研究者詳細 - 髙柴　正悟

Identification and Modification of Porphyromonas gingivalis Cysteine Protease, Gingipain, Ideal for Screening Periodontitis. 査読国際誌

Kimito Hirai, Tomoko Yamaguchi-Tomikawa, Toru Eguchi, Hiroshi Maeda, Shogo Takashiba

Frontiers in immunology 11 1017 - 1017 2020年6月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Chronic periodontitis is an inflammatory disease caused by the formation of oral microbial biofilms. Periodontitis is associated with general health and not only oral diseases. Porphyromonas gingivalis is a well-known keystone pathogen for periodontitis and is associated with several systemic diseases, such as diabetes mellitus and Alzheimer's disease. We previously developed a system for screening periodontitis using P. gingivalis-specific serum immunoglobulin G (IgG) in an enzyme-linked immunosorbent assay with a sensitivity of 0.774 and a specificity of 0.586 and an area under the receiver operating characteristic curve of 0.708. However, the antigens elicited non-specific responses, since they were obtained from whole extracts of sonicated cultured bacteria. The purpose of this study was to identify antigens ideal for a sensitive and specific serum test. We identified the specific antigens using immunoaffinity columns immobilized with IgG antibodies from periodontitis patients. Liquid chromatography-tandem mass spectrometry identified 29 antigens from the elutes. Recombinant proteins for these candidates were synthesized using the wheat germ cell-free translation system and screened by dot blot analysis with serum from the columns. Three of the 16 candidates that reacted showed strongest affinities upon dot blot analysis; they included outer membrane protein 28, cysteine proteases, lysine gingipain Kgp, and arginine gingipain RgpA. Outer membrane protein 28 was not suitable for screening P. gingivalis infection because of its high false-negative rates. Kgp and RgpA were unstable antigens since they underwent self-digestion. They were made stable by substituting the active cysteine residues in Kgp and RgpA with alanine using site-directed mutagenesis. Using the modified antigens, we demonstrated that the patient serum IgG level against RgpA was the highest among all the antigens expressed in P. gingivalis. Moreover, the N-terminus of recombinant RgpA was excellent in differentiating between diseased and non-diseased states (with sensitivity of 0.85, specificity of 0.9, and area under the curve of 0.915). Although dot blot analysis was the only experiment used, the N-terminus of RgpA is an excellent antigen to immunologically test for P. gingivalis infection, especially for estimating the risks for periodontitis-associated systemic diseases. In conclusion, we have developed a P. gingivalis antigen for screening periodontitis.

DOI： 10.3389/fimmu.2020.01017

Tailoring the interaction between graphene oxide and antibacterial pyridinium salts by terminal functional groups 査読国際誌

R. Fujii, K. Okubo, S. Takashiba, A. Bianco, Y. Nishina

Carbon 160 204 - 210 2020年4月

掲載種別：研究論文（学術雑誌）

Nanocarbons, especially two-dimensional carbons, have received considerable attention due to their unique structure and physical and chemical properties, which make them promising candidate materials for biomedical applications. In this study, we focus on graphene oxide (GO), which has many oxygenated functional groups and high affinity with water and biomaterials, and the synthesis of GO complexes with antibacterial agents, like cetylpyridinium chloride (CPC) and its derivatives. We found that the sustained release of CPCs from GO can be controlled by changing the terminal functional group of CPC. The prepared GO-CPC complexes were subjected to antibacterial tests against S. mutans. CPC with the carboxy group was degraded by the oxidizing property of GO, resulting in the loss of antibacterial properties. On the other hand, the other CPC derivatives were released from GO and showed antibacterial activities. Finally, we propose a new mechanism describing how GO and CPC form a functional complex, and how CPC is released from this complex. These findings will lead to pioneering the carbon-based functional antibacterial agents designed at the molecular level.

DOI： 10.1016/j.carbon.2019.11.094

Scopus

Functionalized Graphene Oxide Shields Tooth Dentin from Decalcification. 査読国際誌

MZI Nizami, Y Nishina, T Yamamoto, Y Shinoda-Ito, S Takashiba

Journal of dental research 99 ( 2 ) 182 - 188 2020年2月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

This in vitro study assessed the efficacy of functionalized graphene oxide (f-GO) nanocomposites on the decalcification of dentin, because dental caries of the root surface is becoming one of the new problems in aged society. Hydroxyapatite plates (HAP) and dentin slices were coated with f-GO nanocomposites by comparing them to silver diamine fluoride as a positive control, then treated with decalcification solutions such as ethylenediaminetetraacetic acid and citrate at 37°C for 24 h. Scanning electron microscopy (SEM) revealed significant protection of the surface morphology of HAP and dentin. On the other hand, a cariogenic Streptococcus mutans growth was inhibited by f-GO nanocomposites. In addition, cytotoxicity of them to epithelial cells was much less than that of povidone-iodine, which is commonly used for oral disinfectant. We synthesized 5 different f-GO nanocomposites such as GO-silver (Ag), GO-Ag-calcium fluoride (CaF2), GO-CaF2, GO-zinc, and GO-tricalcium phosphate (Ca3(PO4)2). They were standardized by evaluating under SEM, transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetry analysis (TGA), and Raman spectra after being synthesized in an aseptic technique. The abilities of GO-Ag, GO-Ag-CaF2, and GO-CaF2 nanocomposites were most preventive for decalcification. In addition, GO-Ag and GO-Ag-CaF2 almost completely inhibited S. mutans growth. However, they did not exhibit cytotoxicity to epithelial cells except at the highest concentration (0.1 w/v%) of GO-Ag and GO-Ag-CaF2. Furthermore, these f-GO nanocomposites exhibited less or no discoloration of dentin, although commonly used silver diamine fluoride causes discoloration of dentin to black. Thus, these f-GO nanocomposites are useful to protect dental caries on the tooth root that becomes a social problem in aged society.

DOI： 10.1177/0022034519894583

多施設後ろ向き観察研究による臨床指標としての歯周炎症表面積の基準値査読

井上裕貴, 畑中加珠, 山本直史, 平田貴久, 三辺正人, 山本龍生, 内藤徹, 山本松男, 佐藤秀一, 石幡浩志, 稲垣幸司, 三谷章雄, 中島啓介, 漆原譲治, 高柴正悟

日本歯周病学会会誌 61 ( 4 ) 159 - 167 2019年12月

担当区分：最終著者,　責任著者記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：(NPO)日本歯周病学会

歯周炎症表面積(periodontal inflamed surface area:PISA)は,歯周組織の炎症部の面積を表す新たな歯周病の臨床指標である。従来伝わりづらかった歯周病の炎症程度を歯科以外の医療従事者が理解する上で,有用な指標であると考えられる。しかし,歯周病の程度や治療によるPISAの基準は未だ不明である。そこで,本研究は,日本歯周病学会が設ける歯周病専門医・認定医の電子申請書類のデータから各治療フェーズにおけるPISAの値を調べ,歯周病治療に伴う炎症度の基準値を提案することを目的とした。8施設で取得した113症例を用いて,Nesseらの方法によって歯周ポケット深さとプロービング時の出血からPISAを算出した。その結果,PISAの中央値は,初診時1,271.4mm2,歯周基本治療終了時211.8mm2,SPT移行時52.1mm2,そして最新SPT時30.0mm2であった。また,PISAはBOPと高い相関を示し(p<0.001),BOPよりも鋭敏に治療効果を反映した。以上から,中等度以上の歯周炎においてPISAを用いると,初診時は表面積約1,500mm2の歯周組織の炎症がSPT時は100mm2未満(初診時の約7%)に減少することが明らかになった。今後,更なるデータの蓄積および詳細な分析を行いながらPISAの使用を普及させると,PISAは医科歯科連携の際に歯周病炎症を伝える指標になり得ると考える。(著者抄録)

DOI： 10.2329/perio.61.159

Acceleration of bone regeneration of horizontal bone defect in rats using collagen-binding basic fibroblast growth factor combined with collagen scaffolds. 査読国際誌

Shin Nakamura, Takashi Ito, Kentaro Okamoto, Takehiko Mima, Kentaro Uchida, Yasir D Siddiqui, Masahiro Ito, Masako Tai, Keisuke Okubo, Keisuke Yamashiro, Kazuhiro Omori, Tadashi Yamamoto, Osamu Matsushita, Shogo Takashiba

Journal of periodontology 90 ( 9 ) 1043 - 1052 2019年9月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: Basic fibroblast growth factor (bFGF) has been applied for periodontal regeneration. However, the application depends on bone defect morphology because bFGF diffuses rapidly from defect sites. In a previous study, collagen-binding bFGF (CB-bFGF) has been shown to enhance bone formation by collagen-anchoring in the orthopedic field. The aim of this study is to demonstrate the efficacy of CB-bFGF with collagen scaffolds in bone regeneration of horizontal bone defect. METHODS: Cell proliferation activity and collagen binding activity of CB-bFGF was confirmed by WST-8 assay and collagen binding assay, respectively. The retention of CB-bFGF in the collagen sheet (CS) was measured by fluorescence imaging. The rat horizontal alveolar bone defect model was employed to investigate the efficacy of CB-bFGF with collagen powder (CP). After 4 and 8 weeks, the regenerative efficacy was evaluated by microcomputed tomography, histological, and immunohistochemical analyses. RESULTS: CB-bFGF had a comparable proliferation activity to bFGF and a collagen binding activity. CB-bFGF was retained in CS longer than bFGF. At 8 weeks postoperation, bone volume, bone mineral content, and new bone area in CB-bFGF/CP group were significantly increased compared with those in other groups. Furthermore, epithelial downgrowth was significantly suppressed in CB-bFGF/CP group. At 4 weeks, the numbers of osteocalcin, proliferating cell nuclear antigen, and osteopontin-positive cells at the regeneration site in CB-bFGF/CP group were greater than those in other groups. CONCLUSIONS: CB-bFGF/CP effectively promoted bone regeneration of horizontal bone defect possibly by sustained release of bFGF. The potential of CB-bFGF composite material for improved periodontal regeneration in vertical axis was shown.

DOI： 10.1002/JPER.18-0674

Assessment of the Plasma/Serum IgG Test to Screen for Periodontitis 査読

Resolvin D2 Induces Resolution of Periapical Inflammation and Promotes Healing of Periapical Lesions in Rat Periapical Periodontitis 査読国際共著国際誌

Yasir Dilshad Siddiqui, Kazuhiro Omori, Takashi Ito, Keisuke Yamashiro, Shin Nakamura, Kentaro Okamoto, Mitsuaki Ono, Tadashi Yamamoto, Thomas E. Van Dyke, Shogo Takashiba

Frontiers in Immunology 10 2019年2月

Effects of Lectins on initial attachment of cariogenic Streptococcus mutans 査読国際誌

Takashi Ito, Yasuhiro Yoshida, Yasuyoshi Shiota, Yuki Ito, Tadashi Yamamoto, Shogo Takashiba

Glycoconjugate Journal 35 ( 1 ) 41 - 51 2018年2月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Springer New York LLC

Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.

DOI： 10.1007/s10719-017-9795-2

C. Kudo, K. Naruishi, H. Maeda, Y. Abiko, T. Hino, M. Iwata, C. Mitsuhashi, S. Murakami, T. Nagasawa, T. Nagata, S. Yoneda, Y. Nomura, T. Noguchi, Y. Numabe, Y. Ogata, T. Sato, H. Shimauchi, K. Yamazaki, A. Yoshimura, S. Takashiba

Journal of Dental Research 91 ( 12 ) 1190 - 1195 2012年12月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SAGE Publications

Chronic periodontitis is a silent infectious disease prevalent worldwide and affects lifestyle-related diseases. Therefore, efficient screening of patients is essential for general health. This study was performed to evaluate prospectively the diagnostic utility of a blood IgG antibody titer test against periodontal pathogens. Oral examination was performed, and IgG titers against periodontal pathogens were measured by ELISA in 1,387 individuals. The cut-off value of the IgG titer was determined in receiver operating characteristic curve analysis, and changes in periodontal clinical parameters and IgG titers by periodontal treatment were evaluated. The relationships between IgG titers and severity of periodontitis were analyzed. The best cut-off value of IgG titer against Porphyromonas gingivalis for screening periodontitis was 1.682. Both clinical parameters and IgG titers decreased significantly under periodontal treatment. IgG titers of periodontitis patients were significantly higher than those of healthy controls, especially in those with sites of probing pocket depth over 4 mm. Multiplied cut-off values were useful to select patients with severe periodontitis. A blood IgG antibody titer test for Porphyromonas gingivalis is useful to screen hitherto chronic periodontitis patients (ClinicalTrials.gov number NCT01658475).

DOI： 10.1177/0022034512461796

Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method 査読

その他リンク： http://journals.sagepub.com/doi/full-xml/10.1177/0022034512461796

Gene polymorphisms in periodontal health and disease. 招待査読国際誌

Shogo Takashiba, Koji Naruishi

Periodontology 2000 40 ( 1 ) 94 - 106 2006年2月

Maeda, H., Kokeguchi, S., Fujimoto, C., Tanimoto, I., Yoshizumi, W., Nishimura, F., Takashiba, S.

FEMS Immunology and Medical Microbiology 43 ( 2 ) 233 - 239 2005年2月

Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria 査読

Maeda, H, Fujimoto, C, Haruki, Y, Maeda, T, Kokeguchi, S, Petelin, M, Arai, H, Tanimoto, I, Nishimura, F, Takashiba, S

Fems Immunology and Medical Microbiology 39 ( 1 ) 81 - 86 2003年10月

Perspective of cytokine regulation for periodontal treatment: Fibroblast biology 招待査読

Takashiba, S., Naruishi, K., Murayama, Y.

Journal of Periodontology 74 ( 1 ) 103 - 110 2003年1月

Differentiation of Monocytes to Macrophages Primes Cells for Lipopolysaccharide Stimulation via Accumulation of Cytoplasmic Nuclear Factor κB 査読国際誌

Shogo Takashiba, Thomas E. Van Dyke, Salomon Amar, Yoji Murayama, Aubrey W. Soskolne, Lior Shapira

Infection and Immunity 67 ( 11 ) 5573 - 5578 1999年11月

担当区分：筆頭著者記述言語：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>
During infection, circulating blood monocytes migrate from the vasculature to the extravascular compartments where they mature into tissue macrophages. The maturation process prepares the cell to actively participate in the inflammatory and the immune responses, and many transcription factors have been found to be involved. Here we report on a novel role for nuclear factor κB (NF-κB) in this process. Its accumulation in the cytoplasm of differentiated macrophages is responsible for the enhanced ability of the cell to respond to lipopolysaccharide (LPS) stimulation, as determined by tumor necrosis factor alpha (TNF-α) secretion. Differentiation of the human monocytic cell line THP-1 into macrophage-like cells was induced by exposure of the cells to phorbol myristate acetate. DNA-bindable NF-κB was not detected in the cytoplasm of undifferentiated THP-1 cells but accumulated in the cytoplasm of the cells following differentiation. No TNF-α was detected in the media of resting differentiated and nondifferentiated THP-1 cells. Stimulation with LPS of differentiated cells induced the production of higher levels of TNF-α than stimulation of nondifferentiated cells. This hyperresponsiveness to LPS was found in the mRNA and secreted TNF-α levels. Furthermore, stimulation with LPS induced the translocation of NF-κB from the cytoplasm into the nucleus. This translocation process was more rapid in the differentiated cells than in the nondifferentiated cells, and the resultant accumulated levels of NF-κB in the nucleus were higher. The DNA-bindable NF-κB was identified as a heterodimer of p65 and p50. The results suggest that NF-κB accumulation in the cytoplasm during maturation of monocytes to macrophages primes the cells for enhanced responsiveness to LPS and results in the rapid secretion of inflammatory mediators, such as TNF-α, by mature macrophages following LPS challenge.

DOI： 10.1128/iai.67.11.5573-5578.1999

HLA genetics for diagnosis of susceptibility to early-onset periodontitis 査読国際誌

Shogo Takashiba, Hideki Ohyama, Kosuke Oyaizu, Nahoko Kogoe-Kato, Yoji Murayama

Journal of Periodontal Research 34 ( 7 ) 374 - 378 1999年10月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

Human leukocyte antigens (HLA) are essential in the recognition of foreign antigens in humoral immune response, which is genetically predetermined. Susceptibility to certain diseases that involve the immune response has been studied in relation to distinct HLA types. Although some diseases have been found Co correlate to specific HLA loci positively, it has been difficult to isolate HLA types that predispose patients to periodontal destruction. Here. we review the current knowledge and recent advances in HLA genetics and its biology, which determine susceptibility to early-onset periodontitis (EOP). The HLA-DRB1*1501-DQB1*0602 genotype has been found with increasing frequency in EOP patients. This HLA genotype expresses aspartic acid at position 57 and glycine at position 70 on the DQ beta chain, suggesting a capability to bind certain bacterial antigens. The T cell response against the outer membrane protein (Ag53) of Porphyromonas against gingivalis was examined via this HLA genotype. Strong T cell response against Ag53 p141-161 was inhibited partially by anti-DR antibody, but not by anti-DQ antibody, possible host and bacterial peptides capable of binding DRB1*1501 were elucidated when the peptide sequence was compared to gene and protein databases. These results suggest that patients who have the HLA-DRB1*1501-DQB1*0602 genotype may have an accelerated T cell response to certain periodontapathic bacteria such as P. gingivalis in hyperimmune reactions and thus increased susceptibility to EOP.

DOI： 10.1111/j.1600-0765.1999.tb02269.x

A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor gene expression: Molecular cloning, sequencing, characterization, and chromosomal assignment 査読

F. Myokai, S. Takashiba, R. Lebo, S. Amar

Proceedings of the National Academy of Sciences 96 ( 8 ) 4518 - 4523 1999年4月

HLA Class II Genotypes Associated With Early-Onset Periodontitis: DQB1 Molecule Primarily Confers Susceptibility to the Disease 査読国際誌

Hideki Ohyama, Shogo Takashiba, Kosuke Oyaizu, Atsushi Nagai, Taeko Naruse, Hidetoshi Inoko, Hidemi Kurihara, Yoji Murayama

Journal of Periodontology 67 ( 9 ) 888 - 894 1996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：Wiley

DNA TYPING WAS PERFORMED on 24 Japanese patients with early-onset periodontitis (EOP) using the PCR-RFLP method to investigate an association of the susceptibility to EOP with the particular HLA class II alleles (HLA-DRB1, -DQA1, and -DQB1), DRB1*1401, DRB1*1501, DQB1*0503, and DQB1*0602 were found more frequently (''susceptible'') in the EOP patients than in healthy controls. In contrast, DRB1*0405 and DQB1*0401 were found less frequently (''resistant'') in EOP patients, All patients carrying DQB1*0602 had an atypical BamHI site in the intron upstream of the third exon of the DQB1 gene, which in our previous studies appeared to be a susceptible marker for EOP. A comparative analysis of the amino acid sequences of these susceptible and resistant HLA-DRB1 and DQB1 alleles elucidated some differences in antigen-derived peptide binding sites related to the susceptible or resistant alleles. Especially, DQB1*0503 and DQB1*0602 alleles carrying aspartic acid at position 57 and glycine at position 70 are increased significantly in EOP. Since amino acid residues at positions 57 and 70 on the DQB1 molecule are supposed to be involved in antigen binding, amino acid substitutions at these positions may affect the immune responsiveness to the periodontopathic antigen. Our results suggest that the DQB1 molecule plays a crucial role in the pathogenesis of EOP and that the susceptibility to EOP may be determined by the binding ability between the peptide and HLA-DQ antigens.

DOI： 10.1902/jop.1996.67.9.888

Lipopolysaccharide-inducible and salicylate-sensitive nuclear factor(s) on human tumor necrosis factor alpha promoter 査読国際誌

S Takashiba, T E Van Dyke, L Shapira, S Amar

Infection and Immunity 63 ( 4 ) 1529 - 1534 1995年4月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

Lipopolysaccharide (LPS) is one of the most potent trigger substances for monocytes and macrophages causing secretion of tumor necrosis factor alpha (TNF-alpha) and other inflammatory mediators. The nature of the nuclear factors involved in human TNF-alpha gene regulation is still unknown. Nuclear factor kappa B (NF-kappa B) proteins have been suggested to play an important role in gene transcription of inflammatory mediators when monocytes are stimulated with LPS. However, it remains unclear whether these nuclear factors are the only ones involved in human TNF-alpha gene regulation. In this report, to further the identification of nuclear factor(s) involved in TNF-alpha gene regulation, human monocytic THP-1 cells were transfected with a series of truncated versions of human TNF-alpha promoter. A 98-bp region located from nucleotides -584 to -487 demonstrated strong promoter activity. Electrophoretic mobility shift assays demonstrated that a 64-bp fragment located within the 98-bp region and lacking any potential NF-kappa B-binding sites avidly bound LPS-challenged THP-1 nuclear protein. Although this binding was inhibited in salicylate-treated cells, as was binding of NF-kappa B, the pattern of binding was found to differ from that noted for NF-kappa B. Analysis of this 64-bp fragment disclosed the absence of an NF-kappa B consensus sequence, suggesting a novel nuclear DNA-binding protein necessary for the initiation of human TNF-alpha transcription other than, or in addition to, NF-kappa B.

DOI： 10.1128/iai.63.4.1529-1534.1995

Induced TNF-alpha and IL1-beta Production by Human Monocytes 査読国際共著国際誌

L Shapira, S Takashiba, C Champagne, S Amar, TE Van Dyke

JOURNAL OF IMMUNOLOGY 153 ( 4 ) 1818 - 1824 1994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：AMER ASSOC IMMUNOLOGISTS

Bacterial LPS stimulates human monocytes to secrete inflammatory cytokines, which are involved in several disease processes. However, the mechanism of LPS activation of cytokine expression and secretion is not completely understood. In this study, we investigated the signal transduction pathways involved in LPS-stimulated TNF-alpha and IL-1 beta secretion. TNF-alpha and IL-1 beta secretion were completely blocked by protein kinase C (PKC) and cyclic nucleotide-dependent protein kinase inhibitor, H-7, but were not affected by H-89, a specific cyclic nucleotide-dependent protein kinase inhibitor. In addition, LPS was found to induce activation of PKC, reaching maximal activity at 30 min and returning to unstimulated levels after 60 min. LPS stimulation only slightly increased intracellular levels of diacylglycerol, the natural activator of PKC, and pretreatment of monocytes with the diacylglycerol-kinase inhibitor, R59022, did not affect LPS-stimulated TNF-alpha secretion. LPS-induced PKC activation was found not to be affected by blocking of the LPS receptor, CD14, with mAb or by inhibition of protein tyrosine kinase with herbimycin A. However, these agents suppressed LPS-induced TNF-alpha secretion and TNF-alpha mRNA accumulation. The results suggest that TNF-alpha and IL-1 beta secretion after LPS stimulation of human monocytes requires the activation of protein tyrosine kinase and PKC, upstream to the activation of gene transcription. The activation of PKC by LPS is probably mediated by a diacylglycerol-independent pathway.

Unique Intronic Variations of HLA-DQβ Gene in Early-Onset Periodontitis 査読国際誌

Shogo Takashiba, Sumihare Noji, Fusanori Nishimura, Hideki Ohyama, Hidemi Kurihara, Yoshio Nomura, Shigehiko Taniguchi, Yoji Murayama

Journal of Periodontology 65 ( 5 ) 379 - 386 1994年5月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

HUMAN LEUKOCYTE ANTIGEN (HLA) class II beta chain plays an important role in the recognition of foreign antigens in immune reactions. Different forms of immune reaction may be concerned with initiation and progression of infectious diseases such as periodontitis. In this study we examined the frequency of HLA class II serotype and the variation of HLA class II beta gene in periodontitis patients. HLA serotypic frequencies in 70 Japanese patients with periodontitis and 26 individuals with periodontal health were examined. No HLA serotype specific to any type of periodontitis was observed. In order to detect differences among some HLA serotypes, restriction fragment length polymorphism (RFLP) analysis was undertaken with cDNA probes for HLA-DR beta and HLA-DQ beta genes in 20 subjects (15 patients and 5 healthy individuals). Atypical BamHI and EcoRI restriction sites were found in the HLA-DQ beta gene from 3 patients with early-onset periodontitis. In addition to these 20 subjects, an additional 80 subjects (40 patients and 40 healthy individuals) were screened for the atypical BamHI restriction site using the polymerase chain reaction method. It was detected in 7 patients with early-onset periodontitis, 1 patient with adult periodontitis, and 3 healthy subjects. No clinical differences except age were found between patients with this gene variation and other patients. Interestingly, all 3 healthy subjects with this gene variation were from subjects whose family members developed early-onset periodontitis with the gene variation. Atypical BamHI and EcoRI restriction sites and 41-nt repeated sequence were found in the intron before the third exon of HLA-DQB gene. These results suggest that these intronic gene variations may be useful as gene markers for a subpopulation of early-onset periodontitis and might affect immune reactions such as antigen recognition.

DOI： 10.1902/jop.1994.65.5.379