Publications (8)16.48 Total impact
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Article: Involvement of Toll-like receptor 2 in apoptosis of Aggregatibacter actinomycetemcomitans-infected THP-1 cells.
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ABSTRACT: BACKGROUND/PURPOSE: Aggregatibacter (Actinobacillus) actinomycetemcomitans is a gram-negative bacterium that has been associated with aggressive periodontitis. A actinomycetemcomitans infection induces apoptosis in the human monocytic cell line THP-1, and p38 mitogen-activated protein kinase (p38) activity and tumor necrosis factor-α (TNF-α) production are enhanced by A actinomycetemcomitans infection. However, mechanisms governing the recognition of A actinomycetemcomitans by monocytes during apoptosis have not been elucidated. A actinomycetemcomitans cell wall components stimulate Toll-like receptor (TLR)2 and/or TLR4. The authors examined whether TLR2 and/or TLR4 were involved in the apoptosis of A actinomycetemcomitans-infected THP-1 cells. METHODS: A actinomycetemcomitans-infected THP-1 cells were transferred to six-well culture plates and incubated for 0 to 6 hours. In some experiments, THP-1 cells were incubated with anti-TLR2, anti-TLR4, or isotype control antibody (10 μg/mL) for 30 minutes prior to A actinomycetemcomitans infection. Expression of messenger RNA (mRNA) was examined by reverse transcription-polymerase chain reaction. Intracellular bacteria recovered from A actinomycetemcomitans-infected cells and apoptotic cells were detected by APOPercentage dye (Biocolor Ltd, Northern Ireland, UK) staining. Cellular p38 activity and TNF-α production were assessed with enzyme-linked immunosorbent assay. RESULTS: The expression of TLR2 mRNA was increased by A actinomycetemcomitans infection. Phagocytosis and apoptosis in A actinomycetemcomitans-infected THP-1 cells were inhibited by the addition of anti-TLR2 antibody. Also, anti-TLR2 antibody suppressed the activation of p38 and production of TNF-α in A actinomycetemcomitans-infected THP-1 cells. CONCLUSION: These results suggest that A actinomycetemcomitans induces increased expression of TLR2, leading to phagocytosis and apoptosis of THP-1 cells via p38 activation and TNF-α production.Journal of microbiology, immunology, and infection = Wei mian yu gan ran za zhi 04/2012; · 0.99 Impact Factor -
Article: Actinobacillus actinomycetemcomitans induces apoptosis in human monocytic THP-1 cells.
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ABSTRACT: It has previously been reported that the murine macrophage cell line J774.1 and the human oral epithelial cell line KB undergo apoptosis as a result of Actinobacillus actinomycetemcomitans infection. Recent studies have demonstrated that apoptosis regulation is modulated by multiple phosphorylation of several different protein kinases, including the major subtypes of the mitogen-activated protein kinase (MAPK) family. The MAPK family promotes cell survival and/or proliferation in response to growth factor stimulation, or apoptosis in response to various stress stimuli. The primary objective of the present investigation was to clarify whether human immune cells undergo apoptosis following A. actinomycetemcomitans infection and, if so, to establish the involvement of the MAPK family. Human monocytic THP-1 cells were infected with A. actinomycetemcomitans in microtubes. Lactate dehydrogenase release into the culture supernatant and DNA fragmentation in the cells were monitored. DNA fragmentation was also identified by agarose gel electrophoresis. Cell death following A. actinomycetemcomitans infection occurred by apoptosis, shown by an increase in the proportion of fragmented DNA and the typical ladder pattern of DNA fragmentation indicative of apoptosis. Furthermore, p38 MAPK activity and tumour necrosis factor alpha (TNF-alpha) levels increased following A. actinomycetemcomitans infection. In contrast, cell death and TNF-alpha levels in infected cells decreased upon addition of a p38 inhibitor or an anti-TNF-alpha antibody. However, exogenous TNF-alpha could not induce apoptosis in uninfected THP-1 cells. Interestingly, p38 MAPK activity diminished in the presence of anti-TNF-alpha antibody. These findings indicated that A. actinomycetemcomitans infection induces apoptosis in THP-1 cells and that p38 MAPK activity is directly involved in apoptosis. TNF-alpha may play an indirect role in apoptosis via enhanced p38 MAPK activity. A. actinomycetemcomitans-induced apoptosis of human immune cells may be important in terms of initiation and progression of periodontal diseases.Journal of Medical Microbiology 04/2005; 54(Pt 3):293-8. · 2.50 Impact Factor -
Article: Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin.
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ABSTRACT: Outer membrane derived vesicles (MVs) secreted by Actinobacillus actinomycetemcomitans JP2 contain a membranolytic leukotoxin and are toxic to human HL60 cells. To determine how MVs interact with human target cells, HL60 cells were incubated with vesicles, reacted with anti-vesicle antibodies and a FITC-labelled reporter, and visualized by confocal scanning laser microscopy. Target cells rapidly became reactive with anti-vesicle antibodies upon exposure to vesicles. Confocal microscopy showed that labelling occurred primarily in the cytoplasmic membrane and that very little internal fluorescence was observed. The cytoplasmic membrane of HL60 cells was also strongly labelled after exposure to MVs that contained the fluorescent phospholipid, SP-DiOC18. In contrast, incubation of cells with free SP-DiOC18 resulted primarily in the labelling of internal structures of HL60 cells. These results suggest that A. actinomycetemcomitans MVs associate with, or are incorporated into the cytoplasmic membrane of HL60 cells. The leukotoxin is a membranolytic cytotoxin and cells exposed to MVs were lysed by vesicle-associated toxin in a time and dose-dependent manner. However, cells became reactive with anti-vesicle antibodies when MVs were added in the presence of inhibitors of leukotoxin-mediated lysis or when sublytic doses of MVs were analysed. In addition, MVs produced by an isogenic leukotoxin-deficient strain of A. actinomycetemcomitans JP2 were non-toxic but rapidly interacted with HL60 cells. These results suggest that A. actinomycetemcomitans MVs can deliver leukotoxin to HL60 cells but that the association of vesicles with the cytoplasmic membrane occurs independently of the leukotoxin polypeptide.Cellular Microbiology 03/2003; 5(2):111-21. · 5.46 Impact Factor -
Article: Nitric oxide-mediated protection of A. actinomycetemcomitans-infected murine macrophages against apoptosis.
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ABSTRACT: We investigated apoptotic cell death in murine macrophage cell line J774.1 following Actinobacillus actinomycetemcomitans infection. Infected macrophages generally kill bacteria within phagosomes with nitric oxide (NO). Our previous study demonstrated that DNA fragmentation in infected cells increased significantly on addition of S-Methylisothiourea (SMT), a selective inhibitor of inducible NO synthetase (iNOS). The purpose of the present study was to determine the mechanism via which NO affects apoptosis of infected macrophages. J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1. The infected cells were then cultured in the presence or absence of SMT (400 microM). Culture supernatant was removed 21 h after the infection to measure LDH activity. Additionally, cellular proteins were extracted from the infected cells and measured for histone-associated DNA fragmentation and caspase-1, -3, -5, -6, -8, -9 activities. LDH activity and DNA fragmentation were significantly elevated by the infection; moreover, levels increased further on addition of SMT. Caspase activity of infected cells, particularly caspase-3, was significantly higher than that of uninfected cells. Furthermore, caspase activity increased on addition of SMT. These findings indicate that NO protects infected J774.1 cells, at least in part, against apoptotic cell death via a decrease in caspase activity.Nitric Oxide 03/2002; 6(1):61-8. · 3.55 Impact Factor -
Article: Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin.
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ABSTRACT: Actinobacillus actinomycetemcomitans is associated with early onset periodontal diseases and secretes membranous vesicles that appear to contain several virulence-associated proteins. However, the composition of these vesicles and the process leading to their secretion are not well defined. Electron micrographs of thin sectioned bacterial cells and purified vesicle preparations showed that vesicles are spherical lipid bilayers, 50-100 nm in diameter, that appear to form by budding from the outer membrane of the bacterium. Thin layer chromatography identified the predominant lipid components of vesicles as lipopolysaccharide, phosphatidylethanolamine and cardiolipin, similar to the main lipid constituents of the outer membrane. However, vesicles also contained minor lipids that were not detected in outer membrane samples. The major protein constituents of vesicles co-migrated with proteins in outer membrane extracts of A. actinomycetemcomitans, but the outer membrane preparations possessed polypeptides that were not detected in vesicles. Three vesicle proteins were identified; the heat-modifiable OmpA homologue of A. actinomycetemcomitans, a 28 kDa lipoprotein related to the major outer membrane lipoprotein of Mannheimia haemolytica and leukotoxin. Incubation of leukotoxin-sensitive human HL60 cells with vesicles from A. actinomycetemcomitans strains JP2 and 652 resulted in cell lysis, indicating that vesicle-associated leukotoxin is biologically active. Vesicles from the highly leukotoxic strain JP2 were five- to 10-fold more toxic than vesicles from the minimally leukotoxic 652 strain. Furthermore, the specific leukotoxic activity of JP2 vesicles was approximately four- to five-fold higher than isolated outer membrane preparations from JP2, suggesting that vesicles are enriched in leukotoxin. Together, these results suggest that the formation of A. actinomycetemcomitans vesicles occurs by a process that results in the enrichment of leukotoxin.Microbial Pathogenesis 02/2002; 32(1):1-13. · 1.94 Impact Factor -
Article: Possible involvement of protein kinase C in apoptotic cell death of macrophages infected with Actinobacillus actinomycetemcomitans
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ABSTRACT: We have previously reported the evidence for apoptosis in the mouse macrophage cell line J774.1 by the periodontopathic bacterium Actinobacillus actinomycetemcomitans. In this study, we examined the role of protein kinases in the induction of apoptosis in A. actinomycetemcomitans-infected J774.1 cells by the MTT assay, fluorescence microscopy and flow cytometric analysis. After J774.1 cells were precultured with protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), J774.1 cells infected with A. actinomycetemcomitans showed the increased percentage of apoptotic cells. On the contrary, protein kinase A (PKA) activators, such as forskolin and dibutyryl cAMP, do not mimic the effect of PMA. PKC inhibitors, such as staurosporine, calphostin C, chelerythrine chloride, and H7 were found to suppress apoptotic cell death in J774.1 cells infected with A. actinomycetemcomitans. However, HA1004, known as PKA inhibitor, had no effect on apoptosis in infected macrophages. The results presented here suggest that the signals through PKC may play crucial roles in the modulation of apoptosis in macrophages infected with A. actinomycetemcomitans.FEMS Microbiology Letters 01/1998; 159(2):247 - 254. · 2.04 Impact Factor -
Article: 電子図書館プロジェクトを中心とする協働体制の構築とDilins紹介 (デジタルライブラリー紹介)
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Article: C-DATS・電子図書館プロジェクト : Dilinsにおける史資料の多言語目録構築とデジタル化を中心に (<企画>国際シンポジウム : アジア・アフリカ史資料学の現在と地域文化研究)
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Institutions
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1998–2012
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Health Sciences University of Hokkaido
- School of Dentistry
Ishikari, Hokkaido, Japan
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2003
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Hospital of the University of Pennsylvania
- Department of Biochemistry and Biophysics
Philadelphia, PA, USA
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