Language：English   Publishing type：Research paper (scientific journal)  

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA-treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA-treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA-induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.

DOI： 10.1111/1348-0421.12672

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Language：English  

Increase in antimicrobial resistance (AMR) among pathogenic bacteria is a serious threat to public health. Surveillance studies to monitor shifting trends in resistance are important and guide the selection of appropriate antimicrobial agents for a particular organism. Furthermore, these studies help in dissemination of accurate information regarding AMR to the public. In this study, we investigated the antimicrobial susceptibility patterns of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis clinical isolates from outpatient children with acute otitis media in Japan from 2014 to 2017. A total of 8693 strains (2415 of S. pneumoniae, 3657 of H. influenzae, and 2621 of M. catarrhalis) were clinically isolated, and their antimicrobial susceptibilities to benzylpenicillin (PCG), ampicillin (ABPC), amoxicillin-clavulanic (AMPC/CVA), azithromycin (AZM), ceftriaxone (CTRX), and levofloxacin (LVFX) were investigated. Based on the minimum inhibitory concentration (MIC) breakpoints, the average proportion of S. pneumoniae isolates non-susceptible to PCG and AZM was 38.2% and 82.0% respectively. The average proportion of H. influenzae isolates non-susceptible to ABPC, CVA/AMPC, and CTRX was 61.9%, 43.5%, and 49.4%, respectively. The high prevalence of these resistant organisms is attributed to frequent use of antibiotic agents in Japan. Moreover, the proportion of LVFX-non-susceptible H. influenzae isolates increased in this four-year study. Here, we report updates regarding the AMR trends amongst the major pathogens that cause acute otitis media in Japan. Continuing surveillance of antimicrobial susceptibility and application of control measures against further transmission are required to decrease the emergence of resistant strains.

DOI： 10.1016/j.jiac.2018.08.018

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Language：English   Publishing type：Research paper (scientific journal)  

OBJECTIVE: Food-derived peptides have been reported to exhibit antibacterial activity against periodontal pathogenic bacteria. However, no effect has been shown on inflammation and bone resorption in periodontal pathology. The overall objective of the current study was to investigate how rice peptides influence biological defense mechanisms against periodontitis-induced inflammatory bone loss, and identify their novel functions as a potential anti-inflammatory drug. DESIGN: The expression of inflammatory and osteoclast-related molecules was examined in mouse macrophage-derived RAW 264.7 cell cultures using qPCR. Subsequently, the effect of these peptides on inflammatory bone loss in mouse periodontitis was examined using a mouse model of tooth ligation. Briefly, periodontal bone loss was induced for 7 days in mice by ligating the maxillary second molar and leaving the contralateral tooth un-ligated (baseline control). The mice were microinjected daily with the peptide in the gingiva until the day before euthanization. One week after the ligation, TRAP-positive multinucleated cells (MNCs) were enumerated from five random coronal sections of the ligated sites in each mouse. RESULTS: Rice peptides REP9 and REP11 significantly inhibited transcription activity of inflammatory and osteoclast-related molecules. Local treatment with the rice peptides, in mice subjected to ligature-induced periodontitis, inhibited inflammatory bone loss, explaining the decreased numbers of osteoclasts in bone tissue sections. CONCLUSION: Therefore, these data suggested that the rice peptides possess a protective effect against periodontitis.

DOI： 10.1016/j.archoralbio.2018.11.021

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Language：English   Publishing type：Research paper (scientific journal)  

Vaccination is an effective strategy to prevent pneumococcal diseases. Currently, licensed vaccines include the pneumococcal polysaccharide vaccine (PPSV) and the pneumococcal conjugate vaccine (PCV), which target some of the most common of the 94 serotypes of S. pneumoniae based on their capsular composition. However, it has been reported that PPSV is not effective in children aged less than 2 years old and PCV induces serotype replacement, which means that the pneumococcal population has changed following widespread introduction of these vaccines, and the non-vaccine serotypes have increased in being the cause of invasive pneumococcal disease. Therefore, it is important that there is development of novel pneumococcal vaccines to either replace or complement current polysaccharide-based vaccines. Our previous study suggested that S. pneumoniae releases elongation factor Tu (EF-Tu) through autolysis followed by the induction of proinflammatory cytokines in macrophages via toll-like receptor 4, that may contribute to the development of pneumococcal diseases. In this study, we investigated the expression of EF-Tu in various S. pneumoniae strains and whether EF-Tu could be an antigen candidate for serotype-independent vaccine against pneumococcal infection. Western blotting and flow cytometry analysis revealed that EF-Tu is a common factor expressed on the surface of all pneumococcal strains tested, as well as intracellularly. In addition, we demonstrate that immunization with recombinant (r) EF-Tu induced the production of inflammatory cytokines and the IgG1 and IgG2a antibodies in mice, and increased the CD4+ T-cells proportion in splenocytes. We also reveal that anti-EF-Tu serum increased the phagocytic activity of mouse peritoneal macrophages against S. pneumoniae infection, independent of their serotypes. Finally, our results indicate that mice immunized with rEF-Tu were significantly and non-specifically protected against lethal challenges with S. pneumoniae serotypes (2 and 15A). Therefore, pneumococcal EF-Tu could be an antigen candidate for the serotype-independent vaccine against pneumococcal infection.

DOI： 10.1016/j.vaccine.2018.11.015

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Authorship：Lead author,　Corresponding author  

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Streptococcus pneumoniae is a leading cause of community-acquired pneumonia. Over the past 2 decades, macrolide resistance among S. pneumoniae organisms has been increasing steadily and has escalated at an alarming rate worldwide. However, the use of macrolides in the treatment of community-acquired pneumonia has been reported to be effective regardless of the antibiotic susceptibility of the causative pneumococci. Although previous studies suggested that sub-MICs of macrolides inhibit the production of the pneumococcal pore-forming toxin pneumolysin by macrolide-resistant S. pneumoniae (MRSP), the underlying mechanisms of the inhibitory effect have not been fully elucidated. Here, we show that the release of pneumococcal autolysin, which promotes cell lysis and the release of pneumolysin, was inhibited by treatment with azithromycin and erythromycin, whereas replenishing with recombinant autolysin restored the release of pneumolysin from MRSP. Additionally, macrolides significantly downregulated ply transcription followed by a slight decrease of the intracellular pneumolysin level. These findings suggest the mechanisms involved in the inhibition of pneumolysin in MRSP, which may provide an additional explanation for the benefits of macrolides on the outcome of treatment for pneumococcal diseases.

DOI： 10.1128/AAC.00161-18

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Language：English   Publishing type：Research paper (scientific journal)  

Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis. We demonstrated that recombinant (r) DnaK, rEF-Tu, and rGAPDH induced significantly higher levels of interleukin-6 and tumor necrosis factor production in peritoneal macrophages and THP-1-derived macrophage-like cells via toll-like receptor 4. Furthermore, the DNA-binding activity of these proteins was confirmed by surface plasmon resonance assay. We demonstrated that pneumococcal DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in macrophages, and might cause host tissue damage and affect the development of pneumococcal diseases.

DOI： 10.1016/j.cellimm.2018.01.006

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Excessive activation of neutrophils results in the release of neutrophil elastase (NE), which leads to lung injury in severe pneumonia. Previously, we demonstrated a novel immune subversion mechanism involving microbial exploitation of this NE ability, which eventually promotes disruption of the pulmonary epithelial barrier. In the present study, we investigated the effect of NE on host innate immune response. THP-1-derived macrophages were stimulated with heat-killed Streptococcus pneumoniae or lipopolysaccharide in the presence or absence of NE followed by analysis of toll-like receptor (TLR) and cytokine expression. Additionally, the biological significance of NE was confirmed in an in vivo mouse intratracheal infection model. NE downregulated the gene transcription of multiple cytokines in THP-1-derived macrophages through the cleavage of TLRs and myeloid differentiation factor 2. Additionally, NE cleaved inflammatory cytokines and chemokines. In a mouse model of intratracheal pneumococcal challenge, administration of an NE inhibitor significantly increased proinflammatory cytokine levels in bronchoalveolar lavage fluid, enhanced bacterial clearance, and improved survival rates. Our work indicates that NE subverts the innate immune response and that inhibition of this enzyme may constitute a novel therapeutic option for the treatment of pneumococcal pneumonia.

DOI： 10.3389/fimmu.2018.00732

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Language：Japanese   Publisher：日本歯科衛生学会  

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Language：Japanese   Publisher：(一社)日本口腔衛生学会  

DOI： 10.5834/jdh.66.3_338

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Other Link： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2016&ichushi_jid=J00440&link_issn=&doc_id=20160524380005&doc_link_id=%2Fdk1dehea%2F2016%2F006603%2F005%2F0338-0343%26dl%3D0&url=https%3A%2F%2Fwww.medicalonline.jp%2Fjamas.php%3FGoodsID%3D%2Fdk1dehea%2F2016%2F006603%2F005%2F0338-0343%26dl%3D0&type=MedicalOnline&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00004_2.gif

Language：Japanese   Publisher：日本歯科衛生学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN ATHEROSCLEROSIS SOC  

Aim: Limited epidemiological studies have investigated the relationship between ischemic vascular disease and periodontitis in non-Western populations. We investigated this relationship in a Japanese cohort by measuring serum titers of antibodies to periodontopathic bacteria.
Methods: As part of the Tokamachi-Nakasato cohort study, we followed up 7021 participants regarding cardiovascular events over 5 years, and observed 99 ischemic vascular events: 66 cerebral infarctions and 33 cases of ischemic heart disease (IHD). For a nested case-control study, we selected 495 sex-and age-matched control subjects. Conditional logistic regression analysis was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) of ischemic vascular events associated with antibody titers to Porphyromonas gingivalis FDC381 and SU63. Multivariable models were adjusted for traditional cardiovascular risk factors using propensity scores.
Results: The highest tertile category of antibody titers to P. gingivalis FDC381 in men was significantly associated with an increased risk of cerebral infarction in only the crude model. The 2nd and 3rd tertile categories of antibody titers to P. gingivalis SU63 were significantly associated with an increased risk of cerebral infarction in men (multivariable ORs (95% CIs) were 7.12 (1.51-33.5) and 9.03 (1.97-41.5), respectively). The association was not appreciably modified when we further adjusted for serum high-sensitivity C-reactive protein levels. Antibody titers to P. gingivalis were not dose-dependently associated with the risk of IHD.
Conclusion: High serum antibody titers to P. gingivalis SU63 could be a predictor of cerebral infarction in Japanese men independent of traditional risk factors and inflammation.

DOI： 10.5551/jat.6957

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Background: Individuals with periodontitis have elevated serum levels of IL-6 and C-reactive protein and have been reported to have a significantly increased risk of developing cardiovascular disease. The transcription factor early growth response factor 1 (Egr-1) has been shown to play an important role in the development and progression of atherosclerosis. However, it is not known whether periodontal infection affects the expression of Egr-1 and subsequent endothelial cells expression of monocyte chemoattractant protein (MCP)-1, a key molecule of leukocyte chemoattraction into vessels. Methods: Human coronary artery endothelial cells (HCAECs) were stimulated with either sonicated extracts from Porphyromonas gingivalis strains 381 or SU63, or a combination of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R). The expression of Egr-1, and subsequently MCP-1, was then analyzed. The role of Egr-1 on MCP-1 expression was analyzed by siRNA transfection. Results: Both P. gingivalis anti-gens and IL-6/sIL-6R stimulations upregulated the expression of Egr-1, with a more robust effect by IL-6/sIL-6R. Increased expression of Egr-1 coincided with MCP-1 production, and Egr-1 downregulation by siRNA suppressed this effect. Conclusion: These results clearly suggest that periodontal infection has the potential to affect HCAECs and hence contribute to the development of subsequent atherosclerosis. Copyright (C) 2009 S. Karger AG, Basel

DOI： 10.1159/000265568

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Publishing type：Research paper, summary (national, other academic conference)  

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J-GLOBAL

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：日本歯科衛生学会  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese  

J-GLOBAL

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：日本歯科衛生学会  

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Language：Japanese   Publisher：日本歯科衛生学会  

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Language：Japanese   Publisher：日本歯科衛生学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：新潟歯学会  

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J-GLOBAL

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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