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Background Tocilizumab, anti-IL-6 receptor monoclonal antibody, is widely used for patients with rheumatoid arthritis. IL-6 is essential for production of C-reactive protein (CRP). Tocilizumab fully inhibit the production of CRP. Therefore, we have difficulty in objective assessment of infection and disease activity because the level of CRP is suppressed under tocilizumab therapy. The development of new biomarker alternative to CRP is needed for daily practice. Objectives To discover new biomarkers alternative to CRP under tocilizumab therapy for rheumatoid arthritis. Methods We registered patients with rheumatoid arthritis treated with tocilizumab. We collected serum samples from those patients at baseline, 4 weeks after the first tocilizumab administration when patient9s CRP level is almost normal, and 1 year later. And we measured CRP, ESR and clinical disease activity index (CDAI) score.Serum peptidomic analysis was conducted by newly-established one-step direct transfer technology (BLOTCHIP-MS analysis), a rapid quantitative technology for peptidomic analysis. All sample measurements were repeated four times. Statistical analyses of MS spectral data were conducted using ClinProTools version 2.2 (Bruker Daltonics). Results We registered 14 patients and their background is shown in Table 1. The levels of CRP were 1.16±0.99 mg/dl at baseline, 0.02±0.01 mg/dl at 4 weeks and 0.01±0.01mg/dl at 1 year, respectively. Their CDAI score were 22±9.2 at baseline, 15±8.9 at 4 weeks and 3±2.6 at 1 year, respectively. CRP titer decreased to almost normal level at 4 weeks regardless whether CDAI score did not fully decrease. We detect 6 biomarkers, named as PRSJ01 to PRSJ06, by the peptidomic analysis (Table 2). The AUC of diagnostic value of these markers is from 0.742 to 0.858. For example, the level of PRSJ06 significantly decreased 4 weeks (Wilcoxon singed-rank test, p=0.02) and 1 year (Wilcoxon singed-rank test, p=0.003) after first tocilizumab administration (Figure 1). and it was inversely-correlated with CDAI score. Conclusions We detect new disease activity biomarkers alternative to CRP under tocilizumab therapy for rheumatoid arthritis. It is useful for exact evaluation of disease activity and infection during tocilizumab therapy. Disclosure of Interest None declared

Background Multitarget therapy for lupus nephritis which was reported by Bao et al. 1 is hopeful treatment option for refractory systemic lupus erythematosus (SLE). However, there are few studies about multitarget therapy with combination of prednisolone (PSL), purine synthesis inhibitor and calcineurin inhibitor for SLE. Objectives The aim of this study is to evaluate efficacy and safety of multitarget therapy with combination of PSL, mizoribine (MZR) and tacrolimus (TAC) as induction or add-on therapy for refractory SLE with or without lupus nephritis. Methods We retrospectively studied 26 SLE patients treated with multitarget therapy in our department from April 2009 to December 2013. The mean age was 44.5 years old and 23 patients were female. They were divided into two groups; (A) 10 patients who were initially treated with this therapy as induction, and (B) 16 patients who were additionally treated with this therapy due to minor flares or difficulty in reducing PSL dose. We evaluated efficacy and safety of this combination strategy as (A) induction therapy and (B) add-on therapy respectively. Results (A) Five of 10 patients had lupus nephritis (2 were class IV and 3 were class IV+V). Three of 10 stopped this therapy due to severe adverse events within 12 months. Other 2 patients stopped this therapy due to preparation for pregnancy. Among nephritis patients, complete renal response rate at 6 months was 75%. The mean SLE disease activity index (SLEDAI) was reduced from 25 at baseline to 7.5 at 6 months. In all non-nephritis patients, their symptoms or blood examination data were improved. The mean SLEDAI was reduced from 14.4 at baseline to 2.4 at 6 months. (B) Five of 16 patients had lupus nephritis. One patient stopped this therapy due to preparation for pregnancy. The mean PSL dose was reduced from 14.8mg/day at baseline to 9.2mg/day at 12 months. The mean SLEDAI was reduced from 6.5 at baseline to 2.7 at 12 months. In all 26 patients, adverse events were 28 cases in the observation period. Most of these were mild infections. Three severe adverse events were observed. These were hypertrophic cardiomyopathy due to interaction of TAC and clarithromycin, nocardial soft tissue abscess and cryptococcal meningitis. All three patients who experienced severe adverse events were over 65 years old. Conclusions The combination use of PSL, MZR and TAC is effective as induction therapy and as add-on therapy for refractory SLE regardless of having lupus nephritis. However, elderly patients tend to suffer from sever adverse events. References Bao H et al: Successful treatment of class V+IV lupus nephritis with multitarget therapy, J Am Soc Nephrol 19; 2001-2010, 2008 Disclosure of Interest Y. Kukida Grant/research support: Astellas Pharma Inc. Asahi Kasei Pharma Corp., T. Kida: None declared, T. Inoue: None declared, Y. Isoda: None declared, T. Sagawa: None declared, R. Ishigaki: None declared, A. Kasahara: None declared, A. Nakabayashi: None declared, K. Fujioka: None declared, H. Nagahara: None declared, W. Fujii: None declared, K. Murakami: None declared, T. Seno: None declared, A. Yamamoto Grant/research support: Astellas Pharma Inc. Asahi Kasei Pharma Corp., M. Kohno Grant/research support: Astellas Pharma Inc. Asahi Kasei Pharma Corp., Y. Kawahito Grant/research support: Astellas Pharma Inc. Asahi Kasei Pharma Corp. DOI 10.1136/annrheumdis-2014-eular.3186

Background It is well known that synovial fluid pH is decreased in rheumatoid arthritis (RA) patients (1), but the meaning and mechanism remain unclear. Objectives We investigate the correlation between synovial fluid pH and the disease activity of RA. We reveal the mechanism by which synovial fluid pH is regulated and explore the novel therapeutic strategy of RA by applying this mechanism. Methods We measured the values of pH and the concentration of L-lactate in synovial fluid of RA patients. Next, we investigated the expression of ion transporters regulating intracellular and extracellular pH in RA synovial fibroblasts (RASFs) obtained from the inflamed joints using quantitative RT-PCR and Western Blotting. Finally, we suppressed the ion transporter expression by small interfering RNA (siRNA) against the ion transporter increased in RASFs, and analyzed their viability and proliferative capacity of RASFs. Results Synovial fluid pH was decreased as the disease activity score (DAS)-28 CRP increased in RA patients (graph A, n=17, r = -0.81, p

Background Recent reports demonstrated that somatic cells such as fibroblasts can be directly reprogrammed into other cell types including neurons and cardiomyocytes by introducing critical transcription factors involved in the differentiation of the corresponding cell lineages 1,2 . Such procedures in combination with co-transduction of a particular gene may allow creation of the cells with desired functions. If patient specific osteoblasts can be induced and engineered to produce IL-10, autologous transplantation of such cells may suppress inflammation and bone destruction in rheumatoid arthritis by modulating immune responses and osteoclast development. Objectives To generate IL-10-producing mouse osteoblast-like cells from fibroblasts, and estimate potential effect of the cell supernatants on osteoclast differentiation. Methods Various combinations of transcription factors were transduced into mouse embryonic fibroblasts (MEFs) with retroviral vectors and the efficiency of conversion into osteoblast-like cells were estimated by alizarine red S staining. IL-10 gene was also transduced to the cells that received the most effective combination of the transcription factors, and resultant cells were characterized by qRT-PCR, alkaline phosphatase staining, and alizarine red S staining. IL-10 production was measured by qRT-PCR and ELISA. The supernatant was added to a mouse macrophage cell line Raw264.7 cells that were induced to differentiate into osteoclasts by an addition of RANKL. Results MEFs were successfully induced to massively produce bone matrix that were mineralized by calcium phosphate. Co-transduction of the IL-10 gene by means of a retrovirus vector resulted in establishment of osteoblasts that produced IL-10 at a significant level. The culture supernatant of the cells significantly suppressed osteoclast differentiation from Raw264.7 cells. Conclusions IL-10-secreting osteoblasts were successfully generated from fibroblasts by direct reprogramming procedures. The cells may be useful in novel cell-based therapy against RA in the future. References Ieda, M. et al.: Cell, 142: 375-386, 2010. Vierbuchen T.: Nature, 463: 1035-1041, 2010. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2247

Background Primary Sjogren syndrome (SS) is an autoimmune exocrinopathy. Mucin are present on apical surfaces of all healthy wet surface epithelia of human body and on the glands of simple secretory epithelial tissue. The major and minor salivary glands express the membrane-bound mucin, MUC1. Tn and sialyl-Tn antigens are tumor-associated carbohydrate antigens expressed on mucins in epithelial cancers. Alteration of glycoproteins is related with various immunoreactions. The study of sugar chains in autoimmune diseases is intriguing, however, there have been few reports on abnormalities of sugar chains, especially in the pathogenesis of SS. The functions of Tn and sialyl-Tn antigens, MUC1 expressed in human salivary gland is unknown. Objectives The carbohydrate chains represented by mucins are expressed by a variety of normal and malignant secretory epithelial cells and induce a variety of immunoreactions. The purpose of this study is to investigate the existence and bioactivity of tumor-associated carbohydrate antigens in SS. Methods A total of eleven patients (females) with primary SS (mean (SD) age = 52.4 (13.5) years) were enrolled. Saliva was collected by the gum test. Labial salivary gland biopsies were obtained in 11 patients. We examined the expression of Tn, sialyl Tn antigens, and MUC1 in salivary glands tissues from SS patients by immunohistochemistry. In addition, saliva from SS patients was subjected to gel filtration on Sepharose 6B and collected the fractions including mucins. The tumor necrosis factor (TNF)-α and prostaglandin E 2 (PGE 2 ) production by mucin in human peripheral blood monocytes (PBMC) were also investigated using ELISA. Results We found that Tn and sialyl Tn antigens, MUC-1 were strongly expressed in mucous acinar cells and infiltrating mononuclear cells on the labial salivary gland (p 2 (Image C) in human PBMC. Image/graph Conclusions We revealed that Tn and sialyl Tn antigens typical malignancy-associated mucins, MUC-1 expressed in SS salivary glands. We found an aberrantly glycosylated mucin from saliva in patients with SS, which can produce TNF-α and PGE 2 in human PBMC. This finding suggests that the mucins exhibiting with abnormal glycosylation may be in part responsible for salivary glands inflammation, leading to the salivary glands destruction in the pathogenesis of SS. References Reduced sulfation of muc5b is linked to xerostomia in patients with Sjogren syndrome. Alliende C, et, al. Ann Rheum Dis. 2008 Oct;67(10):1480-7. Expression of Tn and sialyl Tn antigens in synovial tissues in rheumatoid arthritis. Ishino H, et, al. Clin Exp Rheumatol. 2010 Mar-Apr;28(2):246-9. Disclosure of Interest H. Ishino Grant/research support from: Grants-in-Aid for Scientific Research, M. Kohno: None Declared, N. Kajitani: None Declared, A. Yamamoto: None Declared, T. Seno: None Declared, K. Murakami: None Declared, W. Fujii: None Declared, H. Nagahara: None Declared, H. Nakada: None Declared, Y. Kawahito: None Declared

Background Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. MDSCs are characterized by the co-expression of the myeloid differentiation antigens Gr-1 and CD11b in mice (1). MDSCs in cancer have been studied in detail and are known to play roles in tumor associated immune suppression. However, the roles of MDSCs in autoimmune disease remain controversial and little is known about MDSCs in autoimmune arthritis. Objectives We investigate the roles of MDSCs in autoimmune arthritis using collagen-induced arthritis (CIA) mouse models. Methods We determined the number of Gr-1 + CD11b + MDSCs in the spleens of CIA mice by flow cytometry. Next, we isolated MDSCs from CIA mice by magnetic cell sorting and cultured with CD4 + T cells to analyze the functions of MDSCs. We investigated the proliferation of CD4 + T cells by CFSE dye dilution assay and estimated cytokine levels produced by CD4 + T cells using ELISA. Furthermore, we investigated CD4 + T cell differentiation into Th17 cells by flow cytometry. Finally, we performed adoptive transfer of MDSCs into CIA mice and investigated the severity of arthritis. Results MDSCs significantly accumulated in the spleens of mice with CIA when arthritis severity peaked. These MDSCs inhibited CD4 + T cell proliferation and differentiation into Th17 cells. Moreover, MDSCs inhibited the production of IFNγ, IL-2, TNFα, and IL-6 by CD4 + T cells in vitro , whereas they promoted the production of IL-10. Adoptive transfer of MDSCs reduced both clinical (Image A) and histological arthritis scores (Image B) in vivo , which was accompanied by a decrease in the number of CD4 + T cells and Th17 cells in the draining lymph nodes. Image/graph Conclusions MDSCs in CIA suppress the progression of CIA by inhibiting the pro-inflammatory immune response of CD4 + T cells. These observations suggest that MDSCs play crucial roles in the regulation of autoimmune arthritis, which could be exploited in new cell-based therapies for human rheumatoid arthritis (RA). References Gabrilovich, D. I., and S. Nagaraj. 2009. Myeloid-derived suppressor cells as regulators of the immune system. Nat. Rev. Immunol. 9: 162-174. Disclosure of Interest None Declared