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Next-generation DNA sequencing (NGS) in short-read mode has been recently used for genetic testing in various clinical settings. NGS data accuracy is crucial in clinical settings, and several reports regarding quality control of NGS data, focusing mostly on establishing NGS sequence read accuracy, have been published thus far. Variant calling is another critical source of NGS errors that remains mostly unexplored despite its established significance. In this study, we used a machine-learning-based method to establish an exome-wide benchmark of difficult-to-sequence regions using 10 genome sequence features on the basis of real-world NGS data accumulated in The Genome Aggregation Database (gnomAD) of the human reference genome sequence (GRCh38/hg38). We used the obtained metrics, designated “UNMET score,” along with other lines of structural information of the human genome to identify difficult-to-sequence genomic regions using conventional NGS. Thus, the UNMET score could provide appropriate caveats to address potential sequential errors in protein-coding exons of the human reference genome sequence GRCh38/hg38 in clinical sequencing.

ObjectivesTo collect clinical information and NOD2 mutation data on patients with Blau syndrome and to evaluate their prognosis.MethodsFifty patients with NOD2 mutations were analysed. The activity of each NOD2 mutant was evaluated in HEK293 cells by reporter assay. Clinical information was collected from medical records through the attending physicians.ResultsThe study population comprised 26 males and 24 females aged 0–61 years. Thirty-two cases were sporadic, and 18 were familial from 9 unrelated families. Fifteen different mutations in NOD2 were identified, including 2 novel mutations (p.W490S and D512V); all showed spontaneous nuclear factor kappa B activation, and the most common mutation was p.R334W. Twenty-six patients had fever at relatively early timepoints in the disease course. Forty-three of 47 patients had a skin rash. The onset of disease in 9 patients was recognised after BCG vaccination. Forty-five of 49 patients had joint lesions. Thirty-eight of 50 patients had ocular symptoms, 7 of which resulted in blindness. After the diagnosis of Blau syndrome, 26 patients were treated with biologics; all were antitumour necrosis factor agents. Only 3 patients were treated with biologics alone; the others received a biologic in combination with methotrexate and/or prednisolone. None of the patients who became blind received biologic treatment.ConclusionsIn patients with Blau syndrome, severe joint contractures and blindness may occur if diagnosis and appropriate treatment are delayed. Early treatment with a biologic agent may improve the prognosis.

To collect clinical information andFifty patients withThe study population comprised 26 males and 24 females aged 0-61 years. Thirty-two cases were sporadic, and 18 were familial from 9 unrelated families. Fifteen different mutations inIn patients with Blau syndrome, severe joint contractures and blindness may occur if diagnosis and appropriate treatment are delayed. Early treatment with a biologic agent may improve the prognosis.

Numerous studies have clarified the immunological mechanisms of contact hypersensitivity (CHS). In addition, we have recently shown that M2 macrophages play key roles in the development of CHS by producing matrix metalloproteinase-12 (MMP-12). However, regulatory mechanisms of the elicitation phase in CHS remain largely unknown. To determine the roles of suppressor of cytokine signaling (SOCS) family members in M2 macrophages in the regulation of CHS, we investigated the expression of SOCS family members in M2 macrophages at the inflammatory sites of CHS. Transcriptome analysis revealed that among SOCS family members, SOCS3 was highly expressed in M2 macrophages at the site of CHS, and SOCS3 induction was reduced by IFN-? neutralization. 2,4-Dinitrofluorobenzene-induced CHS was significantly enhanced and prolonged in mice lacking SOCS3 expression in monocytes/macrophages (SOCS3(?/?) mice) compared with that in control mice. Importantly, expression of MMP-12 in M2 macrophages was significantly increased in SOCS3(?/?) mice at the site of CHS, and deletion of the MMP-12 gene reduced the exacerbated CHS in SOCS3(?/?) mice. Finally, IFN-? inhibited IL-4-induced MMP-12 expression in a SOCS3-dependent manner. Taken together, these results suggest that SOCS3 expressed in M2 macrophages is involved in the attenuation and/or resolution of CHS, presumably by suppressing MMP-12 production.

Objective We have previously shown that expression of the Bcl-3 gene, a member of the IκB family, is down-regulated in CD4+ T cells from patients with rheumatoid arthritis (RA) following tocilizumab therapy. The objective of this study was to examine the role of Bcl-3 in the pathogenesis of RA. Methods DNA microarray analysis was used to compare the signal intensity of Bcl-3 in CD4+ T cells from untreated RA patients and healthy controls. We examined the roles of interleukin-6 (IL-6)/STAT-3 signaling in the induction of Bcl-3. In addition, we analyzed the gene expression profiles of Bcl-3–transduced CD4+ T cells by RNA sequencing. The effects of enforced expression as well as gene silencing of Bcl-3 on the development of follicular helper T (Tfh) cells were evaluated. Finally, we examined correlations between the signal intensities of Bcl-3 and Tfh cell–related genes in CD4+ T cells from untreated RA patients. Results Bcl-3 levels were significantly higher in RA patients than in healthy controls. IL-6 induced Bcl-3 expression in CD4+ T cells in a STAT-3–dependent manner. Transcriptome analysis revealed that the expression of Bcl-6, a master regulator of Tfh cell differentiation, was significantly up-regulated by the enforced Bcl-3 expression. The enforced Bcl-3 expression increased, but Bcl-3 silencing decreased, the numbers of IL-21–producing Tfh-like cells. Bcl-3 levels in CD4+ T cells from RA patients correlated positively with the levels of Tfh cell–related genes CXCR5, inducible costimulator, and achaete-scute homolog 2. Conclusion Bcl-3 is involved in the development of Tfh cells and the pathogenesis of RA, presumably by inducing IL-21 production.

The synthesis of indole skeleton by using Pd(II)-catalyzed cyclization of the urethane has been achieved. The urethanes with allylic alcohol were converted into vinyl indolines in good yield. The vinyl indoline was transformed into some intermediates of indole alkaloids.

In the past decade, mechanisms underlying allergic contact dermatitis have been intensively investigated by using contact hypersensitivity (CHS) models in mice. However, the regulatory mechanisms, which could be applicable for the treatment of allergic contact dermatitis, are still largely unknown. To determine the roles of B and T lymphocyte attenuator (BTLA), a CD28 family coinhibitory receptor, in hapten-induced CHS, BTLA-deficient (BTLA(-/-)) mice and littermate wild-type (WT) mice were subjected to DNFB-induced CHS, severe combined immunodeficient (SCID) mice were injected with CD4(+) T cells, and CD8(+) T cells from either WT mice or BTLA(-/-) mice were subjected to CHS. BTLA(-/-) mice showed enhanced DNFB-induced CHS and proliferation and IFN-γ production of CD8(+) T cells as compared with WT mice. SCID mice injected with WT CD4(+) T cells and BTLA(-/-) CD8(+) T cells exhibited more severe CHS as compared with those injected with WT CD4(+) T cells and WT CD8(+) T cells. On the other hand, SCID mice injected with BTLA(-/-) CD4(+) T cells and WT CD8(+) T cells exhibited similar CHS to those injected with WT CD4(+) T cells and WT CD8(+) T cells. Finally, to evaluate the therapeutic potential of an agonistic agent for BTLA on CHS, the effects of an agonistic anti-BTLA antibody (6A6) on CHS were examined. In vivo injection of 6A6 suppressed DNFB-induced CHS and IFN-γ production of CD8(+) T cells. Taken together, these results suggest that stimulation of BTLA with agonistic agents has therapeutic potential in CHS.

Background: Mast cells are known to play a pivotal role in allergic diseases by releasing granules containing histamine and other preformed chemical mediators. Cross-linking of high-affinity receptors for IgE (FceRI) on mast cells results in rapid increases in intracellular free calcium concentration [Ca2+]i and consequent activation of many transcription factors, including NFAT, NF-κB, JNK and CREB. Ca2+ signaling is essential for many cellular activities such as proliferation, gene expression and degranulation in mast cells. In addition to Ca2+ signaling, previous reports have shown that IkappaB kinase 2 (IKK2 or IKKß), a central component of the IKK complex mediating NF-κB activation, also plays a crucial role in FceRI-mediated degranulation and cytokine production. Moreover, it has been demonstrated that activation of PKCß, a calcium-dependent PKC isoform, leads to IKK2 activation in many cell types. However, the roles of Ca2+ signaling and PKCß in the activation of IKK2 in mast cells remain largely unknown. Methods: We investigated the effect of PKC inhibitor Gö6976 on calcium ionophore A23187-induced activation of IKK2 in mast cells. We also examined the role of IKK2 in A23187-induced NF-κB-dependent gene induction, degranulation, proinflammatory cytokine production and extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation by using IKK2-deficient (IKK2-/-) fetal liver-derived mast cells (FLMCs). Results: A23187 activated IKK2 and NF-κB even in the presence of Gö6976 in mast cells. A23187-induced degranulation, cytokine production and activation of ERK1/2 were diminished in IKK2-/- FLMCs compared to those in wild-type FLMCs. Conclusions: Ca2+-IKK2 signaling is involved in the degranulation and cytokine production in activated mast cells by a mechanism independent of PKCß.

Pd-catalyzed cyclization of substrates (I) provides vinyl indolines, which are further transformed into intermediates of indole alkaloids such as (III) or (IV).

IκBNS plays an important role in Tfh cell differentiation and function.

We have previously shown that expression of the Bcl-3 gene, a member of the IκB family, is down-regulated in CD4+ T cells from patients with rheumatoid arthritis (RA) following tocilizumab therapy. The objective of this study was to examine the role of Bcl-3 in the pathogenesis of RA.DNA microarray analysis was used to compare the signal intensity of Bcl-3 in CD4+ T cells from untreated RA patients and healthy controls. We examined the roles of interleukin-6 (IL-6)/STAT-3 signaling in the induction of Bcl-3. In addition, we analyzed the gene expression profiles of Bcl-3-transduced CD4+ T cells by RNA sequencing. The effects of enforced expression as well as gene silencing of Bcl-3 on the development of follicular helper T (Tfh) cells were evaluated. Finally, we examined correlations between the signal intensities of Bcl-3 and Tfh cell-related genes in CD4+ T cells from untreated RA patients.Bcl-3 levels were significantly higher in RA patients than in healthy controls. IL-6 induced Bcl-3 expression in CD4+ T cells in a STAT-3-dependent manner. Transcriptome analysis revealed that the expression of Bcl-6, a master regulator of Tfh cell differentiation, was significantly up-regulated by the enforced Bcl-3 expression. The enforced Bcl-3 expression increased, but Bcl-3 silencing decreased, the numbers of IL-21-producing Tfh-like cells. Bcl-3 levels in CD4+ T cells from RA patients correlated positively with the levels of Tfh cell-related genes CXCR5, inducible costimulator, and achaete-scute homolog 2.Bcl-3 is involved in the development of Tfh cells and the pathogenesis of RA, presumably by inducing IL-21 production.

Objective This prospective study aimed to determine whether the comprehensive ultrasonographic assessment of synovial inflammation predicts relapse after discontinuation of treatment with a biologic agent in patients with rheumatoid arthritis (RA) in clinical remission. Methods RA patients in clinical remission (Disease Activity Score in 28 joints [DAS28]

Introduction. This study aimed to investigate the efficacy of abatacept for arthritis in patients with rhupus, an overlap syndrome between rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Methods. Patients who fulfilled both the 2010 ACR/EULAR criteria for RA classification and the 1997 ACR revised criteria for classification of SLE and received abatacept treatment for arthritis were retrospectively studied. Results. Six rhupus patients who fulfilled the inclusion criteria above were identified. All patients had active arthritis despite receiving antirheumatic drugs including methotrexate when abatacept was initiated. Clinical Disease Activity Index (CDAI) significantly decreased between baseline and 12 weeks (P = 0.028) and remained low through 24 weeks. All patients achieved either a good or moderate response according to the EULAR response criteria at 24 weeks. Health Assessment Questionnaire-Disability Index (HAQ-DI) also significantly decreased between baseline and 24 weeks (P = 0.043). In addition, the levels of immunoglobulin G and anti-DNA antibody significantly decreased between baseline and 24 weeks (P = 0.028 and P = 0.043, resp.). Conclusions. Treatment with abatacept is likely to be efficacious in patients with rhupus whose arthritis is refractory to methotrexate. In addition, abatacept may have a moderate effect on abnormal antibody production in rhupus patients.

Background Rheumatoid arthritis (RA) is a chronic inflammatory disease and proinflammatory cytokines such as TNFα and IL-6 play critical roles in the pathogenesis of RA. The blockade of IL-6 signaling by Tocilizumab (TCZ) has shown the clinical efficacy for patients with RA. To clarify the roles of IL-6 signaling in CD4+ T cells in the pathogenesis of RA, we previously compared gene expression profiles of CD4+ T cells by DNA microarray analysis before and after the treatment with TCZ in RA patients who exhibited good clinical responses to the treatment (1, 2), and identified that B cell leukemia/lymphoma 3 (Bcl3), an IκB family member, was down-regulated by TCZ therapy (1). However, the role of Bcl3 expressed in CD4+ T cells in the pathogenesis of RA remains unclear. Objectives The objective of this study is to examine the role of Bcl3 expressed in CD4+ T cells in the pathogenesis of RA. Methods We compared signal intensity of Bcl3 in CD4+ T cells between untreated RA patients and healthy controls by DNA microarray analysis. We examined the roles of IL-6-STAT3 signaling in Bcl3 induction. We also analyzed gene expression profiles of Bcl3-transduced CD4+ T cells by RNA-sequencing analysis. We examined the effect of enforced expression as well as gene silencing of Bcl3 on the development of T follicular helper (Tfh) cells. Finally, we examined a correlation between signal intensities of Bcl3 and Tfh cell-related genes in CD4+ T cells in untreated RA patients. Results Bcl3 levels were significantly higher in RA patients than those in healthy controls. IL-6 induced Bcl3 expression in CD4+ T cells in a STAT3-dependent manner. Transcriptome analysis revealed that the expression of Bcl6, a master regulator of Tfh cell differentiation, was significantly upregulated by the enforced Bcl3 expression ([Figure1][1]). The enforced Bcl3 expression increased but the Bcl3 silencing decreased IL-21-producing Tfh-like cells. Bcl3 levels were positively correlated with those of Tfh cell-related genes such as CXCR5, ICOS, and ASCL2 in CD4+ T cells in RA patients. ![Figure][2] Conclusions Bcl3 is involved in the development of Tfh cells and the pathogenesis of RA presumably by inducing IL-21 production. References 1. Saito Y, Kagami SI, Sanayama Y, Ikeda K, Suto A, Kashiwakuma D, et al. AT-rich interactive domain-containing protein 5a functions as a negative regulator of RORγt-induced Th17 cell differentiation. Arthritis Rheum. 2014;66(5):1185-94. 2. Sanayama Y, Ikeda K, Saito Y, Kagami S, Yamagata M, Furuta S, et al. Prediction of therapeutic responses to tocilizumab in patients with rheumatoid arthritis: biomarkers identified by analysis of gene expression in peripheral blood mononuclear cells using genome-wide DNA microarray. Arthritis Rheum. 2014;66(6):1241-31. Disclosure of Interest K. Meguro: None declared, K. Suzuki: None declared, J. Hosokawa: None declared, Y. Sanayama: None declared, S. Tanaka: None declared, S. Furuta: None declared, K. Ikeda: None declared, H. Takatori: None declared, A. Suto: None declared, O. Ohara: None declared, H. Nakajima Grant/research support from: Chugai Pharmaceutical Co., Ltd, Bristol-Myers Squibb, and Mitsubishi Tanabe Pharma Co. [1]: #F1 [2]: pending:yes

A 1/2.5 inch, 5.2 Mpeixel CMOS image sensor with wide dynamic range operation mode is developed and its effectiveness for high contrast scene pictures is verified. The adopted algorithm for this operation is inherently free from fixed pattern noise generation which often resists the realization of mass production level wide dynamic range image sensors. The attained dynamic range is 96 dB with 12 bit output scheme.

Background Clinical information does not accurately predict relapse after discontinuation of biological agents in patients with rheumatoid arthritis (RA). Although ultrasound is a sensitive tool to detect sub-clinical synovial inflammation in RA patients with low disease activity, the ultrasound findings on the unilateral hand did not discriminate between patients who relapsed and who did not after discontinuation of TNF antagonists in the previous report 1 . Objectives This pilot, single-blinded, prospective study aimed to determine whether the comprehensive ultrasound scan on 40 joints is informative in the prediction of relapse after discontinuation of biological agents. Methods RA patients in remission states (DAS28 ≤ 2.6) receiving biological agents who agreed to discontinue the biological agent were recruited. Patients underwent a comprehensive ultrasound scan on 40 joints (DAS28 joints + ankles + MTP joints) and were prospectively followed up for 26 weeks. The physicians who evaluated the patients during the study period were blinded to the ultrasound findings at baseline. Results Thirty patients receiving either TNF antagonists (n = 24), or tocilizumab (n = 6) were enrolled. The disease activity was very low (median DAS28 1.64 [IQR 1.2-2.3]) before the biological agent was discontinued. Eleven patients had relapse which was defined as DAS28 > 3.2 and restarted receiving the same biological agent within 26 weeks. Total ultrasound scores provided with larger areas under the ROC curves for the prediction of relapse than DAS28 did. Using the optimal cut-off values determined by the ROC analysis, the PPV and NPV of total GS score ≥ 12 to predict flare were 88% and 82%, respectively. On the other hand, the PPV and NPV of total PD score ≥ 3 were 100% and 79%, respectively. Image/graph Conclusions In RA patients with very low disease activity receiving biological agents, a comprehensive ultrasound scan provides good diagnostic values to predict relapse after discontinuation of the biological agent. References Saleem B, Keen H, Goeb V, Parmar R, Nizam S, Hensor EM, et al. Patients with RA in remission on TNF blockers: when and in whom can TNF blocker therapy be stopped? Ann Rheum Dis 2010;69(9):1636-42. Disclosure of Interest T. Iwamoto: None Declared, K. Ikeda Grant/research support from: Mitsubishi-Tanabe Pharma Corporation, Abbott Japan Co., Ltd, Consultant for: Mitsubishi-Tanabe Pharma Corporation, Abbott Japan Co., Ltd, J. Hosokawa: None Declared, M. Yamagata: None Declared, S. Tanaka: None Declared, Y. Sanayama: None Declared, D. Nakagomi: None Declared, A. Okubo: None Declared, K. Takahashi: None Declared, K. Hirose: None Declared, M. Sueishi: None Declared, H. Nakajima Grant/research support from: Mitsubishi-Tanabe Pharma Corporation, Chugai Pharmaceutical Co., Ltd, Consultant for: Mitsubishi-Tanabe Pharma Corporation, Chugai Pharmaceutical Co., Ltd

Introduction In addition to the pivotal roles of mast cells in allergic diseases, recent data suggest that mast cells play crucial roles in a variety of autoimmune responses. However, their roles in the pathogenesis of autoimmune skeletal muscle diseases have not been clarified despite their distribution in skeletal muscle. Therefore, the objective of this study is to determine the roles of mast cells in the development of autoimmune skeletal muscle diseases. Methods The number of mast cells in the affected muscle was examined in patients with dermatomyositis (DM) or polymyositis (PM). The susceptibility of mast cell-deficient WBB6F1-KitW/KitWv mice (W/Wv mice) to a murine model of polymyositis, C protein-induced myositis (CIM), was compared with that of wild-type (WT) mice. The effect of mast cell reconstitution with bone marrow-derived mast cells (BMMCs) on the susceptibility of W/Wv mice to CIM was also evaluated. Results The number of mast cells in the affected muscle increased in patients with PM as compared with patients with DM. W/Wv mice exhibited significantly reduced disease incidence and histological scores of CIM as compared with WT mice. The number of CD8+ T cells and macrophages in the skeletal muscles of CIM decreased in W/Wv mice compared with WT mice. Engraftment of BMMCs restored the incidence and histological scores of CIM in W/Wv mice. Vascular permeability in the skeletal muscle was elevated in WT mice but not in W/Wv mice upon CIM induction. Conclusion Mast cells are involved in the pathogenesis of inflammatory myopathy.