Development of a new diagnostic strategy of periodontal disease based on the gene profile associated with host defense mechanisms.

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The purpose of this study is to develop a new diagnostic strategy based on the gene profile that determine the host-parasite interaction occurring in periodontal disease lesion. We have successfully developed 2 methods applying the sensitive PCR technique ; 1)RT-PCR of 16sRNA genes to detect 25 periodontopathic bacteria from small amount of plaque samples, 2)real-time PCR protocol for quantification of multiple cytokine mRNA levels in the gingival tissue biopsies from periodontal disease patients. For microbial analyis, Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by PCR of the 16S rRNA genes. the detection frequencies of 11 bacteria including Porphyromonas gingivalis, Treponema denticola and Prevotella intermedia in subgingival plaque were significantly higher in Periodontitis (P) group than in Healthy (H) group. P. gingivalis was detected only in P group, suggesting this bacterium as the candidate for identifying periodontal diseases. The bacteria flora of supragingival plaque was also found to reflect that of subginginval plaque. Real-time PCR detection of cytokine mRNA showed a comparable levels of semi-quantitative conventional RT-PCR. We next applied the real-time technique for the quantification of multiple cytokine gene levels, and compared the mRNA levels with clinical parameters. Among the target cytokines in this study, IL-6 and IL-1α showed a positive and strong association with PPD and GI. IL-1ra mRNA levels of gingival specimens from periodontitis patients were consistently high, suggesting the evoke of immunosuppressive activity in periodontal lesion. Our data suggest that these protocols could provide powerful insights into the complexities of the cytokine network and host-parasite interactiuon. The elucidation of the network may clarify the mechanisms of disease sensitivity at individual level and disease activity at site level.