Papers by Kazuyuki Ishihara
Journal of Periodontal Research, Jun 15, 2021
Systemic administration of cytotoxic T lymphocyteassociated antigen 4 (CTLA-4)-Ig abrogates alveo... more Systemic administration of cytotoxic T lymphocyteassociated antigen 4 (CTLA-4)-Ig abrogates alveolar bone resorption in induced periodontitis through inhibition of osteoclast differentiation and activation: An experimental investigation Author(s) Alternative
PLOS ONE, Oct 21, 2014
Background: A number of studies have revealed a link between chronic periodontitis and cardiovasc... more Background: A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg), on pathogenesis of atherosclerosis in obesity. In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD) or normal chow diet (CD), as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1) were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA) induced endothelial cells apoptosis and regulation of cytokine gene expression. Results: Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate. Conclusions: Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury.
Journal of Antimicrobial Chemotherapy, Oct 28, 2006
Objectives: Actinobacillus actinomycetemcomitans is a major causative agent of chronic and aggres... more Objectives: Actinobacillus actinomycetemcomitans is a major causative agent of chronic and aggressive periodontitis. Freshly isolated strains of A. actinomycetemcomitans display rough-type colonies and initiate biofilm formation on glass surfaces. The purpose of this study was to determine the antibiotic susceptibility of A. actinomycetemcomitans biofilm during different phases of maturation. Methods: Using 96-well microtitre plates, we determined the antibiotic susceptibility of rough-type strain 310a to concentrations from 0.1 to 10 mg/L each of erythromycin, ofloxacin, ampicillin, cefalexin, tetracycline and minocycline during biofilm formation. Antibiotics were added at the start of the culture (early phase) and after 24 h of cultivation (mature phase). Results: Adding 10 mg/L of ampicillin, 10 mg/L of cefalexin, 0.1 or 1 mg/L of tetracycline, or 0.1 mg/L of minocycline significantly inhibited 310a biofilm formation in the early phase, but not in the mature phase. Although adding 10 mg/L of erythromycin, tetracycline or minocycline reduced biofilm development in the early phase, it enhanced 310a biofilm development in the mature phase. Ofloxacin exerted a strong inhibitory effect in both the early and mature phases of biofilm formation throughout all experiments. Conclusions: The present study demonstrated that the susceptibility of A. actinomycetemcomitans to many antibiotics decreased after biofilm maturation.
The Bulletin of Tokyo Dental College, 2021
Candida albicans is the primary candidiasis-causing fungal pathogen in humans, and one of its mos... more Candida albicans is the primary candidiasis-causing fungal pathogen in humans, and one of its most important virulence factors is the ability to form biofilms. Moreover, these biofilms are often resistant to antifungal agents, so there is a need to develop alternative elimination strategies and therapeutic agents for such infections. The antifungal activity of resveratrol, a phytoalexin polyphenolic compound, impairs the morphological transition of C. albicans under various hypha-inducing conditions and inhibits growth of the yeast-form and mycelia. The purpose of this study was to investigate the effect of resveratrol against C. albicans biofilm formation. The developmental, sustained, and mature stages of biofilm formation were affected or inhibited by resveratrol. Exposure to resveratrol at the developmental stage inhibited growth of C. albicans in a dose-dependent manner. A >30% reduction was observed in sustained biofilm growth in the presence of 200 μg/ml resveratrol in comparison with in its absence. In terms of disruption of matured biofilm, 6.25-100 μg/ml resveratrol significantly reduced cell viability of C. albicans compared with in a control sample (p<0.05). The present results indicate that resveratrol has the potential to serve as an anti-Candida treatment and preventive tool which functions by inhibiting existing or under-forming C. albicans biofilms.
The Bulletin of Tokyo Dental College, 2016
Periapical periodontitis usually results from microbial infection, with these microorganisms occa... more Periapical periodontitis usually results from microbial infection, with these microorganisms occasionally migrating to the root canal, which can lead to further, potentially life-threatening, complications. Here, the susceptibility of 27 bacterial strains to various antimicrobial agents was evaluated. These strains comprised 13 species; 16 of the strains were clinical isolates from periapical lesions. Each strain was inoculated onto blood agar plates containing one of the antimicrobial agents. The plates were incubated anaerobically at 37°C for 96 hr and the minimal inhibitory concentrations (MICs) determined. Ten strains required an MIC of 32 μg/ml or greater for amoxicillin, 6 for cefmetazole, and 5 for cefcapene among β-lactam antibiotics; 8 strains required an MIC of 32 μg/ml or greater for clindamycin, 4 for azithromycin, and 11 for clarithromycin among macrolide antibiotics; 3 strains required an MIC of 32 μg/ml or greater for ciprofloxacin and 2 for ofloxacin among fluoroquinolones. The effect of cefcapene on 5 strains was evaluated after biofilm formation to investigate the relationship between biofilm formation and susceptibility. All strains showed a decrease in susceptibility after biofilm formation. The results revealed that several antimicrobial agents commonly used in a clinical setting, including amoxicillin, cefmetazole, and clindamycin, are potentially effective in the treatment of orofacial odontogenic infections. The development of resistant strains, however, means that this can no longer be guaranteed. In addition, azithromycin, ciprofloxacin, and ofloxacin were more effective than the 3 β-lactam antibiotics tested. These results suggest that sensitivity testing is needed if odontogenic infections are to be treated safely and effectively.
Infection and Immunity, Dec 1, 1996
A chymotrypsin-like protease from Treponema denticola ATCC 35405 was purified by chromatographic ... more A chymotrypsin-like protease from Treponema denticola ATCC 35405 was purified by chromatographic techniques. The purified enzyme consisted of three polypeptides (38, 43, and 72 kDa). The protease exhibited specificity for peptide bonds containing phenylalanine and proline at the P1 and P2 positions, respectively, and was classified as a serine protease on the basis of inhibition studies. Naturally occurring protease inhibitors such as ␣1-antitrypsin and ␣1-antichymotrypsin had no effect on enzymatic activity. The enzyme degraded fibronectin, ␣1-antitrypsin, and gelatin while weakly degrading the immunoglobulin G heavy chain and type IV collagen. N-terminal amino acid sequences were determined for the 43-and 72-kDa proteins. On the basis of these sequences, the genes coding for the 43-and 72-kDa proteins were isolated and sequenced. The open reading frame which codes for the 72-kDa protein was designated prtP. This gene consists of 2,169 bp and codes for a protein with an M r of 77,471. The protein appeared to be composed of a signal peptide region followed by a prosequence and the mature protein domain. The deduced amino acid sequence exhibited similarity with that of the Bacillus subtilis serine protease subtilisin. The deduced properties of the sequence suggest that the 72-kDa protein is a chymotrypsin-like protease. However, the nature and function of the 43-kDa protein have not yet been determined.
Journal of the Japanese Association of Periodontology, 1990
1990年3月 These findings indicate that the SK-013 activity is a useful indication of T. denticola, ... more 1990年3月 These findings indicate that the SK-013 activity is a useful indication of T. denticola, B. gingivalis and B. forsythus in subgingival plaque, and it could be used to diagnose periodontitis.
PLoS ONE, 2014
Background: A number of studies have revealed a link between chronic periodontitis and cardiovasc... more Background: A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg), on pathogenesis of atherosclerosis in obesity. In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD) or normal chow diet (CD), as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1) were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA) induced endothelial cells apoptosis and regulation of cytokine gene expression. Results: Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate. Conclusions: Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury.
Journal of the Japanese Association of Periodontology, 1990
Environmental Infections, 2006
An in vitro model of periodontopathic bacteria biofilm was established and the killing activity o... more An in vitro model of periodontopathic bacteria biofilm was established and the killing activity of various gargles including povidone-iodine gargle (PVP-I) determined with this model. Actinobacillus actinomycetemcomitan Y4 (ATCC 43718) biofilm was formed by culturing this strain on polycarbonate membrane (PCM). Similarly, mixed biofilms of A. actinomycetemcomitans Y4, Streptococcus oralis ATCC 10557, and Fusobacterium nucleatum ATCC 25586 were formed (co-aggregation) by culturing a mixture of these strains on PCM. Exposure of the A. actinomycetemcomitans biofilm formed by culturing for one day or three days to 0.23% PVP-I for 3 min reduced the count of viable cells by -3.59 and -3.39 (log10 CFU/mL), respectively. The killing activity of PVP-I was stronger than those of 0.02% benzethonium chloride (BEC) and 0.002% chlorhexidine gluconate (CHG) . Repeated exposure of this biofilm to 0.23% PVP-I (for 1 min, 3 times a day for 5 consecutive days) reduced the count of viable cells on the ...
The Effect of Artificial Recombinant Bone Morphogenetic Protein 2 with Titanium-binding Peptide on a Ti Graft in Vivo
Journal of oral tissue engineering, 2014
The Bulletin of Tokyo Dental College, 2021
Capnocytophaga ochracea possesses a type-IX secretion system that exports proteins which have a g... more Capnocytophaga ochracea possesses a type-IX secretion system that exports proteins which have a gliding motility-associated C-terminal (CTD) domain. This system is found in several species of the Bacteroidetes phylum. Hyalin, a large protein encoded by Coch_0033 in C. ochracea ATCC 27872, has a CTD domain and is posited to be involved in quorum sensing according to the database of the Kyoto Encyclopedia of Genes and Genomes. This suggests that it plays a role in biofilm formation via interbacterial communication. The aim of this study was to investigate the potential role of the hyalin-like protein coded by the Coch_0033 gene in gliding and biofilm formation of C. ochracea. A hyalin-like protein-deficient mutant strain of C. ochracea, designated mutant WR-1, was constructed through insertion of the ermF-ermAM cassette into the target gene. The spreading feature at the edge of the colony was lost in the mutant strain. Crystal violet and confocal laser scanning microscopy revealed no ...
The Bulletin of Tokyo Dental College, 2018
Treponema denticola, an anaerobic spirochete found mainly in the oral cavity, is associated with ... more Treponema denticola, an anaerobic spirochete found mainly in the oral cavity, is associated with periodontal disease and has a variety of virulence factors. Although in vitro studies have shown that T. denticola is able to penetrate epithelial cell monolayers, its effect on the epithelial barrier junction is not known. Human gingival epithelial cells are closely associated with adjacent membranes, forming barriers in the presence of tight junction proteins, including zonula occludens-1 (ZO-1), claudin-1, and occludin. Tight junction proteins are also expressed by Madin-Darby canine kidney (MDCK) cells in culture. In this study, the MDCK cell profile was investigated following infection with T. denticola (ATCC 35405) wild-type, as well as with its dentilisin-deficient mutant, K1. Basolateral exposure of MDCK cell monolayers to T. denticola at a multiplicity of infection (MOI) of 10 4 resulted in a decrease in transepithelial electrical resistance (TER). Transepithelial electrical resistance in MDCK cell monolayers also decreased following apical exposure to T. denticola (MOI=10 4), although this took longer with basolateral exposure. The effect on the TER was time-dependent and required the presence of live bacteria. Meanwhile, MDCK cell viability showed a decrease with either basolateral or apical exposure. Immunofluorescence analysis demonstrated decreases in the amounts of immunoreactive ZO-1 and claudin-1 in association with disruption of cell-cell junctions in MDCK cells exposed apically or basolaterally to T. denticola. Western blot analysis demonstrated degradation of ZO-1 and claudin-1 in culture lysates derived from T. denticola-exposed MDCK cells, suggesting a bacteria-induced protease capable of cleaving these tight junction proteins.
Methods in molecular biology, 2021
Treponema denticola is a potent periodontal pathogen that forms a red complex with Porphyromonas ... more Treponema denticola is a potent periodontal pathogen that forms a red complex with Porphyromonas gingivalis and Tannerella forsythia. It has many virulence factors, yet there are only a few reports detailing these factors. Among them, dentilisin is a well-documented surface protease. Dentilisin is reported to be involved in nutrient uptake, bacterial coaggregation, complement activation, evasion of the host immune system, inhibition of the hemostasis system, and cell invasion as a result of its action, in addition to its original proteolysis function. Therefore, characterization of dentilisin, and clarifying the relationship between T. denticola and the onset of periodontal disease will be important to better understanding this disease. In this chapter, we explain the methods for analysis of dentilisin activity and pathogenicity.
The Bulletin of Tokyo Dental College, 2007
The purpose of this study was to evaluate age-related differences in expression of vascular endot... more The purpose of this study was to evaluate age-related differences in expression of vascular endothelial growth factor (VEGF) by periodontal ligament (PDL) cells. PDL cells were obtained from Wistar male rats weighing approximately 150g each in the young group and 350g each in the old group. PDL cells derived from upper and lower incisors were seeded in 35-mm culture dishes after primary culture. For cell proliferation assays, cells were detached and counted at 1, 3, 5, 7, 11 and 14 days after culture. VEGF mRNA expression was analyzed with TaqMan ®. The number of cells in both groups increased day by day, but the rate of increase in the young group was higher than that in the old group. VEGF mRNA expression in the young group increased from 3 to 14 days, but in the old group increased only slightly over the same time period. Expression ratios in the young group were higher than those in the old group, and there were significant differences between the young and old groups at 7 and 14 days of culture. In conclusion, the data revealed that PDL cells varied with age, and suggest that in view of such changes in cell proliferation and VEGF mRNA expression, age should be taken into consideration in periodontal treatment.
FEMS Microbiology Letters, 2018
Treponema denticola controls the expression of ABC transporters and methyl-accepting chemotaxis p... more Treponema denticola controls the expression of ABC transporters and methyl-accepting chemotaxis proteins to uptake serum components as nutrient sources in the gingival crevice or the periodontal lesion.
FEMS Microbiology Letters, 2004
A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43-and 38-... more A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43-and 38-kDa proteins. A sequence encoding a 43-kDa protein was located in the 3P region of the prcA gene upstream of the dentilisin gene (prtP). The 43-kDa protein was apparently generated from digestion of PrcA. To clarify the function of the protein, we constructed a mutant of the 43-kDa protein following homologous recombination. The mutant lacked detectable dentilisin activity. Immunoblot analysis demonstrated that the dentilisin protein was degraded in the mutant. The results of real-time polymerase chain reaction suggested that prtP mRNA expression in the mutant was somewhat decreased compared with the wild-type strain. These data suggest that the 43-kDa protein is involved in the stabilization of the dentilisin protein.
FEMS Immunology & Medical Microbiology, 2008
Invasion by Porphyromonas gingivalis has been proposed as a possible mechanism of pathogenesis in... more Invasion by Porphyromonas gingivalis has been proposed as a possible mechanism of pathogenesis in periodontal and cardiovascular diseases. P. gingivalis have direct access to the systemic circulation and endothelium in periodontitis patients by transient bacteremia. Periodontitis can be described as one of the predominant polymicrobial infections of humans. In the present study, P. gingivalis strains were tested for their ability to invade a human gingival epithelial cell line (Ca9-22) and human aortic endothelial cells (HAEC) in co-infection with Fusobacterium nucleatum using antibiotic protection assays. Co-infection with F. nucleatum resulted in 2-to 20-fold increase in invasion of host cells by P. gingivalis strains. The invasive abilities of P. gingivalis strains were significantly greater when incubated with a F. nucleatum clinical isolate (which possesses strong biofilm-forming ability), than when incubated with F. nucleatum type strain. In inhibition assays with metabolic inhibitors, difference in inhibition profiles was noted between mono-and polymicrobial infections. Collectively, our results suggest that F. nucleatum facilitates invasion of host cells by P. gingivalis. Investigations of polymicrobial infection of host cells should improve our understanding of the role of P. gingivalis in periodontal infection and proatherogenic mechanisms.
FEMS Immunology & Medical Microbiology, 2009
Galectins, a family of animal lectins, are involved not only in development and differentiation b... more Galectins, a family of animal lectins, are involved not only in development and differentiation but also in immunoregulation and host-pathogen interactions. Galectin-3 interacts with lipopolysaccharides in gram-negative bacteria such as Escherichia coli, Salmonella minnesota and Pseudomonas aeruginosa. The present study investigated whether galectin-3 inhibited the cytokine-inducing activity of periodontopathic bacterial lipopolysaccharides using splenocytes derived from mice of different ages. Lipopolysaccharides were extracted from Aggregatibacter actinomycetemcomitans Y4 and Porphyromonas gingivalis ATCC 33277, and then purified. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that galectin-3 adhered to A. actinomycetemcomitans lipopolysaccharides, but not to the lipopolysaccharides of P. gingivalis. Splenocytes were prepared from 1-or 7month-old C57BL/6 mice. Either A. actinomycetemcomitans lipopolysaccharides (200 ng mL À1) alone or lipopolysaccharides and murine galectin-3 (10 mg mL À1) were added to culture solutions, and the release of interleukin-6 (IL-6) and interferon-g (IFNg) from splenocytes was measured by ELISA after a 17-h incubation. In all mice tested, A. actinomycetemcomitans lipopolysaccharide stimulation significantly increased the production of IL-6 and IFNg (P o 0.01). Murine galectin-3 suppressed lipopolysaccharide-induced cytokine production in the splenocytes of the 1-month-old mice (P o 0.02 for IL-6; P o 0.05 for IFNg), but not in the splenocytes of the 7-month-old mice. This suggests that responses change with age.
FEMS Immunology & Medical Microbiology, 2006
We studied the effect of antibodies against Porphyromonas gingivalis gingipain domains, preparing... more We studied the effect of antibodies against Porphyromonas gingivalis gingipain domains, preparing them against three recombinant fragments of RgpA (catalytic domain: r-Rgp CAT; hemagglutinin domains: r-Rgp 44 and r-Rgp 15-27) and one fragment of Kgp (catalytic domain: r-Kgp CAT). Enhancement of opsonization, and killing by human polymorphonuclear leukocytes was measured in the non-invasive FDC 381 and invasive W50 strains of P. gingivalis. Anti-r-Rgp 44 antibody was the most effective in both strains of P. gingivalis. The present findings recommend RgpA 44 as a candidate immunogen for vaccines against P. gingivalis.
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Papers by Kazuyuki Ishihara