Porphyromonas gingivalis Fimbria-Dependent Activation of Inflammatory Genes in Human Aortic Endothelial Cells

Epidemiological and pathological studies have suggested that infection with the oral pathogen Porphyromonas gingivalis can potentiate atherosclerosis and human coronary heart disease. Furthermore, infection with invasive, but not noninvasive P. gingivalis has been demonstrated to accelerate atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice and to accelerate local inflammatory responses in aortic tissue. In the present study, using high-density oligonucleotide microarrays, we have defined the gene expression profile of human aortic endothelial cells (HAEC) after infection with invasive and noninvasive P. gingivalis. After infection of HAEC with invasive P. gingivalis strain 381, we observed the upregulation of 68 genes. Genes coding for the cytokines Gro2 and Gro3; the adhesion molecules intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule (VCAM)-1, and ELAM-1 (E-selectin); the chemokine interleukin-8 (IL-8); and the proinflammatory molecules IL-6 and cyclooxygenase-2 were among the most highly upregulated genes in P. gingivalis 381-infected HAEC compared to uninfected HAEC control. Increased mRNA levels for signaling molecules, transcriptional regulators, and cell surface receptors were also observed. Of note, only 4 of these 68 genes were also upregulated in HAEC infected with the noninvasive P. gingivalis fimA mutant. Reverse transcription-PCR, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorting analysis confirmed the expression of ICAM-1, VCAM-1, E-/P-selectins, IL-6, and IL-8 in HAEC infected with invasive P. gingivalis. We also demonstrated that increased expression of ICAM-1 and VCAM-1 in aortic tissue of ApoE(-/-) mice orally challenged with invasive P. gingivalis but not with the noninvasive P. gingivalis fimA mutant by immunohistochemical analysis. Taken together, these results demonstrate that P. gingivalis fimbria-mediated invasion upregulates inflammatory gene expression in HAEC and in aortic tissue and indicates that invasive P. gingivalis infection accelerates inflammatory responses directly in the aorta.


Three-dimensional quantification of alveolar bone loss in Porphyromonas gingivalis-infected mice using micro-computed tomography

Animal models are routinely used for the study of the pathogenesis of periodontal disease. However, some of the methods used for evaluating alveolar bone loss are limited to linear or two dimension measurements, while other methods, such as histology, are labor consuming. The present study was designed to evaluate a novel method for assessing the volume of alveolar bone loss in mice and to compare it to the traditional morphometric (linear) technique. Seven- to 8-week-old BALB/c mice were divided into three equal groups of six each; two groups were infected orally with Porphyromonas gingivalis (P. gingivalis) 381 or 53,977, while the third group was used as non-infected control. Forty-two days following infection, serum samples were obtained and maxillae were harvested. Bone loss was evaluated by micro-computed tomography (micro-CT) and by the morphometric technique. Significant decrease in residual supportive bone volumes (RSBV) was observed in both P. gingivalis-infected groups compared to the control group (P<0.0001). The P. gingivalis 53,977-infected group showed less residual supportive bone volume compared to the P. gingivalis 381-infected group, but there were no significant differences between these two groups. Using the morphometric technique, the differences between the three groups were not found to be statistically significant (P>0.05). The present study describes a novel micro-CT technique for volumetric evaluation of alveolar bone loss in mice. This technique is relatively simple and accurate, and due to its high sensitivity, enables the investigator to reduce the number of animals needed in each experimental group.


Isolation and characterization of a plasmid DNA from periodontopathogenic bacterium, Eikenella corrodens 1073, which affects pilus formation and colony morphology

Eikenella corrodens (Ec) is one of a group of periodontopathogenic bacteria. A plasmid DNA (8.7 kb) isolated from Ec 1073 was designated pMU1. Agarose gel electrophoresis and Southern analysis suggested that pMU1-like plasmids were carried in 2 Ec strains, including 1073, with higher hemagglutination (HA) activity than other strains. We determined the nucleotide sequence of this plasmid and identified 7 ORFs. A homology search revealed that 4 ORFs of pMU1 were homologous to ORFs in pJTPS1, found in a spontaneous avirulent mutant of the phytopathogenic bacterium, Ralstonia solanacearum. pJTPS1 is a putative hypovirulent plasmid, which is thought to control the virulence of R. solanacearum. We also found the ORF to be homologous to the recombinase specific to the type IV pilin gene. We introduced a part of pMU1 into the Ec 23834 strain, which has a pilus structure on its cell surface and forms corroding colonies on solid medium. No pilus structure was observed on the surface of transformants, most of which formed non-corroding colonies. When such transformants (or Ec 1073) were cured of pMU1 with acridine orange, they remained non-foliated and non-corroding. The results suggest that pMU1 might irreversibly affect pilus formation and colony morphology, and might be involved in the pathogenicity and virulence of Ec.


Innate Immune Recognition of Invasive Bacteria Accelerates Atherosclerosis in Apolipoprotein E-Deficient Mice

Infectious diseases have emerged as potential risk factors for cardiovascular disease (CVD). Epidemiological studies support a connection between periodontal disease, a chronic inflammatory disease of the supporting tissues of the teeth, and CVD. To directly test the connection between periodontal disease and atherosclerosis, apoE-/- mice were orally challenged with the periodontal disease pathogen Porphyromonas gingivalis or an invasion-impaired P gingivalis fimbriae-deficient mutant (FimA-). Both wild-type P gingivalis and the FimA- mutant were detected in blood and aortic arch tissue of apoE-/- mice by PCR after challenge. ApoE-/- mice challenged with wild-type P gingivalis presented with increased atherosclerotic plaque and expressed the innate immune response markers Toll-like receptor (TLR)-2 and TLR-4 in aortic tissue. Despite detection of the FimA- mutant in the blood and in aortic arch tissue, apoE-/- mice challenged with the FimA- mutant did not present with periodontal disease, upregulation of TLRs, or accelerated atherosclerosis. Furthermore, we demonstrate that immunization to control P gingivalis-elicited periodontal disease concomitantly prevents P gingivalis-accelerated atherosclerosis. We conclude that invasive P gingivalis accelerates atherosclerosis.


An immunohistological study of the localization of bacteria invading root pulpal walls of teeth with periapical lesions

We immunohistologically examined the prevalence and localization of bacteria invading dentinal tubules of the roots of teeth with infected canals. Forty extracted teeth with apical lesions were selected and divided into two groups: a group of untreated teeth and a group of canal-enlarged teeth. The bacteria in the specimens were detected by Brown-Brenn stain and the labeled-streptavidin-biotin method with specific antisera for 16-bacteria. Seventy percent of the examined teeth showed bacteria invading the dentinal tubules of the roots. Fusobacterium nucleatum, Eubacterium alactolyticum, E. nodatum, Lactobacillus casei, and Peptostreptococcus micros were abundant. Even in the canal-enlarged group, invasion of bacteria was observed in 65% of teeth. This study revealed the actual condition of bacteria in infected root dentin and suggested that the canal-enlargement procedure could not completely remove all the bacteria in the infected dentinal tubules of the root.


N-acetyl-D-galactosamine specific lectin of Eikenella corrodens induces intercellular adhesion molecule-1 (ICAM-1) production by human oral epithelial cells

During the acute inflammatory response in periodontitis, gingival epithelial cells are considered to play important roles in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known about the expression of molecules that are involved in the interaction between the epithelium and neutrophils following bacterial attachment. Earlier work reported that periodontopathogenic Eikenella corrodens strain 1,073 up-regulated the expression and secretion of chemokines such as interleukin-8 (IL-8) from KB cells (a human oral epithelial cell line derived from a human oral epidermoid carcinoma). To elucidate the mechanism of the transmigration of neutrophils through the epithelium, the present study investigated the expression of adhesion molecules on KB cells in response to E. corrodens attachment. Adhesion molecule gene expression was assessed by RT-PCR and adhesion proteins expressed on KB cell surfaces were determined by cell-based ELISA and FACS. In RT-PCR, ICAM-1 mRNA levels were significantly increased within 1 h in response to exposure to E. corrodens and continued to increase over the 12-h period of study. In ELISA, increased surface ICAM-1 expression was paralleled by increased ICAM-1 mRNA levels. Furthermore, the increases in ICAM-1 expression on epithelial cells infected with E. corrodens were observed to be due to the N-acetyl-D-galactosamine (GalNAc) specific bacterial lectin-like substance of E. corrodens (EcLS), which was one of the adhesins of E. corrodens. This is the first study to report that a bacterial lectin-like substance increased the expression of ICAM-1 on gingival epithelial cells.


Macrophage Inflammatory Protein 3α-CC chemokine receptor 6 interactions play an important role in CD4+ T-cell accumulation in periodontal diseased tissue.

The regulatory role of chemokines and chemokine receptors on specific lymphocyte recruitment into periodontal diseased tissue is poorly characterized. We observed that lymphocytes infiltrating inflamed gingival tissue expressed marked levels of CCR6. In periodontal diseased tissue, the expression of MIP-3alpha mRNA was detected by RT-PCR and further, MIP-3alpha was distributed in the basal layer of gingival epithelial cells, microvascular endothelial cells and the areas of inflammatory cells as shown by immunohistochemistry. Moreover, CCR6-expressing cells infiltrated into periodontal diseased tissue, and the proportion of CCR6-positive CD4+ T cells was significantly elevated in periodontal diseased tissue compared with peripheral blood in the same patients. Furthermore, gingival lymphocytes isolated from patients showed migration toward MIP-3alpha in an in vitro chemotaxis assay in which migration was abrogated by specific antibody to CCR6. Thus, these findings suggested that CCR6 and the corresponding chemokine, MIP-3alpha may have an important regulatory role in specific lymphocyte migration into inflamed periodontal tissue.


Soluble products from Eikenella corrodens stimulate oral epithelial cells to induce inflammatory mediators

In the inflammatory response elicited by bacterial colonization in periodontal pockets, pocket epithelial cells not only serve as a barrier to isolate the pocket microenvironment from external stimuli but also regulate the functions of neighboring cells including fibroblasts and inflammatory cells. To elucidate this mechanism, we characterized the effects of periodontopathic bacterium Eikenella corrodens 1073 components on the production of some inflammatory mediators in a human oral epithelial cell line (KB). In enzyme-linked immunosorbent assay (ELISA), the E. corrodens supernatant induced interleukin-6 (IL-6), IL-8 and prostaglandin E2 but not interferon-gamma (IFN-gamma) production by KB cells. After incubation with E. corrodens supernatant, KB cells showed a marked increase in the levels of IL-6, IL-8 and PG G/H synthase (cyclooxygenase)-2, but not IFN-gamma, gene expression by reverse-transcriptase polymerase chain reaction. All these E. corrodens products responsible for production of these inflammatory mediators resisted freezing and boiling and were present in a 10-kDa filtrate. These results imply that these soluble small-molecular-mass products from E. corrodens stimulate various inflammatory mediator productions by human oral epithelial cells and may play a role in the initiation of periodontal inflammation and subsequently perpetuate the inflammatory response during chronic infection.


Three-dimensional structural analysis of histone-like protein from streptococci

レンサ球菌のヒストン様蛋白質の構造解析


Interleukin-6 (IL-6) and IL-8 are induced in human oral epithelial cells in response to exposure to periodontopathic Eikenella corrodens


X-ray Microanalysis of Resin-dentin Adhesive Interfaces

X線微小分析法による象牙質接着界面の解析

The Japanese Journal of Conservative Dentistry


Cloning, sequencing and expression of an Eikenella corrodens gene encoding a component protein of the lectin-like adhesin complex


Molecular analysis of the gene encoding a protein component of the Eikenella corrodens adhesin complex that is close to the carbohydrate recognition domain


Mitogenic stimulation of murine B lymphocytes by the N-acetyl-D-galactosamine specific bacterial lectin-like substance from Eikenella corrodens


Conception of Infected Root Canal Treatment

It is a well-established fact that bacterial infection of the dental pulp ultimately results in the formation of apical periodontitis. And apical periodontitis may develop periapical leisons consisting granulomas and cysts. In this article, we show the pathologic condition of infected root canal systems and periapical lesions immunohistologically. We also describe the possible causes of refractory apical periodontitis that conventional root canal therapy could not work effectively. Finally,we explain our newly developing treatment for refractory apical periodontitis named electro-magnetic apical treatment, and illustrate clinical efficacy of this treatment.


Streptococcus intermedius biofilm componentsの糖鎖結合能と炎症反応誘導能に関する解析


水溶性MPCポリマーの口腔細菌付着抑制効果

水溶性MPCポリマーの口腔細菌付着抑制効果

口腔細菌によるbiofilm，すなわちdental plaqueの形成にはいくつかのkey stepがあり，dental plaque形成の抑制のためにはそれらのkey stepに効果的に働く方法を選択する必要が有る．我々は，超親水性表面を得ることができる2-methacryloloxethyl phosphorylcholine(MPC)co-polymerを用いてプラスチック表面をコートした界面は微生物の付着抑制に関与することを報告してきた．本実験では5%水溶性MPCポリマーで処理したHydroxyapatite(HY)プレートと培養歯肉上皮細胞(RT-7)へのStreptococcus mutansの付着状況を顕微鏡下にて観察した．その結果，5%水溶性MPCポリマー処理でHYプレート及び培養歯肉上皮細胞上へのS.mutansの付着は抑制されることが示された．以上よりHYプレートや培養歯肉上皮細胞に吸着した水溶性MPCポリマーは，プラスチックへのコーティングと同様にS.mutansの付着抑制効果を発揮することが示唆された．


歯周病患者に対する禁煙指導について --- ニコチネルの応用

歯周病患者に対する禁煙指導について --- ニコチネルの応用


臼歯部の動揺を伴う歯周病患者への対応 --- 特にメインテナンスにおける歯科衛生士の役割


更年期障害を有する歯周病患者の一症例

更年期障害を有する歯周病患者の一症例


口腔がん患者における歯周治療および管理の重要性

口腔がん患者における歯周治療および管理の重要性


喫煙習慣を持つ歯周病患者への対応

喫煙習慣を持つ歯周病患者への対応


A case of severe necrotizing ulcerative gingivitis with feeding difficulty


Biological Roles of Extracellular DNA in Bacterial Infection

Asean Plus and Tokushima Joint International Conference, Program and Proceeding Book


High prevalence of Pseudomonas aeruginosa from oropharyngeal biofilm in patients with cerebrovascular infarction and dysphagia.