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Objective To clarify relevant proteins and clinical characteristics of a phenotype of IgG4-related disease (IgG4-RD) with lymphadenopathy. Methods We enrolled patients newly diagnosed with IgG4-RD in our department between January 2000 and June 2018 and performed proteomic analysis to measure serum concentrations of 1305 proteins. We extracted proteins overexpressed in patients with IgG4-RD with lymphadenopathy by comparing between those with lymphadenopathy, those without lymphadenopathy and healthy controls. We further reviewed all the patients with IgG4-RD in our institution and investigated the characteristics and prognosis of the patients with IgG4-RD with lymphadenopathy. Results Eighty-five patients with IgG4-RD were enrolled, of which, 55% had lymphadenopathy. Proteomic analysis in 31 patients with IgG4-RD and 6 healthy controls revealed that eotaxin-3 was a potential serum biomarker in the patients with lymphadenopathy versus those without lymphadenopathy and healthy controls. A cohort of 85 patients with IgG4-RD demonstrated that patients with lymphadenopathy showed a significantly higher serum IgG4, IgG4:IgG ratio, IgG4-RD responder index and eosinophilia (P Conclusion Lymphadenopathy in IgG4-RD represents a phenotype associated with high disease activities, eosinophilia and relapsing disease. Eotaxin-3 is a novel biomarker related to IgG4-RD with lymphadenopathy.

Interleukin (IL)-21-producing T peripheral helper (Tph) cells are thought to contribute to extra-follicular B cell activation and play a pathogenic role in autoimmune diseases. In this study, we investigated the relationship between Tph cells and interferons (IFNs) in several autoimmune diseases because our previous study demonstrated that type I IFNs promote the differentiation of IL-21-producing Tph-like cells. The frequency of Tph cells in the blood as well as serum IFN-α2a and IFN-λ1 were markedly elevated in patients with active systemic lupus erythematosus (SLE) compared to other autoimmune diseases or healthy controls. Notably, the frequency of Tph cells was positively correlated with the SLE disease activity index, serum IFN-α and serum IFN-λ1 in SLE patients. Additionally, we found that type III IFNs (IFN-λ1, IFN-λ2 and IFN-λ3) promote the differentiation of programmed cell death-1 (PD-1)+ CXCR5 −CD4+ T cells and enhance the secretion of IL-21, IFN-γ and CXCL13. IFN-λ1, like IFN-α, up-regulated the mRNA expression of IL21, IFNG, CXCL13, CD244, SLAMF7, GZMB, PRF1, CCR5 and PRDM1, whereas it down-regulated that of CXCR5 and BCL6, reflecting a Tph-related gene expression pattern. IFN-α in combination with IFN-λ1, IFN-λ2 or IFN-λ3 significantly increased the differentiation of PD-1+CXCR5− Tph-like cells and the secretion of Tph-related cytokines as compared with each IFN alone, suggesting a cooperative interaction. From these findings, it is highly probable that type III IFNs in addition to type I IFNs play a key role in the differentiation of Tph cells and that high levels of IFN-α and IFN-λ1 trigger the differentiation and expansion of Tph cells in SLE.

Background and Purpose We conducted preclinical and clinical studies to examine the pharmacological, particularly cardiac, effects of amiselimod (MT-1303), a second-generation sphingosine 1-phosphate (S1P) receptor modulator, designed to reduce the bradycardia associated with fingolimod and other S1P receptor modulators. Experimental Approach The selectivity of the active metabolite amiselimod phosphate (amiselimod-P) for human S1P receptors and activation of G-protein-coupled inwardly rectifying K+ (GIRK) channels in human atrial myocytes were assessed. Its cardiac distribution was determined in rats, and cardiovascular telemetry was assessed in monkeys. We also examined the pharmacokinetics, pharmacodynamics and safety of amiselimod in healthy humans. Key Results Amiselimod-P showed potent selectivity for S1P1 and high selectivity for S1P5 receptors, with minimal agonist activity for S1P4 and no distinct agonist activity for S1P2 or S1P3 receptors and approximately five-fold weaker GIRK activation than fingolimod-P. After oral administration of amiselimod or fingolimod at 1 mg·kg−1, the concentration of amiselimod-P in rat heart tissue was lower than that of fingolimod-P, potentially contributing to the minimal cardiac effects of amiselimod. A telemetry study in monkeys confirmed that amiselimod did not affect heart rate or ECG parameters. In healthy human subjects, peripheral blood lymphocyte counts gradually reduced over the 21 day dosing period, with similar lymphocyte count profiles with the highest doses by day 21, and no clinically significant bradycardia observed on day 1 or during the study. Conclusions and Implications Amiselimod exhibited potent therapeutic efficacy with minimal cardiac effects at the anticipated clinical dose and is unlikely to require dose titration.

Amiselimod (MT-1303) is a novel and selective sphingosine 1-phosphate receptor-1 (S1P1) modulator with a more favorable cardiac safety profile than other S1P1 receptor modulators. In this study, we evaluated the effects of MT-1303 on the progression of lupus nephritis in two well-known murine systemic lupus erythematosus (SLE) models, MRL/lpr and NZBWF1 mice, compared with those of FK506. Daily oral doses of 0.1 and 0.3 mg/kg MT-1303 not only inhibited the development of lupus nephritis when administered before onset in MRL/lpr and NZBWF1 mice but also improved symptoms of lupus nephritis when administered after onset in MRL/lpr mice. Its efficacy in these models was more potent or comparable to that of FK506 (1 and 3 mg/kg). In histological analysis, treatment with MT-1303 inhibited infiltration of T cells into the kidneys, mesangial expansion, and glomerular sclerosis. MT-1303 treatment resulted in a marked reduction in T cells and B cells in the peripheral blood and significantly inhibited increases in the number of plasma cells in the spleen and T cells in the kidneys. In addition, administration of MT-1303 suppressed elevations in serum anti-dsDNA antibody levels in MRL/lpr mice, but not in NZBWF1 mice. Our findings show that MT-1303 exhibits marked therapeutic effects on lupus nephritis in two SLE models, likely by reducing the infiltration of autoreactive T cells into the kidneys. These results suggest that MT-1303 has the potential to be used as a therapeutic agent for patients suffering from SLE, including lupus nephritis.

Background IgG4-related disease (IgG4-RD) is an immune-mediated systemic disease characterized by the enlargement of multiple organs. IgG4-RD frequently involves the pancreas, lacrimal glands, salivary glands, bile duct, kidneys, lungs, and retroperitoneum, but can potentially affect any organs. Lymphadenopathy is often seen in patients with IgG4-RD along with other organ involvements, but the significance of the exist of lymphadenopathy in IgG4-RD is unclear. Objectives The aim of this study is to clarify the characteristics of patients with IgG4-RD who have lymphadenopathy. Methods We conducted cluster analysis with clinical data in consecutive 85 patients with untreated active IgG4-RD in our institute. We divided the patients into two groups who had lymphadenopathy or not (lymph group and non-lymph group), and compared clinical characteristics between the two groups. We counted the areas of swollen lymph nodes and performed correlation analysis with clinical characteristics. We also comprehensively screened serum protein concentrations in IgG4-RD patients (n=15) and healthy controls (n=5) by using high-throughput proteomic analysis, and differentially expressed proteins were extracted. We compared the concentrations of serum proteins which were extracted between lymph group and non-lymph group. Results Among 85 patients with IgG4-RD, 44 (52%) were male. Median age was 63 years old. Forty-seven (55%) patients were lymph group. Cluster analysis revealed that lymphadenopathy was in a cluster with serum IgG4, IgG4/IgG, IgG4-RD responder index (IgG4-RD RI), soluble interleukin-2 receptor (sIL-2R), eosinophilia, erythrocyte sedimentation rate (ESR), lung involvement, hemoglobin, albumin, C3 and C4. In lymph group, serum IgG4, IgG4/IgG, IgG4-RD RI, eosinophilia, sIL-2R and ESR were significantly higher than non-lymph group (Figure). Spearman’s correlation analysis revealed that areas of the swollen lymph nodes were positively correlated with serum IgG4, IgG4/IgG, sIL-2R, eosinophilia. Proteomics analysis revealed that 68 proteins were significantly higher in IgG4-RD (p Conclusion Lymphadenopathy in IgG4-RD is associated with disease activity and eosinophilia. Proteomics analysis can provide some clues for elucidating the significance of lymphadenopathy in IgG4-RD. References [1] Stone JH, Zen Y, Deshpande V. IgG4-related disease. N Engl J Med2012;366:539–51. [2] Bookhout CE, Rollins-Raval MA. Immunoglobulin G4-Related Lymphadenopathy. Surg Pathol Clin. 2016;9:117-29. Disclosure of Interests Satoshi Takanashi: None declared, Jun Kikuchi: None declared, Takanori Sasaki: None declared, Mitsuhiro Akiyama: None declared, Yuko Kaneko Grant/research support from: Abbvie, Eisai, Speakers bureau: AbbVie, Astellas, Ayumi, Bristol-Myers Squibb, Chugai, Eisai, Eli Lilly, Jansen, Kissei, Pfizer, Sanofi, Takeda, Tanabe-Mitsubishi, UCB, Hidekata Yasuoka: None declared, Katsuya Suzuki: None declared, Kunio Sugahara: None declared, Tsutomu Takeuchi Grant/research support from: Astellas Pharma Inc, Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Takeda Pharmaceutical Co., Ltd., AbbVie GK, Asahikasei Pharma Corp., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Eisai Co., Ltd., AYUMI Pharmaceutical Corporation, Nipponkayaku Co. Ltd., Novartis Pharma K.K., Grant/research support from: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Grant/research support from: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Abbivie GK, Nipponkayaku Co.Ltd, Janssen Pharmaceutical K.K., Astellas Pharma Inc., Taiho Pharmaceutical Co. Ltd., Chugai Pharmaceutical Co. Ltd., Taisho Toyama Pharmaceutical Co. Ltd., GlaxoSmithKline K.K., UCB Japan Co. Ltd., Consultant for: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Asahi Kasei Medical K.K., AbbVie GK, Daiichi Sankyo Co., Ltd., Bristol Myers Squibb, and Nipponkayaku Co. Ltd., Speakers bureau: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Speakers bureau: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Speakers bureau: AbbVie GK., Bristol–Myers K.K., Chugai Pharmaceutical Co. Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Astellas Pharma Inc, Diaichi Sankyo Co. Ltd., Eisai Co. Ltd., Sanofi K.K., Teijin Pharma Ltd., Takeda Pharmaceutical Co. Ltd., Novartis Pharma K.K.

Amiselimod (MT-1303) is a novel sphingosine 1-phosphate receptor-1 (S1P1 receptor) modulator with a more favorable cardiac safety profile than other S1P1 receptor modulators. MT-1303 phosphate (MT-1303-P), an active metabolite of MT-1303, exhibits S1P1 receptor agonism at a lower EC50 value than other S1P1 receptor modulators currently being developed. We aimed to evaluate the efficacy of MT-1303 and its mode of action in chronic colitis using an inflammatory bowel disease (IBD) model. Oral administration of MT-1303 (0.3 mg/kg) once daily for 3 days to mice almost completely abolished S1P1 receptor expression on CD4+ T cells from mesenteric lymph nodes, which corresponded to a marked decrease in CD4+ T cell count in peripheral blood, indicating that MT-1303-P acts as a functional antagonist of the S1P1 receptor. The potential benefit of MT-1303 for IBD was assessed using immunodeficient SCID mice with chronic colitis induced by adoptive transfer of CD4+CD45RBhigh T cells from BALB/c mice. An oral dose of 0.1 and 0.3 mg/kg MT-1303 administered daily one week after the cell transfer inhibited the development of chronic colitis with an efficacy comparable to that of an anti-mTNF-α mAb (250 μg/mouse). In addition, MT-1303 administration significantly reduced the number of infiltrating Th1 and Th17 cells into the lamina propria of the colon in colitis mice. Our results suggest that MT-1303 acts as a functional antagonist of the S1P1 receptor on lymphocytes, regulates lymphocyte trafficking, and inhibits infiltration of colitogenic Th1 and Th17 cells into the colon to inhibit the development of chronic colitis.

Background Herpes virus infections (HVIs) including cytomegalovirus infections (CMVs) and herpes zoster (HZ) remains as major complications during treatments with immunosuppressant (IS) in patients with autoimmune diseases.1 2 Previous reports have suggested the associations between virus infections and characteristics of T cells.3 4 Objectives To elucidate the characteristics of peripheral immune cells associated with risk factors of HVIs during induction therapies in patients with active lupus nephritis (LN). Methods Standardised peripheral immunophenotyping was performed using flow cytometry in active LN and ANCA-associated vasculitis (AAV) patients starting induction therapy and also with inactive LN patients with maintenance therapy between April 2015 to April 2017. The definition of HVIs was the infection necessary to administer anti-viral agents. Results Sixty-two LN patients and 11 AAV patients were enrolled. Among 30 active LN patients, 27 were analysed except for 3 patients (2 died and 1 withdrawn consent). Mean age was 41.7 years, 9 patients (33%) had newly-onset, and mean SLE disease activity index (SLEDAI) was 19.3. All active LN patients were treated with prednisolone (PSL) (mean 51.7 mg/day) and 25 were treated with an additional IS (cyclophosphamide [CYC];13, mycophenolate mofetil;8, tacrolimus;3, rituximab [RTX];1). Six (22.2%) patients developed HVIs (5 CMVs and 1 HZ) within 3 months following induction therapy. None of the 32 LN patients in maintenance phase (mean age, 54.8; SLEDAI, 2.5; PSL 2.5 mg/day) developed HVIs during the mean 2.8 years-observational period. Two (18.2%) AAV patients developed HVIs within 3 months following induction therapy. All AAV patients (mean age, 64.3) were treated with PSL (mean 41.8 mg/day) and 10 with IS (CYC;5, RTX;3, azathioprine;1, methotrexate;1). Among active LN patients, univariate analysis revealed that older age, lower proportions of naive CD8 +T cells, higher proportions of effector CD8 +T cells and HLA-DR +regulatory T cells (Tregs) at baseline and lower naive CD8 +T cells at month 3 associated with HVIs (p=0.011, p Among AAV patients, univariate analysis showed that older age, lower proportions of naive CD8 +T cells, higher proportions of Tregs at baseline associated with HVIs. However, multivariate analysis showed no independent risk factor for HVIs among them. Conclusions Our results suggest that active LN patients with low proportion of naive CD8 +T cells and high HLA-DR +Tregs at the time of induction therapy should be closely monitored for HVIs. The different results between LN and AAV implicated the different risks of HVIs by immunophenotyping. Larger prospective study is desired to confirm our results. References [1] Takizawa Y. et al. Rheumatology (Oxford). 2008;47:1373–8. [2] Chakravarty EF, et al. Lupus. 2013;22:238–44. [3] Weltevrede M, et al. Exp Gerontol. 2016;77:87–95. [4] Egli A, et al. PLoS One. 2012;7:e43937. Disclosure of Interest J. Kikuchi: None declared, M. Ushikubo: None declared, S. Saito: None declared, H. Yasuoka: None declared, K. Yamaoka: None declared, H. Tsujimoto Employee of: Mitsubishi Tanabe Pharma Corporation, K. Sugahara Employee of: Mitsubishi Tanabe Pharma Corporation, T. Takeuchi Grant/research support from: Mitsubishi Tanabe Pharma Corporation

Background We reported that BAFF robustly increases IL-6 production by peripheral monocytes of patients with primary Sjogren’s syndrome (pSS) and that the BAFF-induced IL-6 production and serum IgG levels of the patients were positively correlated with the expression level of a BAFF receptor (BR3) in pSS monocytes. Moreover, we found that IgG production by pSS B cells in vitro was significantly enhanced by BAFF-stimulated monocytes. These results collectively suggest that BAFF signalling via BR3 is involved in IgG overproduction and that is a possible therapeutic target to treat autoimmune diseases, such as pSS. We successfully discovered a pyrroloprymidine derivative, BIK-13, which inhibits BAFF binding to BR3 by our original high throughput screening. Objectives To explored the possibility of BIK-13 as a drug to treat autoimmune diseases. Methods Human PBMC were stimulated in vitro with a mixture of soluble BAFF (sBAFF), recombinant human IL-21, and anti-IgM and anti-CD40 antibodies (‘multiple stimulation’) to differentiate B cells into plasma blasts and/or plasma cells. BIK-13 was added to the culture and the differentiation of B cells was monitored by the expression levels of CD19/CD38/IgD and Activation-induced cytidine deaminase (AID) analysed by FACS and quantitative RT-PCR, respectively. The amounts of IL-6, IL-10 and IgG in the culture supernatants were measured by ELISA. BIK-13 was administered intraperitoneally to MRL/lpr mice and serum levels of an anti-dsDNA antibody, IL-6 and IL-10 were measured by ELISA. The proportion of B cells among splenic lymphocytes of the mice was analysed by FACS. Production of cytokines in vitro by stimulated splenic lymphocytes was measured by ELISA. Infiltration of lymphocytes into organs was analysed by immunohistochemistry. Results FACS analysis of multiple-stimulated human PBMC indicated that differentiation of B cells into plasma blasts and/or plasma cells was inhibited by BIK-13 in a dose dependent manner. Interestingly, increased IgG, IL-6 and IL-10 production by the stimulated PBMC were concomitantly and significantly suppressed by BIK-13. In addition, the expression level of AID in the cells was also suppressed by BIK-13, suggesting that an IgG class switching was impaired. Administration of BIK-13 to MRL/lpr mice for 16 weeks decreased the serum levels of an anti-dsDNA antibody, IL-6 and IL-10 as compared to the control. Notably, immunohistochemical analysis revealed that infiltration of B cells into salivary and lacrimal glands was remarkably suppressed in BIK-13-treated mice. Moreover, the proportion of B cells among splenic lymphocytes was also decreased in BIK-13-treated mice as compared to the control. In addition, production of IL-6 and IL-10 by activated splenic lymphocytes of the BIK-13-treated mice was also remarkably suppressed as compared to control mice. Conclusions Our data collectively suggest that BIK-13, a low molecular weight BAFF-signalling inhibitor, suppresses B cell activation/differentiation in vitro and consequently inhibits production of IgG and cytokines, such as IL-6 and IL-10, by the cells. The compound inhibited infiltration of B cells into organs of model mice of autoimmune diseases. These data suggest that the compound is a promising drug candidate to treat autoimmune diseases, such as pSS. Disclosure of Interest K. Yoshimoto: None declared, N. Seki Employee of: Mitsubishi Tanabe Pharma Corporation, K. Suzuki: None declared, K. Sugahara Employee of: Mitsubishi Tanabe Pharma Corporation, K. Chiba Employee of: Mitsubishi Tanabe Pharma Corporation, T. Takeuchi Grant/research support from: Mitsubishi Tanabe Pharma Corporation

Background In our previous study, we reported that soluble BAFF (sBAFF) robustly enhanced IL-6 production by peripheral monocytes of patients with primary Sjogren9s syndrome (pSS) and that the expression level of a BAFF receptor (BR3) was significantly elevated in pSS monocytes. We also found that the proportion of BR3-positive monocytes to total monocytes was positively and significantly correlated with the serum IgG level of pSS patients. Investigation of the interaction of monocytes and B cells showed that IgG production by B cells was enhanced by sBAFF-stimulated monocytes. These data collectively suggest that the elevated expression of BR3 on monocytes is involved in IgG overproduction by B cells which is often observed in pSS, and that BAFF signaling via BR3 is a possible therapeutic target to treat pSS. A high throughput screening of a low molecular weight compound library successfully discovered two pyrrolopryimidine derivatives, BIK12 and BIK13, which inhibit sBAFF binding to BR3. We found that these compounds inhibited not only IL-6 production by BAFF-stimulated monocytes, but also IgG production by B cells co-cultured with the monocytes. Objectives To elucidate the mechanism of inhibitory activities of these compounds on BAFF signaling pathways, we measured production of IL-10 as well as IL-6 by monocytes in vitro, both of which work for B cell activation. In addition, we analyzed in vivo effects of the compounds on production of autoantibody and cytokines in autoimmune disease model mice. Methods Peripheral monocytes and B cells were prepared from healthy individuals. The monocytes were stimulated with sBAFF and cultured in vitro with or without peripheral B cells in the presence of BIK-12 or BIK-13. The amounts of IL-6, IL-10 and IgG were measured by ELISA. BIK-13 was administered intraperitoneally to MRL/lpr mice, an animal model of autoimmune diseases, three times a week for 6 months. Serum levels of an anti-ds DNA antibody, IL-6 and IL-10 were measured by ELISA. Results sBAFF enhanced the production of not only IL-6, but also IL-10 by peripheral monocytes in vitro. As expected, BIK12 and BIK-13 significantly suppressed production of IL-6 and IL-10 by BAFF-stimulated monocytes in vitro in a dose dependent manner. Notably, IgG production by B cells co-cultured in vitro with sBAFF-stimulated monocytes was significantly suppressed by these compounds. The compounds did not exhibit toxicities to the cells in the dose range. These data suggest that BAFF signaling via BR3 lead to production of IL-6 and IL-10 in monocytes, and the cytokines in turn mediates the signal to B cells resulting in IgG overproduction. Interestingly, serum levels of an anti-dsDNA antibody, IL-6 and IL-10 in MRL/lpr mice received BIK13 simultaneously declined as compared to control mice. These data collectively suggest that the compound was also efficacious in vivo. Conclusions BIK-13, a pyrrolopyrimidine derivative, suppressed IgG production by B cells through affecting monocytes. IL-6 and/or IL-10 may intermediate the effect. Our findings strongly suggest that BAFF signaling via BR3 is a possible therapeutic target for drug discovery to treat pSS or other intractable autoimmune diseases which accompany hypergammaglobulinemia. Moreover, these compounds may provide novel tools to explore the pathological mechanism of the development of these autoimmune diseases. Disclosure of Interest K. Yoshimoto: None declared, K. Suzuki: None declared, K. Sugahara Employee of: Mitsubishi Tanabe Pharma Corporation, T. Takeuchi Grant/research support from: Mitsubishi Tanabe Pharma Corporation

Background Although the mainstream of pathogenesis of systemic lupus erythematosus (SLE) is thought to be interactions between antigen-presenting cells like dendritic cells, helper T cells, B cells and cytokines, previous reports suggested CD8 T cells also involve in the pathogenesis of SLE1,2. However the associations between subsets of CD8 T cells and clinical manifestations remains unclear. Objectives We conducted standard immunophenotyping analysis with peripheral blood from SLE patients and focused on CD8 T cell subsets to elucidate the association with clinical phenotype and serological markers. Methods Peripheral blood was obtained from inactive SLE patients and healthy subjects as controls and also from active SLE before and 3 months after treatment. CD8 T cell subsets were measured by flow cytometry with fresh whole blood samples. Results Thirty-four active SLE patients and 38 inactive patients and 22 healthy controls (HCs) whose age and sex were matched with those in SLE patients were enrolled. Mean SLE disease activity index (SLEDAI) was 14.2 and 1.8 in active and inactive patients, respectively. Among CD8 T cell subsets, the proportion of HLA-DR+ cells was significantly higher in SLE patients than HCs and positively correlated with SLEDAI (p=0.016, rho =0.283), and was also higher in patients with nephritis than patients without nephritis (p=0.074), though it did not reach statistical significance. The proportion of naive CD8 T cells positively correlated with the titer of anti-dsDNA antibody (p=0.011, rho =0.30) and C1q immune complex levels (p=0.043, rho =0.25), and negatively correlated with serum complement levels (p=0.019, rho = -0.34). The proportion of central memory CD8 T cells (Tcm) negatively correlated with SLEDAI (p Conclusions Pathological state of SLE positively correlated with the proportion of naive CD8 T cells and negatively correlated with the proportion of Tem and these subsets were affected by treatment with PSL and immunosuppressant particularly cyclophosphamide. These results indicate that CD8 T cells are involved in pathophysiology and could potentially become a biomarker or a treatment target in SLE patients. References Blanco P, et al. Arthritis Rheum 2005; 52: 201–11. Kim JS, et al. Rheumatology (Oxford) 2012; 51: 1587–94. Disclosure of Interest J. Kikuchi: None declared, M. Ushikubo: None declared, S. Saito: None declared, K. Yamaoka: None declared, K. Sugahara Employee of: Mitsubishi Tanabe Pharma Corporation, K. Chiba Employee of: Mitsubishi Tanabe Pharma Corporation, T. Takeuchi: None declared

Sphingosine 1-phosphate (S1P), a multi-functional phospholipid mediator, is generated from sphingosine by sphingosine kinases and binds to five known G protein-coupled S1P receptors (S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 ). It is widely accepted that S1P receptor 1 (S1P 1 ) plays an essential role in lymphocyte egress from the secondary lymphoid organs (SLO) and thymus, because lymphocyte egress from these organs to periphery is at extremely low levels in mice lacking lymphocytic S1P 1 . Fingolimod hydrochloride (FTY720) is a first-in-class, orally active S1P 1 functional antagonist which was discovered by chemical modification of a natural product, myriocin. Since FTY720 has a structure closely related to sphingosine, the phosphorylated FTY720 (FTY720-P) is converted by sphingosine kinases and binds 4 types of S1P receptors. FTY720-P strongly induces down-regulation of S1P 1 by internalization and degradation of this receptor and acts as a functional antagonist at S1P 1 . Consequently, FTY720 inhibits S1P 1 -dependent lymphocyte egress from the SLO and thymus to reduce circulating lymphocytes including autoreactive Th17 cells, and is highly effective in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). In relapsing remitting MS patients, oral FTY720 shows a superior efficacy when compared to intramuscular interferon-β-1a. Based on these data, it is presumed that modulation of the S1P-S1P 1 axis provides an effective therapy for autoimmune diseases including MS.

Background and Purpose It is well known that BAFF (B cell activating factor) signaling is involved in the pathogenesis of autoimmune diseases. To search for specific inhibitors against BAFF signaling, we have established our original high throughput screening system and discovered a pyrroloprymidine derivative, BIK-13, which inhibits BAFF binding to its receptor, BR3. In this study, we explored the possibility of BIK-13 as a drug to treat autoimmune diseases. Methods Human PBMC were stimulated in vitro with a mixture of BAFF, IL-21, and anti-IgM and anti-CD40 antibodies in the presence of BIK-13. Differentiation of B cells and the expression level of Activation-induced cytidine deaminase (AID) in the cells were analyzed by FACS and qPCR, respectively. The amounts of IL-6, IL-10 and IgG in the culture supernatants were measured by ELISA. Serum levels of an anti-dsDNA antibody, IL-6 and IL-10 in BIK-13-treated autoimmune model mice, MRL/lpr, were measured by ELISA. The proportion of B cells among splenocytes and infiltration of lymphocytes into organs of the mice were analyzed by FACS and immunohistochemistry, respectively. Results In vitro experiments revealed that BIK-13 suppressed 1) differentiation of B cells, 2) AID expression and 3) IL-6, IL-10 and IgG production in a dose dependent manner. BIK-13 decreased the serum levels of an anti-dsDNA antibody, IL-6 and IL-10 in MRL/lpr mice. Notably, infiltration of B cells into lacrimal glands and the proportion of B cells among splenocytes were remarkably suppressed in BIK-13-treated mice. Conclusion Our data collectively suggest that BIK-13, a low molecular weight BAFF-signaling inhibitor, is a promising drug candidate for autoimmune diseases by suppressing B cell activation.

Sphingosine 1-phosphate (S1P) and S1P receptor 1 (S1P1) play an important role in the egress of mature CD4 or CD8 single-positive (SP) thymocytes from the thymus. Fingolimod hydrochloride (FTY720), an S1P1 functional antagonist, induced significant accumulation of CD62LhighCD69low mature SP thymocytes in the thymic medulla. Immunohistochemical staining using anti-S1P1 antibody revealed that S1P1 is predominantly expressed on thymocytes in the thymic medulla and is strongly down-regulated even at 3h after FTY720 administration. 2-Acetyl-4-tetrahydroxybutylimidazole (THI), an S1P lyase inhibitor, also induced accumulation of mature SP thymocytes in the thymic medulla with an enlargement of the perivascular spaces (PVS). At 6h after THI administration, S1P1-expressing thymocytes reduced partially as if to form clusters and hardly existed in the proximity of CD31-expressing blood vessels in the thymic medulla, suggesting S1P lyase expression in the cells constructing thymic medullary PVS. To determine the cells expressing S1P lyase in the thymus, we newly established a mAb (YK19-2) specific for mouse S1P lyase. Immunohistochemical staining with YK19-2 revealed that S1P lyase is predominantly expressed in non-lymphoid thymic stromal cells in the thymic medulla. In the thymic medullary PVS, S1P lyase was expressed in ER-TR7-positive cells (reticular fibroblasts and pericytes) and CD31-positive vascular endothelial cells. Our findings suggest that S1P lyase expressed in the thymic medullary PVS keeps the tissue S1P concentration low around the vessels and promotes thymic egress via up-regulation of S1P1.

Phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 70-kDa ribosomal protein S6 kinase (S6K1) in the rat liver increased in proportion to the amount of leucine administered, ranging from 0.169 to 1.35 g/kg of body weight. In the skeletal muscle, phosphorylation of these factors reached a plateau at 0.675 g/kg of body weight. The sensitivity of mammalian target of rapamycin (mTOR) signaling to leucine in the skeletal muscle appeared to be higher than that in the liver.

Infiltration of myelin-specific helper T (Th) cells into the central nervous system (CNS) plays a key role in pathogenesis of experimental autoimmune encephalomyelitis (EAE). In this study, we investigated the involvement of sphingosine 1-phosphate (S1P)-S1P receptor 1 (S1P1) axis in lymphocytes for EAE development when C57BL/6 mice were immunized with myelin oliogodendrocyte glycoprotein (MOG). The expression of S1P1 mRNA and S1P responsiveness of lymphocytes in draining lymph nodes (DLN) were down-regulated markedly after MOG immunization until onset of EAE. Accompanying with reacquisition of down-regulated S1P1 transcript and S1P responsiveness in DLN lymphocytes, MOG-immunized mice developed EAE symptoms with significant infiltration of Th1 and Th17 cells into the CNS and a marked elevation of IFN-γ, T-bet, IL-17, and RORγt mRNA expressions. Prophylactic administration of an S1P1 functional antagonist, fingolimod hydrochloride (FTY720, 0.3 mg/kg, orally) significantly inhibited EAE development and almost completely prevented infiltration of Th1 and Th17 cells into the CNS with a marked reduction of IFN-γ, T-bet, IL-17, and RORγt mRNA expressions. Similar results were obtained by treatment with an S1P1-selective agonist, SEW2871 or an S1P lyase inhibitor, 2-acetyl-4-tetrahydroxybutylimidazole. Moreover, FTY720-phosphate and SEW2871 inhibited in vitro migration of Th1 and Th17 cells toward S1P but did not affect cytokine production or generation of Th1 or Th17 cells. These results suggest that reacquisition of S1P1 expression in DLN lymphocytes plays a major role in trafficking of myelin antigen-specific Th1/Th17 cells from DLN to the CNS in EAE and that prophylactic effect of FTY720 on EAE is predominantly caused by functional antagonism via lymphocytic S1P1.

We investigated the mechanism underlying central glucagon-induced hyperglycemia and anorexia in chicks. Male 8-day-old chicks (Gallus gallus) were used in all experiments. Intracerebroventricular administration of glucagon in chicks induced hyperglycemia and anorexia from 30 min after administration. However, the plasma insulin level did not increase until 90 min after glucagon administration, suggesting that glucose-stimulated insulin secretion from pancreatic beta cells may be suppressed by central glucagon. The plasma corticosterone concentration significantly increased from 30 min to 120 min after administration, suggesting that central glucagon activates the hypothalamic pituitary adrenal (HPA) axis in chicks. However, central administration of corticotropin-releasing factor (CRF), which activates the HPA axis in chicken hypothalamus, significantly reduced not only food intake but also plasma glucose concentration, suggesting that CRF and the activation of the HPA axis are related to the glucagon-induced anorexia but not hyperglycemia in chicks. Phentolamine, an α-adrenergic receptor antagonist, significantly attenuated the glucagon-induced hyperglycemia, suggesting that glucagon induced hyperglycemia at least partly via α-adrenergic neural pathway. Co-administration of phentolamine and α-helical CRF, a CRF receptor antagonist, significantly attenuated glucagon-induced hyperglycemia and anorexia. It is therefore likely that central administration of glucagon suppresses food intake at least partly via CRF-induced anorexigenic pathway in chicks.

1. This study was conducted to examine whether oral administration of lysine solution affect food intake and the ventromedial hypothalamic (VMH) monoamines in chickens fed on a lysine-free diet. 2. Chickens were assigned to four treatment groups. Two groups of chickens were given two different doses of lysine solution (0.1 g and 0.07 g in 1 ml of saline) exogenously (orally) while being fed on a lysine-free diet, and these results were compared with a control diet plus saline group. Another group of chickens was fed on a lysine-free diet without lysine supplementation, and their results were compared with the lysine treated groups. The extracellular dopamine (DA), norepinephrine (NE) and serotonin (5-HT) in the VMH of freely moving chicken were measured by in vivo microdialysis. 3. There was no significant difference in food intake between the control diet and the lysine supplemented groups during the time-course of the experiments. Food intake significantly decreased at 4, 5 and 6 h in the lysine-free diet plus saline group compared with the lysine supplemented groups. Of the VMH monoamines, the DA concentration remained close to the baseline in the lysine supplemented groups. This DA concentration was significantly lower than the baseline in the lysine-free diet plus saline group at 3.5 h and thereafter. 4. No significant difference from the baseline was observed for NE in the lysine-free diet plus saline group. The 5-HT concentrations were close to the baseline for all groups throughout the experiments. 5. The findings suggest that oral administration of lysine solution to chickens fed on a lysine-free diet restored food intake which was associated with the variations of VMH DA concentration.

Background and Purpose We conducted preclinical and clinical studies to examine the pharmacological, particularly cardiac, effects of amiselimod (MT‐1303), a second‐generation sphingosine 1‐phosphate (S1P) receptor modulator, designed to reduce the bradycardia associated with fingolimod and other S1P receptor modulators. Experimental Approach The selectivity of the active metabolite amiselimod phosphate (amiselimod‐P) for human S1P receptors and activation of G‐protein‐coupled inwardly rectifying K+ (GIRK) channels in human atrial myocytes were assessed. Its cardiac distribution was determined in rats, and cardiovascular telemetry was assessed in monkeys. We also examined the pharmacokinetics, pharmacodynamics and safety of amiselimod in healthy humans. Key Results Amiselimod‐P showed potent selectivity for S1P1 and high selectivity for S1P5 receptors, with minimal agonist activity for S1P4 and no distinct agonist activity for S1P2 or S1P3 receptors and approximately five‐fold weaker GIRK activation than fingolimod‐P. After oral administration of amiselimod or fingolimod at 1 mg·kg−1, the concentration of amiselimod‐P in rat heart tissue was lower than that of fingolimod‐P, potentially contributing to the minimal cardiac effects of amiselimod. A telemetry study in monkeys confirmed that amiselimod did not affect heart rate or ECG parameters. In healthy human subjects, peripheral blood lymphocyte counts gradually reduced over the 21 day dosing period, with similar lymphocyte count profiles with the highest doses by day 21, and no clinically significant bradycardia observed on day 1 or during the study. Conclusions and Implications Amiselimod exhibited potent therapeutic efficacy with minimal cardiac effects at the anticipated clinical dose and is unlikely to require dose titration.

A Research of the situation of Gangi Arcade remained at present time in Japan were analyzed on following 2 aspects.1. Research by Dr. Takesi-Ujiie (1966-85) showed there were 46 cities which had Gangi Arcades. However, in the 2007-2008 years, there are 22 cities in Japan, which has Gangi Arcade.2. Gangi Arcade was disappeared by following reason. 1) Gangi Arcade was changed to modern Arcade 2) Gangi Arcade was used by town peoples 3) Removal of Gangi Arcade

Fingolimod (FTY720), a sphingosine 1-phosphate (S1P) receptor modulator, inhibits S1P-dependent lymphocyte egress from secondary lymphoid organs and is highly effective in experimental autoimmune encephalomyelitis (EAE) in mice. In this study, we directly compared the therapeutic effects of FTY720 and recombinant mouse interferon (rm-IFN)-β on relapse and progression of EAE in mice. When FTY720 at oral dose of 0.03 to 1 mg/kg was administered daily after establishment of EAE induced by myelin proteolipid protein (PLP) in SJL/J mice, relapse of EAE was significantly inhibited during administration period. Subcutaneous injection of rm-IFN-β (10,000 IU/mouse) also inhibited the relapse of EAE at early period; however EAE was relapsed in all the mice within administration period. Therapeutic administration of FTY720 (0.03 to 1 mg/kg) significantly improved the symptoms of chronic EAE induced by myelin oligodendrocyte glycoprotein in C57BL/6 mice whereas rm-IFN-β (10,000 IU/mouse) showed no clear effect. These results indicate that FTY720 is more efficacious in mouse EAE as compared with rm-IFN-β. FTY720 markedly reduced the frequency of PLP-specific Th17 and Th1 cells in the spinal cord of EAE mice. On the contrary, FTY720 increased the frequency of PLP-specific Th17 and Th1 cells in the inguinal lymph nodes, suggesting inhibition of egress of myelin antigen-specific Th cells from draining lymph nodes. From these results, the ameliorating effects of FTY720 on EAE are likely due to reduction of infiltration of myelin antigen-specific Th17 and Th1 cells into the central nervous system.

Backgrounds and Purpose We have demonstrated that the elevated expression of BAFF receptor (BR3) on monocytes is involved in the overproduction of IgG by B cells in patients with primary Sjögren’s syndrome (pSS) which is often accompanied with hypergammaglobulinemia. We discovered a low molecular weight compound, BIK13, which inhibits binding of BAFF to BR3, by our original high-throughput screening system. We found that BIK-13 suppressed IL-6 production by BAFF-stimulated pSS monocytes. In this study, we investigated inhibitory effects of BIK-13 on B cell functions. Methods PBMCs were cultured with a B cell-stimulants cocktail, such as anti-IgM and CD40 antibodies, human IL-21 and human BAFF, in the presence of BIK-13. B cell-differentiation was monitored through analysis of the expression levels of CD19/CD38/IgD and activation-induced cytidine deaminase (AID) by FACS and qPCR, respectively. The amounts of IL-6 and IgG produced in vitro by the cells were measured by ELISA. Results Increased production of IgG and IL-6 by PBMC upon stimulation with B cell-stimulants was suppressed by BIK13 in a dose dependent manner. FACS analysis indicated that differentiation of B cells into plasma blasts and/or plasma cells was inhibited by BIK-13. In addition, the expression level of AID was also suppressed by the compound, suggesting that the IgG class switching was impaired. Conclusion These data collectively suggest that BIK-13 suppresses the B cell functions and may provide a novel therapeutic possibility to treat autoimmune diseases such as pSS.

Sphingosine 1-phosphate (S1P) and its receptor, S1P receptor type 1 (S1P(1)), are essential for lymphocyte egress from secondary lymphoid organs (SLO). Fingolimod (FTY720), the S1P receptor modulator, inhibits lymphocyte egress from SLO and decreases circulating lymphocytes; however, it also induces a significant decrease in the number of peripheral blood lymphocytes in alymphoplasia (aly/aly) mice lacking SLO. In this study, we demonstrated that the administration of FTY720 induced sequestration of mature lymphocytes, particularly T cells, into the bone marrow (BM) in aly/aly mice, implying that the reduction of circulating lymphocytes in these mice by FTY720 was due to inhibition of lymphocyte egress from the BM. Since sequestration of mature T cells into the BM was also induced in normal mice by selective S1P(1) agonist or S1P lyase inhibitor, it is suggested that S1P(1) expression and the S1P gradient play an important role in egress of mature T cells from the BM. Prophylactic administration of FTY720 to ovalbumin (OVA)-immunized mice significantly inhibited footpad swelling induced by OVA challenging with a marked reduction of OVA-specific T(h) cells in the BM, indicating that immunomodulation by FTY720 is likely due to reduced circulation of antigen-specific T(h) cells. On the other hand, OVA-specific T(h) cells, like naive T cells, were also sequestered into the BM and SLO of OVA-immunized mice by a short exposure of FTY720 after OVA challenging. These results suggest that the S1P-S1P(1) axis plays a regulatory role in egress of mature T cells including antigen-specific T(h) cells from the BM.

A series of 2-substituted 2-aminopropane-1,3-diols having a biphenyl moiety and their phosphate esters were synthesized to obtain sphingosine 1-phosphate receptor-1 (S1P(1)) receptor agonists with potent immunomodulatory activity accompanied by little or no effect on heart rate. Many of the synthesized compounds sufficiently decreased the number of peripheral blood lymphocytes. Some of the phosphates had potent agonism at S1P(1) but no agonism at S1P(3), which had been reported to be a receptor responsible for heart rate reduction. Although high S1P(1)/S1P(3) selectivity was considered to be favorable to reduce the effect on heart rate, almost all the phosphates showed a remarkable heart rate lowering effect in vivo. The results suggest that other factors in addition to S1P(3) agonism should be responsible for the heart rate reduction caused by S1P(1) agonists. Only 2-amino-2-[2-[2'-fluoro-4'-(4-methylphenylthio)biphenyl-4-yl]ethyl]propane-1,3-diol (6d) was identified as a desired S1P(1) receptor agonist having both the immunomodulatory activity and an attenuated effect on heart rate by a unique screening flow using in vivo evaluating systems primarily.

1. The effects of a surgical operation, to implant a guide cannula in the chick hypothalamus for microdialysis, on behavioural responses and neural activity in broiler chicks are described. 2. General behavioural activities (feeding, preening, sitting, drinking, cage pecking and beak wiping), open field and locomotor activity tests were conducted to evaluate the effects related to surgery in the immediate 4 d following this procedure. Perfusion of Ringer solution with high K(+) after 4 d of guide cannula implantation was used to estimate the neural activity resulting from surgery through stimulation of monoamine release by in vivo brain dialysis. 3. The results of direct behavioral observations indicated that the stress provoked by surgical guide cannula insertion caused behavioural alterations that are particularly evident in the immediate days following this procedure. Open-field tests on day 4 after surgery showed that, compared to the intact control chickens, the treated chicks had a shorter latency to ambulate and defecate, with more vocalisation. Locomotor activity was less in the treated chicks than in the controls. 4. After 4 d of guide cannula implantation, the serotonin concentration started to increase 30 min after the onset of perfusing high-K(+) Ringer solution. It reached its highest value at one hour, suggesting that the 4 d after surgery is enough to alleviate some neurochemical dysfunction resulting from surgery. The results of behavioural observations, open-field and locomotor activity tests indicate that the surgical operation caused stress and fear in chicks which persisted up to 4 d.

FTY720 (Fingolimod) is the first of a new immunomodulator class: sphingosine 1-phosphate (S1P) receptor modulator. We have found FTY720 by chemical modification of a natural product, myriocin derived from Isaria sinclairii, a kind of vegetative wasp. FTY720 is orally active and is highly effective in various autoimmune disease models including experimental autoimmune encephalomyelitis (EAE), adjuvant- or collagen-induced arthritis, and lupus nephritis. Particularly, oral administration of FTY720 shows marked therapeutic effects on EAE in mice with a significant reduction of demyelination and T cell infiltration in the central nervous system. A most striking feature of FTY720 is the induction of a marked decrease in peripheral blood lymphocytes at doses that show immunomodulating effects. It is revealed that the reduction of circulating lymphocytes by FTY720 is due to sequestration of lymphocytes into secondary lymphoid organs. FTY720 is rapidly converted to FTY720-phosphate (FTY720-P) by sphingosine kinases. FTY720-P acts as a potent agonist at S1P receptor type 1 (S1P1), internalizes S1P1 on lymphocytes, and inhibits migration of lymphocytes toward S1P. It is highly likely that immunomodulating effects of FTY720 are caused by inhibition of S1P/S1P1-dependent lymphocyte egress from secondary lymphoid organs. Moreover, it is suggested that direct effects of FTY720-P on neural cells via S1P receptors promote neuroprotection. Since FTY720 possesses a novel mechanism of action and is highly effective in relapsing remitting multiple sclerosis patients, it is presumed that oral FTY720 provides a new therapeutic approach for autoimmune diseases including multiple sclerosis.

Fingolimod (FTY720), a sphingosine 1-phosphate (S1P) receptor modulator, is highly effective in various autoimmune disease models and decreases circulating mature lymphocytes by inhibiting S1P-dependent lymphocyte egress from secondary lymphoid organs (SLO). On the contrary, FTY720 at an oral dose of 1 mg/kg also induces a significant decrease in the number of peripheral blood lymphocytes (PBL), particularly T cells, in alymphoplasia (aly/aly) mice lacking SLO. We demonstrated that there was no contribution of thymus or spleen for the reduction of PBL by FTY720 because FTY720 could also decrease the number of PBL in thymectomized and/or splenectomized aly/aly mice. When mice were given FTY720 at 1 mg/kg orally, the blood concentration of FTY720 is approximately 0.2 μM or less. On the other hand, no apoptosis was seen in aly/aly lymphocytes when they were treated with FTY720 at a concentration of up to 1 μM. Moreover, we found that oral administration of FTY720 at 1 mg/kg to aly/aly mice induced the accumulation of mature lymphocytes, particularly T cells, into the bone marrow but not spleen or thymus. Consequently, it is highly probable that mature lymphocytes depleted from blood by FTY720 were sequestered into the bone marrow in aly/aly mice. The sequestration of T cells into the bone marrow was also induced by S1P lyase inhibitor that disrupts S1P gradients. These results demonstrate that the reduction of PBL in aly/aly mice by FTY720 is due to inhibition of S1P-dependent lymphocyte egress from the bone marrow, but not induction of lymphocyte apoptosis.

Experimental autoimmune encephalomyelitis (EAE) is a CD4 T cell-mediated disease model for multiple sclerosis (MS). When SJL/J mice are immunized with myelin proteolipid protein (PLP), EAE symptoms were developed within 2 weeks, remitted thereafter, and then relapsed at about 3 weeks after immunization, indicating that EAE induced by PLP in SJL/J mice shares a certain characteristic with relapsing remitting MS. In this study, we evaluated the preventing effect of fingolimod (FTY720), a sphingosine 1-phosphate receptor modulator, on relapse of EAE induced by PLP in SJL/J mice. When FTY720 at oral does of 0.1 and 0.3 mg/kg was administered daily after establishment of EAE, relapse of EAE was markedly inhibited during administration period. The relapse of EAE was significantly inhibited by subcutaneous administration of recombinant mouse interferon-β (rm-IFN-β) at 10000 IU every other day at early period; however EAE was relapsed in the half number of mice in latter period. These results indicate that FTY720 shows a more marked preventing effect on relapse of EAE compared with rm-IFN-β. By immunohistochemical staining, it is revealed that the area of demyelination and the infiltration of CD4 T cells are significantly reduced in the spinal cords of EAE mice by FTY720. Interestingly, FTY720 markedly decreased infiltration of PLP-specific, interleukin 17-expressing CD4 T cells (Th17 cells) into the spinal cords. Consequently, the preventing effect of FTY720 on relapse of EAE is likely due to reduction of infiltration of encephalitogenic CD4 T cells into the central nervous system.

Fingolimod (FTY720), a sphingosine 1-phosphate (S1P) receptor modulator, is highly effective in experimental autoimmune encephalomyelitis (EAE) and decreases circulating mature lymphocytes by inhibiting S1P-dependent lymphocyte egress from secondary lymphoid organs. Infiltration of encephalitogenic CD4 T cells plays a critical role in the development and progression of EAE in mice. In this study, we investigated the effects of FTY720 on infiltration of interleukin 17 (IL-17)-expressing helper T cells (Th17 cells) and interferon-γ-expressing type 1 helper T cells (Th1 cells) into the spinal cords in EAE mice induced by immunization with myelin oligodendrocyte glycoprotein (MOG). Prophylactic treatment with FTY720 at an oral dose of 0.3 mg/kg significantly inhibited the development of EAE and markedly reduced the frequency of Th17 and Th1 cells in the spinal cords of EAE mice. On the contrary, the frequency of Th17 and Th1 cells in draining inguinal lymph nodes was increased to approximately 2-fold by FTY720, suggesting sequestration of myelin antigen-specific Th cells into draining lymph nodes. Moreover, we found that Th17 and Th1 cells could migrate toward 10 nM S1P and that the pretreatment with 1 nM FTY720-phosphate, an active metabolite of FTY720, almost completely inhibited S1P-induced migration of these Th cells. On the other hand, FTY720-phosphate up to 100 nM showed no clear effect on generation of these Th cells or cytokine production by them. From these results, the ameliorating effects of FTY720 on EAE are likely due to reduction of infiltration of encephalitogenic Th17 and Th1 cells into the central nervous system.

This study was conducted to investigate effects of glucagon intracerebroventricularly administered on feed intake and endocrine changes in sheep. Four male sheep (48-55 kg BW) were used. The animals were acclimatized to be fed alfalfa hay cubes at 12.00 hour. Human glucagon (40 and 80 microg/0.5 mL) was injected into the lateral ventricle at 12.00 hour. Blood samples were taken every 10 min from 30 min before to 180 min after the glucagon injection. Soon after the injection, the animals were given alfalfa hay cubes, and the amounts of the feed eaten within 2 h were measured. Feed intakes were significantly (P < 0.05) suppressed by 80 microg of glucagon. Plasma glucose levels in control animals were gradually decreased after the feeding, whilst those in glucagon-treated animals were temporarily elevated just after the feeding and then kept higher than control levels. Plasma insulin was abruptly elevated after the feeding and was maintained at higher levels than before the feeding in all treatments. Plasma NEFA concentrations were decreased after the feeding in all treatments. A tendency of increase in plasma cortisol levels occurred in glucagon-injected animals. The present study provides the first evidence that glucagon directly acts on the brain, then inhibiting feeding behavior and inducing endocrine responses in ruminants.

Amino acid (AA) administration can stimulate heat accumulation in the body, as especially found under anesthetic conditions. To test our hypothesis that marked rise in plasma insulin concentrations following AA administration plays an important role in the heat storage, we intravenously administered either a balanced AA mixture or saline over 3 h, both with and without a primed-constant infusion of somatostatin in propofol-anesthetized rats. Rats on AA but lacking marked rise in plasma insulin by somatostatin treatment failed to show: attenuation of fall in core body temperature; partial increases in oxygen consumption; and stimulated muscle protein synthesis. Furthermore, the AA’s stimulatory effects on phosphorylation of mTOR, 4E-BP1, and S6K1 were partially blocked by somatostatin. Our findings strongly suggest that the marked rise in insulin following AA administration promote translation initiation activities and stimulate muscle protein synthesis, which facilitates heat accumulation in the body.

The purpose of the present study was to determine if central administration of somatostatin influences feeding behavior in layer chicks. Five- to 7-day-old chicks that received intracerebroventricular (ICV) injections of 0.5 or 2 nmol somatostatin increased their food intake at 30 and 60 min after the injection, suggesting that central somatostatin serves as an orexigenic neuropeptide in chicks. This hypothesis was further supported since chicks ICV injected with 0.5 or 2 nmol cortistatin, which binds to somatostatin receptors, also had increased food intake at the same time. Somatostatin-associated feeding behavior was attenuated by co-administration of 20 nmol beta-funaltrexamine (an opioidergic mu-receptor antagonist) (to 31% of the orexigenic effect of somatostatin at 60 min after the injection) but not ICI-174,864 or nor-binaltorphimine (antagonists of opioidergic delta- and kappa-receptors, respectively). Co-administration of 13 nmol yohimbine, an adrenergic alpha-2 receptor antagonist, also attenuated the orexigenic effect of somatostatin (to 31% of the orexigenic effect of somatostatin at 60 min after the injection). These results suggest that somatostatin-associated feeding behavior is mediated by opioidergic mu- and adrenergic alpha-2-receptors in chicks.

Deficiencies of single essential amino acids decreases food intake and this is likely to be involved in the central nervous system. Central mechanism underlying the decreased food intake induced by amino acid deficiency in chickens remains to be elucidated. We determined the hypothalamic interstitial monoamines (dopamine, norepinephrine and serotonin) with brain microdialysis technique to investigate whether hypothalamic monoaminergic system is associated with the reduction of food intake in growing chickens on a lysine devoid (LD) diet (a purified diet with a crystalline amino acid mixture not containing lysine as a sole source of nitrogen). Microdialysis probes had been inserted at the ventromedial (VMH) and lateral hypothalamus (LH) before presenting the diets. Perfusate was collected every 30min from two hours before feeding the LD diet to five hours later. Monoamines in the perfusate were determined using a high performance liquid chromatography. Chickens on the LD diets ate as much as the control chickens for early three hours, but the former ate less amount of food than the control one four and five hours after presenting the diet. Out of the ventromedial hypothalamic monoamines, dopamine level of the LD group started to decrease from the basal line one hour later and was lower than that of the control group 3.5 hours and afterward. Compared with the control group norepinephrine level of the LD group showed no change. Serotonin level was lower in the LD than control groups at one and four hours. The level of monoamines in the lateral hypothalamus did not differ between the dietary treatments. In conclusion, the present results demonstrate that feeding the lysine devoid diet decreased DA release in the VMH not in the LH before food intake started to decrease.

Neuropeptide Y (NPY) is one of the most important orexigenic agents in central regulation of feeding behavior, body weight and energy homeostasis in domestic chickens. To examine differences in the hypothalamic NPY between layer-type and meat-type of chickens, which are two divergent kinds of the domestic chickens in feeding behavior and body weight, we detected mRNA levels of NPY in hypothalamic infundibular nucleus (IN), paraventricular nucleus (PVN) and lateral hypothalamic area (LHA) of these two types of chickens using one-step real time RT-PCR. The meat-type chicken had more food daily (about 1.7 folds) and greater body weights (about 1.5 folds) and brain weights than the layer-type chicken at the age of 14 d. In the meat-type of chicken, NPY mRNA levels of the IN and PVN were significantly greater than those of the LHA, and were not significantly different between the IN and PVN. However, in the layer-type of chicken, NPY mRNA levels were significantly greater in the IN than those in the LHA and PVN, and were not significantly different between the PVN and LHA. In all these hypothalamic regions, the layer-type of chicken had significantly higher NPY mRNA levels than the meat-type chicken did. These results suggest the expression of NPY in the hypothalamus has a type-dependent pattern in domestic chickens.

Dendritic cells (DCs) and lymphocytes are known to show a migratory response to the phospholipid mediator, sphingosine 1-phosphate (S1P). However, it is unclear whether the same S1P receptor subtype mediates the migration of lymphocytes and DCs toward S1P. In this study, we investigated the involvement of S1P receptor subtypes in S1P-induced migration of CD4 T cells and bone marrow-derived DCs in mice. A potent S1P receptor agonist, the (S)-enantiomer of FTY720-phosphate [(S)-FTY720-P], at 0.1 nM or higher and a selective S1P receptor type 1 (S1P1) agonist, SEW2871, at 0.1 μM or higher induced a dose-dependent down-regulation of S1P1. The pretreatment with these compounds resulted in a significant inhibition of mouse CD4 T cell migration toward S1P. Thus, it is revealed that CD4 T cell migration toward S1P is highly dependent on S1P1. Mature DCs, when compared with CD4 T cells or immature DCs, expressed a relatively higher level of S1P3 mRNA. S1P at 10–1000 nM induced a marked migration and significantly enhanced the endocytosis of FITC-dextran in mature but not immature DCs. Pretreatment with (S)-FTY720-P at 0.1 μM or higher resulted in a significant inhibition of S1P-induced migration and endocytosis in mature DCs, whereas SEW2871 up to 100 μM did not show any clear effect. Moreover, we found that S1P-induced migration and endocytosis were at an extremely low level in mature DCs prepared from S1P3-knockout mice. These results indicate that S1P regulates migration and endocytosis of murine mature DCs via S1P3 but not S1P1.

Hepatic asialoglycoprotein receptor, which may mediate the clearance of circulating thyroglobulin, is known to have a high affinity for GalNAc. Recently, the receptor has been reported to be present also in the thyroid, implicating interaction with thyroglobulin. Here, mammalian thyroglobulins were analyzed for GalNAc termini by Western blotting with GalNAc-recognizing lectins labeled with peroxidase or 125 I. Wistaria floribunda lectin was found to bind human thyroglobulin and, to some extent, bovine, but not porcine thyroglobulin. After desialylation, the lectin bound all of the thyroglobulins tested. The binding was inhibited by competitive inhibitor GalNAc. Peptide N -glycanase treatment of human desialylated thyroglobulin resulted in the complete loss of reactivity with W. floribunda lectin, indicating that the binding sites are exclusively on N -glycans. The binding sites on human desialylated thyroglobulin were partly sensitive to β-galactosidase, and the remainder was essentially sensitive to β- N -acetylhexosaminidase. On the other hand, the binding sites of bovine and porcine desialylated thyroglobulins were totally sensitive to β-galactosidase. Thus the lectin binds β-Gal termini, as well as β-GalNAc. GalNAc-specific Dolichos biflorus lectin also bound human thyroglobulin weakly. In contrast to W. floribunda lectin, desialylation diminished binding, suggesting that these two lectins recognize different GalNAc-terminated structures. Again, the binding was inhibited by GalNAc and by treatment with peptide N -glycanase. These results strongly indicate the presence of distinct GalNAc termini of N -glycans on human thyroglobulin.