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The interaction of oral bacteria with host cells may play an important role in the pathogenesis of periodontal disease. The purpose of this study was to investigate the adherence mechanism of Eikenella corrodens 1073 to guinea pig peritoneal macrophages in the absence or presence of anti-bacterial antibodies. The first experiments were focused on the mechanism of E. corrodens adherence to macrophages in the absence of antibodies and complements. The adherence was dependent on time, bacterial concentration and pH. The adherence was inhibited by ethylendiaminetetraacetic acid and restored by adding calcium ions. Heating (100°C for 5 min) or protease (trypsin or pronase) treatment of bacterial cells abolished their capacity to adhere to macrophages. The specificity of E. corrodens adherence to macrophages was also proved using a hapten inhibition assay with saccharides. Among the sugars tested, the adherence was sensitive to N-acetyl-D-galactosamine (GalNAc)- and D-galactose (Gal)-containing sugars. GalNAc was the most potent inhibitor, being 10 times greater than Gal. These results suggest that the adherence of E. corrodens to macrophages in the absence of antibodies and complements may require the interaction between bacterial cell surface lectins and GalNAc-like receptors on the surface of macrophages. The second group of experiments was aimed at comparing the adherence mechanism of anti-body-nontreated E. corrodens to macrophages with that of antibody-treated E. corrodens. The adherence of antibody-nontreated E. corrodens reached a plateau after incubation for 10 mm, whereas that of antibody-nontreated E. corrodens reached a plateau after incubation for 30 mm. The adherence of antibody-nontreated E. corrodens was completely inhibited by GaINAc, whereas the adherence of antibody-treated E. corrodens was only slightly inhibited by GaINAc. Treatment of macrophages with heat aggregated IgG before incubation with antibody-treated E. corrodens caused an 80% decrease in adherence. These results show that E. corrodens adherence to GaINAc-like receptors on the macrophage surface via bacterial lectin in the absence of antibodies is quite different from the mechanism of E. corrodens adherence to Fc receptors in the presence of bacterial antibodies. [ABSTRACT FROM AUTHOR]

The article defines functional T cell subsets by using monoclonal antibodies to obtain more knowledge of T cell regulatory mechanisms on periodontal lesions. Advanced human periodontitis was presumed to be B cell lesion including many plasma cells, but many T cells were detected in conjunction with plasma cells located subjacent to the pocket epithelium. The activated T cells could serve as effector cells with the release of soluble factors including inflammatory lymphokines as well as immune regulation.

Extracts of gingival tissue from periodontal patients can generate chemotactic factors from guinea pig serum for mononuclear leukocytes. Both the classical and alternative pathways of complement activation are triggered. The data do not preclude the fact that other components of the gingival extract (bacterial and/or tissue enzymes, immune complexes and endotoxin) may also play a role in the generation of chemotactic factors from complement. [ABSTRACT FROM AUTHOR]

Extracts of gingival tissue obtained from periodontal patients, but not from patients with clinically healthy tissue, were found to be chemotactic for human mononuclear cells. The chemotactic activity of the extracts could be suppressed by anti-C5 serum. The data obtained suggest that C5-related compounds are major factors in the chemotaxis of mononuclear cells in periodontal disease. [ABSTRACT FROM AUTHOR]

Adenosine has been reported to alter a variety functions of the cells that participate in inflammatory responses. However, the effect(s) of adenosine on human gingival fibroblasts (HGF), one of the immunomodulator cells in inflamed periodontal lesions, remains to be established. In this study, we examined the influence of adenosine on the production of interleukin (IL)-6 by HGF. Ligation of adenosine receptors with adenosine or its related analogue, 2-chloroadenosine (2-CADO), increased IL-6 production by HGF without any other stimuli. In addition, adenosine and 2-CADO enhanced the cyclic AMP (cAMP) level in HGF as did prostaglandin E1 (PGE1) and forskolin. Interestingly, these cAMP-arising reagents and the permeable cAMP analogue, dibutyryl cAMP (dbtcAMP), also increased IL-6 production by HGF. These results suggest that cAMP is involved in adenosine-induced IL-6 production by HGF. Adenosine-induced IL-6 production was suppressed by protein kinase A (PKA) inhibitor, H89, indicating that cAMP/PKA pathway is involved in the induction. Moreover, the experiments using antagonists specific for adenosine receptor subtypes revealed that the adenosine-induced IL-6 production by HGF was, at least in part, mediated by the adenosine A2b receptor. These results provide new evidence for the possible effects of adenosine or its related analogue as an immunomodulator in inflammatory periodontal lesions.

The present study was carried out to examine the antigen-presenting cell (APC) functions of human gingival fibroblasts (HGF) elicited with IFN gamma. Stimulation of HGF with IFN gamma clearly induced HLA-DR expression and enhanced expression of intercellular adhesion molecule-1 (ICAM-1) on HGF. Despite the phenotypical resemblance of IFN gamma-treated HGF to so-called APC, HLA-DR positive HGF were unable to induce proliferation of allo-reactive peripheral blood T cells (PBT) isolated from different donors. The failure of IFN gamma-treated HGF to stimulate unprimed allo-reactive PBT was not due to the lack of production of IL-1 or the immunosuppressive effect of PGE2 from HGF. On the other hand, the fact that detectable expression of CD80, ligand for CD28, was not found on IFN gamma-treated HGF may at least in part explain the ineffective function of HGF as APC. Interestingly, IFN gamma-treated HGF induced proliferation of primed allo-reactive CD4+ T cells in a HLA-DR dependent manner, suggesting that IFN gamma-treated HGF may have the ability to stimulate pre-activated T cells. We then confirmed that high levels of IFN gamma mRNA were detectable in inflamed gingival tissue. Although it cannot be concluded from this study that HGF are incapable of effectively presenting antigenic peptides to autologous T cells bearing appropriate T cell receptors, present results suggest that HGF may be affected by locally-secreted IFN gamma and that the IFN gamma-stimulated HGF may play a role in regulating immune responsiveness in inflammatory periodontal lesions.

A lectin-like substance (EcLS) was purified from the Eikenella corrodens 1073 cell and monoclonal antibodies were produced against it to confirm the role of EcLS in adhesive properties of E. corrodens such as hemagglutination and coaggregation with oral bacteria. Four hybridoma clones were selected. Two of the antibodies were of the IgG1 isotype and the others were of the IgG2b isotype. These monoclonal antibodies inhibited both the hemagglutination of E. corrodens and the coaggregation with Actinomyces viscosus or Streptococcus sanguis. The reactivity of the monoclonal antibody to E. corrodens 1073 was significantly higher than that to E. corrodens 1080 of which adhesive activity was weaker than that of E. corrodens 1073. These findings suggest the difference in adhesive properties is due to the difference in the amount of EcLS expressed on the cell surface. The immunoelectron microscopic study revealed that EcLS of E. corrodens 1073 was localized in the outer space of outer membrane, not in cell surface appendages such as fimbriae where bacteria possessed adhesin. These results suggest that coaggregation of E. corrodens with A. viscosus or S. sanguis was mediated by EcLS.

The mechanism of aggregation of Eikenella corrodens 1073 with E. corrodens aggregating factor (EcAF) which was purified from submandibular-sublingual (SM-SL) saliva was investigated. Heating (100 degrees C, 10 minutes) or protease treatment of E. corrodens cells abolished the aggregating activity. The aggregation was inhibited by adding N-acetyl-D-galactosamine (GalNAc) and saccharides which contain a galactose residue at the non-reducing end. The aggregating activity was sensitive to EDTA and was restored by Ca2+ but not by Mn2+ or Mg2+. Neuraminidase treatment of EcAF increased their ability to aggregate. E. corrodens, suggesting that the sialic acids on EcAF interfere with aggregation. These data suggest that the aggregation of E. corrodens 1073 with EcAF is mediated by specific interactions between a bacterial cell surface lectin-like substance and a complementary GalNAc-like receptor. EcAF also aggregated 16 strains of oral bacteria including periodontopathic bacteria such as Porphyromonas (Bacteroides) gingivalis 381 and Actinobacillus actinomycetemcomitans ATCC29522; however, those aggregations were not inhibited by GalNAc. Therefore, EcAF appears to have more than two types of bacterial binding site and plays important roles in accumulation of dental plaque by forming a complex network of plaque bacteria including periodontopathic strains.

The periodontopathic ability of Eikenella corrodens 1073-R (EcR) on ligature-induced periodontal defects in immunosuppressed and non-immunosuppressed rats was studied bacteriologically and histologically. Silk ligatures were placed around the maxillary second molars of rats, and the rats were divided into eight groups. Four groups received cyclophosphamide (CY), an immunosuppressive agent, the day after ligature placement. On days 3 and 4, the oral cavities of the rats in all four groups were inoculated with 8 x 10(8) cells of live EcR. Rats were sacrificed on d 1, 3, 5, 8, 11 and 18. Alveolar bone resorption of ligated rats was enhanced by the implantation of EcR, although the number of total cultivable bacterial cells from ligated sites was not changed by the implantation of EcR. CY suppressed the number of white blood cells, inflammatory cellular infiltrates in gingival tissue, and repair of periodontal tissue, and enhanced bone destruction. The implantation of EcR in the ligature- and CY-treated rats also enhanced osteoclastic bone resorption. Without ligatures, high doses of CY or EcR inoculum did not result in periodontal destruction. These results indicate that it is possible to establish EcR in conventional rat flora with a ligature and that EcR causes osteoclastic bone resorption in this model.

PT-01, a controlled-release insert, was developed for topical chemotherapy in periodontal disease. It is a soluble insert that consists of fast-release and sustained-release parts containing ofloxacin (OFLX) as an antibacterial agent. In this study, the release profile of OFLX from PT-01 was investigated in vitro. Twelve adult volunteers were administered OFLX as PT-01 or as an aqueous solution into their periodontal pockets, OFLX concentrations in gingival crevicular fluid (GCF) were evaluated from the viewpoint of pharmacokinetics. The in vitro release profile of OFLX from PT-01 showed a biphasic pattern. The release rate of OFLX was relatively rapid in the early phase and slow thereafter. When OFLX aqueous solution was administered into periodontal pockets, the OFLX level in GCF rapidly decreased to be about 1/100 after 30 minutes. When PT-01 was inserted into the pockets, the OFLX level in GCF immediately reached a peak (about 12 mg/ml), and gradually decreased until the 3rd day, and maintained a constant level above 2 micrograms/ml, the effective minimum antibacterial concentration for periodontopathic microorganisms, from the 3rd to 7th day after insertion. No side-effects were observed in the volunteers who received the PT-01 insert. The above results suggest that PT-01 is a suitable pharmaceutical preparation for periodontal chemotherapy.