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BackgroundGlycosylation is an important post-translational modification of proteins. Modification of glycan structure has been reported in some disease conditions such as rheumatoid arthritis (RA). RA is a chronic, systemic autoimmune disease where many inflammatory cytokines play pathological roles. In patients with RA, a significant defect in the galactosyltransferase enzyme activity results in a change in the galactosylation of immunoglobulin G (IgG). Autoantibodies against IgG lacking galactose frequently appear in RA patients and this change has been demonstrated to be associated with disease activities in RA. Although the precise mechanism is still not known, it is likely that the abnormal glycosylation processes may pathologically associate with RA. We hypothesize that this kind of glycosylation abnormalities occurs in the development of immune competent cells in the bone marrow (BM). Ochi et al reported an abnormality of BM cells contributes to the pathogenesis of RA in human and animal models (Arthritis Res Ther. 2007). Especially, they reported the presence of abnormal myeloid cells expressing the difucosyl type 2 chain structure (a specific marker of human undifferentiated cells) in RA patients' BM. This fact encouraged us to focus on bone marrow cells.ObjectivesThis study is to clarify the abnormal expression of glycosylation-related genes in bone marrow cells of RA patients and to analyze the network regulation of immune and inflammatory responses related to glycosylation abnormalities in RA.MethodsComprehensive gene expression analysis was conducted using DNA microarray on the BM cells of 28 RA patients and 11 healthy individuals (HI). According to the gene lists based on the Glyco Gene Database (GGDB) and Lectin Frontier Database (LfDB), glycosylation-related genes were extracted among the differentially expressed genes in RA compared with HI. Network analysis was performed using Ingenuity Pathway AnalysisR (IPA).Results1416 down regulated and 1673 up regulated genes were identified by more than two-fold change in the expression in RA compared to HI. In these differentially expressed genes, 50 glycosylation related genes decreased and 54 genes increased significantly. The decreased genes included A4GALT, B4GALT (encoding galactosyltransferases), UGCG (glucosyltransferase), FUT8 (fucosyltransferase) and EXT1,2 (exostoses) listed as top-five changed genes. On the other hand, the increased genes included many lectin genes such as ITGAL (encoding integrin), LGAL (galectin) and SIGLEC (sialic acid binding lectin). IPA analysis showed that the differentially expressed genes composed networks with inflammatory cytokines including TNF and IL-1. Moreover, they composed a network relevant to a dendritic cells and NK cells functions.ConclusionsThis is the first report that abnormal expression of glycosylation related genes in the bone marrow cells of RA patients. The down-regulation of genes of glycan synthesis and up-regulation of lectin genes might associate with the pathogenesis of RA.Disclosure of InterestNone declared

Background Enhanced expression of CD14+CD15+ cells has been reported in the bone marrow of patient with severe rheumatoid arthritis (RA) (Tomita et al, J. Rheumatology, 1997). CD14+CD15+ cells (the phenotype of previously described “abnormal” myeloid cells) has been also reported to be increased in peripheral blood stem cell harvests on a per CD34+cell basis (Snowden et al, Br. J of Haematol, 1998) suggesting the abnormal process in the early differentiation of bone marrow cells in RA patients. Our study is focusing to clarify the appearance of CD14+CD15+ population in the early differentiation of bone marrow cells from RA patients. As the strategy, we utilize the monocyte differentiation system of induced pluripotent stem (iPS) cells derived from RA patients. Objectives To examine whether the CD14+CD15+ cells appear during hematologic differentiation process of the iPS cells established from RA patients (RA-iPS). Methods iPS cells were established from skin fibroblasts and peripheral blood mononuclear cells from RA patients as described previously. Six days after transduction, the cells were harvested and re-plated onto mitotically inactivated SNL feeder cells. For the healthy controls, non-onset family members (NOF) of the patients are recruited as donors in order to adjust hereditary background as much as possible. Monocytes were induced from each iPS clone culture on feeder-free conditions using a previously reported method (Niwa et al, PLoS One, 2011). The cells collected from each culture were stained with specified antibodies against CDs, including CD14 and CD15 and were analyzed by fluorescence-activated cell sorting (FACS) on day 12, 15, 18, 21 and 25. Results Cultured cells started to express CD14 on the day 12 and more than 90% of the cells expressed CD14 on the day 21 in the monocyte differentiation induction course. According to the expression levels of CD14, the cell population was divided into three groups: CD14 (−), CD14 (+) and CD14 (++). CD15 (+) cells were observed in CD14 (−) and CD14 (+) population but not in CD14 (++) population. The CD15+ cells in CD14 (+) transiently appeared in RA-iPS derived cells at 11.9±2.8% (mean ± SE) on day15. However these cell proportion in NOF was1.7±2.0%. Meanwhile, CD15+ cells in CD14 (−) proportion decreased during monocyte differentiation in RA-iPS cells, but remained in NOF-iPS cells (representative data, RA 31.5, 20.6, 15.6%, NOF 47.3, 46.1, 47.3%, on day15, 18 and 21). Conclusions We successfully differentiated iPS cells into monocytes and found CD14+CD15+cells transiently appeared only in the very early differentiation process of RA-iPS cells into monocytes. Taken together with the evidence that CD14+CD15+cells exist in RA patient bone marrow, abnormal early differentiation of myeloid/monocyte lineage cells may be pathologically involved in RA. Functional analysis will be required to elucidate the role of this distinct CD14+CD15+population in RA. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3073

BackgroundRheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic proliferation of synovial cells and destructive polyarthritis. Bone destruction in RA is mediated by increased numbers of osteoclasts, and the enhanced osteoclastgenesis has been reported in RA. However, it remains unclear whether or not the genetic factors influence the increased osteoclastgenesis in RA. To answer this question, we induced osteoclasts in vitro using induced pluripotent stem cells (iPSC) derived from RA-patients and non-onset family members of the patients (NOR).ObjectivesThe aim of this study is to clarify if the osteoclastgenesis in RA is enhanced genetically compare to NOR.MethodsiPSCs have been established from skin fibroblasts from RA patients and NORs. Under monocytes-induced conditions in vitro, floating cells were sequentially collected and subsequently inoculated onto glass chamber slides with M-CSF and RANKL. An osteoclast was defined as a TRAP-positive cell containing at least three nuclei. To analyze the bone resorption activity, the fluorescent-intensity of FITC-labeled chondroitin sulfate (CS), which is released from the calcium phosphate layer resolved by osteoclasts, was measured in the culture supernatant by a fluorimeter. The expressions of osteoclast markers (cathepsin K and MMP-9) in osteoclasts induced from monocytes collected on day 24 and day 32, and osteoclast-precursor markers (CD115: MCSF-receptor, and RANK) in the monocytes collected on days 24, 28 and 32 after induction from iPSCs were analyzed by either RT-PCR or fluorescence-activated cell sorting using the corresponding antibodies.ResultsOsteoclastgenesis of monocytes derived from RA-iPSCs increased as compared to that of NOR-iPSCs. The TRAP positive cell numbers per well of RA patients were higher than those of NOR in the presence of RANKL (25 ng/mL and 100 ng/mL) at all 6 time points as shown in the figure below. The fluorescent-intensity of FITC-labeled CS in the culture supernatant of osteoclasts differentiated from RA-iPSCs was also higher than that of NOR-iPSCs (fluorescent intensity/well; 4869±838 and 954±252, respectively on day24). The expression of cathepsin K and MMP9 on day24 were also increased in osteoclasts from RA-iPSCs by 20 and 12 times higher than osteoclasts from NOR-iPSCs, respectively. The proportion of CD115-positive cells in CD14-positive cells was increased in monocytes induced from RA-iPSCs. Furthermore, monocytes induced from RA-iPSCs highly expressed RANK mRNA as compared with those of NOR-iPSCs at all day points of harvesting monocytes.ConclusionsThe genetically up-regulated M-CSF receptor and RANK in the osteoclast precursors in RA-iPSC may lead to the enhanced osteoclastgenesis.Disclosure of InterestNone declared

Background Rheumatoid arthritis (RA) is a common immflamatory autoimmune disease characterized by persistent synovitis and progressive destruction of cartilage and bone in multiple joints. Among the autoantibodies detected in RA patients, circulating autoantibodies, anti-citrullinated protein/peptide antibodies (ACPA), are highly specific and useful diagnostic tools for RA. ACPA production seems to depend on antigen-specific CD4 (+) helper T cell activation. Activation of T cells requires costimulatory signals via binding of CD28 receptor with CD80/CD86 located on antigen-presenting cells (APC). Abatacept (ABA) is a biological agent for RA treatment, consisting of extracellular domains from cytotoxic T-lymphocyte antigen-4 and the Fc portion of human immunoglobulin G1 (CTLA-4-Ig) which competes with CD28 for CD80/86 binding. Objectives This study aims to clarify how 48-week ABA treatment affects CD4 (+) T cell activation and ACPA production in RA patients. Methods Peripheral blood mononucleated cells (PBMCs) and plasma were obtained from 30 patients enrolled in abatacept research outcome as a first-line biological agent in the real world (ABROAD study) at baseline, 24 and 48 weeks of ABA treatment. Fifty-three healthy individuals (HIs) were also enrolled as controls. Surface phenotypes and activation markers of T cells were analyzed with flow cytometory. ACPA titers were measured with EliA CCP ELISA kit. Results Twenty-five patients (83.3%) were ACPA (+) (>4.5 U/mL). DAS28-CRP and SDAI were significantly reduced at 24 and 48 weeks compared with those at baseline. The proportion of CD25 (+) in CD4 (+) T cells in ACPA (+) group was significantly higher than those of ACPA (-) group and HIs at baseline (13.9±5.4% in ACPA (+) group; 6.7±2.9% in ACPA (-) group, p=0.0089; 7.1±4.6% in HIs, p<0.0001). The proportion of CD25 (+) in CD4 (+) T cells decreased at 24 and 48 weeks (13.9±5.4% at baseline; 6.6±5.8% at 24 weeks, p<0.0001; 6.1±3.1% at 48 weeks, p<0.0001). However, ACPA titers were not significantly changed at 24 and 48 weeks compared with those at baseline. Conclusions 48-week T cell co-stimulation blockade reduces disease activities and the proportion of CD25 (+) in CD4 (+) T cells but not the ACPA titers in ACPA (+) RA patients. Disclosure of Interest M. Murakami Speakers bureau: Bristol-Myers Squibb Japan, M. Ito: None declared, M. Sekiguchi Grant/research support: Bristol-Myers Squibb Japan, Speakers bureau: Bristol-Myers Squibb Japan, K. Matsui Grant/research support: Bristol-Myers Squibb Japan, M. Kitano Grant/research support: Bristol-Myers Squibb Japan, Y. Imura Grant/research support: Bristol-Myers Squibb Japan, K. Ohmura Grant/research support: Bristol-Myers Squibb Japan, T. Fujii Grant/research support: Bristol-Myers Squibb Japan, T. Kuroiwa: None declared, K. Maeda: None declared, S. Morita: None declared, Y. Kawahito Grant/research support: Bristol-Myers Squibb Japan, T. Mimori Grant/research support: Bristol-Myers Squibb Japan, H. Sano Grant/research support: Bristol-Myers Squibb Japan, N. Nishimoto Grant/research support: Bristol-Myers Squibb Japan DOI 10.1136/annrheumdis-2014-eular.3997

Background Rheumatoid arthritis (RA) is a common immflamatory autoimmune disease characterized by persistent synovitis and progressive destruction of cartilage and bone in multiple joints. Biological agents have shown enhanced efficacy in patients with RA. Beside their beneficial effects, one of the main safety concerns is an increased risk of infection. CD8 (+) T cells play important roles in the immune surveillance system. Abatacept (ABA) is a T cell inhibitor biologic agent, consisting of extracellular domains from cytotoxic T-lymphocyte antigen-4 and the Fc portion of human immunoglobulin G1 (CTLA-4-Ig) which competes with CD28 on T cells for CD80/86 binding. The post marketing surveillance of ABA revealed that the serious infections were less frequent compared with the treatment using other biologic agents (ACR 2013). CD8 (+) T cells could be influenced by ABA through CD80/CD86 co-stimulatory blockade although CD28 expression on CD8 (+) T cells is lower than that on CD4 (+) T cells. Objectives This study aims to clarify how 48-week ABA treatment affects CD8 (+) T cell subsets. Methods Peripheral blood mononucleated cells (PBMCs) were obtained from 30 patients enrolled in abatacept research outcome as a first-line biological agent in the real world (ABROAD study) at baseline, 24 and 48 weeks of ABA treatment. Fifty-three healthy individuals (HIs) were also enrolled as controls. Surface phenotypes and activation markers of T cells were analyzed with flow cytometory. Results DAS28-CRP and SDAI were significantly reduced at 24 and 48 weeks compared with those at baseline. The proportion of CD25 (+) in CD4 (+) T cells decreased at 24 and 48 weeks compared with that at baseline (13.9±5.4% at baseline; 6.6±5.8% at 24 weeks, p<0.0001; 6.1±3.1% at 48 weeks, p<0.0001). On the other hand, no significant reduction in the proportion of CD8 (+) T cells (19.3±11.0% at baseline, 19.0±7.9% at 24 weeks and 20.4±9.1% at 48 weeks), as well as in the proportion of CD25(+) cells in CD8 (+) T cells (3.26±2.7% at baseline, 3.4±2.5% at 24 weeks and 3.0±1.9% at 48 weeks) was observed. Conclusions 48-week T cell co-stimulation blockade does not alter the activation status of CD8 (+) T cells while it suppresses that of CD4 (+) T cells as well as disease activities. Disclosure of Interest M. Murakami Speakers bureau: Bristol-Myers Squibb Japan, M. Ito: None declared, M. Sekiguchi Grant/research support: Bristol-Myers Squibb Japan, Speakers bureau: Bristol-Myers Squibb Japan, K. Matsui Grant/research support: Bristol-Myers Squibb Japan, M. Kitano Grant/research support: Bristol-Myers Squibb Japan, Y. Imura Grant/research support: Bristol-Myers Squibb Japan, K. Ohmura Grant/research support: Bristol-Myers Squibb Japan, T. Fujii Grant/research support: Bristol-Myers Squibb Japan, T. Kuroiwa: None declared, K. Maeda: None declared, S. Morita: None declared, Y. Kawahito Grant/research support: Bristol-Myers Squibb Japan, T. Mimori Grant/research support: Bristol-Myers Squibb Japan, H. Sano Grant/research support: Bristol-Myers Squibb Japan, N. Nishimoto Grant/research support: Bristol-Myers Squibb Japan DOI 10.1136/annrheumdis-2014-eular.4014