Yoshiaki Morita

SOCS-1/SSI-1-Deficient NKT Cells Participate in Severe Hepatitis through Dysregulated Cross-Talk Inhibition of IFN-γ and IL-4 Signaling In Vivo

Suppressor of cytokine signaling-1 (SOCS-1), also known as STAT-induced STAT inhibitor-1 (SSI-1), is a negative feedback molecule for cytokine signaling, and its in vivo deletion induces fulminant hepatitis. However, elimination of the STAT1 or STAT6 gene or deletion of NKT cells substantially prevented severe hepatitis in SOCS-1-deficient mice, while administration of IFN-gamma and IL-4 accelerated its development. SOCS-1 deficiency not only sustained IFN-gamma/IL-4 signaling but also eliminated the cross-inhibitory action of IFN-gamma on IL-4 signaling. These results suggest that SOCS-1 deficiency-induced persistent activation of STAT1 and STAT6, which would be inhibited by SOCS-1 under normal conditions, may induce abnormal activation of NKT cells, thus leading to lethal pathological changes in SOCS-1-deficient mice.

IFN Regulatory Factor-1-Mediated Transcriptional Activation of Mouse STAT-Induced STAT Inhibitor-1 Gene Promoter by IFN-γ

STAT-induced STAT inhibitor-1 (SSI-1), also referred to as suppressor of cytokine signaling-1 and JAK-binding protein, is a member of a new family, the members of which are negative regulators of cytokine signals. SSI-1 is induced by various cytokines; however, the transcriptional mechanism of the SSI-1 gene is not fully understood. Here, we showed that transcription of the mouse SSI-1 gene was initiated from six adjoining sites accompanying three GC boxes and a single GC box-like element near them, but not from the TATA box or an initiator sequence. We also showed that IFN-γ induced SSI-1 mRNA more strongly than IL-6 in NIH-3T3 fibroblasts and that this IFN-γ effect was mediated by Stat1. To determine the signal pathway downstream of Stat1, transcriptional activities of several mutant promoters were examined. The region mediating stimulatory effect of IFN-γ to the gene transcription was localized to the −88/−60 region containing three tandem GAAA units, named variant IFN-γ-responsive element (VIRE), while four IFN-γ activation site (GAS)-like elements located far upstream were not related to the IFN-γ response. Gel-shift assays revealed that IFN-γ induced IFN regulatory factor-1 (IRF-1) binding to VIRE, but not that of IRF-2 or three components of ISGF3. Furthermore, forced expression of IRF-1 mimicked and that of IRF-2 inhibited the stimulatory effect of IFN-γ on SSI-1 gene transcription. Finally, mouse embryonal fibroblasts lacking IRF-1 showed impaired SSI-1 mRNA induction by IFN-γ. These results demonstrated that IRF-1, which is induced by activation of Stat1, mediated transcriptional activation of the SSI-1 gene by IFN-γ via VIRE.

Defective thymocyte development and perturbed homeostasis of T cells in STAT-induced STAT inhibitor-1/suppressors of cytokine signaling-1 transgenic mice.

Previous experiments have shown that STAT-induced STAT inhibitor-1 (SSI-1; also named suppressors of cytokine signaling-1 (SOCS-1) or Janus kinase binding protein) is predominantly expressed in lymphoid organs and functions in vitro as a negative regulator of cytokine signaling. To determine the function of SOCS-1 in vivo, we generated SSI-1 transgenic mice using the lck proximal promoter that drives transgene expression in T cell lineage. In thymocytes expressing SSI-1 transgene, tyrosine phosphorylation of STATs in response to cytokines such as IFN-γ, IL-6, and IL-7 was inhibited, suggesting that SSI-1 suppresses cytokine signaling in primary lymphocytes. In addition, lck-SSI-1 transgenic mice showed a reduction in the number of thymocytes as a result of the developmental blocking during triple-negative stage. They also exhibited a relative increase in the percentage of CD4+ T cells, a reduction in the number of γδ T cells, as well as the spontaneous activation and increased apoptosis of peripheral T cells. Thus, enforced expression of SSI-1 disturbs the development of thymocytes and the homeostasis of peripheral T cells. All these features of lck-SSI-1 transgenic mice strikingly resemble the phenotype of mice lacking common γ-chain or Janus kinase-3, suggesting that transgene-derived SSI-1 inhibits the functions of common γ-chain-using cytokines. Taken together, these results suggest that SSI-1 can also inhibit a wide variety of cytokines in vivo.

Static and dynamic properties of optical second harmonic generation in ferroelectric liquid crystal

Abstract The second harmonic generation (SHG) in the ferroelectric liquid crystal (FLC) has been studied as functions of electric field strength, sample thickness, rotating angle, temperature and molecular structure. A sharp angular phase-matching curve of the SHG controlled by an electric field is observed even in the liquid crystal. The temperature dependences of the phase-matched SHG and Maker fringe in the ferroelectric phase have also been studied and nonlinear optical coefficients are obtained as a function of temperature. The SHG in several kinds of FLCs is measured in the Sm C* and crystalline phase. Anomalous temperature dependence of the SHG is also observed in DOBA-1-MPC.

Pharmacological profile of FK881(ASP6537), a novel potent and selective cyclooxygenase-1 inhibitor.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are now understood to fall into one of two agent classes in clinical use. Traditional NSAIDs inhibit both cyclooxygenases-1 and 2 (COX-1, 2), which act as key enzymes catalyzing the same reaction in the production of prostaglandins (PGs), while the second class of NSAIDs selectively inhibit COX-2. Inhibition of the inducible COX-2 isoform is believed to be responsible for the therapeutic effects of NSAIDs, such as anti-inflammatory, analgesic, and antipyretic effects, while COX-1 inhibition results in side-effects on the gastrointestinal (GI) system. In the present study, however, we changed this notion that inhibiting only COX-1 causes adverse effects. We discovered FK881, a specific COX-1 inhibitor which exhibits a 650-fold ratio for human whole blood COX-1/COX-2 and rats in vivo. In rats, FK881 dose dependently inhibited carrageenan-induced paw edema (ED30: 22 mg/kg; diclofenac ED30: 3.6 mg/kg, rofecoxib ED30: 26 mg/kg) and paw swelling associated with adjuvant arthritis (ED50: 17 mg/kg; diclofenac ED50: 1.4 mg/kg, rofecoxib ED50: 1.8 mg/kg). Further, FK881 dose dependently inhibited acetic acid-induced writhing in mice (ED50: 19 mg/kg; diclofenac ED50: 14 mg/kg, rofecoxib ED50: >100mg/kg) and adjuvant arthritis hyperalgesia in rats (ED50: 1.8 mg/kg; diclofenac ED50: 1.0mg/kg, rofecoxib ED50: 0.8mg/kg). However, unlike traditional NSAIDs, GI tolerability was improved, although the antipyretic effect of FK881 was weak (NOEL: >320 mg/kg; diclofenac NOEL: <1mg/kg, rofecoxib NOEL: 100 mg/kg). These results suggest that FK881 may be useful in treating symptoms of rheumatoid arthritis and osteoarthritis.

Second harmonic generation in ferroelectric liquid crystals and their mixtures

Abstract The second harmonic generation (SHG) in the ferroelectric liquid crystal (FLC) state has been studied as functions of electric field strength, rotating angle, temperature and molecular structure. It has been confirmed that a sharp angularly phase-matching curve of the SHG controlled by an electric field is observed even in the liquid crystal. The temperature dependences of the phase-matched SHG and Maker fringe in the ferroelectric phase have also been studied, and temperature dependences of non-linear optical coefficients obtained. The SHG in several kinds of FLC and dye doped FLC have also been measured, and the enhancement of SHG realized by means of doping the FLC with several kinds of dye.

Constitutive Activation of Integrin α9 Augments Self-Directed Hyperplastic and Proinflammatory Properties of Fibroblast-like Synoviocytes of Rheumatoid Arthritis

Despite advances in the treatment of rheumatoid arthritis (RA), currently approved medications can have significant side effects due to their direct immunosuppressive activities. Additionally, current therapies do not address residual synovial inflammation. In this study, we evaluated the role of integrin α9 and its ligand, tenascin-C (Tn-C), on the proliferative and inflammatory response of fibroblast-like synoviocytes (FLSs) from RA patients grown in three-dimensional (3D)–micromass culture. FLSs from osteoarthritis patients, when grown in the 3D-culture system, formed self-directed lining-like structures, whereas FLSs from RA tissues (RA-FLSs) developed an abnormal structure of condensed cellular accumulation reflective of the pathogenic features of RA synovial tissues. Additionally, RA-FLSs grown in 3D culture showed autonomous production of proinflammatory mediators. Predominant expression of α9 and Tn-C was observed in the condensed lining, and knockdown of these molecules abrogated the abnormal lining-like structure formation and suppressed the spontaneous expression of matrix metalloproteinases, IL-6, TNFSF11/RANKL, and cadherin-11. Disruption of α9 also inhibited expression of Tn-C, suggesting existence of a positive feedback loop in which the engagement of α9 with Tn-C self-amplifies its own signaling and promotes progression of synovial hyperplasia. Depletion of α9 also suppressed the platelet-derived growth factor–induced hyperplastic response of RA-FLSs and blunted the TNF-α–induced expression of matrix metalloproteinases and IL-6. Finally, α9-blocking Ab also suppressed the formation of the condensed cellular lining by RA-FLSs in 3D cultures in a concentration-related manner. This study demonstrates the central role of α9 in pathogenic behaviors of RA-FLSs and highlights the potential of α9-blocking agents as a nonimmunosuppressive treatment for RA-associated synovitis.

Additive antithrombotic effect of ASP6537, a selective cyclooxygenase (COX)-1 inhibitor, in combination with clopidogrel in guinea pigs

ABSTRACT Clopidogrel (Plavix®, Sanofi‐Aventis), the adenosine diphosphate P2Y12 receptor antagonist, is reported to be effective in the prevention of cardiovascular events and is often used in combination with aspirin, particularly in high‐risk patients. ASP6537 is a reversible cyclooxygenase (COX)‐1 inhibitor that is under investigation as an anti‐platelet agent. First, we investigated the reversibility of the antiplatelet effect of ASP6537 and its interaction with ibuprofen to compare the usability of ASP6537 with that of aspirin. We then evaluated the antithrombotic effect of ASP6537 in combination with clopidogrel using a FeCl3‐induced thrombosis model in guinea pigs. ASP6537 exerted reversible antiplatelet activity, and no pharmacodynamic interaction with ibuprofen was noted. When administered as monotherapy, ASP6537 exerted a significant antithrombotic effect at ≥3 mg/kg, while aspirin inhibited thrombosis at 100 mg/kg. ASP6537 exerted significant additive effects in combination with clopidogrel, and the minimum antithrombotic dose was reduced by concomitant administration of clopidogrel. Our study showed that ASP6537 did not interact with ibuprofen and has clear additive effects in combination with clopidogrel. ASP6537 may therefore represent a promising antiplatelet agent for use in clinical settings in combination with clopidogrel.

THU0101 ASP015K: A Novel Jak Inhibitor Demonstrated Potent Efficacy in Adjuvant-Induced Arthritis Model in Rats

Background The Janus kinase (JAK) family of enzymes plays a key role in cytokine signaling, which is involved in the pathogenic events of immune-mediated disorders such as rheumatoid arthritis (RA). Objectives The aim of this study was to identify pharmacological profiles of a newly synthesized JAK inhibitor, ASP015K, and to estimate its therapeutic potential in the treatment of RA patients using an experimental animal model. Methods In vitro enzyme inhibition assays were conducted against JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) enzymes. Cell-based assays were also conducted to assess the selectivity of ASP015K for signaling via JAK1/JAK3 over JAK2/JAK2. JAK1/3 activation was evaluated by interleukin (IL)-2-stimulated T cell proliferation; JAK2/2 action was evaluated by erythropoietin (EPO)-stimulated erythroleukemia cell proliferation. Phosphorylation of signal transducer and activator of transcription5 (STAT5) was quantified by flow cytometry as a biomarker of JAK activity in vitro in human whole blood and ex vivo in rat whole blood. In order to evaluate the potential efficacy of ASP015K to reduce clinical signs and symptoms of RA as well as disease progression, the reduction of paw swelling and ankle bone destruction in adjuvant-induced arthritic (AIA) rats were assessed after both prophylactic and therapeutic dosing regimens of ASP015K. Results ASP015K was shown to inhibit JAK enzymes, with moderate selectivity for JAK3. ASP015K suppressed the IL-2-induced proliferation of human T cells with greater potency than EPO-induced proliferation of human erythroleukemia cells. ASP015K inhibited STAT5 phosphorylation (pSTAT5) in human whole blood in a concentration related manner. Additionally, after oral administration, ASP015K also suppressed pSTAT5 in rat whole blood. In the rat AIA model, the hind paw volume gradually increased after adjuvant injection and ankle bone destruction was established. In a prophylactic dosing regimen, the increase in paw volume was significantly decreased by oral administration of ASP015K in a dose-dependent manner. Similar findings in ankle bone destruction score were observed. In a therapeutic dosing regimen, paw swelling and ankle bone destruction score were also suppressed in a dose-dependent manner. Conclusions Data from the current study demonstrate that ASP015K inhibits human JAK enzymes with moderate selectivity against JAK1/3 over JAK2/2, which may translate to less hematological side effects observed in the clinic. ASP015K improved paw swelling and ankle bone destruction after both prophylactic and therapeutic dosing regimens in the rat AIA model. These data together with the finding that ASP015K suppressed pSTAT5 in whole blood after oral administration in rats suggest that the effects of ASP015K on AIA model are due to JAK inhibition. These data also suggest that ASP015K has the potential to reduce clinical signs and symptoms as well as prevent disease progression in RA patients and warrants further clinical investigation. Disclosure of Interest S. Yamazaki Employee of: Astellas Pharma Inc., H. Morio Employee of: Astellas Pharma Inc., M. Inami Employee of: Astellas Pharma Inc., M. Ito Employee of: Astellas Pharma Inc., Y. Fujii Employee of: Astellas Pharma Inc., K. Hanaoka Employee of: Astellas Pharma Inc., K. Yamagami Employee of: Astellas Pharma Inc., K. Okuma Employee of: Astellas Pharma Inc., Y. Morita Employee of: Astellas Pharma Inc., S. Shirakami Employee of: Astellas Pharma Inc., T. Inoue Employee of: Astellas Pharma Inc., S. Miyata Employee of: Astellas Pharma Inc., Y. Higashi Employee of: Astellas Pharma Inc., N. Seki Employee of: Astellas Pharma Inc.

Inhibitory effects of ASP6537, a selective cyclooxygenase-1 inhibitor, on thrombosis and neointima formation in rats

INTRODUCTION Percutaneous coronary interventions (PCIs), such as balloon angioplasty and stent placement, are effective in the treatment of coronary artery disease. PCI has drawbacks, however, including acute thrombosis after the procedure and restenosis of the vascular lumen due to abnormal neointimal hyperplasia. ASP6537 is a selective COX-1 inhibitor that has been investigated as a possible candidate for clinical development as an antiplatelet agent. In the present study, we evaluated the in vivo antithrombotic effect of ASP6537 and its effect on neointima formation after balloon angioplasty. MATERIAL AND METHODS The antithrombotic effect of ASP6537 was examined using an arteriovenous shunt thrombosis model in rats while the effect of ASP6537 on neointima formation was evaluated in a rat carotid arterial balloon angioplasty model. RESULTS In the thrombosis study, ASP6537 dose-dependently decreased the protein content of the thrombus, while no prolongation of template bleeding time was observed. In the neointima study, ASP6537 reduced neointima formation. CONCLUSIONS ASP6537 may be a promising agent for the prevention of acute thrombosis and restenosis after PCI in place of aspirin.