Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

PURPOSE: Aiming at complete excision of cholesteatoma during trympanomastoidectomy and therefore reducing the risk of recurrence, intraoperative imaging techniques are required to assist the visualization of cholesteatoma residue. Galectin-7 has been demonstrated to be a biomarker for cholesteatoma matrix and used for intraoperatively identifying the excision margins. METHODS: A galectin-7-targeted DNA-aptamer library was generated for labeling the cholesteatoma matrix using cell-systematic evolution of ligands by an exponential enrichment technique. The binding characteristics of the identified aptamers were analyzed, and structure optimization of the identified aptamers was carried out both in silico and in vitro. FINDINGS: A fluorophore-labeled structure-optimized DNA fragment was commercially synthesized as a non-invasive aptamer-based probe for intraoperative lesion detection. Using galectin-7-aptamer-guided molecular imaging, the excision margins of cholesteatoma matrix and surrounding normal tissue were successfully achieved within 15-20 min. CONCLUSIONS: Galectin-7-targeted aptamers could benefit molecular imaging-guided surgical treatment, which would enable clinicians to not only intraoperatively detect the locations of cholesteatoma matrix in the middle ear, but also assess the postoperative response of the expression profile to therapy. It is highly expected that further efforts for rational design and development should be directed towards the development of clinically translatable aptamer-based imaging agents.

DOI： 10.1016/j.jphs.2022.08.002

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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DOI： 10.1016/j.bja.2021.04.009

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DOI： 10.1016/j.bja.2021.04.009

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Mesenchymal stem cell (MSC)-based articular regeneration might be beneficial for both protecting and rebuilding cartilaginous tissues in the management of rheumatoid arthritis. However, it is unclear how current immunosuppressive strategies influence the multipotency of MSCs. The present study was undertaken to profile the direct effectiveness of major antirheumatic drugs including methotrexate, prednisolone, adalimumab, and tocilizumab on the multipotency of MSCs, with a special focus on chondrogenesis. The inhibitory effects of methotrexate on adipogenesis, osteogenesis, and chondrogenesis were observed to occur in a dose-dependent manner in an in vitro differentiation system. Prednisolone enhanced adipogenesis, but reduced alkaline phosphatase activity in osteoprogenitors and suppressed the formation of chondrospheroids. Adalimumab suppressed alkaline phosphatase activity, while tocilizumab diminished osteogenesis and chondrogenesis of MSCs in vitro. Chondrogenesis of antirheumatic drug-treated MSCs was also evaluated in vivo using a scaffolded spheroid-engrafted murine model. The biologics examined appeared to be relatively safe for cartilaginous formation, but methotrexate and prednisolone exhibited opposing influences on chondrogenesis. Taken together, these results reveal the direct efficacy of major antirheumatic agents on the multipotency of MSCs. Therefore, our findings suggest that optimization of medication protocols is further required for therapeutic approaches involving cartilaginous tissue engineering.

DOI： 10.3389/fphar.2020.01004

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Molecular hydrogen is thought to have an inhibitory effect on oxidative stress, thereby attenuating the onset and progression of various diseases including cardiovascular disease; however, few reports have assessed the preventive effect of constitutive inhalation of hydrogen gas on of vascular remodeling. Here, we investigated the effect of constitutive inhalation of hydrogen gas on vascular neointima formation using a cuff-induced vascular injury mouse model. After constitutive inhalation of compressed hydrogen gas (O2 21%, N2 77.7%, hydrogen 1.3%) or compressed air only (O2 21%, N2 79%) by C57BL/6 mice for 2 weeks from 8 weeks of age in a closed chamber, inflammatory cuff injury was induced by polyethylene cuff placement around the femoral artery under anesthesia, and hydrogen gas administration was continued until sampling of the femoral artery. Neointima formation, accompanied by an increase in cell proliferation, was significantly attenuated in the hydrogen group compared with the control group. NADPH oxidase NOX1 downregulation in response to cuff injury was shown in the hydrogen group, but the expression levels of NADPH oxidase subunits, p40phox and p47phox, did not differ significantly between the hydrogen and control groups. Although the increase in superoxide anion production did not significantly differ between the hydrogen and control groups, DNA damage was decreased as a result of reduction of reactive oxygen species such as hydroxyl radical (⋅OH) and peroxynitrite (ONOO-) in the hydrogen group. These results demonstrate that constitutive inhalation of hydrogen gas attenuates vascular remodeling partly via reduction of oxidative stress, suggesting that constitutive inhalation of hydrogen gas at a safe concentration in the living environment could be an effective strategy for prevention of vascular diseases such as atherosclerosis.

DOI： 10.1371/journal.pone.0227582

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Calcium is a ubiquitous intracellular messenger that has a crucial role in determining the proliferation, differentiation, and functions of multipotent mesenchymal stem cells (MSCs). Our study is aimed at elucidating the influence of genetically manipulating Ca2+ release-activated Ca2+ (CRAC) channel-mediated intercellular Ca2+ signaling on the multipotency of MSCs. The abilities of genetically engineered MSCs, including CRAC-overexpressing and CRAC-knockout MSCs, to differentiate into multiple mesenchymal lineages, including adipogenic, osteogenic, and chondrogenic lineages, were evaluated. CRAC channel-mediated Ca2+ influx into these cells was regulated, and the differentiation fate of MSCs was modified. Upregulation of intracellular Ca2+ signals attenuated the adipogenic differentiation ability and slightly increased the osteogenic differentiation potency of MSCs, whereas downregulation of CRACM1 expression promoted chondrogenic differentiation potency. The findings demonstrated the effects of genetically manipulating MSCs by targeting CRACM1. CRAC-modified MSCs had distinct differentiation fates to adipocytes, osteoblasts, and chondrocytes. To aid in the clinical implementation of tissue engineering strategies for joint regeneration, these data may allow us to identify prospective factors for effective treatments and could maximize the therapeutic potential of MSC-based transplantation.

DOI： 10.1155/2019/7510214

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Inc.  

Nitrogen-containing bisphosphonates (NBPs) have been widely used as bone anti-resorptive drugs for the treatment of osteoclast-dependent bone disorders. Zoledronate is currently the most potent NBP, and has potential as an inhibitor of farnesyl pyrophosphate synthase. The present study was undertaken to elucidate the possible effects of zoledronate on FcεRI-dependent mast cell activity in vitro, which is essential for in maintaining homeostasis of the gastrointestinal mucosa. Treatment with zoledronate significantly diminished exocytosis of mast cells, which was reflected by a decrease of FcεRI-dependent histamine release compared to that in vehicle-treated mast cells. Our single-vesicle monitoring and biochemical results suggested that zoledronate modulates intracellular formation of the myosinVa/Rab3a complex and syntaxin4/VAMP7 complex, which are critical in vesicle motility, and therefore disturbs exocytosis via suppression of the velocity of intracellular vesicles and inhibition of membrane fusion. Our findings imply that oral administration of zoledronate could modulate mucosal immune function by blocking mast cell function, and this risk should be of concern in the clinical usage of NBPs.

DOI： 10.1016/j.bcp.2018.02.013

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Ca2+ release-activated calcium channel 3 (CRACM3) is a unique member of the CRAC family of Ca2+-selective channels. In a non-excitable exocytosis model, we found that the extracellular L3 domain and the cytoplasmic C-terminus of CRACM3 interacted in an activity-dependent manner with the N-peptide of syntaxin4, a soluble N-ethylmaleimide-sensitive factor attachment receptor protein. Our biochemical, electrophysiological and single-vesicle studies showed that knockdown of CRACM3 suppressed functional exocytosis by decreasing the open time of the vesicle fusion pore without affecting Ca2+ influx, the activity-dependent membrane capacitance (Cm) change, and the total number of fusion events. Conversely, overexpressing CRACM3 significantly impaired cell exocytosis independent of Ca2 +, led to an impaired Cm change, decreased the number of fusion events, and prolonged the dwell time of the fusion pore. CRACM3 changes the stability of the vesicle fusion pore in a manner consistent with the altered molecular expression. Our findings imply that CRACM3 plays a greater role in exocytosis than simply acting as a compensatory subunit of a Ca2+ channel.

DOI： 10.1038/srep28133

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

Immune cells, including T cells, B cells, and osteoclasts, in conjunction with their associated cytokines, have been studied as primary molecular therapeutic targets for the management of rheumatoid arthritis (RA) patients. The increase in cytosolic Ca2+ levels through the activation of store-operated Ca2+ release-activated channels (CRACs) is involved in mediating a disparate array of cellular responses by these immune cells. This study was undertaken to investigate the feasibility and efficiency of the regulation of Ca2+ entry in the treatment of RA. To moderately suppress Ca2+ entry via CRACs, we gene silenced CRACM3,which was induced by systemic application of specific short hairpin RNAs (shRNAs) using a lentiviral-delivery system, in a murine model of collagen-induced arthritis (CIA). The inflammatory responses were determined by measuring the levels of a panel of cytokines and chemokines in the joints and serum. Ag-specific responses were evaluated by determining the cytokine profile of T cells stimulated with autoantigen. We also analyzed the ability of specific CRACM3-shRNA to regulate mature osteoclast function in CIA mice. The therapeutic effect of lentiviral-delivered CRACM3-shRNA was associated with gene silencing of CRACM3, along with the successful biodistribution of the virus. Extracellular Ca2+ influx in the splenocytes, thymocytes, and knee joint synovial cells was moderately suppressed. Inflammatory responses and autoimmune responses were reduced by CRACM3 gene silencing. A decrease in mature osteoclast activity also was observed in CRACM3-shRNA-treated CIA mice. These results indicate that regulation of Ca2+ entry through lentivirus-mediated CRACM3 gene silencing is beneficial in the treatment of RA.

DOI： 10.4049/jimmunol.1401976

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The regulated control of Ca2+ influx is essential for the activation and function of the adaptive immune response, as Ca2+ is a key regulator of important transcription factors. To determine whether Ca2+ release-activated Ca2+ (CRAG) channels contribute to the abnormal behaviour of T cells in patients with rheumatoid arthritis (RA), we performed a cross-sectional study to characterize the expression and functional status of CRACM1 channels in RA patients. Peripheral blood was obtained from 50 RA patients, 50 osteoarthritis (OA) patients and healthy donors. We measured Ca2+ influx and CRAG currents in naive and memory CD4(+) T cells. CRACM1 expression was evaluated in T cells from each of the three groups. These cells were further characterized by flow cytometric analysis of interleukin-4 (IL-4), IL-17, interferon-gamma and tumour necrosis factor-alpha. These cytokines were also measured in naive CD4(+) T cells following the lentivirus-mediated silencing of CRACM1. There was a significant positive correlation between Ca2+ influx in naive T cells and RA activity. Functionally aberrant naive CD4(+) T cells from patients with active RA showed the different cytokine release pattern and exhibited increased Ca2+ influx as well as increased CRACM1 protein expression and function. Specific lentiviral-induced gene silencing of CRACM1 reversed the alterations in T-cell cytokine production. The data presented here indicate that an upregulation of CRACM1 expression and function may be responsible for the abnormal cytokine release of naive CD4(+) T cells in RA patients. CRACM1 might therefore represent a new molecular target for RA therapies.

DOI： 10.1038/icb.2014.45

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Mucosal mast cells (MMCs) have an important role in allergic inflammation, and effective antagonists are required for their regulation. To discover a possible mechanism of controlling the activation of MMCs, we investigated the expression and function of syntaxin4, one of the soluble membrane N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, in RBL-2H3 cells, which is a rat mucosal mast cell line. Syntaxin4 silencing was induced by transfection of short interfering RNAs (siRNAs). Syntaxin4 was knocked down in mast cells at both the mRNA and protein levels. The release of granule contents that are involved in inflammation, such as histamine and hexosaminidase, was significantly suppressed by the gene silencing of syntaxin4. Silencing of this gene was also induced in the trachea and bronchi of rats by intratracheal application of the siRNAs using an atelocollagen delivery system. The activation of MMCs, which was monitored by the level of rat mast cell protease-II (RMCPII) in the bronchoalveolar lavage fluid (BALF), was inhibited, and asthmatic airway constriction was prevented by administration of the syntaxin/atelocollagen complex. These results indicate that siRNAs targeting syntaxin4 can stabilize mucosal mast cells and may have beneficial therapeutic effects on the asthmatic response. Immunology and Cell Biology (2012) 90, 337-345; doi:10.1038/icb.2011.41; published online 31 May 2011

DOI： 10.1038/icb.2011.41

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DOI： 10.1371/journal.pone.0319103

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DOI： 10.7759/cureus.57555

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Comprehensive profiling of the transcriptome reflects the dynamic changes in complex regulator interactions between genes and proteins in the pathological processes of autoimmune disease. To enable real-time adaptive sampling and run rapid sequencing with maximal expected readings of 50 Gb of data with accessibility to clinicians, basic researchers, and even students, a portable and affordable sequencing device, MinION, was employed in our laboratory for both basic and clinical studies. Here, the workflow of bulk RNA-seq in murine spleen using MinION is introduced. The methodology of both laboratory library preparation and the establishment of a bioinformatic pipeline are included in this chapter.

DOI： 10.1007/978-1-0716-3682-4_30

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The diagnosis of vasculitis in rheumatoid arthritis (RV) is associated with considerable mortality; therefore, understanding the basic mechanisms underlying the pathogenesis of vasculitis is very important. Animal models of vasculitis have contributed to elucidating such mechanisms. We here introduce a Candida albicans water-soluble glycoprotein (CAWS)-induced vasculitis model and the methodological approach to evaluate inflammatory vascular change.

DOI： 10.1007/978-1-0716-3682-4_28

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Nucleic acid aptamers are therapeutic agents consisting of short single-strand DNA or RNA oligonucleotides, which have the ability to bind to target therapeutic molecules with high affinity and specificity and have been developed as potent drugs for the treatment of rheumatoid arthritis. Aptamers have unique and advantageous features over antibodies, such as superior affinity with nano- or pico-molar dissociation constants and ease of chemical synthesis, modification, and inactivation by designing antisense sequences. In this chapter, using a DNA-oligonucleotide pool, the technology of proteoliposome-systematic evolution of ligands by exponential enrichment (SELEX) is introduced. By using this technique, potential therapeutic agents with high affinity and specificity could be obtained.

DOI： 10.1007/978-1-0716-3682-4_13

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To protect subjects who participate in human research, Institutional Review Boards (IRBs) play an important role in reviewing research and determining the validity of a study by comprehensively examining it for ethical issues, including invasiveness and management of personal information. They conduct regular and independent reviews to protect the health, rights, and welfare of research subjects. When we as researchers conduct clinical research, we must obtain IRB approval and submit our research for investigation of ethical issues before we begin.

DOI： 10.1007/978-1-0716-3682-4_31

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Language：Japanese   Publishing type：Research paper (conference, symposium, etc.)   Publisher：(NPO)日本高血圧学会  

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Language：Japanese   Publisher：(一社)日本女性栄養・代謝学会  

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Frailty attracts research as it represents a significant target for intervention to extend the healthy life span. An unanswered question in this field is the time point during the life-course at which an individual becomes predisposed to frailty. Here, we propose that frailty has a fetal origin and should be regarded as part of the spectrum of the developmental origins of health and disease. The developmental origins of health and disease theory originated from findings linking the fetal environment to lifestyle-related disorders such as hypertension and diabetes. Coincidentally, a recent trend in frailty research also centers on vascular dysfunction and metabolic alterations as the causality of lifestyle-related disorders such as sarcopenia and dementia. Here, we explore the relationship between fetal programming, frailty-related disorders (sarcopenia and dementia), and other age-related diseases mainly based on reports on intrauterine growth restriction. We propose a "total" life-course approach to combat frailty. With this viewpoint, not only physicians and gerontologists but also obstetricians and pediatricians should team up to overcome age-related diseases in the elderly. Geriatr Gerontol Int 2023; 23: 263-269.

DOI： 10.1111/ggi.14565

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AIM: Molecular hydrogen is not only expected to be used as an energy-generating resource, but also to have preventive effects on a variety of clinical manifestations related to oxidative stress through scavenging radicals or regulating gene expression. In the current study, we investigated the influence of intermittent environmental exposure to hydrogen gas at a safe concentration (1.3%) on photoaging using an ultraviolet A (UVA)-irradiated murine model. METHODS: To mimic the expected human daily activity cycle, UVA exposure in the daytime and hydrogen exposure in the night-time, an original design, UVA-transmission, hydrogen-exposure system was established. Mice were bred under experimental conditions of UVA irradiation and normal air for 8 h (outdoor time 09.00-17.00 hours), and UVA non-irradiation and inhalation of hydrogen gas for 16 h (indoor time 17.00-09.00 hours), and the daily cycle was continued for up to 6 weeks. The progression of photoaging, including morphological changes, collagen degradation and UVA-related DNA damage, was evaluated. RESULTS: Intermittent administration of hydrogen gas by our system prevented UVA-induced epidermal signs, such as hyperplasia, melanogenesis and appearance of senescence cells, and UVA-induced dermal signs, such as collagen degradation. In addition, we detected attenuation of DNA damage in the hydrogen exposure group as indirect evidence that intermittent exposure to hydrogen gas reduced oxidative stress. CONCLUSIONS: Our findings support the notion that long-term, intermittent environmental exposure to hydrogen gas in daily life has a beneficial effect on UVA-induced photoaging. Geriatr Gerontol Int 2023; ••: ••-••.

DOI： 10.1111/ggi.14562

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Language：Japanese   Publishing type：Research paper (conference, symposium, etc.)   Publisher：日本心脈管作動物質学会  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Part of collection (book)   Publisher：Springer International Publishing  

DOI： 10.1007/16833_2022_108

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Language：Japanese   Publisher：Japanese Pharmacological Society  

The present study is undertaken to investigate the effect of fetal growth restriction (FGR) on retinal neovasculature in a murine premature neonatal oxygen-induced retinopathy (OIR) model. According to the results of histological analysis, a significant decrease of neovasculature, as indicated by the decreases in the number of branch junctions, the vesicular distribution, maximal vesicular radius and microaneurysm-like tufts, were observed in OIR mice with FGR while comparing to OIR neonates with normal birth weight. The results of retinal RNA-sequencing revealed a down-regulation of angiogenic factors that trigger pathologic retina neovascularization, such as MAPK pathway and relative upstream signaling pathways in OIR mice with FGR. These results suggest that FGR neonates may be equipped with a higher capacity for retinal oxygen stress, and the risk of OIR development is lower compared to mature neonates.

DOI： 10.1254/jpssuppl.97.0_1-b-p-022

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Language：Japanese   Publishing type：Research paper (conference, symposium, etc.)   Publisher：Japanese Pharmacological Society  

It has been known that sarcopenia causes the immune dysfunction. In this study, we evaluated the influence of the sarcopenia on the progression of melanoma using a denervation-induced sarcopenic model and the efficacy of leucine-intervention was evaluated. 

Initially, the influences of leucine-intake on maintaining immune homeostasis were observed. The expression of PD1 on CD4-positive T cells and CD8-positive T cells increased in the sarcopenia mice comparing those in control mice. Then, a diet-intake leucine was orally administrated to both sarcopenic mice and melanoma-implanted sarcopenic mice. The administration of leucine caused an elevation of total CD4-positive cells and significantly decreased the number of PD1+CD4+ fraction and PD1+CD8+ fraction in sarcopenic mice. The subpopulation of PD1+CD8+ fraction in leucine-treated sarcopenic mice was even much lower than that in non-sarcopenic group. The Kaplan-Meier curve also suggests that leucine-intake potentially increased the survival rates in sarcopenic mice. 

Taken together, the results obtained from the present study suggests that oral administration of leucine can restore the skeletal muscle mass and may affect the onset of cancer progression via improving the function of immunological defense. It would benefit the management of melanoma in sarcopenic patients.

DOI： 10.1254/jpssuppl.97.0_2-b-p-085

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Language：Japanese   Publisher：日本女性栄養・代謝学会  

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Language：Japanese   Publisher：(一社)日本老年医学会  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：Japanese Pharmacological Society  

Aiming at complete excision of cholesteatoma during trympanomastoidectomy and therefore reducing the risk of recurrence, the current study was undertaken to develop a seed DNA-aptamer-based fluorophore-probe, which targets human galectin-7, as an intraoperative lesion-identifying indicator for the surgical treatment of cholesteatoma. A galectin-7-targeted DNA-aptamer library was generated for labeling the cholesteatoma matrix using cell-based systematic evolution of ligands by an exponential enrichment technique. The binding characteristics of the identified aptamers were analyzed, and structure optimization of the identified aptamers was carried out both in silico and in vitro. Using galectin-7-aptamer guided molecular imaging, the excision margins of cholesteatoma matrix and surrounding normal tissue were successfully observed in a xenografted cholesteatoma model. It is highly expected that specific galectin-7-aptamers could progress to future clinical trials for both imaging and therapeutic applications and therefore benefit cholesteatoma patients.

DOI： 10.1254/jpssuppl.96.0_2-b-p-169

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Language：Japanese   Publisher：Japanese Pharmacological Society  

It is well known that chronic inflammation causes the sarcopenia. Whether the efforts to prevent skeletal muscle loss benefit the immune homeostasis remains unclear. In the present study, the efficacy of leucine supplementation on the maintenance of systemic immune function was evaluated. A sciatic nerve denervation-induced sarcopenic model was established and the volume and thickness of skeletal muscle on the hindlimb were evaluated by magnetic resonance imaging. Oral administration of leucine was carried on for 56 weeks. The skeletal muscle mass and the subsets or function of immune cells were analyzed. 

In leucine-treated sarcopenic mice, skeletal muscle mass on the hindlimb was significantly increased compared to that in non-treated sarcopenic mice. Leucine treatment repaired the mitochondria dysfunction in splenocytes from sarcopenic mice both <i>in vitro</i> and <i>in vivo</i>. In sarcopenic mice, an increase of the PD-1 expression was observed in CD4+ and CD8+ T cells. However, oral leucine administration restored the expression of PD-1 in the lymphocytes to the level of non-sarcopenic mice. 

In conclusion, the administration of leucine exhibits beneficial effects on sarcopenia and may influence the anti-cancer immune responses via adaptive immune resistance mechanism.

DOI： 10.1254/jpssuppl.96.0_2-b-ss11-2

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Language：Japanese   Publisher：Japanese Pharmacological Society  

Mast cells are important sentinel cells in the first line of host defense against viral and bacterial entry. SARS-CoV-2 can activate mast cells present in the respiratory tract in the initial stage of the disease. In the present study, we attempted to dissect the mechanisms underlying the interaction of the function of mast cells, especially focusing on the mast cell-derived chymase, with the cell entry of SRAS-CoV-2 by using a well-established mast cell line, rat basophil leukemia (RBL-2H3) cells. RBL-2H3 cells were infected by pseudovirons and the viral entry was evaluated. The interactions between SRAS-CoV-2 spike and mast cell-derived chymase were observed and predicated binding-site were determined by using spike-truncating variants. During the viral infection, the interaction of chymase with SRAS-CoV-2 may have both detrimental and positive impacts. Whether SRAS-CoV-2 spike is a potential substrate of chymase, and if its cleavage modulates biological properties of spike-bearing virus would be focused on in future planned study.

DOI： 10.1254/jpssuppl.95.0_1-p-059

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Mast cell (MC) exocytosis is organized by prenylated protein, including Rab families. Among Rab proteins, Rab3a, Rab27a, and Rab11 are responsible for exocytosis arrangement. Rab3a and Rab27a are contributed to exocytosis by interacting with other exocytosis proteins. Zoledronate administration disrupted the Rab prenylation process that affected its interaction with other proteins, and finally, its function. The present study has investigated the effect of zoledronate on the histamine release (HR) from RBL-2H3 cells. The main focus is to answer the question of whether zoledronate affects Rab27a/Doc2a interaction. Histamine release on RBL-2H3 cells after zoledronate or clodronate administration was measured using HPLC-fluorometry. Dinitrophenylated bovine serum albumin (DNP-BSA) (20 ng/mL) or ionomycin (1 µM) are used as secretagogues. Calcium (Ca2+) influx observation was performed using Fura-2A/M. In situ proximity ligation assay (PLA) is used to investigate Rab27a/Doc2a interaction after bisphosphonates (BPs) treatment. Histamine concentration measurement with HPLC-fluorometry showed that zoledronate (30, 100 µM) inhibited HR from antigen-activated RBL-2H3 cells. Zoledronate showed less inhibition in cells activated with ionomycin. Intracellular Ca2+ concentration and Ca2+ flux rate from the extracellular compartment was not changed by zoledronate administration. No changes in Rab27a/Doc2a interaction after zoledronate treatment. Histamine release inhibition by zoledronate in DNP-BSA-activated RBL-2H3 cells is not related to the disruption of Rab27a/Doc2a interaction and is not involve the change in Ca2+ influx.

DOI： 10.1248/bpb.b21-00717

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Language：Japanese   Publisher：(一社)日本サルコペニア・フレイル学会  

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Allergic diseases, including allergic rhinitis and asthma, reduce the quality of life when severe. Allergic disease is conventionally characterized by an increase in serum immunoglobulin E and activation of type 2 helper T cells and eosinophils. There is accumulated evidence that Enterococcus faecalis, a kind of lactobacillus preparation, has a significant effect on various allergic diseases through regulation of the systemic immune response. Since E. faecalis is a gram-positive, facultative anaerobic organism and causes infectious diseases such as bacteremia, E. faecalis preparations have been investigated and developed as a tyndallized probiotic. In addition, tyndallized treatment with E. faecalis FK-23 augments the effect on health promotion. It has been reported that lysed E. faecalis FK-23 (LFK) as a tyndallized probiotic has health-promoting effects by activation of Treg cells. We also reported that oral administration of LFK provides therapeutic benefit in ovalbumin-induced asthma model mice through regulation of the Treg/Th17 balance. Many of the studies were conducted by a group at Nichinichi Pharmaceutical Co., Ltd. The effects of LFK preparation on allergic diseases are introduced in this article.

DOI： 10.1016/B978-0-12-819815-5.00032-X

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Patients with systemic autoimmune disease are predisposed to developing sarcopenia associated with their underlying proinflammatory condition and a decrease in motility. How sarcopenic status affects disease progression remains unknown. The present study explored the influence of sarcopenia on immune homeostasis and investigated related biological pathways that might be important for future disease management. A denervation-induced skeletal muscle loss model was established and the function of the immune system was evaluated. The results of immune status profiling and functional assessment demonstrated that the loss of skeletal muscle impaired the function of mitochondrial respiration and activation in T cells, and therefore influenced the balance of T helper cell subsets. Consequently, sarcopenia might promote the progression of autoimmune diseases, such as lupus nephritis, as well as the enhancement of Th1/Th17 functions. These results suggest that physicians should be aware of the impact of the loss of skeletal muscle on the management of autoimmune disease, and that a multidisciplinary approach is required to minimize the overall adverse impact of sarcopenia.

DOI： 10.1254/jpssuppl.94.0_1-p2-22

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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DOI： 10.1016/j.bja.2020.05.046

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Language：Japanese   Publisher：(一社)日本抗加齢医学会  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ejphar.2020.173104

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In atopic diseases, the epithelium releases cytokines and chemokines that initiate skin inflammation. Atopic dermatitis (AD) is characterized by a disrupted epidermal barrier and is triggered or exacerbated by environmental stimuli such as house dust mite (HDM) allergens. The proinflammatory cytokine interleukin 33 (IL-33) plays an important role in the pathogenesis of AD, but how IL-33 production in keratinocytes is elicited by HDM is unknown. To that end, here we stimulated monolayer-cultured human keratinocytes and human living skin equivalents with Dermatophagoides pteronyssinus HDM extract to investigate its effects on IL-33 production from keratinocytes. The HDM extract induced intracellular expression of IL-33 and modulated its processing and maturation, triggering rapid IL-33 release from keratinocytes. Group 1 HDM allergen but not group 2 HDM allergen elicited IL-33 production. An ATP assay of keratinocyte culture supernatants revealed an acute and transient accumulation of extracellular ATP immediately after the HDM extract stimulation. Using the broad-spectrum P2 antagonist suramin, the specific purinergic receptor P2Y2 (P2RY2) antagonist AR-C118925XX, and P2RY2-specific siRNA, we discovered that the HDM extract-induced IL-33 expression was mainly dependent on extracellular ATP/P2Y2 signaling mediated by transactivation of epidermal growth factor receptor, followed by activation of the ERK kinase signaling pathway. Moreover, HDM extract–induced release of 25-kDa IL-33 from the keratinocytes depended on an extracellular ATP/P2 signaling–mediated intracellular Ca2+ increase. Our study demonstrates the new mechanism controlling the induction and maturation of keratinocyte-produced IL-33 by HDM allergens, an innate immune process that might play a role in AD development or severity.

DOI： 10.1016/j.bbadis.2020.165719

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OBJECTIVE: Mice and rats are important animal models for mast cell (MC) study. However, rat Mas-related-GPCR-B3 receptor (MRGPRB3) has been less studied than its mouse counterpart. Therefore, we aimed to characterize rat MRGPRB3. METHODS: Mrgprb3 mRNA expression was assessed in peritoneal cells (RPCs) and peritoneal MCs (RPMCs) of wild-type rats, RPCs of MC-deficient rats, and RBL-2H3 cells by reverse-transcriptase polymerase chain reaction (RT-PCR). RPMCs, MRGPRX2-transfected and non-transfected RBL-2H3 cells were activated by 15-30 min incubation with DNP-BSA, substance-P (SP), or compound-48/80. L732138 or CP96344 was used as a tachykinin/neurokinin-1-receptor antagonist. Histamine release from MCs was measured by HPLC fluorometry. RESULTS: Mrgprb3 mRNA expression was found in all cells, with the highest level in wild-type RPCs. All cells responded to DNP-BSA, but only MRGPRX2-transfected-RBL-2H3 cells and RPMCs responded to all activators. L732138 (0.1-10 μM) and CP96344 (1-100 μM) suppressed SP (10 μM)-induced RPMC activation. L732138 inhibition was dose independent, whereas CP96344 inhibition occurred in a dose-dependent manner. Additionally, only CP96344 suppressed SP (100 μM)- and compound-48/80 (10 μg/mL)-induced RPMC activation. CONCLUSIONS: RPMCs expressing functional MRGPRB3 response upon MRGPRX2 ligands to regulated MC-mediated activities. It`s provide novel insights for future pseudo-allergic studies in rodents.

DOI： 10.1007/s00011-020-01319-z

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Language：Japanese   Publisher：Japanese Pharmacological Society  

Defects in articular cartilage ultimately results in loss of joint function in rheumatoid arthritis. To investigate the influences of anti-rheumatoid drugs on chondrogenic differentiation of mesenchymal stem cell (MSC), <i>in vitro</i> and <i>in vivo</i> screening system were established. MSCs were collected and palleted in 4-microtube strips for chondrogenic induction. Quantification of formed micromasses was performed by three dimensional T2-weighted magnetic resonance imaging. For <i>in vivo</i> screening, scaffold or scaffoldless cartilaginous tissue were transplanted to NOD/ShiJic-scid mice for 2 months. The cartilaginous tissues were then explanted and the expressions of chondrogenic markers, including aggrecan and CD44, were assessed. We examined influence on inducted differentiation cartilages by methotrexate (MTX) and prednisolone (PSL). The volume of the chondrogenic spheroid in the presence of MTX was decreased in a dose-dependent manner, whereas no significant effect of PSL on chondrogenic differential potency were observed. The MSC-derived cartilaginous spheroid provides an effective screening tool to get the impact of anti-rheumatoid drugs on cartilaginous regeneration.

DOI： 10.1254/jpssuppl.93.0_1-ss-39

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Language：Japanese   Publisher：Japanese Pharmacological Society  

In present study, we aimed to investigate the feasibility of machine-learning-based classification using clinical features of patients for risk predication of anesthesia-related anaphylaxis.

After data pre-processing, the performance of four classification methods, which were integrated with four feature selection methods, were evaluated using two-layer cross-validation. Linear Discriminate Analysis in conjunction with Recursive Feature Elimination presented the best performance, with accuracy of 0.867 and Matthews correlation coefficient of 0.558 with 25 features used in the classification.

This study presents initial proof of the capability of a machine-learning-based strategy for forecasting low-prevalence anesthesia-related anaphylaxis. In future, we plan to utilize an extended database including preoperative information and vital-sign streams to define personalized risk status for anaphylaxis.

DOI： 10.1254/jpssuppl.93.0_3-p-385

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We investigated the effects of variations in anesthesia exposure time prior to conducting anxiety response behavioral testing in sham controls from an experimental murine model. The staying time in the center area of the Open Field test in the "long exposure" group was significantly decreased compared to that of the "short exposure" group. Significant correlation was found between anesthesia time and the duration of staying time in the center area. We conclude that anesthesia time may have a significant impact on behavioral anxiety testing in this context, and advise careful control of this parameter in protocol optimization in related surgical animal models.

DOI： 10.1016/j.jphs.2019.03.007

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Language：Japanese   Publisher：(一社)日本集中治療医学会  

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The resources of released histamine from activated mast cells, as initial effectors of allergic disease, include not only endogenous prepackaged histamine and newly synthesized histamine, but also histamine that is obtained through the de novo reuptake pathway. To investigate the de novo histamine production pathway, a mast cell line, RBL-2H3 Sc98 in which endogenous histamine production is lacking and only the de novo histamine release pathway via transporters is maintained, was used to dissect histamine reuptake in the present study. Histamine content measurements indicated that RBL-2H3 Sc98 cells took up extracellular histamine for storage in granules and subsequent release after stimulation by an antigen. Profiling and inhibition analysis of possible transporters suggested that the plasma membrane monoamine transporter and organic cation transporter 1 may be candidate transporters for histamine uptake from extracellular spaces, and that vesicular monoamine transporter 2 was responsible for intracellular vesicle uptake. These results may provide the foundation to understand the contribution of exogenous histamine to outward histamine release that is mediated by mechanisms other than conventional exocytosis.

DOI： 10.1016/j.ejphar.2019.01.050

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Language：Japanese   Publisher：Japanese Pharmacological Society  

Mas-related G protein coupled receptor-X2 (MRGPRX2) is a novel G protein-coupledreceptor with unique features including selective expression and response topeptidergic drug-induced activation in mast cells. With the aim of inhibitingMRGPRX2-dependent mast cell activation, a specific functional aptamer wasgenerated using the proteoliposome-based systematic evolution of ligands byexponential enrichment. After multiple cycles of selection and evolution,potential functional aptamers with high affinity and specificity were selectedby binding assay and functional screening and enriched for sequencing. Severalaptamers showed dose-dependent inhibitory potency on MRGPRX-dependent mast cellactivation. The feasibility and efficacy of these aptamers on drug-inducedanaphylactoid reactions will be investigated in our future planned study.

DOI： 10.1254/jpssuppl.92.0_3-p-104

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Muscle atrophy is a result of aging and disease, and can compromise physical function and impair vital metabolic processes. Low levels of muscular fitness, known as ‘flail', contribute greatly to weakness, disability, and immobility, and lead to increased hospitalization and loss of independence.

To investigate the correlation between muscle atrophy and aging-related immune deterioration, a dissected sciatic nerve sarcopenic mouse model was established and the fraction of immunological cells, including T cells, B cells, macrophages, natural killer cells, and neutrophils, was determined. Eight weeks after sciatic nerve dissection, the volume of both hind legs was evaluated based on acquired magnetic resonance imaging. Primary splenocytes and biceps femoris muscle-derived cells were isolated and labeled by cellular markers. The subpopulation of the cells was then detected using flow cytometry. T and B cell lineages were significantly suppressed in the sarcopenic model compared with control mice, and the fraction of macrophages and natural killer cells also tended to increase.

In conclusion, muscular loss appears to affect the immunological system by modulating humoral immunity and the cell-based immunology response. Further functional analysis of individual cell subsets could further determine the influence of sarcopenia on immune system improvement.

DOI： 10.1254/jpssuppl.92.0_1-ss-54

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Mast cells, in addition to endocrine cells and neurons, are typical secretory cells. Their function in allergic inflammation is to secrete inflammatory mediators from secretory vesicles. Intracellular synthesized inflammatory mediators are transported by vesicular monoamine transporters (VMATs) to vesicles where they are stored. After stimulation, the contents of the secretory vesicles are released via exocytosis. This study established a high throughput imaging screening system to monitor the functions of secretory vesicles in mast cells, including molecular uptake via VMAT2 and the exocytotic process, by using a novel fluorescent probe, FFN206, which was developed as a VMAT2 substrate. After loading with FFN206, the rapid uptake of FFN206 was observed and secretory vesicles in mouse bone marrow derived mast cells and a cultured mast cell line were clearly visualized. FFN206 uptake by secretory vesicles was time-dependent and was blocked by reserpine. Furthermore, exocytotic trafficking was monitored dynamically by real-time high-throughput fluorescence quantitation. In the present study, we verified the application of FFN206 for the monitoring of functional vesicles. This high-throughput screening system may benefit instinctive drug evaluation.

DOI： 10.1371/journal.pone.0198785

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

The aim of this study was to investigate the efficacy and safety of YM-58483, a small molecular antagonist of Ca2+ release-activated Ca2+ (CRAC) channels, for the treatment of rheumatoid arthritis (RA), in vivo and ex vivo. YM-58483 was continuously injected subcutaneously in a collagen-induced arthritis (CIA) mouS.E.M.odel using an implanted osmotic pump. The severity of CIA was evaluated using the following parameters: body weight, hind paw volume, clinical score, histological analysis, cytokine levels, Ca2+ influx, and specific IgG production. The efficacy of long-term application of YM-58483 was also verified ex vivo in RA patient-derived peripheral blood monocytes. Assessment of the clinical severity of CIA, cytokine profile in serum and joint protein extracts, and specific IgG production showed that continuous application of YM-58483 suppressed synovial inflammation by inhibiting immune cell activity. Chemical screening and hepatography indicated that long-term subcutaneous delivery of YM-58483 was safer than oral administration for systemic application. Moreover, constant preincubation with YM-58483 at an IC50 of 0.1–1 nM altered proinflammatory cytokine production ex vivo in peripheral T cells derived from RA patients. Our findings suggest that continuous long-term application of appropriate CRAC inhibitors such as YM-58483 is a potential therapeutic strategy for global immunosuppression in RA.

DOI： 10.1016/j.ejphar.2018.02.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media S.A.  

G-protein-coupled receptors (GPCRs) are membrane proteins distributed on the cell surface, and they may be potential drug targets. However, synthesizing GPCRs in vitro can be challenging. Recently, some cell-free protein synthesis systems have been shown to produce a large amount of membrane protein combined with chemical chaperones that include liposomes and glycerol. Liposomes containing high concentrations of glycerol are known as glycerosomes, which are used in new drug delivery systems. Glycerosomes have greater morphological stability than liposomes. Proteoglycerosomes are defined as glycerosomes that contain membrane proteins. Human histamine H1 receptor (HRH1) is one of the most studied GPCRs. In this study, we synthesized wild-type HRH1 (WT-HRH1) proteoglycerosomes and D107A-HRH1, (in which Asp107 was replaced by Ala) in a wheat germ cell-free protein synthesis system combined with asolectin glycerosomes. The mutant HRH1 has been reported to have low affinity for the H1 antagonist. In this study, the amount of synthesized WT-HRH1 in one synthesis reaction was 434 ± 66.6 μg (7.75 ± 1.19 × 103pmol). The specific binding of [3H]pyrilamine to the WT-HRH1 proteoglycerosomes became saturated as the concentration of the radioligand increased. The dissociation constant (Kd) and maximum density (Bmax) of the synthesized WT-HRH1 were 9.76 ± 1.25 nM and 21.4 ± 0.936 pmol/mg protein, respectively. However, specific binding to D107A-HRH1 was reduced compared with WT-HRH1 and the binding did not become saturated. The findings of this study highlight that HRH1 synthesized using a wheat germ cell-free protein synthesis system combined with glycerosomes has the ability to bind to H1 antagonists.

DOI： 10.3389/fphar.2018.00038

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Language：English   Publishing type：Part of collection (book)   Publisher：Humana Press Inc.  

The Ca 2+ ion is an important second messenger in lymphocytes, similarly to its function in other mammalian cells. The generation of long-lasting intracellular Ca 2+ elevations is essential for Ca 2+ -dependent gene transcription, proliferation, differentiation, and cytokine production in lymphocytes. Since store-operated Ca 2+ entry (SOCE) is considered the predominant mode of Ca 2+ influx in lymphocytes, the activation and function of lymphocytes can be generally predicted by monitoring SOCE. A method suitable for dynamic monitoring of Ca 2+ influx using fura-2 labeling in lymphocytes is introduced in this chapter. Using this technique, large-scale screening of the activation status of primary or cultured lymphocytes can be realized.

DOI： 10.1007/978-1-4939-8802-0_16

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DOI： 10.1007/978-1-4939-8802-0_9

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Nucleic acid aptamers are therapeutic agents consisting of short single-strand DNA or RNA oligonucleotides, which have the ability to bind to target therapeutic molecules with high affinity and specificity, and have been developed as potent drugs for the treatment of rheumatoid arthritis. Aptamers have unique and advantageous features over antibodies, such as superior affinity with nano- or pico-molar dissociation constants, and ease of chemical synthesis, modification, and inactivation by designing antisense sequences. In this chapter, using a DNA-oligonucleotide pool, the technology of proteoliposome-systematic evolution of ligands by exponential enrichment (SELEX) is introduced. By using this technique, potential therapeutic agents with high affinity and specificity could be obtained.

DOI： 10.1007/978-1-4939-8802-0_11

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The diagnosis of vasculitis in rheumatoid arthritis (RV) is associated with considerable mortality; therefore, understanding the basic mechanisms underlying the pathogenesis of vasculitis is very important. Animal models of vasculitis have contributed to elucidating such mechanisms. We here introduce a Candida albicans water-soluble (CAWS) glycoprotein-induced vasculitis model and the methodological approach to evaluate inflammatory vascular change.

DOI： 10.1007/978-1-4939-8802-0_23

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Language：English   Publishing type：Part of collection (book)   Publisher：Humana Press Inc.  

Since mice are widely used to establish rheumatoid arthritis models, assessment of the pathogenesis of local arthritis is fundamental. Proteins are the most diverse group of biologically important molecules and are essential for cellular structure and function. The first step in pathogenesis-related protein analysis is joint tissue extraction. Unlike other large rodents, obtaining synovium from model mice is challenging, since it is so small and fragile. In this chapter, methods for harvesting synovium through a quadriceps approach and preparing protein extracts are introduced.

DOI： 10.1007/978-1-4939-8802-0_6

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To achieve the most accurate assessment of functional Ca 2+ channel or modulator properties and their regulation, a patch clamp technique to record membrane currents is required. This technique has wide applications ranging from recording the activity of native channels in their natural environment to that of recombinant channels expressed in heterologous cells. This chapter introduces the methods that have been used for the detection of calcium release-activated calcium (CRAC) currents, one of the store-operated calcium entry pathways, in human primary T cells. This standard protocol is for laboratories already equipped with a full patch clamp setup or for investigators collaborating with laboratories experienced in patch clamp.

DOI： 10.1007/978-1-4939-8802-0_18

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which block the mevalonate pathway. The activity of statins not only decreases cholesterol levels but also ameliorates inflammation and modulates the immune system. In this study, we investigated the effects of simvastatin on histamine release using rat basophilic leukaemia (RBL-2H3) cells, and examined its interaction with proteins involved in the exocytosis process. Treatment with simvastatin for 24 h inhibited histamine release in RBL-2H3 cells in a concentration-dependent manner after stimulation with dinitrophenylated bovine serum albumin (DNP-BSA, as an antigen), ionomycin (a calcium ion [Ca2+] ionophore), and thapsigargin (an inhibitor of Ca(2+)ATPase in the endoplasmic reticulum). Simvastatin-induced inhibition was counteracted by co-administration of mevalonolactone or geranylgeraniol, but not farnesol. Indeed, several exocytotic proteins were post-translationally modified by isoprenylation, which is required for proper localization in the lipid membrane. RBL-2H3 cells express proteins involved in the fusion of granules and the plasma membrane, such as Ras-like protein in the brain 27a (Rab27a), synaptosome-associated protein 23 (SNAP23), and vesicle-associated membrane protein 7 (VAMP7), as well as Ca2+ binding proteins, such as double C2 alpha (Doc2a), synaptotagmin2, and mammalian uncoordinated13-4 (munc13-4). The interaction of Rab27a and Doc2a proteins was detected using proximity ligation assays. Antigen stimulation caused these proteins to interact, and this interaction could be disrupted by co-administration of simvastatin. In conclusion, simvastatin inhibited the mevalonate pathway, which suppressed the geranylgeranylation of Rab27a by depleting geranylgeranyl pyrophosphate and interfering with the Rab27a-Doc2a interaction. This activity resulted in the inhibition of exocytosis in RBL-2H3 cells.

DOI： 10.1016/j.ejphar.2017.08.026

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

Abnormal store-operated calcium uptake has been observed in peripheral T lymphocytes of rheumatoid arthritis (RA) patients, and sustained intracellular calcium signalling is known to mediate the functions of many types of immune cells. Thus, it is hypothesized that regulating calcium entry through CRACM1 (the pore-forming subunit of calcium release-activated calcium (CRAC) channels; also known as ORAI1) may be beneficial for the management of RA. Localized CRACM1 knockdown in the joints and draining lymph nodes (DLNs) of mice with collagen-induced arthritis (CIA) was achieved via lentiviral-based delivery of shRNA targeting mouse CRACM1. Consistent with CRACM1 knockdown, calcium influx in synovial cells and the histopathological features of CIA were reduced. These effects were also associated with reduced levels of several notable inflammatory cytokines, such as IL-6, IL-17A, and IFN-gamma, in the joints. Additionally, CRACM1-shRNA reduced the number of bone marrow-derived osteoclasts in vitro as well as osteoclasts in CIA joints, which was associated with reduced RANKL levels in the serum and joints. In summary, inhibiting calcium entry by CRACM1 knockdown suppressed arthritis development and may be therapeutically beneficial for RA patients. (C) 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

DOI： 10.1016/j.jphs.2017.02.001

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Language：English   Publisher：(公社)日本薬理学会  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BEGELL HOUSE INC  

With the aim of controlling disease relapse and bone deformation of individual joints, the application of gene therapy in rheumatoid arthritis (RA) has slowly progressed on a trial-and-error basis. Several new therapeutic targets have been identified in preclinical studies in animal models, although a limited number of gene-based clinical trials have been conducted. In this article, we summarize the status of gene therapy for RA by addressing issues related to innovating drug development. More disease-and target-specific preclinical tests are required to overcome the insufficient information regarding pharmacokinetics and toxicokinetics, which are related to safety issues in the field of RA gene therapy.

DOI： 10.1615/CritRevImmunol.2016017062

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Language：English   Publisher：(公社)日本薬理学会  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPANDIDOS PUBL LTD  

Evidence is increasing that oral administration of probiotics can attenuate asthmatic responses both in murine models and clinical trials. T-helper 17 (Th17) cells, a subset of CD4(+) T cells have been implicated as having an important role in the development of several allergic disorders, but the relationship between oral administration of probiotics and Th17 development has not been well studied. BALB/c mice were given lysed Enterococcus faecalis FK-23 (LFK) orally for 28 days. After sensitization by subcutaneous injection of ovalbumin (OVA) on Days 14 and 21 and 1% OVA inhalation on Days 25,26 and 27, they were challenged with a 5% OVA aerosol on Day 28. Twenty-four hours later, airway resistance and accumulation of inflammatory cells in bronchoalveolar lavage fluid (BALF) and lung tissues were determined. Interleukin (IL)-17-expressing CD4(+) lymphocytes isolated from lung, spleen and lamina propria of the intestine were detected by flow cytometry. The expression of IL-6 and TGF-beta mRNA was assessed by real-time PCR. Increases in airway hyperresponsiveness, and numbers of total leukocytes and mast cells in BALF induced by OVA challenge were significantly suppressed by oral administration of LFK. The increased percentage of IL-17-expressing CD4(+) cells from lung, spleen and intestine in OVA-challenged mice was reduced following LFK treatment. We conclude that the oral administration of LFK suppresses the asthmatic response and that this is associated with attenuation of Th17 cell development.

DOI： 10.3892/ijmm.2012.1010

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

During an allergic inflammatory response in the airway, if a failure of the epithelial cell barrier occurs before the systemic immune response is triggered by allergens, more allergens can invade. Using a rat model of asthma, we previously found that mucosal mast cells, which localise to the epithelial layer of the airways, are activated to promote a pro-asthmatic immune response. In this study, we developed a neonatal rat model of allergic airway hypersensitivity that mimics some features of childhood asthma. Airway hypersensitivity was measured using unrestrained whole-body plethysmography after analysis of the serum IgE titre. Inflammatory cells and inflammatory mediators in bronchoalveolar lavage fluid samples were examined. Two mast cell-specific proteases were detected using PCR. In addition, we analysed the phenotype and the number of mast cells in the airways by immunohistochemistry, and we found that the number of mucosal mast cells and the expression level of the proteases increased 2 weeks after sensitisation. Changes in the IgE titre, airway hypersensitivity and the activation of other inflammatory cells were delayed, appearing during the 4 weeks after sensitisation. Our results indicate that the activation of mucosal mast cells contributes to the pro-asthmatic immune response. This activation may be a biomarker allowing early intervention that could help prevent allergic airway inflammation. Immunology and Cell Biology (2011) 89, 239-245; doi:10.1038/icb.2010.90; published online 27 July 2010

DOI： 10.1038/icb.2010.90

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The pathology of non-immuno logical airway contraction is not well understood. To define the activation of different phenotypes of mast cells, a rat non-immunological asthmatic model was prepared. Air-way contraction in rats was measured by an unrestrained whole-body plethysmographic system following a 10-min inhalation challenge with a 5% solution of compound 48/80. Histamine, leukotrein C-4 (LTC4) and tumor necrosis factor (TNF)-alpha levels in bronchoalveolar lavage fluid, as well as tissue histamine content were quantified. Mast cells and eosinophils were detected by histology. Both the early and late phase of airway responses were induced by inhalation of compound 48/80. Histamine and TNF-alpha levels increased significantly 30 min after challenge, but no increases were detected at either 8 or 24 h after challenge. A high LTC4 level was detected in 30 min and 8 h after challenge. Tissue histamine content decreased at 30 min after challenge and returned to the unstimulated level by 8 h. Connective tissue mast cells in rat trachea showed a degranulation response. Along with the increase in numbers of mucosal mast cells, rat mast cell protease 11 at both mRNA and protein levels in the trachea epithelial layer was also increased significantly at 30 min after challenge. We conclude that compound 48/80 inhalation causes both the early and late phase of airway contraction in rats. Mast cell degranulation is responsible for the early phase of airway response, which subsequently triggers the late phase of airway response. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejphar.2005.10.067

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIRKHAUSER VERLAG AG  

Introduction: Mast cells are thought to be the main cause of an immediate asthmatic response, but their contribution to the late-phase of asthma is unknown.
Objective: To prove the contribution of preactivated mast cells to the late phase of allergic asthma by advanced activation.
Methods: Mast cell function in the late-phase of asthma was studied. Rats (wild, +/+ and mast cell deficient, Ws/Ws) were challenged with OVA to investigate the relationship between the contraction of airways and the population of inflammatory cells in the trachea.
Result: During the entire asthmatic period, the contraction of the airway after OVA challenge in +/+ rats was enhanced significantly compared to Ws/Ws rats, especially in the late phase. The bronchoalveolar lavage fluid histamine in +/+, but not Ws/Ws, rats increased 5.3-fold in 30 min and 3.4-fold in 8 h after challenge, significantly. The number of mucosal mast cells in the tracheal epithelial layer in +/+ rats increased significantly 2.2-fold over controls at 8 h after challenge, as demonstrated by in situ hybridization.
Conclusions: Mast cells may contribute to the late phase of asthmatic response by continuous mast cell activation and the mucosal mast cell number increased in the late phase of asthmatic response.

DOI： 10.1007/s00011-005-1346-9

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