Tannerella forsythia-induced alveolar bone loss in mice involves leucine-rich-repeat BspA protein.

Tannerella forsythia (formerly Bacteroides forsythus) is one of the periodontal pathogens recently implicated in the development of periodontal disease. The cell-surface-associated, as well as the secreted, leucine-rich-repeat protein (BspA) of this bacterium have been suggested to play roles in bacterial adherence, and also in inflammation, by triggering release of pro-inflammatory cytokines from monocytes and chemokines from osteoblasts, leading to inflammation and bone resorption. In this study, we sought to determine the pathogenic potential of T. forsythia and the in vivo role of the BspA protein in pathogenesis in the mouse model of infection-induced alveolar bone loss. The results showed alveolar bone loss in mice infected with the T. forsythia wild-type strain, whereas the BspA mutant was impaired. In conclusion, evidence is presented in support of T. forsythia as an important organism involved in inducing alveolar bone loss, and the BspA protein is an important virulence factor of this bacterium.

Immunostimulatory activity of LT-IIa, a type II heat-labile enterotoxin of Escherichia coli.

Certain bacterial molecules potentiate immune responses to parenterally administered antigens. One such molecule that has been intensely investigated is cholera toxin, a type I heat-labile enterotoxin produced by the Gram-negative bacterium Vibrio cholerae. Immunization with a mixture of a foreign antigen and cholera toxin enhances the immune response to the antigen. Similar adjuvant activity is associated with LT-I, a closely related type I heat-labile enterotoxin produced by Escherichia coli. The adjuvant activities of LT-IIa, a member of the type II heat-labile enterotoxins produced by E. coli, have not been described. LT-IIa and CT differ significantly in amino acid sequence of the B polypeptides and in receptor binding affinity. In this study, rats were subcutaneously immunized with fimbrillin, a protein isolated from the bacterium Porphyromonas gingivalis, and with fimbrillin in combination with LT-IIa, the prototypical type II enterotoxin. Previous studies documented that fimbrillin administered alone is a poor immunogen. Animals immunized with the mixture of fimbrillin and LT-IIa produced high titers of specific IgG antibody directed against fimbrillin. Anti-fimbrillin antibody titers in sera from animals receiving the combination of LT-IIa + fimbrillin were comparable to those obtained from sera of animals immunized with cholera toxin + fimbrillin. The results of these experiments demonstrate that LT-IIa exhibits an adjuvant activity that is equal to that of cholera toxin. Recombinant methods have been established for producing large amounts of LT-IIa, an advantage that will likely provide an economic impetus to consider incorporating the enterotoxin as an immunostimulatory agent in future vaccines.

Periodontopathic potential of two strains of Porphyromonas gingivalis in gnotobiotic rats.

Germ-free rats were monoinfected with Porphyromonas gingivalis strains 381 or A7A1-28 for 42 or 84 days. Both strains induced substantial destruction of alveolar bone and soft tissue when compared to non-infected controls, but the patterns were different. Strain A7A1-28 was associated with increased activity of host collagenase and gelatinase at 42 days, whereas the activity was elevated to a lesser extent at 84 days. Strain 381 showed a moderate increase in host proteinase activity at 42 days, and this remained unchanged until day 84. Strain A7A1-28 was associated with more bone loss than strain 381 by a morphometric analysis that detects horizontal bone loss in the maxilla. Strain 381 was associated with more bone loss than strain A7A1-28 by a radiographic method that detects vertical intrabony defects in the mandible. Infection with one strain gave rise to serum and salivary antibodies strongly reactive to the infecting strain and moderately reactive to antigens from the other strain. This indicates that some antigenic similarity exists between the strains and that there are also strain or perhaps serotype differences in antibody responses induced by infection. Thus two strains of P. gingivalis differing in antigenicity and pathogenicity in the mouse model of the subcutaneous abscess cause substantial periodontal destruction in the germ-free rat. The disease pattern is, however, different, with strain A7A1-28 inducing mostly horizontal bone loss and strain 381 mostly vertical.

Immunization with Porphyromonas (Bacteroides) gingivalis fimbriae protects against periodontal destruction.

Adhesive fimbriae from Porphyromonas gingivalis are cell surface structures which may be important in the virulence of this oral pathogen and thus may serve as a critical or target antigen. Immunization with highly purified 43-kDa fimbrial protein protected against periodontal tissue destruction when tested in the P. gingivalis-infected gnotobiotic rat model. A similarly highly purified 75-kDa cell surface component did not provide protection. Heat-killed whole-cell and sonicated cell surface extracts which contain the 43-kDa protein as well as the 75-kDa component were protective also. This study indicates that the fimbrial protein may serve as a model for the development of effective vaccines against periodontitis, a major human oral disease.

Periodontal bone level and gingival proteinase activity in gnotobiotic rats immunized with Bacteroides gingivalis.

Bacteroides gingivalis is associated with various forms of periodontal disease. To assess the role of the immune response in modulating B. gingivalis-associated periodontal disease, the effect of immunization of B. gingivalis-induced periodontal bone loss was evaluated in gnotobiotic rats. Male Sprague-Dawley rats immunized with various doses of whole cells or sham-immunized with incomplete Freund's adjuvant were monoinfected with B. gingivalis in carboxymethylcellulose by gavage. Two additional groups served as either sham-immunized or untreated germ-free controls. Forty-two days after infection, all rats were killed, periodontal bone level was assessed morphometrically and radiographically, and gingival proteinase (mammalian collagenase and acid cathepsin) activity was assessed biochemically. B. gingivalis was present in oral samples from all monoinfected rats, and no contaminating bacteria were detected in any oral or fecal sample. Animals immunized with B. gingivalis cells had elevated serum and saliva antibodies to whole cells and partially purified fimbriae from B. gingivalis. Infected sham-immunized rats had significantly more periodontal bone loss than noninfected controls, whereas the periodontal bone level in infected rats immunized with 10(10) B. gingivalis cells was similar to that of the noninfected controls. The activities of gingival collagenase and cathepsin B and L were high in sham-immunized infected rats and low in all other animal groups. In conclusion, it is possible to reduce B. gingivalis-induced periodontal tissue loss in gnotobiotic rats by immunization.

Asymmetry in periodontal bone loss of gnotobiotic Sprague-Dawley rats.

In five consecutive experiments involving 78 gnotobiotic rats, significant bone loss was seen in the maxillae of those mono-infected with strains of Porphyromonas gingivalis. No significant bone loss was seen in the mandibles, and when data from both jaws were combined, the significant loss in the maxillae was occasionally concealed. It is recommended, therefore, that the levels of maxillary and mandibular bone in rats be analysed separately. A possible lateral bias of periodontal bone level was investigated in the same rats. In a highly significant number of cases the right-hand side was more severely affected than the left. This asymmetry was found in both germ-free and infected rats, and consequently could not be ascribed to P. gingivalis infection. Asymmetry of bone loss may contribute to the total random variation in bone level in rats and should be accounted for in future studies of experimental periodontitis in this model.

Two complementary methods of assessing periodontal bone level in rats.

Several methods have been applied to measure periodontal disease in rats. The purpose of the present study was to test the reproducibility of a morphometric and a radiographic method and to describe the correlation between the two methods. Periodontal bone loss on 25 defleshed rat heads was assessed under microscopic by measuring the distances from the cementoenamel junction to the alveolar bone crest at 36 buccal sites in each animal. On magnified radiographs from 25 rat mandibles periodontal bone support was expressed by the ratio apex-deepest bony defect: apex-cusp tip distally on first molars. All measurements were performed blind and in duplicate on two separate occasions. The bilateral 95% confidence limits for the error of method of measurement were estimated from the t-distribution. In a second experiment 50 rat heads were assessed by both methods, and the correlation between the recordings was estimated by the Spearman rank correlation analysis. Compared to the considerable total variation in the material, the variations due to error of methods of measurement were small, i.e. the reproducibility of both methods was satisfactory. A significant correlation was found between the methods. Since the morphometric method mainly measures horizontal bone loss, whereas the radiographic method detects intrabony interproximal defects, it is concluded that future studies would benefit from applying both methods to assess alveolar bone loss in rats.