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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.

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...

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.

Objective SKG mice develop interstitial lung disease (ILD) resembling rheumatoid arthritis–associated ILD in humans. The aim of this study was to clarify the mechanism underlying the lung pathology by analyzing lung-infiltrating cells in SKG mice with ILD. Methods We assessed the severity of zymosan A (ZyA)–induced ILD in SKG mice histologically, and we examined lung-infiltrating cells by flow cytometry. Total lung cells and isolated monocytic myeloid-derived suppressor cells (MDSCs) were cultured in vitro with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-4. The proliferation of 5,6-carboxyfluorescein diacetate N-succinimidyl ester–labeled naive T cells cocultured with isolated CD11b+Gr-1dim cells and MDSCs was evaluated by flow cytometry. CD11b+Gr-1dim cells were adoptively transferred to ZyA-treated SKG mice. 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+Gr-1dim cells, was expanded in the severely inflamed lungs. Approximately half of the CD11b+Gr-1dim cells expressed CD11c. CD11b+Gr-1dim cells were induced from monocytic MDSCs with GM-CSF in vitro and were considered tolerogenic because they suppressed T cell proliferation. These CD11b+Gr-1dim cells have never been described previously, and we termed them CD11b+Gr-1dim tolerogenic dendritic cell (DC)–like cells. Th17 cells, ILC1s, and ILC3s in the inflamed lung produced GM-CSF, which may have expanded CD11b+Gr-1dim tolerogenic DC–like cells in vivo. Furthermore, adoptive transfer of CD11b+Gr-1dim tolerogenic DC–like cells significantly suppressed progression of ILD in SKG mice. Conclusion We identified unique suppressive myeloid cells that were differentiated from monocytic MDSCs in SKG mice with ILD, and we termed them CD11b+Gr-1dim tolerogenic DC–like cells.

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.

Purpose To evaluate the incidence of fragility fractures associated with high-dose glucocorticoid therapy in patients with systemic rheumatic disease. Methods: A retrospective study of patients who were treated with high-dose prednisolone (> 0.8 mg/kg) for systemic rheumatic disease at Kobe University Hospital from April 1988 to March 2012. The primary outcome was a major osteoporotic fracture (defined as a clinical vertebral, hip, forearm, or proximal humerus fracture) after high-dose glucocorticoid therapy. For postmenopausal women and men over 40 of age, the patient's fracture risk at the beginning of high-dose glucocorticoid therapy was assessed by the World Health Organization's Fracture Risk Assessment Tool (FRAX®). Results Of 229 patients (median age: 49 years), 57 suffered a fragility fracture during the observation period (median observation period: 1558 days). Of 84 premenopausal patients, 5 suffered a fracture. In contrast, of 86 postmenopausal female, 36 suffered a fracture. Fragility fractures were far more frequent than predicted by the FRAX® score. Patients with FRAX® scores over 8.3% had a particularly high risk of fracture. Conclusions Fragility fractures associated with high-dose glucocorticoid therapy are common among postmenopausal women. Extreme care should be taken especially for postmenopausal women when high-dose glucocorticoid therapy is required., Highlights • We evaluated the incidence of the fragility fractures associated with high-dose glucocorticoid therapy. • Of 86 postmenopausal female, 36 suffered a fracture during the observation period. • Patients with FRAX scores over 8.3% had a particularly high risk of fracture.

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.

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

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 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.

Summary The recent development of salivary proteomics has led to the identification of potential biomarkers for diagnosing patients with primary Sjögren's syndrome (pSS). Here we sought to identify differentially produced salivary metabolites from pSS patients and healthy controls (HCs) that might be used to characterize this disease. We obtained salivary samples from 12 female pSS patients (mean age 44.2 ± 13.01) and 21 age-matched female HCs. The metabolite profiles of saliva were analysed by gas chromatography-mass spectrometry. The total metabolite levels in each of the samples were calculated and compared across the study participants. A total of 88 metabolites were detected across the study samples, 41 of which were observed at reduced levels in the samples frompSS patients. Principal component analysis (PCA) revealed a loss in salivary metabolite diversity in the pSS patient samples compared to the HC samples. The reduced presence of glycine, tyrosine, uric acid and fucose, which may reflect salivary gland destruction due to chronic sialoadenitis, contributed to the loss of diversity. Comparative PCA of the pSS patients revealed the presence of two subpopulations based on their metabolite profiles, and these two subpopulations showed a significant difference in the prevalence of major salivary glanditis (P = 0·014). In this study, we found that the salivary metabolite profile of pSS patients was less diverse than that of HCs and that the metabolite profiles in pSS patients were affected by the presence of major salivary glanditis.

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.

ETV6–ABL1 fusion is a rare but recurrent oncogenic lesion found in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), without an established chromosomal abnormality, and is associated with poor outcome. In ETV6–ABL1-positive cases, an in-frame fusion produced by a complex rearrangement results in constitutive chimeric tyrosine kinase activity. Monosomy 7 is also a rare and unfavorable chromosomal abnormality in childhood BCP-ALL. Here, we report a 14-year-old female BCP-ALL patient with ETV6–ABL1 fusion combined with monosomy 7. She was admitted to our hospital because of persistent fever. Bone marrow nuclear cell count on admission was 855,000/µL with 90.0% blastic cells of lymphoid morphology. Blasts were positive for CD10, CD19, CD20, CD34, cyCD79a, cyTdT, HLA-DR, and CD66c, had a karyotype of 45, XX, − 7 [18/20] and a split signal for ABL1 FISH probe (92.7%), and were sensitive to tyrosine kinase inhibitors, imatinib and dasatinib, in vitro. ETV6–ABL1 fusion transcript was identified by whole transcriptome sequencing and confirmed by RT-PCR. She was treated with the high-risk protocol based on ALL-BFM 95, achieved complete remission (CR) after induction chemotherapy, and maintained CR for 4 months. To our knowledge, this is the first report of ETV6–ABL1 fusion combined with monosomy 7 in childhood BCP-ALL.

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 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 38-year-old woman was diagnosed with systemic lupus erythematosus and received immunosuppressive therapy. After 6 months of treatment, workup for low-grade fever yielded elevated enzyme-linked immunosorbent assay titers for Aspergillus antigen in serum and ascites, leading to the diagnosis of disseminated aspergillosis. Transthoracic echocardiography revealed a claviform vegetation attached to the left ventricular anterior septum. Two days after the start of antifungal Amphotericin-B therapy, the patient suffered from several neurologic disorders. A second transthoracic echocardiography revealed that the vegetation decreased in size. Two weeks later, the vegetation increased again. Combination therapy of Amphotericin-B and Voriconazole was initiated, and the vegetation eventually disappeared completely. © 2017 Wiley Periodicals, Inc. J Clin Ultrasound 46:231-232, 2018.

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.

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.

Galectin-3 belongs to a family of beta-galactoside-binding animal lectins expressed in several cell types, including epithelial and immune cells. To establish the role of galectin-3 in the development of allergic skin inflammation, we compared inflammatory skin responses of galectin-3-deficient (gal3(-/-)) and wild-type (gal3(+/+)) mice to epicutaneous sensitization with ovalbumin (OVA). OVA-treated gal3(-/-) mice exhibited markedly reduced epidermal thickening, lower eosinophil infiltration, and lower serum IgE levels compared with gal3(+/+) mice. The former evoked lower interleukin-4, but higher interferon-gamma, mRNA expression at OVA-treated skin sites. Moreover, gal3(-/-) splenocytes from OVA-sensitized mice secreted more interleukin-12 compared with gal3(+/+) splenocytes. In addition, antigen presentation by gal3(-/-) dendritic cells to T cells in vitro were T helper cell (Th1)-polarized relative to presentation by gal3(+/+) dendritic cells. When exposed to OVA, recipients engrafted with T cells from gal3(-/-) OVA-specific T cell receptor transgenic mice developed significantly reduced dermatitis and a markedly lower Th2 response compared with recipients of comparable gal3(+/+) T cells. We conclude that galectin-3 is critical for the development of inflammatory Th2 responses to epicutaneously administered antigens; in its absence, mice develop a Th1-polarized response. This regulatory effect of galectin-3 on Th development is exerted at both the dendritic cell and T cell levels. Our studies suggest that galectin-3 may play an important role in the acute phase of human atopic dermatitis.

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.

Background Endothelin-1 (ET-1) is important in the pathogenesis of systemic sclerosis (SSc). It has been reported that plasma concentration of ET-1 are higher in SSc patients, and that endothelin receptor expression are also increased in lungs and skins in the patients. ET-1 binds two receptors, endothelin type A (ET A ) and endothelin type B (ET B ). Dual ET A /ET B receptor antagonists and a selective ET A receptor antagonist are used clinically to treat SSc, and the effect of these antagonists on fibroblast activation has been described. However, the role of ET B receptor signaling in fibrogenesis is less clear. Objectives This study was conducted to evaluate the profibrotic function of ET B receptor signaling in a murine model of bleomycin (BLM)-induced scleroderma. Methods We used ET B receptor–knockout (ET B KO) mice, which are genetically rescued from lethal intestinal aganglionosis by an ET B receptor transgene driven by the human dopamine β-hydroxylase (DβH)-gene promoter, and wild-type mice with DβH-ETB (WT). BLM or phosphate-buffered saline (PBS) was administered subcutaneously by osmotic minipump, and skin fibrosis was assessed by dermal thickness, subcutaneous fat atrophy, and alpha-smooth muscle actin (αSMA)-expressing myofibroblast count in the dermis. Dermal fibroblasts isolated from ET B KO and WT mice were cultured in vitro , stimulated with BLM or ET-1, and the expression of profibrotic genes was compared by quantitative PCR. Results Dermal thickness, subcutaneous fat atrophy, and myofibroblast counts in the dermis were significantly reduced in ET B KO mice compared to WT mice, after BLM treatment. Compared with wild-type, dermal fibroblasts isolated from ET B KO mice showed lower gene expressions of αSMA and collagen 1α1 in response to BLM or ET-1 stimulation in vitro. Conclusions ET-1–ET B receptor signaling is involved in skin sclerosis and in collagen synthesis by dermal fibroblasts. Disclosure of Interest None declared

Background Endothelin-1 (ET-1) is important in the pathogenesis of systemic sclerosis (SSc). ET-1 binds two receptors, endothelin type A (ETA) and endothelin type B (ETB). Dual ETA/ETB receptor antagonists and a selective ETA receptor antagonist are used clinically to treat SSc, and the effect of these antagonists on fibroblast activation has been described. However, the role of ETB receptor signaling in fibrogenesis is less clear. This study was conducted to evaluate the profibrotic function of ETB receptor signaling in a murine model of bleomycin (BLM)-induced scleroderma. Methods We used ETB receptor–knockout (ETBKO) mice, which are genetically rescued from lethal intestinal aganglionosis by an ETB receptor transgene driven by the human dopamine β-hydroxylase (DβH)-gene promoter, and wild-type mice with DβH-ETB (WT). BLM or phosphate-buffered saline (PBS) was administered subcutaneously by osmotic minipump, and skin fibrosis was assessed by dermal thickness, subcutaneous fat atrophy, and myofibroblast count in the dermis. Dermal fibroblasts isolated from ETBKO and WT mice were cultured in vitro, stimulated with BLM or ET-1, and the expression of profibrotic genes was compared by quantitative PCR. Results Dermal thickness, subcutaneous fat atrophy, and myofibroblast counts in the dermis were significantly reduced in ETBKO mice compared to WT mice, after BLM treatment. Compared with wild-type, dermal fibroblasts isolated from ETBKO mice showed lower gene expressions of α-smooth muscle actin and collagen 1α1 in response to BLM or ET-1 stimulation in vitro. Conclusions ET-1–ETB receptor signaling is involved in skin sclerosis and in collagen synthesis by dermal fibroblasts. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1011-4) contains supplementary material, which is available to authorized users.

Objective.To clarify the role of platelet-derived microparticles (PDMP), which are small vesicles with thrombotic and immunological properties, in systemic lupus erythematosus (SLE), systemic sclerosis (SSc), dermatomyositis/polymyositis (PM/DM), and mixed connective tissue disease (MCTD).Methods.Plasma levels of PDMP were measured by ELISA, and compared among patients with one of the 4 diseases. Association of PDMP levels with clinical characteristics and medication of the patients was also examined.Results.PDMP levels were higher in patients with MCTD and SSc than in controls. Multiple linear regression analysis revealed that patients with Raynaud’s phenomenon (RP) showed higher PDMP levels than those without. PDMP levels in individual patients did not fluctuate significantly over several months.Conclusion.PDMP level is associated with MCTD, SSc, and RP, and could be a novel marker for RP.

The purpose of this study was to elucidate the effects of histone deacetylase inhibition on the phenotype and function of dendritic cells and on arthritis in SKG mice. Arthritis was induced in SKG mice by zymosan A injection. Trichostatin A, a histone deacetylase inhibitor, was administered and its effects on arthritis were evaluated by joint swelling and histological evaluation. Interleukin-17 production in lymph node cells was determined by an enzyme-linked immunosorbent assay (ELISA). Foxp3 expression in lymph node cells and the phenotypes of splenic dendritic cells were examined by fluorescence-activated cell sorting (FACS). Bone marrow-derived dendritic cells (BM-DC) were generated with granulocyte macrophage colony-stimulating factor. The effects of trichostatin A on cell surface molecules, cytokine production, indoleamine 2,3-dioxygenase (IDO) expression and T cell stimulatory capacity were examined by FACS, ELISA, quantitative real-time polymerase chain reaction and Western blot, and the allo-mixed lymphocyte reaction, respectively. Trichostatin A, when administered before the onset of arthritis, prevented SKG mice from getting arthritis. Trichostatin A treatment also showed therapeutic effects on arthritis in SKG mice, when it was administered after the onset of arthritis. Trichostatin A treatment reduced Th17 cells and induced regulatory T cells in lymph node, and also decreased co-stimulatory molecule expression on splenic dendritic cells in vivo. In vitro, trichostatin A markedly suppressed zymosan A-induced interleukin-12 and interleukin-6 production by BM-DC and up-regulated IDO expression at mRNA and protein levels. Trichostatin A-treated BM-DC also showed less T cell stimulatory capacity. Histone deacetylase inhibition changes dendritic cells to a tolerogenic phenotype and ameliorates arthritis in SKG mice.

Background MicroRNAs (miRNAs) are small (∼22 nt) endogenous, noncoding RNAs that act as post-transcriptional regulators of gene expression.[1] We previously reported that miR-124a expression decreases proliferative RA synoviocytes as compared to osteoarthritis synoviocytes. The transfection of precursor -miR-124a into RA synoviocytes significantly suppresses synoviocyte proliferation and suppresses the secretion of monocyte chemoattractant protein 1 (MCP-1). Furthermore, miR-124a directly downregulates the production of cyclin-dependent kinase 2 (CDK-2) and MCP1 proteins at translational level. [2] Objectives The aim of this study is to investigate the in vivo effect of miRNA-124 (miR-124, the rat analog of human miR-124a) on adjuvant-induced arthritis (AIA) in rats. Methods 1) Experimental arthritis was induced in rats by injecting 0.1 ml of incomplete Freund9s adjuvant (IFA) and 1.0 mg of a heat-killed Mycobacterium tuberculosis (Difco), into the base of the tail on day 0. On day 9, Pre-miR-124 (Life Technologies) or Pre-miR Negative Control #2 (Life Technologies) was mixed with AteloGene Reagent (Koken), and 50μl of the mixture (14.3 pmol/μl of miRNA) was injected into rats at the right ankle joints. The severity of arthritis was observed from day 1 to day 18. Rats were sacrificed on day 18. The hind feet were used for micro-computed tomography (micro CT) analysis, and the ankles were used for both histopathological analysis and quantitative real-time RT-PCR. For western blotting analysis, rats were sacrificed on day 12, and the ankle tissues were collected for protein extraction. 2) For luciferase reporter assay, the 39-UTR of mRNAs and their site-directed mutants were PCR-amplified, and cloned into psiCHECK-2 vector (Promega). Pre-miRNAs were transfected into E11 synovial fibroblast cell line, and the cells were transfected with these vectors on the next day. After 48 h, the cells were lysed and luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer9s instructions. 3) The effect of pre-miR-124 or pre-miR-124a on the differentiation of human osteoclasts was examined by tartrate-resistant acid phosphatase (TRAP) staining. Results 1) MiR-124 suppressed AIA in rats, as demonstrated by decreased synoviocyte proliferation, leukocyte infiltration, and cartilage/ bone destruction at ankle. Osteoclast counts and expression level of RANKL, ITGB1 and NFATc1 were reduced in AIA rats treated with pre-miR-124 at ankle. 2) Luciferase analysis showed that miR-124 directly targeted the 39-UTR of the rat NFATc1, ITGB1, SP1, and CEBPA mRNAs. Pre-miR-124 also suppressed NFATc1 expression in RAW264.7 cells. 3) Both miR-124 and miR-124a directly targeted the 39-UTR of human NFATc1 mRNA, and both pre-miR-124 and pre-miR-124a suppressed the differentiation of human osteoclasts. Conclusions We showed 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. References Chu CY et al. J Cell Physiol 2007:412-9. Nakamachi Y et al. Arthritis Rheum 2009; 1294-304. Disclosure of Interest None declared

Exposure to ultraviolet radiation exacerbates the skin lesions of autoimmune diseases, and is known to induce cell surface expression of SS-A/Ro antigen on keratinocytes in vitro. Following up on recent reports on ultraviolet-B (UVB)-induced oxidative stress, we examined the role of oxidative stress in the surface expression of SS-A/Ro antigen on human keratinocytes. First, the exclusive induction by UVB irradiation of the 52-kDa protein (Ro52) but not of the 60-kDa protein (Ro60) of SS-A/Ro antigen was demonstrated by means of indirect immunofluorescence. The surface expression of Ro52 induced by UVB irradiation was concentration-dependently inhibited by N-acetyl-L-cysteine, an antioxidant. Furthermore, surface expression of Ro52 was similarly induced by diamide, a chemical oxidant. We next used Hoechst 33342 staining and the TUNEL assay to demonstrate that a low dose (20 mJ/cm(2)) of UVB did not induce apoptosis but induced the surface expression of Ro52. Moreover, zVAD-fmk, a pan-caspase inhibitor, did not inhibit UVB-induced surface expression of Ro52 even at a high dose (200 mJ/cm(2)) of UVB, which was sufficient to induce apoptosis in keratinocytes in the absence of zVAD-fmk. Taken together, we concluded that UVB-induced surface expression of Ro52 on keratinocytes is mediated by oxidative stress through a pathway other than apoptosis.

Background Primary Sjogren9s syndrome (pSS) is an autoimmune exocrinopathy characterised by the infiltration of mononuclear cells into salivary and lacrimal glands leading to destruction of the parenchymal tissue and causing oral and ocular dryness. Omics studies such as genomics, proteomics and metabolomics have been extensively used to elucidate biological mechanisms of diseases through collective characterization and quantification of pools of biological molecules and generating molecular profiles for biospecimens. Saliva contains a variety of informative substances which may closely reflect the pathogenetic process of pSS in salivary glands. In fact, salivary proteomics from pSS patients have detected some proteins as biomarkers for pSS diagnosis. To date, metabolomics has been widely conducted and is regarded as the end point of “omics cascade” and most predictive of phenotype. However, salivary metabolomics from pSS patients has not been reported up-to-date. Objectives To compare the salivary metabolite profile between patients with pSS and healthy control (HC) subjects. Methods We obtained saliva from 12 female pSS patients (mean age 44.2±13.01) and 21 age-matched female HCs by spitting for 15 minutes. The metabolite profile of saliva specimens were analyzed by gas chromatography-mass spectrometry. The levels of metabolites were calibrated by saliva volume per 15 minutes enabling us to compare the metabolite production level of whole salivary glands for 15 minutes. Results 88 metabolites were detected by our system. 32 metabolites were decreased and only one metabolite was increased in pSS patients. Principal component analysis (PCA) revealed a loss in salivary metabolite diversity in pSS patients compared to HCs. The decrease of Glysine, Tyrosine, Uric Acid and Fucose which may reflect the destruction of salivary gland due to chronic sialoadenitis contributed to the loss of diversity. PCA amongst pSS patients revealed that pSS could be divided into two subpopulations according to their metabolite profiles and the prevalence of major salivary glanditis was different between the two subpopulations (p=0.014). Conclusions In this preliminary study, the salivary metabolite profile of pSS patients was less diverse compared to controls. Salivary metabolite profile was affected by the presence of major salivary glanditis suggesting that salivary metabolomics may serve as a prospective approach to discover the underlying pathogenesis of pSS. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.1201

Background Rheumatoid arthritis (RA) is a chronic inflammatory disorder, characterized by cellular infiltration of and proliferation in the synovium, leading to the progressive destruction of the joints. Dendritic cells (DC) play a pivotal role by triggering the immune responses in RA pathogenesis. Trichostatin A (TSA) plays a significant role of controlling gene transcription as a histone deacetylase inhibitor, and has been reported to regulate immune responses. Objectives We examined to elucidate the effects of TSA on the phenotype and function of DC and on arthritis in SKG mice. Methods Arthritis was induced in SKG mice by Zymosan A (ZyA) injection. TSA was administered and its effects on arthritis were evaluated by joint swelling and histological evaluation. IL-17A production in lymph node cells was determined by ELISA. Foxp3 expression in lymph node cells and the phenotypes of splenic conventional DC (cDC) were examined by flow cytometry. Bone marrow-derived DC (BM-DC) were generated with granulocyte macrophage colony-stimulating factor. The effects of TSA on cytokine production, cell surface molecules, indoleamine 2,3-dioxygenase (IDO) expression and T cell stimulatory capacity of BM-DC were examined by flow cytometry, ELISA, quantitative real-time polymerase chain reaction and Western blot, and the allo-mixed lymphocyte reaction, respectively. Results TSA, when administered before the onset of arthritis, prevented SKG mice from arthritis. TSA treatment also showed therapeutic effects on arthritis in SKG mice, when administered after the onset of arthritis (daily treatment and twice-a- week treatment). TSA treatment reduced IL-17A production and increased in the ratio of CD4+CD25+Foxp3+ cells among CD4+ cells in lymph node, suggesting that regulatory T cells are involved in the prevention of arthritis in SKG mice with TSA. In the CD8α+ splenic cDC subset, the expressions of CD86, CD80, and CD40 were significantly decreased in the TSA-treated group compared to the control group. In contrast, there were no significant differences in the expression of these molecules in the CD8α- cDC subset. Thus, TSA predominantly affects CD8α+ cDC in vivo . In vitro , TSA markedly suppressed ZyA-induced IL-12 and IL-6 productions, cell surface molecules by BM-DC and up-regulated IDO expression at mRNA and protein levels. TSA-treated BM-DC also showed less T cell stimulatory capacity. Conclusions TSA changes DC to a tolerogenic phenotype, induces regulatory T cells and ameliorates arthritis in SKG mice. Disclosure of Interest None Declared

Keratinocytes undergo apoptosis in a variety of physiological and pathological conditions. Galectin-3 is a member of a family of beta-galactoside-binding animal lectins expressed abundantly in keratinocytes and other epithelial cells. Here, we have studied the regulatory role of galectin-3 in keratinocyte apoptosis by using cells from gene-targeted galectin-3 null (gal3(-/-)) mice. We showed that galectin-3 mRNA was transiently upregulated in ultraviolet-B (UVB)-irradiated wild-type keratinocytes. We found that gal3(-/-) keratinocytes were significantly more sensitive to apoptosis induced by UVB as well as various other stimuli, both in vitro and in vivo, than wild-type cells. Moreover, we demonstrated that increased apoptosis in gal3(-/-) keratinocytes was attributable to higher extracellular signal-regulated kinase (ERK) activation and lower AKT activation after UVB irradiation. We conclude that endogenous galectin-3 is an anti-apoptotic molecule in keratinocytes functioning by suppressing ERK activation and enhancing AKT activation and may play a role in the development of apoptosis-related skin diseases.

A 63-year-old man was admitted to our hospital with a complaint of right lateroabdominal pain. He was diagnosed with metastatic colon cancer, and then developed multiple brain embolic infarctions 7 days after admission. Transesophageal echocardiography showed that mobile, echo-dense masses were attached to the anterior and posterior mitral valve leaflet. Furthermore, there was a thrombus in the left auricular appendage despite sinus rhythm. These findings led to a diagnosis of suspected infectious endocarditis with subsequent multiple brain infarctions. The patient's general condition worsened and he died 13 days after admission. An autopsy was performed, and, while poorly differentiated cancer was observed in multiple organs, no primary tumor could be identified. Histological analysis showed that the masses of the mitral valve consisted mainly of fibrin without bacteria or oncocytes. This patient was therefore diagnosed with nonbacterial thrombotic endocarditis associated with cancer of unknown origin complicated with thrombus in the left auricular appendage.