Monoclonal antibody-mediated modulation of the humoral immune response against mucosally applied Streptococcus mutans.

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.

Expression and immunogenicity of hemagglutinin A from Porphyromonas gingivalis in an avirulent Salmonella enterica serovar typhimurium vaccine strain.

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.

Oral diseases, mycology and periodontal microbiology of HIV-1-infected women.

HIV-1 infection is increasing more rapidly among heterosexual women. Relatively limited information is available on HIV-related oral pathoses in these individuals. To gain insight into the type and occurrence of oral lesions in this population, 25 HIV-1 infected women including asymptomatic, symptomatic and AIDS patients were examined clinically and sampled for detection of oral yeast and characterization of their subgingival microbial flora. Sixty percent of the subjects were African-American, with 80% infected via heterosexual contact. Oral candidiasis was the most common nonperiodontal oral lesion, observed in 44% of the patients. Oral yeast was cultured from all women with candidiasis and 76% of the total subjects. Oral hairy leukoplakia was clinically diagnosed in 16% of the subjects. Clinically mild to moderate gingivitis and periodontitis were observed in 84% and 52% of the patients, respectively. Candidiasis and the presence of cultivable yeast were observed in patients with low, intermediate, and high CD4+ T lymphocyte numbers. Plaque samples were collected from each subject and enumerated by predominant cultivable methods, selective media and microscopy. No differences were detected in the microflora associated with seropositive women with existing periodontitis relative to those without periodontitis or to seronegative women with periodontitis. Candidiasis was the most notable oral clinical manifestation in the HIV-1-infected women and may be a useful clinical indicator of early immune dysfunction mediated by HIV-1.

Peripheral blood leukocyte populations in the elderly with and without periodontal disease.

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.

Differentiation of the serotype b and species-specific antigens of Actinobacillus actinobacillus actinomycetemcomitans recognized by monoclonal antibodies.

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.

Inhibition of adherence of Actinomyces naeslundii (Actinomyces viscosus) T14V-J1 to saliva-treated hydroxyapatite by a monoclonal antibody to type 1 fimbriae.

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.

Antibody responses to suspected periodontal pathogens in elderly subjects with periodontal disease.

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.

Treatment of subjects with refractory periodontal disease.

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.

Modeling the relationship between clinical, microbiologic, and immunologic parameters and alveolar bone levels in an elderly population.

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.

Specific antibodies and their potential role in periodontal diseases.

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.

Subgingival microbiota in squirrel monkeys with naturally occurring periodontal diseases.

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.

Immune modulation of Prevotella intermedia colonization in squirrel monkeys.

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.

Clinical, microbiological and immunological characteristics of subjects with "refractory" periodontal disease.

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.

Host responses in the etiology and pathogenesis of periodontal disease.

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.

Geriatric house calls: relic of the past or challenge of the future?

Historically, house calls played an important role in mainstream medical practice, but since the early part of the 20th century, medical care has been increasingly focused on the acute care hospital. This focus appears to have contributed to increasing physician frustration, patient alienation, and a growing public dissatisfaction with service. The geriatric population, and in particular the frail elderly, are a unique and growing proportion of the general population, and they are also the major consumers of health care resources. Patient-oriented and professional factors point to a need for physicians to return to making more house calls. Active participation by primary care physicians in developing standards and conducting research in this area of practice is necessary.

Transfer of plasmid pE5-2 from Escherichia coli to Bacteroides gingivalis and B. intermedius.

A unique shuttle plasmid, pE5-2, previously constructed to mediate gene transfer from Escherichia coli to colonic Bacteroides spp. has also been transferred via conjugation from E. coli to isolates of Bacteroides gingivalis and B. intermedius. The transfer occurred at a frequency of 1.4 to 2 x 10(-7) per recipient. The presence of the plasmid in transconjugants was verified by hybridization of the total DNA of B. gingivalis recipients with sequences of the pE5-2 plasmid, as well as by standard plasmid isolation techniques.

Modulation of colonization by black-pigmented Bacteroides species in squirrel monkeys by immunization with Bacteroides gingivalis.

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.

Natural occurrence of black-pigmented Bacteroides species in the gingival crevice of the squirrel monkey.

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.

Monoclonal antibodies to Actinobacillus actinomycetemcomitans.

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.