Search

Streptococcus mutans antigen I/II (AgI/II) has been widely studied as a candidate vaccine antigen against human dental caries. In this report we follow up on prior studies that indicated that anti-AgI/II immunomodulatory monoclonal antibodies (MAbs) exerted their effects by destabilizing the native protein structure and exposing cryptic epitopes. We show here that similar results can be obtained by immunizing mice with truncated polypeptides out of the context of an intra-molecular interaction that occurs within the full-length molecule and that appears to dampen the functional response against at least two important target epitopes. Putative T cell epitopes that influenced antibody specificity were identified immediately upstream of the alanine-rich repeat domain. Adherence inhibiting antibodies could be induced against two discrete domains of the protein, one corresponding to the central portion of the molecule and the other corresponding to the C-terminus., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The adhesin known as Antigen I/II, P1 or PAc of the cariogenic dental pathogen Streptococcus mutans is a target of protective immunity and candidate vaccine antigen. Previously we demonstrated that immunization of mice with S. mutans complexed with anti-AgI/II monoclonal antibodies (MAbs) resulted in changes in the specificity, isotype and functionality of elicited anti-AgI/II antibodies in the serum of immunized mice compared to administration of bacteria alone. In the current study, an anti-AgI/II MAb reported in the literature to confer unexplained long term protection against S. mutans re-colonization following passive immunization in human clinical trials (MAb Guy's 13), and expressed in tobacco plants (MAb Guy's 13 plantibody), was evaluated for its potential immunomodulatory properties. Immunization of BALB/c mice with immune complexes of Guy's 13 plantibody bound to S. mutans whole cells resulted in a similar change in specificity, isotype, and functionality of elicited anti-AgI/II antibodies as had been observed for other immunomodulatory MAbs. This new information, coupled with the recently solved crystal structure of the adhesin, now provides a rational explanation and plausible mechanism of action of passively administered Guy's 13/Guy's 13 plantibody in human clinical trials, and how long-term prevention of S. mutans carriage well past the application period of the therapeutic antibody could have been achieved., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We showed previously that deliberate immunization of BALB/c mice with immune complexes (IC) of the cariogenic bacterium Streptococcus mutans and mAbs against its surface adhesin P1 results in changes in the specificity and isotype of elicited anti-P1 Abs. Depending on the mAb, changes were beneficial, neutral, or detrimental, as measured by the ability of the serum from immunized mice to inhibit bacterial adherence to human salivary agglutinin by a BIAcore surface plasmon resonance assay. The current study further defined changes in the host response that result from immunization with IC containing beneficial mAbs, and evaluated mechanisms by which beneficial immunomodulation could occur in this system. Immunomodulatory effects varied depending upon genetic background, with differing results in C57BL/6 and BALB/c mice. Desirable effects following IC immunization were observed in the absence of activating FcRs in BALB/c Fcer1g transgenic mice. mAb F(ab')(2) mediated desirable changes similar to those observed using intact IgG. Sera from IC-immunized BALB/c mice that were better able to inhibit bacterial adherence demonstrated an increase in Abs able to compete with an adherence-inhibiting anti-P1 mAb, and binding of a beneficial immumomodulatory mAb to S. mutans increased exposure of that epitope. Consistent with a mechanism involving a mAb-mediated structural alteration of P1 on the cell surface, immunization with truncated P1 derivatives lacking segments that contribute to recognition by beneficial immunomodulatory mAbs resulted in an improvement in the ability of elicited serum Abs to inhibit bacterial adherence compared with immunization with the full-length protein.

In this report, we define requirements for the successful translocation and functional maturation of the adhesin P1 of Streptococcus mutans. Conformational epitopes recognized by anti-P1 monoclonal antibodies (MAbs) were further characterized, thus facilitating the use of particular MAbs as tools to monitor the locations of various forms of the protein. We show that correct localization of P1 is dependent on structural features of the molecule itself, including a requisite A region-P region intramolecular interaction that occurs within the cell prior to secretion. P1 also was shown to be affected by several members of the protein-folding-secretion-turnover apparatus. It does not achieve a fully functional form in the absence of the trigger factor PPIase homolog RopA, and its translocation is delayed when DnaK levels are limited. In addition, dnaK message levels are differentially altered in the presence of P1 lacking the alanine-rich compared to the proline-rich repeat domains. Lastly, nonsecreted P1 lacking the P region accumulates within the cell in the absence of htrA, implying an intracellular HtrA protease function in the degradation and turnover of this particular internal-deletion polypeptide. However, the opposite effect is seen for full-length P1, suggesting a sensing mechanism and substrate-dependent alteration in HtrA's function and effect that is consistent with its known ability to switch between chaperone and protease, depending on environmental perturbations.

We previously identified five monoclonal antibodies (MAbs) against Streptococcus mutans adhesin P1 that modulate the humoral response when bound to whole bacteria and immune complexes (ICs) are administered to BALB/c mice. The two MAbs that redirected the response towards increased efficacy recognize discontinuous epitopes involving pre-alanine-rich domain sequence; therefore, to evaluate whether epitope specificity contributes to a desirable outcome a further MAb with this characteristic was tested. A beneficial immune response was promoted. None of the three MAbs that promoted a beneficial response was opsonic, suggesting that increased uptake of ICs by phagocytes does not mediate the improvement of the IC-elicited antibodies to inhibit bacterial adherence. Finally, two of the six anti-P1 MAbs activated complement but did not partition according to desirable vs. nondesirable effects.

Sequences contributing to epitopes recognized by a panel of monoclonal antibodies (mAbs) against the Streptococcus mutans surface protein P1 were delineated by Western blot and enzyme-linked immunosorbent assay using a battery of deletion constructs and recombinant polypeptides. mAbs that recognize complex discontinuous epitopes reconstituted by combining the alanine-rich and proline-rich repeat domains and varying degrees of flanking sequence were identified as well as mAbs that bound epitopes contained within contiguous segments of P1. Cross-reactivity with SspA and SspB from Streptococcus gordonii is also reported. This information enables insight into the structure and function of a streptococcal adhesin and its correlates of protection and furthers our understanding of the immunomodulatory and bacterial-adherence inhibition activities of anti-P1 mAbs.

Researchers now recognize the utility of surface plasmon resonance technology to evaluate interactions of microbial pathogens with host components. The surface adhesin and candidate vaccine antigen P1 of Streptococcus mutans, the main causative agent of dental caries, interacts with a high molecular weight glycoprotein called salivary agglutinin, or gp340, in the salivary pellicle. We optimized a BIAcore assay to measure P1-mediated Ca(2+) dependent binding of S. mutans whole cells to this physiological ligand immobilized on a Pioneer F1 sensor chip. Regeneration conditions allowed cells to be eluted from the sensor chip permitting multiple reuse of the agglutinin-coated surface. An isogenic P1-deficient S. mutans mutant did not bind to immobilized agglutinin demonstrating specificity of the detected interaction. Glutaraldehyde-fixation of bacterial cells showed the assay measured a whole cell-ligand interaction and was not an artifact of solubilized or leached proteins. Adherence inhibition assays demonstrated varying degrees of disruption of the S. mutans-agglutinin interaction by anti-P1 monoclonal antibodies recognizing different epitopes, whereas a polyclonal reagent demonstrated more complete inhibition. This report describes an improved method to assess salivary agglutinin-mediated adherence of S. mutans in vitro under physiological-like conditions and to evaluate the effectiveness of antibodies of differing specificities to inhibit binding.

The adhesin P1 of Streptococcus mutans has been studied as an anticaries vaccine antigen. An anti-P1 monoclonal antibody (MAb) bound to S. mutans prior to mucosal immunization of mice was shown previously to alter the amount, specificity, isotype, and biological activity of anti-P1 antibodies. The present study was undertaken to screen this and four additional anti-P1 MAbs for immunomodulatory activity when complexed with S. mutans and administered by a systemic route and to evaluate sera from immunized mice for the ability to inhibit adherence of S. mutans to immobilized human salivary agglutinin. All five MAbs tested influenced murine anti-P1 serum antibody responses in terms of subclass distribution and/or specificity. The effects varied depending on which MAb was used and its coating concentration. Two MAbs promoted a more effective, and two others a less effective, adherence inhibition response. An inverse relationship was observed between the ability of the MAbs themselves to inhibit adherence and the ability of antibodies elicited following immunization with immune complexes to inhibit adherence. Statistically significant correlations were demonstrated between the levels of anti-P1 serum immunoglobulin G2a (IgG2a) and IgG2b, but not of IgG1 or IgG3, and the ability of sera from immunized animals to inhibit bacterial adherence. These results indicate that multiple anti-P1 MAbs can mediate changes in the immune response and that certain alterations are potentially more biologically relevant than others. Immunomodulation by anti-P1 MAbs represents a useful strategy to improve the beneficial immune response against S. mutans.

Monoclonal antibody (MAb) 6-11A directed against Streptococcus mutans surface adhesin P1 was shown previously to influence the mucosal immunogenicity of this organism in BALB/c mice. The specificity of anti-P1 serum immunoglobulin G (IgG) and secretory IgA antibodies and the subclass distribution of anti-P1 serum IgG antibodies were altered, and the ability of elicited serum antibodies to inhibit S. mutans adherence in vitro was in certain cases increased. MAb 6-11A is known to recognize an epitope dependent on the presence of the proline-rich region of the protein, although it does not bind directly to the isolated P-region domain. In this report, we show that MAb 6-11A recognizes a complex discontinuous epitope that requires the simultaneous presence of the alanine-rich repeat domain (A-region) and the P-region. Formation of the core epitope requires the interaction of these segments of P1. Residues amino terminal to the A-region also contributed to recognition by MAb 6-11A but were not essential for binding. Characterization of the MAb 6-11A epitope will enable insight into potential mechanisms of immunomodulation and broaden our understanding of the tertiary structure of P1.

We demonstrated previously that mucosal immunization of mice with Streptococcus mutans coated with the monoclonal antibody (MAb) 6-11A directed against the major surface adhesin protein P1 results in changes in the amount, isotype distribution, and specificity of serum antibodies compared with animals immunized with bacteria only. We now show that the specificity of the mucosal secretory IgA response was also influenced by this MAb. Changes in antibody specificity were associated with changes in biological activity. Serum samples which differed in antibody reactivity with P1 polypeptides generated by partial digestion with N-chlorosuccinimide but not in isotype distribution or overall reactivity with S. mutans or intact P1 demonstrated a statistically significant difference in the ability to inhibit bacterial adherence to salivary-agglutinin-coated hydroxyapatite beads. Serum IgG antibodies against P1 from mice immunized with either S. mutans alone or S. mutans coated with 6-11A were shown to recognize antigenic determinants dependent on the presence of the central proline-rich repeat domain, a segment necessary for the structural integrity of the molecule. However, no statistically significant differences were observed in antibody reactivity with a panel of six partial P1 polypeptides encoded by overlapping spaP subclones, suggesting that the targets of biologically relevant antibodies involve complex epitopes not reconstituted by the recombinant products tested. Lastly, we show that binding of MAb 6-11A to P1 on the surface of S. mutans alters P1's susceptibility to proteolytic digestion. Hence, changes in antigen processing and presentation may contribute to the immunomodulatory effects of this MAb.

During characterization of the surface antigens of serotype III group B streptococci (GBS), a protein with an apparent M(r) of approximately 173,500 migrating on a SDS--polyacrylamide gel was found to have an N-terminal amino acid sequence identical to that of the plasmin receptor (Plr) of group A streptococci, a surface-localized glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work begins to characterize GBS GAPDH and to assess its functional activity on the cell surface. The 1.0-kb gapC gene of GBS was amplified by PCR. plr and gapC demonstrated 87% homology. An anti-Plr monoclonal antibody reacted with GBS whole cells, suggesting GBS GAPDH is surface localized. Multiple serotypes of GBS demonstrated functional GAPDH on their surfaces. The anti-Plr monoclonal antibody recognized GBS protein bands of approximately 41 and 173.5 kDa, by Western blot. Presumably, these represent monomeric and tetrameric forms of the GAPDH molecule. GBS GAPDH was demonstrated by Western blot analysis to interact with lys- and glu-plasminogens. Fluid-phase GBS GAPDH interacted, by means of ELISA, with immobilized lys-plasminogen, glu-plasminogen, actin, and fibrinogen. Enzymatically active GAPDH, capable of binding cytoskeletal and extracellular matrix proteins, is expressed on the surface of GBS.

Systemic immunization with antigen coupled to monoclonal antibody (MAb) has been used by several investigators to increase the number of MAb-producing hybridomas against an antigen and to elicit antibodies specific for poorly immunogenic epitopes. This strategy has implications for vaccine design in that protective immunity is not necessarily directed at immunodominant epitopes of pathogens and may be improved by deliberately shifting the immune response toward subdominant epitopes. To our knowledge, no studies to date have addressed the potential for immunomodulatory activity mediated by MAbs bound to mucosally applied antigen. To test whether administration of an exogenous MAb directed against a streptococcal surface protein could influence the humoral immune response, BALB/c mice were immunized orally by gastric intubation or intranasally with Streptococcus mutans alone or S. mutans complexed with a MAb directed against the major surface protein P1. Significant changes in the subclass distribution, as well as the specificity, of anti-P1 serum immunoglobulin G antibodies were demonstrated in groups of mice which received S. mutans coated with the anti-P1 MAb versus those which received S. mutans alone. Alterations in the humoral immune response were dependent on the amount of anti-P1 MAb used to coat the bacteria. In addition, differences in the anti-P1 immune responses were observed between groups of mice immunized via oral versus intranasal routes. In summary, an exogenous MAb complexed with a streptococcal antigen prior to mucosal immunization can influence the immunoglobulin isotype and specificity of the host humoral immune response against the antigen.

Porphyromonas gingivalis is a major etiologic agent of periodontitis, a chronic inflammatory disease that ultimately results in the loss of the supporting tissues of the teeth. Previous work has demonstrated the usefulness of avirulent Salmonella enterica serovar Typhimurium strains as antigen delivery systems for protective antigens of pathogens that colonize or cross mucosal surfaces. In this study, we constructed and characterized a recombinant S. enterica serovar Typhimurium avirulent vaccine strain which expresses hemagglutinin A and carries no antibiotic resistance markers. HagA, a major virulence-associated surface protein, is a potentially useful immunogen that contains an antigenic epitope which, in humans, elicits an immune response that is protective against subsequent colonization by P. gingivalis. The hagA gene, including its promoter, was cloned into a balanced-lethal Salmonella vector and transferred to the vaccine strain. Heterologous expression of HagA was demonstrated in both Escherichia coli JM109 and S. enterica serovar Typhimurium vaccine strain chi4072. The HagA epitope was present in its native configuration as determined by immunochemistry and immunoelectron microscopy. Purified recombinant HagA was recognized by sera from mice immunized with the S. enterica serovar Typhimurium vaccine strain. The HagA-specific antigen of the vaccine was also found to be recognized by serum from a periodontal patient. This vaccine strain, which expresses the functional hemagglutinin protein, induces a humoral immune response against HagA and may be useful for developing a protective vaccine against periodontal diseases associated with P. gingivalis.

Periodontal disease in the elderly has not been characterized. Initial reports suggest that the disease is common and severe. Deficiencies in the immune response have also been reported to occur in the elderly. Consequently it was hypothesized that aging-related changes in the immune response may contribute to the severity and occurrence of periodontal disease in the elderly. To test that hypothesis, the % and number of leukocytes and leukocyte subsets in the peripheral blood of elderly (65-75 years) subjects were tested and used as indicators of the immune potential of those individuals. Age and gender effects on several of the parameters tested were identified. With the exception of increased number of leukocytes in the elderly group with severe periodontal disease, no other alteration in the leukocyte parameters tested were identified. These results suggest that periodontal disease in the elderly was not associated with obvious changes in the leukocyte and leukocyte subsets in the peripheral blood due to aging.

The serotype b antigens have been reported to be associated with lipopolysaccharide. Using murine monoclonal antibodies specific for either a serotype b antigen or the Actinobacillus actinomycetemcomitans species, the relationship of the two epitopes to lipopolysaccharide was determined. Both the species-specific and serotype b-specific monoclonal antibodies bound to whole cells, vesicles and conventionally isolated lipopolysaccharide and polysaccharide material derived from A. actinomycetemcomitans culture supernatants. Serotype b-specific monoclonal antibodies bound to the polysaccharide of acid-hydrolyzed lipopolysaccharide. Species-specific monoclonal antibodies bound to both the polysaccharide and the lipid A fraction of lipopolysaccharide after acid hydrolysis. Polymyxin b partially inhibited the binding of the species-specific monoclonal antibodies to lipopolysaccharide and had no effect on the binding of the serotype b-specific monoclonal antibodies to lipopolysaccharide. Lipopolysaccharide from whole bacteria and polysaccharide material isolated from culture supernatants were separated by gel filtration chromatography in deoxycholate into fractions that contained serotype b antigen, both serotype b and species-specific antigens, or species-specific antigen. SDS-polyacrylamide gel electrophoresis and Western blotting analysis of the fractions revealed that the serotype b antigen was on a high-molecular-weight polysaccharide material. The species-specific antigen was on a ladder of lower-molecular-weight polysaccharides identical to the blot pattern of lipopolysaccharide molecules separated by polyacrylamide gel electrophoresis and stained with silver stain. Chemical analysis of the polysaccharide containing serotype b antigen revealed 85% ribose, 11% glucose, and no lipid. Chemical content of the species-specific antigenic material revealed a composition typical of lipopolysaccharide. Immunoelectron microscopy using the species- or serotype b-specific monoclonal antibodies confirmed the biochemical and immunological characterization of the two antigens, showing that the species-specific epitopes were on the surface of the A. actinomycetemcomitans cell membrane and the serotype b-specific epitopes on the amorphous material extending from the cell surface. The data indicated that the serotype b antigen, detected by the antibody, was separable from lipopolysaccharide and was an A. actinomycetemcomitans capsular material. The species-specific antigen, being more conserved than the serotype antigen, was on all the lipopolysaccharide molecular species.

A monoclonal antibody to Actinomyces naeslundii (A. viscosus) T14V-J1 type 1 fimbriae, capable of inhibiting the adherence of this bacterium to salivary proline-rich protein-treated hydroxyapatite, was generated by immunization of SWR mice with A. naeslundii 55-19, a strain derived from T14V-J1 that possess only type 1 fimbriae. Supernatants of hybridomas were screened for reactivity with purified type 1 fimbriae. An IgG monoclonal antibody, 86-49E, blocked the adsorption of the parent strain to proline-rich protein-treated hydroxyapatite by 77% with 1.0 microgram/ml of the monoclonal antibody; the Fab fragment derived from this monoclonal antibody inhibited adherence by 38% at the same concentration. Similarly, the adherence of strain 55-19 was inhibited by 100% and 64% to proline-rich protein-treated hydroxyapatite with 1.0 micrograms/ml of IgG and Fab fragments respectively. Control monoclonal antibody to the subunit of type 1 fimbriae, as well as to Actinobacillus actinomycetemcomitans caused only minimal adherence inhibition. Monoclonal antibody 86-49E also agglutinated both type 1 fimbriae-bearing strains of A. naeslundii T14V-J1 and 55-19 but not strains 59-51 and 147, which lack type 1 fimbriae. Further confirmation of the specificity of monoclonal antibody 86-49E was obtained using these fimbria-deficient mutant strains in an enzyme-linked immunosorbent assay, with the monoclonal antibody binding only to strains possessing type 1 fimbriae. Immunogold labeling in conjunction with electron microscopy suggested binding of monoclonal antibody 86-49E occurring near the distal end of the fimbriae. In contrast, when a monoclonal antibody specific for the type 1 fimbrial subunit but not capable of adherence inhibition was used together with 86-49E in double-labeling experiments, extensive labeling of the fimbriae by the subunit antibody was noted. These data suggest that a monoclonal antibody specific for the type 1 fimbriae of A. naeslundii that is capable of binding to a discrete site on the fimbriae has the capacity to inhibit the adsorption of this organism to saliva-treated hydroxyapatite.

Little is known about the relationship of aging to periodontal disease. The immune response undergoes aging-related changes resulting in loss of functional capacity. The aim of this study was to investigate the relationship between levels of serum IgG antibodies against suspected periodontal pathogenic microorganisms to the presence or absence of periodontal disease in an elderly (65-75 yrs) population. From this study, we obtained information concerning: (1) the ability to differentiate elderly individuals without disease from those with disease by their levels of antibodies against periodontal pathogens and (2) which periodontal pathogen(s) triggered those responses. IgG anti- Porphyromonas gingivalis (strains W83 and 381) levels in the serum of elderly patients with severe periodontal disease were the only antibody responses measured which were elevated compared to the elderly control group of subjects with no periodontal disease. Anti- Prevotella intermedia IgG levels in both elderly patient groups were depressed compared to anti- P. intermedia levels in the young normal control subjects. Serum IgG antibody levels to six other plaque microorganisms did not differentiate between diseased and normal, elderly or young subjects. This data suggested that P. gingivalis was associated with periodontal disease in this elderly group of individuals and that those elderly individuals were able to respond with a normal IgG immune response.

The aim of the present study was to evaluate the effect of non-surgical periodontal therapy with the adjunct of a selected antibiotic in subjects diagnosed with refractory periodontal disease. 21 subjects were selected for the study; all had a history of periodontal surgery, tetracycline therapy, and regular maintenance by a periodontist. When disease activity was detected, a bacterial sample was taken and a whole plaque susceptibility test was performed. Before the outcome of the susceptibility test the subjects were assigned to either antibiotic or placebo therapy. All subjects received scaling and rootplaning prior to antibiotic or placebo therapy. Based on the susceptibility test, subjects in the antibiotic group were treated either with Augmentin or clindamycin. The results demonstrated that in subjects with refractory periodontal disease there was no significant difference (N.S.) in the proportion of sites losing attachment before and after treatment (11.3% and 12.4%, respectively) over a 2-year post therapy observation period. However, the proportion of sites showing gain of attachment increased from 0.9% before therapy to 5.1% (p = 0.029) following selective antibiotic therapy when combined with scaling and rootplaning. The remainder of sites showed no change between pre- and post-therapy monitoring periods. The progression of attachment loss in the active sites could not be completely stopped over the entire 2-year period. After 12-15 months following therapy, there was a tendency towards new loss of attachment and an increase of pocket depth. However, all 4 subjects treated with placebo drug demonstrated continuous deterioration and had to be retreated. Although the proportion of sites losing attachment decreased from 5.1% to 2.3% (N.S.), the proportion of sites gaining attachment also decreased from 2.0% to 1.0% (N.S.). The results suggest that scaling and rootplaning together with selected antibiotic therapy repeated every 12-15 months may be beneficial for these subjects although it may not completely stop progressive attachment loss.

A cross-sectional periodontal study of 74 subjects aged 65 to 75 years was performed. Clinical data were collected and related to microbiological and immunological data. A statistical model (step-wise multiple regression) of factors related to bone loss was created initially using clinical data only; then by adding either the microbiologic or immunologic data; and then by using clinical, microbiologic, and immunologic data together. When only clinical data were considered, three factors were found to have significant positive correlations with bone loss. Tooth mobility accounted for 17% of the variability in the alveolar bone level measurements, probing depth for 12%(r2), and plaque index for 3%, for a total of 32% of the variability explained by these clinical factors. Tooth mobility and probing depth were clinical factors which remained significant in the model when the microbiological data were also considered. As percentages of the total cultivable microbiota, E. corrodens (r2 = 14%) and black-pigmenting Prevotella intermedia (r2 = 4%) correlated positively with alveolar bone loss. The addition of the microbiologic data only increased the r2 to 33%. When immunological data were considered with the clinical data, pocket depth and tooth mobility were the clinical parameters which remained in the model. IgG antibody levels to P. gingivalis W83 and/or 381 (r2 = 24%) A. actinomycetemcomitans 627 (r2 = 2%) were the significant immunologic measures having a positive correlation with bone loss. Anti-F. nucleatum levels had a significant negative correlation. A total of 50% of the variability in alveolar bone level was accounted for in the model by the addition of specific serum antibody levels to subgingival plaque microorganisms. When clinical, microbiological, and immunological measurements were all considered together, antibody to P. gingivalis W83 and/or 381 (r2 = 42%), percentage of B-lymphocytes (r2 = 3%), probing depth (r2 = 4%), anti-E. corrodens levels (r2 = 2%), and anti-P. gingivalis 33277 levels (r2 = 4%) all had significant positive correlation with loss of alveolar bone. The number of enteric bacteria, anti-F. nucleatum levels, and anti-P. intermedia levels each had a significant negative correlation with alveolar bone heights. The r2 for this model was 75%. These results indicated that antibody levels to subgingival plaque microorganisms and tooth mobility were the best predictors of bone loss in the elderly patients tested in this study.

Periodontal diseases are thought to result from inflammatory responses to bacterial challenges in the gingival crevicular area. Antibodies are a major host-protective mechanism in many bacterial infections. Consequently, the antibody responses to suspected periodontal pathogenic bacteria have been extensively measured as to their relationship to diseases and specificity for suspected pathogens associated with progressing disease sites. Recently, studies on the bacterial immunogen characterization, antibodysubclass identification, and antibody biological capabilities have been reported. Although increased antibody levels to certain suspected periodontal pathogens were associated with periodontal diseases in humans, little evidence exists as to the role of these antibodies in the infectious process. In vivo experiments in animals indicated that specific antibodies against certain suspected periodontal pathogens were associated with suppression of bacterial colonization, limiting the spread of infection, and a decrease in alveolar bone loss. However, in vitro as well as in vivo experiments suggested that phagocytic cells are required for efficient bactericidal activity of antibodies and that the presence of other sensitized immune cells may either have inhibited or enhanced the infectivity of certain periodontal pathogens. Possible explanations for the observed inconsistencies are presented and the potential for utilization of specific anti-periodontal pathogen responses in the understanding and prevention of diseases is discussed. J Periodontol 1993; 64:807- 818., (© 1993 American Academy of Periodontology.)

Periodontal diseases are thought to result from inflammatory responses to bacterial challenges in the gingival crevicular area. Antibodies are a major host-protective mechanism in many bacterial infections. Consequently, the antibody responses to suspected periodontal pathogenic bacteria have been extensively measured as to their relationship to diseases and specificity for suspected pathogens associated with progressing disease sites. Recently, studies on the bacterial immunogen characterization, antibody-subclass identification, and antibody biological capabilities have been reported. Although increased antibody levels to certain suspected periodontal pathogens were associated with periodontal diseases in humans, little evidence exists as to the role of these antibodies in the infectious process. In vivo experiments in animals indicated that specific antibodies against certain suspected periodontal pathogens were associated with suppression of bacterial colonization, limiting the spread of infection, and a decrease in alveolar bone loss. However, in vitro as well as in vivo experiments suggested that phagocytic cells are required for efficient bactericidal activity of antibodies and that the presence of other sensitized immune cells may either have inhibited or enhanced the infectivity of certain periodontal pathogens. Possible explanations for the observed inconsistencies are presented and the potential for utilization of specific anti-periodontal pathogen responses in the understanding and prevention of diseases is discussed.

The squirrel monkey (Saimiri sciureus) has been proposed as an in vivo model for the study of subgingival colonization by suspected periodontopathogens, such as black-pigmented porphyromonads and prevotellas (BP/P). However, the indigenous microbiota of the squirrel monkey has not been well described. Therefore, in order to more fully characterize the oral microbiota of these animals, we studied two groups of squirrel monkeys from widely different sources. Group I consisted of 50 breeding colony monkeys ranging in age from 9 months to over 6 years which had been raised in captivity; group II consisted of 16 young sexually mature monkeys recently captured in the wild in Guyana. Group I animals in captivity had developed moderate to severe gingivitis, with a mean gingival index (GI) of 2.6; 52% of the sites bled, 26% had detectable calculus, and 83% had detectable BP/P. A group I subset (six animals), for which predominant cultivable microbiota was described, had a mean GI of 2.4. Colony morphology enumeration revealed that five of the six subset animals were detectably colonized with BP/P (range, 0 to 16.9%) and Actinobacillus actinomycetemcomitans (range, 0 to 3.9%); all subset animals were colonized with Fusobacterium species (range, 0.8 to 3.6%), Actinomyces species (range, 2.3 to 11%), and gram-positive cocci (range, 1.4 to 21.4%). Predominant cultivable microbiota results revealed the presence of many bacterial species commonly found in the human gingival sulcus. At baseline, group II animals were clinically healthy and had a mean GI of 1.4; 67% of the sites bled and 2.1% had calculus, and none of the animals had detectable BP/P. Neisseriae were very common in noninflamed sites. Subsequently, when inflamed sites were compared with noninflamed sites in group II animals after they had been maintained in captivity for 6 months, inflamed sites exhibited a more complex microbiota and increased proportions of gram-negative rods and asaccharolytic bacteria.

Colonization of the gingival crevice by black-pigmented Porphyromonas or Prevotella spp. (BP/P), including Porphyromonas gingivalis (formerly Bacteroides gingivalis) and Prevotella intermedia (formerly Bacteroides intermedius), is thought to be an important ecological event which may result in the destruction of connective tissues supporting the teeth. Theoretically, periodontal diseases could be prevented if these or other periodontal pathogenic microorganisms did not colonize the subgingival area. The humoral immune response is one mechanism which may modulate bacterial colonization in the gingival crevice. In the present study, we tested the effect of systemic humoral immunity on subgingival colonization by indigenous P. intermedia in squirrel monkeys (Saimiri sciureus). Animals rendered essentially free of detectable BP/P by a single scaling, 10 days of tetracycline therapy, and toothbrushing three times per week were immunized with P. intermedia 1447 or were sham immunized with phosphate-buffered saline. Subsequently, all oral hygiene procedures were discontinued and five teeth in one quadrant were ligated with bacterium-soaked suture material to facilitate BP/P colonization. Immunization resulted in a significant increase in the level of immunoglobulin G anti-P. intermedia antibody in serum. Two weeks after ligation was initiated, P. intermedia could be detected in five of six sham-immunized and three of six immunized animals. Immunization was associated with a reduction in the emergence of indigenous P. intermedia in the gingival crevice.

The aim of the present study was to analyze the relationship between attachment loss and clinical, microbiological, and immunological parameters in a group of 21 human subjects exhibiting poor response to previous periodontal therapy. All had been treated with periodontal surgery, tetracycline, and subsequent maintenance recalls to periodontists who, upon detection of disease progression, referred the subjects to our clinic. In our clinic, each subject received oral hygiene instruction and a thorough subgingival scaling and root planing utilizing as many appointments as necessary. Clinical indices, including gingival index, bleeding on probing, suppuration, plaque index, pocket depth, and duplicate measurements of attachment level from an acrylic stent, were collected at monthly intervals. Probing measurements were performed using the Florida Probe. When significant attachment loss (0.8-1.2 mm) was detected in at least 1 site, a bacterial sample was taken from that site and from a comparably deep, but non-progressing, control site. Microbial samples were enumerated by darkfield microscopy, on selective and non-selective media, and by predominant cultivable technique. Blood samples were also collected to determine antibody levels against potential pathogens. There was no difference in the amount of plaque present in sites gaining or losing attachment, but losing sites exhibited more bleeding and suppuration. 20 of the 21 subjects were tested; of these, 17 exhibited elevated serum antibody against one or more of the following microorganisms: Actinobacillus actinomycetemcomitans, Bacteroides, gingivalis, and Eikenella corrodens. However, few, if any, of the "classical" pathogens were detected in the plaque samples obtained at the time progressive disease was diagnosed. The only exception was Streptococcus intermedius, which occurred in slightly higher numbers in active sites.

Periodontal disease is an inflammatory condition of the supporting structures of teeth. This condition is actually considered to be caused by a number of different diseases, possibly associated with different etiologies, rather than a single disease. Due to limitations in our present understanding of cellular and molecular events involved in the pathogenesis of various periodontal diseases, many therapeutic failures still occur. For this reason, studies concerning the responses of the host to pathogenic bacteria are thought to be critically important. This review presents current opinions on the role of host responses in periodontal disease.

In the absence of antiserum, rabbit polymorphonuclear leukocytes (PMNs) released lysosomal enzymes in response to Actinomyces viscosus (19246) but not to Streptococcus mutans (6715). Antibodies had a marked modulating influence on these reactions. PMN hydrolase release was significantly enhanced to both organisms when specific rabbit antiserum and isolated immunoglobulin G (IgG) were included in the incubations. Immune complex F(ab')2 fragments of IgG directed against S. mutans agglutinated bacteria. Immune complexes consisting of S. mutans and F(ab')2 fragments of IgG directed against this organism were not effective as bacteria-IgG complexes in stimulating PMN release. The intensity of the release response to bacteria-IgG complexes was also diminished when PMNs were preincubated with isolated Fc fragments derived from IgG. Fresh serum as a source of complement components had no demonstrable effect on PMN release either alone or in conjuction with antiserum in these experiments. These data may be relevant to the mechanisms and consequences of the interaction of PMNs and plaque bacteria in the pathogenesis of periodontal disease.

The objective of this study was to determine whether the squirrel monkey (Saimiri scuireus) is indigenously colonized with black-pigmented bacteroides (BPB) resembling human Bacteroides gingivalis and Bacteroides intermedius (suspected periodontal pathogens) and to determine the usefulness of the squirrel monkey as an in vivo model for studying colonization by putative pathogens. We assayed the subgingival plaques of 138 monkeys of various ages and in four different colonies for the presence of anaerobic BPB microorganisms. We also tested half the animals for the presence of Actinobacillus actinomycetemcomitans. Clinical indices and levels of serum antibody to B. gingivalis were recorded. We detected BPB in 50% of the animals and A. actinomycetemcomitans in 69% of the animals. The presence of BPB was generally associated with increased age, increased gingival index, presence of calculus, and increased levels of serum antibody. These data indicate that the squirrel monkey may be a good model for studying the parameters of natural infection of the gingival crevice with suspected periodontopathogenic BPB microorganisms.

In the present study we identified a gram-negative anaerobic rod referred to as Y4 which was cytotoxic for human polymorphonuclear leukocytes. Y4 was isolated from dental plaque of a patient with juvenile periodontitis and presented most of the taxonomic characteristics of Actinobacillus species. Under experimental conditions, viable Y4 were cytotoxic for human peripheral blood polymorphonuclear leukocytes in serum-free cultures. Cytotoxicity was dependent on bacterial concentrations and was enhanced in the presence of a fresh or heat-inactivated (56 degrees C, 30 min) autologous serum. Leukotoxicity was independent of phagocytosis. Y4 leukotoxic effect was abolished when bacteria were heat treated (56 degrees C, 30 min) or when incubations were carried out at 4 degrees C instead of at 37 degrees C. The leukotoxicity was monitored by electron microscopy and biochemically by measuring lactate dehydrogenase indicator of cell viability. No cytotoxic effects of Y4 on human mononuclear cells, chicken fibroblasts, or mouse macrophages were detected under the conditions studied. Polymorphonuclear leukocytes may play an important role in the host defense against bacteria in periodontal disease. The cytotoxic effect of Y4 for polymorphonuclear leukocytes presented in this study is the first report of a direct offensive microbial vector in a plaque-derived microorganism and may prove to be relevant in the pathogenesis of juvenile periodontitis.

Both virulent (V) and avirulent (AV) strains of Actinomyces viscosus T14 are capable of colonizing the oral cavity of gnotobiotic rats, but only T14-V causes destructive periodontal disease. The basis for this difference in in vivo pathogenicity has not been adequately defined. In the present study we compared the capacities of T14-AV and T14-V to provoke in vitro extracellular release of lysosomal constituents from human polymorphonuclear leukocytes (PMNs). In serum-free cultures, viable T14-V but not T14-AV stimulated discharge of PMN lysosomes. The release response was correlated with PMN phagocytic activity; thus, PMNs readily ingested T14-V but not T14-AV. To explain these differences in PMN-bacteria interactions, subcellular fractions of T14-AV or T14-V were incubated with PMNs. A crude, insoluble sonic extract derived from T14-V caused PMN lysosome release, but a similar fraction from T14-AV was inactive. However, following extensive washing and treatment with deoxyribonuclease or sodium dodecyl sulfate, cell wall fractions of T14-AV stimulated lysosome release. These procedures apparently removed an extracellular polysaccharide slime which is synthesized by T14-AV but not by T14-V. There was a significant reduction in the capacities of viable T14-V or cell wall fractions of T14-V or T14-AV to provoke PMN lysosome release when these agents were preincubated with a slime material isolated from T14-AV. This inhibitory influence of slime was overcome by the addition of fresh or heated (56 degrees C, 30 min) serum to the PMN-bacteria cultures. The data suggest a relationship between the abilities of the avirulent and virulent strains of A. viscosus T14 to act as periodontal pathogens in vivo and to serve as stimuli for PMN lysosome release in vitro.

An endotoxin was isolated from Capnocytophaga sputigena strain 4 by a modification of the hot phenol-water method. The extraction procedure yielded a lipopolysaccharide which accounted for approximately 1.5% of the dry weight of the cells. The material was composed of 18.6% lipid (as C(15) fatty acid), 46.5% neutral sugar including 9.6% hexose, 18.3% 6-deoxy sugar, 1.0% 2-keto-3-deoxy sugar, and 4.8% heptose. Hexosamine, protein, and phosphorus were found in quantities amounting to 9.0, 2.9, and 2.0% of the dry weight, respectively. No pentose or nucleic acid was detected. Acid hydrolysis resulted in the release of the constituent sugars and the formation of an insoluble precipitate. The lipopolysaccharide was tested for numerous biological activities characteristic of endotoxins. The pyrogenicity was relatively low; the fever index 40 was 17 mug, and 10 mug was required to give the characteristic biphasic fever response. The toxicity of the extract was very low, with a 50% chicken embryo lethal dose of 15.6 mug and a 50% mouse embryo lethal dose of greater than 8 mg. Similarly, the C. sputigena endotoxin had modest effects on leukocytes when compared with endotoxin standards from other organisms. The extract exhibited little or no mitogenicity when tested on mouse spleen lymphocytes. It was not toxic to human peripheral polymorphonuclear leukocytes and caused the release of only a small (13%) portion of lysosomal enzymes. Although the C. sputigena lipopolysaccharide caused significant activation of mouse peritoneal macrophages, the dose required was twice that of an Escherichia coli endotoxic standard. However, the Limulus amoebocyte lysate clotting activity of the lipopolysaccharide was comparable to that of an Serratia marcescens lipopolysaccharide standard, and passive hemagglutination tests revealed that 1 mug of the lipopolysaccharide was capable of sensitizing 1 ml of a 2% sheep erythrocyte suspension for agglutination with an antiserum prepared against C. sputigena whole cells.

The trichloroacetic acid (TCA) extractable molecules in Propionibacterium acnes cell wall were tested for the ability to activate the alternative pathway of complement in human serum treated with ethyleneglycol-bis (beta-aminoethyl ether) N,N-tetracetic acid (EGTA). The extracted molecules failed to consume hemolytic activity against antibody-coated sheep erythrocytes but gave a dose-dependent consumption of rabbit erythrocyte (RE) lytic activity. Similarly, the extract produced significant cleavage of Factor B, but failed to cleave C3, as detected by immunoelectrophoresis. Adsorption of the extracted material to sheep erythrocytes did not render the cells susceptible to lysis via the alternative pathway. Sephadex G-25 chromatography yielded several fractions which were able to consume RE lytic activity from EGTA-treated serum. These fractions were analyzed and found to contain glucose, mannose, and galactose. No teichoic acid or protein was detected. The alternative pathway activator in P. acnes is thus a nonteichoic acid cell wall carbohydrate which, in its extractable form is capable of activating only alternative pathway reactants prior to C3.

Dapsone at doses of 0.5 to 5.0 micrograms/ml was found to produce a dose-dependent inhibition of opsonized zymosan-induced human polymorphonuclear leukocyte (PMN) chemiluminescence (CL) in vitro. Simultaneous exposure of PMN to dapsone and zymosan was as effective in reducing CL as preincubation of PMN with dapsone. Preincubation of PMN with dapsone followed by washing, resulted in the loss of dapsone-mediated CL inhibition, indicating that dapsone did not permanently alter the CL-generating mechanism and that the drug had to be present to inhibit CL. Dapsone did not absorb light at the wavelength of CL and was not toxic to PMN at concentrations tested. Sodium azide, an inhibitor of myeloperoxidase-mediated CL inhibited PMN CL to the same degree as dapsone. When incubated together with PMN, dapsone and azide did not produce an additive inhibition of CL. These data suggest that inhibition of myeloperoxidase may be the mechanism by which dapsone inhibits PMN CL.

The ability of pyran copolymer to interact with the classical and alternative pathways of complement was assessed in human and C4-deficient guinea pig serum. Pyran induced a dose-dependent inhibition of hemolytic activity in both serum systems. Immuno-electrophoretic analysis of pyran-treated human serum revealed that C3 was not cleaved. Factor B was altered into a more anionic mobility which was not similar to biologically cleaved Ba or Bb fragments. Pyran-treated serum was unable to lyse antibody-coated erythrocytes (EA or EA coated with C1 and C4 and EA coated with C1, C4 and C2. Pretreatment of serum with ethylenediaminetetraacetic acid did not prevent inhibition of hemolytic activity by pyran. Cobra venom factor did not cleave C3 in the presence of pyran. These data indicate that pyran does not activate complement by standard mechanisms but does inhibit one or more of its components.

Periodontal diseases are inflammatory responses thought to be triggered by specific microorganisms colonizing in the gingival crevice. Theoretically, periodontal diseases could be prevented if the etiologic organisms were not allowed to colonize the subgingival area. The humoral immune response is one mechanism which may modulate bacterial colonization in the gingival crevice. To test the effect of systemic humoral immunity on subgingival colonization by bacteria, squirrel monkeys (Saimiri sciureus) were immunized with Bacteroides gingivalis, a black-pigmented Bacteroides sp. and putative periodontal pathogen. Immunized and sham-immunized monkeys were orally inoculated with 10(10) viable B. gingivalis during ligation of five teeth in one quadrant with bacterium-soaked suture material and distribution over the entire dentogingival margin. Immunization resulted in an increased level of immunoglobulin G anti-B. gingivalis in serum and was associated with a strong trend toward a statistically significant reduction in colonization of the gingival crevice by black-pigmented bacteroides.

Murine hybridoma cell lines were developed which synthesized monoclonal antibodies against Actinobacillus actinomycetemcomitans-associated antigens. Monoclonal antibodies specific for an antigen(s) common to all A. actinomycetemcomitans isolates tested but not detected on other gram-negative oral plaque microorganisms or other Actinobacillus species were identified. Monoclonal antibodies specific for each serotype group of A. actinomycetemcomitans which did not bind to other Actinobacillus species or oral plaque microorganisms were also identified.

Activation of the alternative pathway of complement is known to be initiated by bacterial structures. We have fractionated Propionibacterium acnes cells, purified various cell fractions, and tested their complement-activating ability in human serum chelated with ethyleneglycol bis-(beta-aminoethylether)-N,N1-tetraacetic acid. The majority of complement-activating activity was localized in the wall fraction. This activity was resistant to lipid extraction, protease, RNAse, DNAse and lysozyme treatment. NaIO4, formamide, and hot (but not cold) trichloroacetic acid (TCA) extraction ablated the complement-activating capacity of cell walls. Compounds removed by extraction failed to consume significant hemolytic activity against antibody-coated sheep erythrocytes (EA). Addition of TCA-extracted soluble material to cell wall suspensions resulted in an inhibition of hemolytic consumption by the cell wall. These results indicate that, in P. acnes, complement-activating molecules are located in the cell wall and are carbohydrate in nature. Peptidoglycan, lipid, protein, and nucleic acid do not appear to contribute to the cell wall's ability to activate complement.

A relatively high incidence of infection by Actinobacillus actionomycetemcomitans can be shown in subgingival plaque samples obtained from patients with juvenile periodontitis. These organisms possess a potent leukotoxin(s) which rapidly destroys isolated human polymorphonuclear leukocytes (PMNs) and monocytes. If such leukotoxins operate in vivo, they could deprive the gingival crevice area of an essential antibacterial defense mechanism. We have found that sera from juvenile periodontitis patients consistently (greater than 90%) contain antibodies which neutralize Actinobacillus actinomycetemcomitans leukotoxin(s). On the other hand, sera from normal individuals or patients with other types of periodontal disease usually amplified rather than inhibited the leukotoxic reaction. Many patients with juvenile periodontitis have demonstrable defects in PMN or monocyte chemotaxis and this may place them at risk to gingival infection by Actinobacillus actinomycetemcomitans. The immune response against these organisms could be a crucial determinant in the course of juvenile periodontitis. While this disease is relatively rare, it does cause immeasurable emotional, physical and economic hardship for patients and their families. The identification of Actinobacillus actinomycetemcomitans as a potential pathogen in this disorder may eventually lead to specific forms of therapy to prevent and eliminate infection by this organism in these patients.

The deposition of bacterial plaques on tooth surfaces appears to be responsible for the initiation and progression of periodontal disease. In this study, human peripheral blood polymorphonuclear leukocytes (PMNs) actively released lysosomal constituents upon in vitro exposure to either viable or irradiated, supragingival or subgingival dental plaque. Plaques were obtained from the PMN donors (autologous plaque) or from pooled samples (homologous plaque) secured from patients with periodontal lesions. Fresh sera from PMN donors amplified the release reactions to supragingival and subgingival plaques. Heated (56 degrees C, 30 min) sera also enhanced release reactions, but not as consistently as fresh serum. It was postulated that modulation of PMN release by serum is mediated by complement components and/or antibodies to plaque bacteria. Electron microscopic observations indicated that degranulation and discharge of PMN lysosomal enzymes may be associated with phagocytosis of gram-positive and gram-negative plaque bacteria and with reverse endocytosis of lysosomes from cells contacting relatively large masses of aggregated plaque bacteria. These data suggest that PMN lysosome release in response to plaque may serve as a potential mechanism of tissue injury in the pathogenesis of gingival and periodontal inflammation.