Molecular and antigenic similarities of the fimbrial major components between Porphyromonas gulae and P. gingivalis.

Porphyromonas gulae is black-pigmented anaerobic bacteria associated with canine periodontitis. There is little information available about the specific identify and relative occurrence of pigmented anaerobes in companion animals. Our aim was to clarify the factor involved in the adherence and colonization of the organism in the oral cavity. Fimbrial protein was purified from P. gulae ATCC 51700. The molecular mass of this protein was approximately 41kDa as estimated by SDS-PAGE. An antibody against 41-kDa fimbrial protein from P. gingivalis ATCC 33277 reacted with fimbrillin of P. gulae ATCC 51700. Immunogold electron microscopy revealed that the anti-41kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. Thus, fimbrial protein of P. gulae ATCC 51700 had the same size and antigenicity as 41-kDa fimbriae of P. gingivalis ATCC 33277. The nucleotide sequence of the fimA gene from P. gulae ATCC 51700 showed 94% homology with that of P. gingivalis ATCC 33277. Moreover, the deduced amino acid sequences have 96.8% identity. P. gulae has adherent ability to gingival epithelial cells. The properties of P. gulae fimbriae are similar to those of P. gingivalis fimbriae. We suggest that the surface structure of P. gulae may play a role in the colonization of this organism in periodontal pockets in companion animals.

Expression of bone extracellular matrix proteins on osteoblast cells in the presence of mineral trioxide.

The biocompatibility of periapical tissue with mineral trioxide aggregate (MTA) affects its ability to repair and regenerate itself. Here we report the cytotoxicity of MTA and how it affects the expression of bone extracellular matrix protein in MC3T3-E1 osteoblast cells. We quantified the cytotoxicity of MTA, amalgam, and Dycal (Dentsply/Caulk, Milford, DE) on MC3T3-E1 cells by measuring the ability of cells to cleave a tetrazolium salt to produce formazan dye during a period of 24, 48, or 96 hours. We used reverse-transcriptase polymerase chain reaction with primer sets for type I collagen, osteocalcin, and bone sialoprotein to measure the gene-expression response of MC3T3-E1 cells treated with MTA. MTA, amalgam, and Dycal were less toxic after 48 hours. MC3T3-E1 cell growth with MTA and Dycal was greater than nonstimulated controls. MTA caused an upregulation of type I collagen and osteocalcin messenger RNA expression after 24 hours. These results showed that, in the presence of MTA, cells grow faster and produce more mineralized matrix gene expression in osteoblasts.

Antifungal effect of acrylic resin containing apatite-coated TiO2 photocatalyst.

The purpose of this study was to develop an acrylic resin with antifungal properties by leveraging the photocatalytic activity of apatite-coated titanium dioxide (Ap-TiO2). Candida albicans was used for antifungal activity assay of the specimen plates under ultraviolet A (UVA) with a black light source. Statistically significant decreases in cell viability in acrylic resins containing 5 wt% and 10 wt% Ap-TiO2 were observed after irradiation for two, four, and six hours (P<0.01), when compared to the control. As for the flexural strength and modulus values of acrylic resins mixed with Ap-TiO2 and TiO2 particles, they varied before and after irradiation. Among the tested specimens, a 5 wt% content of Ap-TiO2 in acrylic resin exceeded the requirements of ISO 1567. It was thus suggested that acrylic resin containing 5 wt% Ap-TiO2 could exert antifungal effects on C. albicans, while at the same time maintain adequate mechanical properties for clinical use.

The r40-kDa outer membrane protein human monoclonal antibody protects against Porphyromonas gingivalis-induced bone loss in rats.

BACKGROUND: Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. We recently reported the construction and characterization of human immunoglobulin G isotype clones, which were specifically reactive with recombinant (r) 40-kDa outer membrane protein (OMP) of P. gingivalis. The aim of this study was to investigate the efficacy of human monoclonal antibody (hMAb) against r40-kDa OMP of P. gingivalis to the protection alveolar bone loss by P. gingivalis in rats. METHODS: The role of 40-kDa OMP in the adherence of P. gingivalis to human gingival epithelial cells (HGECs) was examined by preincubating with r40-kDa OMP hMAb before adding the HGECs. Moreover, we used a rat model to examine the effect of the anti-r40-kDa OMP hMAb in alveolar bone loss by oral infection. Forty-six days after the last infection, the periodontal bone level was assessed morphometrically on defleshed rat jaws. RESULTS: The adherence to HGECs was reduced by 84% compared to adherence levels without the antibody. P. gingivalis could not be detected from rats in a P. gingivalis-non-infected group and a group that was administered the anti-r40-kDa OMP hMAb. The bone loss in P. gingivalis-infected animals that were administered the anti-r40-kDa OMP hMAb was significantly lower than that of P. gingivalis-infected rats. CONCLUSIONS: Our results suggest that transchromosomic mouse-derived hMAb against r40-kDa OMP of P. gingivalis protects against periodontal bone loss. This newly constructed anti-r40-kDa OMP hMAb was used to protect against periodontal diseases caused by P. gingivalis infection.

Porphyromonas gingivalis 67-kDa fimbriae induced cytokine production and osteoclast differentiation utilizing TLR2.

Porphyromonas gingivalis, a major etiological agent of adult periodontitis, has two distinctly different types of fimbriae on the cell surface. The major fimbriae, which consist of a 41-kDa fimbrillin of P. gingivalis ATCC 33277, have been known to induce inflammatory cytokine production in murine peritoneal macrophages. In this study, we examined the effects of the minor fimbriae of P. gingivalis, composed of a 67-kDa fimbrillin, on cytokine production in murine peritoneal macrophages and the ability to induce osteoclast differentiation. Murine peritoneal macrophages were stimulated with P. gingivalis 67-kDa minor fimbriae for 24 h, then the levels of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and IL-6 production were determined by enzyme-linked immunosorbent assay (ELISA). To estimate osteoclast differentiation, mouse osteoclast precursors were placed on dentine slices, and cultured with or without P. gingivalis 67-kDa minor fimbriae for 7 days. P. gingivalis 67-kDa minor fimbriae clearly induced IL-1beta, TNF-alpha and IL-6 production in mouse macrophages. Furthermore, pit formations on the dentine slices were significantly extended when the osteoclast precursors were incubated with P. gingivalis 67-kDa minor fimbriae. Pretreatment with anti-Toll-like receptor 2 (TLR2) antibody significantly inhibited IL-1beta, TNF-alpha and IL-6 induction (P<0.05) in mouse macrophages and pit-forming activity of osteoclast precursor cells stimulated with P. gingivalis 67-kDa minor fimbriae. These results suggest that P. gingivalis 67-kDa minor fimbriae may provoke host inflammatory response and be involved in periodontal tissue breakdown.

Serum antibodies of periodontitis patients compared to the lipopolysaccharides of Porphyromonas gingivalis and Fusobacterium nucleatum.

Serum antibody titers against the lipopolysaccharides (LPSs) of Porphyromonas gingivalis and Fusobacterium nucleatum were compared between 9 periodontitis patients and 24 healthy persons. The IgG titers against the LPSs of P. gingivalis ATCC 33277(T) and W50 were clearly higher in the patients than in the healthy persons. However, IgM titers against the LPSs of P. gingivalis strains were relatively low, and no significant difference was observed between the patients and healthy persons. On the other hand, IgG and IgM titers against the LPS of Fusobacterium nucleatum JCM 8532(T) in some patients were significantly higher than those in the healthy persons, although the difference in IgG titers was not large compared to that of the LPS of P. gingivalis. These results suggest that the antibody measurement of patients' sera against the LPS of periodontal bacteria can be applied for the diagnosis of periodontitis.

Characterization of biologically active cell surface components of a periodontal pathogen. The roles of major and minor fimbriae of Porphyromonas gingivalis.

BACKGROUND: We previously reported that the presence of 2 different types of fimbriae expressed on the cell surface of Porphyromonas gingivalis ATCC 33277. The initial event in most infectious diseases involves adhesion of pathogens to host tissues and subsequent invasion by the pathogens. To define the role of fimbriae in Porphyromonas gingivalis adherence to and invasion of epithelial cells, we have constructed fimbrial mutants. The involvement of P. gingivalis fimbriae in the invasion process and alveolar bone resorption in rats was examined. METHODS: Inactivated mutants of 41-K fimbrillin gene (fimA) and/or the 67-K fimbrillin gene (mfa1) were constructed by a homologous recombination technique and compared among fimA mutant (MPG1), mfa1 mutant (MPG67), and double knockout mutant (MPG4167). Adherence and invasion of P. gingivalis was assessed in human oral epithelial KB cells. We used a rat model to examine the role of each type of fimbriae in alveolar bone loss by oral infection. RESULTS: The adherence and invasion levels of the mutants were lower than the wild-type strain. The bone loss of rats infected with the MPG1 was higher than that of those infected with MPG67. Moreover, the bone loss of rats infected with the double knockout mutant was significantly decreased compared to that of rats infected with the wild-type strain. CONCLUSIONS: Data from this study suggest that not only the 41-K fimbrial protein, but also the 67-K fimbrial protein, play important roles in the pathogenesis of periodontal disease.

Endotoxic properties of free lipid A from Porphyromonas gingivalis.

The relationship between chemical structure and biological activity of the lipid A from Porphyromonas gingivalis, which we recently isolated and whose complete chemical structure was determined [Kumada et al. (1995). J Bacteriol 177, 2098-2106], was studied. The lipid A exhibited endotoxic activity in all the assay systems tested: Limulus gelation activity, lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells and induction of nitric oxide (NO) and tumour necrosis factor alpha (TNF) release from both mouse peritoneal macrophages and the J774-1 mouse macrophage-like cell line. The activity was, however, about 100-fold less than that of Salmonella minnesota LPS used as a control. The moderate activity of the lipid A may be partially explained by its unique fatty acid composition and the lack of a phosphate group in position 4. In contrast, the lipid A as well as whole LPS of P. gingivalis unexpectedly exhibited an even stronger induction of TNF from the human monocytic THP-1 cell line than control LPS when measured by the minimum stimulatory dose. The difference in sensitivity of human and mouse cells to P. gingivalis lipid A suggests that the recognition mechanism, including that for the receptor for endotoxin, may be regulated in different ways in the two cells.