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Background/aims: Clinical studies indicate that primary proinflammatory cytokines, such as interleukin-1β (IL-1β) are elevated in the gingival crevice around teeth with periodontitis but the secondary cytokines and chemokines, IL-6 and IL-8, are not. The human gingival epithelial cells (HGECs) lining the gingival sulcus respond to perturbation by microbes of dental plaque by releasing a wide range of cytokines. Porphyromonas gingivalis, a putative periodontal pathogen, possesses numerous virulence factors some of which directly impact on the host response. In the present study, we sought to determine how P. gingivalis influences the inflammatory cytokine responses. Methods: HGECs were challenged with P. gingivalis and other putative periodontal pathogens, and the resultant production of IL-1β, IL-6, and IL-8 was assayed by enzyme-linked immunosorbent assay (ELISA). Culture supernatants and recombinant human cytokines were challenged with live P. gingivalis wild-type and gingipain-deficient strains and the resultant cytokine profile was assessed by ELISA and Western blot. Results: We show here that primary HGECs challenged with live P. gingivalis result in high levels of IL-1β but not the related secondary cytokines IL-6 and IL-8. We further demonstrate that cytokine response differences are the result of the action of P. gingivalis proteases, with lysine gingipain being the most effective. Conclusion: We conclude that P. gingivalis, through lysine gingipain, can subvert the protective host proinflammatory response by direct cytokine degradation. Changes in the crevicular cytokine profile have consequences in periodontal disease pathogenesis that should be considered in the development of diagnostic and therapeutic modalities. [ABSTRACT FROM AUTHOR]

Introduction: The program for mammalian cell growth and division consists of four successive phases; G1, S, G2, and M. Porphyromonas gingivalis may manipulate the host cell cycle to benefit bacterial virulence expression, which likely causes the cell and tissue tropism observed in chronic periodontal infections. We examined P. gingivalis for its effects on cell-cycle modulation in mouse ST2 osteoblastic/stromal cells. Methods: Synchronized ST2 cells were infected with P. gingivalis ATCC33277 (wild-type, WT), gingipain-mutants [KDP136 (Δ rgpAΔ rgpBΔ kgp), KDP129 (Δ rgpAΔ rgpB), and KDP133 (Δ kgp)], and a fimbria-deficient mutant (KDP150) for 24 h, then the cell cycle was evaluated using flow cytometry. Cell-cycle-related molecule expression was examined with a microarray, as well as with quantitative real-time polymerase chain reaction and Western blotting assays. Results: Both the WT and KDP150 strains significantly inhibited cellular proliferation and arrested the cell cycle in the G0/G1 phase, while the expression levels of the cell-cycle regulatory molecules cyclin D and cyclin E were also decreased. In contrast, KDP136 did not show any effects. G1 arrest was also clearly induced by KDP129 and KDP133, with KDP129 being more effective. Conclusion: The present findings suggest that P. gingivalis gingipains reduce cyclin expression and cause early G1 arrest, leading to the inhibition of cellular proliferation. [ABSTRACT FROM AUTHOR]

Introduction: We investigated the effect of anti- Porphyromonas gingivalis egg yolk antibody against gingipains [immunoglobulin Y (IgY)-GP] on gingipain activity in vitro. Methods: IgY-GP was isolated from the yolks of White Leghorn hens immunized with purified gingipains. Control antibody (IgY) was isolated from the yolks of non-immunized hens. Gingipain activity was assessed according to the rate of enzymatic substrate hydrolysis. Human epithelial cells were cultured with or without gingipains and with gingipains pretreated with either IgY-GP or IgY. Results: Hydrolytic activity decreased in the presence of IgY-GP. Cells incubated with gingipains showed a dose-dependent loss of adhesion activity. Pretreatment of gingipains with IgY-GP was associated with strong inhibition of cell detachment, whereas pretreatment with IgY was not. Conclusion: Our findings suggest that IgY-GP may be an effective immunotherapeutic agent in the treatment of periodontitis. [ABSTRACT FROM AUTHOR]

Introduction: Porphyromonas gingivalis is one of the most important bacteria in the progression of chronic periodontal disease. We hypothesized that the major outer membrane proteins Pgm6/7, which are homologous to the OmpA protein in Escherichia coli, might contribute to the stabilization of the cell surface. In this study, the effects of Pgm6/7 on the cell surface were examined morphologically. Methods: Deletion mutants of Pgm6/7 (Δ 694, Δ 695 and Δ 695–694) were constructed using the polymerase chain reaction-based overlap extension method. Wild-type ATCC 33277 and Pgm6/7 mutants were grown under anaerobic conditions. Whole cells and thin sections of fixed cells were stained and examined by transmission electron microscopy. Results: Compared with the wild-type, numerous vesicles released from cells were observed in each deletion mutant. The outer membrane appeared wavy and irregular. Increased numbers of vesicles were confirmed after their preparation from the culture supernatant. Total gingipain activity in vesicles was increased five- to 10-fold in the deletion mutants. Conclusion: This report provides further evidence that Pgm6/7 proteins in P. gingivalis play an important role in the maintenance of bacterial outer membrane integrity. [ABSTRACT FROM AUTHOR]

Background/aims: Gingipains, proteolytic enzymes produced by the periodontal pathogen Porphyromonas gingivalis, are regarded as virulence factors in the pathogenesis of periodontitis. Inhibition of gingipain activity therefore may have therapeutic potential, and it has been suggested that chlorhexidine may inhibit the activities of these enzymes. The purposes of the present study were to examine systematically the inhibitory effects of chlorhexidine on three purified gingipains and to determine the effect of Zn(II) on chlorhexidine inhibition. Methods: The activities of lys-gingipain (Kgp) and two forms of arg-gingipain (RgpB and HRgpA) were measured in the presence of varying concentrations of chlorhexidine and with chlorhexidine supplemented with Zn(II). Inhibition constants ( Ki's) were determined for chlorhexidine alone and in the presence of Zn(II). Fractional inhibitory constant indices were calculated to assess the synergy of the chlorhexidine–Zn(II) inhibition. Results: RgpB, HRgpA, and Kgp were all inhibited by chlorhexidine with Ki's in the micromolar range. For RgpB and HRgpA, the inhibitory effects of chlorhexidine were enhanced 3–30-fold by Zn(II). The chlorhexidine–Zn(II) interaction was synergistic for inhibition of HRgpA and RgpB. For Kgp, the effect of Zn(II) on chlorhexidine inhibition was antagonistic. Conclusions: Chlorhexidine is an effective inhibitor of gingipains, and the inhibition of R-gingipains is enhanced by Zn(II). A mixture of chlorhexidine and Zn(II) may be useful as an adjunct in the treatment of periodontitis and in the post-treatment maintenance of periodontitis patients. [ABSTRACT FROM AUTHOR]