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Sagamihara, Japan

Negoro T.,Showa University | Shimizu S.,Showa University | Narushima M.,Showa University | Banham A.H.,University of Oxford | And 11 more authors.
Clinical and Experimental Allergy | Year: 2014

Summary: Background: Regulatory T cells (Tregs) are activated during anergy in response to T cell receptor (TCR) activation and functional immune suppression. Anergy of paediatric Tregs is partially dependent on intracellular calcium mobility; following TCR activation, Tregs do not exhibit increased intracellular Ca2+ concentration ([Ca2+]i). Objective: We determined whether [Ca2+]i in adult Tregs defined their anergy, if intracellular Ca2+ movement was linked to regulatory functions, whether [Ca2+]i was indicative of asthma pathology, and the potential molecular mechanism responsible for Ca2+ movement in Tregs. Methods: Tregs were purified by the magnetic bead method, and their regulatory functions were assessed by monitoring carboxyfluorescein succinimidyl ester-labelled responder T cell proliferation. The Ca2+ response of Fura-2-labelled cells was measured using a video image analysis system. To analyse the functions of Tregs at the molecular level, we generated Jurkat Tet-On® clones with doxycycline (Dox)-induced forkhead box P3 (FOXP3) protein expression. Results: CD4+CD25+CD127-/low Tregs from participants without asthma did not elicit Ca2+ influx in response to TCR activation, exhibited little proliferation and suppressed proliferation of CD4+CD25- T cells. In contrast, under similar conditions, Tregs from patients with asthma exhibited increased [Ca2+]i and robust proliferation with partial loss of regulatory functions. FOXP3 protein levels in Tet-On® clones were high after both 2- and 5-day Dox treatment; however, 5-day cells were comparable with Tregs from patients with asthma, whereas 2-day cells were similar to Tregs from participants without asthma. Increasing [Ca2+]i induced a high level of receptor for activated C kinase 1 (RACK1) expression in 5-day cells. Conclusions and Clinical Relevance: We confirmed that Tregs in patients with asthma are functionally impaired and that the abnormal regulatory functions of these cells can be analysed by [Ca2+]i following TCR engagement. Furthermore, the impaired functioning of Tregs evident in patients with asthma may be due to a high level of RACK1. © 2014 John Wiley & Sons Ltd.

Shimizu S.,Yokohama College of Pharmacy | Shimizu S.,Showa University | Yonezawa R.,Showa University | Hagiwara T.,Sagami Womens Junior College | And 8 more authors.
European Journal of Pharmacology | Year: 2014

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca2+-permeable channel that controls Ca2+signalling. The activation of Janus kinase 2 (Jak2) by oxidative stress is implicated in the production of inflammatory mediators. We found that AG490, a Jak2 inhibitor, had an inhibitory effect on H2O2-induced TRPM2 activation. The purpose of this study was to examine the underlying mechanisms of the inhibitory effects of AG490. Activation of TRPM2 in TRPM2-expressing human embryonic kidney 293 (TRPM2/HEK) cells or the human monocytic cell line U937 was monitored by fluorescence-based Ca2+imaging and patch-clamp techniques. Treatment with AG490 almost completely blocked H2O2-induced increase in intracellular Ca2+in TRPM2/HEK and U937 cells. In the patch-clamp study, AG490 inhibited the H2O2-evoked inward current but not the ADP-ribose-induced inward current in TRPM2/HEK cells. In contrast, Jak inhibitor 1 (pyridone 6) and staurosporine, both of which inhibit Jak2, had no effect on H2O2-induced increase in intracellular Ca2+. Moreover, AG490 decreased intracellular reactive oxygen species level, which was measured by using a hydroperoxide-sensitive fluorescent dye, on incubation with H2O2. In the cell-free assay system, AG490 scavenged hydroxyl radicals but not H2O2. These findings indicate that AG490 significantly reduces H2O2-induced TRPM2 activation, presumably by scavenging hydroxyl radicals rather than Jak2-dependent mechanisms. Although transient receptor potential ankyrin 1 (TRPA1) channel is also activated by H2O2, the H2O2-induced Ca2+entry through TRPA1 was only slightly delayed by AG490. This validates the potential use of AG490, as one of the materials for characterizing the role of TRPM2 channels in pathological models. © 2014 Elsevier B.V.

Kameda N.,Sagami Womens Junior College | Okigawa T.,Kyoto University | Kimura T.,Kyoto University | Fujibayashi M.,Kyoto University | And 6 more authors.
Journal of Physiological Anthropology | Year: 2011

The purpose of this study was to investigate the effect of L-citrulline ingestion on ECG QT interval. To accomplish this purpose, nine male subjects (age: 23.4±0.5 years, weight: 57.7±5.6kg) participated, who had no history of cardiovascular or respiratory function disorders. Each subject performed two tests (L-citrulline and placebo ingestion) on a separate day. ECG was taken before, and 60 min and 90 min after L-citrulline ingestion. The heart-rate-corrected QT interval (QTc) decreased significantly 60 min (p=0.0004) and 90 min (p=0.011) after L-citrulline ingestion. However, there were no significant changes after placebo ingestion. In addition, the interaction between L-citrulline ingestion and placebo was significant (two-way ANOVA: interaction, p=0.010). Our major finding was that L-citrulline ingestion decreases the QT interval in healthy subjects. This result suggests that transient L-citrulline ingestion may shorten the time required to complete myocardial depolarization and repolarization. © 2011 Japan Society of Physiological Anthropology.

Seike M.,Sagami Womens Junior College | Furuya K.,Sagami Womens Junior College | Omura M.,Sagami Womens Junior College | Hamada-Watanabe K.,Sagami Womens Junior College | And 2 more authors.
Allergy: European Journal of Allergy and Clinical Immunology | Year: 2010

Background: The present study observed effects of the histamine H 4 receptor on chronic allergic contact dermatitis induced by repeated challenge in mice. Methods: Acute contact dermatitis was induced by single epicutaneous challenge of 2,4,6-trinitro-1-chlorobenzene (TNCB) to the ear. Chronic allergic contact dermatitis was developed by repeated epicutaneous challenge using TNCB on the dorsal back skin. H4 receptor antagonist JNJ7777120 was administered to wild-type mice, while H4 receptor agonist 4-methylhistamine was administered to histidine decarboxylase (HDC) (-/-) mice that synthesized no histamine. Results: HDC (-/-) mice did not differ phenotypically from HDC (+/+) mice, and H4 receptor antagonist/agonist did not have clinical effects in terms of acute contact dermatitis reactions. H4 receptor antagonist ameliorated skin eczematous lesions induced by repeated TNCB challenge in HDC (+/+) mice. On the contrary, H4 receptor agonist exacerbated skin lesions exclusively in HDC (-/-) mice. Application of H4 receptor agonist induced migration of mast cells and eosinophils in skin lesions, and H4 receptor antagonist suppressed these changes. H4 receptor was immunohistochemically detected on mast cells in eczematous lesions. Levels of interleukin (IL)-4, -5, and -6 in lesions were decreased, whereas levels of interferon-γ and IL-12 were increased by H4 receptor antagonistic activity. Serum Immunoglobulin E levels rapidly increased with repeated challenge, but decreased with H4 receptor antagonist. Conclusion: Because chronic allergic contact dermatitis is developed by H 4 receptor stimulation, H4 receptor antagonists might represent new candidate drugs for treating chronic allergic contact dermatitis. © 2009 John Wiley & Sons A/S.

Matsushita A.,Sagami Womens Junior College | Seike M.,Sagami Womens Junior College | Okawa H.,Sagami Womens Junior College | Kadawaki Y.,Sagami Womens Junior College | Ohtsu H.,Tohoku University
Experimental Dermatology | Year: 2012

Histamine facilitates development of eczematous lesions in chronic allergic contact dermatitis. In addition to the well-known corticosteroid treatments, H1 receptor (H1R) antagonists also have been used. This study observed effects of histamine H4 receptor (H4R) antagonist usage with H1R antagonist in a murine chronic allergic contact dermatitis model, developed by repeated percutaneous challenge to the dorsal skin with 2,4,6-trinitro-1-chlorobenzene (TNCB). The H1R antagonist olopatadine hydrochloride and/or the H4R antagonist JNJ7777120 was then administered. Combination therapy was more effective than H1R antagonist monotherapy. Serum IgE and levels of interleukin (IL)-4, IL-5 and IL-6 (Th2 cytokines) in eczematous lesions decreased with combined therapy. Combined therapy with H1R and H4R antagonists counteracts the disadvantages of each as monotherapeutic agents and potentially represents a new strategy for the treatment of chronic allergic contact dermatitis. © 2012 John Wiley & Sons A/S.

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