after failure of disease-modifying antirheumatic drugs (DMARDs). However, despite significant improvements in disease management, most patients have persistent disease activity, significant joint damage and disability. The risk of serious infections could be one of the concerns about using biologics earlier in the treatment paradigm. Objectives To explore the efficacy and safety of earlier intervention, we reviewed the frequency of remission and serious infection events (SIEs) in DMARD-naive and DMARD-inadequate responders (IR) in randomised, controlled clinical trials of approved biologics. Methods A PubMed search up to January 2015 was performed for randomised, double-blind, placebo-controlled, multi-centre trials of all approved biologics in combination with methotrexate (MTX), in RA subjects that were either DMARD-naive or DMARD-IR. All trials had to include disease activity score (DAS)28(CRP or ESR) remission (DAS28 Results 15 DMARD-naive and 9 DMARD-IR trials were identified. Subjects in DMARD-naive trials tended to be younger with shorter disease duration and lower baseline DAS28 than those in DMARD-IR trials. In DMARD-naive subjects, the frequency of DAS28-ESR at 6 months was 39% for biologic + MTX and 21% for placebo + MTX; and 16% and 3% in DMARD-IR subjects, respectively. SIEs were slightly higher in both populations when using biologic + MTX, compared with MTX alone, but there was no apparent difference between DMARD-naive and DMARD-IR populations, although the numbers are relatively small and duration of treatment relatively short. Conclusions Overall most patients do not achieve remission with biologics and there is much scope for improvement. The frequency of remission was higher in DMARD-naive subjects than DMARD-IR subjects; however there were no apparent differences in serious infections across the two populations. These findings demonstrate that there remains a significant unmet need for more effective therapeutic strategies for RA in both DMARD-naive and DMARD-IR populations. Disclosure of Interest P. Mehta: None declared, K. Davy Shareholder of: GSK, Employee of: GSK, R. Williamson Shareholder of: GSK, Employee of: GSK, R. Rao Shareholder of: GSK, Employee of: GSK, P.-P. Tak Shareholder of: GSK, Employee of: GSK

Background In last decade rituximab (RTX), a B-cell targeted monoclonal antibody, has been introduced to treat rheumatoid arthritis (RA) patients but with variable response rates (30-50%). To increase treatment efficacy and reduce costs, treatment should be individualized to match the spectrum of RA. B-cell targeted therapy has been hypothesized to be most effective in a more B-cell mediated RA disease, possibly correlated to serological status (1). Whole body Positron Emission Tomography (PET) is capable to show B-cells targeting with high specificity in lymphoma patients using 89Zr-RTX (2). In addition, our group previously demonstrated the potential of PET for non-invasive visualization of immunological targets in RA (3). Objectives To investigate the feasibility of non-invasive imaging of B-cells in RA patients who initiate RTX treatment by [89Zr]RTX PET-CT. Methods Anti-B cell therapy naive RA patients (n=20; female18/20; age 53±11; 65% IgM RF/a-CCP +) with clinical arthritis in at least two hand joints, who were eligible for RTX treatment, were included. Directly after the first therapeutic RTX infusion (1000 mg, without methylprednisolone), 18MBq [89Zr]-10 mg RTX was administered. Whole body PET-CT and detailed images of wrists and hands (22 joints/patient) were acquired at 3, and in addition, in a subpopulation at 6 days post-injection (p.i.). Thereafter, RTX treatment was continued according to standard clinical protocol. Areas of enhanced uptake on PET were defined and quantified as maximum standardized uptake values (SUVmax). Wrist and hand joints were clinically assessed for swelling and tenderness. Results Visually, all patients showed at least one hand joint with increased focal tracer uptake (87/440 joints; mean/patient ± SD 4.4±4.9) (Figure 1) with distribution: metacarpophalangeal (n=44/200), proximal interphalangeal (n=22/200) and wrist joints (n=21/40). Interestingly, 66% of PET-positive joints (57/87) corresponded to clinical findings of arthritis, while PET additionally displayed possible subclinical disease activity in another 30 joints (34%). Quantitative analysis showed high mean SUVmax values of hotspots in hand joints (2.98±1.46) which were up to 4 times higher than maximum background uptake, but varying between patients (range SUVmax 1.2-8.0), regardless of serological status. Stability of joint uptake was found over time (3-6 days p.i.) while the tracer cleared from circulation, pointing at specific binding in joints. Whole body PET-CT also demonstrated tracer uptake in extra-articular tissues especially in liver, spleen and in 5/20 patients slightly enhanced uptake in at least one peripheral lymph node. Conclusions [89Zr]RTX PET-CT seems to be a sensitive tool for in vivo identification of B-cells in arthritic joints and extra-articular tissues in RA patients. Whether quantitative differences in uptake (articular and body distribution) correlate to clinical and RTX response data is currently investigated in a prospective study. References Isaacs JD et al; Ann Rheum Dis 2013. Muylle K et al; Ann Oncol 2008. van der Laken CJ et al; Arthritis Rheum 2008. Acknowledgements This study was financially supported by Hoffmann-La-Roche,The Netherlands Disclosure of Interest S. Bruijnen: None declared, M. Tsang-A-Sjoe: None declared, H. Raterman: None declared, T. Ramwadhdoebe: None declared, D. Vugts: None declared, G. Van Dongen: None declared, M. Huisman: None declared, O. Hoekstra: None declared, P. Tak Grant/research support from: P.P. Tak participated in this study from his position at the Academic Medical Center, Amsterdam, The Netherlands. GSK did neither sponsor this study nor supported this study otherwise. P.P.Tak also has affiliations at Cambridge and Ghent but his activities at these locations were not involved in the current study., A. Voskuyl: None declared, C. van der Laken: None declared

Introduction Activation of the cholinergic anti-inflammatory (CAP) reduces inflammation in different animal models, while abrogation of the pathway has been shown to increase inflammation. Sjogren’s syndrome (SjS) is a chronic autoimmune disease of the salivary glands, which can currently only be treated symptomatically. The pathogenesis of SjS can be studied in the non-obese diabetic (NOD) mouse model; these mice spontaneously develop immune cell infiltration in the salivary glands (SG) in addition to type I diabetes due to pancreatic immune cell infiltration (insulitis). Methods The CAP was activated in the NOD mouse model for 4 weeks (10–14 weeks of age) with two agonists of the α7 subunit of nicotinic acetylcholine receptor (α7nAChR), AR-R17779 and nicotine. In the next study the pathway was abrogated by unilateral cervical vagotomy performed at 12 weeks of age; animals were monitored until 20 weeks of age. Alpha-7nAChR expression and focus score (FS) were evaluated in the SG in addition to the insulitis score in the pancreas. Salivary function was evaluated by measuring stimulated salivary flow. Cytokines levels were measured in serum and SG. Results Alpha-7nAChR was expressed on myoepithelial cells in the SG surrounding the ducts. There were no changes in cytokine production in the serum, but in the SG monocyte chemotactic protein (MCP)-1 was reduced after AR-R17779 treatment (p = 0.023) and tumour necrosis factor (TNF) production was increased in the vagotomy group (p = 0.042). This however did not change the FS or stimulated salivary flow in the two CAP intervention studies. The NOD mice developed diabetes more rapidly in the vagotomy group, but at completion of the study the same number of animals had developed diabetes and there were no significant differences in insulitis scores. Conclusion The cholinergic anti-inflammatory pathway does not influence the inflammatory processes in the NOD mouse model of SjS and type I diabetes.

Background and objectives The NF-κB family of transcription factors is strongly involved in synovial inflammation. We have previously shown that NF-κB-inducing kinase (NIK) is a key regulator of inflammation-induced angiogenesis in rheumatoid arthritis (RA) synovial tissue (ST). Here, we investigated synovial NIK expression in early arthritis patients and in autoantibody-positive individuals at risk for developing RA. Materials and methods ST biopsies were obtained via arthroscopy from 154 early arthritis patients (arthritis duration Results In early arthritis patients, NIK was predominantly expressed in EC of small blood vessels. Furthermore, NIK expression correlated with ESR (r = 0.184; p = 0.024), CRP (r = 0.194; p = 0.017), joint swelling (r = 0.297; p + lining and sublining macrophages r = 0.585; p + T cells r = 0.733; p + B cells r = 0.264; p = 0.040), MRI effusion (r = 0.665; p + EC were present, but this was not predictive for the development of arthritis. Conclusions NIK + EC are present in the earliest phase of synovial inflammation and may be indicative of high angiogenic activity in the inflamed ST. Therefore, NIK + EC may play an important role in the persistence of synovitis. Collectively, our data underscore the importance of angiogenesis in synovial inflammation and identify NIK as a potential therapeutic target in arthritis.

Objective: To explore whether pretreatment features of synovial tissue in patients with gonarthritis could predict the clinical effect of radiation synovectomy with yttrium-90 ( 90 Y) and glucocorticoids or with intra-articular glucocorticoids alone. Methods: A synovial biopsy was carried out blindly 2 weeks before treatment in 66 patients with persistent gonarthritis, who were randomised to treatment either with 90 Y and triamcinolone or with placebo and triamcinolone. Immunohistochemistry was used to detect T cells, macrophages, B cells, plasma cells, fibroblast-like synoviocytes, adhesion molecules and pro-inflammatory cytokines. Stained sections were evaluated by digital image analysis. Individual patient improvement was expressed using a composite change index (CCI; range 0–12). Successful treatment was defined as CCI ⩾6 after 6 months. Results: Patients with rheumatoid arthritis, psoriatic arthritis, undifferentiated arthritis and other causes of gonarthritis were included. The overall response rate was 47%. Clinical efficacy in both therapeutic groups was similar and not dependent on diagnosis. No significant differences were noted between baseline microscopic features of synovial tissue inflammation in patients with rheumatoid arthritis and in those with non-rheumatoid arthritis (ie, all diagnoses other than rheumatoid arthritis). The number of macrophages in the synovial sublining was significantly higher in responders than in non-responders (p = 0.002), independent of treatment group and diagnosis. The clinical effect was positively correlated with pretreatment total macrophage numbers (r = 0.28; p = 0.03), sublining macrophage numbers (r = 0.34; p = 0.005) and vascular cell adhesion molecule 1 expression (r = 0.25; p = 0.04). Conclusion: The observations support the view that intra-articular treatment either with 90 Y and glucocorticoids or with glucocorticoids alone is especially successful in patients with marked synovial inflammation.

Cobalamine (vitamine B12) deficientie komt vooral bij ouderen relatief frequent voor (1). Oorzaken zijn onder andere een tekort aan het zogenaamde intrinsic factor dat noodzakelijk is voor de resorptie van cobalamine, achloorhydrie, afwijkingen aan de dunne darm, bacteriele overgroei en een insufficient dieet.

Many of the following questions reflect items on the research agenda of the Group for Research and Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA) for Outcome Measures in Rheumatology (OMERACT) 8 (May 2006) and will have task forces addressing them in the form of research projects, questionnaires, and consensus exercises. ### Which joints should be included in psoriatic arthritis (PsA) assessment: carpometacarpal joints, distal interphalangeal (DIP) joints, feet DIPs? Should we perform 76, 68, 44, or 28 joint counts? Can feet DIPs be distinguished from proximal interphalangeal (PIP) joints clinically and/or radiologically? Can they be distinguished from osteoarthritis radiologically? Should we count or score? Mease : When we set about to assess joints in the original etanercept trial, our assumption was that we needed to capture a larger number of joints than in typical rheumatoid arthritis (RA) trials, including the DIP joints of both hands and feet, as well as the carpometacarpal (CMC) joints which can be commonly affected, thus using a 76/74 joint count. Some have countered that it is difficult in many patients to assess the DIP joints of the feet, especially the smaller ones, and that assessment of the CMC joint may not be valid. Further, radiologists have grumbled that it is often difficult to distinguish the DIP joints, especially in the feet. Thus the perennial question is: Is it sufficient to count the joints, or does it add more information to score relative degree of tenderness and swelling in each joint? The Erlangen analysis of raw data from the etanercept and infliximab phase II trials suggests that counts are as distinguishing as scores, and that 76, 68, 44, and 28 joint counts are all adequate to detect differences between treatment and placebo. The only caveat is that it is worthwhile to assess at least 68 joints at study entry in order to qualify a patient, especially oligoarticular patients. FitzGerald would omit the second through fifth proximal interphalangeal (PIP) joints of the toes as difficult to assess. Gladman : However, this may be important not only at study entry. If a patient happens to have a number of feet or distal …

Treatment with rituximab may reduce disease activity in patients with rheumatoid arthritis (RA). The current dosing schedule of rituximab 2×1000 mg has been shown to induce and maintain a clinical response in initial responders and is also protective against progression of joint destruction.1,–,4 Recently, the treatment schedules of 2×1000 mg and 2×500 mg rituximab were compared side-by-side in early active RA patients.4 It was shown that only initial treatment with 2×1000 mg rituximab resulted in statistically significant protection against progression of structural damage, whereas 2×500 mg and 2×1000 mg resulted in comparable clinical efficacy. Exploratory analysis suggested that re-treatment with 2×500 mg rituximab after 6 months might be protective in terms of inhibition of structural damage. Induction therapy with 2×1000 mg rituximab followed by re-treatment with 2×500 mg or 1×1000 mg after …

Background and objectives Interleukin 6 (IL-6) is a key inflammatory cytokine in the pathobiology of rheumatoid arthritis (RA) and biological therapies targeting the IL-6 receptor have shown clinical benefit. The production of inflammatory cytokines is tightly regulated by reversible acetylation and deacetylation of histones and transcription factors. Histone deacetylase (HDAC) inhibitors (HDACi) are effective therapeutics in animal models of arthritis and have anti-inflammatory effects in RA synovial macrophages and tissue. Here, we examined the effects of HDACi on IL-6 production by RA FLS, and the molecular mechanisms underlying regulation of IL-6 expression by HDACi. Materials and methods RA FLS and macrophages were treated with IL-1β, TNFα, LPS or poly (I:C) in the absence or presence of the HDACi trichostatin A (TSA). IL-6 production was measured by ELISA. The expression and stability of IL-6 mRNA was analysed by quantitative PCR. Activation of NF-κB and MAP kinase signalling pathways was assessed by immunoblotting. The nuclear translocation and DNA binding activity of NF-κB and AP-1 components was assessed by immunoblotting and ELISA-based DNA-binding assays. Results TSA potently suppressed RA FLS IL-6 production induced by IL-1β, TNF and TLR ligands LPS and poly (I-C) (p Conclusions Inhibition of HDAC activity in RA FLS efficiently blocks the production of IL-6, and modulation of IL-6 mRNA stability is a potential mechanism underlying this regulation both in RA FLS and macrophages. Enhanced nuclear export of NF-κB mediated by HDACi might lead to suppressed transcription of other inflammatory mediators. Our findings suggest that therapies targeting HDAC activity may be useful in suppressing inflammation in RA.

Background Treatment with rituximab may reduce disease activity in patients with rheumatoid arthritis (RA). The current dosing schedule of rituximab 2×1000 mg has been shown to induce and maintain a clinical response in initial responders, and is also protective against progression of joint destruction. Recently, the treatment schedules of 2×1000 and 2×500 mg rituximab were compared side-by-side in early active RA patients.1 It was shown that only initial treatment with 2×1000 mg rituximab resulted in statistically significant protection against progression of structural damage, whereas 2×500 mg and 2×1000 mg resulted in comparable clinical efficacy. Exploratory analysis suggested that retreatment with 2×500 mg rituximab after 6 months might be protective in terms of inhibition of structural damage. Induction therapy with 2×1000 mg rituximab followed by retreatment with 2×500 mg or 1×1000 mg after 6 months could be a very cost effective approach. Objective To investigate if clinical response induced by treatment of RA patients with 2×1000 mg rituximab can be sustained following retreatment after 24 weeks with 1×1000 mg rituximab. Methods 11 patients with RA, who were previous tumour necrosis factor inadequate responders with a good or moderate response to initial treatment with 2×1000 mg rituximab according to the European League Against Rheumatism response criteria, were retreated after at least 24 weeks with 1×1000 mg rituximab when their Disease Activity Score of 28 joints (DAS28) was ≤2.6. Patients were followed-up for 48 weeks with monthly assessment of the DAS28. At baseline and at week 48, radiographs of hand and feet were taken and the change in Total Van der Heijde Modified Sharp Score (TvdHS) was determined. Results Nine out of 11 patients completed the 48 weeks of follow-up, 2 patients were lost to follow-up after 24 weeks. The mean DAS28 did not reach the baseline level at any visit, meaning that the clinical response was sustained (figure 1A,B). The mean change in TvdHS after 1 year was −0.60 (±2.9), indicating there was no progression of structural damage after retreatment with 1×1000 mg rituximab on the group level. 3 out of 11 patients did show progression of structural damage: two patients showed an increase in TvdHS of two points in 1 year and one patient an increase of three points in 6 months. Conclusion The clinical response and the inhibition of structural damage induced by 2×1000 mg rituximab can be sustained in most RA patients by retreatment with 1×1000 mg rituximab.

Background and objectives Rap1 is an important signalling molecule downstream of the T-cell receptor (TCR), which can modulate T lymphocyte function upon antigen stimulation. The authors recently demonstrated that transgenic mice with constitutively active Rap1 (RapV12) are protected from experimental arthritis ( Abreu, Arthritis Rheum, 2010 ). Here, the authors aimed to identify the mechanisms of protection by using a TCR transgenic model of myelin oligodendrocyte glycoprotein (MOG)–induced experimental autoimmune encephalomyelitis (EAE) (2D2 mice). Methods Flow cytometry and ELISA were used to analyse the phenotype and cytokine profile of MOG-specific autoreactive T cells in 2D2xRapV12, 2D2, RapV12 and wild type (WT) mice (n=5 per genotype), either in basal conditions or after in vivo priming with MOG peptide. In addition, clinical EAE was monitored for 30 days (n=19 mice per genotype) Results In basal conditions, there was a strong reduction in the number of autoreactive T cells in 2D2xRapV12 versus 2D2 animals in the naive and memory compartment (42 vs 24%, p=0.03), indicating that constitutive Rap1 activation enforces central tolerance. Qualitative analysis of the autoreactive T cells that escaped tolerance showed no differences in T cell subsets, proliferation, apoptosis, expression of costimulatory molecules and production of pro-inflammatory cytokines. After in vivo priming with MOG, however, the authors observed a profound inhibition of tumour necrosis factor production by T cells constitutively expressing Rap1 (19% of 2D2 RapV12 CD4s vs 0.4% in 2D2 controls, p=0.002), whereas there was a slight increase in IFN-γ and IL-17 production. To evaluate the pathophysiological relevance of these quantitative and qualitative alterations of the autoreactive T cells, EAE was induced in 2D2xRapV12 versus 2D2 and in RapV12 and WT littermates. In the 2D2 model, where a significant number of autoreactive T cells are still present despite the constitutive RapV12, the authors observed no difference in EAE scores but an improved survival in the 2D2xRapV12 versus 2D2 mice (42 vs 16%; p=0.04). In wild-type mice, constitutive Rap1 expression led to increased survival (100 vs 74%; p=0.02) as well as lower EAE scores (p=0.07). Conclusion Constitutive activation of Rap1 reduces the autoreactive T cell pool and affects pro-inflammatory cytokine production by the remaining autoreactive T lymphocytes. As in experimental arthritis, these quantitative and qualitative effects are associated with a protection from autoimmune disease in EAE.

Background and objectives The aetiology of rheumatoid arthritis (RA), a prototype immune-mediated inflammatory disease, is poorly understood. Increased serum levels of acute phase reactants and autoantibodies (IgM rheumatoid factor (IgM-RF) and anticitrullinated protein antibodies (ACPA)) may be detected several years before the development of clinical signs and symptoms. Moreover, animal models of RA as well as examination of clinically uninvolved joints from established RA patients have suggested that clinically manifest arthritis may be preceded by an asymptomatic phase characterised by synovial inflammation. However, it is unknown whether the disease process starts in the synovium or at other sites in the body. Materials and methods Fifty five individuals without any evidence of arthritis upon physical examination who were positive for IgM-RF and/or ACPA were included in the study and prospectively followed to assess arthritis development. To evaluate synovial inflammation, all participants underwent dynamic contrast enhanced MRI and arthroscopic synovial biopsy sampling of a knee joint at inclusion. An interim analysis of the first 13 individuals has been performed; these data are shown in this abstract. Results were compared with knee MRI data and synovial biopsy data of 6 and 10 healthy controls, respectively. Results MRI findings evaluated by measurement of maximal enhancement, rate of enhancement, synovial volume and enhancement shape curve distribution were similar in the autoantibody positive individuals compared to the healthy controls. Consistent with these findings, all but one autoantibody positive individuals showed very low scores for T cells, B cells, intimal macrophages, sublining macrophages, fibroblast-like synoviocytes, plasma cells, E-selectin, ICAM-1, VCAM-1, and von WiIllebrand factor, all in the same range as those in normal controls. The patient with higher scores showed patellofemoral joint space narrowing on conventional x-ray consistent with osteoarthritis. During follow-up of a median of 37 (range 25–45) months, four individuals developed clinical signs and symptoms of arthritis. In these patients the baseline synovium was completely comparable with the synovium of healthy controls and of the individuals who did not develop arthritis. Data will be presented on the larger cohort. Conclusions Subclinical inflammation of the synovium does not coincide with the appearance of serum autoantibodies during the preclinical arthritis stage. Thus, systemic autoimmunity precedes the development of synovitis, suggesting that a ‘second hit’ is involved. Moreover, even if there are no signs of synovial inflammation, autoantibody positive individuals may suffer from arthralgia.

Background and objectives Rheumatoid arthritis (RA) synovium is characterised by hyperplasia of fibroblast-like synoviocytes (FLS), which display properties of transformed cells. Regulation of proto-oncogenic phosphatidylinositol 3-kinase (PI3-K) signaling by components of the NF-κB signal transduction pathway has emerged as a candidate molecular mechanism linking inflammation to cancer. Conversely, activation of PI3-K pathways in genetically engineered mice, via reducing expression of PTEN or FoxO transcription factors or transgenically expressing constitutively active PKB, can drive NF-κB-mediated autoimmunity. Here we examined potential functional relationships between PI3-K and NF-κB signaling in RA FLS and synovial tissue. Materials and methods Expression and/or phosphorylation of IKKβ, IkBα, PTEN, a negative regulator of PI3-kinase, PKB and FoxO1, downstream targets of PI3-kinase, was detected by immunohistochemistry combined with digital image analysis in synovial tissue from 15 disease-modifying antirheumatic drug (DMARD)-naive RA patients. Biopsies were obtained at baseline, and x-rays made of hands and feet at baseline and at 2 years follow-up. At 2 years, patients were classified as having non-erosive or erosive disease (Sharp van der Heijde score > 2). Effects of the pan-PI3-K inhibitor LY294002 and inhibitors specific for α/β, γ and λ isoforms of PI3-K on RA FLS IL-1β-induced IkBα phosphorylation and NF-κB p65 activation were assessed by immunoblotting and ELISA, respectively. Results In vivo we observed a strong correlation between expression of IKKβ and phosphorylation of IκBα (r = 0.83, p Conclusions Our studies provide evidence that PI3-K signaling pathways may contribute to joint destruction in RA through enhancement of NF-κB-dependent gene transcription and that PI3-kinase inhibitors may be effective in preventing disease progression.

Background T-cells play a key role in psoriatic arthritis (PsA). Nevertheless, the distribution and exact role of these cells remains unclear. Here, we study whether expanded T-cell clones are present in synovial tissue (ST), and whether such clones are also detectable in peripheral blood (PB) and synovial fluid (SF). Methods ST and SF from inflamed knees was sampled in 2 PsA patients together with paired PB samples. Using next generation sequencing T-cell clones were identified by their unique T-cell receptor β-chain sequence. Clones with a frequency of ≥0.5% were arbitrarily considered to be expanded. Results ST and SF samples from the same joint shared many of the expanded clones (see Table 1). Ninety percent (median, range 71-100%) of the expanded clones in ST were retrieved in SF, 33% of these being expanded in both samples. ST clones accounted for 14% of the SF T-cell repertoire. In PB 83% of the expanded ST clones were retrieved, 17% being expanded in both samples. Analysis of both inflamed knees in patient 1 showed that 83% of the expanded ST clones in the left knee were retrieved in the right knee, 80% of these being expanded in both. Conclusions In PsA synovial tissue harbors expanded T-cell clones, which are not fully represented in synovial fluid or blood samples. Preliminary analysis indicates that different joints contain identical expanded T cell clones. Our results suggest that in PsA comprehensive analysis of inflammation-related expanded clones best focuses on ST, rather than PB and SF. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4456

Background Lymph node stromal cells (LNSC) build the scaffold that enables migration and interaction of lymphocytes in the lymph node. More recently, it has been shown that during inflammation LNSC play a crucial role in shaping the immune response and maintaining tolerance. However, little is known about the underlying molecular mechanisms. Objectives In the current study we analyzed the expression of microRNAs in LNSC from healthy individuals, RA patients as well as anti-CCP positive arthralgia patients with a high risk to develop RA. Methods Needle biopsies of inguinal lymph nodes were strained through a 70μm nylon mesh and the resulting stromal part was cultured in DMEM with 10% FCS. Adherent cells were passaged 4 times before total RNA was isolated. RNA was reverse transcribed with specific microRNA stem loop primers and loaded onto TaqMan® Low Density Arrays (Life technologies, MicroRNA Panel A). The data were analyzed on the Expression Suite software using RNU48 as endogenous control, and differentially expressed microRNAs were re-measured in single-assay reactions. Results 4 microRNAs appeared differentially regulated between healthy individuals (n=5) and RA patients (n=6) with a p-value Conclusions LNSC of RA patients have specific changes in their microRNA profile with upregulation of a microRNA cluster on chromosome X q26.3. Studying these miRNAs in a larger cohort of patients, further follow up of the arthralgia patients and functional analysis of these microRNAs will clarify the role of the lymph node microenvironment on arthritis development and bring valuable insights into pathophysiological mechanisms of disease development. Acknowledgements IMI BTCure n° 115142, EURO-TEAM n° 305549, IAR, Dutch Arthritis Foundation grant 11-1-308, Netherlands Organisation for Health Research and Development (ZonMw) Veni project 916.12.109 Disclosure of Interest C. Ospelt Grant/research support: IMI BTCure, IAR, EURO-Team, J. Hahnlein: None declared, R. Gay Grant/research support: IMI BTCure, IAR, EURO-Team, P. Tak Employee of: GSK, D. Gerlag Employee of: GSK, S. Gay Grant/research support: IMI BTCure, IAR, EURO-Team, L. Van Baarsen: None declared DOI 10.1136/annrheumdis-2014-eular.4383

Background Rheumatoid arthritis (RA) is characterized by proliferation of synovial fibroblasts (FLS), activation of these cells by synovial macrophages (Mϕ), and production of pro-inflammatory proteins. As genetic backgrounds do not completely explain the etiology of RA, a growing attention has focused on potential epigenetic contributions to inflammation. Objectives Here we examined the role of the closely related epigenetic regulatory proteins sirtuin 1 (Sirt1) and sirtuin 2 (Sirt2), class III histone deacetylases, in the inflammatory activation of RA FLS and human macrophages. Methods Sirt1 and Sirt2 mRNA and protein expression was measured in RA FLS following stimulation with IL-1β and TNF-α. RA-FLS production of IL-6, IL-8, MMP-1 and MMP-3 mRNA and protein expression in response to IL-1β, in the absence or presence of specific inhibitors for Sirt1 and Sirt2, was measured by quantitative PCR (qPCR) and ELISA. The expression of 84 genes regulated by IL-1β in RA FLS were analyzed by qPCR array in the presence or absence of Sirt2-specific inhibitor. Monocytes obtained from healthy volunteers were differentiated into either M1 or M2 macrophages with human recombinant IFN-γ or IL-10 for 7 days. Macrophage production of IL-6 and IL-8 in response to LPS stimulation in the absence or presence of Sirt inhibitors was assessed by ELISA. Results The expression of Sirt1 was enhanced following RA FLS IL-1β stimulation, while Sirt2 expression was significantly reduced (p Conclusions Our studies identify Sirt2 as a novel epigenetic regulator of inflammatory activation of macrophages and RA FLS. Disclosure of Interest E. Mohammadi: None declared, C. Angiolilli: None declared, P. Kabala: None declared, P. Tak Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, D. Baeten: None declared, F. Husseini Shirazi: None declared, K. Reedquist: None declared DOI 10.1136/annrheumdis-2014-eular.4012

Background Phosphatidylinositol 3-kinase (PI3K) –dependent activation of PKB, and subsequent PKB phosphorylation and inactivation of FoxO transcription factors, is a critical regulator of cellular proliferation and survival. Constitutive activation of the PI3K/PKB pathway, and inactivation of FoxO proteins is observed in rheumatoid arthritis (RA) patient synovial tissue, and rescue of FoxO1 activity induces RA fibroblast-like synoviocyte (FLS) apoptosis. However, SGK, an AGC kinase related to PKB, can also target the same residues of FoxO proteins. Objectives Here, we sought to determine the relative contributions of PKB and SGK to RA FLS survival and inflammatory activation. Methods PKB and SGK isoform expression was detected by qRT-PCR and immunoblotting experiments on isolated cell populations. Effects of specific PKB (Akt inhibitor VIII) and SGK (GSK 650394) inhibitors on metabolic activity and IL6 production was assessed by MTT assay and ELISA, respectively. The influence of pharmacological inhibition of PKB or SGK on expression of IL1 responsive genes in RA FLS and human macrophages was determined using low density qPCR arrays. Furthermore, effects of pharmacological targeting of PKB or SGK on RA FLS IL-1β- or PDGF- induced FoxO1 phosphorylation was assessed by immunoblotting. Results All PKB isoforms ( AKT1, AKT2 , and AKT3 ) were expressed at equivalent levels in RA FLS, while only SGK1 and SGK3 , but not SGK2 , were detected. PKBi, but not SGKi, significantly decreased FLS mitochondrial activity. In contrast, only SGKi significantly reduced IL-1β-induced IL-6 production. Examining 84 genes induced by IL-1β in RA FLS, we found that PKBi suppressed transcription of CCL7 , CXCL6 CXCL9 , CXCL10 , CXCL11 , IL1RN , and MMP13 , but enhanced IL23A and TNFα expression. SGKi enhanced EREG expression, but suppressed CXCL10, CCL4, CCL5 , CSF3 , IL6 , MMP3 , MMP7 and VCAM1 expression. In human macrophages we found that PKBi suppressed transcription of IL36A and PGLYRP1 , SGKi suppressed IL6 and TNF . Neither compound interfered with IL-1β-induced FoxO1 phosphorylation at AGC kinase consensus sites. Conclusions Our results provide the first evidence for a role of SGK in the inflammatory activation of RA FLS and human macrophages, point to an unidentified third AGC kinase regulating FoxO1, and suggest that targeting distinct PI3K-dependent AGC kinases can preferentially modulate specific components of cellular activation. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5096

Angiogenesis contributes to inflammation and joint destruction in rheumatoid arthritis (RA). Angiopoietins 1 (Ang-1) and Ang-2, which mediate blood vessel remodelling, as well as their receptor Tie2, are expressed in inflamed synovial tissue. The authors have recently shown that Ang-1 expression relative to Ang-2 is significantly higher in RA compared to psoriatic arthritis, corresponding with enhanced activation …

Activation of the small GTPase Rap1 in T lymphocytes regulates cell adhesion, cytokine production and reactive oxygen species production. The authors have recently identified blocked Rap1 activation as one of the T cell receptor proximal signalling defects in rheumatoid arthritis (RA) synovial fluid T cells. The goal of this study was to determine if maintaining Rap1 signalling in murine T cells could modify disease in collagen-induced arthritis. Arthritis was induced in wild-type (WT) and RapV12 transgenic (expressing constitutively active Rap1 in …

Background Disease activity and response to therapy in RA correlates with changes in synovial macrophage numbers and their products. M-CSF or interleukin (IL)-34 stimulation of their common receptor CSFR1 promotes macrophage differentiation, activation and osteoclastogenesis, but M-CSF and IL-34 play distinct roles in murine development. Pharmacological inhibition of CSFR1 is beneficial in animal models of arthritis but the relative contributions of M-CSF, IL-34 and their receptor to inflammation in rheumatoid arthritis (RA) are unknown. Objectives To determine the roles of M-CSF and IL-34 in huma macrophage activation, and human and murine models of RA. Methods M-CSF, IL-34, and cellular markers were detected by immunohistochemistry and digital image analysis in synovial tissue from 18 biological-naive RA patients, 14 PsA patients, and 4 controls without inflammatory disease (2 healthy donors and 2 OA patients). Gene expression in M-CSF and IL-34 –differentiated human macrophages was assessed by FACS analysis and q-PCR arrays. RA synovial explants were incubated with M-CSF, IL-34, control antibody (Ab), or a humanized CSFR1-blocking Ab. Prophylactic effects of control and CSFR1-blocking Ab were examined in murine collagen-induced arthritis (CIA). Results Expression of M-CSF and IL-34 was similar in RA and PsA synovial tissue, but lower in controls (p CXCL5 , CXCL6 , FN1 , COL1A1 , COL6A1-2 , COL14A1 , COL15A1 , MMP7 , MMP8 and MMP9 mRNA expression was significantly upregulated in macrophages differentiated in M-CSF compared to IL-34, while CXCL7 , CXCL9 , CXCL10 , CXCL11 , CXCL12 , LAMA1 , and MMP2 were downregulated. M-CSF or IL-34 had no effect on RA synovial explant IL-6 production, but anti-CSFR1 Ab dose-dependently reduced IL-6 production. Treatment with anti-CSFR1 Ab in CIA significantly reduced paw swelling and joint destruction. Conclusions M-CSF and IL-34 are expressed in topographically distinct regions of inflamed synovial tissue and differentially effect macrophage capacity to attract inflammatory cells and remodel tissue. Simultaneous inhibition of CSFR1 interactions with both M-CSF and IL-34 suppresses inflammatory activation of RA synovial tissue and pathology in CIA, suggesting a novel therapeutic strategy for RA. Disclosure of Interest S. Garcia: None Declared, L. Hartkamp: None Declared, M. W. Tang: None Declared, I. van Es: None Declared, H. Lin Shareholder of: stock options in Five Prime Therapeutics, Inc., Employee of: Five Prime Therapeutics, Inc., L. Long Shareholder of: stock options in Five Prime Therapeutics, Inc., Employee of: Five Prime Therapeutics, Inc., E. Masteller Shareholder of: stock options in Five Prime Therapeutics, Inc., Employee of: Five Prime Therapeutics, Inc., B. Wong Shareholder of: stock options in Five Prime Therapeutics, Inc., Employee of: Five Prime Therapeutics, Inc., P. Tak Employee of: GlaxoSmithKline, K. Reedquist Grant/research support from: Five Prime Therapeutics, Inc.

Background The NF-κB family of transcription factors is strongly involved in synovial inflammation. We have previously shown that NF-κB-inducing kinase (NIK) is a key regulator of inflammation-induced angiogenesis in rheumatoid arthritis (RA) synovial tissue (ST). Objectives In this study we investigated the expression of NIK in ST of early arthritis patients and individuals at risk of developing RA. Methods Arthroscopic synovial biopsy samples were obtained from early arthritis patients (arthritis duration Arthroscopic synovial biopsy samples were also obtained from 46 IgM-rheumatoid factor and/or anti-citrullinated peptide antibody positive individuals at risk for developing RA, who never had any evidence of arthritis upon physical examination. ST sections were stained for NIK and evaluated by semi quantitative analyses (positive or negative). Results 154 Early arthritis patients were included (RA (n=64), unclassified arthritis (n=61), crystal arthropathy (n=11), osteoarthritis (n=4) and spondyloarthritis (n=14)). NIK was predominantly expressed in endothelial cells of small blood vessels as described before. No significant difference was observed in baseline NIK expression between different classification groups. NIK expression correlated with ESR (r=0.214; p=0.006), CRP (r=0.207; p=0.008), swelling of the biopsied joint (r=0.297; p Conclusions We established that synovial NIK expression is associated both with systemic markers of disease activity (ESR and CRP) and with local disease activity in DMARD-naive early arthritis patients, independent of the diagnosis. These findings underscore the importance of angiogenesis in synovial inflammation and identify NIK as a potential therapeutic target in arthritis. Disclosure of Interest None Declared

Background TNFα blockers have had a significant impact on the management of psoriatic arthritis (PsA). Despite their effectiveness, patient response is heterogeneous, underscoring the need for markers to assist in therapy selection for likely responders. The 14-3-3 family of proteins represents intracellular chaperones that regulate various biological processes. The η (eta) isoform, is detectable in the synovial fluid and serum of patients with arthritis, is up-regulated by TNFα and correlates with measures of clinical improvement in rheumatoid arthritis (RA). Objectives To study the serum levels of 14-3-3η in PsA patients in relationship to clinical response to adalimumab therapy. Methods Serum 14-3-3η levels of 24 patients (15 males and 9 females) with active PsA fulfilling the CASPAR classification criteria were measured before receiving adalimumab therapy for either 8- or 12-weeks. Clinical assessment included: SJC68, PASI, HAQ, CRP, ESR, DAS28 and ACR response. Two-tailed Mann-Whitney U-tests were run to compare differences between ACR50 responders and non-responders. Pearson correlations were generated to determine relationships between 14-3-3η and clinical variables. Contingency and regression analyses were performed to identify which variables were predictors of an ACR50 response. Receiver operating characteristic (ROC) curves were used to examine differential expression for response and to determine the accuracy of the regression model. Results Baseline 14-3-3η serum concentrations were significantly higher in ACR50 responders (0.46ng/ml) versus non-responders (0ng/ml, p=0.02) delivering a ROC AUC of 0.75, p=0.04. Consistent with published data [1] median CRP levels were also higher in ACR50 responders; 18.3mg/l versus 5.0mg/l in non-responders, p=0.05. There were no statistically significant differences in any other clinical variables between the two response groups. Across the entire cohort, 14-3-3η titres correlated moderately with CRP levels at baseline (r=0.429, p=0.03). No correlations with other clinical measures were observed. Regression analysis revealed that 14-3-3η titres were predictive of an ACR50 response delivering a significant likelihood ratio (LR) of 5.53 (p=0.018), an odds ratio (OR) of 4.62 with a ROC AUC=0.75. Similarly, a contingency analysis showed that 14-3-3η positivity reflected likelihood of ACR50 response to adalimumab therapy [LR=5.83 p=0.016; OR=8.56 and a relative risk (RR) of 3.27]. Combining 14-3-3η positivity with CRP titres strengthens the model wherein the ROC AUC for CRP as a single variable is 0.74, LR=5.86 (p=0.015) while combining the two markers delivers a ROC AUC of 0.86, LR=10.3 (p=0.006) increasing the prediction value of CRP nearly 2-fold when 14-3-3η is added. The PPV and NPV for CRP are 0.35 and 0.65 whereas with 14-3-3η they are 0.54 and 0.46, respectively. Conclusions Serum 14-3-3η titres and positivity both predict ACR50 response to adalimumab therapy in PsA patients. 14-3-3η sero-positivity when taken together with CRP titres significantly improves prediction of response. References F Van den Bosch et al. Effectiveness of adalimumab in treating patients with active psoriatic arthritis and predictors of good clinical responses for arthritis, skin and nail lesions. Ann Rheum Dis 2010;69:394-399 Disclosure of Interest A. Marotta Shareholder of: Co-founder, A. Kuijk: None Declared, W. Maksymowych: None Declared, P. Tak: None Declared