Alteration of PTGS2 promoter methylation in chronic periodontitis.

Levels of prostaglandin E(2) and the prostaglandin-endoperoxide synthase-2 (PTGS2, or COX-2) increase in actively progressing periodontal lesions, but decrease in chronic disease. We hypothesized that chronic inflammation is associated with altered DNA methylation levels within the PTGS2 promoter, with effects on COX-2 mRNA expression. PTGS2 promoter methylation levels from periodontally inflamed gingival biopsies showed a 5.06-fold increase as compared with non-inflamed samples (p = 0.03), and the odds of methylation in a CpG site in the inflamed gingival group is 4.46 times higher than in the same site in the non-inflamed group (p = 0.016). The level of methylation at -458 bp was inversely associated with transcriptional levels of PTGS2 (RT-PCR) (p = 0.01). Analysis of the data suggests that, in chronically inflamed tissues, there is a hypermethylation pattern of the PTGS2 promoter in association with a lower level of PTGS2 transcription, consistent with a dampening of COX-2 expression in chronic periodontitis. These findings suggest that the chronic persistence of the biofilm and inflammation may be associated with epigenetic changes in local tissues at the biofilm-gingival interface.

Porphyromonas gingivalis lipopolysaccharide induces tumor necrosis factor-alpha and interleukin-6 secretion, and CCL25 gene expression, in mouse primary gingival cell lines: interleukin-6-driven activation of CCL2.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis infection is strongly associated with periodontitis. Although P. gingivalis is known to elicit a strong inflammatory response, details of that remain fragmentary. To understand the local response to P. gingivalis, primary cell lines derived from mouse gingival tissues were exposed to P. gingivalis or Escherichia coli lipopolysaccharide, and the production of interleukin-6 and tumor necrosis factor-alpha was measured. CCL25 gene expression was measured by real-time polymerase chain reaction. Cells stimulated with combinations of interleukin-6, soluble interleukin-6 receptor and/or soluble gp130 were assayed for CCL2 and tumor necrosis factor-alpha secretion. MATERIAL AND METHODS: Primary cell lines were generated from mouse gingival tissues. Enzyme-linked immunosorbent assays were used to determine cytokine levels, and real-time polymerase chain reaction was used to quantify CCL25 gene expression. RESULTS: Exposure to P. gingivalis lipopolysaccharide but not to E. coli lipopolysaccharide resulted in significantly elevated levels of both interleukin-6 and tumor necrosis factor-alpha, and stimulation with P. gingivalis lipopolysaccharide also upregulated CCL25 gene expression. In one of three experiments, interleukin-6 induced CCL2 secretion, whereas interleukin-6 plus soluble interleukin-6 receptor induced CCL2 secretion in all three experiments, suggesting that both direct interleukin-6 signaling and interleukin-6 trans-signaling may be involved. However, because soluble gp130 did not inhibit trans-signaling, and because direct stimulation of gingival cells with soluble gp130 resulted in CCL2 secretion, the possibility exists that soluble gp130 forms binary complexes with soluble interleukin-6 receptor that promote direct interleukin-6 stimulation. CONCLUSION: These findings define a pathway in which exposure of gingival cells to P. gingivalis induces the release of interleukin-6 and tumor necrosis factor-alpha; interleukin-6, in turn, induces CCL2 secretion.

Biochemical and mutational analyses of the cathepsin c gene (CTSC) in three North American families with Papillon Lefèvre syndrome.

Papillon Lefèvre syndrome (PLS) is an autosomal recessive disorder characterized by palmoplantar hyperkeratosis and severe periodontitis. The disease is caused by mutations in the cathepsin C gene (CTSC) that maps to chromosome 11q14. CTSC gene mutations associated with PLS have been correlated with significantly decreased enzyme activity. Mutational analysis of the CTSC gene in three North American families segregating PLS identified four mutations, including a novel mutation p.G139R. All mutations were associated with dramatically reduced CTSC protease enzyme activity. A homozygous c.96T>G transversion resulting in a p.Y32X change was present in a Mexican PLS proband, while one Caucasian PLS proband was a compound heterozygote for the p.Y32X and p.R272P (c.815G>C) mutations. The other Caucasian PLS proband was a compound heterozygote for c.415G>A transition and c.1141delC mutations that resulted in a p.G139R and a frameshift and premature termination (p.L381fsX393), respectively. The c.415G>A was not present in more than 300 controls, suggesting it is not a CTSC polymorphism. Biochemical analysis demonstrated almost no detectable CTSC activity in leukocytes of all three probands. These mutations altered restriction enzyme sites in the highly conserved CTSC gene. Sequence analysis of CTSC exon 3 confirmed the previously reported p.T153I polymorphism in 4 of the 5 ethnically diverse populations studied.

CD40 mediated activation of gingival and periodontal ligament fibroblasts.

CD40 is a 50 kDa transmembrane protein important for regulating B lymphocyte proliferation and differentiation. This novel activation antigen is primarily expressed by hematopoietic cells including B lymphocytes, follicular dendritic cells, and monocytes. Recently, human fibroblasts from a variety of tissues were shown to display CD40; however, its function was unknown. Cellular responses mediated by CD40 are naturally triggered by its counter-receptor, the CD40 ligand, which is displayed on activated T cells, mast cells, eosinophils, basophils, and B lineage cells. This study investigated the functional significance of CD40 expression on periodontal fibroblasts, in the context of periodontal inflammation. The experiments reported herein demonstrate constitutive CD40 expression on cultured periodontal ligament (PDL) and gingival fibroblasts. Interestingly, cells of gingival origin displayed up to 13-fold higher constitutive levels of CD40, versus fibroblasts from PDL. Interferon gamma (IFN gamma) treatment enhanced CD40 expression on PDL and gingival fibroblasts, with up to 61-fold induction of expression. Immunohistochemical staining was used to detect CD40 on fibroblastic cells in both normal and acutely inflamed gingival tissue. Expression of CD40 in inflamed tissue was significantly higher than in uninflamed tissue. Western blot analysis of anti-CD40 triggered cells revealed the induction of tyrosine phosphorylation on a 50 kDa protein in PDL and gingival fibroblasts. These results indicate that CD40 is an active signaling conduit in periodontal fibroblasts. This concept was further substantiated by the fact that CD40 engagement stimulated interleukin 6 (IL-6) production by gingival fibroblasts, but not periodontal ligament fibroblasts. Overall, these results demonstrate that CD40 on periodontal fibroblasts may functionally interact with CD40L-expressing cells. This CD40/CD40L interaction can stimulate fibroblast activation and synthesis of the proinflammatory cytokine IL-6.

Treatment of retrograde peri-implantitis: clinical report.

Six months after implant placement to restore a maxillary lateral incisor, a 55-year-old patient developed a sinus tract associated with the apical area. Radiographic examination revealed a periapical radiolucency at the apex of the implant, and a diagnosis of retrograde peri-implantitis was made. Treatment consisted of elevation of a full-thickness flap, curettage of the apical lesion, irrigation with chlorhexidine gluconate, placement of demineralized freeze-dried bone, and coverage of the site with an absorbable collagen would dressing before primary closure of the flap. The prosthesis is functioning satisfactorily 17 months after apical treatment was performed.