研究者詳細 - 髙柴　正悟

Antibody responses against Porphyromonas gingivalis infection in patients with early-onset periodontitis 査読国際誌

Shujuan Guo, Keiso Takahashi, Susumu Kokeguchi, Shogo Takashiba, Denis F. Kinane, Yoji Murayama

Journal of Clinical Periodontology 27 ( 10 ) 769 - 777 2000年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

Background, aims: The aim of this study was to evaluate antibody responses against Porphyromonas gingivalis (P. gingivalis) infection in early-onset periodontitis (EOP) patients to elucidate further the host-parasite interactions in the pathogenesis of EOP.
Method: 16 P. gingivalis-infected EOP and 20 adult periodontitis (AP) patients, and 18 periodontally healthy subjects (HS) participated in this study. Serum immunoglobulin G (IgG) antibody levels and avidities against extracted P. gingivalis whole cells were measured. The components of P. gingivalis outer membrane antigens (OMA) reacting to patients' sera were analysed from the molecular weights by Western blotting. Serum antibody levels against P. gingivalis lipopolysaccharide (LPS) were also measured. The ability of the patients' sera to block interleukin-1 beta (IL-1 beta) production by human mononuclear cells in response to P. gingivalis LPS was examined.
Results: Antibody levels were positively correlated with antibody avidities in both EOP and AP patients (r=0.91, r=0.72, p<0.0005, respectively), while not significantly so in HS (r=0.09). There was variability in the antigen recognition of P. gingivalis OMA in EOP and AP patients. Smear and 53-kDa protein were more frequently recognized by sera of EOP and AP patients rather than that of HS (p<0.05). The smear was partly diminished by absorption with P. gingivalis LPS, indicating the smear antigen was partly composed of LPS. There was high correlation between antibody levels against P. gingivalis whole-cell extracts and LPS in EOP and AP patients (r=0.81, p=0.0002, r=0.87, p<0.0001, respectively), while not significant in HS (r=0.22). The sera of EOP and AP patients with high IgG titre to P. gingivalis LPS blocked IL-1 beta production more effectively than that of the patients with low IgG titre to P. gingivalis LPS.
Conclusions: These results indicate that EOP patients' antibody response against P. gingivalis infection does not differ significantly from that of AP patients. The person-to-person heterogeneous antibody production against P. gingivalis LPS could contribute to our understanding of the relationship between the defensive ability of EOP patients and their chronic infection with this pathogen.

DOI： 10.1034/j.1600-051x.2000.027010769.x

Induction of Intracellular Interleukin-1 β Signals via Type II Interleukin-1 Receptor in Human Gingival Fibroblasts 査読国際誌

H.-H. Chou, S. Takashiba, H. Maeda, K. Naruishi, F. Nishimura, H. Arai, H.-k. Lu, Y. Murayama

Journal of Dental Research 79 ( 9 ) 1683 - 1688 2000年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SAGE Publications

The type II interleukin-1 receptor (IL-1RII) has been thought to be incapable of transducing signals to cells because of its short intracellular domain, while type I IL-1 receptor (IL-1RI) does transduce signals. Since over-expression of IL-1RII has been demonstrated to inhibit cytokine production in the fibroblastic cell line, it has been proposed to use IL-1RII to prevent IL-1-induced inflammation in connective tissue. In this study, trace amounts of IL-1RII mRNA expression were detected in human gingival fibroblasts (HGFs), which are affected by cytokines in inflammatory periodontal disease. Cloning of the cDNA encoding IL-1RII expressed in HGFs revealed 3 amino acid substitutions in the extracellular domain, when compared with the 408 residues predicted from human B-cells. Over-expression of IL-1RII on HGFs by gene transfer down-regulated the expression of IL-1β mRNA and IL-6 mRNA in response to IL-1β stimulation, while the expression of IL-8 mRNA was not affected. In the IL-1RII-transfected HGFs, phosphorylation of 25-and 74-kDa proteins was up-regulated upon IL-1β stimulation in the transfected HGFs. The phosphorylation of these proteins was suppressed by the addition of a neutralizing antibody against IL-1RII. These results suggest that the IL-1RII may regulate HGFs expression of cytokine mRNA upon IL-1β stimulation, possibly by altering the IL-1RI-dependent signals.

DOI： 10.1177/00220345000790090801

High Glucose Suppresses Cathepsin Activity in Periodontal-ligament-derived Fibroblastic Cells 査読国際誌

F. Nishimura, K. Naruishi, H. Yamada, T. Kono, S. Takashiba, Y. Murayama

Journal of Dental Research 79 ( 8 ) 1614 - 1617 2000年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SAGE Publications

The accumulation of extracellular matrices and integrins by high glucose has been reported in relation to diabetic complications. We previously reported that PDL cells expressed a higher amount of VLA-5 when cultured in high-glucose (4500 mg/L) medium than those cultured in low-glucose (1100 mg/L) medium. In this study, we aimed to address (1) whether this effect was mediated by the transcriptional level of the gene or the degradative level of the protein, and (2) whether this effect was mediated by TGF-β. The results indicated that the level of mRNA encoding a5 integrin did not change in PDL cells regardless of the concentration of glucose. Alternatively, high glucose suppressed cathepsin B+L activity. Additionally, the level of mRNA encoding TGF-β was not affected by high glucose, nor did an anti-TGF-β neutralizing antibody have an effect on the expression of β5 gene or cathepsin activity. Therefore, the effects of high glucose appeared to be mediated by impaired protein degradation, but not by autocrine TGF-β.

DOI： 10.1177/00220345000790081501

Epitope mapping of heat shock protein 60 (GroEL) from Porphyromonas gingivalis 査読

Maeda, H, Miyamoto, M, Kokeguchi, S, Kono, T, Nishimura, F, Takashiba, S, Murayama, Y

Fems Immunology and Medical Microbiology 28 ( 3 ) 219 - 224 2000年7月

Development of 16S rDNA-based PCR assay for detecting Centipeda periodontii and Selenomonas sputigena 査読

S. Sawada, S. Kokeguchi, S. Takashiba, Y. Murayama

Letters in Applied Microbiology 30 ( 6 ) 423 - 426 2000年6月

Phenytoin and Cyclosporin A Suppress the Expression of MMP-1, TIMP-1, and Cathepsin L, But Not Cathepsin B in Cultured Gingival Fibroblasts 査読国際誌

Hisa Yamada, Fusanori Nishimura, Koji Naruishi, Hsin-Hua Chou, Shogo Takashiba, George M. Albright, Salvador Nares, Anthony M. Iacopino, Yoji Murayama

Journal of Periodontology 71 ( 6 ) 955 - 960 2000年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

Background: Fibroblasts are known not only to synthesize and secrete extracellular matrix proteins, but also to degrade them for connective tissue remodeling. Drug-induced gingival overgrowth is characterized by a massive accumulation of extracellular matrix components in gingival connective tissues. Although some previous reports suggested that causative drugs stimulated the fibroblast proliferation, the results are not conclusive yet. In this study, we hypothesized that drug-induced gingival overgrowth could be a consequence of impaired ability of matrix degradation rather than an enhanced proliferation of gingival fibroblasts induced by these drugs.
Methods: Normal human gingival fibroblasts were cultured with or without either 20 mug/ml of phenytoin or 200 ng/ml of cyclosporin A. Total RNA and cellular proteins were collected every day for RT-PCR analyses and for measuring lysosomal enzyme activity. In addition, an immunohistochemical study was performed to detect lysosomal enzymes in cells from enlarged gingiva of the patients with phenytoin-induced gingival overgrowth.
Results: RT-PCR analyses revealed that these drugs suppressed the expression of MMP-1, TIMP-1, and cathepsin L, but not that of cathepsin B in a time-dependent manner. Then, we measured the activity of lysosomal enzymes and cathepsin B and L. The results indicated that although cathepsin B activity was not observed to be impaired, regardless of the drugs used in these cells, both total and active forms of combined activity of cathepsins B and L were suppressed in a time-dependent manner.
Conclusions: The results indicate that, besides suggested effects of these drugs on gingival fibroblasts and/or on accumulated cells in the gingival tissues, extracellular matrix-degrading ability, particularly that by cathepsin L, is also suppressed by cyclosporin A and phenytoin in gingival fibroblasts, and that lysosomal enzyme plays an important role in the pathogenesis of drug-induced gingival hyperplasia.

DOI： 10.1902/jop.2000.71.6.955

Cell surface-associated enolase in Actinobacillus actinomycetemcomitans 査読

Hara, H., Ohta, H., Inoue, T., Ohashi, T., Takashiba, S., Murayama, Y., Fukui, K.

Microbiology and Immunology 44 ( 5 ) 349 - 356 2000年5月

Erratum: Identification by subtractive hybridization of a novel insertion sequence specific for virulent strains of Porphyromonas gingivalis (Infection and Immunity (1999) 67:11 (5621-5625))

Sawada, K., Kokeguchi, S., Hongyo, H., Sawada, S., Miyamoto, M., Maeda, H., Nishimura, F., Takashiba, S., Murayama, Y.

Infection and Immunity 68 ( 9 ) 5470 - 5470 2000年

細菌16SリボソームRNA遺伝子を用いた歯周病細菌の同定 PCR法査読

綿城哲二, 苔口進, 宮本学, 前田博史, 藤本千代, 澤田聡子, 澤田弘一, 弘末勝, 清水明美, 片山知子, 杉典子, 西村英紀, 新井英雄, 高柴正悟, 村山洋二

日本歯科保存学雑誌 42 ( 6 ) 1108 - 1115 1999年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：(NPO)日本歯科保存学会

1)A.actinomycetemcomitans,P.gingivalis,P.intermedia,P.nigrescens,C.rectus,及びF.nucleatumの6菌種について検出限界は各々10^2個であった. 2)歯周病細菌3菌種を一度のPCRで検出・同定することができた. 3)歯周病患者の歯肉縁下プラークからも,各々の歯周病細菌を高感度に,かつ特異的に検出・同定することが可能であった

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Identification by Subtractive Hybridization of a Novel Insertion Sequence Specific for Virulent Strains of Porphyromonas gingivalis 査読国際誌

Koichi Sawada, Susumu Kokeguchi, Hiroshi Hongyo, Satoko Sawada, Manabu Miyamoto, Hiroshi Maeda, Fusanori Nishimura, Shogo Takashiba, Yoji Murayama

Infection and Immunity 67 ( 11 ) 5621 - 5625 1999年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

<title>ABSTRACT</title>

Subtractive hybridization was employed to isolate specific genes from virulent
<italic>Porphyromonas gingivalis</italic>
strains that are possibly related to abscess formation. The genomic DNA from the virulent strain
<italic>P. gingivalis</italic>
W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only
<italic>Bacillus stearothermophilus</italic>
IS
<italic>5377</italic>
, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS
<italic>1598</italic>
, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS
<italic>1598</italic>
was detected in
<italic>P. gingivalis</italic>
W83, W50, and FDC 381 by Southern hybridization. All three
<italic>P. gingivalis</italic>
strains have been shown to possess abscess-forming ability in animal models. However, IS
<italic>1598</italic>
was not detected in avirulent strains of
<italic>P. gingivalis</italic>
, including ATCC 33277. The IS
<italic>1598</italic>
may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS
<italic>1598</italic>
isolated in this study is a novel insertion element which might be a specific marker for virulent
<italic>P. gingivalis</italic>
strains.

DOI： 10.1128/iai.67.11.5621-5625.1999

Role of soluble interleukin-6 receptor in inflamed gingiva for binding of interleukin-6 to gingival fibroblasts 査読国際誌

Koji Naruishi, Shogo Takashiba, Hsin-Hua Chou, Hideo Arai, Fusanori Nishimura, Yoji Murayama

Journal of Periodontal Research 34 ( 6 ) 296 - 300 1999年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

Interleukin-6 (IL-6), frequently detected in periodontitis, is known to mediate important signals in the inflammatory cytokine network. Gingival fibroblasts (GF) secrete cytokines upon stimulation with inflammatory mediators. However, it is not clear if GF respond to IL-6. We examined the lL-6 receptor gene expression in GF. Furthermore, we tested whether GF are target cells for IL-6 by examination of binding of IL-6. GF were found to contain trace amounts of mRNA for IL-6 receptor (IL-GR), but had high levels of mRNA for 130-kDa glycoprotein (gp130), which is a signal transducer for IL-6/IL-6R complex. Based on this observation, we hypothesized that IL-6 could bind GF if exogenous soluble forms of IL-6R (sIL-6R) existed in the gingiva or culture condition. Thus, we investigated the existence of sIL-6R in gingiva using enzyme-linked immunosorbent assay and whether sIL-6R influenced the binding of IL-6 to GF in vitro. In inflamed gingiva, sIL-6R was detected and its concentration ranged from 150 to 700 pg/mu g protein. The sIL-6R enhanced the binding of IL-6 to GF in a dose-dependent manner. This enhancement was inhibited by an antibody against gp130, suggesting that the IL-6/sIL-6R complex bound to the fibroblasts via gp130. These data demonstrated that gingival fibroblasts can be target cells for IL-6 in the presence of appropriate amounts of sIL-6R. This situation may exist during inflammation in periodontal tissue.

DOI： 10.1111/j.1600-0765.1999.tb02257.x

In Vitro Induction of Activation-Induced Cell Death in Lymphocytes from Chronic Periodontal Lesions by Exogenous Fas Ligand 査読国際誌

Takamasa Sawa, Fusanori Nishimura, Hideki Ohyama, Keiso Takahashi, Shogo Takashiba, Yoji Murayama

Infection and Immunity 67 ( 3 ) 1450 - 1454 1999年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

<title>ABSTRACT</title>
Periodontitis is a chronic inflammatory disease which gradually destroys the supporting tissues of the teeth, leading to tooth loss in adults. The lesions are characterized by a persistence of inflammatory cells in gingival and periodontal connective tissues. To understand what mechanisms are involved in the establishment of chronic lesions, we hypothesized that infiltrating lymphocytes might be resistant to apoptosis. However, both Bcl-2 and Bcl-xL were weakly detected in lymphocytes from the lesions, compared with those from peripheral blood, suggesting that these cells are susceptible to apoptosis. Nevertheless, very few apoptotic cells were observed in tissue sections from the lesions. Lymphocytes from the lesions expressed mRNA encoding Fas, whereas Fas-ligand mRNA was very weakly expressed in lymphocytes from the lesions and in periodontal tissues. Since the results indicated that lymphocytes in the lesions might be susceptible to Fas-mediated apoptosis but lack the death signal, we next investigated if these lymphocytes actually undergo apoptosis by the addition of anti-Fas antibodies in vitro. Fas-positive lymphocytes from the lesions underwent apoptosis by these antibodies, but Fas-negative lymphocytes and Fas-positive peripheral lymphocytes did not undergo apoptosis by these antibodies. These results indicate that lymphocytes in the lesions are susceptible to activation-induced cell death and are induced to die by apoptosis after the addition of exogenous Fas ligand.

DOI： 10.1128/iai.67.3.1450-1454.1999

慢性歯科疾患病巣が全身の健康を影響する機序の解析招待査読

西村英紀, 滝川雅之, 苔口進, 高柴正悟, 村山洋二

日本歯科医学会誌 18 133 - 133 1999年3月

Phylogenetic characterization of Centipeda periodontii, Selenomonas sputigena and Selenomonas species by 16S rRNA gene sequence analysis 査読国際誌

S Sawada, S Kokeguchi, F Nishimura, S Takashiba, Y Murayama

MICROBIOS 98 ( 391 ) 133 - 140 1999年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：FACULTY PRESS

The nearly complete 16S rRNA gene sequences for oral Gram-negative anaerobic motile bacteria, Centipeda periodontii, Selenomonas sputigena and Selenomonas species (formerly S. sputigena type strain), were determined in order to unveil their relationship to other oral motile bacteria. To determine the phylogenetic characterization of these bacteria, their 16S rRNA gene sequences were obtained and compared with those from the ribosomal sequence databases previously reported. The 16S rRNA gene sequences of these bacteria were similar to those of Selenomonas ruminantium and Schwartzia succinivorans isolated from rumens, and to Pectinatus cerevisiiphilus isolated from spoiled beer. Among oral bacteria, the nucleotide sequence analysis of these bacteria revealed high nucleotide similarity to Veillonella species, whereas low similarity to oral motile bacteria such as Campylobacter species. Phylogenetic analysis clearly confirmed that C. periodontii and two Selenomonas species were classified as relatives of a group besides Selenomonas, Schwartzia, and Pectinatus species, and not as close relatives to oral motile bacteria, such as Campylobacter species. These results suggest that such oral Gram-negative anaerobic motile bacteria are close relatives of oral bacteria.