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Methanobrevibacter oralis is an archaeal species frequently isolated from sites of severe periodontitis. However, its pathogenic roles remain unclear. Here, we aimed to isolate group II chaperonin from M.?oralisand examine its antigenicity. The genes encoding two chaperonin subunits (Cpn-1 and Cpn-2) were cloned from M.?oralisusing polymerase chain reaction and genome walking procedures. Recombinant proteins Cpn-1 and Cpn-2 were generated, and the reactivities of sera from patients with periodontitis were examined by Western immunoblotting. The open reading frames of Cpn-1 and Cpn-2 genes consisted of 1641 and 1614 base pairs, respectively. Putative ATP-binding domains conserved among the chaperonin family were observed in both genes. The deduced amino acid sequences of the two genes showed 28.8-40.0 identity to each of the subunits of human CCT (CCT1-8). Thirty and 29 of 36 patients' sera reacted with the recombinant Cpn-1 and recombinant Cpn-2, respectively. Western immunoblotting using antiserum against human CCT subunits indicated that anti-CCT3 and anti-CCT8 antibodies recognized recombinant Cpn-1. In addition, anti-CCT1, CCT3, CCT6, and CCT8 antibodies recognized an antigen of approximately 60?kDa in M.?oralis. The results suggested that the chaperonin subunits of M.?oraliswere antigenic molecules that were recognized by periodontitis patients and that may cross-react with human chaperonin CCT.

To detect oral motile bacteria directly from dental plaque, specific PCR primers for Centipeda periodontii and Selenomonas sputigenawere designed based on the sequence analysis of their 16S rDNA. The primers were specific and sensitive enough to amplify DNA fragments from the available oral bacteria. The detection limit was fewer than 10 bacterial cells per sample. It was also possible to detect these bacteria in dental plaque. The prevalence of these bacteria varied in each sample. The specific primers designed in this study may clarify the epidemiology of periodontal disease.

Subtractive hybridization was employed to isolate specific genes from virulent Porphyromonas gingivalis strains that are possibly related to abscess formation. The genomic DNA from the virulent strain P. gingivalis 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 Bacillus stearothermophilus IS5377, 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 IS1598, 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. IS1598 was detected in P. gingivalis W83, W50, and FDC 381 by Southern hybridization. All three P. gingivalis strains have been shown to possess abscess-forming ability in animal models. However, IS1598 was not detected in avirulent strains of P. gingivalis, including ATCC 33277. The IS1598 may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS1598 isolated in this study is a novel insertion element which might be a specific marker for virulent P. gingivalis strains.

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 kappaB (NF-kappaB) 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-alpha) 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-kappaB was not detected in the cytoplasm of undifferentiated THP-1 cells but accumulated in the cytoplasm of the cells following differentiation. No TNF-alpha 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-alpha than stimulation of nondifferentiated cells. This hyperresponsiveness to LPS was found in the mRNA and secreted TNF-alpha levels. Furthermore, stimulation with LPS induced the translocation of NF-kappaB 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-kappaB in the nucleus were higher. The DNA-bindable NF-kappaB was identified as a heterodimer of p65 and p50. The results suggest that NF-kappaB 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-alpha, by mature macrophages following LPS challenge.

OBJECTIVE: To understand the effects of aging on cellular motility of human periodontal ligament (PDL) cells, and to determine the possible link between cell proliferation and migration in relation to cellular aging.

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.

OBJECTIVE: The aim of this study is to compare the in vitroproliferative capacity of periodontal ligament (PDL) cells from aged and juvenile donors.

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.

Inflammatory mediators produced by cells in the gingiva have been implicated in the initiation and progression of periodontal disease, a common infectious disease. In this study, we examined the biological activity of neutrophil chemotactic factors and the kinetics of expression of interleukin-8 (IL-8) mRNA derived from normal gingival fibroblasts in response to inflammatory mediators in an in vitro model. Gingival fibroblasts stimulated by either recombinant human interleukin-1 beta or recombinant human tumor necrosis factor alpha produced neutrophil chemotactic factors after 4 h, whereas expression of cell-derived IL-8 mRNA was detected within 1 h after stimulation. Furthermore, in a neutralization assay, rabbit anti-recombinant human IL-8 antiserum inhibited neutrophil chemotactic activity to basal levels. These results provide evidence that gingival fibroblasts synthesize potent chemotactic factors such as IL-8 in the presence of the inflammatory mediators interleukin-1 beta and tumor necrosis factor alpha. The activity of these factors may contribute to neutrophil-mediated processes in the pathogenesis of periodontal disease.

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.