Accurate quantification of bacterial species in dental plaque is needed for microbiological diagnosis of periodontal diseases. The present study was designed to assess the sensitivity, specificity and quantitativity of the real-time PCR using the GeneAmp Sequence Detection System with two fluorescence chemistries. TaqMan probe with reporter and quencher dye, and SYBR Green dye were used for sources of the fluorescence. Primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and total bacteria based on the nucleotide sequences of the respective 16S ribosomal RNA genes. Since spread of antibiotic resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confers resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans was linear over a range of 10-10(7) cells (10-10(7) copies for tetQ gene), while the quantitative range for total bacteria was 10(2)-10(7) cells. Species-specific amplifications were observed for the three periodontal bacteria, and there was no significant difference between the TaqMan and SYBR Green chemistry in their specificity, quantitativity and sensitivity. The SYBR Green assay, which was simpler than TaqMan assay in its manipulations, was applied to the clinical plaque samples. The plaque samples were obtained from eight patients (eight periodontal pockets) before and 1 week after the local drug delivery of minocycline. Although the number of P. gingivalis, P. intermedia and A. actinomycetemcomitans markedly decreased after the antibiotic therapy in most cases, higher copy numbers of the tetQ gene were detectable. The real-time PCR demonstrated sufficient sensitivity, specificity and quantitativity to be a powerful tool for microbiological examination in periodontal disease, and the quantitative monitoring of antibiotic resistance gene accompanied with the antibiotic therapy should be included in the examination.

Three colonial variants of Actinobacillus actinomycetemcomitans, which formed transparent rough (TR)-, transparent smooth (TS)-, and opaque smooth (OS)-surfaced colonies, were described in relation to their fimbriation. TR- and TS-cells were adhesive to agar and glass surfaces but not the OS-cells. The examination by electron microscopy revealed that TR-cells were highly fimbriated but not TS- and OS-cells. Thus, TS-cells seemed to be an intermediate type. The fimbriae were isolated from TR-cells by suspending in 0.15 M ethanolamine-HCl buffer (pH 10.5) and purified by dissolving non-fimbrial components in 0.5% deoxycholate and 0.7% n-octyl-beta-D-glucopyranoside. The relative molecular mass of the fimbrial subunit protein was 54,000.

There is controversy regarding the existence of archaeal pathogens. Periodontitis is one of the human diseases in which Archaea have been suggested to have roles as pathogens. This study was performed to investigate the distribution of Archaea in Japanese patients with periodontitis and to examine the serum IgG responses to archaeal components. Subgingival plaque samples were collected from 111 periodontal pockets of 49 patients (17 with aggressive periodontitis and 32 with chronic periodontitis), and 30 subgingival plaque samples were collected from 17 healthy subjects. By PCR targeting the 16S rRNA gene, Archaea were detected in 15 plaque samples (13.5% of total samples) from 11 patients (29.4% of patients with aggressive periodontitis and 18.8% of patients with chronic periodontitis). Archaea were detected mostly (14/15) in severe diseased sites (pocket depth > or =6 mm), while no amplicons were observed in any samples from healthy controls. Sequence analysis of the PCR products revealed that the majority of Archaea in periodontal pockets were a Methanobrevibacter oralis-like phylotype. Western immunoblotting detected IgG antibodies against M. oralis in eight of the 11 sera from patients. These results suggest the potential of Archaea (M. oralis) as an antigenic pathogen of periodontitis.

The purpose of this study was to assess the serum antibody levels to periodontal bacteria in patients with periodontal disease, and to explore the diagnostic uses of the serum antibody assessment and its potential as a therapeutic guide. One hundred twenty-nine patients were clinically examined for the type and extent of periodontal destruction and serum IgG antibody levels to Actinobacillus actinomycetemcomitans (Aa), Actinomyces israelii (Ai), A. viscosus (Av), Bacteroides asaccharolyticus (Ba), B. corporis (Bc), B. denticola (Bd), B. gingivalis (Bg), B. intermedius (Bi), B. loescheii (BI), Capnocytophaga gingivalis (Cg), C. ochracea (Co), and Fusobacterium nucleatum (Fn). Clinical and serological data were subjected to correlation analyses. A small group of patients was monitored during the progress of periodontal treatments. The IgG antibody levels were assessed with an enzyme-linked immunosorbent assay (ELISA). Significantly elevated IgG antibody levels were manifested to Aa, Ai, Bg, and Fn in all forms of periodontal disease, additionally to Cg and Co in juvenile periodontitis, and to Bi in adult periodontitis. There were some correlations between a few clinical parameters and the antibody levels. Successful periodontal treatment significantly decreased the antibody levels to all of the micro-organisms; however, during periodontal treatment, there were no marked differences between pre- and post-treatment levels. The antibody reactivities to the periodontopathic micro-organisms may be of diagnostic and predictive value in patients.

SummarySeveral reports have demonstrated a possible association of periodontal infections with coronary heart disease (CHD) by elevated antibody titre to periodontopathic bacteria in CHD patients compared with non-diseased controls. Although each periodontopathic bacterium may vary in virulence for periodontitis and atherosclerosis, antibody response to multiple bacteria in CHD patients has not been understood fully. Therefore, serum levels of antibody to 12 periodontopathic bacteria together with other atherosclerotic risk markers were compared among 51 patients with CHD, 55 patients with moderate to severe chronic periodontitis and 37 healthy individuals. The antibody response was the most prevalent for Porphyromonas gingivalis, a major causative organism, in CHD as well as periodontitis patients. However, antibody positivity was different between CHD and periodontitis if the response was analysed for two different strains of P. gingivalis, namely FDC381 and Su63. While periodontitis patients were positive for both P. gingivalis FDC381 and Su63, a high frequency of antibody positivity for P. gingivalis Su63 but not for FDC381 was observed in CHD patients. The results indicate that the presence of particular periodontopathic bacteria with high virulence may affect atherogenesis. Identifying the virulence factors of P. gingivalis Su63 may gain insight into the new therapeutic modality for infection-induced deterioration of atherosclerosis.

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Objective: Recent studies suggested macrophages were integrated in adipose tissues, interacting with adipocytes, thereby exacerbating inflammatory responses. Persistent low-grade infection by gram-negative bacteria appears to promote atherogenesis. We hypothesized a ligand for tolllike receptor 4 (TLR4), bacterial lipopolysaccharide (LPS), would further exaggerate macrophage-adipocyte interaction. Research Methods and Procedures: RAW264.7 macrophage cell line and differentiated 3T3-L1 preadipocytes were co-cultured using transwell system. As a control, each cell was cultured independently. After incubation of the cells with or without Escherichia coli LPS, tumor necrosis factor (TNF)-␣ and interleukin (IL)-6 production was evaluated. Results: Co-culture of macrophages and adipocytes with low concentration of Escherichia coli LPS (1 ng/mL) markedly up-regulated IL-6 production (nearly 100-fold higher than that of adipocyte culture alone, p Ͻ 0.01), whereas TNF-␣ production was not significantly influenced. This increase was partially inhibited by anti-TNF-␣ neutralizing antibody. Recombinant TNF-␣ and LPS synergistically upregulated IL-6 production in adipocytes. However, this increase did not reach the level of production observed in co-cultures stimulated with LPS. Discussion: A ligand for TLR-4 stimulates macrophages to produce TNF-␣. TNF-␣, thus produced, cooperatively upregulates IL-6 production with other soluble factors secreted either from adipocytes or macrophages in these cells. Markedly up-regulated IL-6 would greatly influence the pathophysiology of diabetes and its vascular complications.

A leukotoxin of Actinobacilus actinomycetemcomitans 301-b was solubilized from cell-associated membrane vesicles by treatment with externally added DNase and RNase and was further purified by a procedure which included ammonium sulfate fractionation, gel filtration chromatography, and ion-exchange chromatography. The purified toxin had a molecular mass of 113,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a high isoelectric point (approximately 8.8). From these characteristics, it was to be expected that the membrane vesicle toxin was almost identical to the leukotoxin extracted with polymyxin B in an earlier study (C.-C.