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Objectives We aimed to identify disease-specific surface proteins on extracellular vesicles (EVs) as novel serum biomarkers of PM/DM. Methods We performed liquid chromatography–tandem mass spectrometry (LC/MS) on purified EVs from sera of 10 PM/DM patients, 23 patients with other autoimmune diseases and 10 healthy controls (HCs). We identified membrane proteins preferentially present in EVs of PM/DM patients by bioinformatics and biostatistical analyses. We developed an EV sandwich ELISA for directly detecting serum EVs expressing disease-specific membrane proteins and evaluated their clinical utility using sera from 54 PM/DM, 24 RA, 20 SLE, 13 SSc and 25 Duchenne and Becker types of muscular dystrophy (DMD/BMD) patients and 36 HCs. Results LC/MS analysis identified 1220 proteins in serum EVs. Of these, plexin D1 was enriched in those from PM/DM patients relative to HCs or patients without PM/DM. Using a specific EV sandwich ELISA, we found that levels of plexin D1+ EVs in serum were significantly greater in PM/DM patients than in HCs or RA, SLE or DMD/BMD patients. Serum levels of plexin D1+ EVs were greater in those PM/DM patients with muscle pain or weakness. Serum levels of plexin D1+ EVs were significantly correlated with levels of aldolase (rs = 0.481), white blood cells (rs = 0.381), neutrophils (rs = 0.450) and platelets (rs = 0.408) in PM/DM patients. Finally, serum levels of plexin D1+ EVs decreased significantly in patients with PM/DM in clinical remission after treatment. Conclusion We identified levels of circulating plexin D1+ EVs as a novel serum biomarker for PM/DM.

Summary The gene mutation profiles of gastric neuroendocrine neoplasms are incompletely understood. The purpose of this study was to characterize the molecular pathology of poorly differentiated neuroendocrine carcinoma (NEC) and mixed neuroendocrine‒non-neuroendocrine neoplasm (MiNEN) of the stomach. Surgical cases of gastric NEC (n = 7) and MiNEN (n = 6) were examined by clinical review, immunohistochemistry, microsatellite instability (MSI) analysis and whole-exome sequencing. NEC cases consisted of small- (n = 2) and large-cell types (n = 4). All cases of MiNEN were histologically composed of large-cell type NEC and tubular adenocarcinoma. Whole-exome sequencing analysis detected recurrent mutations in TP53 in 8 cases (62%), and they were more frequently observed in MiNEN than in NEC (100% vs. 29%). Frameshift mutations of APC were observed in two cases of MiNEN. One case of large-cell type NEC had a frameshift mutation with loss of heterozygosity in RB1. The other mutated genes (e.g., ARID1 and KRAS) were detected in a single case each. A high level of MSI was confirmed in one case of MiNEN, which harbored mutations in two well-differentiated neuroendocrine tumor (NET)-related genes (MEN1 and ATRX1). In cases of MiNEN, two histological components shared mutations in TP53, APC and ZNF521, whereas alterations in CTNNB1, KMT2C, PTEN and SPEN were observed in neuroendocrine components only. In conclusion, TP53 is a single, frequently mutated gene in gastric NEC and MiNEN, and alterations in other genes are less common, resembling the mutation profiles of gastric adenocarcinomas. Gene mutations frequently observed in well-differentiated NET were uncommon but not entirely exclusive.

Neonatal jaundice has been suggested as a perinatal risk factor for autism spectrum disorder (ASD). We examined UGT1A1 polymorphisms to assess the potential of neonatal jaundice as a risk factor for ASD in children by using DNA extracted from preserved umbilical cord. In total, 79 children with ASD were genotyped for UGT1A1*28 (c.-41-40dup), UGT1A1*6 (c.211 G > A), and UGT1A1*27 (c.686 C > A). The allele frequency of UGT1A1*6 (OR = 1.34, p = 0.26) and UGT1A1*28 (OR = 0.80, p = 0.54) and the prevalence of UGT1A1*28/*6 diplotypes did not differ significantly from those in the control population. No UGT1A1*27 allele was detected in the subjects. ASD symptom assessment scores were not associated with UGT1A1*28/*6/*27 genotypes or UGT1A1*28/*6 diplotypes. These results suggest that neonatal jaundice is not significantly associated with ASD.

The objective of the study was to compare the efficacy and safety of biological disease-modifying antirheumatic drugs (bDMARDs) between elderly-onset rheumatoid arthritis (EORA) and young-onset rheumatoid arthritis (YORA) patients. Patients with rheumatoid arthritis (RA) aged ≧18 years enrolled in a Japanese multicenter observational registry between 2009 and 2018 who had moderate or high disease activity when initiating bDMARDs were included. EORA was defined as RA with onset at 60 or over. After propensity score weighting for differences in confounding factors, generalized estimating equations were used to assess the relationship between the age of RA onset and bDMARD clinical effectiveness at 48 weeks after starting a bDMARD. Among a total of 7183 patients in the registry, 2815 (39.2%) were identified as EORA. The proportion of patients on bDMARDs was lower in the EORA as compared to the YORA (18.3% vs 28.0%, p

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disease characterized by benign skin hamartomas, pulmonary cysts leading to spontaneous pneumothorax, and an increased risk of renal cancer. BHD syndrome is caused by germline mutations in the folliculin (FLCN) gene, a putative tumor suppressor, which result in loss of function of the folliculin protein and may cause cancer predisposition. In a 45-year-old woman with anemia, lymphadenopathy, and a history of recurrent spontaneous pneumothorax,F-18-FDG PET/CT detected diffuse and slight (18)F-FDG accumulation in the bone marrow, enlarged spleen, and systemic multiple enlarged lymph nodes. Genetic examination identified a germline nonsense mutation [c.998C > G (p.Ser333*)] on exon 9 of FLCN. Pathological examination of the lymph node revealed a diffuse neoplastic proliferation of plasmacytoid lymphocytes. The neoplastic lymphoid cells were positive for CD20, CD138, and light chain kappa as per immunohistochemistry and mRNA in situ hybridization, and a MYD88g ene mutation [c.755T > C (p.L252P)] was identified. Accordingly, she was diagnosed with lymphoplasmacytic lymphoma concomitant with BHD syndrome. To the best of our knowledge, this is the first report describing the development of hematological malignancy in a patient with BHD syndrome. The FLCN mutation might contribute lymphomagenesis as an additional mutation cooperating with the MYD88 mutation.

Background: We compared the prophylactic effect of trimethoprim-sulfamethoxazole (TMP-SMX) with atovaquone for pneumocystis pneumonia (PCP) in patients with connective tissue diseases (CTDs) receiving high-dose glucocorticoids.Methods: Patients with CTDs aged ≥ 18 years who were treated with a prolonged course (≥ 4 weeks) of glucocorticoids (≥ 20 mg/day prednisone) in a Japanese tertiary center between 2013 and 2017 were included. The patients were categorized into two groups: TMP-SMX and atovaquone group. Adjusted cumulative incidence of PCP was compared between the two groups after propensity score weighting for differences in confounding factors. Results: A total of 480 patients with a prolonged high-dose glucocorticoid treatment were identified. Out of 383 patients with TMP-SMX prophylaxis, 102 (26.8%) patients experienced adverse events leading to discontinuation within 4 weeks of initiation while no patient in the atovaquone discontinued the therapy. Two hundred eighty-one patients received TMP-SMX while 107 received atovaquone for PCP prophylaxis. During a total of 397.0 person-years, 7 PCP cases (2 in the TMP-SMX, 5 in the atovaquone) occurred with a mortality rate of 54.5%. After adjusting for differences in baseline characteristics, the adjusted cumulative incidence of PCP was similar between the two group (HR= 0.97, 95% CI = 0.19 to 5.09, p = 0.97).Conclusion: Prophylactic effects for PCP in CTDs patients receiving prolonged high-dose glucocorticoids were similar between TMP-SMX and atovaquone. Atovaquone was well-tolerated with no side effects.

Chromosomal rearrangements involving the MECOM locus, which consists of two genes MDS1 and EVI1 located at 3q26.2, are found in different types of myeloid malignancies [1,2]. Among these, the t(3;1...

Background This multi-center, retrospective study aimed to clarify retention rates and reasons for discontinuation of either tumor necrosis factor inhibitors (TNFi) or interleukin-6 inhibitors (IL-6i) in patients with elderly-onset rheumatoid arthritis (EORA). Methods Patients with rheumatoid arthritis (RA) enrolled in a Japanese multicenter observational registry between 2011 and 2020 were included. EORA was defined as RA with onset at 60 or over. To adjust confounding by indication for treatment with TNFi or IL-6i, a propensity score based on multiple baseline characteristics variables was used to compare the drug retention and causes for discontinuation between TNFi and IL-6i. Adjusted cumulative incidence of drug discontinuation for each reason was compared between the two groups using the Fine-Gray model. Results Among a total of 9,550 patients in the registry, 674 TNFi and 297 IL-6i initiators with EORA were identified. Age, the proportion of females, disease duration, and baseline disease activity at the time of TNFi or IL-6i initiation were similar between the two groups. After adjusting for differences in baseline characteristics between the two groups, overall drug discontinuation was significantly lower in the IL-6i as compared to the TNFi (HR = 0.71, 95%CI = 0.59–0.86, p p p = 0.28) or achievement of clinical remission (HR = 1.09, 95%CI = 0.62–1.91, p = 0.76) were similar between the two groups. Conclusions In EORA patients initiating a TNFi or IL-6i, significantly higher drug retention was observed with IL-6i. Discontinuation due to lack of effectiveness was significantly less frequent in IL-6i while discontinuations due to adverse event or achievement of clinical remission were similar between the two groups.

Recent studies have revealed a relationship between cellular metabolism and cell function in immune cells. Cellular metabolism not only provides supplemental ATP, but also supports dynamic changes in cell proliferation and differentiation. For example, T cells exhibit subset-specific metabolic profiles, and require certain types of metabolism for their functions. Determining the metabolic profiles that support inflammatory immune responses may lead to novel treatment strategies for chronic inflammatory diseases such as rheumatoid arthritis (RA). However, the mechanisms by which metabolism modulates cell function have been unclear. Recent studies have begun to unveil unexpected non-metabolic functions for metabolic enzymes in the context of inflammation, including roles in signaling and gene regulation. Here we describe recent findings related to immunometabolism, the metabolome of RA patients, and the metabolically independent functions of glycolytic enzymes. We discuss how metabolic processes impact immune cells, especially T cells and fibroblast like synoviocytes, which are considered the orchestrators of autoimmune arthritis.

Resolvins, are specialized pro-resolving mediators (SPMs) derived from n-3 polyunsaturated fatty acids. They contribute actively to the resolution of inflammation, but little is known concerning their role in chronic inflammation, such as in rheumatoid arthritis (RA). Here, we performed lipid mediator (LM) profiling in tissues from the paws of SKG arthritic mice using lipid chromatography (LC)/mass spectrometry (MS)/MS-based LM metabololipidomics. We found elevated levels of SPMs including resolvin D5 (RvD5) in these tissues. Moreover, RvD5 levels were significantly correlated with arthritis disease activity. From experiments to assess the role of RvD5 in the pathology of RA, we concluded that RvD5 suppressed Th17 cell differentiation and facilitated regulatory T cell differentiation, as well as inhibiting CD4+ T cell proliferation. Furthermore, RvD5 attenuated osteoclast differentiation and interfered with osteoclastogenesis. Targeting the resolution of inflammation could be promising as a novel treatment for RA.

Background One of the main complications in patients with muscular dystrophies is cardiac dysfunction. The literature on cardiac involvement in patients with Fukuyama congenital muscular dystrophy (FCMD) is limited. Aim To compare cardiac involvement between patients with FCMD and Duchenne muscular dystrophy (DMD). Methods We compared cardiac involvement between 30 patients with FCMD and 181 patients with DMD using echocardiography and serum biomarkers. All patients were receiving regular checkups at Kobe University Hospital. We used single regression analysis to compare echocardiographic parameters, age, and serum biomarkers. Results Almost all clinical and echocardiographic parameters were lower in patients with FCMD than DMD. The brain natriuretic peptide concentration in patients with FCMD showed no correlation with age or left ventricular ejection fraction ( r = 0.231, p = 0.22 and r = 0.058, p = 0.76, respectively). A log-rank test revealed that the risk of left ventricular systolic dysfunction was lower in patients with FCMD than DMD ( p = 0.046, hazard ratio = 0.348). Conclusion The clinical progression of cardiac dysfunction is significantly milder in patients with FCMD than DMD, while skeletal muscle involvement is significantly worse in patients with FCMD. These data suggest that the pathophysiological findings of FCMD can be explained by less severe cardiac dysfunction in FCMD than DMD.

A 78-year-old female with massive pericardial effusion fulfilled diagnostic criteria for immunoglobulin G4 (IgG4)-related disease. Although her adenosine deaminase (ADA) level in the pericardial effusion was high, all the tests for tuberculosis infection were negative. Immunostaining of the pericardium biopsy specimen revealed remarkably increased IgG4-positive cells. This is the first report describing IgG4-related pericarditis with elevated ADA level. We also demonstrate the elevated interleukin-10 (IL-10) level in pericardial fluid and IL-10-producing T-cells in the pericardium.

Background Anti-TIF1gamma antibody positive dermatomyositis(TIF1γ-DM) has greater risk of complication with cancer. Objectives We aimed to analyze the clinical features of TIF1γ-DM and find the difference between cancer-associated TIF1γ-DM (CA-TIF1γ-DM) and non-cancer-associated TIF1γ-DM (nCA-TIF1γ-DM). Methods We investigated the clinical data of idiopathic inflammatory myositis (IIM) patients received remission induction therapy in out hospital between January 2006 and December 2018. Results We treated 148 patients with IIM in this period, 9 cases of them were TIF1γ-DM. Patients with CA-TIF1γ-DM and nCA-TIF1γ-DM were 4 and 5 cases, respectively. Average age at onset of 9 patients with TIF1γ-DM was 63 years old. CA-TIF1γ-DM was significantly older than nCA-TIF1γ-DM (average:78.0 vs 57.4). All of 9 TIF1γ-DM had distinctive skin rash and muscle weakness. Seven cases of them suffered from dysphagia. Interstitial lung disease was not seen in all patients with TIF1γ-DM. Serum creatine kinase (CK) levels of CA-TIF1γ-DM was significantly higher than those of nCA-TIF1γ-DM (average: 3405.5IU/L vs 719.8IU/L) (Fig 1.). Among the TIF1γ-DM patients, we administered glucocorticoid (GCs) for 8 cases, immunosuppresants for 4 cases, and intravenous immunoglobulins (IVIG) for 7 cases. All 4 cases of CA-TIF1γ-DM were treated with GC and IVIG. All TIF1γ-DM patients, especially 4 CA-TIF1γ-DM patients, seemed to have good response to treatment in serum CK levels. Although serum CK levels of these 4 cases were normalized within a month (Fig1.), their muscle weakness, skin rash and dysphagia which was associated with quality of life and the prognosis were poorly improved. Conclusion In our investigation, the age of onset and serum CK levels were significantly different between CA-TIF1γ-DM and nCA-TIF1γ-DM. We report our investigation of the phenotype and treatment of anti TIF1gamma antibody positive dermatomyositis. TIF1γ-DM with high serum CK levels, we should perform appropriate tests to check for cancer throughout our treatment, and the improvement of serum CK levels is not equal to the success of our treatment. Disclosure of Interests None declared

Rationale: Immunoglobulin G4 related disease (IgG4-RD) rarely coexists with other autoimmune diseases, though we had a patient whose primary clinical problem was shifted from IgG4-RD to systemic lupus erythematosus (SLE) after gastrectomy. The present paper aimed to report pathological findings and clinical course of the patient. Patient concerns: The patient was a male aged 74 years old with gastric cancer characterized by the following symptoms: Raynaud phenomenon, polyarthralgia, and swollen parotid glands on both sides. Before gastrectomy, laboratory examination results showed renal dysfunction, hypocomplementemia, antinuclear antibodies (ANAs) positivity, and elevated serum IgG and IgG4 levels. Diagnosis: Based on postoperative renal biopsy showing severe plasma cell infiltration with tubulointerstitial fibrosclerosis, the patient was diagnosed with IgG4-RD. Despite significant improvement in renal function and reduction in parotid gland swelling during the postoperative follow-up period, after 7 months of the gastrectomy, anti-DNA antibody levels were increased and serositis was detected, which indicated the onset of SLE. IgG4-type ANA were also detected in the sera of the patient. Interventions: Treatment by oral prednisolone at 30 mg/day was initiated. Outcomes: Pericardial fluid, pleural effusions, and thickening of the gallbladder wall improved after 3 months of treatment according to computed tomography. Lessons: This study presented a rare case of comorbidity, wherein the patient's primary problem progressed from IgG4-type ANA positive IgG4-RD to SLE after excision of gastric cancer. Abbreviations: ANAs = antinuclear antibodies, Cr = creatinine, CT = computed tomography, IgG4-RD = immunoglobulin G4-related disease, IgG4-RKD = immunoglobulin G4-related kidney disease, PSL = prednisolone, SLE = systemic lupus erythematosus.

Background Systemic sclerosis (SSc) is a chronic autoimmune-mediated connective tissue disorder. Although the etiology of the disease remains undetermined, SSc is characterized by fibrosis and proliferative vascular lesions of the skin and internal organs. SSc involves the gastrointestinal tract in more than 90 % of patients. Soluble guanylate cyclase (sGC) stimulator is used to treat pulmonary artery hypertension (PAH) and has been shown to inhibit experimental skin fibrosis. Methods Female C57BL/6J mice were treated with BLM or normal saline by subcutaneous implantation of osmotic minipump. These mice were sacrificed on day 28 or day 42. Gastrointestinal pathologies were examined by Masson Trichrome staining. The expression of fibrosis-related genes in gastrointestinal tract was analyzed by real-time PCR, and the levels of collagen in the tissue were measured by Sircol collagen assay. To evaluate peristaltic movement, the small intestinal transport (ITR%) was calculated as [dyeing distance × (duodenum − appendix)] − 1 × 100 (%). We treated BLM-treated mice with sGC stimulator or DMSO orally and analyzed them on day 42. Results Histological examination revealed that fibrosis from lamina propria to muscularis mucosa in the esophagus was significantly increased in BLM-treated mice, suggesting that BLM induces esophageal hyperproliferative and prefibrotic response in C57BL/6J mice. In addition, the gene expression levels of Col3a1, CCN2, MMP-2, MMP-9, TIMP-1, and TIMP-2 in the esophagus were significantly increased in BLM-treated mice. More severe hyperproliferative and prefibrotic response was observed in the mice sacrificed on day 42 than the mice sacrificed on day 28. The ITR% was found to be significantly lower in BLM-treated mice, suggesting that gastrointestinal peristaltic movement was reduced in BLM-treated mice. Furthermore, we demonstrated that sGC stimulator treatment significantly reduced hyperproliferative and prefibrotic response of esophagus and intestine in BLM-treated mice, by histological examination and Sircol collagen assay. Conclusions These findings suggest that BLM induces gastrointestinal hyperproliferative and prefibrotic response in C57BL/6J mice, and treatment with sGC stimulator improves the BLM-induced gastrointestinal lesion.

Previous studies from various countries have reported anti-dense fine speckled pattern (DFS)70 antibody prevalence but few studies have been from Asia. We investigated the prevalence of anti-DFS70 autoantibodies in a Japanese cohort of healthy individuals (HI) and patients with antinuclear antibody-associated autoimmune rheumatic diseases (AARD). Enzyme-linked immunosorbent assay and indirect immunofluorescence were performed using samples from 250 HI and 276 AARD patients. The overall anti-DFS70 antibody prevalence in HI was 16.4%, with 12.8% for males and 20.0% for females (sex difference; P = .12). In AARD patients, the anti-DFS70 antibody prevalence in systemic lupus erythematosus, mixed connective tissue disease, systemic sclerosis, dermatomyositis and polymyositis (DM/PM), Sjögren syndrome, and rheumatoid arthritis (RA) was 22.1%, 14.3%, 14.3%, 3.0%, 21.3%, and 18.1%, respectively (no significant difference between AARD patients except DM/PM and HI). The prevalence of isolated anti-DFS70 antibody in HI and all AARD patients excluding RA was 14.8% (37/250) and 4.4% (9/204), respectively (P

Background:Systemic lupus erythematosus (SLE) patients present with variable clinical features ranging from mild joint and skin involvement to life-threatening organ involvement such as nephritis, neuropsychiatric involvement, diffuse alveolar hemorrhage, and hemophagocytic lymphohistiocytosis (HLH). HLH is rare, but fatal complication of SLE. Recently, European League Against Rheumatism, the American College of Rheumatology, and the Pediatric Rheumatology International Trials Organization was to develop a set of classification criteria for MAS complicating systemic juvenile idiopathic arthritis (PRINTO criteria) [1]. Sung Soo Ahn and his colleagues reported PRINTO criteria predicted mortality of adult SLE patient, but they followed only one year [2].Objectives:To reveal association PRINTO criteria with long term mortalities in SLE patient in our Hospital.Methods:We performed a retrospective analysis of SLE patients who received moderate dose glucocorticoid therapy (>0.4mg/kg/d) in our hospital between April 2008 and April 2019. Patients were evaluated for HLH using the 2016 PRINTO classification criteria for MAS. Clinical features and laboratory findings were compared and overall survival rate was analyzed.Results:Among 164 episode (144 patients) with SLE, 31 episode (31 patients) 5.2% were considered to have MAS on admission.The overall survival rate was significantly lower in patients with MAS than without MAS (86.2% vs. 95.3%, p = 0.048). Interestingly, SLEDAI had no association with mortality, relapse rate, and MAS complication. SLEDAI more focused on renal and neuropsychiatric symptoms than hematologic features. So SLEDAI might not be associated with MAS secondary to SLE. Furthermore, we observed no death patient with MAS after one year, and only 1 case relapse in MAS patient. So MAS might have fatal but less relapsing property compared with other lupus cases.Conclusion:PRINTO criteria may be useful to differentiated fatal MAS patients from others. Further investigations are required to confirm our findings.Limitation The main limitations of our study include its retrospective design, single center site, and that the number of admitted patients with SLE was small.Limitation:The main limitations of our study include its retrospective design, single center site, and that the number of admitted patients with SLE was small.Limitation:Limitation The main limitations of our study include its retrospective design, single center site, and that the number of admitted patients with SLE was small.References:[1]Wulffraat N, Schneider R, Filipovic L, et al. 2016 Classification Criteria for Macrophage Activation Syndrome Complicating Systemic Juvenile Idiopathic Arthritis. Ann Rheum Dis 2016;75:481–9.[2]Ahn SS, Yoo BW, Jung SM, et al. In-hospital mortality in febrile lupus patients based on 2016 EULAR/ACR/PRINTO classification criteria for macrophage activation syndrome. Semin Arthritis Rheum 2017;47:216–21.Disclosure of Interests:None declared

Background: Vagus nerve (VN) stimulation has shown the potential to improve the disease development in animal models of arthritis and in patients with RA. However, the VN can affect respiratory, cardiovascular, endocrine and gastro-intestinal physiology. The splenic nerve (SpN) has been confirmed to be the principal effector nerve for the VN-mediated immune control. Previous studies have shown that stimulating the splenic nerve resulted in an increase of norephiniphrine in the spleen, as well as a significant reduction in LPS-induced TNF (1). Objectives: To test the therapeutic efficiency of splenic nerve stimulation (SNS) in collagen-induced arthritis (CIA) in mice alone or in combination with anti-TNF treatment. Methods: CIA was induced in DBA1/J mice by immunization with bovine type II collagen at days 0 and 21. At day 11, mice were implanted with micro-cuff electrode (CorTec) onto the SpN or VN. From day 16 to day 45, SNS were applied as rectangular charged-balanced biphasic pulses with 650 μA pulse amplitude, 200 µs pulse width at 10 Hz frequency for 2 min 1 or 6 times a day using a Plexon stimulator. In order to investigatedthe mechanism of action in more detail, propranolol, a beta-adrenergic receptor (β-AR) antagonist, was added to the drinking water of mice receiving SNS. In addition, a control group was treated with anti-TNF (etanercept, 3 times/week; 10mg/kg i.p.). In curative settings, SNS and/or anti-TNF treatment was applied starting when mice scored positive for 3 consecutive days. Clinical arthritis was determined by visual examination of swelling and redness of the paws and measurement of paw thickness. Sham mice were undergoing the same procedure but did not receive stimulation. Results: In CIA in mice all sham animals developed arthritis, compared to only 14% following six times per day SNS (p Conclusion: These studies demonstrate that SNS suppresses pro-inflammatory cytokine production, and reduces clinical symptoms in mice with CIA which is at least partially mediated by the β-AR. The additive effect of anti-TNF in reducing the clinical scores demonstrates that that mechanism of action of SNS is not primarilys mediated by reducing TNF levels. Moreover, anti-TNF potentiating the inhibitory effect of SNS is supporting a combined use of these treatments, or even a combination of SNS with other biologicals, to treat RA, potentially getting more patients closer to remission. In conclusion, the data is providing compelling scientific rationale and pre-clinical evidence that splenic neuromodulation might be a new treatment modality for RA. References: [1] Guyot M et al, Brain Behav Immun. 2019;80:238. Disclosure of Interests: Thomas Simon Grant/research support from: research grant from Galvani Bioelectronics, Clara Panzolini Grant/research support from: Working on research grant Galvani bioelectronics, Julien Lavergne Grant/research support from: working on research grant Galvani Bioelectrocnics, Nicolas Hypolite Grant/research support from: Working on research grant Galvani Bioelectronics, Nicolas Glaichenhaus: None declared, Margriet Vervoordeldonk Employee of: I am an employee of Galvani Bioelectronics, Philippe Blancou Grant/research support from: Received research grant from Galvani Bioelectronics

Background:Resolution phase of acute inflammation has been recognized not passive but active process during the last 2 decades. This active process is highly regulated by novel families of potent bioactive lipid mediators, which are coined as specialized proresolving mediators (SPMs) including resolvins. Little has been known, however, about how resolvins are involved in chronic inflammation, such as rheumatoid arthritis (RA).Objectives:To investigate whether lipid mediators (LM) are involved in the pathogenesis of RA.Methods:We investigated lipid mediator profiling in the paws of SKG arthritis mice by using lipid chromatography (LC) /mass spectrometry (MS) /MS-based LM metabololipidomics. CD4+T cells from spleens of SKG mice were cultured on anti-CD3/ CD28Abs precoated plate with IL-6/TGF-β, anti-IFNγ/IL-4 and analyzed by flow cytometry. CD4+T cells were labeled with CFSE, and cell proliferation was analyzed by flow cytometry. Mouse bone marrow cells were cultured with M-CSF and RANKL, and TRAP-positive multinucleated cells were defined as osteoclasts. Osteoclast differentiation markers were analyzed by qRT-PCR. RvD5 or normal saline was administered daily into the peritoneal cavity of arthritic SKG mice.Results:RvE3, RvD1, RvD3, RvD5 and Maresin2 were significantly elevated on the paws of arthritic SKG mice. Among the elevated SPMs, only RvD5 levels on arthritic paws were significantly correlated with arthritis disease activity (Figure 1). We demonstrated that RvD5 suppressed Th17 cell differentiation, and facilitated Treg cell differentiationin vitro. In addition, RvD5 inhibited CD4+T cell proliferation. Furthermore, RvD5 attenuated osteoclast differentiation (Figure 2) and interfered osteoclastogenesis at the molecular level. In thein vivoexperiment, incidence of arthritis tended to be lower in RvD5-treated mice than that in control group, although there was no significant difference.Conclusion:RvD5 is increased in the paws of arthritic mice, and that RvD5 suppresses Th17 cell differentiation and CD4+T cell proliferation, facilitates Treg cell differentiation, and suppresses osteoclastogenesis.References:[1]Serhan, C. N. 2014. Pro-resolving lipid mediators are leads for resolution physiology. Nature 510: 92-101.[2]Buckley, C. D., Gilroy, D. W., Serhan, C. N. 2014. Proresolving lipid mediators and mechanisms in the resolution of acute inflammation. Immunity 40: 315-27.[3]Colas, R. A., Shinohara, M., Dalli, J., Chiang, N., Serhan, C. N. 2014. Identification and signature profiles for pro-resolving and inflammatory lipid mediators in human tissue. Am J Physiol Cell Physiol 307: C39-54.Acknowledgments:The authors thank Shino Natsui (Department Rheumatology and Clinical Immunology, Kobe University Graduate School of Medicine) for providing technical assistance.Disclosure of Interests:None declared

Background:Systemic scleroderma (SSc) is a chronic autoimmune-mediated connective tissue disorder. Although the etiology of the disease remains undermined, SSc is characterized by fibrosis and proliferative vascular lesions of the skin and internal organs. SSc involves the gastrointestinal tract in more than 90% of patients1. Soluble guanylate cyclase (sGC) is used to treat pulmonary artery hypertension (PAH), and has been shown to inhibit experimental skin fibrosis2.Objectives:The aim of this study is to investigate whether bleomycin (BLM)-treated mice show gastrointestinal fibrosis, and find a therapeutic strategy to the lesion.Methods:Female C57BL/6J mice were treated with BLM or normal saline by subcutaneous implantation of osmotic minipump. These mice were sacrificed on day 28 or day 42. Gastrointestinal pathologies were examined by Masson Trichrome staining. The expression of fibrosis-related genes in gastrointestinal tract were analyzed by real-time PCR, and the levels of collagen in the tissue was measured by Sircol collagen assay. To evaluate peristaltic movement, the small intestinal transport (ITR%) was calculated as [Dyeing distance×(Duodenum- Appendix)] -1 ×100 (%). We treated BLM-treated mice with soluble guanylate cyclase (sGC) or DMSO orally and analyzed them on day 42.Results:Histological examination revealed that fibrosis from lamina propria to muscularis mucosa in the esophagus was significantly increased in BLM-treated mice, suggesting that BLM induces esophageal fibrosis in C57BL/6J mice. In addition, the levels of Col3a1 and CTGF were significantly increased in BLM-treated mice. More severe fibrosis was observed in the mice sacrificed on day 42 than the mice sacrificed on day 28. The ITR% was found to be significantly lower in BLM-treated mice, suggesting that gastrointestinal peristaltic movement was reduced in BLM-treated mice. Furthermore, we demonstrated that sGC treatment significantly reduced fibrosis of esophagus and intestine in BLM-treated mice, by histological examination and Sircol collagen assay.Esophagus (Masson’s trichrome stain×100)* One way ANOVA Newman-KeulsConclusion:These findings suggest that BLM induces gastrointestinal fibrosis in C57BL/6J mice, and treatment with sGC improves the BLM-induced gastrointestinal lesion.References:[1]Anton Emmanuel. Current management of the gastrointestinal complications of systemic sclerosis. Nat Rev Gastroenterol Hepatol. 2016; 13: 461-472.[2]Clara Dees, et al. Stimulators of Soluble Guanylate Cyclase (sGC) Inhibit experimental Skin Fibrosis of Different Aetiologies. Ann Rheum Dis. 2015;74 (8): 1621-5.Acknowledgments: :None.Disclosure of Interests:None declared

Objectives Biologic treatment has recently revolutionized the management of RA. Despite this success, ∼30–40% of the patients undergoing biologic treatment respond insufficiently. The aim of this study was to identify several specific reliable metabolites for predicting the response of RA patients to TNF-α inhibitors (TNFi) and abatacept (ABT), using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). Methods We collected serum from RA patients with moderate or high disease activity prior to biologic treatment, and obtained the serum metabolomic profiles of these samples using CE-TOFMS. The patients' response was determined 12 weeks after starting biologic treatment, according to the EULAR response criteria. We compared the metabolites between the response and non-response patient groups and analysed their discriminative ability. Results Among 43 total patients, 14 of 26 patients in the TNFi group and 6 of 17 patients in the ABT group responded to the biologic treatment. Of the metabolites separated by CE-TOFMS, 196 were identified as known substances. Using an orthogonal partial least-squares discriminant analysis, we identified five metabolites as potential predictors of TNFi responders and three as predictors of ABT responders. Receiver operating characteristic analyses for multiple biomarkers revealed an area under the curve (AUC) of 0.941, with a sensitivity of 85.7% and specificity of 100% for TNFi, and an AUC of 0.985, with a sensitivity of 100% and specificity of 90.9% for ABT. Conclusion By metabolomic analysis, we identified serum biomarkers that have a high ability to predict the response of RA patients to TNFi or ABT treatment.

Background Myeloid-derived suppressor cells (MDSCs) are a highly heterogeneous population of immature myeloid cells with immunosuppressive function. Although their function in tumor-bearing conditions is well studied, less is known about the role of MDSCs in various organs under non-neoplastic inflammatory conditions. Main body MDSCs are divided into two subpopulations, G-MDSCs and M-MDSCs, and their distribution varies between organs. MDSCs negatively control inflammation in inflamed organs such as the lungs, joints, liver, kidneys, intestines, central nervous system (CNS), and eyes by suppressing T cells and myeloid cells. MDSCs also regulate fibrosis in the lungs, liver, and kidneys and help repair CNS injuries. MDSCs in organs are plastic and can differentiate into osteoclasts and tolerogenic dendritic cells according to the microenvironment under non-neoplastic inflammatory conditions. Conclusion This article summarizes recent findings about MDSCs under inflammatory conditions, especially with respect to their function and differentiation in specific organs.

Background Polymyositis/dermatomyositis (PM/DM) is a chronic autoimmune disease that is often complicated by interstitial lung disease (ILD). Anti-aminoacyl-transfer RNA synthetase antibody (ARS-Ab) and anti-melanoma differentiation-associated gene 5 antibody (MDA5-Ab) are highly detected in PM/DM with ILD. It was reported that ARS-Ab-positive-ILD (ARS-ILD) is often recurrent1, and patients with MDA5-Ab-positive-ILD (MDA5-ILD) develop fatal rapidly progressive ILD2. Objectives To evaluate the differences in clinical courses between ARS-ILD and MDA5-ILD, including the changes in serum ILD markers. Methods We retrospectively investigated 25 patients with ARS-ILD and 26 patients with MDA5-ILD who received induction therapy between April 2002 and September 2017 at Kobe University Hospital. The survival rate and relapse-free survival rate were analysed with Kaplan-Meier estimation and the log-rank test. The differences in serum ILD markers between patients with ARS-ILD and MDA5-ILD were evaluated with the Student’s t-test. Results Disease subtypes at diagnosis with PM/DM-associated ILD were as follows: Eleven ARS-ILD and no MDA5-ILD patients had PM, 10 ARS-ILD and 5 MDA5-ILD patients had DM, and 4 ARS and 21 MDA5 patients had amyopathic DM. The survival rate for MDA5-ILD was significantly lower than that for ARS-ILD (p Conclusions MDA5-ILD patients should monitored for both rapidly progressive disease and relapsing disease. A normal SP-D level is a feature of MDA5-ILD. References [1] Nakazawa M, Kaneko Y, Takeuchi T. Risk factors for the recurrence of interstitial lung disease in patients with polymyositis and dermatomyositis: a retrospective cohort study. Clin Rheumatol2017. Epub ahead of print. [2] Hozumi H, Fujisawa T, Nakashima R, et al. Comprehensive assessment of myositis-specific autoantibodies in polymyositis/dermatomyositis-associated interstitial lung disease. Respir Med2016;121:91–9. Acknowledgements none. Disclosure of Interest None declared

Background Some experimental models suggested bacterial infection, such as periodontal disease and gut microbiota, might be an inciting and aggravating factor in rheumatoid arthritis (RA)1. Moreover, medications that have an antibacterial effect such as minocycline and sulfasalazine are used as DMARDs. However, the antibacterial effect itself on disease activity are unknown in patients with RA whereas trimethoprim-sulfamethoxazole (TMP-SMX) is often used for Pneumocystis jirovecii pneumonia (PCP) prophylaxis because PCP is a prevalent and potentially life-threatening opportunistic infection among patients with RA, especially in Japan2.3. Objectives To identify the effect of TMP-SMX on disease activity in patients with RA in a multi-center cohort study (ANSWER cohort study). Methods RA patients with a sampling interval of less than 1 year and at least two assessment of disease activity were enrolled. Disease activity was assessed using disease activity score 28-CRP (DAS28-CRP). Linear mixed effect models were used to evaluate the trajectories of disease activity in RA patients. Time from baseline, TMP-SMX administration, and their interaction were included as fixed effects while participant identification number and time from baseline were included as random factors. Age, sex, disease duration, RF, ACPA, HAQ, and DMARDs were included as covariates. Results A total of 49878 samples (mean sampling interval: 49 days) from 3255 patients was included. The median age at baseline was 64.0 years (interquartile range, 53.0 to 71.0 years) with 78.2 % of women (ACPA positivity, 79.2%; RF positivity, 70.8 %). The median DAS28-CRP was 2.83 with 33.8 % of patients taking TMP-SMX at baseline. Patients with taking TMP-SMX had a significantly but minimally better longitudinal trajectory on disease activity than patients without (-0.00012/month, P=0.009). This result was similar even when patients taking sulfasalazine were excluded from analysis. Conclusions TMP-SMX has minimal impact on disease activity, and therefore clinical utility of TMP-SMX for controlling disease besides PCP prophylaxis is limited. These results did not support a theoretical effect of bacterial infection on disease activity in RA, although the effect of the other antibiotics on disease activity should be examined. References: [1] Roszyk E, Puszczewicz M. Role of human microbiome and selected bacterial infections in the pathogenesis of rheumatoid arthritis. Reumatologia 2017;55:242-50. [2] Galli M, Antinori S, Atzeni F, et al. Recommendations for the management of pulmonary fungal infections in patients with rheumatoid arthritis. Clin Exp Rheumatol 2017;35:1018-28. [3] Harigai M, Koike R, Miyasaka N, et al. Pneumocystis pneumonia associated with infliximab in Japan. N Engl J Med 2007;357:1874-6. Acknowledgements: None. Disclosure of Interest: None declared

Background Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells that increase in the pathological state such as tumour or inflammation and have the immunosuppressive ability. MDSCs have been reported to ameliorate arthritis in several mice models. Mechanistic target of rapamycin (mTOR) pathway and glutaminolysis activate cooperatively in the differentiation from myeloid progenitors to mature myeloid cells such as dendritic cells, macrophages, or osteoclasts as well as the activation of effector T cells and the differentiation of Th1 and Th17 cells. Although rapamycin has reported to facilitate the expansion of MDSCs and their immunosuppressive ability, the effect of the inhibitor of glutaminolysis on MDSCs is still unknown. Objectives The aim of this study is to evaluate the facilitative effects of the inhibition of mTOR pathway and glutaminolysis on MDSCs in a mouse model of rheumatoid arthritis. Methods Bone marrow (BM) cells from untreated Balb/c mice were cultured for 5 days under granulocyte–macrophage colony-stimulating factor (GM-CSF) stimulation with four patterns of drugs; 1) DMSO (control), 2) rapamycin (Rapa), 3) 6-Diazo-5-oxo-l-norleucine (DON; a glutamine antagonist), or 4) the combination of rapamycin and DON (Rapa +DON). Cultured BM cells were analysed by flow cytometry. Cultured MDSCs were isolated by manual MACS and analysed their immunosuppressive characters by co-culture with CFSE-dyed CD4+ T cells. Rapa or Rapa +DON were administered intraperitoneally to arthritic SKG mice induced by Zymosan A injection. Results We found that DON suppressed the differentiation of dendritic cells (DC) in a dose-dependent manner and the addition of Rapa on DON inhibited the differentiation of macrophages in vitro. The proportions of the phenotype of MDSCs were increased with administrations of Rapa or DON, and large part of them were Ly6G+ cells (the phenotype of polymorphonuclear MDSCs; PMN-MDSCs). Rapa ±DON significantly increased the expressions of TGF-β and PD-L1 and the inhibitory capacity of Ly6G+ PMN-MDSCs. Rapa +DON significantly suppressed arthritis more efficiently in SKG mice than Rapa in vivo. (see figure 1) Conclusions The combination of rapamycin and a glutamine antagonist facilitates the expansion of PMN-MDSCs in vitro and ameliorates arthritis in SKG mice in vivo. Disclosure of Interest None declared

Osteoclasts play a critical role not only in bone homeostasis but also in inflammatory osteolysis, such as that occurring in inflammatory arthritis and systemic inflammation. In both inflammation conditions, inflammatory cytokines like Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α induce RANKL expression in osteoblasts, but the roles of these cytokines in osteoclast activation remain unclear. S100A12, an S100 family member, is a low-molecular-weight calcium-binding protein. Although it has a pro-inflammatory role, its effects on osteoclast differentiation have been unclear. Here we examined the direct effects of S100A12 on human osteoclasts in vitro. S100A12 facilitated osteoclast formation in the presence of RANKL, as judged by the cells' morphology and elevated expression of osteoclast-related molecules, including NFATc1, ACP5, CALCR, and ITGβ3. In addition, S100A12 administration markedly enhanced the osteoclasts' bone resorption ability, consistent with their increased expression levels of CTSK and CA2. Blocking RAGE and TLR4 cancelled the effects of S100A12. Our results indicate that S100A12 is a potential therapeutic target for inflammatory osteolysis.

Objective Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that have the ability to suppress T cell responses. The aim of this study was to evaluate the effects of the JAK inhibitor tofacitinib on MDSCs in a mouse model of rheumatoid arthritis. Methods Arthritis was induced in SKG mice by zymosan A (ZyA) injection. MDSCs isolated from the bone marrow (BM) of donor SKG mice with arthritis were adoptively transferred to recipient mice with arthritis. In a separate experiment, tofacitinib was administered to arthritic SKG mice subcutaneously via osmotic pump, in some cases followed by injection of an anti–Gr-1 monoclonal antibody (mAb). BM cells from untreated mice were cultured for 5 days with granulocyte–macrophage colony-stimulating factor, with or without tofacitinib, and then analyzed by flow cytometry. Results The numbers of MDSCs and polymorphonuclear MDSCs (PMN-MDSCs) were significantly increased in the spleens of SKG mice following ZyA injection. Adoptive transfer of MDSCs to recipient arthritic mice reduced the severity of arthritis compared to that in untreated control mice. Treatment with tofacitinib also ameliorated the progression of arthritis in SKG mice and induced significantly higher numbers of MDSCs and PMN-MDSCs in the BM of these animals. Furthermore, administration of an anti–Gr-1 mAb reduced the antiarthritic effect of tofacitinib in SKG mice. In vitro, tofacitinib facilitated the differentiation of BM cells to MDSCs, and inhibited their differentiation to dendritic cells. Conclusion Tofacitinib facilitates the expansion of MDSCs and ameliorates arthritis in SKG mice.

ObjectiveMicroRNAs (miRNAs) are small endogenous, non-coding RNAs that act as post-transcriptional regulators. We analysed the in vivo effect of miRNA-124 (miR-124, the rat analogue of human miR-124a) on adjuvant-induced arthritis (AIA) in rats.MethodsAIA was induced in Lewis rats by injecting incomplete Freund's adjuvant with heat-killedMycobacterium tuberculosis. Precursor (pre)-miR-124 was injected into the right hind ankle on day 9. Morphological changes in the ankle joint were assessed by micro-CT and histopathology. Cytokine expression was examined by western blotting and real-time RT-PCR. The effect of miR-124 on predicted target messenger RNAs (mRNAs) was examined by luciferase reporter assays. The effect of pre-miR-124 or pre-miR-124a on the differentiation of human osteoclasts was examined by tartrate-resistant acid phosphatase staining.ResultsWe found that miR-124 suppressed AIA in rats, as demonstrated by decreased synoviocyte proliferation, leucocyte infiltration and cartilage or bone destruction. Osteoclast counts and expression level of receptor activator of the nuclear factor κB ligand (RANKL), integrin β1 (ITGB1) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were reduced in AIA rats treated with pre-miR-124. Luciferase analysis showed that miR-124 directly targeted the 3′UTR of the rat NFATc1, ITGB1, specificity protein 1 and CCAAT/enhancer-binding protein α mRNAs. Pre-miR-124 also suppressed NFATc1 expression in RAW264.7 cells. Both miR-124 and miR-124a directly targeted the 3′-UTR of human NFATc1 mRNA, and both pre-miR-124 and pre-miR-124a suppressed the differentiation of human osteoclasts.ConclusionsWe found that miR-124 ameliorated AIA by suppressing critical prerequisites for arthritis development, such as RANKL and NFATc1. Thus, miR-124a is a candidate for therapeutic use for human rheumatoid arthritis.

Background Patient9s global assessment (PtGA) is one of the most widely used patient reported outcomes in rheumatoid arthritis (RA) that reflects both disease activity and other factors. PtGA is onymous and obtained at hospital, which may cause a conscious or unconscious bias, whereas PtGA obtained anonymously may be free from any bias. The credibility of PtGA to report RA patient outcomes has been usually investigated by assessing test-retest reliability. There has been no study comparing routine PtGA and PtGA where patients answered anonymously. Objectives The aim of this study was to compare routinely obtained in-hospital PtGA before clinical examination with those answered anonymously at home. Additionally, physician9s global assessment (PhyGA) was compared with routine PtGA and anonymized PtGA Methods We asked RA patients (n=389) to answer and mail the PtGA test anonymously, Clinical data regarding disease duration, stage, class, swollen joint counts, tender joint counts, pain visual analog scale (VAS), PhyGA, Health Assessment Questionnaire (HAQ), EuroQOL five dimensions questionnaire (EQ5D), Kessler 6 scale (anxiety/ depression), treatment data, laboratory data, and socioeconomic factors were collected simultaneously. We compared the PtGA that is routinely surveyed at hospital before clinical examination with those surveyed anonymously at home. We calculated a discrepancy score by subtracting anonymized PtGA from routine in-hospital PtGA. We defined (1) positive discrepancy when routine PtGA was over-rated relative to the anonymized PtGA; (2) negative discrepancy when routine in-hospital PtGA was under-rated relative to the anonymized PtGA. Results The anonymized PtGA score was higher than routinely evaluated in-hospital PtGA (p Conclusions Discrepancy exists between routine in-hospital PtGA and anonymized PtGA. The high pain VAS scale and low QOL are risk factors that could make the difference between routine PtGA from anonymized PtGA. There is a possibility that high pain VAS score and low QOL influence the reliability of PtGA. Disclosure of Interest None declared

Background Osteoporosis is one of the important adverse effects in the glucocorticoids treatment for the patients with rheumatoid arthritis (RA) and connective tissue diseases (CTDs). Although the usefulness of denosmab for primary osteoporosis has been well-established, the efficacy for GIOP remains unclear. Objectives This study aimed to clarify the therapeutic effects of denosumab for GIOP. Methods We evaluated bone mineral density (BMD) and serum markers of bone metabolism (BAP, NTx, TRACP-5b and P1NP) of patients who had been treated with over 5mg of predonisolone for RA and CTDs, and denosumab for GIOP, for two years in Kobe University Hospital. BMD and serum markers were evaluated every six months for 2 years from the baseline. The changes of those data from baseline were analyzed by Student9s t test using GraphPad Prism 5 software and p Results Number of the patients were 53 (male: 4 cases, female: 49 cases), and their characteristics at the beginning of denosumab treatment were as below; age: 64.19±12.0 years old, dose of prednisolon: 10.59±9.97mg/day, BMD of lumber spine: 0.768±0.112g/cm3, T-score of lumber spine: -2.28±1.01, BMD of femoral neck: 0.540±0.085g/cm3, T-score of femoral neck: -2.28±0.76. After 2-years denosumab treatment, T-scores of lumber spine (0.54±0.39 gain) and femoral neck (0.13±0.26 gain) were significantly increased from baseline (Figure; mean ± SEM. *:p Conclusions Denosumab can suppress bone metabolic turnover, and increase lumber spine and femoral neck T-scores of GIOP patients. Disclosure of Interest None declared

Background Behcet9s disease is a systemic vasculitis disease with oral and genital aphthous ulcers, ocular involvements, skin manifestations, arthritis, gastrointestinal manifestations, neurogenic diseases and vascular involvements. Patients with Behcet9s disease are known to distribute along the ancient Silk Road, including Japan. Objectives We evaluate the clinical features of Behcet9s disease in Japan. Methods We retrospectively investigated 223 patients (108 males and 115 females) who fulfilled the International Criteria for Behcet9s Disease (ICBD) from January, 2006 until May, 2015.We examined sex, onset age, disease type, clinical symptoms, laboratory data and medications. Results Median age at diagnosis was 36.0±12.8 years old. Oral ulcers were the most common manifestation (98.2%), followed by genital ulcers (62.4%), ocular involvements (53.2%), erythema nodosum (53.2%), acneiform lesions (51.8%), arthritis (38.6%), gastrointestinal manifestations (25.1%), neurogenic diseases (9.0%), and vascular involvements (8.1%). The relationship of HLA and disease manifestations was studied in 123 patients (41.5% with HLA-B51 and 24.1% with HLA-A26). The frequency of acneiform lesions, ocular involvements and HLA-B51 was significantly higher in male, while genital ulcers and arthritis were significantly higher in female. Patients with ocular involvements showed a higher assosiation rate with neurogenic diseases and HLA-B51, and lower wih gastrointestinal manifestations. TNFα inhibitor (infliximab or adalimumab) were used for 66 cases (30%), and it could be continued for 1 year in 91%, and for 2 years in 83%. Conclusions A higher incidence of gastrointestinal manifestations was observed in patients with Behcet9s disease in Japan. Patients with ocular involvements showed a higher assosiation rate with neurogenic diseases, and lower wih gastrointestinal manifestation. Most patients could continue TNFα inhibitor safety and effectively. Disclosure of Interest None declared

Background Patients with RA have higher risk of developing chronic kidney disease compared to the general population. Although some causes of renal impairment in RA include nephrotoxic medications, amyloidosis, and glomerulonephritis, specific causes are not determined among most patients with RA. While experimental researches showed inflammatory process per se might also contribute to renal dysfunction, the results of previous clinical studies that assessed the effects of disease activity or C-reactive protein on renal function were inconsistent. This inconsistency might be attributable to their cross-sectional design, small sample size, and assessment of association between only baseline characteristics and renal outcomes without consideration for the fact that disease activity and medications had changed over time. Objectives To identify the effects of disease activity on renal function in RA in a multi-center cohort study. Methods RA patients with a sampling interval of less than 150 days were enrolled because wide sampling intervals could not take into consideration changes in disease activity and medications during their follow-up. An estimated glomerular filtration rate (eGFR) was calculated using an equation approved by the Japanese Society of Nephrology and used as an outcome variable. Linear mixed effect models were used to evaluate the renal trajectories of patients. Time from baseline (months), disease activity, and their interaction were included as fixed effects while participant identification number and time from baseline were included as random factors. Age, sex, disease duration, RF, ACPA, NSAIDs, and DMARDs that were known as a cause of renal impairment, such as tacrolimus, iguratimod, and tofacitinib, were included as covariates. Results A total of 25661 samples (mean sampling interval: 2.0 months) from 2104 patients was included. Patients with lower DAS28-CRP had worse renal function at inclusion, but a significantly better longitudinal trajectory on eGFR (0.0079 ml/min/1.73m2 per month, P=0.025). Although all RA patients had naturally progressive renal impairment as they got older, patients who achieved remission or low disease activity had slower renal impairment rate of -0.068 ml/min/1.73m2 per month compared to patients with moderate or high disease activity (-0.084 ml/min/1.73m2 per month; P=0.037). These results were also similar using SDAI or CDAI. Conclusions Lower disease activity results in slower renal impairment. Because the effects of disease activity on renal function are mild, additional measures to protect renal function, such as avoiding nephrotoxic medications and treating cardiovascular risk factors are important. Disclosure of Interest None declared

Background Many signaling pathways activated under inflammatory and hypoxic conditions have profound effects on intracellular metabolism to support cell growth and survival. The recent findings of cancer-specific metabolic changes, including increased glucose and glutamine consumption, has provided new therapeutic targets for consideration. The microenvironment in inflamed joints in RA is also characterized by hypoxia and low concentration of nutrients, and fibroblast-like synoviocytes from RA patients (RA-FLS) is known to have several tumor-like characteristics. However, the role of glucose and glutamine metabolism in the aberrant proliferation of these cells is unclear. Objectives We evaluated the role of these metabolic pathways in RA-FLS proliferation and in autoimmune arthritis in SKG mice. Methods The expression of glycolysis- or glutaminolysis-related enzymes was evaluated by real-time PCR and Western blotting, and the intracellular metabolites were evaluated by metabolomic analyses. The effects of glucose or glutamine on RA-FLS cell growth were investigated using glucose- or glutamine-free medium. Glutaminase 1 (GLS1) siRNA and the GLS1 inhibitor compound 968 were used to inhibit GLS1 in RA-FLS. Arthritis was induced in SKG mice by zymosan A injection. Compound 968 was used to study the effect of GLS1 inhibition on Zymosan A-injected SKG mice. Ki-67-positive cells were analyzed by immunohistochemistry. Results GLS1 expression was increased in RA-FLS, and metabolomic analyses revealed that glutamine and glutamate consumption were increased in RA-FLS. RA-FLS proliferation was reduced under glutamine-deprived, but not glucose-deprived conditions. Cell growth of RA-FLS was inhibited by GLS1 siRNA transfection or GLS1 inhibitor treatment. Silencing of GLS1 in RA-FLS did not affect IL-6 or MMP-3 production in supernatants. GLS1 expression in RA-FLS was not affected by pro-inflammatory cytokine stimulation. Compound 968 ameliorated the autoimmune arthritis and decreased the number of Ki-67-positive synovial cells in SKG mice. Conclusions Our findings suggested that glutamine metabolism plays an important role in regulating RA-FLS proliferation, without being affected by pro-inflammatory cytokine stimulation or affecting cytokine production, and may be a novel therapeutic target for RA. Disclosure of Interest None declared

Background SKG mice develop interstitial lung disease (ILD) resembling human rheumatoid arthritis (RA)–associated ILD. We identified a unique cell population of CD11b + Gr1 dim cells in the severely inflamed lungs in SKG mice. Objectives The aims of this study are to clarify the mechanism behind the lung pathology, and to elucidate the phenotype and function of CD11b + Gr1 dim cells in ILD-induced SKG mice. Methods We assessed the severity of zymosan A-induced ILD in SKG mice histologically, and examined lung-infiltrating cells by Giemsa stain and flow cytometry. Total lung cells and isolated monocytic myeloid-derived suppressor cells (M-MDSCs) were cultured in vitro with GM-CSF (and IL-4). The proliferation of CSFE-labeled naive T cells co-cultured with isolated CD11b + Gr1 dim cells and MDSCs was evaluated by flow cytometry. Results MDSCs, Th17 cells, and group 1 and 3 innate lymphoid cells (ILC1s and ILC3s) were increased in the lungs; the proportion of these cells varied with ILD severity. In this process, we found that a unique cell population, CD11b + Gr1 dim cells, were expanded in the severely inflamed lungs (Figure). About half of the CD11b + Gr1 dim cells expressed CD11c and Giemsa stain revealed that they were morphologically dendritic cell (DC)-like. The CD11b + Gr1 dim cells were induced from M-MDSCs with GM-CSF in vitro and were considered tolerogenic because they expressed high levels of PD-L1 and suppressed T-cell proliferation. The CD11b + Gr1 dim cells have never described previously and termed CD11b + Gr1 dim tolerogenic DC-like cells (CD11b + Gr1 dim tolDC-LCs). Th17 cells, ILC1s and ILC3s in the inflamed lung produced GM-CSF, which in turn could induce CD11b + Gr1 dim tolDC-LCs to reduce inflammation. Conclusions We identified a unique cell population, termed CD11b + Gr1 dim tolDC-LCs, in ILD-induced lungs in SKG mice. References Nishimura K, Saegusa J, Matsuki F, Akashi K, Kageyama G, Morinobu A. Tofacitinib facilitates the expansion of myeloid-derived suppressor cells and ameliorates arthritis in SKG mice. Arthritis Rheumatol 2015;67:893–902. Shiomi A, Usui T, Ishikawa Y, Shimuzu M, Murakami K, Mimori T. GM-CSF but not IL-17 is critical for the development of severe interstitial lung disease in SKG mice. J. Immunol 2014;193:849–59. Takenaka MC, Quintana FJ. Tolerogenic dendritic cells. Semin. Immunopahol 2016. doi:10.1007/s00281–016–0587–8. Acknowledgements The authors thank Shino Tanaka (Department Rheumatology and Clinical Immunology, Kobe University Graduate School of Medicine) for providing technical assistance. Disclosure of Interest None declared

Recent studies have shown that cellular metabolism plays an important role in regulating immune cell functions. In immune cell differentiation, both interleukin-17-producing T (Th17) cells and dendritic cells (DCs) exhibit increased glycolysis through the upregulation of glycolytic enzymes, such as hexokinase-2 (HK2). Blocking glycolysis with 2-deoxyglucose was recently shown to inhibit Th17 cell differentiation while promoting regulatory T (Treg) cell generation. However, 2-DG inhibits all isoforms of HK. Thus, it is unclear which isoform has a critical role in Th17 cell differentiation and in rheumatoid arthritis (RA) pathogenesis. Here we demonstrated that 3-bromopyruvate (BrPA), a specific HK2 inhibitor, significantly decreased the arthritis scores and the histological scores in SKG mice, with a significant increase in Treg cells, decrease in Th17 cells, and decrease in activated DCs in the spleen. In vitro, BrPA facilitated the differentiation of Treg cells, suppressed Th17 cells, and inhibited the activation of DCs. These results suggested that BrPA may be a therapeutic target of murine arthritis. Although the role of IL-17 is not clarified in the treatment of RA, targeting cell metabolism to alter the immune cell functions might lead to a new therapeutic strategy for RA.

Background Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a sometimes life-threatening complication in RA patients. SKG mice develop not only arthritis but also an ILD resembling RA-ILD. We previously reported that tofacitinib, a JAK inhibitor, facilitates the expansion of myeloid-derived suppressor cells (MDSCs) and ameliorates arthritis in SKG mice. The aim of this study was to elucidate the effect of tofacitinib on the ILD in SKG mice. Methods We assessed the effect of tofacitinib on the zymosan (Zym)-induced ILD in SKG mice histologically and examined the cells infiltrating the lung by flow cytometry. The effects of lung MDSCs on T cell proliferation and Th17 cell differentiation were assessed in vitro. We also evaluated the effects of tofacitinib on MDSCs and dendritic cells in vitro. Results Tofacitinib significantly suppressed the progression of ILD compared to the control SKG mice. The MDSCs were increased, while Th17 cells, group 1 innate lymphoid cells (ILC1s), and GM-CSF+ILCs were decreased in the lungs of tofacitinib-treated mice. MDSCs isolated from the inflamed lungs suppressed T cell proliferation and Th17 cell differentiation in vitro. Tofacitinib promoted MDSC expansion and suppressed bone marrow-derived dendritic cell (BMDC) differentiation in vitro. Conclusion Tofacitinib facilitates the expansion of MDSCs in the lung and ameliorates ILD in SKG mice. Electronic supplementary material The online version of this article (10.1186/s13075-019-1963-2) contains supplementary material, which is available to authorized users.

Glutamine metabolism and the mechanistic target of rapamycin (mTOR) pathway are activated cooperatively in the differentiation and activation of inflammatory immune cells. But the combined inhibition of both pathways was rarely investigated. This study investigated how inhibiting both glutamine metabolism with 6-diazo-5-oxo-L-norleucine (DON) and mTOR with rapamycin affects immune cells and the arthritis in a mouse model. We revealed that rapamycin and DON additively suppressed CD4+ T cell proliferation, and both of them inhibited Th17 cell differentiation. While DON inhibited the differentiation of dendritic cells and macrophages and facilitated that of Ly6G+ granulocytic (G)-MDSCs more strongly than did rapamycin, G-MDSCs treated with rapamycin but not DON suppressed CD4+ T cell proliferation in vitro. The combination of rapamycin and DON significantly suppressed the arthritis in SKG mice more strongly than did each monotherapy in vivo. The numbers of CD4+ T and Th17 cells in the spleen were lowest in mice treated with the combination therapy. Thus, combined treatment with rapamycin and DON additively ameliorated the arthritis in SKG mice, possibly by suppressing CD4+ T cell proliferation and Th17 differentiation. These results suggest the combination of rapamycin and DON may be a potential novel therapy for arthritis.

Background Duchenne muscular dystrophy (DMD), the most common inherited muscular disease in childhood, is caused by dystrophin deficiency because of mutations in the DMD gene. Although DMD is characterized by fatal progressive muscle wasting, cardiomyopathy is the most important nonmuscle symptom threatening the life of patients with DMD. The relationship between cardiac involvement and dystrophin isoforms has not been analyzed. Methods and Results The results of 1109 echocardiograms obtained from 181 Japanese DMD patients with confirmed mutations in the DMD gene were retrospectively analyzed. Patients showed an age-related decline in left ventricular ejection fraction. Patients were divided by patterns of dystrophin isoform deficiency into 5 groups. The cardiac dysfunction-free survival was significantly higher in the group with mutations in the Dp116 coding region than the others, whereas no significant differences in the other 3 groups. At age 25 years, the cardiac dysfunction-free rate was 0.6 in the Dp116 group, but only 0.1 in others. PCR amplification of Dp116 transcript in human cardiac muscle indicated promoter activation. Conclusions Left ventricular ejection fraction in DMD declined stepwise with age. Cardiac dysfunction was less frequent in Dp116-deficient than other patients with DMD. Dp116 transcript was identified in human cardiac muscle for the first time. These results indicate that Dp116 is associated with cardiac involvement in DMD.

In rheumatoid arthritis (RA), synovial fibroblasts (RA-SFs) accumulate in affected joints, where they play roles in inflammation and joint destruction. RA-SFs exhibit tumor-like proliferation and are resistant to apoptosis. Although RA-SF activation is well described, negative regulators of RA-SF activation are unknown. We previously reported that histone deacetylase (HDAC) inhibitors facilitate apoptosis in RA-SFs. Here we found that RA-SFs treated with the HDAC inhibitor Trichostatin A (TSA) exhibited an upregulation of the immediate early response gene X-1 (IEX-1). IEX-1 has roles in apoptosis sensitivity, cell-cycle progression, and proliferation, and is reported to be involved in immune responses, inflammation, and tumorigenesis, and to have anti-arthritic properties. To investigate IEX-1’s role in RA-SFs, we used in vitro-cultured synovial fibroblasts from RA and osteoarthritis (OA) patients. We confirmed that TSA upregulated the IEX-1 protein and mRNA expressions in RA-SFs by western blotting and quantitative RT-PCR. Inhibiting HDAC1, 2, and 3 (but not 6 or 8) also upregulated IEX-1. The IEX-1 mRNA levels were higher in RA-SFs than in OA-SFs, and were further upregulated in RA-SFs by the pro-inflammatory cytokines TNFα and IL-1β. The staining of surgical specimens showed that IEX-1 was present in the pannus from affected RA joints. Si-RNA-mediated IEX-1 knockdown upregulated the lipopolysaccharide (LPS)-induced expression of TNFα and various chemokine mRNAs, indicating that IEX-1 downregulates TNFα and chemokines. Furthermore, apoptosis analysis showed that IEX-1 knockdown protected RA-SFs from apoptosis induced by TSA or by an anti-Fas mAb, indicating that IEX-1 is pro-apoptotic in RA-SFs. Collectively, our results showed that IEX-1 is induced by TNFα and IL-1β in RA-SFs, in which it suppresses TNFα and chemokine production and induces apoptosis; thus, IEX-1 negatively regulates RA-SF activation. Further investigation of IEX1’s functions in RA-SFs may lead to new therapeutic approaches for RA.

MicroRNAs (miRNAs) are non-coding RNAs, approximately 22 nucleotides in length, that act as post-transcriptional regulators. Thousands of miRNAs have been identified in animals, and they are well conserved across species. MicroRNAs play essential regulatory roles in cellular processes, and changes in miRNA expression are associated with human diseases. Originally, miR-124 was identified as a brain-enriched miRNA and shown to be involved in brain and neuronal development. MiR-124 has since been reported to be expressed in other organs and to be involved in various biological phenomena. MiRNA-124 plays roles in various pathologic conditions, including cancers, acute stress, cardiovascular disorders, inflammatory responses, chronic pain, and osteoclast differentiation. MiR-124 has also been shown to suppress various tumor functions, including proliferation, activation, survival, invasion, metastasis, and migration. Rheumatoid arthritis (RA) is a chronic auto-inflammatory disorder of unknown etiology, whose treatment has been significantly improved by the advent of biological drugs. Even so, some RA patients show little or no response to these therapies, suggesting the need for additional treatments. In a study comparing miRNA expression in RA and osteoarthritis (OA) fibroblast-like synoviocytes (FLS), we found that miR-124a was the only miRNA whose expression was lower in RA than in OA FLS.MiR-124a was found to directly downregulate the production of CDK-2 and MCP-1. In the rat adjuvant-induced arthritis (AIA) model, a single injection of pre-miR-124 into one ankle joint suppressed joint swelling in all of the limbs. Histological examination showed that AIA rats treated with pre-miR-124 exhibited reduced synoviocyte proliferation, less leucocyte infiltration into synovial tissue, and less cartilage and bone destruction than untreated AIA rats. The joints of the pre-miR-124-treated AIA rats also showed reduced osteoclast numbers and reduced RANKL, integrin β1 (ITGB1), and NFATc1 expression levels. MiR-124 was shown to directly target the 3’UTRs of the rat NFATc1, ITGB1, SP1, and CEBPα mRNAs. Both miR-124 and miR-124a were also found to directly target human NFATc1 mRNA and to suppress the differentiation of human osteoclasts from monocytes. Taken together, recent studies suggest that MiR-124 may be a promising therapeutic agent for RA and other diseases.

A 72-year-old man was diagnosed with rheumatoid arthritis (RA) and prior hepatitis B virus (HBV) infection. He began treatment with salazosulfapyridine (SASP). Several months later, his blood tests reflected a slightly elevated liver function. Serum tests were positive for hepatitis B surface antigen and HBV-DNA, and the diagnosis of de novo HBV hepatitis was made. A genetic analysis showed that he had polymorphisms of ABCG2 and NAT2, which could lead to high plasma concentrations of SASP and sulfapyridine. To the best of our knowledge, this is the first report of de novo hepatitis developing during SASP monotherapy for RA.

Procalcitonin (PCT) levels are elevated early after birth in newborn infants; however, the physiological features and reference of serum PCT concentrations have not been fully studied in preterm infants. The aims of the current study were to establish an age-specific percentile-based reference curve of serum PCT concentrations in preterm infants and determine the features. The PCT concentration peaked in infants at 1 day old and decreased thereafter. At 1 day old, serum PCT concentrations in preterm infants

Recent studies have shown that mycophenolate mofetil (MMF) is similar to intravenous cyclophosphamide (IVC) for the treatment of lupus nephritis (LN), but that treatment response may vary according to location and race/ethnicity. Moreover, no studies have been conducted to compare the efficacy of MMF with that of IVC for a Japanese population. We therefore conducted a retrospective study to clarify the efficacy and safety of MMF compared with that of IVC for induction therapy for active LN, classes III and IV, in a Japanese population of 21 patients, 11 of whom received MMF and 10 IVC.The primary endpoint was expressed as the percentage of responders, who in turn were defined as the patients who met complete or partial response criteria according to the European consensus statement. The secondary endpoints comprised the renal activity component and serological activity.The primary endpoint was achieved in nine (81.8 %) patients receiving MMF and in four (40.0 %) receiving IVC, with no significant difference between the two groups (p = 0.081), while there was also no significant difference between them in terms of secondary endpoints. However, the MMF group suffered significantly fewer hematologic toxic effects than the IVC group.MMF may be used as an alternative to IVC for inducing renal remission of LN in Japanese patients.

Serum 1,3-β- d -glucan (BG) assay may be helpful as a marker for the diagnosis of Pneumocystis jiroveci pneumonia (PJP) and invasive fungal infection (IFI). We conducted a systematic review to assess the diagnostic accuracy of this assay. We searched MEDLINE, Web of Science, Cochrane Collaboration databases, Ichushi-Web, reference lists of retrieved studies, and review articles. Our search included studies of serum BG assay that used (i) positive cytological or direct microscopic examination of sputum or bronchoalveolar lavage fluid for PJP and (ii) European Organization for Research and Treatment of Cancer or similar criteria for IFI as a reference standard and provided data to calculate sensitivity and specificity. Only major fungal infections such as invasive candidiasis and invasive aspergillosis were included in the IFI group. Twelve studies for PJP and 31 studies for IFI were included from January 1966 to November 2010. The pooled sensitivity, specificity, diagnostic odds ratio (DOR), and area under the summary receiver operating characteristic curve (AUC-SROC) for PJP were 96% (95% confidence interval [95% CI], 92% to 98%), 84% (95% CI, 83% to 86%), 102.3 (95% CI, 59.2 to 176.6) and 0.96 (95% CI, 0.94 to 0.99), respectively. No heterogeneity was found. For IFI, the values were 80% (95% CI, 77% to 82%), 82% (95% CI, 81% to 83%), 25.7 (95% CI, 15.0 to 44.1), and 0.88 (95% CI, 0.82 to 0.93). Heterogeneity was significant. The diagnostic accuracy of the BG assay is high for PJP and moderate for IFI. Because the sensitivity for PJP is particularly high, the BG assay can be used as a screening tool for PJP.

Galectin-3 is highly expressed in epithelial cells including keratinocytes and is involved in the pathogenesis of inflammatory skin diseases by affecting the functions of immune cells. For example, galectin-3 can contribute to atopic dermatitis (AD) by promoting polarization toward a Th2 immune response by regulating dendritic cell (DC) and T cell functions. In addition, galectin-3 may be involved in the development of contact hypersensitivity by regulating the migratory capacity of antigen presenting cells. Galectin-3 may act as a regulator of epithelial tumor progression and development through various signaling pathways, such as inhibiting keratinocyte apoptosis through regulation of the activation status of extracellular signal-regulated kinase (ERK) and activated protein kinase B (AKT). Galectin-3 is detected at different stages of melanoma development. In contrast, a marked decrease in the expression of galectin-3 is observed in non-melanoma skin cancers, such as squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). Galectin-3 may play an important role in tumor cell growth, apoptosis, cell motility, invasion, and metastasis. Galectin-3 may be a novel therapeutic target for a variety of skin diseases.

The objective of this study is to clarify the characteristics and imaging results of Japanese patients with giant cell arteritis (GCA). Eight patients with biopsy-proven GCA were enrolled. Their clinical data and imaging results were retrospectively examined from their medical records. All the patients met the criteria for the classification of GCA by the American College of Rheumatology. Although the clinical manifestations are similar to those previously reported, none of the eight patients presented ocular symptoms, and half of them presented jaw claudication. Ultrasonography (US) of temporal artery showed the halo sign in all the patients. Fluorodeoxyglucose positron emission tomography (FDG-PET) was performed in four patients and indicated the presence of aortitis of the patients. US is a quick and noninvasive test to detect inflammation of temporal artery, and FDG-PET is very helpful for early diagnosis of aortitis in GCA. Awareness of the disease and appropriate imaging tests will result in diagnosis of GCA.

This case report describes findings in a 61-year-old woman who manifested scleritis, small pulmonary nodules, otitis media, periaortitis, and progressive epidural spinal tumor, associated with elevated serum myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA) levels. She was clinically diagnosed with Wegener's granulomatosis, although vasculitis was not diagnosed due to the lack of typical histological findings. We discuss the differential diagnosis in this patient, and the association of MPO-ANCA with periaortitis or epidural spinal tumor.

Background Most patients with spinal muscular atrophy lack the survival motor neuron 1 gene ( SMN1 ) in the telomeric region of the spinal muscular atrophy locus on chromosome 5q13. On the other hand, the copy number of SMN2 , a centromeric homolog of SMN1 , is increased in many of these patients. This study aimed to clarify the mechanism underlying these structural variations. Methods We determined the copy numbers of telomeric and centromeric genes in the spinal muscular atrophy locus of 86 patients and 22 control subjects using multiplex ligation-dependent probe amplification analysis. Then, we chose 74 patients lacking SMN1 exons 7 and 8, and compared their dataset with that of 22 control subjects retaining SMN1 exons 7 and 8. Results The SMN2 copy number was shown to vary widely and to correlate with the disease severity of the patients. Interestingly, telomeric NAIP and telomeric GTF2H2 showed similar tendencies. We also noted positive correlations among the copy number of SMN2 and the telomeric genes of the spinal muscular atrophy locus. However, the copy numbers of centromeric NAIP and centromeric GTF2H2 were stable among the patients, with both approximating a value of two. Conclusion Our findings suggested that the telomeric region of the spinal muscular atrophy locus appears to be susceptible to structural variation, whereas the centromeric region is stable. Moreover, according to our results, new SMN2 copies may be generated in the telomeric region of the spinal muscular atrophy locus, supporting the SMN1 -to- SMN2 gene conversion theory.