Rapid tissue factor induction by oral streptococci and monocyte-IL-1beta.

The ability of pro-inflammatory cytokines to promote coagulation prompted the hypothesis that pro-inflammatory cytokines induced by oral streptococci might play a role in the pathogenesis of viridans endocarditis. We used supernatant fluids from peripheral blood mononuclear monocyte (PBMC) cultures, stimulated for just 4-6 hrs with representative streptococcal isolates, to study cytokines that promoted endothelial tissue factor (TF) activity. Neutralizing antibodies demonstrated that interleukin-1beta (IL-1beta) was a major early endothelial TF inducer, and that recombinant IL-1beta was comparable with the supernatant fluid in activity. IL-1beta-rich supernatant fluids from oral streptococci-stimulated or lipopolysaccharide-stimulated PBMC cultures up-regulated the expression of endothelial ICAM-1 and E-selectin. These molecules could help trap TF-producing monocytes or dendritic cells bearing streptococci at the site. Thus, the rapid IL-1beta-inducing capacity of oral streptococci could facilitate the early deposition of bacteria in fibrin clots and promote endocarditis.

Dendritic cells stimulated with Actinobacillus actinomycetemcomitans elicit rapid gamma interferon responses by natural killer cells.

Human immunoglobulin G2 (IgG2) responses are gamma interferon (IFN-gamma) dependent, and monocyte-derived dendritic cells (mDCs) promote IgG2 production. DCs spontaneously emerge from monocytes in cultures prepared from localized aggressive periodontitis (LagP) patients, and these patients have high levels of IgG2 that is reactive with Actinobacillus actinomycetemcomitans. These results prompted the hypothesis that an interaction between mDCs and A. actinomycetemcomitans promotes IFN-gamma production, and IFN-gamma is known to promote both immunopathology and protective IgG2. A. actinomycetemcomitans induced mDCs to produce interleukin-12 (IL-12), and the addition of A. actinomycetemcomitans and DCs to cultured peripheral blood lymphocytes elicited high levels of IFN-gamma within just 24 h. In contrast, IL-4 was not detectable although DC-derived IL-10 production was apparent. A. actinomycetemcomitans-stimulated macrophages prepared from the same monocytes lacked the ability to induce IL-12 or IFN-gamma responses. NK cells of the innate immune system were the primary source of this early IFN-gamma, although CD8 T cells also contributed some. The NK cell-derived IFN-gamma was IL-12 dependent, and A. actinomycetemcomitans-DC interactions were Toll-like receptor 4 dependent. A. actinomycetemcomitans and A. actinomycetemcomitans lipopolysaccharide (LPS) were more potent than Escherichia coli and E. coli LPS in the ability to induce DC IL-12 and IFN-gamma. The ability of A. actinomycetemcomitans-stimulated DCs to induce NK cells to rapidly produce IFN-gamma in the absence of detectable IL-4 suggests their potential for skewing responses toward Th1. This may help explain the presence of Th1-associated cytokines in gingival crevicular fluid (GCF) from LagP patients and the high levels of IgG2 in their serum and GCF that is reactive with A. actinomycetemcomitans.

Cytokine induction by Streptococcus mutans and pulpal pathogenesis.

Chronic pulpal inflammation under caries appears to be elicited by bacterial antigens that diffuse into the pulp through dentinal tubules. This prompted the hypothesis that cytokines elicited by antigens from Streptococcus mutans, which frequently dominates shallow lesions, could play a major role in eliciting the initial T-cell response in the pulp. To test this, we examined the ability of S. mutans to stimulate T cells and elicit cytokines and used Lactobacillus casei, which often predominates in deep carious lesions where B cells and plasma cells predominate, as a control. In addition, the presence of cytokines in the pulp was analyzed at the mRNA level. S. mutans elicited potent gamma interferon (IFN-gamma) responses in peripheral blood mononuclear cell cultures and reduced the CD4/CD8 ratio by promoting CD8(+) T cells. Multiple inflammatory cytokine mRNAs (IFN-gamma, interleukin 4 [IL-4], and IL-10) were detected in human dental pulp. A higher prevalence of IFN-gamma (67%) than IL-4 (19%) or IL-10 (29%) was obtained in shallow caries, suggesting a type 1 cytokine mechanism in early pulpitis where S. mutans predominates. In contrast, in deep caries no differences in cytokine frequency were observed. Furthermore, the presence of IFN-gamma in the pulp correlated with the presence of S. mutans. The extraordinary induction of type 1 cytokines and the preferential activation of CD8(+) T cells by S. mutans offers an explanation for the etiology of the CD8(+) T-cell-dominant lesion in early pulpitis and suggests that S. mutans may have a major impact on the initial lesion and pulpal pathology.

Antiphosphorylcholine antibody levels are elevated in humans with periodontal diseases.

Human immunoglobulin G2 (IgG2) serum concentrations and the IgG2 antibody response to Actinobacillus actinomycetemcomitans can be influenced by genes, by environmental factors such as smoking, and by periodontal disease status. Examination of the IgG2 response to phosphorylcholine (PC), a response thought to be mainly induced by the C polysaccharide of Streptococcus pneumoniae, suggested that periodontal disease status was also associated with this response. This prompted the hypothesis that PC is an important oral antigen associated with organisms in the periodontal flora and that anti-PC antibody is elevated as a consequence of periodontal disease. Subjects in various periodontal disease diagnostic categories in which attachment loss is exhibited were tested for anti-PC in serum. Those with adult periodontitis, localized juvenile periodontitis, generalized early-onset periodontitis, and gingival recession all had similar levels of anti-PC IgG2 serum antibody which were significantly greater than in the group of subjects with no attachment loss. Analysis of plaque samples from subgingival and supragingival sites in all diseases categories for reactivity with the anti-PC specific monoclonal antibody TEPC-15 revealed that a substantial proportion of the bacteria in dental plaque (30 to 40%) bear PC antigen; this antigen was not restricted to morphotypes resembling only cocci but was also present on rods and branched filamentous organisms. We found that S. mitis, S. oralis, and S. sanguis, as well as oral actinomycetes, including A. viscosus, A. odontolyticus, and A. israelii, incorporated substantial amounts of [(3)H]choline from culture media. Further analysis of antigens derived from these organisms by Western blot indicated that S. oralis, S. sanguis, A. viscosus, A. odontolyticus, and A. israelii contained TEPC-15-reactive antigens. The data show that many commonly occurring bacterial species found in dental plaque contain PC antigen and that immunization with plaque-derived PC antigens as a consequence of inflammation and periodontal attachment loss may influence systemic anti-PC antibody concentrations.

Polyclonal B cell activators and in vitro induction of auto-antibody reactive with collagen.

Cells producing autoantibodies are known to be present in chronically inflamed periodontal tissues. In sites of chronic inflammation, polyclonal B cell activators (PBA) are known to exhibit adjuvant activity when combined with foreign antigens. These results prompted an examination of PBA in eliciting an antibody response to an autoantigen (i.e. collagen type I). Rat lymphocytes were stimulated with rat collagen (type I), microbial PBA (LPS) or the combination of LPS plus rat collagen in vitro. Anti-collagen antibody-forming cells (AFC) were enumerated using an ELISPOT assay. Collagen or LPS alone elicited few anti-collagen AFC but the addition of LPS to collagen resulted in a substantial adjuvant effect and yielded maximal responses to collagen. Comparisons of anti-collagen AFC from short-term immunized (2-6 wk after booster), non-immunized and long-term immunized (3-4 months after booster) animals were performed. It revealed that cells from recently immunized rats were significantly easier to activate than the other 2 groups. The adjuvant effect of microbial PBA may be important in anti-collagen antibody production and thus the localization of PBA in periodontal pockets may explain why anti-collagen AFC are restricted to the chronically inflamed periodontal tissues.

Tobacco and smoking: environmental factors that modify the host response (immune system) and have an impact on periodontal health.

This review summarizes the current data on the effects of smoking and tobacco on the immune system and its potential impact on periodontal health. Smokers are 2.5-6 times more likely to develop periodontal disease than non-smokers, and there is evidence for a direct correlation between the number of cigarettes smoked and the risk of developing disease. Tobacco users also tend to exhibit increased severity of periodontal disease. Direct correlations between tobacco use and increased attachment loss and pocket depth and reduced bone crest height have been reported. Although the correlation between tobacco use and periodontal disease is quite strong, the role of tobacco in the pathogenesis of periodontal disease is uncertain. Recent studies indicate that one potential mechanism is that tobacco use exacerbates periodontal disease because it alters the immune response to periodontal pathogens. Indeed, smokers exhibit increased numbers of peripheral blood mononuclear phagocytes which appear to be functionally compromised. Inadequate phagocyte activity could reduce the clearance of pathogens from the oral cavity and thereby facilitate the development of periodontal disease. Tobacco-exposed B- and T-lymphocytes exhibit reduced proliferative capacities which could limit the production of protective immunoglobulins against oral pathogens. The risk factors for periodontal disease can be broadly classified as genetic, environmental, host-response factors, and host-related factors such as age. Tobacco, an environmental factor, undermines the host response and may facilitate the development and progression of periodontal disease. This review highlights the inter-relatedness of two of the risk factors associated with periodontal disease.

The effects of immunoglobulins on the convective permeability of human dentine in vitro.

Immunoglobulin molecules are localized in the dentinal tubules of non-carious and carious teeth, but their possible role in caries invasion is not understood. This study sought to examine the effects of immunoglobulin molecules on dentine permeability using a fluid-filtration method. Crown segments cut from impacted human third molars were treated by filtration with 100 micrograms/ml IgG, 100 micrograms/ml IgA or 30 micrograms/ml IgM under a constant pressure. Flow rates were recorded and percent changes in flow rate analysed over time. Filtrates collected at various times were tested for changes in immunoglobulin concentrations by an enzyme-linked immunosorbent assay, and the percent retention of immunoglobulins to dentine was calculated. There was a decreasing non-linear exponential relation between the percent changes in flow rate and filtration time for all three immunoglobulins. The percentage of retained immunoglobulins was significantly related to the filtration time for all three classes of immunoglobulins. Immunoglobulin retention contributed to significant changes in flow rate with time. These in vitro results indicate the potential mechanism of immunoglobulins in decreasing tabular permeability.

Correlation between thermal sensitivity and microorganisms isolated from deep carious dentin.

The correlation between thermal sensitivity and the microorganisms present in 29 deep carious lesions was studied. The numbers of lactobacilli and total Gram-positive rods in the carious lesions were found to be negatively related to the length of pain triggered by cold and heat stimulants. The presence of Gram-positive cocci and non-black-pigmented Bacteroides were positively associated with both cold and heat sensitivities. Black-pigmented Bacteroides, Streptococcus mutans, and total anaerobic colony counts were positively related to the heat sensitivity. Recovery of Fusobacterium nucleatum, Actinomyces viscosus, and enterics on the selective plates was associated with cold sensitivity. Total counts of Gram-positive cocci and Gram-negative rods on the anaerobic nonselective medium were positively related to the cold sensitivity. It appeared that teeth with low numbers of lactobacilli in the carious lesions usually responded to thermal tests with longer duration of pain. Conversely, teeth with high numbers of lactobacilli in the carious lesions usually responded with shorter duration of pain.

Antibodies in normal and diseased pulps reactive with microorganisms isolated from deep caries.

Immunoglobulin molecules in the supernatant fluids (SF) from pulpal explant cultures have been observed to react with microorganisms implicated in infections of root canals. In this study, the reactivity of immunoglobulin molecules in the SF from normal and irreversible pulpitis pulps to six strains of predominant microorganisms isolated from the immediate layer of carious lesions above the pulps used for explant cultures was investigated using an enzyme-linked immunosorbent assay. Two ATCC strains of Eubacterium were also included in this assay. Specific antibodies to Lactobacillus casei subsp. casei, Lactobacillus casei subsp. rhamnosus, Lactobacillus acidophilus (I), (II), Streptococcus mutans, Bacteroides intermedius, Eubacterium brachy, and Eubacterium alactolyticum in the SF from the normal and irreversible pulpitis tissues were observed with a large variation of antibody levels in both groups. Immunodiffusion assays of the SF revealed that IgG was the major class of immunoglobulin in the normal as well as the irreversible groups. The presence of natural antibodies in the normal pulps suggested a possible protective role of antibodies during the invasive process of caries.

Microbiological studies of carious dentine from human teeth with irreversible pulpitis.

Aerobic and anaerobic cultures were made from 29 extracted teeth with irreversible pulpitis to identify the predominant flora in different parts of deep carious lesions. Most isolates were Gram-positive rods, in which lactobacilli were the most frequent organisms, then other Gram-positive, non-branching rods. Gram-positive cocci were the next most common; only a low number of Streptococcus mutans was recovered. Two types of carious lesions were found, one with high numbers of lactobacilli, the other with low. In the 15 lesions with high numbers, lactobacilli constituted 91.9% of the total flora at the pulpal site and gradually decreased in number as the sampling moved away from the pulp. Strep. mutans and alpha-haemolytic streptococci were not recovered from pulpal or deep carious sites in this group. In the 14 lesions with low numbers of lactobacilli, the flora was diverse. Gram-positive cocci, anaerobic Gram-positive non-branching rods, branching rods and/or Bacteroides were the main isolates in a few of this group.

A study of T and B cells in pulpal pathosis.

A study was undertaken using monoclonal antibodies to determine the types of lymphocytes present in pulpal tissues. Pulps were extirpated from teeth clinically diagnosed as normal, reversibly inflamed, or irreversibly inflamed and stained with hematoxylin and eosin and an indirect immunoperoxidase technique using monoclonal antibodies reactive to pan-B lymphocytes (B), pan-T lymphocytes (T1), and helper (T4) and suppressor (T8) T lymphocytes. T and/or B lymphocytes were observed in normal pulpal tissues with T8 lymphocytes being predominant. The pulpal tissue in the reversible group demonstrated that more than 90% of the lymphocyte population were T lymphocytes, with a T4/T8 ratio of 0.56. Higher numbers of T1, T4, T8; and B lymphocytes were observed in the pulp from teeth in the irreversible group. A ratio of 1.14 of T4/T8 lymphocytes was observed in the irreversible group. A B/T1 lymphocyte ration of 1.60 suggested this ratio might be used as an index in the immunohistological diagnosis of irreversible pulpal pathosis. There appeared to be no association between the periodontal status of the teeth and the number of immunocompetent cells observed in the pulps. An hypothesis on the regulatory functions of T4 and T8 lymphocytes as well as the interaction of T and B lymphocytes and their products in the pathogenesis of pulpal disease is presented.

Pulsatile heparin administration in pregnancy: a new approach.

The role of anticoagulation during pregnancy for women with a past history of thromboembolism is a controversial issue. The risk of recurrence of thromboembolism, however, is of the magnitude of about 15%, which warrants intervention, especially when one is dealing with such a potentially lethal disease. The analysis of plasma heparin levels is suggested as the best way to monitor treatment. As an alternative to subcutaneous injections twice or three times daily, 15 women used a portable infusion pump (Zyklomat, Ferring) that delivered heparin either subcutaneously or intravenously for periods of up to 25 weeks. There were no recurrencies of thromboembolism and no clinical signs of osteoporosis. The patient acceptability of the pump was excellent as was the ability to maintain adequate heparin levels. Although there are some theoretical benefits with intermittent infusion of heparin evaluation of the advantages from a medical point of view must await further studies.