Yuji Kukida

Monocarboxylate Transporter 4, Associated With the Acidification of Synovial Fluid, Is a Novel Therapeutic Target for Inflammatory Arthritis

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.

Allograft inflammatory factor-1 in the pathogenesis of bleomycin-induced acute lung injury

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.

Roles of high-mobility group box 1 and thrombin in murine pulmonary fibrosis and the therapeutic potential of thrombomodulin

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.

AB0256 Very Early Response To Abatacept Could Be A Predictive Factor for Repair of Bone Erosion in Patients with Rheumatoid Arthritis Assessed by MRI

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<0.0001) and from 5.1±8.2 to 1.9±2.5 (p=0.003), respectively). On the other hand, mean MRI erosion scores showed no change throughout the study period (from 28.2±38.6 to 28.7±39.2). Mean CR erosion scores also showed no change (from 17.3±42.5 to 17.6±42.6). However, at the patient level, reductions of MRI erosion score were observed in 13% of patients, whereas no patient showed reduction of CR erosion score. The functional remission rates, which is defined as HAQ-DI≤0.5, of patients with regressive, unchanged and progressive MRI erosion score were 100%, 71% and 33%, respectively. On multiple regression analysis, SDAI response rate at month 1 and CRP at baseline were identified as the predicting factors for changes of erosion score (regression coefficient; -0.70 (p<0.001)/0.42 (p=0.02), respectively). Optimal cutoff value of SDAI response rate at month 1 was 0.524 (p=0.019). It is interesting that patients with regression of MRI erosion had high titer of rheumatoid factor (median: 591IU/ml) and anti-CCP antibody (median: 254U/ml) at baseline. 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

Role of allograft inflammatory factor-1 in bleomycin-induced lung fibrosis

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.

Efficacy of abatacept in patients with rheumatoid arthritis, as assessed by magnetic resonance imaging of bilateral hands

To examine the efficacy of abatacept in patients with rheumatoid arthritis (RA) using magnetic resonance imaging (MRI) of bilateral hands.

AB0247 A New Disease Activity Biomarker Alternative To CRP under Tocilizumab Therapy for Rheumatoid Arthritis via Peptidomic Analysis

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 patients 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

AB0910 Once-Weekly Teriparatide is Effective for Glucocorticoid-Induced Osteoporosis Patients with Collagen Diseases

Background Glucocorticoid-induced osteoporosis (GIOP) patients are at very high risk of fractures. Unfortunately, only a small minority of GC users receive effective preventive, diagnostic, and therapeutic interventions. Intermittent administration of teriparatide (TPTD) is the only currently available anabolic agent to stimulate osteoblast activity. Daily TPTD was more efficacious than alendronate in increasing lumbar spine BMD in men and pre- and postmenopausal women. Recently, once-weekly TPTD was provided, which had both rapid and powerful anti-fracture efficacy. It has unique feature; serum NTx is decreasing after administration for patients with primary osteoporosis contrast to daily TPTD. Once-weekly TPTD is expected to be effective for GIOP, but it has not been reported. Objectives To examine the efficacy of once-weekly TPTD for patients with GIOP and collagen diseases. Methods We registered GIOP patients with collagen diseases treated with once-weekly TPTD at our department. They were treated with PSL for at least 6 months and had inadequate response for bisphosphonates. We measured YAM (Young Adult Mean), serum NTx (cross-linked N-terminal telopeptides of type I collagen), BAP (Bone alkaline phosphatase), Ca, FRAX at baseline, 6 months, 12months and 18 months after starting once-weekly TPTD. We calculated the change rate of those values to baseline. The continuous variables were compared using ANOVA and Wilcoxon matched-pairs single rank test. Results 12 GIOP patients with collagen diseases were registered into this study, and 9 patients completed (6 systemic lupus erythematosus, 2 rheumatoid arthritis, 1 adult onset still disease; 7 female, 2 male; Age 57.4±11.1). Only one new fracture event, that was lumbar compression fracture, occurred during the study period despite the patient had many fractures before this treatment. The change rate of lumbar spine YAM was significantly improved at 18 months (p=0.041, ANOVA; p=0.019, Wilcoxon matched-pairs single rank test). Femoral neck YAM (p=0.477, ANOVA), serum NTx (p=0.555), BAP (p=0.936), Ca and FRAX were not significantly changed. Interestingly, the change rate of serum NTx was increased during this GIOP study despite the rate was decreased in primary osteoporosis previously reported. Conclusions Our study shows that once-weekly teriparatide is effective for GIOP patients with collagen diseases, and its dosing period needs 18 months. Mean % change of serum NTx was increased in GIOP patients in spite of primary osteoporosis. References Bone 2006;39:244–52. J Bone Miner Res 2010;25:472–81. Osteoporos Int 2009;20:2095–104. J Bone Miner Metab 2004;22:569–76. Disclosure of Interest None declared

SAT0562 Directly Reprogrammed Osteoblasts Genetically Engineered to Produce Interleukin-10 Significantly Suppress Osteoclastgenesis

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 lineages1,2. 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

AB0501 Retrospective Study of Multitarget Therapy with Combination of Mizoribine and Tacrolimus for Systemic Lupus Erythematosus with or without Nephritis

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