Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Advanced Life Science　Functional Life Sciences　Embryonic and Genetic Engineering

Affiliation (Master)

• Faculty of Advanced Life Science　Functional Life Sciences　Embryonic and Genetic Engineering

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Profile and Settings

Degree

• Ph.D（2000/03 Health Sciences University of Hokkaido）

Profile and Settings

• Name (Japanese)

  Kiminori

• Name (Kana)

  Nakamura

• Name

  200901056393405370

Alternate Names

http://altair.sci.hokudai.ac.jp/infsig/

Achievement

Research Interests

• 腸内環境   Regenerative medicine   Inflammatory bowel disease   Infectious disease   Stem cell   Paneth cell   Intestine   defensin   Antimicrobial peptides   Innate immunity   Mucosal immunology   

Research Areas

• Life sciences / Immunology
• Life sciences / Oral medicine

Research Experience

• 2023/04 - Today 北海道大学大学院先端生命科学研究院 教授
• 2013 Hokkaido University Faculty of Advanced Life Science
• 2011 Hokkaido University Faculty of Advanced Life Science
• 2009 Hokkaido University Faculty of Advanced Life Science
• 2005 札幌医科大学医学部分子医学研究部門 講師
• 2004 札幌医科大学医学部分子医学研究部門 助手

Education

• 2000 -   Health Sciences University of Hokkaido  Graduate School of Dentistry
• 1996 -   Health Sciences University of Hokkaido  School of Dentistry

Published Papers

• Potential consequences of phototoxicity on cell function during live imaging of intestinal organoids

  Yuki Yokoi, Ryu Nakamura, Shuya Ohira, Shota Takemi, Tokiyoshi Ayabe, Kiminori Nakamura

  PLOS ONE 19 (11) e0313213 - e0313213 2024/11/15 [Refereed]
   

  Live imaging visualizes the structure, dynamics, and function of cells and tissues to reveal the molecular mechanisms, and has contributed to the advancement of life science. In live imaging, it has been well known that there is a trade-off between higher-resolution analysis and cell damage caused by light illumination, i.e., phototoxicity. However, despite the risk of unknowingly distorting experimental results, phototoxicity is an unresolved issue in live imaging because overall consequences occurring inside cells due to phototoxicity remains unknown. Here, we determined the molecular process of phototoxicity-induced cell damage systematically under low- and high-dose light illumination conditions by analyzing differential gene expression using RNA-sequencing in a three-dimensional organoid of small intestinal epithelial cells, enteroid. The low-dose light illumination already induced various abnormalities in functional molecules involved in the response to reactive oxygen species generated by the excitation of fluorescent dyes, intracellular metabolism, mitosis, immune responses, etc., at mRNA expression level. Together with the behavior toward apoptosis caused by high-dose light illumination, the light dose-dependent progression of intracellular damage was revealed. About visible impairment of intestinal epithelial function, failures in both the structure-forming ability of enteroids and Paneth cell granule secretion were observed under high-dose light illumination, while the drug efflux was not disturbed despite abnormal drug efflux transporter mRNA expression. Based on the gene expression profiles, we comprehensively clarified phenomena in the cells at mRNA level that cannot be recognized both morphologically and functionally during live imaging, further providing a new insight into the risk of phototoxicity. This study warns from the aspect of mRNA expression that awareness of phototoxic artifacts is needed when analyzing cellular function and the mechanism in live imaging.
• Association between oral frailty and Prevotella percentage in the oral microbiota of community-dwelling older adults who participated in the CHEER Iwamizawa project, Japan.

  Chizuru Kimura, Kazuhito Miura, Yutaka Watanabe, Haruhisa Baba, Kimiya Ozaki, Akira Hasebe, Tokiyoshi Ayabe, Kiminori Nakamura, Shinji Nakaoka, Katsuhiko Ogasawara, Teppei Suzuki, Hiroshi Saito, Takashi Kimura, Akiko Tamakoshi, Yutaka Yamazaki

  Journal of oral rehabilitation 2024/06/08 [Refereed]
   

  BACKGROUND: Prevotella bacteria are associated with inherent diseases of the oral cavity, such as periodontal disease, and systemic diseases. Oral frailty (OF) has been associated with nursing necessity and death. However, the relationship between OF and oral microbiota has not been fully clarified. OBJECTIVE: This cross-sectional study investigated the association between OF and Prevotella percentage in the oral microbiota of community-dwelling older adults. METHODS: Oral bacteria species from saliva were identified in 208 community-dwelling older individuals aged ≥60 years in Japan. The proportion of Prevotella in the oral microbiota was classified into three tertile groups, and its relationship with each test item for OF (number of remaining teeth, masticatory performance, oral diadochokinesis, tongue pressure, difficulties eating tough foods, difficulties swallowing tea or soup, number of applicable OF judgement items, and existence of OF) was examined using ordinal logistic regression analysis. RESULTS: The Prevotella proportions were classified into lower, middle and upper groups, comprising 70, 69 and 69 participants, respectively. The three groups showed a significant relationship between the number of remaining teeth (odds ratio [OR]: 0.946, 95% confidence interval [CI]: 0.915-0.977), masticatory performance (OR: 0.897, 95% CI: 0.844-0.953), number of applicable OF judgement items (OR: 1.477, 95% CI: 1.14-1.915), and existence of OF (OR: 4.194, 95% CI: 1.519-11.576). CONCLUSION: The proportion of Prevotella in oral microbiota was high in individuals with OF. Among the older adults, the type of oral microbiota and systemic diseases may be related to the examination and management of oral function decline.
• Development of an Evaluation System Using Intestinal Organoids for Drug Efflux Transport Analysis by an Imaging Approach

  Chihiro Koseki, Takehiko Ishikawa, Yuki Sato, Mikiko Shimada, Yuki Yokoi, Kiminori Nakamura, Naoyuki Honma, Takanori Moriyama, Hitoshi Kashiwagi, Mitsuru Sugawara

  Journal of Pharmaceutical Sciences 0022-3549 2024/06 [Refereed]
  
• Effects of exposure to the neonicotinoid pesticide clothianidin on α-defensin secretion and gut microbiota in mice

  Sakura YONOICHI, Yukako HARA, Yuya ISHIDA, Asuka SHODA, Mako KIMURA, Midori MURATA, Sarika NUNOBIKI, Makiko ITO, Ayano YOSHIMOTO, Youhei MANTANI, Toshifumi YOKOYAMA, Tetsushi HIRANO, Yoshinori IKENAKA, Yuki YOKOI, Tokiyoshi AYABE, Kiminori NAKAMURA, Nobuhiko HOSHI

  Journal of Veterinary Medical Science 0916-7250 2024 [Refereed]
  
• A Basic Study of the Effects of Mulberry Leaf Administration to Healthy C57BL/6 Mice on Gut Microbiota and Metabolites

  Li Gan, Yuga Inamura, Yu Shimizu, Yuki Yokoi, Yuki Ohnishi, Zihao Song, Yasuhiro Kumaki, Takashi Kikukawa, Makoto Demura, Masaaki Ito, Tokiyoshi Ayabe, Kiminori Nakamura, Tomoyasu Aizawa

  Metabolites 13 (9) 1003 - 1003 2023/09/10 [Refereed]
   

  Mulberry leaves contain α-glucosidase inhibitors, which have hypoglycemic effects and are considered functional foods. However, few reports have covered the effects of mulberry leaf components on normal gut microbiota and gut metabolites. Herein, gut microbiota analysis and NMR-based metabolomics were performed on the feces of mulberry leaf powder (MLP)-treated mice to determine the effects of long-term MLP consumption. Gut microbiota in the mouse were analyzed using 16S-rRNA gene sequencing, and no significant differences were revealed in the diversity and community structure of the gut microbiota in the C57BL/6 mice with or without MLP supplementation. Thirty-nine metabolites were identified via 1H-NMR analysis, and carbohydrates and amino acids were significantly (p < 0.01–0.05) altered upon MLP treatment. In the MLP-treated group, there was a marked increase and decrease in maltose and glucose concentrations, respectively, possibly due to the degradation inhibitory activity of oligosaccharides. After 5 weeks, all amino acid concentrations decreased. Furthermore, despite clear fluctuations in fecal saccharide concentrations, short-chain fatty acid production via intestinal bacterial metabolism was not strongly affected. This study provides the knowledge that MLP administration can alter the gut metabolites without affecting the normal gut microbiota, which is useful for considering MLP as a healthy food source.
• Antimicrobial Properties and Mode of Action of Cryptdin-4, a Mouse α-Defensin Regulated by Peptide Redox Structures and Bacterial Cultivation Conditions

  Yi Wang, Yuchi Song, Shaonan Yan, Rina Hiramine, Yuki Ohnishi, Yuki Yokoi, Kiminori Nakamura, Takashi Kikukawa, Tokiyoshi Ayabe, Tomoyasu Aizawa

  Antibiotics 12 (6) 1047 - 1047 2023/06/14 [Refereed]
   

  Cryptdin-4 (crp4) is an enteric α-defensin derived from mice, and is a main mediator of immunity to oral infections and a determinant of the composition of the intestinal microbiota. Structurally, crp4 exists in two states: the oxidized form (crp4oxi), constrained by three invariant disulfide bonds, and the reduced form (crp4red) with six free thiol groups, both of which exist in the intestinal tract. In this study, the antibacterial mechanisms of crp4 in both forms under aerobic and anaerobic conditions were investigated using Escherichia coli (E. coli), an anaerobic facultative bacterium, as a model. Fluorescent dye studies revealed that both crp4oxi and crp4red exhibited antimicrobial activity against cells cultured under aerobic conditions via rapid membrane depolarization. Furthermore, the antioxidant treatment experiments suggested that only crp4oxi exhibited antimicrobial activity by the induction and accumulation of reactive oxygen species (ROS). However, under anaerobic culture conditions, the ability of both forms to disrupt the function of bacterial membranes decreased and activity was greatly reduced, but crp4red maintained some antimicrobial activity. This activity may be due to the inhibition of intracellular functions by DNA binding. Altogether, these data indicate that, according to its redox structure and the environmental redox conditions, crp4 could perform different antimicrobial activities via different mechanisms.
• Application of Benchtop NMR for Metabolomics Study Using Feces of Mice with DSS-Induced Colitis

  Zihao Song, Yuki Ohnishi, Seiji Osada, Li Gan, Jiaxi Jiang, Zhiyan Hu, Hiroyuki Kumeta, Yasuhiro Kumaki, Yuki Yokoi, Kiminori Nakamura, Tokiyoshi Ayabe, Kazuo Yamauchi, Tomoyasu Aizawa

  Metabolites 13 (5) 611 - 611 2023/04/28 [Refereed]
   

  Nuclear magnetic resonance (NMR)-based metabolomics, which comprehensively measures metabolites in biological systems and investigates their response to various perturbations, is widely used in research to identify biomarkers and investigate the pathogenesis of underlying diseases. However, further applications of high-field superconducting NMR for medical purposes and field research are restricted by its high cost and low accessibility. In this study, we applied a low-field, benchtop NMR spectrometer (60 MHz) employing a permanent magnet to characterize the alterations in the metabolic profile of fecal extracts obtained from dextran sodium sulfate (DSS)-induced ulcerative colitis model mice and compared them with the data acquired from high-field NMR (800 MHz). Nineteen metabolites were assigned to the 60 MHz 1H NMR spectra. Non-targeted multivariate analysis successfully discriminated the DSS-induced group from the healthy control group and showed high comparability with high-field NMR. In addition, the concentration of acetate, identified as a metabolite with characteristic behavior, could be accurately quantified using a generalized Lorentzian curve fitting method based on the 60 MHz NMR spectra.
• Shorter sleep time relates to lower human defensin 5 secretion and compositional disturbance of the intestinal microbiota accompanied by decreased short-chain fatty acid production

  Yu Shimizu, Ryodai Yamamura, Yuki Yokoi, Tokiyoshi Ayabe, Shigekazu Ukawa, Koshi Nakamura, Emiko Okada, Akihiro Imae, Takafumi Nakagawa, Akiko Tamakoshi, Kiminori Nakamura

  Gut Microbes 15 (1) 1949-0976 2023/03/21 [Refereed]
  
• Decreased Paneth cell α-defensins promote fibrosis in a choline-deficient L-amino acid-defined high-fat diet-induced mouse model of nonalcoholic steatohepatitis via disrupting intestinal microbiota

  Shunta Nakamura, Kiminori Nakamura, Yuki Yokoi, Yu Shimizu, Shuya Ohira, Mizu Hagiwara, Zihao Song, Li Gan, Tomoyasu Aizawa, Daigo Hashimoto, Takanori Teshima, Andre J. Ouellette, Tokiyoshi Ayabe

  Scientific Reports 13 (1) 2023/03/09 [Refereed]
   

  Abstract Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fibrosis that develops from fatty liver. Disruption of intestinal microbiota homeostasis, dysbiosis, is associated with fibrosis development in NASH. An antimicrobial peptide α-defensin secreted by Paneth cells in the small intestine is known to regulate composition of the intestinal microbiota. However, involvement of α-defensin in NASH remains unknown. Here, we show that in diet-induced NASH model mice, decrease of fecal α-defensin along with dysbiosis occurs before NASH onset. When α-defensin levels in the intestinal lumen are restored by intravenous administration of R-Spondin1 to induce Paneth cell regeneration or by oral administration of α-defensins, liver fibrosis is ameliorated with dissolving dysbiosis. Furthermore, R-Spondin1 and α-defensin improved liver pathologies together with different features in the intestinal microbiota. These results indicate that decreased α-defensin secretion induces liver fibrosis through dysbiosis, further suggesting Paneth cell α-defensin as a potential therapeutic target for NASH.
• Associations between Maternal Diet, Human Milk Macronutrients, and Breast-Fed Infant Growth during the First Month of Life in the SMILE Iwamizawa in Japan.

  Yosuke Komatsu, Yasuaki Wada, Fuka Tabata, Satomi Kawakami, Yasuhiro Takeda, Kiminori Nakamura, Tokiyoshi Ayabe, Koshi Nakamura, Takashi Kimura, Akiko Tamakoshi

  Nutrients 15 (3) 2023/01/28 [Refereed]
   

  Maternal diet may affect human milk macronutrients, but it remains to be elucidated whether this is also influential in infant growth. This study aimed to examine (1) how maternal diet influences human milk macronutrients, and (2) to what extent the variation in milk macronutrients affects infant growth during the first month of life. In 71 Japanese lactating women, maternal dietary information was collected from the brief-type self-administered diet history questionnaire, and anthropometry of mother-infant dyads was collected from medical records. Macronutrients in milk were analyzed by a Human Milk Analyzer. Maternal retinol intake was associated with the carbohydrate content in human milk at 1-month postpartum (standardized β coefficient: 0.287; p = 0.038). Moreover, the energy content in human milk was associated with an increase in the weight standard deviation score based on the WHO growth standard at 1 month of age (standardized β coefficient: 0.399; p = 0.046). Nevertheless, the milk macronutrient was not associated with the risk of infant growth abnormalities. In conclusion, a part of the maternal diet impacts macronutrient contents in human milk, but milk macronutrients have a limited effect on infant growth only within the normal growth curve during the first month of life.
• Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation.

  Yuchi Song, Yi Wang, Shaonan Yan, Kiminori Nakamura, Takashi Kikukawa, Tokiyoshi Ayabe, Tomoyasu Aizawa

  Microbial cell factories 22 (1) 9 - 9 2023/01/13 [Refereed]
   

  BACKGROUND: A number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods. RESULTS: To avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences. CONCLUSION: In this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.
• Efficient and simple genetic engineering of enteroids using mouse isolated crypts for investigating intestinal functions

  Shuya Ohira, Yuki Yokoi, Tokiyoshi Ayabe, Kiminori Nakamura

  Biochemical and Biophysical Research Communications 637 153 - 160 0006-291X 2022/12 [Refereed]
  
• Reactive granulopoiesis depends on T-cell production of IL-17A and neutropenia-associated alteration of gut microbiota.

  Xuanzhong Chen, Daigo Hashimoto, Ko Ebata, Shuichiro Takahashi, Yu Shimizu, Ryuga Shinozaki, Yuta Hasegawa, Ryo Kikuchi, Hajime Senjo, Kazuki Yoneda, Zixuan Zhang, Shinpei Harada, Eiko Hayase, Takahide Ara, Hiroyuki Ohigashi, Yoichiro Iwakura, Kiminori Nakamura, Tokiyoshi Ayabe, Takanori Teshima

  Proceedings of the National Academy of Sciences of the United States of America 119 (48) e2211230119  2022/11/29 [Refereed]
   

  Granulopoiesis in the bone marrow adjusts cellular output as demand for neutrophils changes. Reactive granulopoiesis is induced by profound neutropenia, but its mechanism remains to be clarified. We herein explored its mechanisms using mouse models of syngeneic hematopoietic stem cell transplantation (SCT) and 5-fluorouracil-induced neutropenia. After SCT, T cell production of IL-17A was up-regulated. Neutrophil recovery was significantly delayed in IL-17A-deficient or T cell-deficient RAG1-/- mice, and adoptive transfer of wild-type (WT) T cells facilitated neutrophil engraftment. Gut decontamination with oral antibiotics suppressed T cell production of IL-17A and impaired neutrophil recovery. Transplantation of fecal microbiota collected from neutropenic, not naive, mice promoted neutrophil recovery in these mice, suggesting that neutropenia-associated microbiota had a potential to stimulate reactive granulopoiesis. Our study uncovered a cross talk between gut microbiota and neutropenia after SCT and chemotherapy.
• Hemodynamic response to intestinal pH stimulation measured with spectroscopic video imaging

  Tsukasa Funane, Yuki Yokoi, Masashi Kikuchi, Ryuga Shinozaki, Tokiyoshi Ayabe, Hirokazu Atsumori, Ayako Nishimura, Kiminori Nakamura, Akihiko Kandori

  Biomedical Physics & Engineering Express 2022/11/11 [Refereed]
   

  Abstract To investigate the relationship between the gut and skin (gut-skin axis), head skin hemodynamic responses to gut stimulation including the injection of acetic acid in nude mice were measured by spectroscopic video imaging, which was calculated using a modified Beer-Lambert formula. The relationship with blood proteins was also analyzed. The blood volume changes in three mice injected with acetic acid were highly reproducible in the mathematical model equation. Four proteins correlated with blood volume changes were all related to immunity. These results suggest that intestinal pH can alter the blood volume in the skin and induce immune-related responses.
• Oral frailty and carriage of oral Candida in community‐dwelling older adults (Check‐up to discover Health with Energy for senior Residents in Iwamizawa; CHEER Iwamizawa)

  Haruhisa Baba, Yutaka Watanabe, Kazuhito Miura, Kimiya Ozaki, Takae Matsushita, Miyako Kondoh, Kazutaka Okada, Akira Hasebe, Tokiyoshi Ayabe, Kiminori Nakamura, Shinji Nakaoka, Katsuhiko Ogasawara, Teppei Suzuki, Hiroshi Saito, Takashi Kimura, Akiko Tamakoshi, Yutaka Yamazaki

  Gerodontology 39 (1) 49 - 58 0734-0664 2022/03 [Refereed]
  
• Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption

  Yuji Sato, Yi Wang, Yuchi Song, Weiming Geng, Shaonan Yan, Kiminori Nakamura, Takashi Kikukawa, Makoto Demura, Tokiyoshi Ayabe, Tomoyasu Aizawa

  Amino Acids 54 (2) 289 - 297 0939-4451 2022/02 [Refereed]
  
• Ingestion of miso regulates immunological robustness in mice

  Kunihiko Kotake, Toshihiko Kumazawa, Kiminori Nakamura, Yu Shimizu, Tokiyoshi Ayabe, Takahiro Adachi

  PLOS ONE 17 (1) e0261680 - e0261680 2022/01 [Refereed]
   

  In Japan, there is a long history of consumption of miso, a fermented soybean paste, which possesses beneficial effects on human health. However, the mechanism behind these effects is not fully understood. To clarify the effects of miso on immune cells, we evaluated its immunomodulatory activity in mice. Miso did not alter the percentage of B and T cells in the spleen; however, it increased CD69⁺ B cells, germinal center B cells and regulatory T cells. Anti-DNA immunoglobulin M antibodies, which prevent autoimmune disease, were increased following ingestion of miso. Transcriptome analysis of mouse spleen cells cultured with miso and its raw material revealed that the expression of genes, including interleukin (IL)-10, IL-22 and CD86, was upregulated. Furthermore, intravital imaging of the small intestinal epithelium using a calcium biosensor mouse line indicated that miso induced Ca²⁺ signaling in a manner similar to that of probiotics. Thus, ingestion of miso strengthened the immune response and tolerance in mice. These results appear to account, at least in part, to the salubrious effects of miso.
• Intestinal commensal microbiota and cytokines regulate Fut2 ⁺ Paneth cells for gut defense

  Mariko Kamioka, Yoshiyuki Goto, Kiminori Nakamura, Yuki Yokoi, Rina Sugimoto, Shuya Ohira, Yosuke Kurashima, Shingo Umemoto, Shintaro Sato, Jun Kunisawa, Yu Takahashi, Steven E. Domino, Jean-Christophe Renauld, Susumu Nakae, Yoichiro Iwakura, Peter B. Ernst, Tokiyoshi Ayabe, Hiroshi Kiyono

  Proceedings of the National Academy of Sciences 119 (3) 0027-8424 2022/01 [Refereed]
   

  Significance Paneth cells produce granules containing antimicrobial peptides, such as α-defensin for host defense. Examination of the production and utilization of fucosyltransferase 2 (Fut2) by Paneth cells revealed two distinct subsets: Fut2 ⁺ Paneth cells and Fut2 ⁻ Paneth cells. Compared with Fut2 ⁻ Paneth cells, Fut2 ⁺ Paneth cells were enriched in granules containing antimicrobial peptides. IL-22 and IL-17a were found to be essential for commensal bacteria-dependent Fut2 ⁺ Paneth cell development and function as part of gut defense.
• Lower human defensin 5 in elderly people compared to middle-aged is associated with differences in the intestinal microbiota composition: the DOSANCO Health Study.

  Yu Shimizu, Kiminori Nakamura, Mani Kikuchi, Shigekazu Ukawa, Koshi Nakamura, Emiko Okada, Akihiro Imae, Takafumi Nakagawa, Ryodai Yamamura, Akiko Tamakoshi, Tokiyoshi Ayabe

  GeroScience 44 (2) 997 - 1009 2509-2715 2021/06/08 [Refereed]
   

  Recently, aging is considered a risk factor for various diseases. Although changes in the intestinal microbiota along with aging are thought to associate with the increased disease risk, mechanisms that cause age-related transition of the intestinal microbiota remain unknown. This study aims to clarify relationships between the amount of human defensin 5 (HD5), a Paneth cell α-defensin, which is known to regulate the intestinal microbiota, and age-related differences of the intestinal microbiota composition. Fecal samples from 196 healthy Japanese (35 to 81 years old) were collected and measured HD5 concentration. HD5 concentration in the elderly group (age > 70 years old) was significantly lower than the middle-aged group (age ≤ 70 years old). Furthermore, individual age was negatively correlated with HD5 concentration (r =  − 0.307, p < 0.001). In β-diversity, the intestinal microbiota of the elderly showed a significantly different composition compared to the middle-aged. At the genus level, relative abundance of Collinsella, Alistipes, Peptococcaceae; unassigned, Lactobacillus, Lactococcus, Weissella, Christensenellaceae R-7 group, Megasphaera, and [Eubacterium] eligens group was significantly higher, and Lachnospiraceae; unassigned, Blautia, Anaerostipes, Fusicatenibacter, Dorea, and Faecalibacterium was significantly lower in the elderly compared to the middle-aged. In addition, HD5 concentration was negatively correlated with Alistipes, Peptococcaceae; unassigned, and Christensenellaceae R-7 group and positively correlated with Lachnospiraceae; unassigned and Dorea. These results provide novel insights into the immunosenescence of enteric innate immunity, indicating low HD5 is suggested to contribute to the age-related differences in the intestinal microbiota and may relate to increased risk of diseases in elderly people.
• Decrease of α-defensin impairs intestinal metabolite homeostasis via dysbiosis in mouse chronic social defeat stress model.

  Kosuke Suzuki, Kiminori Nakamura, Yu Shimizu, Yuki Yokoi, Shuya Ohira, Mizu Hagiwara, Yi Wang, Yuchi Song, Tomoyasu Aizawa, Tokiyoshi Ayabe

  Scientific reports 11 (1) 9915 - 9915 2021/05/10 [Refereed]
   

  Psychological stress has been reported to relate to dysbiosis, imbalance of the intestinal microbiota composition, and contribute to the onset and exacerbation of depression, though, underlying mechanisms of psychological stress-related dysbiosis have been unknown. It has been previously established that α-defensins, which are effector peptides of innate enteric immunity produced by Paneth cells in the small intestine, play an important role in regulation of the intestinal microbiota. However, the relationship between disruption of intestinal ecosystem and α-defensin under psychological stress is yet to be determined. Here we show using chronic social defeat stress (CSDS), a mouse depression model that (1) the exposure to CSDS significantly reduces α-defensin secretion by Paneth cells and (2) induces dysbiosis and significant composition changes in the intestinal metabolites. Furthermore, (3) they are recovered by administration of α-defensin. These results indicate that α-defensin plays an important role in maintaining homeostasis of the intestinal ecosystem under psychological stress, providing novel insights into the onset mechanism of stress-induced depression, and may further contribute to discovery of treatment targets for depression.
• Simultaneous real-time analysis of Paneth cell and intestinal stem cell response to interferon-γ by a novel stem cell niche tracking method

  Yuki Yokoi, Takahiro Adachi, Rina Sugimoto, Mani Kikuchi, Tokiyoshi Ayabe, Kiminori Nakamura

  Biochemical and Biophysical Research Communications 545 14 - 19 0006-291X 2021/03 [Refereed]
  
• Mycotoxin Deoxynivalenol Has Different Impacts on Intestinal Barrier and Stem Cells by Its Route of Exposure

  Hikaru Hanyu, Yuki Yokoi, Kiminori Nakamura, Tokiyoshi Ayabe, Keisuke Tanaka, Kinuko Uno, Katsuhiro Miyajima, Yuki Saito, Ken Iwatsuki, Makoto Shimizu, Miki Tadaishi, Kazuo Kobayashi-Hattori

  Toxins 12 (10) 610 - 610 2020/09/24 [Refereed]
   

  The different effects of deoxynivalenol (DON) on intestinal barrier and stem cells by its route of exposure remain less known. We explored the toxic effects of DON on intestinal barrier functions and stem cells after DON microinjection (luminal exposure) or addition to a culture medium (basolateral exposure) using three-dimensional mouse intestinal organoids (enteroids). The influx test using fluorescein-labeled dextran showed that basolateral DON exposure (1 micromolar (µM) disrupted intestinal barrier functions in enteroids compared with luminal DON exposure at the same concentration. Moreover, an immunofluorescence experiment of intestinal epithelial proteins, such as E-cadherin, claudin, zonula occludens-1 (ZO-1), and occludin, exhibited that only basolateral DON exposure broke down intestinal epithelial integrity. A time-lapse analysis using enteroids from leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescence protein (EGFP) transgenic mice and 5-ethynyl-2-deoxyuridine (EdU) assay indicated that only the basolateral DON exposure, but not luminal DON exposure, suppressed Lgr5+ stem cell count and proliferative cell ratio, respectively. These results revealed that basolateral DON exposure has larger impacts on intestinal barrier function and stem cells than luminal DON exposure. This is the first report that DON had different impacts on intestinal stem cells depending on the administration route. In addition, RNA sequencing analysis showed different expression of genes among enteroids after basolateral and luminal DON exposure.
• Paneth cell α-defensin misfolding correlates with dysbiosis and ileitis in Crohn's disease model mice.

  Yu Shimizu, Kiminori Nakamura, Aki Yoshii, Yuki Yokoi, Mani Kikuchi, Ryuga Shinozaki, Shunta Nakamura, Shuya Ohira, Rina Sugimoto, Tokiyoshi Ayabe

  Life science alliance 3 (6) e201900592 - e201900592 2020/06 [Refereed][Not invited]
   

  Crohn’s disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and α-defensin misfolding have been reported in CD patients. Because α-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, α-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form α-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form α-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form α-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell α-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets.
• Disease progression-associated alterations in fecal metabolites in SAMP1/YitFc mice, a Crohn's disease model.

  Yosuke Komatsu, Yu Shimizu, Megumi Yamano, Mani Kikuchi, Kiminori Nakamura, Tokiyoshi Ayabe, Tomoyasu Aizawa

  Metabolomics. 16 (4) 48 - 48 2020/04/10 [Refereed][Not invited]
   

  INTRODUCTION: Crohn's disease (CD) is a chronic, relapsing inflammatory bowel disease affecting the gastrointestinal tract. Although its precise etiology has not been fully elucidated, an imbalance of the intestinal microbiota has been known to play a role in CD. Fecal metabolites derived from microbiota may be related to the onset and progression of CD OBJECTIVES: This study aimed to clarify the transition of gut microbiota and fecal metabolites associated with disease progression using SAMP1/YitFc mice, a model of spontaneous CD METHODS: The ileum tissues isolated from SAMP1/YitFc mice at different ages were stained with hematoxylin-eosin for histologic characterization with CD progression. Feces from control, Institute of Cancer Research (ICR; n = 6), and SAMP1/YitFc (n = 8) mice at different ages were subjected to microbial analysis and 1H nuclear magnetic resonance (NMR) analysis to investigate fluctuations in gut microbiota and fecal metabolites with CD progression RESULTS: Relative abundance of the Lachnospiraceae, Ruminococcaceae, Bacteroidaceae, and Bacteroidales S24-7 at family-level gut microbiota and fecal metabolites, such as short-chain fatty acids, lactate, glucose, xylose, and choline, dramatically fluctuated with histologic progression of intestinal inflammation in SAMP1/YitFc mice. Unlike the other metabolites, fecal taurine concentration in SAMP1/YitFc mice was higher than ICR mice regardless of age CONCLUSION: The fecal metabolites showing characteristic fluctuations may help to understand the inflammatory mechanism associated with CD, and might be utilized as potential biomarkers in predicting CD pathology.
• Analysis of Serotonin in Human Feces Using Solid Phase Extraction and Column-Switching LC-MS/MS.

  Yukiko Hirabayashi, Kiminori Nakamura, Tsuyoshi Sonehara, Daisuke Suzuki, Satoru Hanzawa, Yu Shimizu, Tomoyasu Aizawa, Koshi Nakamura, Akiko Tamakoshi, Tokiyoshi Ayabe

  Mass spectrometry (Tokyo). 9 (1) A0081  2020 [Refereed]
   

  Serotonin, an important neurotransmitter, is produced mainly in intestines, and serotonin levels in feces can be an indicator of the intestinal environment. Human feces, however, contain a large amount of contaminants, which vary widely owing to food contents and the intestinal environment, and these contaminants would be expected to interfere with the determination of serotonin levels in human feces. To remove these contaminants and determine serotonin levels, we developed a new method using solid phase extraction (SPE) and column-switching LC-MS/MS. Serotonin, labeled with a stable isotope, was added to human feces samples prior to SPE as an internal standard to correct for individual differences in matrix effects. The recovery rate for SPE was 55.9-81.0% (intraday) and 56.5-78.1% (interday) for feces from two subjects. We analyzed 220 fecal samples from 96 subjects including 76 pregnant and post-delivery women. The endogenous serotonin content per unit weight of dried feces was 0.09-14.13 ng/mg for pregnant and post-delivery women and 0.30-9.93 ng/mg for the remaining subjects.
• Expression and Localization of Paneth Cells and Their α-Defensins in the Small Intestine of Adult Mouse.

  Kiminori Nakamura, Yuki Yokoi, Rie Fukaya, Shuya Ohira, Ryuga Shinozaki, Takuto Nishida, Mani Kikuchi, Tokiyoshi Ayabe

  Frontiers in immunology 11 570296 - 570296 2020 [Refereed]
   

  Paneth cells contribute to intestinal innate immunity by sensing bacteria and secreting α-defensin. In Institute of Cancer Research (ICR) mice, α-defensin termed cryptdin (Crp) in Paneth cells consists of six major isoforms, Crp1 to 6. Despite accumulating evidences that α-defensin functions in controlling the intestinal microbiota, topographical localization of Paneth cells in the small intestine in relation to functions of α-defensin remains to be determined. In this study, we examined the expression level of messenger RNA (mRNA) encoding six Crp-isoforms and Crp immunoreactivities using singly isolated crypts together with bactericidal activities of Paneth cell secretions from isolated crypts of duodenum, jejunum, and ileum. Here we showed that levels of Crp mRNAs in the single crypt ranged from 5 x 103 to 1 x 106 copies per 5 ng RNA. For each Crp isoform, the expression level in ileum was 4 to 50 times higher than that in duodenum and jejunum. Furthermore, immunohistochemical analysis of isolated crypts revealed that the average number of Paneth cell per crypt in the small intestine increased from proximal to distal, three to seven-fold, respectively. Both Crp1 and 4 expressed greater in ileal Paneth cells than those in duodenum or jejunum. Bactericidal activities in secretions of ileal Paneth cell exposed to bacteria were significantly higher than those of duodenum or jejunum. In germ-free mice, Crp expression in each site of the small intestine was attenuated and bactericidal activities released by ileal Paneth cells were decreased compared to those in conventional mice. Taken together, Paneth cells and their α-defensin in adult mouse appeared to be regulated topographically in innate immunity to control intestinal integrity.
• Associations of gut microbiota, dietary intake, and serum short-chain fatty acids with fecal short-chain fatty acids.

  Ryodai YAMAMURA, Koshi NAKAMURA, Naoya KITADA, Tomoyasu AIZAWA, Yu SHIMIZU, Kiminori NAKAMURA, Tokiyoshi AYABE, Takashi KIMURA, Akiko TAMAKOSHI

  Bioscience of Microbiota, Food and Health 39 (1) 11 - 17 2020 [Refereed]
  
• Butyric Acid and Leucine Induce α-Defensin Secretion from Small Intestinal Paneth Cells.

  Akiko Takakuwa, Kiminori Nakamura, Mani Kikuchi, Rina Sugimoto, Shuya Ohira, Yuki Yokoi, Tokiyoshi Ayabe

  Nutrients 11 (11) 2019/11/18 [Refereed][Not invited]
   

  The intestine not only plays a role in fundamental processes in digestion and nutrient absorption, but it also has a role in eliminating ingested pathogenic bacteria and viruses. Paneth cells, which reside at the base of small intestinal crypts, secrete α-defensins and contribute to enteric innate immunity through potent microbicidal activities. However, the relationship between food factors and the innate immune functions of Paneth cells remains unknown. Here, we examined whether short-chain fatty acids and amino acids induce α-defensin secretion from Paneth cells in the isolated crypts of small intestine. Butyric acid and leucine elicit α-defensin secretion by Paneth cells, which kills Salmonella typhimurium. We further measured Paneth cell secretion in response to butyric acid and leucine using enteroids, a three-dimensional ex vivo culture system of small intestinal epithelial cells. Paneth cells expressed short-chain fatty acid receptors, Gpr41, Gpr43, and Gpr109a mRNAs for butyric acid, and amino acid transporter Slc7a8 mRNA for leucine. Antagonists of Gpr41 and Slc7a8 inhibited granule secretion by Paneth cells, indicating that these receptor and transporter on Paneth cells induce granule secretion. Our findings suggest that Paneth cells may contribute to intestinal homeostasis by secreting α-defensins in response to certain nutrients or metabolites.
• A simple culture method for liver and intestinal tissue-resident macrophages from neonatal mice.

  Yu Shimizu, Naoya Sakuragi, Kiminori Nakamura, Toshio Taira, Tokiyoshi Ayabe, Akimasa Fukui

  In vitro cellular & developmental biology. Animal 55 (6) 436 - 444 2019/06 [Refereed][Not invited]
   

  The liver and intestine contain a remarkably large portion of tissue-resident macrophage cells representing a phenotype that downregulates inflammation and initiates tissue repair. Here, liver and intestinal tissues obtained from neonatal mice were minced, enzymatically digested, and incubated in RPMI1640-based media. In a 2-wk culture, spherical floating cells emerged on a fibroblastic sheet. These cells showed phagocytic activity and F4/80+-CD11b+-CD206+-Arg1+-iNOS--CD209a- phenotype, suggesting that these cells are tissue-resident macrophages. These macrophages proliferated in the co-culture system in the presence of fibroblastic feeder cell layer and absence of supplemental cytokines; the co-culture system did not cause a significant change in the phenotype of cells grown in a 4-wk culture. On the feeder cells, macrophage density was approximately 1.5 × 104/cm2 and the doubling time was approximately 70 h. Based on these observations, we present a simple method for the isolation and propagation of tissue-resident macrophages resembling M2 macrophage from neonatal mice, and this method provides a useful platform for in vitro studies of tissue-resident macrophages.
• Paneth cell granule dynamics on secretory responses to bacterial stimuli in enteroids.

  Yuki Yokoi, Kiminori Nakamura, Tsukasa Yoneda, Mani Kikuchi, Rina Sugimoto, Yu Shimizu, Tokiyoshi Ayabe

  Scientific reports 9 (1) 2710 - 2710 2019/02/25 [Refereed][Not invited]
   

  Paneth cells at the base of small intestinal crypts secrete granules containing α-defensins in response to bacteria and maintain the intestinal environment by clearing enteric pathogens and regulating the composition of the intestinal microbiota. However, Paneth cell secretory responses remain debatable and the mechanisms that regulate the secretion are not well understood. Although enteroids, three-dimensional cultures of small intestinal epithelial cells, have proven useful for analyzing intestinal epithelial cell functions including ion transport, their closed structures have imposed limitations to investigating interactions between Paneth cells and the intestinal microbiota. Here, we report that microinjection of bacteria or lipopolysaccharide (LPS) into the enteroid lumen provides an ex vivo system for studying Paneth cell secretion in real-time. The results show that Paneth cells released granules immediately when the apical surfaces of enteroid epithelial cells were exposed to LPS or live bacteria by microinjection. However, Paneth cells did not respond to LPS delivered in culture media to enteroid exterior basolateral surface, although they responded to basolateral carbamyl choline. In addition, Paneth cells replenished their granules after secretion, enabling responses to second stimulation. These findings provide new insight for apically-induced Paneth cell secretory responses in regulating the intestinal environment.
• Myc-induced nuclear antigen constrains a latent intestinal epithelial cell-intrinsic anthelmintic pathway.

  Meenu R Pillai, Belgacem Mihi, Kenji Ishiwata, Kiminori Nakamura, Naoya Sakuragi, David B Finkelstein, Maureen A McGargill, Toshinori Nakayama, Tokiyoshi Ayabe, Mathew L Coleman, Mark Bix

  PloS one 14 (2) e0211244  2019/02 [Refereed][Not invited]
   

  Expulsion of parasitic gastrointestinal nematodes requires diverse effector mechanisms coordinated by a Th2-type response. The evolutionarily conserved JmjC protein; Myc Induced Nuclear Antigen (Mina) has been shown to repress IL4, a key Th2 cytokine, suggesting Mina may negatively regulate nematode expulsion. Here we report that expulsion of the parasitic nematode Trichuris muris was indeed accelerated in Mina deficient mice. Unexpectedly, this was associated not with an elevated Th2- but rather an impaired Th1-type response. Further reciprocal bone marrow chimera and conditional KO experiments demonstrated that retarded parasite expulsion and a normal Th1-type response both required Mina in intestinal epithelial cells (IECs). Transcriptional profiling experiments in IECs revealed anti-microbial α-defensin peptides to be the major target of Mina-dependent retention of worms in infected mice. In vitro exposure to recombinant α-defensin peptides caused cytotoxic damage to whipworms. These results identify a latent IEC-intrinsic anthelmintic pathway actively constrained by Mina and point to α-defensins as important effectors that together with Mina may be attractive therapeutic targets for the control of nematode infection.
• Bifidogenic and butyrogenic effects of young barely leaf extract in an in vitro human colonic microbiota model

  Daisuke Sasaki, Kengo Sasaki, Yasushi Kadowaki, Yasuyuki Aotsuka, Akihiko Kondo

  AMB Express 9 (1) 2191-0855 2019 

  Young barley leaf extract (YBL) contains beneficial substances such as fructans, minerals, and vitamins. The effects of YBL administration on the human colonic microbiota and its production of metabolites were evaluated using an in vitro model culture system. Fermentations were started by inoculating fecal samples from nine healthy subjects, with or without 1.5% YBL. Bacterial 16S rRNA sequencing results confirmed that YBL administration significantly increased the relative abundances of bacteria related to the genus Bifidobacterium (p = 0.001, paired t-test) and those of the genera Faecalibacterium, Roseburia, Unclassified Ruminococcaceae, and Lachnospira (p = 0.013, p = 0.019, p = 0.028, and p = 0.034, respectively, paired t-test). Increased abundances of the latter genera corresponded to increased butyrate production in human colonic microbiota models following fermentation with 1.5% YBL, when compared to fermentation without 1.5% YBL (p = 0.006, Dunnett’s test). In addition, YBL administration significantly increased the production levels of amino acids such as lysine, glutamate, serine, threonine, alanine, isoleucine, leucine, valine, and phenylalanine. Therefore, our results showed the health-promoting bifidogenic and butyrogenic effects of YBL.
• Correction: Entamoeba histolytica alters ileal paneth cell functions in intact and Muc2 Mucin deficiency (Infection and Immunity (2018) 86:7 (e00208-18) DOI: 10.1128/IAI.00208-18)

  Eduardo R. Cobo, Ravi Holani, France Moreau, Kiminori Nakamura, Tokiyoshi Ayabe, Jennifer R. Mastroianni, Yoshihiro Eriguchi, Andre Ouellette, Kris Chadee

  Infection and Immunity 86 (10) 1098-5522 2018/10/01 

  Volume 86, no. 7, e00208-18, 2018, https://doi.org/10.1128/IAI.00208-18. Page 1: This article was published on 21 June 2018 with a byline that lacked Yoshihiro Eriguchi. The byline was updated in the current version, posted on 25 July 2018.
• Essential role of IFN-γ in T cell–associated intestinal inflammation.

  Yoshihiro Eriguchi, Kiminori Nakamura, Yuki Yokoi, Rina Sugimoto, Shuichiro Takahashi, Daigo Hashimoto, Takanori Teshima, Tokiyoshi Ayabe, Michael E. Selsted, André J. Ouellette

  JCI Insight 3 (18) 2018/09/20 [Refereed]
  
• Ploidy-dependent change in cyclin D2 expression and sensitization to cdk4/6 inhibition in human somatic haploid cells

  Kan Yaguchi, Takahiro Yamamoto, Masaya Shimada, Rina Sugimoto, Kiminori Nakamura, Tokiyoshi Ayabe, Ryota Uehara

  Biochemical and Biophysical Research Communications 504 (1) 231 - 237 0006-291X 2018/09 [Refereed]
   

  Near-haploidy is observed in certain cancer types, but ploidy-dependent alterations in gene regulation in the haploid state remain elusive. Here, by comparative transcriptome analysis between human isogenic haploid and diploid cell lines, we found lowering of cyclin D2 level in haploids. Acute genome duplication in haploids restored cyclin D2 expression to diploid level, indicating that the regulation of cyclin D2 expression is directly linked to ploidy. Downstream pathways of cyclin D2, such as Rb phosphorylation and p27 sequestration remained intact in haploids, suggesting that they adapt to lowered cyclin D level. Interestingly, however, haploid cells were more susceptible to cdk4/6 inhibition compared to diploids. Our finding indicates feasibility of selective growth suppression of haploid cells based on ploidy-linked gene regulation.
• Entamoeba histolytica Alters Ileal Paneth Cell Functions in Intact and Muc2 Mucin Deficiency.

  Eduardo R Cobo, Ravi Holani, France Moreau, Kiminori Nakamura, Tokiyoshi Ayabe, Jennifer R Mastroianni, Yoshihiro Eriguchi, Andre Ouellette, Kris Chadee

  Infection and immunity 86 (7) e00208 - 18 2018/07 [Refereed][Not invited]
   

  Enteric α-defensins, termed cryptdins (Crps) in mice, and lysozymes secreted by Paneth cells contribute to innate host defense in the ileum. Antimicrobial factors, including lysozymes and β-defensins, are often embedded in luminal glycosylated colonic Muc2 mucin secreted by goblet cells that form the protective mucus layer critical for gut homeostasis and pathogen invasion. In this study, we investigated ileal innate immunity against Entamoeba histolytica, the causative agent of intestinal amebiasis, by inoculating parasites in closed ileal loops in Muc2+/+ and Muc2-/- littermates and quantifying Paneth cell localization (lysozyme expression) and function (Crp secretion). Relative to Muc2+/+ littermates, Muc2-/- littermates showed a disorganized mislocalization of Paneth cells that was diffusely distributed, with elevated lysozyme secretion in the crypts and on villi in response to E. histolytica Inhibition of E. histolytica Gal/GalNAc lectin (Gal-lectin) binding with exogenous galactose and Entamoeba histolytica cysteine proteinase 5 (EhCP5)-negative E. histolytica had no effect on parasite-induced erratic Paneth cell lysozyme synthesis. Although the basal ileal expression of Crp genes was unaffected in Muc2-/- mice in response to E. histolytica, there was a robust release of proinflammatory cytokines and Crp peptide secretions in luminal exudates that was also present in the colon. Interestingly, E. histolytica-secreted cysteine proteinases cleaved the proregion of Crp4 but not the active form. These findings define Muc2 mucin as an essential component of ileal barrier function that regulates the localization and function of Paneth cells critical for host defense against microbes.
• R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease.

  Eiko Hayase, Daigo Hashimoto, Kiminori Nakamura, Clara Noizat, Reiki Ogasawara, Shuichiro Takahashi, Hiroyuki Ohigashi, Yuki Yokoi, Rina Sugimoto, Satomi Matsuoka, Takahide Ara, Emi Yokoyama, Tomohiro Yamakawa, Ko Ebata, Takeshi Kondo, Rina Hiramine, Tomoyasu Aizawa, Yoshitoshi Ogura, Tetsuya Hayashi, Hiroshi Mori, Ken Kurokawa, Kazuma Tomizuka, Tokiyoshi Ayabe, Takanori Teshima

  The Journal of experimental medicine 214 (12) 3507 - 3518 0022-1007 2017/12/04 [Refereed][Not invited]
   

  The intestinal microbial ecosystem is actively regulated by Paneth cell-derived antimicrobial peptides such as alpha-defensins. Various disorders, including graft-versus-host disease (GVHD), disrupt Paneth cell functions, resulting in unfavorably altered intestinal microbiota (dysbiosis), which further accelerates the underlying diseases. Current strategies to restore the gut ecosystem are bacteriotherapy such as fecal microbiota transplantation and probiotics, and no physiological approach has been developed so far. In this study, we demonstrate a novel approach to restore gut microbial ecology by Wnt agonist R-Spondin1 (R-Spo1) or recombinant a-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of a-defensins. Administration of R-Spo1 or recombinant a-defensin prevents GVHD-mediated dysbiosis, thus representing a novel and physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and host-microbiota cross talk toward therapeutic benefits.
• Paneth cell α-defensins and enteric microbiota in health and disease.

  Kiminori NAKAMURA, Naoya SAKURAGI, Akiko TAKAKUWA, Tokiyoshi AYABE

  Bioscience of Microbiota, Food and Health 35 (2) 57 - 67 2016 [Refereed]
  
• Decreased secretion of Paneth cell -defensins in graft-versus-host disease

  Y. Eriguchi, K. Nakamura, D. Hashimoto, S. Shimoda, N. Shimono, K. Akashi, T. Ayabe, T. Teshima

  TRANSPLANT INFECTIOUS DISEASE 17 (5) 702 - 706 1398-2273 2015/10 [Refereed][Not invited]
   

  BackgroundIntestinal microbial ecology is actively regulated by Paneth cell-derived antimicrobial peptides, -defensins. Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (SCT). We previously demonstrated that Paneth cells are targeted by GVHD, and their expression of antimicrobial peptide -defensins is impaired, leading to a loss of physiological diversity among the microflora and development of bloodstream infection. Herein, we evaluated whether fecal levels of -defensins could be surrogate marker of intestinal dysbiosis. MethodsWe directly measured -defensin cryptdin-1 (Crp1) in fecal pellets of mice with GVHD by using a novel enzyme-linked immunosorbent assay. ResultsFecal levels of Crp1 were significantly decreased in mice with GVHD but unchanged in mice without GVHD after SCT. These were correlated with intestinal flora diversity. ConclusionWe demonstrate a link between reduced secretion of Paneth cell -defensins and dysbiosis of intestinal flora in GVHD. Fecal levels of -defensins could be surrogate markers for intestinal microbial homeostasis.
• Stiff substrates increase YAP-signaling-mediated matrix metalloproteinase-7 expression

  A Nukuda, C Sasaki, S Ishihara, T Mizutani, K Nakamura, T Ayabe, K Kawabata, H Haga

  Oncogenesis Nature Publishing Group 4 (9) e165 - e165 2157-9024 2015/09 [Refereed]
   

  Abnormally stiff substrates have been shown to trigger cancer progression. However, the detailed molecular mechanisms underlying this trigger are not clear. In this study, we cultured T84 human colorectal cancer cells on plastic dishes to create a stiff substrate or on collagen-I gel to create a soft substrate. The stiff substrate enhanced the expression of matrix metalloproteinase-7 (MMP-7), an indicator of poor prognosis. In addition, we used polyacrylamide gels (2, 67 and 126 kPa) so that the MMP-7 expression on the 126-kPa gel was higher compared with that on the 2-kPa gel. Next, we investigated whether yes-associated protein (YAP) affected the MMP-7 expression. YAP knockdown decreased MMP-7 expression. Treatment with inhibitors of epidermal growth factor receptor (EGFR) and myosin regulatory light chain (MRLC) and integrin-alpha 2 or integrin-beta 1 knockdown downregulated MMP-7 expression. Finally, we demonstrated that YAP, EGFR, integrin-alpha 2 beta 1 and MRLC produced a positive feedback loop that enhanced MMP-7 expression. These findings suggest that stiff substrates enhanced colorectal cancer cell viability by upregulating MMP-7 expression through a positive feedback loop.
• Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization.

  Satoshi Tomisawa, Yuji Sato, Masakatsu Kamiya, Yasuhiro Kumaki, Takashi Kikukawa, Keiichi Kawano, Makoto Demura, Kiminori Nakamura, Tokiyoshi Ayabe, Tomoyasu Aizawa

  Protein expression and purification 112 21 - 8 1046-5928 2015/08 [Refereed][Not invited]
   

  Mammalian alpha-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of alpha-defensins, large amounts of alpha-defensins are essential. Although many expression systems for the production of recombinant alpha-defensins have been developed, attempts to obtain large amounts of alpha-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse alpha-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in Escherichia coil expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies. Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of alpha-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step. (C) 2015 Elsevier Inc. All rights reserved.
• Pr1E11, a novel anti-TROP-2 antibody isolated by adenovirus-based antibody screening, recognizes a unique epitope.

  Masahiro Ikeda, Miki Yamaguchi, Kazunori Kato, Kiminori Nakamura, Sagano Shiina, Takako Ichikawa-Ando, Hirofumi Misaka, Kensuke Myojo, Kazuyasu Nakamura, Yoshiyuki Sugimoto, Hirofumi Hamada

  Biochemical and biophysical research communications 458 (4) 877 - 82 0006-291X 2015/03/20 [Refereed][Not invited]
   

  TROP-2 is a type I transmembrane glycoprotein that is highly expressed in various epithelial cancer cells, and its increased expression correlates with poor prognosis. Although several anti-TROP-2 antibodies have been described, they were found unsuitable for antitumor therapy use in vivo as naked antibodies. In this study, we established a novel anti-TROP-2 antibody, designated Pr1 E11, from mice immunized with primary prostate cancer cells. Antibody screening was based on the infection activity of Adv-LacZ-FZ33, which displays an immunoglobulin G binding domain in the adenoviral fiber protein. We found that Pr1 E11 specifically binds to TROP-2 with high affinity and recognizes diverse epithelial cancer cell lines and primary pancreatic cancer tissues. Epitope analysis using TROP-2 deletion mutants revealed that binding site of Pr1 E11 is a cysteine-rich domain, a unique epitope compared with other available anti-TROP-2 antibodies. In addition, Pr1 E11 exhibited low internalization activity, which may make it suitable for naked antibody therapeutics. Our results suggest that Pr1 E11 may stimulate different biological activities from other anti-TROP-2 antibodies based on its unique binding epitope, and is a potential candidate for naked antibody therapeutics for various epithelial cancer treatments. (C) 2015 Elsevier Inc. All rights reserved.
• Bacterial cell wall components regulate adipokine secretion from visceral adipocytes.

  Risa Taira, Sayori Yamaguchi, Kyoko Shimizu, Kiminori Nakamura, Tokiyoshi Ayabe, Toshio Taira

  Journal of clinical biochemistry and nutrition 56 (2) 149 - 54 0912-0009 2015/03 [Refereed][Not invited]
   

  Recent studies suggest a relationship between intestinal microbiota and metabolic syndromes; however, the underlying mechanism remains unclear. To clarify this issue, we assessed the effects of bacterial cell wall components on adiponectin, leptin and resistin secretion from rat visceral adipocytes in vitro. We also measured the relative population of Firmicutes and Bacteroidetes in fecal microbiota and the amount of fecal mucin as an intestinal barrier function, when mice were fed a high-fat diet. In the present study, we demonstrated that bacterial cell wall components affect the secretion of adipokines, depending on the presence of antigens from gram-positive or gram-negative bacteria. Lipopolysaccharide markedly inhibited adiponectin, leptin, and resistin secretion, whereas peptidoglycan increased adiponectin secretion and decreased resistin secretion in vitro. In vivo experiments showed that the high-fat diet increased the population of Firmicutes and decreased that of Bacteroidetes. In contrast, the high-fat diet downregulated the stool output and fecal mucin content. These results demonstrate that bacterial cell wall components affect the onset of metabolic syndromes by mediating the secretion of adipokines from visceral adipose tissue. Furthermore, we believe that metabolic endotoxemia is not due to the increasing dominance of gram-negative bacteria, Bacteroidetes, but due to the depression of intestinal barrier function.
• Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells.

  Miki Yamaguchi, Yukari Nishii, Kiminori Nakamura, Haruka Aoki, Sachie Hirai, Hiroaki Uchida, Yuji Sakuma, Hirofumi Hamada

  Biochemical and biophysical research communications 454 (4) 600 - 3 0006-291X 2014/11/28 [Refereed][Not invited]
   

  Antibody-drug conjugates (ADCs), drugs developed by conjugation of an anticancer agent to a monoclonal antibody (mAb), have lately attracted attention in cancer therapy because ADCs can directly bind cancer cells and kill them. Although mAbs for ADCs must be internalized by the target cells, few methods are available for screening mAbs for their ability to be internalized by cells. We have developed a recombinant protein, termed DT3C, which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the Cl, C2, and C3 domains of Streptococcus protein G (3C). When a mAb-DT3C conjugate, which functions in vitro like an ADC, reduces the viability of cancer cells, the mAb being tested must have been internalized by the target cells. DT3C can thus be a tool to identify efficiently and easily mAbs that can be internalized by cells, thereby enhancing the development of promising ADCs. (C) 2014 Elsevier Inc. All rights reserved.
• A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin.

  Kiminori Nakamura, Naoya Sakuragi, Tokiyoshi Ayabe

  Analytical biochemistry 2 443 (2) 124 - 31 0003-2697 2013/12/15 [Refereed][Not invited]
   

  Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8 ± 5.2 ng/ml in proximal small intestine to 54.3 ± 10.3 ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota. © 2013 Elsevier Inc. All rights reserved.
• Reciprocal expression of enteric antimicrobial proteins in intestinal graft-versus-host disease.

  Yoshihiro Eriguchi, Hidetaka Uryu, Kiminori Nakamura, Sonoko Shimoji, Shuichiro Takashima, Hiromi Iwasaki, Toshihiro Miyamoto, Nobuyuki Shimono, Daigo Hashimoto, Koichi Akashi, Tokiyoshi Ayabe, Takanori Teshima

  Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation 19 (10) 1525 - 9 1083-8791 2013/10 [Refereed][Not invited]
   

  We recently demonstrated that expression of alpha-defensins, the major antimicrobial peptides produced by Paneth cells, was severely suppressed in mice with graft-versus-host disease (GVHD). In this study, we found that antibacterial lectin, regenerating islet-derived III gamma (RegIII gamma) was upregulated in villous enterocytes, thus demonstrating the reciprocal control of enteric antimicrobial proteins in GVHD. Upregulation of RegIII gamma was mediated by a mechanism independent upon radiation-induced intestinal tract damage. MyD88-mediated signaling in intestinal epithelium was required for RegIII gamma upregulation in GVHD and antibiotic therapy downregulated RegIII gamma expression. These results suggest that MyD88-mediated sensing of the intestinal microbes disregulated in GVHD induces RegIII gamma upregulation in GVHD and argue a role for RegIII gamma in the pathogenesis of GVHD. (C) 2013 American Society for Blood and Marrow Transplantation.
• Molecular Mechanisms of the Cytotoxicity of Human alpha-Lactalbumin Made Lethal to Tumor Cells (HAMLET) and Other Protein-Oleic Acid Complexes

  Takashi Nakamura, Tomoyasu Aizawa, Ryusho Kariya, Seiji Okada, Makoto Demura, Keiichi Kawano, Koki Makabe, Kunihiro Kuwajima

  JOURNAL OF BIOLOGICAL CHEMISTRY 20 288 (20) 14408 - 14416 0021-9258 2013/05 [Refereed][Not invited]
   

  Although HAMLET human alpha-lactalbumin made lethal to tumor cells), a complex formed by human alpha-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat alpha-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 degrees C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and beta(2)-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.
• Intracellular superoxide dismutase activity defines invasiveness of the murine T-lymphoma cell line L5187Y-ML25 in vitro and in vivo.

  Maki Tanaka, Kageaki Kuribayashi, Kastuhisa Kogawa, Kiminori Nakamura, Naoki Watanabe

  Leukemia research 37 (1) 89 - 92 0145-2126 2013/01 [Refereed][Not invited]
   

  Superoxide anion in the tumor microenvironment promotes a malignant phenotype. Here, we examined superoxide in a murine T-lymphoma cell line L5187Y-ML25. Clones with high and low intracellular superoxide dismutase (SOD) activities were obtained from parental L5187Y-ML25 cells and were subjected to assays determining cell invasiveness, motility, and in vivo dissemination. Cells with lower SOD activity exhibited higher invasiveness in vitro and in vivo. NADPH oxidase inhibitor suppressed intracellular free radical levels and cell motility, suggesting NADPH oxidase as a source of superoxides that stimulates cell motility. These results implicate superoxide as a potential anti-metastatic therapy for hematopoietic cell malignancies. © 2012 Elsevier Ltd.
• Therapeutic effect of suicide gene-transferred mesenchymal stem cells in a rat model of glioma

  H. Kosaka, T. Ichikawa, K. Kurozumi, H. Kambara, S. Inoue, T. Maruo, K. Nakamura, H. Hamada, I. Date

  CANCER GENE THERAPY 19 (8) 572 - 578 0929-1903 2012/08 [Refereed][Not invited]
   

  We evaluated a new therapeutic strategy for malignant glioma, which combines intratumoral inoculation of mesenchymal stem cells (MSCs) expressing cytosine deaminase gene with 5-fluorocytosine (5-FC) administration. For in vitro and in vivo experiments, MSCs were transfected with adenovirus carrying either enhanced green fluorescent protein gene (AdexCAEGFP) or cytosine deaminase gene (AdexCACD), to establish MSC-expressing EGFP (MSC-EGFP) or CD (MSC-CD). Co-culture of 9L glioma cells with MSC-CD in a medium containing 5-FC resulted in a remarkable reduction in 9L cell viability. The migratory ability of MSC-EGFP toward 9L cells was demonstrated by double-chamber assay. For the in vivo study, rats harboring 9L brain tumors were inoculated with MSC-EGFP or MSC-CD. Immunohistochemistry of rat brain tumors inoculated with MSC-EGFP showed intratumoral distribution of MSC-EGFP. Survival analysis of rats bearing 9L gliomas treated with intratumoral MSC-CD and intraperitoneal 5-FC resulted in significant prolongation of survival compared with control animals. In conclusion, molecular therapy combining suicide gene therapy and MSCs as a targeting vehicle represents a potential new therapeutic approach for malignant glioma, both with respect to the antitumor potential of this system and its neuroprotective effect on normal brain tissue.
• Graft-versus-host disease disrupts intestinal microbial ecology by inhibiting Paneth cell production of α-defensins.

  Yoshihiro Eriguchi, Shuichiro Takashima, Hideyo Oka, Sonoko Shimoji, Kiminori Nakamura, Hidetaka Uryu, Shinji Shimoda, Hiromi Iwasaki, Nobuyuki Shimono, Tokiyoshi Ayabe, Koichi Akashi, Takanori Teshima

  Blood 1 120 (1) 223 - 31 0006-4971 2012/07/05 [Refereed][Not invited]
   

  Allogeneic hematopoietic stem cell transplantation (SCT) is a curative therapy for various hematologic disorders. Graft-versus-host disease (GVHD) and infections are the major complications of SCT, and their close relationship has been suggested. In this study, we evaluated a link between 2 complications in mouse models. The intestinal microbial communities are actively regulated by Paneth cells through their secretion of antimicrobial peptides, alpha-defensins. We discovered that Paneth cells are targeted by GVHD, resulting in marked reduction in the expression of alpha-defensins, which selectively kill noncommensals, while preserving commensals. Molecular profiling of intestinal microbial communities showed loss of physiologic diversity among the microflora and the overwhelming expansion of otherwise rare bacteria Escherichia coli, which caused septicemia. These changes occurred only in mice with GVHD, independently on conditioning-induced intestinal injury, and there was a significant correlation between alteration in the intestinal microbiota and GVHD severity. Oral administration of polymyxin B inhibited outgrowth of E coli and ameliorated GVHD. These results reveal the novel mechanism responsible for shift in the gut flora from commensals toward the widespread prevalence of pathogens and the previously unrecognized association between GVHD and infection after allogeneic SCT. (Blood. 2012;120(1):223-231)
• Paneth cells and stem cells in the intestinal stem cell niche and their association with inflammatory bowel disease.

  Nakamura K, Ayabe T

  Inflammation Regeneration. 32 (2) 53 - 60 2012 [Refereed][Not invited]
  
• EpCAM- and EGFR-targeted selective gene therapy for biliary cancers using Z33-fiber-modified adenovirus.

  Rei Kawashima, Masato Abei, Kuniaki Fukuda, Kiminori Nakamura, Takehide Murata, Mariko Wakayama, Emiko Seo, Naoyuki Hasegawa, Hiroyuki Mizuguchi, Yuichi Obata, Ichinosuke Hyodo, Hirofumi Hamada, Kazunari K Yokoyama

  International journal of cancer 129 (5) 1244 - 53 0020-7136 2011/09/01 [Refereed][Not invited]
   

  A critical issue in adenovirus (Ad)-based cancer gene therapy is to improve the specificity of gene delivery to cancer cells for better efficacy and safety. We explored methods of retargeting Ad vectors for selective gene therapy of human biliary cancers using the Ad incorporating an IgG Fc-binding motif (Z33) from the Staphylococcus protein A (Ad-FZ33) combined with tumor-specific antibodies. Flow cytometry analysis revealed high-expression levels of epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) on human biliary cancer cells. Ad-FZ33 expressing LacZ combined with antibodies against EpCAM or EGFR, followed by beta-gal assay, demonstrated highly efficient gene transduction in these biliary cancer cells, compared to the treatment with control antibody or without antibody. Ad-FZ33 expressing uracil phosphoribosyl transferase (UPRT), an enzyme which greatly enhances the toxicity of 5-fluorouracil (FU), combined with antibodies against EpCAM or EGFR, remarkably enhanced the sensitivity of biliary cancer cells to 5-FU. By contrast, the treatment did not affect the 5-FU sensitivity of the cells not expressing EpCAM or EGFR including normal hepatocytes. Finally, treatments with the UPRT-expressing Ad-FZ33 with antibodies against EpCAM or EGFR, followed by 5-FU administration, significantly suppressed the growth of biliary cancer xenografts in nude mice. These results indicate that the gene therapy mediated by the Z33 fiber modified Ad with anti-EpCAM or anti-EGFR antibodies offers a potentially effective therapeutic modality against biliary cancers.
• Neural cell adhesion molecule 2 as a target molecule for prostate and breast cancer gene therapy.

  Shu Takahashi, Kazunori Kato, Kiminori Nakamura, Rika Nakano, Kazuishi Kubota, Hirofumi Hamada

  Cancer science 102 (4) 808 - 14 1347-9032 2011/04 [Refereed][Not invited]
   

  In adenovirus-derived gene therapy, one of the problems is the difficulty in specific targeting. We have recently demonstrated that monoclonal antibody (mAb) libraries screened by fiber-modified adenovirus vector (Adv-FZ33), which is capable of binding to immunoglobulin-G (IgG), provide a powerful approach for the identification of suitable target antigens for prostate cancer therapy. Hybridoma libraries from mice immunized with androgen-dependent prostate cancer cell line LNCaP were screened and mAb were selected. Through this screening, we obtained one mAb, designated LNI-29, that recognizes a glycoprotein with an apparent molecular mass of 100 kD. It was identified as neural cell adhesion molecule 2 (NCAM2). Some prostate and breast cancer cell lines highly expressed NCAM2 whereas normal prostate cell lines expressed NCAM2 at low levels. In contrast to the low efficiency of gene transduction by Adv-FZ33 with a control antibody, LNI-29-mediated Adv-FZ33 infection induces high rates of gene delivery in NCAM2-positive cancers. NCAM2-mediated therapeutic gene transduction of uracil phosphoribosyltransferase (UPRT) had a highly effective cytotoxic effect on NCAM2-positive cancer cells, whereas it had less of an effect in cases with a control antibody. In conclusion, NCAM2 should be a novel gene therapy target for the treatment of prostate and breast cancer. (Cancer Sci 2011; 102: 808-814)
• Bactericidal activity of mouse α-defensin cryptdin-4 predominantly affects noncommensal bacteria.

  Koji Masuda, Naoki Sakai, Kiminori Nakamura, Sawako Yoshioka, Tokiyoshi Ayabe

  Journal of innate immunity 3 3 (3) 315 - 26 1662-811X 2011 [Refereed][Not invited]
   

  Mouse Paneth cell alpha-defensins, termed cryptdins, are secreted into the intestinal lumen, have microbicidal activity, and contribute to intestinal innate immunity. Among them, cryptdin-4 (Crp4) has the most potent microbicidal activity. In the intestinal lumen, commensal bacteria colonize and elicit beneficial effects in the host. However, the effects of Crp4 against commensal bacteria are poorly understood. Thus, we investigated the bactericidal activities of Crp4 against commensal bacteria compared to noncommensal bacteria. Oxidized Crp4 showed only minimal or no bactericidal activity against 8 out of 12 commensal bacterial species, including Bifidobacterium bifidum and Lactobacillus Casei. We further addressed a role of the conserved disulfide bonds of Crp4 by analyzing reduced Crp4 (r-Crp4). r-Crp4 demonstrated significantly greater bactericidal activities against 7 of 12 commensal bacteria than did oxidized Crp4. Oxidized Crp4 and r-Crp4 elicited equivalently potent bactericidal activities against 11 of the 11 noncommensal bacteria tested, such as Salmonella enterica serovar Typhimurium, and against 5 of 12 commensal bacteria. Furthermore, when r-Crp4 was exposed to a processing enzyme of cryptdins, i.e. MMP-7, r-Crp4 was degraded and the bactericidal activities disappeared. These findings suggest that Crp4 has selective bactericidal activities against intestinal microbiota and that the activities are dependent on the disulfide bonds. Copyright (C) 2010 S. Karger AG, Basel
• Regulation of Microbiota by Antimicrobial Peptides in the Gut.

  Koji Masuda, Kiminori Nakamura, Sawako Yoshioka, Rie Fukaya, Naoki Sakai, Tokiyoshi Ayabe

  Recent Advances in Tonsils and Mucosal Barriers of the Upper Airways 72 97 - 99 1662-2847 2011 [Refereed]
   

  The antimicrobial peptide is one of major effectors of the innate immunity, and is common in the entire multicellular organisms. In mammals, one family of antibacterial peptide named defensins plays a central role in host defense, especially in the epithelial surface such as oral cavity, skin and the intestine. Recently, the importance of the antimicrobial peptides has been widely recognized. The epithelium of the gut is a largest surface that is exposed to various pathogens in the environment. It is the Paneth cells that produce antimicrobial peptides, α-defensins in the small intestine. Paneth cells contribute to mucosal innate immunity by sensing bacteria and releasing microbicidal activities mostly from activated α-defensins. In mice, α-defensins, named cryptdins, consisted of six major isoforms (cryptdin-1 to cryptdin-6), and among those cryptdin-4 is the most microbicidal, suggesting that cryptdin-4 has a pivotal role in innate immunity. Paneth cell α-defensins have selective activities against commensal bacteria which may be associated with compositions of intestinal microbiota in vivo and homeostasis of the entire intestine. In addition, Paneth cell α-defensins appeared to be regulated topographically to control intestinal integrity.
• Interleukin-2 gene transfer potentiates the alpha-galactosylceramide-stimulated antitumor effect by the induction of TRAIL in NKT and NK cells in mouse models of subcutaneous and metastatic carcinoma.

  Yoshiki Nishihori, Kazunori Kato, Maki Tanaka, Tetsuro Okamoto, Seiya Hagiwara, Naoko Araki, Katsuhisa Kogawa, Kageaki Kuribayashi, Kiminori Nakamura, Yoshiro Niitsu

  Cancer biology & therapy 8 (18) 1763 - 70 1538-4047 2009/09 [Refereed][Not invited]
   

  alpha-Galactosylceramide (alpha-GalCer) is a potent CD1d ligand that activates natural killer like T-cells (NKT), leading to the production of helper T (Th) 1 and Th2 cytokines that mediate various immunemodulatory and antitumor effects. Here, we determined whether the administration of adenovirus-vector-encoding mouse interleukin-2 (AdmIL-2) can augment the antitumor effect of alpha-GalCer on subcutaneous and metastatic tumors in mice. Mice were intraperitoneally injected with alpha-GalCer on days 7, 10 and 13 after tumor inoculation, with or without intratumoral injection of AdmIL-2 on day 7. alpha-GalCer treatment increased the serum levels of interferon-gamma, while intratumoral injection of AdmIL-2 elevated serum IL-2 levels. A combination of alpha-GalCer and AdmIL-2 (alpha-GalCer/AdmIL-2) inhibited the in vivo tumor growth and improved the survival of tumor-bearing mice, as compared to the use of a single agent. Experiments on spontaneous metastasis models revealed that alpha-GalCer/AdmIL-2 reduced lung metastasis and prolonged survival, as compared to control groups. In addition, the splenic and liver mononuclear cells from mice treated with alpha-GalCer/AdmIL-2 showed enhanced cytolytic activity against NK-sensitive YAC-1 and NK-resistant 3LL tumors. Moreover, alpha-GalCer/AdmIL-2 treatment expanded the absolute numbers of lung and liver NK, NKT and T-cells as well as the TNF-related apoptosis-inducing ligand (TRAIL) expression of these cells. This study shows the efficacy of alpha-GalCer/AdmIL-2 immunomodulatory therapy, and provides a cellular mechanism on how it exerts the antitumor effects.
• Emerging concepts in biomarker discovery; the US-Japan Workshop on Immunological Molecular Markers in Oncology.

  Hideaki Tahara, Marimo Sato, Magdalena Thurin, Ena Wang, Lisa H Butterfield, Mary L Disis, Bernard A Fox, Peter P Lee, Samir N Khleif, Jon M Wigginton, Stefan Ambs, Yasunori Akutsu, Damien Chaussabel, Yuichiro Doki, Oleg Eremin, Wolf Hervé Fridman, Yoshihiko Hirohashi, Kohzoh Imai, James Jacobson, Masahisa Jinushi, Akira Kanamoto, Mohammed Kashani-Sabet, Kazunori Kato, Yutaka Kawakami, John M Kirkwood, Thomas O Kleen, Paul V Lehmann, Lance Liotta, Michael T Lotze, Michele Maio, Anatoli Malyguine, Giuseppe Masucci, Hisahiro Matsubara, Shawmarie Mayrand-Chung, Kiminori Nakamura, Hiroyoshi Nishikawa, A Karolina Palucka, Emanuel F Petricoin, Zoltan Pos, Antoni Ribas, Licia Rivoltini, Noriyuki Sato, Hiroshi Shiku, Craig L Slingluff, Howard Streicher, David F Stroncek, Hiroya Takeuchi, Minoru Toyota, Hisashi Wada, Xifeng Wu, Julia Wulfkuhle, Tomonori Yaguchi, Benjamin Zeskind, Yingdong Zhao, Mai-Britt Zocca, Francesco M Marincola

  Journal of translational medicine 7 45 - 45 2009/06/17 

  Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations. Converging concepts were identified: enhanced knowledge of interferon-related pathways was found to be central to the understanding of immune-mediated tissue-specific destruction (TSD) of which tumor rejection is a representative facet. Although the expression of interferon-stimulated genes (ISGs) likely mediates the inflammatory process leading to tumor rejection, it is insufficient by itself and the associated mechanisms need to be identified. It is likely that adaptive immune responses play a broader role in tumor rejection than those strictly related to their antigen-specificity; likely, their primary role is to trigger an acute and tissue-specific inflammatory response at the tumor site that leads to rejection upon recruitment of additional innate and adaptive immune mechanisms. Other candidate systemic and/or tissue-specific biomarkers were recognized that might be added to the list of known entities applicable in immunotherapy trials. The need for a systematic approach to biomarker discovery that takes advantage of powerful high-throughput technologies was recognized; it was clear from the current state of the science that immunotherapy is still in a discovery phase and only a few of the current biomarkers warrant extensive validation. It was, finally, clear that, while current technologies have almost limitless potential, inadequate study design, limited standardization and cross-validation among laboratories and suboptimal comparability of data remain major road blocks. The institution of an interactive consortium for high throughput molecular monitoring of clinical trials with voluntary participation might provide cost-effective solutions.
• Effect of transplantation of bone marrow-derived mesenchymal stem cells on mice infected with prions.

  Chang-Hyun Song, Osamu Honmou, Natsuo Ohsawa, Kiminori Nakamura, Hirofumi Hamada, Hidefumi Furuoka, Rie Hasebe, Motohiro Horiuchi

  Journal of virology 83 (11) 5918 - 27 0022-538X 2009/06 [Refereed][Not invited]
   

  Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to migrate to brain lesions in experimental models of ischemia, tumors, and neurodegenerative diseases and to ameliorate functional deficits. In this study, we attempted to evaluate the therapeutic potential of MSCs for treating prion diseases. Immortalized human MSCs (hMSCs) that express the LacZ gene were transplanted into the unilateral hippocampi or thalami of mice, and their distributions were monitored by the expression of beta-galactosidase. In mice infected with prions, hMSCs transplanted at 120 days postinoculation (dpi) were detected on the contralateral side at 2 days after transplantation and existed there even at 3 weeks after transplantation. In contrast, few hMSCs were detected on the contralateral side for mock-infected mice. Interestingly, the migration of hMSCs appeared to correlate with the severity of neuropathological lesions, including disease-specific prion protein deposition. The hMSCs also migrated to a prion-specific lesion in the brain, even when intravenously injected. Although the effects were modest, intrahippocampal and intravenous transplantation of hMSCs prolonged the survival of mice infected with prions. A subpopulation of hMSCs in the brains of prion-infected mice produced various trophic factors and differentiated into cells of neuronal and glial lineages. These results suggest that MSCs have promise as a cellular vehicle for the delivery of therapeutic genes to brain lesions associated with prion diseases and, furthermore, that they may help to regenerate neuronal tissues damaged by prion propagation.
• Targeting vectors for cancer gene therapy.

  Kiminori Nakamura, Kazunori Kato, Toshihiro Tanaka, Hirofumi Hamada

  Frontiers in bioscience (Elite edition) 1 (1) 332 - 40 1945-0494 2009/06/01 [Refereed][Not invited]
   

  Establishment of a system that allows selective gene transfer to a tumor is expected to enable targeted therapy. Using a combination of fiber-modified adenovirus and antibody to a cell surface antigen, we have explored methods to enhance the selectivity of gene transfer. In addition, we aimed to establish a systematic screening method to search for antibody and cell surface target candidates for providing highly selective gene transfer to a variety of malignant tumors.
• Selective gene transfer into neurons via Na,K-ATPase beta1. Targeting gene transfer with monoclonal antibody and adenovirus vector.

  Keiji Ishii, Kiminori Nakamura, Satoshi Kawaguchi, Rong Li, Sachie Hirai, Naoya Sakuragi, Takuro Wada, Kazunori Kato, Toshihiko Yamashita, Hirofumi Hamada

  The journal of gene medicine 10 (6) 597 - 609 1099-498X 2008/06 [Refereed][Not invited]
   

  Background Neuron-selective gene transfer is an attractive therapeutic strategy for neurological disorders. However, optimal targets and gene delivery systems remain to be determined. Methods Following immunization of mice with PC12 cells, hybridomas were screened by p-Gal reporter gene assay using FZ33 fiber-modified adenovirus vectors. Subsequently, the efficacy and specificity of monoclonal antibody (mAb)-mediated gene transfer via FZ33 and FdZ adenovirus vectors were evaluated by flow cytometry, chemiluminescent beta-Gal reporter gene assay, and immunocytochemistry. Finally, the antigen recognized by the mAb was identified by mass spectrometry and transfection analysis. Results A hybridoma clone 6E3 producing monoclonal antibody, mAb6E3, was screened. Flow cytometry, chemiluminescent beta-Gal reporter gene assay, and immunocytochemistry with mAb6E3 and the fiber mutant adenovirus demonstrated efficient gene transfer into the PC12 cells. Treatment of neuron-glia cocultures with mAb6E3 and FdZ adenovirus resulted in neuronselective gene transfer. immunohistochemical images of rat spinal cord tissue showed that mAb6E3 reacts specifically with neurons. Finally, Na,K-ATPase beta 1 was identified as the antigen of mAb6E3. Conclusions Hybridoma screening using FZ33 fiber-modified adenovirus vectors serves as an efficient approach to detect antigens in mAb-targeted gene transfer. Neuronal tropism in the central nervous system through mAb6E3 represents an important initial step towards neuron- selective gene transfer in the treatment of local neurological disorders, such as spinal cord injury. Copyright (C) 2008 John Wiley & Sons, Ltd.
• Gene transfer of CD40-ligand to dendritic cells stimulates interferon-gamma production to induce growth arrest and apoptosis of tumor cells.

  K. Tomihara, K. Kato, Y. Masuta, K. Nakamura, H. Uchida, K. Sasaki, T. Tanaka, J. Huang, H. Hiratsuka, H. Hamada

  GENE THERAPY 15 (3) 203 - 213 0969-7128 2008/02 [Refereed][Not invited]
   

  In this study, we present evidence that gene transfer of the CD40-ligand (CD154) into human immature dendritic cells (DC) imparts direct antitumor effects on tumor cells. DC infected with adenovirus directed to express human CD154 on the cell surface (CD154-DC) elicited significantly higher levels of immune accessory molecules commonly found on mature DC. We found that co-cultivation with a human squamous cell carcinoma cell line (OSC-70) with CD154-DC significantly inhibited cell growth. We further demonstrate that OSC-70 cells stimulated with CD154-DC were more susceptible to apoptosis via TNF-related apoptosis inducing ligand (TRAIL). Importantly, tumor cells co-cultured with CD154-DC in transwell plates expressed upregulated cell surface TRAIL-R2. CD154-DC produced higher levels of interferon (IFN)-gamma, IL-12p70 and soluble CD154, but the ability of CD154-DC to inhibit tumor cell growth was significantly abrogated by a neutralizing antibody to IFN-gamma, indicating that this was mainly mediated by IFN-gamma. Furthermore, intratumoral injection of CD154-DC significantly suppressed OSC-70 tumor growth in a xenograft model. Overall, these results reveal that CD154-DC have potential as an anti-cancer therapy by producing IFN-gamma to arrest adjacent tumor cell growth and increase the susceptibility of apoptosis via TRAIL.
• Gene transfer of noncleavable cell surface mutants of human CD154 induces the immune response and diminishes systemic inflammatory reactions.

  Yukari Masuta, Kazunori Kato, Kei Tomihara, Kiminori Nakamura, Katsunori Sasaki, Satoshi Takahashi, Hirofumi Hamada

  Journal of immunotherapy 30 (7) 694 - 704 1524-9557 2007/10 [Refereed][Not invited]
   

  CD154 (CD40-ligand) is a critical transmembrane molecule with potent immune-stimulatory properties that is used in clinical applications of gene therapy for leukemia and lymphoma. However, CD154 is cleaved into a soluble form, and high levels of sCD154 contribute to systemic inflammatory and cardiovascular diseases, suggesting a deleterious side effect of CD154 gene therapy. In this study, we engineered noncleavable mutants of human CD154 with point mutations to develop a potentially less toxic molecule in vivo. In contrast to wild-type CD154 (CD154-WT) subsequently released as sCD154, both mutants CD154-M3 and CD154-M4 were resistant to cleavage in tumor cells. Also, CD40-expressing leukemia B cells transfected with CD154-M3 mutant were highly effective stimulators in a mixed lymphocyte-leukemia reaction, indicating that CD154-M3 mutant did not lose biologic activity. In mice transplanted with tumors expressing CD154-WT, we found increased plasma levels of human sCD154 followed by various systemic inflammatory reactions such as glomerulonephritis and an increased number of infiltrating mononuclear cells in the liver. However, CD154-M3 mutant did not induce any systemic inflammatory effects in vivo. As such, the noncleavable mutant of CD154 is fully capable of inducing the immune response with less toxic properties and is a useful tool for CD154 immune gene therapy.
• Exploration of target molecules for prostate cancer gene therapy.

  Kazuhiro Suzuki, Kiminori Nakamura, Kazunori Kato, Hirofumi Hamada, Taiji Tsukamoto

  The Prostate 67 (11) 1163 - 73 0270-4137 2007/08/01 [Refereed][Not invited]
   

  BACKGROUND. Focusing on Adv-FZ33, a modified adenovirus in which a synthetic 33-amino-acid immunoglobulin G-binding domain was inserted into the adenoviral fiber protein, we tried to identify suitable target molecules for prostate cancer-specific gene therapy. METHODS. Hybridomas were established from mice immunized with prostate cancer cell lines. The hybridomas were screened using Adv-FZ33 to create monoclonal antibodies (mAbs) that induced high gene transfer efficiency for PC-3 cells. Furthermore, we identified target antigens of the mAbs by immunoprecipitation and mass spectrometry, and investigated the expression of target molecules by flow cytometry and immunocytochemistry. RESULTS. Using Adv-FZ33, we established four different mouse mAbs that increased transduction efficiency for PC-3. The target antigens identified were Ep-CAM, CD155, HAI-1, and Na,K-ATPase beta 1. These antigens were expressed in several cancer cell lines, including prostate cancer. Human prostatic myofibroblast cells lacked expression of Ep-CAM and HAI-1. CONCLUSIONS. We established anti-Ep-CAM mAb and anti- HAM mAbs. Gene transduction via Ep-CAM and HAI-1 maybe a novel strategy for treatment of prostate cancer. Prostate 67: 1163-1173,2007. (c) 2007Wiley-Liss, Inc.
• Wnt3/RhoA/ROCK signaling pathway is involved in adhesion-mediated drug resistance of multiple myeloma in an autocrine mechanism.

  Masayoshi Kobune, Hiroki Chiba, Junji Kato, Kazunori Kato, Kiminori Nakamura, Yutaka Kawano, Kohichi Takada, Rishu Takimoto, Tetsuji Takayama, Hirofumi Hamada, Yoshiro Niitsu

  Molecular cancer therapeutics 6 (6) 1774 - 84 1535-7163 2007/06 [Refereed][Not invited]
   

  Adhesion of myeloma cells to bone marrow stromal cells is now considered to play a critical role in chemoresistance. However, little is known about the molecular mechanism governing cell adhesion-mediated drug resistance (CAM-DR) of myeloma cells. In this study, we focused our interests on the implication of the Wnt signal in CAM-DR. We first screened the expression of Wnt family in myeloma cell lines and found that Wnt3 was overexpressed in all the myeloma cells examined. KMS-5 and ARH77, which highly expressed Wnt3 protein, tightly adhered to human bone marrow stromal cells, and accumulation of (3-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. Conversely, RPM18226 and MM1S, which modestly expressed Wnt3 protein, rather weakly adhered to human bone marrow stromal. We then examined the relevance of Wnt3 expression to adhesive property to stromal cells and to CAM-DR of myeloma cells. KMS-5 and ARH-77 exhibited apparent CAM-DR against doxorubicin. This CAM-DR was significantly reduced by anti-integrin (it antibody, antiintegrin as antibody and a Wnt-receptor competitor, secreted Frizzled-related protein-1, and Rho kinase inhibitor Y27632, but not by the specific inhibitor of canonical signaling (Dickkopf-1), indicating that Wnt-mediated CAMDR that is dependent on integrin alpha(6)/beta(1) (VLA-6)-mediated attachment to stromal cells is induced by the Wnt/RhoA/Rho kinase pathway signal. This CAM-DR was also significantly reduced by Wnt3 small interfering RNA transfer to KMS-5. These results indicate that Wnt3 contributes to VLA-6-mediated CAM-DR via the Wnt/RhoA/ROCK pathway of myeloma cells in an autocrine manner. Thus, the Wnt3 signaling pathway could be a promising molecular target to overcome CAM-DR of myeloma cells.
• Gene transfer of the CD40-ligand to human dendritic cells induces NK-mediated antitumor effects against human carcinoma cells.

  Kei Tomihara, Kazunori Kato, Yukari Masuta, Kiminori Nakamura, Toshihiro Tanaka, Hiroyoshi Hiratsuka, Hirofumi Hamada

  International journal of cancer 120 (7) 1491 - 8 0020-7136 2007/04/01 [Refereed][Not invited]
   

  The CD40-ligand (CD40L) is a key molecule for the activation of dendritic cells (DCs), followed by the induction of DC maturation and cytokine production. Here we found that DC infected with adenovirus vector encoding human CD40L (CD40L-DC) displayed significantly higher levels of immune accessory molecules and IL-12 production than did uninfected cells, and that CD40L-DC produced much higher levels of IFN-gamma. To investigate whether CD40L-DC-derived these soluble factors could stimulate NK cells without physical cell-to-cell contact, we cocultured NK cells with CD40L-DC in transwell culture plates. NK cells showed up-regulated cytotoxic activity toward various oral squamous cell carcinoma (OSC-70, HSC-2, HSC-3), and we determined that both IL-12 and IFN-gamma contributed to the CD40L-DC-mediated NK cell activation. NK cells stimulated with CD40L-DC resulted in the induction of the cell surface expression of TRAIL, the production of IFN-gamma and intracellular accumulation of granzyme B. The cytotoxic activity of NK cells stimulated with CD40L-DC could be mostly inhibited by neutralizing antibody for TRAIL and completely abrogated by the combination of antibody and exocytosis inhibitor, indicating that this was mainly mediated by a TRAIL-TRAIL-receptor interaction and granule exocytosis. Moreover., CD40L-DC-activated NK cells could induce up-regulation of a death-receptor TRAIL-R2 (DR5) and down-regulation of a decoy receptor TRAIL-R3 (DcR1) on carcinoma cells. Overall, these results have revealed that CD40L-DC could activate an innate immune reaction by stimulating NK cells followed by carcinoma cells, supporting that administration of CD40L-DC may have potential as an anticancer therapy. (c) 2007 Wiley-Liss. Inc.
• Essential role of protein kinase C zeta in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP.

  Kageaki Kuribayashi, Kiminori Nakamura, Maki Tanaka, Tsutomu Sato, Junji Kato, Katsunori Sasaki, Rishu Takimoto, Katsuhisa Kogawa, Takeshi Terui, Tetsuji Takayama, Takayuki Onuma, Takuya Matsunaga, Yoshiro Niitsu

  The Journal of cell biology 176 (7) 1049 - 60 0021-9525 2007/03/26 [Refereed][Not invited]
   

  Under various pathological conditions, including infection, malignancy, and autoimmune diseases, tissues are incessantly exposed to reactive oxygen species produced by infiltrating inflammatory cells. We show augmentation of motility associated with morphological changes of human squamous carcinoma SASH1 cells, human peripheral monocytes (hPMs), and murine macrophage-like cell line J774.1 by superoxide stimulation. We also disclose that motility of hPMs and J774.1 induced by a chemotactic peptide (N-formyl-methionylleucyl-phenylalanine [fMLP]) was inhibited by superoxide dismutase or N-acetylcystein, indicating stimulation of motility by superoxide generated by fMLP stimulation. In these cells, protein kinase C (PKC) zeta was activated to phosphorylate RhoGDI-1, which liberated RhoGTPases, leading to their activation. These events were inhibited by dominant-negative PKC zeta in SASH1 cells, myristoylated PKC zeta peptides in hPMs and J774.1, or a specific inhibitor of RhoGTPase in SASH1, hPMs, and J774.1. These results suggest a new approach for manipulation of inflammation as well as tumor cell invasion by targeting this novel signaling pathway.
• Neuroprotection by PlGF gene-modified human mesenchymal stem cells after cerebral ischaemia.

  H. Liu, O. Honmou, K. Harada, K. Nakamura, K. Houkin, H. Hamada, J. D. Kocsis

  BRAIN 129 2734 - 2745 0006-8950 2006/10 [Refereed][Not invited]
   

  Intravenous delivery of mesenchymal stem cells (MSCs) prepared from adult bone marrow reduces infarction size and ameliorates functional deficits in rat cerebral ischaemia models. Placental growth factor (PlGF) is angiogenic to impaired non-neural tissue. To test the hypothesis that PlGF contributes to the therapeutic benefits of MSC delivery in cerebral ischaemia, we compared the efficacy of systemic delivery of human MSCs (hMSCs) and hMSCs transfected with a fibre-mutant F/RGD adenovirus vector with a PlGF gene (PlGF-hMSCs). A permanent middle cerebral artery occlusion (MCAO) was induced by intraluminal vascular occlusion with a microfilament. hMSCs and PlGF-hMSCs were intravenously injected into the rats 3 h after MCAO. Lesion size was assessed at 3 and 6 h, and 1, 3, 4 and 7 days using MR imaging and histology. Functional outcome was assessed using the limb placement test and the treadmill stress test. Both hMSCs and PlGF-hMSCs reduced lesion volume, induced angiogenesis and elicited functional improvement compared with the control sham group, but the effect was greater in the PlGF-hMSC group. Enzyme-linked immunosorbent assay of the infarcted hemisphere revealed an increase in PlGF in both hMSC groups, but a greater increase in the PlGF-hMSC group. These data support the hypothesis that PlGF contributes to neuroprotection and angiogenesis in cerebral ischaemia, and cellular delivery of PlGF to the brain can be achieved by intravenous delivery of hMSCs.
• Ex vivo expansion of G-CSF-mobilized peripheral blood CD133+ progenitor cells on coculture with human stromal cells.

  Yutaka Kawano, Masayoshi Kobune, Hiroki Chiba, Kiminori Nakamura, Rishu Takimoto, Kohichi Takada, Yoshinori Ito, Junji Kato, Hirofumi Hamada, Yoshiro Niitsu

  Experimental hematology 34 (2) 150 - 8 0301-472X 2006/02 [Refereed][Not invited]
   

  Objective. The pentaspan molecule CD133 has been shown to be a marker of more primitive hematopoietic progenitors in mobilized peripheral blood (PB). Our objective was to assess the efficacy of PB CD133(+) cells in our coculture system using human telomerized stromal (HTS) cells. Methods. Five thousand PB CD133(+) cells or conventional cord blood (CB) CD34(+) cells were expanded with or without HTS cells in the presence or absence of stem cell factor, thrombopoietin, and Flk-2/Flt-3 ligand. Results. The coculture was significantly superior in expanding PB clonogenic cells as compared with the stroma-free culture (CFU-C, 2 +/- 0 vs 111 +/- 15-fold of initial cell number p < 0.01), and the fold increase of PB clonogenic cells was comparable to that for CB cells after two weeks of coculture (BFU-E, 54 +/- 3 vs 56 +/- 4-fold; CFU-GM, 156 +/- 26 vs 83 +/- 9-fold; CFU-Mix, 30 +/- 11 vs 80 +/- 36-fold). However, proliferation of CFU-Mk from PB on coculture with HTS cells was modest as compared with stroma-free culture. Concomitantly, multiple hematopoietic cells transmigrated below the stromal layer and formed cobblestone areas (CAs). The production of hematopoietic progenitor cells from CAs after coculture with PB was significantly lower than that seen in cells cocultured with CB for four weeks (CFU-Mix, 0 +/- 0 vs 9 +/- 5-fold on day 28, p < 0.01), although a similar number of CAs derived from PB and CB were observed. Conclusion. PB CD133(+) cells proliferated efficiently above the stromal layer, while the characteristics of PB CD133(+) cells underneath the human stromal layer were likely to be maintained, even after long-term hematopoietic-stromal interaction. (C) 2006 International Society for Experimental Hematology. Published by Elsevier Inc.
• Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion.

  Yasushi Sato, Hironobu Araki, Junji Kato, Kiminori Nakamura, Yutaka Kawano, Masayoshi Kobune, Tsutomu Sato, Koji Miyanishi, Tetsuji Takayama, Minoru Takahashi, Rishu Takimoto, Satoshi Iyama, Takuya Matsunaga, Seiji Ohtani, Akihiro Matsuura, Hirofumi Hamada, Yoshiro Niitsu

  Blood 106 (2) 756 - 63 0006-4971 2005/07/15 [Refereed][Not invited]
   

  Hepatic transdifferentiation of bone marrow cells has been previously demonstrated by intravenous administration of donor cells, which may recirculate to the liver after undergoing proliferation and differentiation in the recipient's bone marrow. In the present study, to elucidate which cellular components of human bone marrow more potently differentiate into hepatocytes, we fractionated human bone marrow cells into mesenchymal stem cells (MSCs), CD34(+) cells, and non-MSCs/ CD34(-) cells and examined them by directly xenografting to allylalcohol (AA)- treated rat liver. Hepatocyte-like cells, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), albumin (Alb), cytokeratin 19 (CK19), cytokeratin 18 (CK18), and asialoglycoprotein receptor (AGPR), and by reverse transcription-polymerase chain reaction (RTPCR) for expression of AFP and Alb mRNA, were observed only in recipient livers with MSC fractions. Cell fusion was not likely involved since both human and rat chromosomes were independently identified by fluorescence in situ hybridization (FISH). The differentiation appeared to follow the process of hepatic ontogeny, reprogramming of gene expression in the genome of MSCs, as evidenced by expression of the AFP gene at an early stage and the albumin gene at a later stage. In conclusion, we have demonstrated that MSCs are the most potent component in hepatic differentiation, as revealed by directly xenografting into rat livers.
• Bcl-xL gene transfer inhibits Bax translocation and prolongs cardiac cold preservation time in rats.

  Jianhua Huang, Kiminori Nakamura, Yoshinori Ito, Takeshi Uzuka, Masayuki Morikawa, Sachie Hirai, Kei Tomihara, Toshihiro Tanaka, Yukari Masuta, Keiji Ishii, Kazunori Kato, Hirofumi Hamada

  Circulation 112 (1) 76 - 83 0009-7322 2005/07/05 [Refereed][Not invited]
   

  Background - Apoptosis is an important cause of early graft loss after heart transplantation. Bcl- xL was reported to protect the heart against normothermic ischemia and reperfusion injury. In this study, we determined whether overexpression of Bcl- xL could inhibit tissue injury resulting from prolonged cold preservation followed by warm reperfusion of heart transplants. Methods and Results - Lewis rat hearts were transduced with an adenovirus vector harboring Bcl- xL cDNA ( AxCAhBclxL) 4 days before collection of tissue. After preservation in University of Wisconsin solution at 4 degrees C for 24 hours, the heart was either perfused with a Langendorff device ex vivo or used for heterotopic heart transplantation in vivo. Bcl- xL gene transfer significantly reduced the infarct size (23.0 +/- 2.6% versus 47.7 +/- 7.0% in saline control and 48.6 +/- 6.1% in vector control, P < 0.01) after 2- hour reperfusion at 37 degrees C with the Langendorff device and significantly decreased creatine kinase release (0.82 +/- 0.27 IU, versus 1.57 +/- 0.33 and 1.50 +/- 0.37 IU in saline and vector controls, respectively; P < 0.05). In heart transplantation, overexpresson of Bcl-xL inhibited Bax translocation from the cytosol to the mitochondria, resulting in decreased cytochrome c release from the mitochondria; it also significantly decreased cardiac cell apoptosis and improved graft survival rate after long cold preservation, followed by warm reperfusion. Conclusions - Bcl- xL gene transfer inhibited the translocation of Bax and prolonged the cold preservation time of cardiac transplants. This may be a potential therapeutic method in clinical practice.
• Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy.

  Hirofumi Hamada, Masayoshi Kobune, Kiminori Nakamura, Yutaka Kawano, Kazunori Kato, Osamu Honmou, Kiyohiro Houkin, Takuya Matsunaga, Yoshiro Niitsu

  Cancer science 96 (3) 149 - 56 1347-9032 2005/03 [Refereed][Not invited]
   

  We developed human mesenchymal stem cell (MSC) lines that could differentiate into various tissue cells including bone, neural cells, bone marrow (BM) stromal cells supporting the growth of hematopoietic stem cell (HSC), and so-called 'tumor stromal cells' mixing with tumor cells. We investigated the applicability of MSC as therapeutic cell transplanting reagents (cytoreagents). Telomerized human BM derived stromal cells exhibited a prolonged lifespan and supported the growth of hematopoietic clonogenic cells. The gene transfer of Indian hedgehog (Ihh) remarkably enhanced the HSC expansion supported by the human BM stromal cells. Gene-modified MSC are useful as therapeutic tools for brain tissue damage (e.g. brain infarction) and malignant brain neoplasms. MSC transplantation protected the brain tissue from acute ischemic damage in the midcerebral artery occlusion (MCAO) animal model. Brain-derived neurotrophic factor (BDNF)-gene transduction further enhanced the protective efficacy against the ischemic damage. MSC possessed excellent migratory ability and exerted inhibitory effects on the proliferation of glioma cells. Gene-modification of MSC with therapeutic cytokines clearly augmented the antitumor effect and prolonged the survival of tumor-bearing animals. Gene therapy employing MSC as a tissue-protecting and targeting cytoreagent would be a promising approach.
• Expansion of CD34+ cells on telomerized human stromal cells without losing erythroid-differentiation potential in a serum-free condition.

  Masayoshi Kobune, Yutaka Kawano, Junji Kato, Yoshinori Ito, Hiroki Chiba, Kiminori Nakamura, Akihito Fujimi, Takuya Matsunaga, Hirofumi Hamada, Yoshiro Niitsu

  International journal of hematology 81 (1) 18 - 25 0925-5710 2005/01 [Refereed][Not invited]
   

  Erythropoiesis progresses from stem cell expansion on stromal cells through the formation of an erythroblastic island. Our aim was to assess the feasibility of using human stromal cells for erythroid production and differentiation. When cord blood CD34(+) cells were cocultured with telomerized human stromal cells (hTERT-stromal cells) for 2 weeks, the CD34(+) cells and burst-forming units-erythroid (BFU-E) significantly expanded, and a few hematopoietic cells transmigrated below the stromal layer. When nonadherent hematopoietic progenitor cells that had expanded above the hTERT-stromal cells (group B) were collected and subjected to our erythroid-differentiation protocol, they differentiated into erythroblasts with a slight hemoglobin synthesis. When the few hematopoietic cells that had transmigrated below the stromal layer were expanded for an additional 2 to 6 weeks, they exhibited a cobblestone-like appearance, and a large amount of BFU-E clambered weekly from the underside of the stromal layer to above the stromal layer (group C). When the hematopoietic progenitor cells in group C were subjected to the erythroid-differentiation protocol, large numbers of mature erythroblasts (more than 300,000 times the initial CD34(+) cell number) were produced. Our hTERT-stromal expansion protocol may contribute to the construction of a system for large-scale, long-term production of erythroid cells. (C) 2005 The Japanese Society of Hematology.
• Mesenchymal stem cells that produce neurotrophic factors reduce ischemic damage in the rat middle cerebral artery occlusion model.

  Kazuhiko Kurozumi, Kiminori Nakamura, Takashi Tamiya, Yutaka Kawano, Keiji Ishii, Masayoshi Kobune, Sachie Hirai, Hiroaki Uchida, Katsunori Sasaki, Yoshinori Ito, Kazunori Kato, Osamu Honmou, Kiyohiro Houkin, Isao Date, Hirofumi Hamada

  Molecular therapy : the journal of the American Society of Gene Therapy 11 (1) 96 - 104 1525-0016 2005/01 [Refereed][Not invited]
   

  Mesenchymal stem cells (MSC) were reported to ameliorate functional deficits after stroke in rats, with some of this improvement possibly resulting from the action of cytokines secreted by these cells. To enhance such cytokine effects, we previously transfected the telomerized human MSC with the BDNF gene using a fiber-mutant adenovirus vector and reported that such treatment contributed to improved ischemic recovery in a rat transient middle cerebral artery occlusion (MCAO) model. In the present study, we investigated whether other cytokines in addition to BDNF, i.e., GDNF, CNTF, or NT3, might have a similar or greater effect in this model. Rats that received MSC-BDNF (P < 0.05) or MSC-GDNF (P < 0.05) showed significantly more functional recovery as demonstrated by improved behavioral test results and reduced ischemic damage on MRI than did control rats 7 and 14 days following MCAO. On the other hand, rats that received MSC-CNTF or MSC-NT3 showed neither functional recovery nor ischemic damage reduction compared to control rats. Thus, MSC transfected with the BDNF or GDNF gene resulted in improved function and reduced ischemic damage in a rat model of MCAO. These data suggest that gene-modified cell therapy may be a useful approach for the treatment of stroke.
• Wnt3 modulates the characteristics and cobblestone area-supporting activity of human stromal cells.

  Hiroki Chiba, Masayoshi Kobune, Junji Kato, Yutaka Kawano, Yoshinori Ito, Kiminori Nakamura, Sumiyo Asakura, Hirofumi Hamada, Yoshiro Niitsu

  Experimental hematology 32 (12) 1194 - 203 0301-472X 2004/12 [Refereed][Not invited]
   

  Objective. Our objective was to investigate the expression and significance of Wnt proteins in adult human hematopoietic-supporting stromal cells. Methods. Degenerate reverse transcription-polymerase chain reaction was performed to screen telomerized human stromal cells (hTERT-stromal cells) and multipotent mesenchymal cells (hTERT-MSCs) for expression of Wnt genes. We studied the actions of Wnt proteins by overexpressing them in stromal cells and MSCs by retrovirus-mediated gene transfer. Results. The hTERT-stromal and primary stromal cells expressed Wnt5A, while hTERT-MSCs and primary MSCs expressed Wnt3 and Wnt5A. Gene transfer of Wnt5A slightly reduced the growth rate of hTERT-stromal cells, but did not affect their morphology. In contrast, gene transfer of Wnt3 into both hTERT-stromal cells and hTERT-MSCs enhanced Wnt-beta-catenin signaling, and caused remarkable morphological changes and growth retardation. Upon 2-week co-culture, expansion of clonogenic cells on Wnt5A-stromal cells was superior to that on control stromal cells. However, expansion of CD34(+) cells on Wnt3-stromal cells did not differ from that on control stromal cells. Moreover, there was a drastic reduction in the formation of cobblestone area (CA) underneath Wnt3-stromal cells compared with that underneath control stromal cells. Conclusion. These results suggest that Wnt3 plays an important role in regulating characteristics and CA support activity of stromal cells. (C) 2004 International Society for Experimental Hematology. Published by Elsevier Inc.
• Interleukin-11 as an osteoprotegerin-inducing factor in culture medium of blastic cells from a patient with acute megakaryocytic leukemia complicated with osteosclerosis.

  Tsutomu Sato, Takuya Matsunaga, Masaya Kida, Kazuhiro Morii, Takuro Machida, Yutaka Kawano, Kiminori Nakamura, Kageaki Kuribayashi, Kohichi Takada, Satoshi Iyama, Yasushi Sato, Tetsuji Takayama, Minoru Takahashi, Junji Kato, Manabu Chokki, Yoshiro Niitsu

  American journal of hematology 77 (1) 62 - 6 0361-8609 2004/09 [Refereed][Not invited]
   

  We came across a rare case of acute megalkaryocytic leukemia, the clinical course of which was relatively chronic and nonaggressive. This case was complicated with generalized severe osteosclerosis (OS). The medium in which blastic cells from the patient were cultured showed a strong activity to enhance the expression of an osteosclerotic cytokine, osteoprotegerin (OPG), as revealed by real-time quantitative RT-PCR and Western blot analysis. The OPG-inducing activity of the culture medium was neutralized by the anti-interieukin-11 (IL-11) antibody. These results indicate that IL-11 produced by the blasts was a causative factor of the OS observed in this patient. (C) 2004 Wiley-Liss, Inc.
• Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID-beta2m-/- repopulating cells.

  Masayoshi Kobune, Yoshinori Ito, Yutaka Kawano, Katsunori Sasaki, Hiroaki Uchida, Kiminori Nakamura, Hironari Dehari, Hiroki Chiba, Rishu Takimoto, Takuya Matsunaga, Takeshi Terui, Junji Kato, Yoshiro Niitsu, Hirofumi Hamada

  Blood 104 (4) 1002 - 9 0006-4971 2004/08/15 [Refereed][Not invited]
   

  Hematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of giving rise to all types of blood cells. However, the mechanisms involved in controlling the number and abilities of HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has an essential role in inducing hematopoietic tissue during embryogenesis. We investigated the roles of the Ihh in coculture with CD34(+) cells and human stromal cells. Ihh mRNA was expressed in primary and telomerized human (hTERT) stromal cells, and its receptor molecules were detected in CD34+ cells. Ihh gene transfer into hTERT stromal cells enhanced their hematopoietic supporting potential, which was elevated compared with control stromal cells, as indicated by the colony-forming units in culture (CFU-Cs) (26-fold 2-fold versus 59-fold +/- 3-fold of the initial cell number; mixed colony-forming units [CFU-Mix's], 63-fold +/- 37-fold versus 349-fold +/- 116-fold). Engraftments of nonobese diabetic/severe combined immunodeficiency-beta(2)m(-/-) (NOD/SCID-beta(2)m(-/-)) repopulating cells (RCs) expanded on Ihh stromal cells were significantly higher compared with control coculture results, and engraftment was neutralized by addition of an antihedgehog antibody. Limiting dilution analysis indicated that NOD/SCID-beta(2)m(-/-) RCs proliferated efficiently on lhh stromal cells, compared with control stromal cells. These results indicate that lhh gene transfer could enhance the primitive hematopoietic support ability of human stromal cells. (C) 2004 by The American Society of Hematology.
• Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model.

  K Nakamura, Y Ito, Y Kawano, K Kurozumi, M Kobune, H Tsuda, A Bizen, O Honmou, Y Niitsu, H Hamada

  Gene Therapy 11 (14) 1155 - 1164 0969-7128 2004/07 [Refereed]
  
• BDNF gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebral artery occlusion model.

  Kazuhiko Kurozumi, Kiminori Nakamura, Takashi Tamiya, Yutaka Kawano, Masayoshi Kobune, Sachie Hirai, Hiroaki Uchida, Katsunori Sasaki, Yoshinori Ito, Kazunori Kato, Osamu Honmou, Kiyohiro Houkin, Isao Date, Hirofumi Hamada

  Molecular therapy : the journal of the American Society of Gene Therapy 9 (2) 189 - 97 1525-0016 2004/02 [Refereed][Not invited]
   

  Examination of the clinical therapeutic efficacy of using bone marrow stromal cells, including mesenchymal stem cells (MSC), has recently been the focus of much investigation. MSC were reported to ameliorate functional deficits after stroke in rats, with some of this improvement possibly resulting from the action of cytokines secreted by these cells. To enhance such cytokine effects, we transfected telomerized human MSC with the BDNF gene using a fiber-mutant F/RGD adenovirus vector and investigated whether these cells contributed to improved functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model. BDNF production by MSC-BDNF cells was 23-fold greater than that seen in uninfected MSC. Rats that received MSC-BDNF showed significantly more functional recovery than did control rats following MCAO. Specifically, MRI analysis revealed that the rats in the MSC-BDNF group exhibited more significant recovery from ischemia after 7 and 14 days. The number of TUNEL-positive cells in the ischemic boundary zone was significantly smaller in animals treated with MSC-BDNF compared to animals in the control group. These data suggest that MSC transfected with the BDNF gene may be useful in the treatment of cerebral ischemia and may represent a new strategy for the treatment of stroke.
• Pre-administration of angiopoietin-1 followed by VEGF induces functional and mature vascular formation in a rabbit ischemic model.

  Akihiko Yamauchi, Yoshinori Ito, Masayuki Morikawa, Masayoshi Kobune, Jianhua Huang, Katsunori Sasaki, Kazuhiro Takahashi, Kiminori Nakamura, Hironari Dehari, Yoshiro Niitsu, Tomio Abe, Hirofumi Hamada

  The journal of gene medicine 5 (11) 994 - 1004 1099-498X 2003/11 [Refereed][Not invited]
   

  Background Angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF) play important roles in vascular formation and maturation, suggesting that the combination of these two would be a promising therapy for ischemia. However, it remains unclear what the best schedule of administration of these cytokines might be. Methods Six experimental groups were used to prepare the rabbit ischemic hindlimb model following naked plasmid intramuscular administration as follows: empty vector (C), single gene (Ang1, A; VEGF, V), Ang-1 followed by VEGF (A - V), co-administration of Ang1 and VEGF (A + V), and VEGF followed by Ang1 (V - A). Results Thirty days after gene administration, A - V showed a significantly increased blood pressure and blood-flow recovery in the ischemic limb compared with the control group. Histological findings by alpha-smooth muscle-actin (alpha-SNM) staining revealed that the two combination groups had more mature vessels as compared with the control group. Significantly, A - V revealed the highest density of alpha-SMA-positive vessels compared with VEGF alone or Ang1 alone. Angiographic assessment revealed that A - V had a greater increased arterial diameter compared with VEGF alone. Edema, one of the major adverse effects induced by VEGF, was not found in A - V throughout the experiments, while VEGF alone and V - A showed severe edema induced by VEGF. Conclusions The pre-administration of Ang1 followed by VEGF resulted in an improvement of hemodynamic status, an increased number of vessels covered with a-actin-positive mural cells, and prevention of VEGF-mediated edema. Thus, priming by Ang1 gene administration would be beneficial for therapeutic angiogenesis in VEGF gene therapy. Copyright (C) 2003 John Wiley Sons, Ltd.
• Adenoviral-delivered angiopoietin-1 reduces the infarction and attenuates the progression of cardiac dysfunction in the rat model of acute myocardial infarction.

  Kazuhiro Takahashi, Yoshinori Ito, Masayuki Morikawa, Masayoshi Kobune, Jianhua Huang, Masaru Tsukamoto, Katsunori Sasaki, Kiminori Nakamura, Hironari Dehari, Katsuya Ikeda, Hiroaki Uchida, Sachie Hirai, Tomio Abe, Hirofumi Hamada

  Molecular therapy : the journal of the American Society of Gene Therapy 8 (4) 584 - 92 1525-0016 2003/10 [Refereed][Not invited]
   

  In acute myocardial infarction (AMI), prognosis and mortality rate are closely related to the infarct size and the progression of postinfarction cardiac failure. Angiogenic gene therapy has presented a new approach for the treatment of AMI. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. We hypothesized that gene therapy using Ang1 for AMI might promote angiogenesis cooperatively with intrinsic VEGF, since high concentrations of circulating VEGF have been reported in AMI. To evaluate our hypothesis, we employed a rat AMI model and adenoviral Ang1 (HGMW-approved gene symbol ANGPT1) gene transfer to the heart. A significant increase in capillary density and reduction in infarct sizes were noted in the infarcted hearts with adenoviral Ang1 gene treatment compared with control infarcted hearts treated with saline or adenoviral vector containing the beta-galactosidase gene. Furthermore, the Ang1 group showed significantly higher cardiac performance in echocardiography (55.0% of ejection fraction, P < 0.05 vs control) than the saline or adenoviral controls (36.0 or 40.5%, respectively) 4 weeks after myocardial infarction. The adenoviral delivery of Ang1 during the acute phase of myocardial infarction would be feasible to attenuate the progression of cardiac dysfunction in the rat model.
• Myocardial injection of CA promoter-based plasmid mediates efficient transgene expression in rat heart.

  Jianhua Huang, Yoshinori Ito, Masayoshi Kobune, Katsunori Sasaki, Kiminori Nakamura, Hironari Dehari, Kazuhiro Takahashi, Katsuya Ikeda, Hiroaki Uchida, Kazunori Kato, Hirofumi Hamada

  The journal of gene medicine 5 (10) 900 - 908 1099-498X 2003/10 [Refereed][Not invited]
   

  Background Although naked plasmid injection is the safest and most convenient method for gene delivery, a major limitation of this approach is currently poor transgene expression. The CA promoter (chicken beta-actin promoter with cytomegalovirus, CMV, enhancer) is one of the strongest transcriptional control modules found; however, it is uncertain whether a CA promoter-based vector is efficient enough for naked gene therapy in a cardiovascular context. Methods The beta-galactosidase (LacZ) expression provided by CA promoter plasmid (pCAZ2) injection into the skeletal muscle or the heart of Lewis rats was compared with CMV promoter plasmid or adenoviral vector (AxCAZ3). The effect of Simian virus 40 of the replication origin (SV40ori) deletion from pCAZ2 on transgene expression was also evaluated. Results pCAZ2 showed the highest LacZ expression in both skeletal muscle and heart in comparison with the CMV promoter-based vector 5 days after naked plasmid injection. LacZ expression in the heart obtained using 20 mug of pCAZ2 was almost equivalent to that shown with AxCAZ3 at 6.0 x 10(9) optical particle units. The time course of transgene expression driven by CMV and CA promoters in the heart were similar, with the CA promoter providing significantly higher gene expression than the CMV promoter across all time points examined. SV40ori deletion from pCAZ2 did not affect transgene expression in either skeletal muscle or heart. Conclusions Transgene expression mediated by naked CA promoter-based plasmid injection was shown to be quite efficient in the heart. We propose that the CA promoter vector is suitable for myocardial gene therapy. Copyright (C) 2003 John Wiley Sons, Ltd.
• Inhibition of type I procollagen production by tRNAVal CTE-HSP47 ribozyme.

  Seiya Hagiwara, Kiminori Nakamura, Hirofumi Hamada, Katsunori Sasaki, Yoshinori Ito, Kageaki Kuribayashi, Tsutomu Sato, Yasushi Sato, Minoru Takahashi, Katsuhisa Kogawa, Junji Kato, Takeshi Terui, Tetsuji Takayama, Takuya Matsunaga, Kazunari Taira, Yoshiro Niitsu

  The journal of gene medicine 5 (9) 784 - 94 1099-498X 2003/09 [Refereed][Not invited]
   

  Background Fibrosis characteristically occurs in the advanced stages of chronic inflammatory diseases, occasionally as the primary lesion, and frequently determines the disease prognosis. Fibrotic lesions consist mostly of collagen, and therefore it may be possible to prevent or treat fibrosis by inhibiting collagen production. Of the currently available therapeutic approaches, however, none is sufficiently effective and specific for inhibition of collagen. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been reported to play a pivotal role in secretion of procollagen molecules. Therefore, we have tried to suppress its function to inhibit these various types of collagen. Methods We have developed a novel type of ribozyme by ligating a v hammerhead sequence to a tRNA promoter to facilitate displacing the ribozyme from nucleus to cytoplasm and to constitutive transport element, a binding motif of helicase which unwinds mRNA to render the target sequence on the mRNA accessible to the ribozyme. Results The ribozyme thus constructed showed strong activity to cleave HSP47 mRNA and suppress the secretion of type I procollagen in the human primary fibroblast. Conclusion We suggest applicability of this ribozyme as a new modality for antifibrosis therapy. Copyright (C) 2003 John Wiley Sons, Ltd.
• Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells.

  Masayoshi Kobune, Yutaka Kawano, Yoshinori Ito, Hiroki Chiba, Kiminori Nakamura, Hajime Tsuda, Katsunori Sasaki, Hironari Dehari, Hiroaki Uchida, Osamu Honmou, Sho Takahashi, Akiko Bizen, Rishu Takimoto, Takuya Matsunaga, Junji Kato, Kazunori Kato, Kiyohiro Houkin, Yoshiro Niitsu, Hirofumi Hamada

  Experimental hematology 31 (8) 715 - 22 0301-472X 2003/08 [Refereed][Not invited]
   

  Objective. To compare the hematopoietic support provided by telomerized human mesenchymal stem cells (MSCs) and telomerized MSC-derived stromal cells. Methods. We transfected the human telomerase catalytic subunit (hTERT) gene into primary MSCs to establish hTERT-transduced MSCs (hTERT-MSCs). Stromal induction of hTERT-MSCs was performed by replacing the culture medium with Dexter-type culture medium. Hematopoietic support was examined by coculture with cord blood CD34(+) cells. Results. The hTERT-MSCs were morphologically identical with the primary MSCs and expressed surface antigens including CD105, CD73, and CD166. hTERT-MSCs showed a similar doubling time as primary MSCs and continued to proliferate to over 80 population doublings (PD), although the primary MSCs underwent crisis in vitro at 16 PD. The osteogenic, chondrogenic, adipogenic, neurogenic, and stromal differentiation potential of hTERT-MSCs were maintained up to at least 40 PD. The degree of expansion of CD34+ cells and total number of colony-forming units in culture (CFU-C) upon 12-day coculture with the hTERT-MSC-derived stromal cells were nearly the same as those upon 12-day coculture with hTERT-MSCs (CD34, 33.0-fold +/- 2.8-fold vs 36.1-fold +/- 1.7-fold of the initial cell number; CFUs, 344.4-fold +/- 62.5-fold vs 239.3-fold +/- 87.0-fold; CFU-mix, 368.4-fold +/- 113.7-fold vs 341.3-fold +/- 234.3-fold). However, on day 18 of coculture, the number of cobblestone areas (CA) observed beneath the stromal cells was 15 times higher than that beneath hTERT-MSCs (CA, 146.9 +/- 54.6 vs 9.4 +/- 8.1, p < 0.01). Conclusion. Stromal induction of hTERT-MSCs exclusively enhanced the support of CA formation provided by hTERT-MSCs. Our human hTERT-MSCs will be useful for elucidating the mechanism of the formation of CAs. (C) 2003 International Society for Experimental Hematology. Published by Elsevier Inc.
• Efficient BMP2 gene transfer and bone formation of mesenchymal stem cells by a fiber-mutant adenoviral vector.

  Hajime Tsuda, Takuro Wada, Yoshinori Ito, Hiroaki Uchida, Hironari Dehari, Kiminori Nakamura, Katsunori Sasaki, Masayoshi Kobune, Toshihiko Yamashita, Hirofumi Hamada

  Molecular therapy : the journal of the American Society of Gene Therapy 7 (3) 354 - 65 1525-0016 2003/03 [Refereed][Not invited]
   

  Strategies using mesenchymal stem cell (MSC)-mediated gene therapy have been developed to improve bone healing. However, transduction efficiency into MSCS by each vector is not always high. To overcome this problem, we used a modified adenoviral vector (Adv-F/RGD) with an RGD-containing peptide in the HI loop of the fiber knob domain of adenovirus type 5 (Ad5). Transduction efficiency into bone marrow-derived MSCs with Adv-F/RGD increased 12-fold compared with a vector containing the wild-type fiber (Adv-F/wt) by beta-galactosidase chemiluminescent assay. As a next step, we constructed AxCAhBMP2-F/RGD and AxCAhBMP2-F/wt carrying human bone morphogenetic protein 2 (BMP2). At the same multiplicity of infection, MSCs infected with AxCAhBMP2-F/RGD produced higher amounts of BMP2 than cells infected with AxCAhBMP2-F/wt, and also differentiated towards the osteogenic lineage more efficiently in vitro. Furthermore, using ex vivo gene transduction, we evaluated the potential for ectopic bone formation by the transduced MSCs in vivo. Transduction with AxCAhBMP2-F/RGD exhibited greatly enhanced new bone formation. These data suggest that Adv-F/RGD is useful for introducing foreign genes into MSCs and that it will be a powerful gene therapy tool for bone regeneration and other tissue engineering.
• Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT)-transfected human stromal cells.

  Yutaka Kawano, Masayoshi Kobune, Miki Yamaguchi, Kiminori Nakamura, Yoshinori Ito, Katsunori Sasaki, Sho Takahashi, Takafumi Nakamura, Hiroki Chiba, Tsutomu Sato, Takuya Matsunaga, Hiroshi Azuma, Kenji Ikebuchi, Hisami Ikeda, Junji Kato, Yoshiro Niitsu, Hirofumi Hamada

  Blood 101 (2) 532 - 40 0006-4971 2003/01/15 [Refereed][Not invited]
   

  We developed a new human stromal cell line that could expand human hematopoietic progenitor/stem cells. Primary human bone marrow stromal cells were infected with retrovirus containing the human telomerase catalytic subunit (hTERT) gene, resulting in increased population doubling and the acquisition of cell immortalization. Characteristics of the hTERT-transduced stromal (hTERT-stromal) cells were identical with those of the primary stromal cells in terms of morphologic appearance and expression of surface antigens. Human cord blood (CB) CD34(+) cells were expanded by coculture with primary stromal or hTERT-stromal cells in the presence of stem cell factor, thrombopoietin, and Fik-2/Fit-3 ligand under serum-free condition. The degree of expansion of CD34(+) cells and total number of colony-forming units in culture (CFU-Cs) after 2 weeks' coculture with the hTERT-stromal cells were nearly the same as those after 2 weeks' coculture with primary stromal cells (CD34(+) cells, 118-fold +/- 8-fold versus 117-fold 13-fold; CFU-Cs, 71-fold +/- 5-fold versus 67-fold +/- 5-fold of initial cell number). CB expansion on hTERT-stromal cells occurred at a similar rate through 7 weeks. In contrast, the rate of CB expansion on primary stromal cells had drastically declined at 7 weeks. In nonobese diabetic/severe combined immunodeficiency (SCID) mice, the degree of engraftment of SCID-repopulating cells that had been cocultured with hTERT-stromal cells for 4 weeks was significantly higher than that of precocultured CB cells. These results indicate that this hTERT-stromal cell line could be useful for ex vivo expansion of hematopoietic progenitor/stem cells and for analyzing the microenvironment of human bone marrow. (C) 2003 by The American Society of Hematology.
• Enhanced expression of type IV collagen-binding protein (p29) in Fyn-transfected murine fibrosarcoma cells.

  Kazuhiko Koike, Katsuhisa Kogawa, Tetsuji Takayama, Naohito Yoshizaki, Hirohito Muramatsu, Kiminori Nakamura, Sumio Sakamaki, Yoshiro Niitsu

  Japanese journal of cancer research : Gann 93 (10) 1090 - 9 0910-5050 2002/10 [Refereed][Not invited]
   

  We investigated the mechanism of the enhancement of metastatic potential induced by transfection of the fyn gene, a member of the src family. We employed two murine fyn cDNA-transfected clones, ML-SN1 and ML-SN2, which were previously established from an ML-01 low-metastatic clone of Meth A sarcoma of BALB/c mice and were proven to have higher metastatic ability than ML-01 and the mock-transfected clone ML-MT-neo (Takayama et al, 1993). Our present investigation revealed that the two transfectants showed higher metastatic ability and higher rates of adherence to type IV collagen than ML-MT-neo. However, no difference was found in in vitro or in vivo growth rates, attachment to laminin or endothelial cells or cell motility through a reconstituted basement membrane. Analysis of surface membrane proteins labeled with I-125 on SDS-PAGE showed that a 29 kD band specifically bound to type IV collagen-coupled beads was more intense in ML-SN2 than in ML-MT-neo. Genistein, a protein tyrosine kinase inhibitor, dramatically reduced protein tyrosine kinase (PTK) activity of ML-SN2 in a dose-dependent fashion, corresponding to the reduction of adhesiveness to type IV collagen. The expression of the type IV collagen-binding protein (p29) of ML-SN2 was also reduced significantly by genistein treatment. These results suggested that the fyn product in Meth A cells augments the expression of a type IV collagen-binding protein through elevation of the PTK activity of the membrane fraction and thus facilitates the metastasis of Meth A.
• Anti-metastatic gene therapy utilizing subcutaneous inoculation of EC-SOD gene transduced autologous fibroblast suppressed lung metastasis of Meth-A cells and 3LL cells in mice.

  M Tanaka, K Kogawa, K Nakamura, Y Nishihori, K Kuribayashi, S Hagiwara, H Muramatsu, S Sakamaki, Y Niitsu

  GENE THERAPY 8 (2) 149 - 156 0969-7128 2001/01 [Refereed][Not invited]
   

  We have previously reported that superoxide stimulates the motility of tumor cells and the administration of Cu-Zn superoxide dismutase (SOD) significantly suppresses metastasis. However, ideally, anti-metastatic therapy should be longlasting, systemically effective and have low toxicity. The half-life of Cu-Zn SOD in plasma is so short that it cannot provide long-lasting effects. Therefore, in this study we have developed a gene therapy in a mouse model utilizing extracellular SOD (EC-SOD), which is the most prevalent SOD isoenzyme in extracellular fluids. We retrovirally transfected fibroblasts (syngeneic) with the EC-SOD gene and established EC-SOD-secreting fibroblasts. Inoculation of EC-SOD-secreting fibroblasts suppressed both artificial and spontaneous metastatic lung nodules in mouse metastasis models. These data indicate the feasibility of anti-metastatic gene therapy utilizing the EC-SOD gene.
• Enhanced inhibition of experimental metastasis by the combination chemotherapy of Cu-ZnSOD and adriamycin

  K Kogawa, H Muramatsu, M Tanaka, Y Nishihori, S Hagiwara, K Kuribayashi, K Nakamura, K Koike, S Sakamaki, Y Niitsu

  CLINICAL & EXPERIMENTAL METASTASIS 17 (3) 239 - 244 0262-0898 1999/05 [Refereed][Not invited]
   

  We previously reported that reactive oxygen species (ROS) enhance tumor cell metastasis, and by administration of recombinant human superoxide dismutase (rh SOD), an enzyme which scavenges O-2(-) successfully reduced lung metastasis of mouse MethA sarcoma and Lewis lung carcinoma. These observations suggested that rh SOD suppressed tumor cell invasion by eliminating O-2(-), the primary source of ROS. However, for the clinical application of the drug as an anti metastatic agent, rh SOD needs to be administered in combination with other cytotoxic agents, since SOD by itself has no cytotoxic activity. In this paper, we investigated the effectiveness of the combination chemotherapy of rh SOD and adriamycin (ADR), an anti-cancer agent against the experimental metastasis of highly metastatic clone, MH-02, which was derived from murine Meth A sarcoma. The present metastasis experiment clearly indicates that the administration of rh SOD enhances the anti-metastatic effect of ADR. On the other hand, we found that the inhibition rate of metastasis exhibited by the combination chemotherapy of rh SOD and a certain dose (5 mg/ml) of ADR was inferior to that of rh SOD. This apparent paradoxical phenomenon was presumably explained by our finding that tumor cells themselves augment their invasive capacity and platelet aggregation, both of which are causative factors for metastasis formation, by generation of O-2(-) when they were treated with ADR. Nevertheless, the combination chemotherapy of SOD with anticancer drugs such as ADR can be a practical anti-metastasis strategy.
• Activation of phosphatdylinositol 3-kinase by serum stimulation enhances cell adhesion and cell motility in highly-invasive tongue squamous cell carcinoma cells, SAS-HI

  Kazuhiko OKUMURA, Atsushi KONISHI, Tomomi YAMASHITA, Ken OMURA, Maki TANAKA, Tsukasa HAGINO, Kiminori NAKAMURA, Masaaki KANAZAWA

  Jpn. J. Ord Maxillofac. Surg. 45 (11) 655 - 665 0021-5163 1999 [Not refereed][Not invited]
  
• Suppression of intracellular Cu-Zn SOD results in enhanced motility and metastasis of Meth A sarcoma cells.

  M Tanaka, K Kogawa, Y Nishihori, K Kuribayashi, K Nakamura, H Muramatsu, K Koike, S Sakamaki, Y Niitsu

  INTERNATIONAL JOURNAL OF CANCER 73 (2) 187 - 192 0020-7136 1997/10 [Refereed][Not invited]
   

  We have previously described an inverse relationship between Cu-Zn superoxide dismutase (SOD) activity and invasiveness of a clone of human tongue cancer cells. In these cells, suppression of Cu-Zn SOD activity by transfection with anti-sense cDNA enhanced motility in vitro. The present studies were undertaken to determine whether the inverse relationship between intracellular Cu-Zn SOD activity and motility is a general property of other tumor cells and whether this enzyme indeed defines in vivo metastatic potential. Murine Meth A sarcoma-derived ML-01 cells, which have low metastatic activity, were transfected with anti-sense Cu-Zn SOD cDNA. Two clones with very different SOD activities-ML-AS2, with the most suppressed, and ML-AS5, with the least suppressed activity-were analyzed for their motility and metastatic capability. Compared to the mock-transfectant ML-neo, the metastatic potential and motility of the ML-AS2 and ML-AS5 were increased 4.5-and 2.1-fold, respectively. Superoxide treatment enhanced the motility of the AS clones but not that of the ML-neo cells, Our results clearly show that there is an inverse relationship between the intracellular level of Cu-Zn SOD, cell motility and in vivo metastatic potential. (C) 1997 Wiley-Liss, Inc.

MISC

• Paneth 細胞・αディフェンシンによる自然免疫機能を介した腸内細菌叢制御

  中村公則  臨床化学 特集：消化管細菌と全身性疾患  52-  (3)  170  2023/07
• 【腸と健康:腸オルガノイドが挑む次世代バイオモデル】自然免疫と共生を紐解く小腸オルガノイドenteroid

  綾部 時芳, 中村 公則  医学のあゆみ  270-  (11)  1059  -1063  2019/09  

  Paneth細胞は、抗菌ペプチドα-defensinを含む細胞質顆粒を小腸内腔へ分泌することにより自然免疫に貢献する。分泌されたα-defensinは病原菌選択的な殺菌活性で腸内細菌叢を制御する。一方、腸内細菌叢の組成破綻と、肥満や炎症性腸疾患などさまざまな疾患との関与が報告されている。α-defensinが腸内細菌叢を介して腸内環境の恒常性を維持するとともに、その破綻は疾患に関与することがわかってきた。小腸上皮細胞の三次元培養系であるenteroidを用いて、これまで不可能だった細胞極性に依存したPaneth細胞の顆粒分泌応答と脱顆粒後の顆粒再形成が証明された。Paneth細胞は培地に添加したカルバコール(CCh)に対して顆粒分泌応答を示したが、リポポリサッカライド(LPS)刺激には分泌応答を示さず、一方、マイクロインジェクションでenteroid内腔に物質を導入し、内腔側からLPSおよびSalmonella生菌で刺激すると顆粒を分泌する。さらに、脱顆粒後のPaneth細胞は新しい顆粒を再形成する。著者らの確立した系で、さまざまな腸内環境因子によるPaneth細胞分泌応答のメカニズムや疾患との関係解明に迫ることが期待される。(著者抄録)
• 【腸内菌叢はコントロールできるか?】抗菌ペプチドαディフェンシンによる腸内細菌叢の制御

  中村 公則, 菊池 摩仁, 綾部 時芳  腸内細菌学雑誌  33-  (3)  129  -135  2019/07  

  抗菌ペプチドは、ヒトを含む多細胞生物において殺微生物活性を持つ自然免疫の主要な作用因子であり、その生体防御における重要性は広く認識されている。腸上皮細胞は広大な表面積を構成し、たえず病原体および食物を含む外部環境や常在する腸内細菌に暴露されている。腸上皮細胞の1系統であるPaneth細胞は抗菌ペプチド・αディフェンシンを分泌し、病原体の排除と常在菌との共生により腸管自然免疫を担っている。このPaneth細胞αディフェンシンは病原菌を強く殺菌する一方、常在菌にはほとんど殺菌活性を示さない特徴を持つことから、生体において腸内細菌叢の制御を介して腸内環境の恒常性維持に重要な役割を果たすと考えられる。近年、腸内細菌叢の構成異常"disbiosis"と、炎症性腸疾患および肥満や動脈硬化などの生活習慣病や癌など様々な疾病との関係が報告されてきているが、いまだその詳細なメカニズムは不明である。抗菌ペプチドα-ディフェンシンの腸内細菌叢制御メカニズムを理解することは、腸内細菌叢と疾病との関連解明に重要な役割を果たすと考えられる。(著者抄録)
• キーワード(No.16) パネート細胞

  中村 公則  消化器病学サイエンス  3-  (1)  43  -43  2019/03
• クローン病自然発症モデルSAMP1/YitFcマウスの病状進行に伴う腸内細菌叢および糞便メタボライトの変動

  小松陽介, 小松陽介, 清水由宇, 山野めぐみ, 中村公則, 綾部時芳, 相沢智康, 相沢智康  日本生物工学会大会講演要旨集  71st-  2019
• 【生体バリア 粘膜や皮膚を舞台とした健康と疾患のダイナミクス】(第1章)生体バリアを支える分子・細胞・組織基盤 細胞分泌物による化学的バリア 抗菌ペプチドによる自然免疫

  綾部 時芳, 中村 公則  実験医学  35-  (7)  1078  -1083  2017/05  

  抗菌ペプチドは殺微生物活性をもつ自然免疫の主要な作用因子である。哺乳類の代表的な抗菌ペプチドにはディフェンシンとカセリシジンがある。小腸陰窩の基底部に位置するPaneth細胞はαディフェンシンを産生し、分泌する。αディフェンシンは病原菌を強く殺菌するが、常在菌にはほとんど殺菌活性を示さず、腸内細菌叢を制御する。αディフェンシンの異常は、肥満、クローン病の病態や移植片対宿主病などのdysbiosisに関与することが示された。抗菌ペプチドの異常がさまざまな疾患の発症や病態に関与している可能性がある。(著者抄録)
• 腸における排除と共生 抗菌ペプチドと腸内細菌

  綾部 時芳, 櫻木 直也, 中村 公則  家畜感染症学会誌  4-  (4)  133  -142  2015/12  

  消化管は、成長と生存に必要な栄養を消化と吸収によって取り入れる場であると同時に、経口的に侵入する様々な病原体を排除する場である。腸管自然免疫は病原体に対してすばやく強力に応答して感染防御に貢献している。一方で腸は、腸内細菌叢を構成する莫大な数の常在菌と共生する場でもある。この、腸における排除と共生が腸内環境の恒常性を維持し、延いては疾病の発症や進展に関与することがわかってきた。腸内細菌叢の構成異常"dysbiosis"と、感染症はもちろん、炎症性腸疾患、生活習慣病や癌など様々な疾病との関係が報告されており、機序の解明が待たれている。粘膜免疫と共生の両方において、腸上皮細胞の1系統であるPaneth細胞が関与している。Paneth細胞は小腸陰窩の最基底部に位置し、細胞内顆粒の抗菌ペプチドαデイフェンシン(α-defensin)を感染刺激にすばやく反応し分泌する。Paneth細胞α-defensinは病原菌を強く殺菌する一方、常在菌にはほとんど殺菌活性を示さず、腸における排除と共生に直接関与し、腸内細菌叢を制御している。腸炎モデルIL10欠損マウスは糞便中α-defensinが有意に低下し、移植片対宿主病モデルマウスでは、Paneth細胞が傷害されてα-defensinが消失し、著しいdysbiosisを生じて感染死に至る。寄生体である腸内細菌叢と宿主のPaneth細胞が担う自然免疫が形成する"腸内環境"から疾病を論じる。(著者抄録)
• 【生体バリアと生体防御・免疫系】腸上皮細胞の抗菌ペプチドによる自然免疫

  中村 公則, 綾部 時芳  臨床免疫・アレルギー科  64-  (1)  20  -25  2015/07
• 【みえてきた小腸の多彩な機能制御にせまる】抗菌ペプチドは腸内環境を制御できるか

  綾部 時芳, 中村 公則  分子消化器病  10-  (1)  27  -32  2013/03  

  抗菌ペプチドは自然免疫の主要なエフェクターである。小腸のパネト細胞が分泌する抗菌ペプチドのαディフェンシンは、腸における病原体の排除と腸内細菌との共生の両方に重要な役割を果たしていることが明らかになってきた。αディフェンシンは病原菌と常在菌を選択し、腸内常在菌の組成を制御している。いま、抗菌ペプチドは腸内環境を制御できるか、を問う時期がきた。本稿では、「腸内環境」を、経口摂取される食品や微生物などの外来性物質、寄生体である腸内細菌(腸内微生物)、および宿主の免疫機構-とくにパネト細胞から分泌されるαディフェンシンが代表する腸管自然免疫-の三者で規定して、αディフェンシンの炎症性腸疾患(IBD)やメタボリック症候群など疾患とのかかわりまで論じ、抗菌ペプチドが腸内環境を制御する可能性にせまる。(著者抄録)
• 抗体工学による次世代バイオ創薬 薬剤運搬に適した腫瘍標的化抗体のスクリーニング法と創薬シーズ開発

  加藤 和則, 中村 公則, 山口 美樹, 濱田 洋文  日本薬学会年会要旨集  132年会-  (1)  164  -164  2012/03  [Not refereed][Not invited]
  
• 【消化管における免疫応答】腸内環境を制御するPaneth細胞αディフェンシン

  綾部 時芳, 中村 公則  臨床免疫・アレルギー科  57-  (1)  25  -31  2012/01
• 【体の中の"小さな生態系"腸内フローラによる免疫ホメオスタシス】αディフェンシンによる腸内細菌制御

  中村 公則, 綾部 時芳  実験医学  29-  (18)  2955  -2961  2011/11  

  自然免疫の主要なエフェクターである抗菌ペプチドの1つであり、小腸上皮Paneth細胞から分泌されるαディフェンシンは腸に侵入してくる病原体から宿主を防御するだけではなく、細菌叢の組成に影響を及ぼし腸内細菌生態系の恒常性を維持していることが明らかになってきた。腸内細菌叢の変化は健康と疾患に密接な関係があり、αディフェンシンが炎症性腸疾患や肥満をはじめとして多くの疾患の発症リスクにかかわるという構図が考えられることから、その機序解明が注目されている。(著者抄録)
• 【IBDの疾患感受性遺伝子を知り、治療につなげる】Paneth細胞とクローン病UPDATE

  綾部 時芳, 中村 公則  IBD Research  5-  (2)  108  -112  2011/06  

  小腸陰窩の基底部に位置するPaneth(パネト)細胞は感染防御に重要な役割を果たしている。近年、クローン病(Crohn's disease)におけるPaneth細胞の異常が報告されている。Paneth細胞の細胞内顆粒に存在する抗菌ペプチドのαディフェンシンは腸内腔に分泌され、微生物排除のみならず腸内細菌との共生にも関与している。さらに、Paneth細胞はいくつかのクローン病感受性遺伝子の発現部位として注目されている。クローン病とPaneth細胞の関係を、Paneth細胞ホメオスターシスの維持と破綻という視点からとらえて理解することができる。(著者抄録)
• 【肝癌 基礎・臨床研究のアップデート】肝癌の治療 遺伝子治療 腫瘍の特異的標的化を目指した遺伝子治療法の開発

  中村 公則, 濱田 洋文  日本臨床  67-  (増刊3 肝癌)  560  -564  2009/05
• Z33アデノウイルスと抗MCSP抗体を介したメラノーマに対する遺伝子治療(Targeted gene therapy for melanoma using anti-MCSP antibody and Z33 fiber modified adenovirus vectors)

  桜木 直也, 中村 公則, 平井 幸恵, 加藤 和則, 濱田 洋文  日本癌学会総会記事  67回-  92  -92  2008/09  [Not refereed][Not invited]
  
• 前立腺癌に対する遺伝子治療標的としてのNCAM2(Neural cell adhesion molecule 2 as a target molecule for prostate cancer gene therapy)

  高橋 秀, 加藤 和則, 中村 公則, 濱田 洋文  日本癌学会総会記事  67回-  93  -93  2008/09  [Not refereed][Not invited]
  
• Interleukin-13 Receptor alpha2 Chain メラノーマの新規標的分子としての検討(Interleukin-13 Receptor alpha2 Chain as a Biomarker and Molecular Target for Melanoma)

  中村 公則, 桜木 直也, 加藤 和則, 濱田 洋文  日本癌学会総会記事  67回-  498  -498  2008/09  [Not refereed][Not invited]
  
• ここまで進んだ抗体のトランスレーショナル・リサーチ、臨床応用 創薬・診断シーズとしての癌標的抗体の作製・選別と応用

  加藤 和則, 中村 公則, 濱田 洋文  基盤的癌免疫研究会総会抄録  12回-  22  -22  2008/06  [Not refereed][Not invited]
  
• スーパー標的抗体によって認識される新規メラノーマ抗原IL-13受容体α2(Super-targeting antibody NS-66 recognizes a novel tumor antigen interleukin-13 receptor alpha 2 on human melamoma)

  桜木 直也, 中村 公則, 加藤 和則, 濱田 洋文  日本癌学会総会記事  66回-  241  -242  2007/08  [Not refereed][Not invited]
  
• 前立腺癌遺伝子療法の標的遺伝子の探索(EXPLORATION OF TARGET MOLECULES FOR PROSTATE CANCER GENE THERAPY)

  中村 公則, 加藤 和則, 鈴木 一弘, 塚本 泰司, 濱田 洋文  日本癌学会総会記事  66回-  246  -246  2007/08  [Not refereed][Not invited]
  
• ヒトメラノーマに対する標的化抗体NS-66の樹立と認識抗原解析

  加藤 和則, 中村 公則, 桜木 直也, 濱田 洋文  基盤的癌免疫研究会総会抄録  11回-  87  -87  2007/05  [Not refereed][Not invited]
  
• 【がん領域におけるドラッグデリバリーシステム(DDS)】ベッドサイドから見たDDS開発の進歩 DDSを視点とした標的化遺伝子治療

  中村 公則, 加藤 和則, 濱田 洋文  最新医学  61-  (6)  1130  -1137  2006/06  

  腫瘍だけを見つけて選択的に遺伝子導入や薬剤投与が可能なDDSシステムを作ることができれば,標的化治療が達成できる.我々は,ファイバー変異型アデノウイルスと細胞表面抗原を認識する抗体を組み合わせて,遺伝子導入の選択性強化を目指した.系統的スクリーニング方法が樹立され,前立腺がんや膵がんなどの悪性腫瘍に対して選択性の高い遺伝子導入・薬剤投与を可能とする標的候補が見つかってきた(著者抄録)
• Wnt3/Rho経路は多発性骨髄腫における骨髄間質依存性の抗癌剤耐性を惹起する分子機構の一つである

  千葉 大樹, 小船 雅義, 河野 豊, 中村 公則, 加藤 和則, 瀧本 理修, 松永 卓也, 加藤 淳二, 濱田 洋文, 新津 洋司郎  日本血液学会・日本臨床血液学会総会プログラム・抄録集  67回・47回-  830  -830  2005/09  [Not refereed][Not invited]
  
• Wnt3はnon-canonical経路を介して多発性骨髄腫の抗癌剤耐性を増強させる

  千葉 大樹, 小船 雅義, 河野 豊, 中村 公則, 加藤 和則, 瀧本 理修, 松永 卓也, 加藤 淳二, 濱田 洋文, 新津 洋司郎  日本癌学会総会記事  64回-  371  -371  2005/09  [Not refereed][Not invited]
  
• 抗PAP2a抗体を用いた膵癌に対する標的化遺伝子治療

  中村 公則, 加藤 和則, 濱田 洋文  日本癌学会総会記事  64回-  436  -436  2005/09  [Not refereed][Not invited]
  
• ヒト多発性骨髄腫に対する抗体を介した標的化遺伝子導入と免疫応答誘導

  増田 ゆかり, 加藤 和則, 冨原 圭, 中村 公則, 濱田 洋文  基盤的癌免疫研究会総会抄録  9回-  54  -54  2005/06  [Not refereed][Not invited]
  
• 抗PAP2a抗体を用いた膵癌に対する標的化遺伝子治療

  加藤 和則, 中村 公則, 濱田 洋文  基盤的癌免疫研究会総会抄録  9回-  55  -55  2005/06  [Not refereed][Not invited]
  
• α-GalactosylceramideとアデノIL-2遺伝子治療の併用による抗腫瘍効果および抗転移効果増強の検討

  西堀 佳樹, 古川 勝久, 加藤 和則, 田中 真樹, 岡本 哲朗, 竹内 直子, 萩原 誠也, 栗林 景晶, 中村 公則, 新津 洋司郎  基盤的癌免疫研究会総会抄録  8回-  67  -67  2004/07  [Not refereed][Not invited]
  
• 【癌転移のメカニズムと癌治療 その新しいチャレンジ】転移の治療 転移の遺伝子治療

  新津 洋司郎, 古川 勝久, 田中 真樹, 中村 公則  実験医学  16-  (16)  2135  -2138  1998/10

Books etc

• 食品免疫学事典 日本食品免疫学会(編)

  (Contributor抗菌ペプチド／ディフェンシン)
  朝倉書店 2021/11

Presentations

• Paneth 細胞･αディフェンシンの腸内細菌叢制御による健康と疾患  [Invited]

  中村公則

  第28回腸内細菌学会学術集会 シンポジウム「腸内環境研究の最前線」  2024/06
• Paneth cell･α-defensin to regulate gut microbiota in health and disease  [Invited]

  中村公則

  第24回日本抗加齢医学会総会 シンポジウム 「口腔細菌から始める アンチエイジング医学」  2024/06  熊本城ホール
• Selective Paneth cell granule secretion in response to pathogenic and commensal bacteria in enteric mucosal immunity

  Yuki Yokoi, Shuya Ohir, Haruto Matsuoka, Shota Takemi, Tokiyoshi Ayabe, Kiminori Nakamura

  The 52nd Annual Meeting of the Japanese Society for Immunology  2024/01
• Establishment of a high-efficient method for generating transgenic enteroid revealing intestinal epithelial function

  松下稜, 横井友樹, 大平修也, 松岡温音, 竹見祥大, 中村公則

  第46回日本分子生物学会年会  2023/12
• Paneth細胞・αディフェンシンによる腸内環境制御と健康維持  [Invited]

  中村公則

  iSeminar（異分野融合研究セミナー）  2023/11
• Paneth細胞・αディフェンシンによる自然免疫機能を介した腸内細菌叢制御  [Invited]

  中村公則

  日本食品免疫学会第19回学術大会 シンポジウム「腸内環境と免疫システム」  2023/11
• 授乳期の食事・母乳栄養成分・乳児の発育」の関連性～岩見沢母子健康調査（SMILE Iwamizawa）における産後1ヶ月時点の解析～

  小松陽介, 和田泰明, 田畑風華, 川上智美, 武田安弘, 中村公則, 綾部時芳, 中村幸志, 木村尚史, 玉腰暁子

  第11回日本DOHaD学会学術集会  2023/08
• Paneth細胞αディフェンシンが担う腸内細菌叢の形成からみた母子の疾患リスク上昇メカニズムの理解  [Invited]

  中村公則

  第77回日本栄養･食糧学会大会 シンポジウム「栄養環境による腸内細菌叢変動から見た母子の健康～北海道大学 COI・岩見沢母子健康調査における取り組み～」  2023/05
• Paneth細胞・αディフェンシンによる自然免疫機能を介した腸内細菌叢制御  [Invited]

  中村公則

  第11回自然免疫シンポジウム「アンチエイジングと自然免疫」  2023/03
• High-efficient genetic engineering of enteroids using mouse isolated crypts revealing the intestinal function,

  大平修也, 横井友樹, 海堀唯人, 清水由宇, 綾部時芳, 中村公則

  第51回日本免疫学会学術集会  2022/12
• 腸管恒常性維持における病原菌と常在菌の選択的センシングによるPaneth細胞顆粒分泌調節

  横井友樹, 大平修也, 松岡温音, 海堀唯人, 篠崎竜我, 綾部時芳, 中村公則

  第45回日本分子生物学会  2022/12
• 食品が誘導するPaneth細胞αディフェンシンによる免疫と共生

  綾部 時芳, 横井 友樹, 高桑 暁子, 中村 公則

  第18回日本食品免疫学会学術大会  2022/11
• 抗菌ペプチドαディフェンシンによる腸内細菌制御と健康

  綾部時芳, 横井友樹, 清水由宇, 中村公則

  日本食品・機械研究会  2022/02
• Paneth細胞αディフェンシンによる腸内細菌形成からみた健康と病気  [Invited]

  中村公則

  東洋大学 ライフイノベーション研究所2021年シンポジウム「免疫と健康維持・増進」  2021/11
• Simple and efficient genetic engineering of enteroids by using mouse isolated crypts．

  Shuya Ohira, Yuki Yokoi, Mani Kikuchi, Natsumi Yatsuzuka, Tokiyoshi Ayabe, Kiminori Nakamura

  第44回日本分子生物学会年会  2021/12
• The mechanism for effects of maternal high-fat diet on the development of Paneth cells in offspring

  八塚夏美, 杉本 理菜, 大平 修也, 横井友樹, 菊池摩仁, 綾部時芳, 中村公則

  第44回日本分子生物学会年会  2021/12
• Paneth細胞顆粒分泌応答からみた食機能評価系の確立

  横井友樹, 中村公則, 高桑暁子, 菊池摩仁, 綾部時芳

  第17回日本食品免疫学会学術大会  2021/11  東京都
• Paneth細胞αディフェンシンが担う腸内細菌叢の形成からみた生体恒常性の維持機構  [Invited]

  中村 公則, 綾部 時芳

  第24回 日本臨床腸内微生物学会総会  2021/08
• 抗菌ペプチドから粘膜免疫を理解する

  綾部時芳, 横井友樹, 中村公則

  第58回日本消化器免疫学会  2021/07
• ER-Stress-Associated Misfolding of Paneth cell α-defensin Induces Dysbiosis and ileitis in a Murine Model of Crohn’s Disease

  Yu Shimizu, Kiminori Nakamura, Aki Yoshii, Yuki Yokoi, Mani Kikuchi, Ryuga Shinozaki, Shunta Nakamura, Shuya Ohira, Rina Sugimoto, Tokiyoshi Ayabe

  World Microbe Forum  2021/06
• Dynamics of Paneth Cell Granule Secretory Responses to Bacteria in Innate Enteric Immunity

  Yuki Yokoi, Kiminori Nakamura, Shuya Ohira, Ryuga Shinozaki, Mani Kikuchi, Tokiyoshi Ayabe

  World Microbe Forum  2021/06
• 霊長類T1R1およびT1R2抗体の作製

  小松さゆり, 中安亜希, 大島永心, 新藤壮剛, 今井啓雄, 伯川美穂, 杉山宗太郎, 綾部時芳, 中村公則, 山根拓実, 大石祐一, 岩槻健

  日本農芸化学会 2021年度大会  2021/03
• Immunopathological features of chronic ileitis in Crohn’s disease model SAMP1/YitFc mouse

  村山綾菜, 中村公則, 菊池摩仁, 横井友樹, 綾部時芳

  The 43th Annual Meeting of Molecular Biology Society of Japan  2020/12
• Paneth細胞αディフェンシンによる腸内細菌叢制御を介した生体恒常性の維持機構  [Invited]

  中村公則, 綾部時芳

  第75回 日本体力医学会大会  2020/09
• Alpha-defensin異常による腸内細菌叢の破綻とIBD

  綾部時芳, 清水由宇, 中村公則

  第127回日本消化器病学会北海道支部例会  2020/09
• Understanding mechanisms of disease development via disrupted Paneth cell development involving maturation and formation of intestinal microbiota in early life stage  [Invited]

  中村公則, 綾部時芳

  第24回 腸内細菌学会学術集会  2020/06
• Impact of sample storage conditions on NMR-based human fecal metabolomics

  宋 子豪, 包 克非, 北田直也, 清水由宇, 菊池摩仁, 熊木康裕, 大西裕季, 塚本 卓, 菊川峰志, 出村 誠, 中村公則, 綾部時芳, 山村凌大, 中村幸志, 玉腰暁子, 相沢智康

  第24回 腸内細菌学会学術集会  2020/06
• Association between lean, obesity and gut microbiota in a general Japanese population: The DOSANCO Health Study

  木村尚史, 山村凌大, 檜森亮吾, 鵜川重和, 中村幸志, 中川貴史, 今江章宏, 國弘忠生, 朴鐘旭, Attayeb Mohsen, 川島和, 清水由宇, 中村公則, 綾部時芳, 玉腰暁子

  第24回 腸内細菌学会学術集会  2020/06
• Association between archaea and obesity in a general Japanese population: The DOSANCO Health Study

  山村凌大, 鵜川重和, 中村幸志, 木村尚史, 中川貴史, 今江章宏, 國弘忠生, 朴鐘旭, Attayeb Mohsen, 川島和, 清水由宇, 中村公則, 綾部時芳, 玉腰暁子

  第24回 腸内細菌学会学術集会  2020/06
• Statistical analysis for the effect of mulberry leaf containing diet on the temporal change of α-defensin and intestinal microbiota

  子安惟, 中岡慎治, 菊池摩仁, 中村公則, 綾部時芳

  第24回 腸内細菌学会学術集会  2020/06
• 膵癌初期発生における膵腸相関

  小野裕介, 中村公則, 清水由宇, 横井友樹, 杉本理菜, 早川祐子, 佐藤裕基, 水上裕輔, 奥村利勝, 綾部時芳

  第24回 腸内細菌学会学術集会  2020/06
• Paneth cell dysfunction involved in development of nonalcoholic steatohepatitis through disruption of the intestinal microbiota

  中村駿太, 中村公則, 菊池摩仁, 横井友樹, 大平修也, 清水由宇, 西田琢人, 綾部時芳

  第24回 腸内細菌学会学術集会  2020/06
• Influence of intestinal environment on bactericidal activity of α-defensin isoform

  篠崎竜我, 中村公則, 清水由宇, 綾部時芳

  第24回 腸内細菌学会学術集会  2020/06
• Mechanisms of microbial colonization regulated by Paneth cell development from early postnatal period

  西田琢人, 中村公則, 横井友樹, 綾部時芳

  第24回 腸内細菌学会学術集会  2020/06
• 多様なα-defensin isoformに対するin vitroにおける効率的なS-S結合導入法の確立

  篠崎竜我, 中村公則, 清水由宇, 綾部時芳

  第42 回日本分子生物学会年会  2019/12
• 桑葉のα-defensin分泌促進効果と腸内細菌叢制御

  菊池摩仁, 中村公則, 綾部時芳

  日本食品免疫学会 設立15周年記念学術大会  2019/11
• The influence of maternal high-fat intake on the regulation of intestinal environment by Paneth cell α-defensin in offsprings

  杉本理菜, 中村公則, 嶋 七海, 清水由宇, 横井友樹, 綾部時芳

  日本農芸化学会 2019年度大会  2019/03
• Involvement of Paneth cell alpha-defensin misfolding in disease progression of SAMP1/YitFc, a murine model of Crohn's disease.

  Yu Shimizu, Kiminori Nakamura, Mani Kikuchi, Tokiyoshi Ayabe

  第47回 日本免疫学会学術集会  2018/12
• 腸管organoidを用いたPaneth細胞分化制御機構の解析

  大平修也, 中村公則, 菊池摩仁, 横井友樹, 杉本理菜, 綾部時芳

  第41回 日本分子生物学会年会  2018/11
• レスベラトロール摂取によるα-defensin 分泌量と腸内細菌叢への影響

  中村公則, 杉本理菜, 横井友樹, 清水由宇, 菊池摩仁, 綾部時芳

  第14回 日本食品免疫学会学術大会  2018/11
• 封入体共発現を利用した大量発現系において抗菌ペプチドcryptdin familyの生産性を決定する因子の解析

  保本美穂子, 平峰里菜, SONG Yuchi, 佐藤優次, 塚本卓, 塚本卓, 菊川峰志, 菊川峰志, 出村誠, 出村誠, 中村公則, 綾部時芳, 相沢智康, 相沢智康

  第18回 日本蛋白質科学会年会  2018/06
• Paneth細胞が制御する腸内環境の機能解明  [Invited]

  中村公則

  第95回 北海道腸疾患研究会  2018/06
• 小腸オルガノイドを利用した排出系トランスポーターの基質関与探索ツールの構築

  佐藤夕紀, 中村公則, 小関千尋, 石川岳彦, 島田美紀子, 横井友樹, 本間直幸, 森山隆則, 菅原満

  第13回 トランスポーター研究会年会  2018/05
• Enteroidを用いたPaneth細胞が制御する腸内環境の 機能解明  [Invited]

  中村公則, 横井友樹, 米田司, 綾部時芳

  日本農芸化学会 2018年度大会  2018/03
• エンテロイドを用いた新規・食品機能性成分評価法の構築  [Invited]

  中村公則

  北大リサーチ&ビジネスパークセミナー ~北海道発！「食」から「健康」へ~  2018/02
• Evaluation system for receptor expression and function in the intestinal stem cell niche

  Rina Sugimoto, Naoya Sakuragi, Yuki Yokoi, Kiminori Nakamura, Tokiyoshi Ayabe

  The 46th Annual Meeting of The Japanese Society for Immunology  2017/12
• Paneth cell α-defensin misfolding in SAMP1/YitFc, a murine model of Crohn's disease

  清水 由宇, 中村 公則, 菊池 摩仁, 櫻木 直也, 綾部 時芳

  第40回 日本分子生物学会年会  2017/12
• Establishment of the evaluation system for Paneth cell granule secretion by using enteroid with lumen microinjection and its application

  米田司, 中村公則, 横井友樹, 大平修也, 菊池摩仁, 櫻木直也, 綾部時芳

  第40回 日本分子生物学会年会  2017/12
• 高純度ソーティング由来cDNAライブラリーを用いたPaneth細胞の新規機能探索

  櫻木 直也, 中村 公則, 横井 友樹, 菊池 摩仁, 綾部 時芳

  第13回 日本食品免疫学会学術大会  2017/11
• 腸内環境を制御する抗菌ペプチドα-defensinと疾患  [Invited]

  中村公則

  日本薬局協励会北海道合同支部「学術繋栄講座」  2017/07
• Mutation analysis of cryptdin-4, antimicrobial peptide from mouse  [Not invited]

  平峰里菜, 久米田博之, 久米田博之, 熊木康裕, 菊川峰志, 菊川峰志, 出村誠, 出村誠, 中村公則, 綾部時芳, 相沢智康

  日本農芸化学会 2017年度大会  2017/03
• Enteroidを用いたPaneth細胞顆粒分泌反応の可視化解析による機序解明

  横井友樹, 中村公則, 櫻木直也, 菊池摩仁, 綾部時芳

  第12回 日本食品免疫学会学術大会  2016/11
• Enteroidを用いたPaneth細胞の機能可視化～顆粒分泌･再形成および免疫系クロストーク

  綾部時芳, 横井友樹, 櫻木直也, 中村公則

  日本農芸化学会 2016年度大会  2016/03
• アミノ酸がパネト細胞αディフェンシンに与える影響

  高桑暁子, 中村公則, 櫻木直也, 綾部時芳

  日本農芸化学会 2016年度大会  2016/03
• 腸からみれば！ 食品と免疫と腸内細菌がつくる“腸内環境”の解明と実用化

  綾部時芳, 櫻木直也, 中村公則

  日本農芸化学会 2016年度大会  2016/03
• クローン病モデルマウスSAMP1/YitFcの病態形成におけるα-defensinの関与

  吉井彩季, 中村公則, 櫻木直也, 綾部時芳

  第38回 日本分子生物学会年会  2015/12
• 腸管星細胞株を用いた腸管線維化機序の解析

  菅原徹也, 中村公則, 櫻木直也, 綾部時芳

  第38回 日本分子生物学会年会  2015/12
• 放射線腸炎の指標としての便中αディフェンシンの可能性～マウスでの研究結果から～

  小野寺俊輔, 中村公則, 西山典明, 綾部時芳, 白𡈽博樹

  第133回 日本医学放射線学会北日本地方会・第78回 日本核医学会北日本地方会  2015/10
• 大麦若葉末がαディフェンシン分泌に与える影響

  森川琢海, 北村整一, 鍔田仁人, 山口和也, 高橋欣也, 櫻木直也, 中村公則, 綾部時芳

  日本食品化学工学会 第62回大会  2015/08
• αディフェンシンが規定する新しい腸内環境の可能性  [Invited]

  中村 公則

  第5回 オルソオルガノジェネシス検討会  2015/08
• 成長に伴うマウスの腸内細菌叢や抗菌ペプチドα－ディフェンシンの変化及びその関係

  佐藤拓海, 中村公則, 菅原宏佑, 加藤久美子, 岩淵紀介, 小田巻俊孝, 清水(肖)金忠, 阿部文明, 綾部時芳

  日本乳酸菌学会 2015年度大会  2015/07
• 基質の硬さによって促進される大腸がん細胞の悪性化:転写因子YAPを介した基質分解酵素MMP-7の発現亢進

  温田晃弘, 石原誠一郎, 水谷武臣, 中村公則, 綾部時芳, 川端和重, 芳賀永

  第67回 日本細胞生物学会大会  2015/06
• クローン病モデルマウスSAMP1/YitFcの病態形成におけるα-defensinの関与

  吉井 彩季, 中村 公則, 櫻木 直也, 綾部 時芳

  第19回 腸内細菌学会  2015/06
• αディフェンシンが規定する新しい腸内環境の可能性  [Invited]

  中村 公則

  北海道大学フード＆メディカルイノベーション国際拠点開設記念 食と腸内環境 学術講演会「新しい腸内環境から考える健康」  2015/06
• クローン病モデルマウスSAMP1/Yitの病態進行とα-defensinの分泌異常  [Not invited]

  中村 公則, 櫻木 直也, 綾部 時芳

  第101回 日本消化器病学会総  2015/04
• 組織常在型M2マクロファージの培養系の確立と間葉系細胞への影響の検討

  藤岡明博, 櫻木直也, 中村公則, 古澤和也, 綾部時芳, 佐々木直樹, 福井彰雅

  平成26年度北大細胞生物研究集会  2015/03
• 同位体顕微鏡で小腸の吸収機能を視る

  櫻木直也, 中村公則, 綾部時芳, 永田康祐, 坂本直哉, 圦本尚義

  第6回 安定同位元素イメージング技術による産業イノベーションシンポジウム  2015/03
• 抗菌ペプチドαディフェンシンによる腸内環境の恒常性制御と疾病

  綾部時芳, 櫻木直也, 中村公則

  第20回日本エンドトキシン・自然免疫研究会  2014/12
• 高脂肪食による肥満における抗菌ペプチドα-defensin分泌の関与

  第37回 日本分子生物学会年会  2014/11
• 組織常在型M2マクロファージ細胞の培養系の確立と間葉系細胞への影響について

  藤岡明博, 櫻木直也, 中村公則, 古澤和也, 佐々木直樹, 綾部時芳, 福井彰雅

  第37回 日本分子生物学会年会  2014/11
• Paneth細胞α-defensinによる腸内細菌叢制御と疾患との関連の解明  [Invited]

  中村 公則, 櫻木 直也, 綾部 時芳

  2014年度 生理学研究所研究会 「粘膜免疫学と膜輸送生理学の融合」  2014/10
• 短鎖脂肪酸がパネト細胞αディフェンシン分泌に与える影響

  高桑暁子, 中村公則, 櫻木直也, 綾部時芳

  日本食品免疫学会 第10回学術大会  2014/10
• 大腸がん細胞において基質の硬さはミオシンのリン酸化を通じて基質分解酵素MMP7の発現を亢進する

  温田晃弘, 石原誠一郎, 中村公則, 綾部時芳, 芳賀永

  第73回日本癌学会学術総会  2014/09
• NMR analysis of the oligomerization mechanism of antimicrobial peptide human defensin 5

  Hashimoto Arata, Tomisawa Satoshi, Kamiya Masakatsu, Kikukawa Takashi, Kumaki Yasuhiro, Nakamura Kiminori, Ayabe Tokiyoshi, Aizawa Tomoyasu, Demura Makoto

  第52回 日本生物物理学会年会  2014/09  The Biophysical Society of Japan General Incorporated Association
  
• 腸内細菌を制御するαディフェンシンから疾病を眺める.

  綾部時芳, 櫻木直也, 中村公則

  第51回 日本消化器免疫学会総会  2014/07
• Novel methods for evaluating Paneth cell functions using small intestinal organoids and sandwich ELISA for -defensin in mouse.

  Sasaki K, Nakamura K, Sakuragi N, Ayabe T

  第36回 日本分子生物学会年会  2013/12
• αディフェンシンによる腸内環境の制御 －その破綻は疾病を招くか－

  綾部時芳, 櫻木直也, 中村公則

  第8回遺伝子栄養学研究会学術集会  2013/08
• Development of antifibrotic therapy targeted to stellate cells for intestinal fibrosis in Crohn's disease.

  金野真奈, 中村公則, 中村公則, 丸山景資, 綾部時芳, 綾部時芳

  第35回 日本分子生物学会年会  2012/12
• Deficiency of secreted cryptdin-4 detected in a mouse model of Crohn’s disease using a new sandwich ELISA.  [Not invited]

  Nakamura K, Kuroishi A, Kono M, Kobayashi M, Ayabe T

  Experimental Biology 2012  2012/04  San Diego
• Development of antifibrotic therapy for intestinal fibrosis targeted to stellate cells.

  Yoshioka S, Nakamura K, Maruyama K, Ayabe T

  第34回 日本分子生物学会年会  2011/12
• Foods-induced innate immune responses of Paneth cell -defensin uncover novel functions of foods.

  Ayabe T, Kuroishi A, Kobayashi M, Nakamura K

  International Society for Nutraceuticals and Functional Foods Annual Conference (ISNFF2011).  2011/11
• 佐藤真希、平 敏夫、中村公則、綾部時芳、福井彰雅

  佐藤真希, 平 敏夫, 中村公則, 綾部時芳, 福井彰雅

  日本動物学会第82回大会  2011/09
• Selection and regulation of intestinal microbiota by Paneth cell -defensins.

  Ayabe T, Nakamura K

  XIII International Union of Microbiological Societies 2011 Congress (IUMS2011)  2011/09
• パネト細胞αディフェンシンによる腸内細菌の制御からみた共生の新展開

  綾部時芳, 吉岡佐和子, 小林美智代, 中村公則

  第6回核酸・核タンパク機能性研究会学術集会（恵庭フォーラム）  2011/08
• Isolation of intermediate stellate cells in the mouse small intestine.

  Yoshioka S, Nakamura K, Sakai N, Ayabe T

  第33回 日本分子生物学会年会  2010/12
• Regulation of microbiota by antimicrobial peptides in the gut.

  Ayabe T, Masuda K, Nakamura K, Sakai N

  7th. International Symposium on Tonsil and Mucosal Barriers of the Upper Airways (ISTMB), Symposium-1 (Innate and Adoposal Immunology)  2010/07
• Selective bactericidal activity of the mouse α-defensin, cryptdin-4 against commensal and non-commensal bacteria.

  Masuda K, Sakai N, Nakamura K, Ayabe T

  14th International Congress of Immunology.  2010/07
• Paneth cells and antimicrobial peptides, α-defensins, in enteric innate immunity.

  Ayabe T, Masuda K, Nakamura K, Sakai N

  6th. Annual Meeting of Japanese Association for Food Immunology, Symposium-2 (International Symposium on Dietary modulation of mucosal barrier and infection)  2010/06
• Regulation of intestinal microbiota by Paneth cell α-defensins.

  Ayabe T, Masuda K, Nakamura K, Sakai N

  The83rd Annual Meeting of Japanese Society for Bacteriology, International Symposium  2010/03
• マウス小腸における星細胞の同定

  吉岡佐和子, 中村公則, 坂井直樹, 綾部時芳

  平成21年度 北大細胞生物研究集会  2009/03
• Interleukin-13 Receptor alpha2 Chain メラノーマの新規標的分子としての検討(Interleukin-13 Receptor alpha2 Chain as a Biomarker and Molecular Target for Melanoma)  [Not invited]

  中村 公則, 桜木 直也, 加藤 和則, 濱田 洋文

  日本癌学会総会記事  2008/09  日本癌学会
  
• 前立腺癌に対する遺伝子治療標的としてのNCAM2(Neural cell adhesion molecule 2 as a target molecule for prostate cancer gene therapy)  [Not invited]

  高橋 秀, 加藤 和則, 中村 公則, 濱田 洋文

  日本癌学会総会記事  2008/09  日本癌学会
  
• Z33アデノウイルスと抗MCSP抗体を介したメラノーマに対する遺伝子治療(Targeted gene therapy for melanoma using anti-MCSP antibody and Z33 fiber modified adenovirus vectors)  [Not invited]

  桜木 直也, 中村 公則, 平井 幸恵, 加藤 和則, 濱田 洋文

  日本癌学会総会記事  2008/09  日本癌学会
  
• Bystander effect between mesenchymal stem cells and brain tumor cells in the HSVTK/GCV system is not species specific  [Not invited]

  Hiroshi Kosaka, Tomotsugu Ichikawa, Hirokazu Kambara, Satoshi Inoue, Tomoko Maruo, Kazuhiko Kurozumi, Kimi-nori Nakamura, Hirofumi Hamada, Isao Date

  JOURNAL OF GENE MEDICINE  2008/04  JOHN WILEY & SONS LTD
  
• 前立腺癌遺伝子療法の標的遺伝子の探索(EXPLORATION OF TARGET MOLECULES FOR PROSTATE CANCER GENE THERAPY)  [Not invited]

  中村 公則, 加藤 和則, 鈴木 一弘, 塚本 泰司, 濱田 洋文

  日本癌学会総会記事  2007/08  日本癌学会
  
• スーパー標的抗体によって認識される新規メラノーマ抗原IL-13受容体α2(Super-targeting antibody NS-66 recognizes a novel tumor antigen interleukin-13 receptor alpha 2 on human melamoma)  [Not invited]

  桜木 直也, 中村 公則, 加藤 和則, 濱田 洋文

  日本癌学会総会記事  2007/08  日本癌学会
  
• Superoxide and metastasis  [Not invited]

  Y. Niitsu, K. Kuribayashi, K. Nakamura, M. Tanaka Araki, T. Sato, R. Takimoto, T. Matsunaga, T. Takayama, J. Kato

  CLINICAL & EXPERIMENTAL METASTASIS  2007  SPRINGER
  
• Na,K-ATPase beta 1-Mediated Selective Gene Transfer into Neurons.  [Not invited]

  Keiji Ishii, Kiminori Nakamura, Satoshi Kawaguchi, Rong Li, Sachie Hirai, Naoya Sakuragi, Takuro Wada, Kazunori Kato, Toshihiko Yamashita, Hirofumi Hamada

  MOLECULAR THERAPY  2006/05  NATURE PUBLISHING GROUP
  
• PAP2a-targeted selective gene therapy for pancreatic, prostate, and lung cancers  [Not invited]

  Kiminori Nakamura, Kazunori Kato, Hirofumi Hamada

  JOURNAL OF GENE MEDICINE  2006/03  JOHN WILEY & SONS LTD
  
• Efficient and selective gene delivery of a fiber-modified adenovirus vector to human myeloma with anti-CD38 antibody  [Not invited]

  K Kato, Y Masuta, K Tomihara, K Nakamura, H Hamada

  JOURNAL OF GENE MEDICINE  2006/03  JOHN WILEY & SONS LTD
  
• ErbB2-targeted selective cancer gene therapy through FZ33 fiber-modified adenoviral vectors  [Not invited]

  Kiminori Nakamura, Rong Li, Sachie Hirai, Kazunori Kato, Takashi Masuko, Kazunari K. Yokoyama, Hirofumi Hamada

  JOURNAL OF GENE MEDICINE  2006/03  JOHN WILEY & SONS LTD
  
• Non-cleavable cell surface mutants of CD40 ligand induce immune response and prevent systemic inflammatory reaction  [Not invited]

  K Kato, Y Masuta, K Tornihara, K Nakamura, H Hamada

  JOURNAL OF GENE MEDICINE  2006/03  JOHN WILEY & SONS LTD
  
• Anti-tumor effect against human carcinoma cells by dendritic cells transfected with CD40 ligand  [Not invited]

  K Tomihara, K Kato, Y Masuta, K Nakamura, H Hiratsuka, H Hamada

  JOURNAL OF GENE MEDICINE  2006/03  JOHN WILEY & SONS LTD
  
• 各種IFNを用いた扁平上皮癌細胞に対する形質変化とアポトーシスの誘導

  冨原圭, 加藤和則, 中村公則, 出張裕也, 濱田洋文, 平塚博義

  日本癌学会学術総会記事  2006
• The Journal of Gene Medicine Japanese Society of Gene Therapy Young Investigator Award 2005 - Kazuhiko Kurozumi, Department of Neurological Surgery, Ohio State University Medical Center, USA  [Not invited]

  K Kurozumi, K Nakamura, T Ichikawa, T Tamiya, Y Ito, O Honmou, K Houkin, H Hamada, Date, I

  JOURNAL OF GENE MEDICINE  2005/12  JOHN WILEY & SONS LTD
  
• Wnt3 augments adhesion mediated drug resistance of myeloma cells via Wnt/RhoA pathway.  [Not invited]

  H Chiba, M Kobune, K Kato, K Nakamura, Y Kawano, R Takimoto, T Matsunaga, J Kato, Y Niitsu, H Hamada

  BLOOD  2005/11  AMER SOC HEMATOLOGY
  
• ヒト樹状細胞に対するCD40リガンド遺伝子導入による抗腫瘍効果の検討  [Not invited]

  冨原 圭, 加藤 和則, 増田 ゆかり, 中村 公則, 野口 誠, 平塚 博義, 濱田 洋文

  日本癌学会総会記事  2005/09  日本癌学会
  
• 抗CD38抗体とファイバー変異型アデノウイルスを用いたヒト骨髄腫細胞への標的化遺伝子導入  [Not invited]

  増田 ゆかり, 加藤 和則, 冨原 圭, 中村 公則, 濱田 洋文

  日本癌学会総会記事  2005/09  日本癌学会
  
• Wnt3はnon-canonical経路を介して多発性骨髄腫の抗癌剤耐性を増強させる  [Not invited]

  千葉 大樹, 小船 雅義, 河野 豊, 中村 公則, 加藤 和則, 瀧本 理修, 松永 卓也, 加藤 淳二, 濱田 洋文, 新津 洋司郎

  日本癌学会総会記事  2005/09  日本癌学会
  
• Wnt3/Rho経路は多発性骨髄腫における骨髄間質依存性の抗癌剤耐性を惹起する分子機構の一つである  [Not invited]

  千葉 大樹, 小船 雅義, 河野 豊, 中村 公則, 加藤 和則, 瀧本 理修, 松永 卓也, 加藤 淳二, 濱田 洋文, 新津 洋司郎

  日本血液学会・日本臨床血液学会総会プログラム・抄録集  2005/09  日本臨床血液学会
  
• Gene-modified mesenchymal stem cells (MSCS) as a therapeutic tool for malignant brain neoplasms  [Not invited]

  H Hamada, K Nakamura, K Kato

  CANCER GENE THERAPY  2004/12  NATURE PUBLISHING GROUP
  
• Selective and efficient gene delivery of CD40-ligand by a fiber-modified adenovirus vector with specific antibody to human leukemia and myeloma  [Not invited]

  K Kato, S Hirai, Y Masuta, A Kuroishi, K Nakamura, H Hamada

  BLOOD  2004/11  AMER SOC HEMATOLOGY
  
• Sendai virus vector can efficiently introduce transgene into cardiomyocytes in vitro and in vivo  [Not invited]

  Y Honma, Y Ito, H Dehari, M Kobune, K Nakamura, J Huang, T Uzuka, T Kobayashi, M Morikawa, M Inoue, M Hasegawa, N Tohse, Y Niitsu, T Abe, H Hamada

  GENE THERAPY  2004/10  NATURE PUBLISHING GROUP
  
• α-GalactosylceramideとアデノIL-2遺伝子治療の併用による抗腫瘍効果および抗転移効果増強の検討  [Not invited]

  西堀 佳樹, 古川 勝久, 加藤 和則, 田中 真樹, 岡本 哲朗, 竹内 直子, 萩原 誠也, 栗林 景晶, 中村 公則, 新津 洋司郎

  基盤的癌免疫研究会総会抄録  2004/07  日本がん免疫学会
  
• BDNF gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebral artery occlusion model (vol 9, pg 189, 2004)  [Not invited]

  K Kurozumi, K Nakamura, T Tamiya, Y Kawano, M Kobune, S Hirai, H Uchida, K Sasaki, Y Ito, K Kato, O Honmou, K Houkin, Date, I, H Hamada

  MOLECULAR THERAPY  2004/05  ACADEMIC PRESS INC ELSEVIER SCIENCE
  
• ヒト骨髄間葉系幹細胞を用いたヒト肝細胞への分化誘導  [Not invited]

  荒木 啓伸, 佐藤 康史, 加藤 淳二, 中村 公則, 河野 豊, 佐藤 勉, 宮西 浩嗣, 高橋 稔, 濱田 洋文, 新津 洋司郎

  肝臓  2004/04  (一社)日本肝臓学会
  
• Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID-repopulating cells.  [Not invited]

  M Kobune, Y Ito, Y Kawano, K Sasaki, H Uchida, K Nakamura, H Dehari, H Chiba, R Takimoto, T Matsunaga, T Terui, J Kato, H Hamada, Y Niitsu

  BLOOD  2003/11  AMER SOC HEMATOLOGY
  
• Wnt3 differentially regulates growth and hematopoietic support in mesenchymal and stromal cells.  [Not invited]

  H Chiba, M Kobune, Y Ito, Y Kawano, K Sasaki, K Nakamura, H Dehari, T Matsunaga, J Kato, H Hamada, Y Niitsu

  BLOOD  2003/11  AMER SOC HEMATOLOGY
  
• ヒト・ストローマ及び間葉系幹細胞におけるモルフォーゲン遺伝子の発現を応用した造血幹細胞増幅の試み  [Not invited]

  小船 雅義, 千葉 大樹, 河野 豊, 伊藤 克礼, 中村 公則, 出張 裕也, 松永 卓也, 加藤 淳二, 濱田 洋文, 新津 洋司郎

  炎症・再生  2003/11  (一社)日本炎症・再生医学会
  
• 間葉系幹細胞(MSC)を用いた浸潤性グリオーマ遺伝子治療の開発  [Not invited]

  中村 公則, 伊藤 克礼, 河野 豊, 黒住 和彦, 佐々木 勝則, 備前 明子, 本望 修, 宝金 清博, 濱田 洋文

  日本癌学会総会記事  2003/08  日本癌学会
  
• 不死化ヒト・ストローマ及び間葉系幹細胞におけるWnt遺伝子の発現とその造血支持能に対する効果  [Not invited]

  千葉 大樹, 小船 雅義, 河野 豊, 伊藤 克礼, 中村 公則, 出張 裕也, 内田 宏昭, 松永 卓也, 加藤 淳二, 濱田 洋文, 新津 洋司郎

  臨床血液  2003/08  (一社)日本血液学会-東京事務局
  
• テロメレース(TERT)遺伝子導入・間葉系幹細胞(MSC)のcobblestone area(CA)支持細胞への分化  [Not invited]

  小船 雅義, 河野 豊, 伊藤 克礼, 千葉 大樹, 中村 公則, 佐々木 勝則, 津田 肇, 出張 裕也, 内田 宏昭, 松永 卓也, 加藤 淳二, 新津 洋司郎, 濱田 洋文

  臨床血液  2003/08  (一社)日本血液学会-東京事務局
  
• Adenoviral Bcl-xL gene trasfer reduces the infarct size and preserves the cardiac function after ischemia and reperfusion in the rat heart  [Not invited]

  JH Huang, Y Ito, M Morikawa, M Kobune, H Uchida, K Nakamura, H Dehari, K Takahashi, T Abe, H Hamada

  MOLECULAR THERAPY  2003/05  ACADEMIC PRESS INC ELSEVIER SCIENCE
  
• Human stromal cells transfected with TERT (Telomerase catalytic subunit) gene supports proliferation of SRCs (Scid repopulating cells).  [Not invited]

  Y Kawano, M Kobune, M Yamaguchi, K Nakamura, Y Ito, T Matsunaga, H Chiba, S Takahashi, K Sasaki, K Kato, J Kato, H Azuma, H Ikeda, Y Niitsu, H Hamada

  BLOOD  2002/11  AMER SOC HEMATOLOGY
  
• Human stromal cells transfected with TERT (telomerasae catalytic subunit) gene supports proliferation of hematopoietic progenitor cells.  [Not invited]

  Y Kawano, M Kobune, M Yamaguchi, K Nakamura, Y Ito, K Sasaki, T Matsunaga, S Sakamaki, J Kato, S Hirai, K Ikebuchi, Y Niitsu, H Hamada

  BLOOD  2001/11  AMER SOC HEMATOLOGY
  
• 口腔扁平上皮癌細胞SASはHGF/SFを自己分泌し浸潤能を促進する

  奥村 一彦, 萩野 司, 中村 公則, 田中 真樹, 金澤 正昭

  千葉医学雑誌  2000/04  千葉医学会
  
• TNFのアポトーシスシグナルにおけるcaspaseとoxygen free radical(OFR)の関連

  荻野 司, 奥村 一彦, 中村 公則, 茂尾 公晴, 山内 尚文, 佐藤 勉, 井上 諭, 田中 真樹, 新津 洋司郎, 金澤 正昭

  日本口腔外科学会雑誌  1999/12  (公社)日本口腔外科学会
  
• 浸潤,転移マーカーとしての細胞内SOD

  中村 公則, 田中 真樹, 萩野 司, 奥村 一彦, 金澤 正昭

  第53回日本口腔科学会総会・学術大会  1999/11  (NPO)日本口腔科学会
  
• 高浸潤性舌扁平上皮癌細胞株SAS-H1の血清刺激によるPI3K活性化を介した細胞接着と細胞運動の検討

  奥村 一彦, 小西 亮, 山下 知巳, 小村 健, 田中 真樹, 萩野 司, 中村 公則, 金澤 正昭

  日本口腔外科学会雑誌  1999/11  (公社)日本口腔外科学会
   

  浸潤性の高い口腔扁平上皮癌細胞は,接着や細胞運動性のシグナルとして脂質キナーゼであるPI3Kが重要な役割を担っており,PI3Kの活性化を介して接着斑におけるFAKのチロシンリン酸化を亢進することで細胞接着性を発現して,引き続いて生じる細胞運動を制御していると推察された
• Superoxideによるマウスマクロファージ細胞株J774.1の運動能亢進はPKCを介する

  中村 公則, 田中 真樹, 萩野 司, 奥村 一彦, 金澤 正昭

  日本口腔科学会雑誌  1999/01  (NPO)日本口腔科学会
  
• 細胞内TNFは口腔癌細胞のTNFアポトーシスシグナルに対し抵抗性因子として働いている

  荻野 司, 奥村 一彦, 中村 公則, 田中 真樹, 金澤 正昭

  日本口腔外科学会雑誌  1998/12  (公社)日本口腔外科学会
  
• 血清刺激による舌扁平上皮癌細胞SASの運動促進シグナルはPI3-K活性化を介している

  奥村 一彦, 田中 真樹, 中村 公則, 萩野 司, 金澤 正昭

  日本口腔科学会雑誌  1998/12  (NPO)日本口腔科学会
  
• 舌扁平上皮癌SASのPI13 Kinaseを介するFAKのチロシンリン酸化と運動促進

  奥村 一彦, 中村 公則, 田中 真樹, 荻野 司, 金澤 正昭

  第955回千葉医学会例会・第18回歯科口腔外科例会  1998/08  千葉医学会
  
• Superoxideによるマウス・マクロファージ細胞株J774.1の運動能亢進はPKCを介する

  中村 公則, 田中 真樹, 萩野 司, 奥村 一彦, 金澤 正昭

  東日本歯学会第16回学術大会（平成10年度総会）  1998/06  北海道医療大学歯学会
  

Association Memberships

• 日本食品免疫学会   THE JAPANESE SOCIETY FOR IMMUNOLOGY   JAPANESE SOCIETY OF GASTROENTEROLOGY   THE JAPANESE CANCER ASSOCIATION   THE MOLECULAR BIOLOGY SOCIETY OF JAPAN   腸内細菌学会   

Research Projects

• 母乳オリゴ糖の腸管上皮幹細胞ニッシェを介した腸内環境発達メカニズムの解明

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)

  Date (from‐to) : 2022/06 -2024/03 

  Author : 中村 公則
• PHRを活用した機械学習モデルによる心血管病の重症化予防を目指した研究

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  Date (from‐to) : 2021/04 -2024/03 

  Author : 横田 卓, 中岡 慎治, 中村 公則

   

  わが国は超高齢社会を迎え、心血管病の患者数は増加の一途を辿っており、早急に患者本人が主体的にセルフモニタリングを行い疾病予防に取り組む「患者中心の医療 (patient-centered care)」 を実現する必要に迫られている。心血管病の予防・治療の基本は食事・運動をはじめとする生活習慣の是正であるが、在宅で取得可能なパーソナル・ヘルス・レコード (PHR) の活用が必要不可欠である。さらにCOVID-19感染拡大をきっかけにオンライン診療が広く推奨されるようになり、とりわけ情報通信技術 (ICT) を活用したPHRのニーズが高まっている。そこで我々は、在宅で取得するPHRを活用し適切なセルフケアの実践を促すスマートフォン対応セルフケアサポートアプリを開発した。本研究の目的は、このアプリで収集する血圧・体重・体脂肪率・体温・酸素飽和度・塩分摂取量・身体活動量・睡眠時間などのバイタルサインや食事・運動内容、さらには便を用いた腸内フローラ解析データなどの多様なPHRを活用し、機械学習モデルを用いて、心血管病の重症化予測を行うとともに重症化予防のための個々に最適な食事・運動療法を提案することである。 臨床試験『スマートフォンアプリを活用した統合型高血圧セルフケアサポートシステムの有効性の検証 (AppCare-HT Study)』については、2022年3月までに目標症例数 (360名) に達したため、募集を終了し、現在12か月間のフォローアップを実施中である。また、心不全患者を対象にした臨床試験についても研究実施中で、順次データ解析を進めている。
• Paneth細胞が担う腸内細菌叢の形成からみた疾患リスク上昇メカニズムの理解

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  Date (from‐to) : 2020/04 -2023/03 

  Author : 中村 公則

   

  本研究は、胎児期から乳幼児期における腸内細菌叢制御に関与する因子であるαディフェンシンを分泌するPaneth細胞の発達異常が、腸内細菌叢形成の破綻を誘導し疾患リスク上昇に関与するとの仮説を立て、そのメカニズムを栄養制御モデルマウスや腸内細菌叢破綻モデルを用いて細胞・分子レベルで証明し、Paneth細胞機能の修復による腸内環境制御機構の正常化を標的とした疾患予防・治療戦略を提案することを目的とする。2021年度は、高脂肪食を摂取した母親由来の子マウス群で示されたαディフェンシン低下に伴う腸内細菌叢の破綻が、αディフェンシン低下そのものに起因するのかを明確にするために、遺伝子的にαディフェンシンを低下させた母親マウス（αDLowM）より生まれた子供（αDLowM-C）のαディフェンシン分泌量測定と腸内細菌叢解析および体重測定を行った。αDLowMの腸内細菌叢は、αディフェンシン正常母親マウス（αDNorM）と比較してβ多様性において異なる分布を示し、腸内細菌叢の破綻（dysbiosis）が誘導されていた。αDLowM-Cのαディフェンシン分泌量は、αDNorMから生まれた子（αDNorM-C）と比較して著しく低下していた。また、αDLowM-Cの腸内細菌叢はαDNorM-Cと比較してβ多様性において異なる分布を示すdysbiosisが誘導されていた。αDLowM-Cの体重は6週齢おいてαDNorM-Cと比較して低かった。以上より、母親のαディフェンシン低下による腸内細菌叢破綻は、子のαディフェンシンを低下させdysbiosiを誘導することが示され、さらに子の低体重誘導に関与する可能性を示した。また、Paneth細胞の顆粒分泌機能評価可能なエンテロイド生体外可視化ライブイメージング解析法により、母親のαディフェンシン分泌量を増加させる食成分の候補が複数抽出された。
• Elucidation of homeostatic mechanism via intestinal environment regulated by oral bacteria

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2017/04 -2020/03 

  Author : Nakamura Kiminori

   

  The purpose of this study was to elucidate the mechanism that commensal bacteria including oral bacteria and pathogenic bacteria are involved in the maintenance of intestinal homeostasis via interaction with Paneth cells. In this study, a novel ex vivo evaluation system to visualize and quantify Paneth cell granule secretion was established by microinjection method of bacteria into the lumen of enteroids, three-dimensional cultures of small intestinal epithelial cells. Paneth cells secreted granules immediately when live non-commensal bacterium S. Typhimurium or commensal bacterium B. bifidum was introduced into the enteroid lumen by microinjection, whereas Paneth cells did not secrete granules when live commensal bacterium L. casei was introduced.
• 口腔細菌が規定する腸内環境を介した生体恒常性維持機能の解明

  文部科学省：科学研究費補助金(基盤研究(C))

  Date (from‐to) : 2017 -2019 

  Author : 中村 公則
• Pancreatic-Intestinal Crosstalk: a novel approach to elucidate the tumorigenic process of PDAC

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

  Date (from‐to) : 2015/04 -2018/03 

  Author : ONO Yusuke

   

  The tumorigenic process of pancreatic ductal carcinoma (PDA) may be closely associated with homeostasis of the intestinal tract, e.g., innate immune system. To elucidate the "Pancreatic-Intestinal Crosstalk" during the tumor initiation, we analyzed the alterations in an intestinal environment using a mouse strain which spontaneously develops acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasias (PanINs), offering a model of the early-stage of the pancreatic tumorigenesis. 16S rRNA gene-based metagenomics assay of stool DNA provided a specific microflora profile during pancreatitis-induced progression from ADM into PanIN. Besides, the morphological study demonstrated the close link between the tumorigenic process in the pancreas and the intestinal tract where Paneth cells may play an essential role.
• Elucidation of the interplay between oral bacteria and intestinal innate immunity using Paneth cells in organoid cultures

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2014/04 -2017/03 

  Author : Nakamura Kiminori

   

  To clarify the relationship between oral bacteria and intestinal epithelia cells in terms of innate immunity, the aim of this study was to establish an evaluation system for intestinal innate immunity focused on Paneth cell α-defensin secretory functions.New analysis method for Paneth cell secretory functions using cultured organoids from mouse small intestine and newly developed sandwich ELISA for α-defensin were established. These methods will contribute to clarifying links between oral bacteria and intestinal innate immunity.
• Paneth cells and their networks as headquarters for clarifying food function

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

  Date (from‐to) : 2014/04 -2016/03 

  Author : Sakuragi Naoya, NAKAMURA Kiminori

   

  This study was focused on "How Paneth Cell Network is organized" that may work as headquarters in the intestine. To invistigate this possibility, we established high purity and large quantity Paneth cell purification method, and analyzed membrane and secretion related gene expression in Paneth cells. Total 37 genes were identified and classfied into absorption of nutrition, nervous system, metabolism and so on, in addition to innate immunology, renewal and differentiation. These results suggest that Paneth cells may have many important functions and work as headquarters in the intestine.
• To investigate the clinical application of alpha-defensin in stool in the objective evaluation of radiation enteritis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2013/04 -2016/03 

  Author : Onodera Shunsuke, Nakamura Kiminori

   

  The purpose of this study was to investigate the clinical application of α-defensin in stool in the objective evaluation of radiation enteritis.As the first step, we experimented on mice and found that the amount of α-defensin significantly decreased in stool of mice irradiated to the abdomen.In addition, a significant negative correlation was observed between the amount of α-defensin and Firmicutes at three days after irradiation.These results suggested that decrease of α-defensin correlated with an altered intestinal flora of the mice irradiated to the abdomen.We could not investigate the clinical application due to insufficient number of patients in this period.
• αディフェンシンによる腸内細菌の統御機構からみた炎症性腸疾患の病因解明と新規治療

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  Date (from‐to) : 2011/04 -2014/03 

  Author : 綾部 時芳, 中村 公則

   

  本研究は、小腸のPaneth細胞が分泌するαディフェンシンの作用から炎症性腸疾患の病因・病態を解明して新規治療を提案することが目的である。本年度は、αディフェンシンと腸内細菌からみた殺菌及び共生メカニズムを、cryptdin およびHD5と腸内細菌との会合から解析した。Acid-Urea PAGE、western blot法、sandwich ELISA等を用いた生化学的・免疫学的解析および殺菌活性測定によって、腸内常在細菌であるLactobacillus、Bifidobacterium等と、非常在菌（病原菌）であるSalmonella、Staphylococcus等でαディフェンシンとの会合状態が異なることを示した。また、クローン病モデルマウスを用いてαディフェンシン異常による腸内共生環境の破壊 (dysbiosis)の可能性について解析した。クローン病モデルマウスのパネト細胞顆粒のcryptdinおよびcryptdin分泌量についてRP-HPLC、Tris-Tricine SDS-PAGE、Acid Urea-PAGE、westen blot法およびsandwich ELISAで解析・測定して、病理病態進展におけるαディフェンシン異常の関与を明らかにした。さらに、小腸絨毛･陰窩におけるcryptdin、MMP7、CD24等の免疫局在を解析し、幹細胞とPaneth細胞が形成するニッチ相互作用の可能性を示した。以上のように、αディフェンシンの病態関与から、クローン病の新規治療法に繋がる重要な成果を得た。本研究で得られた新知見から、αディフェンシン異常によるdysbiosisがクローン病以外にも多くの疾患に寄与すると考え、実際に移植片対宿主病 (GVHD)における病態関与を証明した。
• Effect of oral bacteria on intestinal innate immunity

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2010 -2012 

  Author : NAKAMURA Kiminori

   

  To analyze the interaction of oral bacteria and intestinal epithelia cells, the aim of this study is to establish a intestinal epithelial cell line containing Paneth cells which secrete α -defensins. In conventional monolayer cell culture, intestinal epithelial cells were observed, however, Paneth cells were not identified. Next, organoid culture were performed by using mouse intestinal crypts. It was analyzed whetherobtained organoids were maintaining the function of a Paneth cell.
• 口腔癌の分子標的化よる新規治療法・診断法の開発

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  Date (from‐to) : 2008 -2010 

  Author : 中村 公則

   

  口腔癌の標的化を月指した診断・治療法の開発を目的として、新たな口腔癌細胞に特異的な表面分子の同定するために、ファイバー改変型アデノウイルスAdv-FZ33と癌細胞に対する抗体ライブラリーを用いて、癌細胞に特異的な細胞表面分子をスクリーニング・同定と同時に高性能なモノクローナル抗体の樹立を実施した。抗体ライブラリーの作製:口腔癌細胞(ヒトHSC-2、HSC-3、OSC-70などの混合)でBa1b/cマウスを腹腔へ免役し、P3U1ミエローマ細胞と免疫マウスの脾細胞とをPEG法により細胞融合させ、腫瘍に対する抗体を産生するハイブリドーマ・ライブラリーを作製した。細胞融合後、抗体を含有する培養上清を回収し、以下のスクリーニングを行った。スクリーニングとハイブリドーマ・クローンの樹立:Adv-FZ33を用いて、抗体と腫瘍細胞と架橋することで、遺伝子導入効率が高まるような抗体のスクリーニングを行った。方法:96wellに播種した標的細胞(口腔癌細胞株)に抗体(ハイブリドーマの上清)を反応させる、その後レポーター遺伝子LacZを発現するAx3CAZ3-FZ33を感染させる。24時間後に、Chemiluminescent β-Ga1レポーター遺伝子発現アッセイを行い、LacZ遺伝子産物の発現量を評価する。コントロールに比べて100倍程度、遺伝子導入発現効率が高まっているクローンを選別する。アッセイは3回行い、擬陽性のクローンは除いた。単一のハイブリドーマから単一の抗体産生クローンを樹立するため、96wellを用いたハイブリドーマ細胞の限界希釈クローニングを行った。結果、Z33-Advを介した遺伝子導入が高効率なモノクローナル抗体を12種類樹立する事が出来た。今後、得られた抗体の抗原同定を行って行く。
• Selective targeted drug and gene delivery for cancer treatment

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas

  Date (from‐to) : 2005 -2009 

  Author : HAMADA Hirohumi, KATO Kazunori, NAKAMURA Kiminori, HAMA Susumu, YAMAGUCHI Miki

   

  In order to systematically search for the target molecules for cancer gene therapy, we generated a fiber-modified recombinant adenovirus, Adv-FZ33, which contains a Z33 domain with a high affinity for immunoglobulin Fc. By screening with Adv-FZ33, we established a large number of high affinity targeting antibodies (Ab), and identified their target antigen (Ag) molecules, e.g., a panel of 60 Ag species. During these efforts, we also established a unique method (EZiTox) to generate immunotoxins (iTox), which consists of the targeting Ab conjugated with ribosome-inactivating enzyme toxins. EZiTox method will be a strong screening method to find a powerful combination of Ag/Ab for cancer iTox-chemotherapy.
• 腫瘍の特異的標的化を目指した遺伝子治療法の開発

  日本学術振興会：科学研究費助成事業 特定領域研究

  Date (from‐to) : 2000 -2004 

  Author : 濱田 洋文, 加藤 和則, 中村 公則, 本望 修, 伊藤 克礼, 佐々木 勝則

   

  腫瘍に対する選択的標的化の候補分子を探索するために、抗体のFcドメインに結合するProtein AのZ33モチーフをAd5ファイバーのHIループに持つAdv-FZ33アデノウイルスを作成した。CARをほとんど発現しないヒト膵癌細胞AsPc1やヒトメラノーマ細胞A375に対する遺伝子導入効率をEGFPないしb-gal遺伝子発現で測定した。AsPc1やA375に発現する表面分子(CD29、CD54など)に対する抗体を付着させたAdv-FZ33による遺伝子導入・遺伝子発現は、コントロール(抗体の非存在下またはコントロールIgG併用でのAdv-FZ33、ならびに野生型Ad5ファイバーAdv-Fwtのウイルス)による遺伝子導入・発現の数十倍に増強できた。また、ErbB2を高発現するヒト卵巣癌細胞(SK-OV3など)への遺伝子導入は、ErbB2抗体の併用により、選択的に著明に(EGFP遺伝子導入細胞%で、5%から90%へ)増強できた。腫瘍細胞とZ33アデノウイルスとを架橋することによって遺伝子導入効率が高まるモノクローナル抗体をスクリーニングすることにより、腫瘍細胞に対して標的化の可能な表面分子と抗体の組み合わせの探索を開始している。先行しているヒト膵癌を標的化できる新規抗体作製のプロジェクトでは、すでに4種類の抗体産生ハイブリドーマが樹立されている。このうちの一つクローンA^*によって得られるモノクローナル抗体は、AdvFZ33アデノウイルスの膵癌細胞AsPc1への遺伝子導入効率を、非常に強く(200倍)増強し、有望である。
• 口腔癌の分子標的化よる新規治療法・診断法の開発

  文部科学省：科学研究費補助金(基盤研究(C))

  Author : 中村 公則

Industrial Property Rights

• 特開2021-117062:ヒトαディフェンシンHD5を検出する方法及びキット、並びにこれらにおいて用いられる抗体  2021/08/10

  綾部 時芳, 中村 公則  国立大学法人北海道大学  202103000796879859
• 特許第6607380号:αディフェンシン分泌促進剤及び抗菌剤  

  森川 琢海, 北村 整一, 鍔田 仁人, 綾部 時芳, 中村 公則  株式会社東洋新薬  201903019006558815
• 特許第6209062号:抗菌性ペプチドの分泌促進剤  

  石川 純嗣, 綾部 時芳, 中村 公則  株式会社ファンケル  201703015437482832
• 特許第5873419号:腸管線維症処置剤  

  綾部 時芳, 中村 公則, 味呑 憲二郎, 田中 康進  日東電工株式会社  201603002689572050
• 特許第5088911号:抗微生物物質産生・分泌促進剤  

  綾部 時芳, 中村 公則, 吉成 篤四郎, 石田 真己  国立大学法人北海道大学, 株式会社スリービー  201303081047870680
• 特開2011-078318:パネト細胞群の製造方法、パネト細胞の抗微生物物質の分泌を促進する対象のスクリーニング方法およびパネト細胞からの分泌物の製造方法  2011/04/21

  綾部 時芳, 坂井 直樹, 中村 公則  国立大学法人北海道大学  201103050366618393
• 特開2010-190572:IL13Ra2に対する抗体およびこれを含む診断・治療薬  2010/09/02

  濱田 洋文, 加藤 和則, 中村 公則  北海道公立大学法人 札幌医科大学  201003007136997115
• 特許第4097041号:PAP2aに対する抗体ならびにその診断的および治療的使用  

  濱田 洋文, 中村 公則, 加藤 和則  北海道公立大学法人 札幌医科大学  201103007632735411
• WO2003-038076:不死化間葉系細胞及びその利用  2003/05/08

  濱田 洋文, 河野 豊, 中村 公則, 小船 雅義, 本望 修, 田之岡 篤, 岡 真一, 佐々木 勝則, 津田 肇, 伊藤 克礼, 加藤 淳二, 松永 卓也, 新津 洋司郎  株式会社レノメディクス研究所, 帝国臓器製薬株式会社, 三井住友海上ケアネット株式会社, 株式会社日立製作所  200903020398824560
• パネト細胞群の製造方法、パネト細胞の抗微生物物質の分泌を促進する対象のスクリーニング方法

Social Contribution

• NoMaps WELLNESS 肥満、うつ、アレルギー…カラダの悩みは”腸内細菌”で解決！？

  Date (from-to) : 2023/09/15

  Role : Appearance

  Sponser, Organizer, Publisher  : NoMaps
• 腸はスゴイ！意外と知らない腸の働き

  Date (from-to) : 2021/12/24

  Role : Lecturer

  Event, Program, Title : 第8回北大道新アカデミー
• 親子で楽しむ科学体験「消化管体験ツアー」

  Date (from-to) : 2015/06

  Role : Advisor 北海道大学フード＆メディカルイノベーション国際拠点 「食と健康」に関する消化管の最新の研究成果を分かりやすく社会に伝えるアウトリーチ活動
• サイエンスパーク 「消化管体験ツアー」

  Date (from-to) : 2012/08

  Role : Advisor

  Sponser, Organizer, Publisher  : 札幌駅前通地下歩行空間

  札幌駅前通地下歩行空間 「食と健康」に関する消化管の最新の研究成果を分かりやすく社会に伝えるアウトリーチ活動
• 科学コミュニケーション 「消化管体験ツアー」

  Date (from-to) : 2012/08

  Role : Advisor

  Sponser, Organizer, Publisher  : 子ども霞ヶ関見学デー（文部科学省）
• 科学であそぼ「おもしろ実験室：消化管体験ツアー」

  Date (from-to) : 2010/03

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道電力

  Schoolchildren 札幌市

Media Coverage

• Factors and cells controlling the gut-brain axis

  Date : 2022/09

  Publisher, broadcasting station: 株式会社ヤクルト本社 広報室 ヘルシスト

  Program, newspaper magazine: HEALTHIST Special Issue in 2022

  Pr
• 「良い腸内細菌」を選び取るカギ？ 注目の免疫物質「αディフェンシン」とは、加齢やうつ状態で減少！ 「脳腸相関」で注目の免疫物質

  Date : 2022/08/30

  Program, newspaper magazine: 日経 Gooday

  知られざる脳と腸の関係、「脳と腸」の驚きの関係！ 腸内環境がストレス耐性や性格に影響！？ Internet
• 「腸の守り神、αディフェンシンって？」

  Date : 2022/08/30

  Program, newspaper magazine: マガジンハウス Tarzan

  「食物繊維で、腸を整える!」 Paper
• 「脳と腸」が相互に作用し合う「脳腸相関」のメカニズム

  Date : 2022/08/25

  Publisher, broadcasting station: 日経 Gooday

  知られざる脳と腸の関係、「脳と腸」の驚きの関係！ 腸内環境がストレス耐性や性格に影響！？ Internet
• 健常な腸内細菌叢を保つことで「うつ病」などの改善に繋げる

  Date : 2022/02/14

  Publisher, broadcasting station: 小学館

  Program, newspaper magazine: 週刊ポスト

  Paper
• 病気の予防に繋がる自然免疫成分「αディフェンシン」を発見

  Date : 2022/02/07

  Publisher, broadcasting station: 小学館

  Program, newspaper magazine: 週刊ポスト

  Paper
• カオスな腸内細菌叢を新たな指標「αディフェンシン」で制御する

  Date : 2022/01/05

  Publisher, broadcasting station: 日経 Beyond Health

  Internet
• Factors and cells controlling the gut–brain axis

  Date : 2021/11/10

  Publisher, broadcasting station: 株式会社ヤクルト本社 広報室

  Program, newspaper magazine: HEALTHIST No.270 (Published November 10, 2021)

  Pr
• 鬱病発症と腸内環境の乱れの深い関係

  Date : 2021/05/22

  Program, newspaper magazine: 産経新聞 びっくりサイエンス

  「腸はスゴイ！意外と知らない腸の働き」 Internet