Michelle J. Steffen

Rat Model of Polymicrobial Infection, Immunity, and Alveolar Bone Resorption in Periodontal Disease

ABSTRACT One of the predominant polymicrobial infections of humans is expressed clinically as periodontal disease. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia have been strongly implicated as members of a pathogenic consortium in the etiology of adult periodontitis. In this study we hypothesized that P. gingivalis, T. denticola, and T. forsythia are synergistic in terms of virulence potential and induce chronic periodontal inflammation that leads to alveolar bone resorption in a polymicrobial infection in rats. Groups of rats were infected with either P. gingivalis, T. denticola, or T. forsythia in monomicrobial infections or with all three species in polymicrobial oral infections with or without Fusobacterium nucleatum. PCR analyses of oral microbial samples demonstrated that rats infected with one bacterium were orally colonized by each of the bacteria during the study interval, and increased serum immunoglobulin G (IgG) antibody levels substantiated the interaction of the host with the infecting bacteria. PCR analyses of the rats with polymicrobial infections demonstrated that most rats were infected with P. gingivalis, T. denticola, and T. forsythia as a consortium. Furthermore, all rats exhibited a significant increase in the level of IgG antibody to the polymicrobial consortium. Radiographic measurement of alveolar bone resorption showed that rats infected with the polymicrobial consortium with or without F. nucleatum exhibited significantly increased alveolar bone resorption compared to the resorption in uninfected control rats, as well as the resorption in rats infected with one of the microbes. These results documented that P. gingivalis, T. denticola, and T. forsythia not only exist as a consortium that is associated with chronic periodontitis but also exhibit synergistic virulence resulting in the immunoinflammatory bone resorption characteristic of periodontitis.

Serum antibodies to periodontal pathogens are a risk factor for Alzheimer’s disease

Chronic inflammation in periodontal disease has been suggested as a potential risk factor in Alzheimers disease (AD). The purpose of this study was to examine serum antibody levels to bacteria of periodontal disease in participants who eventually converted to AD compared with the antibody levels in control subjects.

Omega-3 Fatty Acid Effect on Alveolar Bone Loss in Rats

Gingival inflammation and alveolar bone resorption are hallmarks of adult periodontitis, elicited in response to oral micro-organisms such as Porphyromonas gingivalis. We hypothesized that omega (ω)-3 fatty acids (FA) dietary supplementation would modulate inflammatory reactions leading to periodontal disease in infected rats. Rats were fed fish oil (ω-3 FA) or corn oil (n-6 FA) diets for 22 weeks and were infected with P. gingivalis. Rats on the ω-3 FA diet exhibited elevated serum levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), documenting diet-induced changes. PCR analyses demonstrated that rats were orally colonized by P. gingivalis; increased IgG antibody levels substantiated this infection. P. gingivalis-infected rats treated with ω-3 FA had significantly less alveolar bone resorption. These results demonstrated the effectiveness of an ω-3 FA-supplemented diet in modulating alveolar bone resorption following P. gingivalis infection, and supported that ω-3 FA may be a useful adjunct in the treatment of periodontal disease. Abbreviations: PUFA, polyunsaturated fatty acid; EPA, eicosapentanoic acid; DHA, docosahexanoic acid; and PCR, polymerase chain-reaction.

Serum Inflammatory Mediators in Pregnancy: Changes After Periodontal Treatment and Association With Pregnancy Outcomes

BACKGROUND The purposes of this study were to determine: 1) if periodontal treatment in pregnant women before 21 weeks of gestation alters levels of inflammatory mediators in serum; and 2) if changes in these mediators are associated with birth outcomes. METHODS A total of 823 pregnant women with periodontitis were randomly assigned to receive scaling and root planing before 21 weeks of gestation or after delivery. Serum obtained between 13 and 16 weeks, 6 days (study baseline) and 29 to 32 weeks of gestation was analyzed for C-reactive protein; prostaglandin E(2); matrix metalloproteinase-9; fibrinogen; endotoxin; interleukin (IL)-1 beta, -6, and -8, and tumor necrosis factor-alpha. Cox regression, multiple linear regression, and the t, chi(2), and Fisher exact tests were used to examine associations among the biomarkers, periodontal treatment, and gestational age at delivery and birth weight. RESULTS A total of 796 women had baseline serum data, and 620 women had baseline and follow-up serum and birth data. Periodontal treatment did not significantly alter the level of any biomarker (P >0.05). Neither baseline levels nor the change from baseline in any biomarker were significantly associated with preterm birth or infant birth weight (P >0.05). In treatment subjects, the change in endotoxin was negatively associated with the change in probing depth (P <0.05). CONCLUSIONS Non-surgical mechanical periodontal treatment in pregnant women, delivered before 21 weeks of gestation, did not reduce systemic (serum) markers of inflammation. In pregnant women with periodontitis, levels of these markers at 13 to 17 weeks and 29 to 32 weeks of gestation were not associated with infant birth weight or a risk for preterm birth.

Systemic Immune Responses in Pregnancy and Periodontitis: Relationship to Pregnancy Outcomes in the Obstetrics and Periodontal Therapy (OPT) Study

BACKGROUND Our previous studies reported on the obstetric, periodontal, and microbiologic outcomes of women participating in the Obstetrics and Periodontal Therapy (OPT) Study. This article describes the systemic antibody responses to selected periodontal bacteria in the same patients. METHODS Serum samples, obtained from pregnant women at baseline (13 to 16 weeks; 6 days of gestation) and 29 to 32 weeks, were analyzed by enzyme-linked immunosorbent assay for serum immunoglobulin G (IgG) antibody to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Campylobacter rectus, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia (previously T. forsythensis), and Treponema denticola. RESULTS At baseline, women who delivered live preterm infants had significantly lower total serum levels of IgG antibody to the panel of periodontal pathogens (P = 0.0018), to P. gingivalis (P = 0.0013), and to F. nucleatum (P = 0.0200) than women who delivered at term. These differences were not significant at 29 to 32 weeks. Changes in IgG levels between baseline and 29 to 32 weeks were not associated with preterm birth when adjusted for treatment group, clinical center, race, or age. In addition, delivery of low birth weight infants was not associated with levels of antibody at baseline or with antibody changes during pregnancy. CONCLUSIONS Live preterm birth is associated with decreased levels of IgG antibody to periodontal pathogens in women with periodontitis when assessed during the second trimester. Changes in IgG antibody during pregnancy are not associated with birth outcomes.

Characteristics and Utilization of Antibody Measurements in Clinical Studies of Periodontal Disease

The detection and quantitation of immune responses to infections have long been used as a diagnostic tool in medical infections. Recently, increasing evidence has supported that active, specific antibody responses to selected members of the subgingival microbiota are noted in periodontitis patients. This report describes the various specificities of this antibody as they relate to periodontitis classification and prognosis. The functional aspects of the serum antibody have come under increasing scrutiny to understand better the potential immunologic mechanisms acting in the periodontium. Data are available that describe opsonizing potential, complement fixing ability, blocking functions, and anti-toxic capacity for the antibody. Longitudinal alterations in specific antibody levels are shown to relate to infection and accompany changes in the burden of a specific microorganism in the subgingival plaque. Thus, these antibody changes could be useful indicators of altered host-parasite interactions that presage a disease-active episode. Finally, studies were designed to examine the ability of antibody to reflect the effects of treatment on the disease. The results indicated that specific antibody levels change with mechanical, antimicrobial, and anti-inflammatory treatments. The findings described in this report suggest that evaluation of the level and specificity of serum antibody can be a beneficial adjunct in designing and implementing clinical studies delineating the initiation, progression, and treatment of periodontitis. J Periodontol 1992; 63:1110-1116.

Cytokine Responses to Treponema pectinovorum and Treponema denticola in Human Gingival Fibroblasts

ABSTRACT Human gingival fibroblasts were challenged with Treponema pectinovorum and Treponema denticola to test three specific hypotheses: (i) these treponemes induce different cytokine profiles from the fibroblasts, (ii) differences in cytokine profiles are observed after challenge with live versus killed treponemes, and (iii) differences in cytokine profiles are noted from different gingival fibroblast cell lines when challenged with these treponemes. Three normal gingival fibroblast cell cultures were challenged withT. pectinovorum and T. denticola strains, and the supernatants were analyzed for cytokine production (i.e., interleukin-1α [IL-1α], IL-1β, IL-6, IL-8, IL-10, gamma interferon, macrophage chemotactic protein 1 [MCP-1], platelet-derived growth factor, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor). Unstimulated fibroblast cell lines produced IL-6, IL-8, and MCP-1. T. pectinovorum routinely elicited the greatest production of these cytokines from the fibroblast cell lines, increasing 10- to 50-fold over basal production. While T. denticola also induced IL-6 and IL-8 production, these levels were generally lower than those elicited by challenge with T. pectinovorum. MCP-1 levels were significantly lower after T. denticola challenge, and the kinetics suggested that this microorganism actually inhibited basal production by the fibroblasts. No basal or stimulated production of the other cytokines was observed. Significant differences were noted in the responsiveness of the various cell lines with respect to the two species of treponemes and the individual cytokines produced. Finally, dead T. pectinovorum generally induced a twofold-greater level of IL-6 and IL-8 than the live bacteria. These results supported the idea that different species of oral treponemes can elicit proinflammatory cytokine production by gingival cells and that this stimulation did not require live microorganisms. Importantly, a unique difference was noted in the ability of T. pectinovorum to induce a robust MCP-1 production, while T. denticolaappeared to inhibit this activity of the fibroblasts. While the general cytokine profiles of the fibroblast cell cultures were similar, significant differences were noted in the quantity of individual cytokines produced, which could relate to individual patient variation in local inflammatory responses in the periodontium.

Antigen Specificity of Serum Antibody in A. actinomycetemcomitans-infected Periodontitis Patients:

We hypothesized that serum antibody with selected antigen specificities would relate to infection and disease in the patients and, thus, describe the characteristics of potential protective antibody. This study used serum samples from 24 periodontitis patients with subgingival infection and elevated serum IgG antibody to A. actinomycetemcomitans to define the antigenic specificities of IgG, IgM, IgA, and IgGl-4 antibody to A. actinomycetemcomitans strain Y4 outer membrane antigens (OMA). Uniform IgG antibody (> 70% of the patients) was noted to antigens with Mr of 65, 38, 29, and 17 kDa. Both IgA and IgM specificities reflected those shown for IgG in each patient. IgGl and IgG2 antibody reacted with several OMA bands in each patient, while IgG3 antibodies were directed to numerous OMA bands in many patients and represented the most broad-based response. The IgG4 response patterns were limited to a few OMA bands. We noted a prominent occurrence of IgG reactions with OMA bands that were characteristic for individual patients. The frequency of responses to OMA of higher Mr (i.e., > 80 kDa) and to the 34-, 31-, and 24-kDa antigens was positively related to the total IgG antibody levels. Antibody reactive with OMA bands at 65-, 38-, 29-, 17-, 15-, and 11-kDa antigens was detected in patients with few to many teeth infected with A. actinomycetemcomitans. Furthermore, patients with a high percentage of teeth with ≥ 6 mm pockets had a decreased frequency of responses to the high-Mr antigens (i.e. > 90 kDa) as well as to the 58-kDa antigen. These findings indicate that human antibody reactivities with specific OMA bands of A. actinomycetemcomitans: (i) are positively correlated with the level of serum antibody, (ii) are associated with the number of teeth infected, and (iii) describe differences in the severity of the disease as measured by the frequency of teeth with deep pockets.

Systemic inflammatory responses in progressing periodontitis during pregnancy in a baboon model

This study tested the hypothesis that pregnant female baboons exhibit increased levels of various inflammatory mediators in serum resulting from ligature‐induced periodontitis, and that these profiles would relate to periodontal disease severity/extent in the animals. The animals were sampled at baseline (B), mid‐pregnancy (MP; two quadrants ligated) and at delivery (D; four quadrants ligated). All baboons developed increased plaque, gingival inflammation and bleeding, pocket depths and attachment loss following placement of the ligatures. By MP, both prostaglandin E2 (PGE2) and bactericidal permeability inducing factor (BPI) were greater than baseline, while increased levels of interleukin (IL)‐6 occurred in the experimental animals by the time of delivery. IL‐8, MCP‐1 and LBP all decreased from baseline through the ligation phase of the study. Stratification of the animals by baseline clinical presentation demonstrated that PGE2, LBP, IL‐8 and MCP‐1 levels were altered throughout the ligation interval, irrespective of baseline clinical values. IL‐6, IL‐8 and LBP were significantly lower in the subset of animals that demonstrated the least clinical response to ligation, indicative of progressing periodontal disease. PGE2, macrophage chemotactic protein (MCP)‐1, regulated upon activation, normal T cell expressed and secreted (RANTES) and LBP were decreased in the most diseased subset of animals at delivery. Systemic antibody responses to Fusobacterium nucleatum, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Campylobacter rectus were associated most frequently with variations in inflammatory mediator levels. These results provide a profile of systemic inflammatory mediators during ligature‐induced periodontitis in pregnant baboons. The relationship of the oral clinical parameters to systemic inflammatory responses is consistent with a contribution to adverse pregnancy outcomes in a subset of the animals.

Systemic endotoxin levels in chronic indolent periodontal infections.

BACKGROUND AND OBJECTIVE Periodontal disease has been linked with an increased risk of various systemic diseases. A plausible biologic explanation for this link includes the opportunity for oral pathogens to translocate to the circulation as a result of breakdown in integrity of the oral epithelium. This study refined a methodology used to detect endotoxin activity in the serum of subjects with indolent periodontal infections. MATERIAL AND METHODS The QCL Kinetic Chromogenic Assay (Cambrex) is a kinetic measure of endotoxin activity. Sera from 211 pregnant women with periodontitis enrolled in the Obstetrics and Periodontal Therapy Trial were used to develop the assay further and to evaluate the detection of endotoxin activity that might accompany a low-level bacteremia in chronic periodontitis. RESULTS We optimized the system to increase the sensitivity and reproducibility of the assay. The refined system was able to detect endotoxin activity in serum at > 0.0125 EU/mL. At baseline (13-16 wk of gestation), 35.5% of the women were positive for endotoxin activity (1.62 +/- 2.21; range: 0.38-15 EU/mL). CONCLUSION This report describes a sensitive measure of endotoxin activity in serum. The procedure allowed us to document levels of this microbial virulence factor in serum of individuals with indolent infections such as periodontal disease.