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BackgroundHypertrophic pachymeningitis (HP), characterized by an inflammatory disorder indicating intracranial or spinal thickening of dura mater, is found to develop as a neurological involvement in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Meanwhile, the previous studies focusing on HP in AAV have been reported as a single-institution study, and the analyses were performed in a small number of patients because HP is a rare neurological disorder. Therefore, neither etiological nor clinical characteristics of HP in AAV have been adequately elucidated.ObjectivesThis study clarified the characteristics of HP in AAV by analyzing the information of multicenter study in Japan (Japan collaborative registry of ANCA-associated vasculitis: J-CANVAS).MethodsWe analyzed the clinical information from 541 Asian patients with AAV enrolled in J-CANVAS. Of them, newly diagnosed and relapsed AAV were included in 448 and 93, respectively. The epidemiological and clinical findings were compared between patients with and without HP. Clinical manifestations related to AAV were evaluated based on the Birmingham Vasculitis Activity Score version 3. To elucidate independent factors in HP development, logistic regression analyses were additionally performed.ResultsOf the total 541 patients (mean age: 71±14 years, M:F = 1:1.2), HP was demonstrated in 28 (5.17%), including 17 (3.79%) in newly diagnosed AAV and 11 (11.8%) in relapsed AAV. The classification of granulomatosis with polyangiitis (GPA) was significantly higher in patients with HP than those without HP (50% vs. 21%, p = 0.0007). In newly diagnosed AAV, patients with HP significantly had higher GPA classification and higher positivity for PR3-ANCA than those without HP (53% vs. 17%, p = 0.001; 29% vs. 9%, p = 0.015, respectively). Conversely, positivity for MPO-ANCA was significantly higher in patients with HP than those without HP in relapsed AAV (91% vs. 55%, p = 0.025), despite not significantly different in the classification of AAV. Headache and cranial neuropathies were significant neurological symptoms in patients with HP compared to those without HP (82% vs. 6.6%, p < 0.0001; 32% vs. 2.9%, p < 0.0001, respectively). Besides, ear, nose and throat (ENT) and mucous membranes/eyes were significantly higher involvements in patients with HP than in those without HP (54% vs. 26%, p = 0.003; 29% vs. 9%, p = 0.003, respectively). Moreover, higher complications of “conjunctive hearing loss” and “sudden visual loss”, which are included in the categories of ENT and mucous membranes/eyes involvement, respectively, were significantly indicated in patients with HP than those without HP (39% vs. 7.2%, p < 0.0001; 21% vs. 1.2%, p < 0.0001, respectively). Multivariable logistic regression analysis identified that ENT (odds ratio [OR] 1.28, 95% confident interval [CI] 1.09 to 1.49, p = 0.002) and mucous membranes/eyes involvement (OR 1.37, CI 1.14 to 1.65, p = 0.0006), as well as conjunctive hearing loss (OR 4.52, CI 1.56 to 13.05, p = 0.005) and sudden visual loss (OR 1.84, CI 1.12 to 3.00, p = 0.015), were independent related factors in patients with HP.ConclusionGPA could be significantly classified in patients with HP. Notably, patients with HP significantly showed higher positivity for PR3-ANCA than those without HP in newly diagnosed AAV. Furthermore, sudden visual loss and conjunctive hearing loss might be implicated in HP development.Disclosure of InterestsNone declared

Cross talk between inflammation and coagulation plays important roles in acute or subacute progressive pulmonary fibrosis characterized by diffuse alveolar damage. Thrombomodulin is a physiological inhibitor of high-mobility group box 1 (HMGB1), and thrombin and may be effective for this condition. This study investigated the roles of HMGB1 and thrombin in the pathophysiology of bleomycin-induced pulmonary fibrosis and the efficacy of recombinant human soluble thrombomodulin (rhTM). Pulmonary fibrosis was induced in wild-type C57BL/6 mice by intratracheal instillation of bleomycin. We first assessed HMGB1, thrombin, transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA) levels in bronchoalveolar lavage fluid and lung tissue sections over time. Expression of HMGB1 and thrombin was elevated before that of TGF-β1 and α-SMA and remained high during the fibrotic phase after bleomycin instillation. We next examined whether in vitro stimulation with HMGB1 and thrombin induced expression of TGF-β1 and α-SMA in cultured alveolar macrophages and lung fibroblasts, respectively, by performing quantitative PCR, enzyme-linked immunosorbent assay, Western blot, and immunofluorescence analyses. HMGB1 and thrombin stimulation induced TGF-β1 production by alveolar macrophages, and thrombin stimulation also induced α-SMA expression in lung fibroblasts. Finally, we evaluated the effect of rhTM on bleomycin-induced pulmonary fibrosis. Compared with the vehicle control, both early and late-phase administration of rhTM suppressed the fibrotic process. Our results suggest that HMGB1 and thrombin were involved in the pathophysiology of pulmonary fibrosis via production of profibrotic proteins and that rhTM attenuated bleomycin-induced pulmonary fibrosis. rhTM may be a therapeutic option for acute or subacute pulmonary fibrosis.

Aim To examine the efficacy of abatacept in patients with rheumatoid arthritis (RA) using magnetic resonance imaging (MRI) of bilateral hands. Method This prospective study included 35 RA patients. MRI of bilateral hands was performed at baseline and after 12 months of treatment with intravenous abatacept. MRI images were scored for synovitis, osteitis, erosion and joint space narrowing (JSN) according to the RA MRI Scoring System (RAMRIS). The primary endpoint was the change in RAMRIS score from baseline. Repair of erosion was defined as a negative change in the erosion score that was greater than the smallest detectable changes (SDCs). Results Thirty-one patients completed the study. Median synovitis and osteitis scores showed statistically significant reductions at Month 12 when compared to baseline (synovitis score, −5.5 [P

Allograft inflammatory factor-1 (AIF-1) is a protein expressed by macrophages infiltrating the area around the coronary arteries of rats with an ectopic cardiac allograft. Some studies have shown that expression of AIF-1 increased in a mouse model of trinitrobenzene sulfonic acid-induced acute colitis and in acute cellular rejection of human cardiac allografts. These results suggest that AIF-1 is related to acute inflammation. The current study used bleomycin-induced acute lung injury to analyze the expression of AIF-1 and to examine its function in acute lung injury. Results showed that AIF-1 was significantly expressed in lung macrophages and increased in bronchoalveolar lavage fluid from mice with bleomycin-induced acute lung injury in comparison to control mice. Recombinant AIF-1 increased the production of IL-6 and TNF-α from RAW264.7 (a mouse macrophage cell line) and primary lung fibroblasts, and it also increased the production of KC (CXCL1) from lung fibroblasts. These results suggest that AIF-1 plays an important role in the mechanism underlying acute lung injury.

Background Sphingosine-1-phosphate (S1P) is a biologically active phospholipid, which is derived from membrane lipid. It binds to the receptors, named S1P1–5, and regulates several signalling pathways involved in inflammation, cell survival, angiogenesis and cell migration. Concentration of S1P and expression of S1P receptors can vary according to local tissue conditions. RA is a chronic inflammatory disorder of joints and the concentration of S1P in synovial fluid is higher in RA patient than in OA patient. In vitro, S1P3 expression in RA synoviocyte is upregulated by TNFa treatment. On the other hand, it is not clarified whether S1P/S1P3 signalling pathway contributes to arthritis in RA. Objectives The objective of this study is to investigate the role of S1P/S1P3 signalling in inflammatory arthritis. Methods Collagen-induced arthritis (CIA) was induced by subcutaneous injection of bovine type II collagen emulsified in complete Freund’s adjuvant in wild-type (WT) or S1P3-knock-out (S1P3-KO) 7–9 week-old DBA/1J mice. Arthritis severity were evaluated by visual scoring and histological analysis. The severity was assessed over time by using the arthritis score, in which each paw was scored on a scale of 0–4 and the scores of all four paws were cumulated, resulting in a maximum possible score of 16 per mouse. For histopathological examination, mice were sacrificed on the 42nd day and the hindlimbs were removed and fixed in 4% buffered formaldehyde. Paraffin embedded sections of the knee joints stained with hematoxylin and eosin were systematically scanned in a microscope and scored based on cell infiltration, cartilage destruction and bone erosion parameters. S1P3 mRNA expression was examined by realtime PCR method with total RNA extracted from knee joint capsules of CIA or normal WT mice. Murine primary fibroblast like synoviocytes (FLS) were obtained from CIA mice. We examined S1P3 expression after TNFa treatment and measured cytokine produced after S1P treatment with or without TNFa pretreatment in FLS. Results S1P3 deficiency resulted in modest symptoms of arthritis and a significant reduction in synovial inflammation and bone erosions in histological analysis. S1P3 mRNA expression in knee joint capsule in CIA mice was about five times as high as that in normal mice. TNFa treatment upregulated S1P3 expression and S1P treatment enhanced IL-6 production in WT-FLS significantly. TNFa-priming enhanced S1P-induced IL-6 production, which is significantly higher in WT-FLS than in KO-FLS. This effect was not observed in MCP-1 production of WT-FLS. Conclusions S1P3-KO reduced severity of arthritis, inflammation and bone erosions in CIA. S1P3 mRNA was upregulated in inflamed joint capsule. S1P induces IL-6 production via S1P3 upregulation by TNFa in CIA-FLS. S1P3 inhibition could be a good target of the therapy for arthritis. References [1] Blaho VA and Hla T. An update on the biology of sphingosine 1-phosphate receptors. J Lipid Res55:1596–608. [2] Zhao C, Fernandes MJ, and Bourgoin SG, et al. Specific and overlapping sphingosine-1-phosphate receptor functions in human synoviocytes: impact of TNF-alpha. J Lipid Res49: 2323–37. Disclosure of Interest None declared

Allograft inflammatory factor-1 (AIF-1) is a protein expressed by macrophages infiltrating the area around the coronary arteries in a rat ectopic cardiac allograft model. We previously reported that AIF-1 is associated with the pathogenesis of rheumatoid arthritis and skin fibrosis in sclerodermatous graft-versus-host disease mice. Here, we used an animal model of bleomycin-induced lung fibrosis to analyze the expression of AIF-1 and examine its function in lung fibrosis. The results showed that AIF-1 was expressed on lung tissues, specifically macrophages, from mice with bleomycin-induced lung fibrosis. Recombinant AIF-1 increased the production of TGF-β which plays crucial roles in the mechanism of fibrosis by mouse macrophage cell line RAW264.7. Recombinant AIF-1 also increased both the proliferation and migration of lung fibroblasts compared with control group. These results suggest that AIF-1 plays an important role in the mechanism underlying lung fibrosis, and may provide an attractive new therapeutic target.

Objective Synovial fluid pH is decreased in patients with rheumatoid arthritis (RA); however, the underlying mechanisms are unclear. We undertook this study to examine the mechanism by which synovial fluid pH is regulated and to explore the possibility of a therapeutic strategy by manipulating this mechanism. Methods We determined the pH and lactate concentration in synovial fluid from 16 RA patients. Cultured synovial fibroblasts (SFs) from the inflamed joints of 9 RA patients (RASFs) were examined for the expression of ion transporters that regulate intracellular and extracellular pH. The ion transporter up-regulated in RASF lines was then suppressed in RASFs by small interfering RNA (siRNA), and the effect of transfection on viability and proliferation was investigated. Finally, we examined the therapeutic effect of electrotransfer of monocarboxylate transporter 4 (MCT4)–specific siRNA into the articular synovium of mice with collagen-induced arthritis (CIA). Results Synovial fluid pH correlated inversely with both the Disease Activity Score in 28 joints using the C-reactive protein level and the synovial fluid lactate levels. RASFs exhibited up-regulated transcription of MCT4 messenger RNA. MCT4 exported intracellular lactate into the extracellular space. RASFs had significantly higher MCT4 protein levels than did SFs from patients with osteoarthritis. Knockdown of MCT4 induced intrinsic apoptosis of RASFs, thereby inhibiting their proliferation. Moreover, electrotransfer of MCT4-specific siRNA into the articular synovium of mice with CIA significantly reduced the severity of arthritis. Conclusion RA activity correlated with decreased synovial fluid pH. This may be due to increased MCT4 expression in RASFs. Silencing MCT4 induced apoptosis in RASFs and reduced the severity of CIA, suggesting that MCT4 is a potential therapeutic target for inflammatory arthritis.

Background Patients with glucocorticoid-induced osteoporosis (GIOP) are at very high risk of fracture, and patients with severe GIOP often experience fractures during treatment with bisphosphonates. Teriparatide (TPTD) is the only currently available anabolic agent expected to be effective for GIOP. Once-weekly TPTD decreased bone resorption marker with primary osteoporosis different from daily TPTD, but it has not yet been tested with GIOP. Objectives To evaluate the efficacy of once-weekly TPTD for patients with GIOP and inadequate response to bisphosphonates. Methods Patients with GIOP and collagen diseases treated with prednisolone for at least 6 months with inadequate responses to bisphosphonates were administered once-weekly TPTD. Bone density of the lumbar spine and femoral neck, measured as percent young adult mean (YAM); serum concentrations of cross-linked N-terminal telopeptides of type I collagen (NTx), bone alkaline phosphatase (BAP), and calcium; and FRAX were measured at baseline and 6, 12 and 18 months after starting TPTD. Results Of the 12 GIOP patients with collagen diseases enrolled, nine (seven females, two males; mean age 57.4 ± 11.1 years) completed treatment, including six with systemic lupus erythematosus, two with rheumatoid arthritis, and one with adult onset still disease. Only one new fracture event, a lumbar compression fracture, occurred during the study period, although seven patients experienced eight fracture events within 18 months before starting TPTD (p = 0.04). Lumbar spine YAM significantly improved at 18 months (p = 0.04), whereas femoral neck YAM did not (p = 0.477). Serum NTx, BAP, Ca, and FRAX were not significantly affected by TPTD treatment. Conclusions Once-weekly TPTD reduces fracture events and increases bone density of the lumbar spine of GIOP patients with inadequate response to bisphosphonates.

Background There is no report investigating predictive factors for MRI erosion change in treatment of rheumatoid arthritis (RA) with abatacept (ABT), because very little data are available on the efficacy of ABT assessed by MRI. Objectives The aim of this study is to examine the efficacy of ABT in patients with RA assessed by MRI and identify factors associated with change of bone erosion score. Methods Thirty-five RA patients were included in this prospective study. MRI of bilateral hands and conventional radiographs (CR) of bilateral hands and feet were performed at baseline and month 12 of treatment with intravenous ABT. MRI images were scored for synovitis, osteitis and bone erosion according to the Rheumatoid Arthritis MRI Scoring System. CR images were assessed according to the van der Heijde modified total Sharp score. Results Thirty-one patients completed this study. Mean Simplified Disease Activity Index (SDAI) and Health Assessment Questionnaire Disability Index (HAQ-DI) at baseline were 23.4±12.0 and 1.1±0.6 respectively. At the group level, mean MRI synovitis and osteitis scores showed statistically significant reductions through 12 months of treatment with ABT (from 17.1±7.0 to 11.4±6.2 (p Conclusions This study demonstrated ABT has a strong inhibitory effect on joint damage reducing synovitis and osteitis and not progressing bone erosion in RA patients. Over 10% patients showed repair of bone erosion assessed by MRI, which resulted in functional remission. SDAI response rate at month 1 could be a predictive factor for repair of bone erosion. Disclosure of Interest Y. Kukida Grant/research support from: Bristol-Myers Squibb, A. Kasahara: None declared, T. Seno: None declared, T. Inoue: None declared, N. Kamio: None declared, R. Sagawa: None declared, T. Kida: None declared, A. Nakabayashi: None declared, H. Nagahara: None declared, A. Yamamoto: None declared, S. Morita: None declared, H. Ito: None declared, M. Kohno: None declared, Y. Kawahito Grant/research support from: Bristol-Myers Squibb

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