Deficiency of iNOS contributes to Porphyromonas gingivalis-induced tissue damage

Periodontitis is a chronic inflammatory disease that results in extensive soft and hard tissue destruction of the periodontium. Porphyromonas gingivalis possesses an array of virulence factors and has been shown to induce expression of inducible nitric oxide synthase (iNOS) in inflammatory cells. The aim of this study was to investigate the effect of eliminating iNOS in a murine model of P. gingivalis infection. This was achieved by utilizing a P. gingivalis-induced skin abscess model, and an alveolar bone loss model employing an oral infection of P. gingivalis in iNOS knockout mice. The results indicated that iNOS knockout mice exhibit more extensive soft tissue damage and alveolar bone loss in response to P. gingivalis infection compared to wild-type mice. The local immune response to P. gingivalis in iNOS knockout mice was characterized by increased numbers of polymorphonuclear monocytes, while the systemic immune response was characterized by high levels of interleukin-12. The iNOS is required for an appropriate response to P. gingivalis infection.

Chemokines in human periodontal disease tissues

An immunoperoxidase technique was used to examine IP-10 (interferon-gamma inducible protein 10), RANTES (regulated on activation normal T cell expressed and secreted), MCP-1 (monocyte chemoattractant protein-1), and MIP-1alpha (macrophage inflammatory protein-1alpha) in gingival biopsies from 21 healthy/gingivitis and 26 periodontitis subjects. The samples were placed into 3 groups according to the size of infiltrate. MIP-1alpha+ cells were more abundant than the other chemokines with few MCP-1+ cells. The mean percent MIP-1alpha+ cells was higher than the percent MCP-1+ cells (P = 0.02) in group 2 (intermediate size infiltrates) lesions from periodontitis subjects, other differences not being significant due to the large variations between tissue samples. Analysis of positive cells in relation to CD4/CD8 ratios showed that with an increased proportion of CD8+ cells, the mean percent MIP-1alpha+ cells was significantly higher in comparison with the mean percent RANTES+ and MCP-1+ cells (P < 0.015). Endothelial cells were MCP-1+ although positive capillaries were found on the periphery of infiltrates only. Keratinocyte expression of chemokines was weak and while the numbers of healthy/gingivitis and periodontitis tissue sections positive for IP-10, RANTES and MCP-1 reduced with increasing inflammation, those positive for MIP-1alpha remained constant for all groups. In conclusion, fewer leucocytes expressed MCP-1 in gingival tissue sections, however, the percent MIP-1alpha+ cells was increased particularly in tissues with increased proportions of CD8 cells and B cells with increasing inflammation and also in tissues with higher numbers of macrophages with little inflammation. Further studies are required to determine the significance of MIP-1alpha in periodontal disease.

Cytokines in periodontal disease: where to from here?

Numerous studies have attempted to elucidate the cytokine networks involved in chronic periodontitis, often with conflicting results. A variety of techniques were used to study cells in situ, cells extracted from gingival tissues, peripheral blood mononuclear cells, purified cell populations, and T cell lines and clones. Bacterial components, including sonicates, killed cells, outer membrane components, and purified antigens, have all been used to stimulate cells in vitro, making comparisons of cytokine profiles difficult. As it is likely that different cells are present at different disease stages, the inability to determine disease activity clinically is a major limitation of all these studies. In the context of tissue destruction, cytokines such as IL-1, IL-6 and IL-18 are likely to be important, as are their regulating cytokines IL-10 and IL-11. In terms of the nature of the inflammatory infiltrate, two apparently conflicting hypotheses have emerged: one based on direct observations of human lesions, the other based on animal experimentation and the inability to demonstrate IL-4 mRNA in gingival extracts. In the first of these, Th1 responses are responsible for the stable lesion, while in the second Th2 responses are considered protective. Using Porphyromonas gingivalis specific T cell lines we have shown a tendency for IFN-gamma production rather than LL-I or IL-10 when antigen is presented with peripheral blood mononuclear cells which may contain dendritic cells. It is likely that the nature of the antigen-presenting cell is fundamental in determining the nature of the cytokine profile, which may in turn open up possibilities for new therapeutic modalities.

Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts

Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) are important proteolysis factors present in inflamed human periodontal tissues. The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) on the synthesis: of t-PA and PAI-2 by human gingival fibroblasts (HGF). LPS from different periodontal pathogens including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum were extracted by the hot phenol water method. The levels of t-PA and PAI-2 secreted into the cell culture media were measured by enzyme-linked immunosorbent assays (ELISA). The mRNA for t-PA and PAI-2 were measured by RT-PCR. The results showed t-PA synthesis was increased in response to all types of LPS studied and PAI-2 level was increased by LPS from A. actinomycetemcomitans and F. nucleatum, but not P. gingivalis. When comparing the effects of LPS from non-periodontal bacteria (Escherichia coli and Salmonella enteritidis) with the LPS from periodontal pathogens, we found that the ratio of t-PA to PAI-2 was greater following exposure of the cells to LPS from periodontal pathogens. The highest ratio of t-PA to PAI-2 was found in those cells exposed to LPS from P. gingivalis. These results indicate that LPS derived from periodontal pathogens may cause unbalanced regulation of plasminogen activator and plasminogen activator inhibitor by HGF and such an effect may, in part, contribute to the destruction of periodontal connective tissue through dysregulated pericellular proteolysis.

Immune response to Bacteroides forsythus in a murine model

A murine skin abscess model was used to study the immune response to an acute infection with Bacteroides forsythus. BALB/c mice were given subcutaneous injections of either viable or heat-killed B. forsythus, while a third sham-immunized control group received phosphate-buffered saline. Weights and lesion sizes were measured. Blood was collected from the heart and specific antibodies to B. forsythus measured by an ELISA. Swabs taken from the lesions and also from pooled blood were cultured anaerobically for viable B. forsythus. Viable B. forsythus-induced lesions reached maximum size at day 7. B. forsythus cells were recovered from lesions up to day 4 although none were cultured from blood samples. Heat-killed bacteria induced much smaller lesions. Serum antibody levels increased during the 9-day study period, being significantly higher in mice injected with viable compared with heat-killed B. forsythus. Antibody levels in sham control mice were significantly lower than those seen in the other two groups. These results showed that a subcutaneous injection of viable cells of B. forsythus elicited a pronounced abscess formation and induce higher levels of specific antibodies compared with that produced by an injection of dead bacteria. This suggests that, as with other periodontopathic organisms, this mouse model can be used to study the immune response to B. forsythus.

A longitudinal study of interleukin-1 gene polymorphisms and periodontal disease in a general adult population

Background: Cross-sectional studies have demonstrated that a specific polymorphism (allele 2 of both IL-1A +4845 and IL-1B +3954) in the IL-1 gene cluster has been associated with an increased susceptibility to severe periodontal disease and to an increased bleeding tendency during periodontal maintenance. The aim of the present study was to investigate the relationship between IL-1 genotype and periodontitis in a prospective longitudinal study in an adult population of essentially European heritage. Methods: From an ongoing study of the Oral Care Research Programme of The University of Queensland, 295 subjects consented to genotyping for IL-1 allele 2 polymorphisms. Probing depths and relative attachment levels were recorded at baseline, 6, 12, 24, 36, 48 and 60 months using the Florida probe. Periodontitis progression at a given site was defined as attachment loss greater than or equal to2 mm at any observation period during the 5 years of the study and the extent of disease progression determined by the number of sites showing attachment loss. Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Prevotella intermedia were detected using ELISA. Results: 38.9% of the subjects were positive for the composite IL-1 genotype. A relationship between the IL-1 positive genotype and increased mean probing pocket depth in non-smokers greater than 50 years of age was found. Further, IL-1 genotype positive smokers and genotype positive subjects with P. gingivalis in their plaque had an increase in the number of probing depths greater than or equal to3.5 mm, There was a consistent trend for IL-1 genotype positive subjects to experience attachment loss when compared with IL-1 genotype negative subjects. Conclusion: The results of this study have shown an interaction of the IL-1 positive genotype with age, smoking and P. gingivalis which suggests that IL-1 genotype is a contributory but non-essential risk factor for periodontal disease progression in this population.

A microbiological study of Papillon-LeFevre syndrome in two patients

Aim-To analyse the microflora of subgingival plaque from patients with Papillon-Lefevre syndrome (PLS), which is a very rare disease characterised by palmar-plantar hyperkeratosis with precocious periodontal destruction. Methods-Bacterial isolates were identified using a combination of commercial identification kits, traditional laboratory tests, and gas liquid chromatography. Some isolates were also subjected to partial 16S rDNA sequencing. Plaque samples were also assayed for the presence of Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in a quantitative enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies. Results-The culture results showed that most isolates were capnophilic and facultatively anaerobic species-mainly Capnocytophaga spp and Streptococcus spp. The latter included S constellatus, S oralis, and S sanguis. Other facultative bacteria belonged to the genera gemella, kingella, leuconostoc, and stomatococcus. The aerobic bacteria isolated were species of neisseria and bacillus. Anaerobic species included Prevotella intermedia, P melaninogenica, and P nigrescens, as well as Peptostreptococcus spp. ELISA detected P gingivalis in one patient in all sites sampled, whereas A actinomycetemcomitans was detected in only one site from the other patient. Prevotella intermedia was present in low numbers. Conclusions-Patients with PLS have a very complex subgingival flora including recognised periodontal pathogens. However, no particular periodontopathogen is invariably associated with PLS.