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Treponema medium Glycoconjugate Inhibits Activation of Human Gingival Fibroblasts Stimulated with Phenol-Water Extracts of Periodontopathic Bacteria

Department of Oral Microbiology, Asahi University School of Dentistry, 1851-1 Hozumi, Mizuho, Gifu 501-0296, Japan;

View larger version (17K): [in a new window]   Figure 1. CD14 expression and IL-8 production in human gingival fibroblasts. (A) Cell-surface expression of CD14 on human gingival fibroblasts was determined with a specific antibody (bold line) or its isotype control (thin line), as described in MATERIALS & METHODS. (B) IL-8 production in human gingival fibroblasts stimulated with various periodontopathic bacterial phenol-water extracts. The cells were stimulated with the indicated doses of the phenol-water extracts or with E. coli LPS in the presence of 5% FBS. The results are shown as the mean ± SEM. Representative results from 3 independent experiments are presented.

View larger version (42K): [in a new window]   Figure 2. Inhibitory effects of T. medium glycoconjugate on phenol-water extract-induced activation of human gingival fibroblasts. Cells were stimulated with the phenol-water extracts or with E. coli LPS with (closed bar) or without (open bar) T. medium glycoconjugate in the presence of 5% FBS. (A) The results of IL-8 production are presented as the mean ± SEM. Data were analyzed by a one-way analysis of variance (ANOVA) with the Bonferroni or Dunn method, and asterisks indicate statistically significant (p < 0.01) inhibition by T. medium glycoconjugate. Representative results from 3 independent experiments are presented. (B) IL-8 mRNA expression. The ß-actin gene was assayed as a positive control, and PCR products of non-RT samples were used as a negative control. (C) p38 MAPK phosphorylation (pp38). (D) The cell-surface expression of ICAM-1 on human gingival fibroblasts was determined with a specific antibody (bold line) or its isotype control (thin line) as described in MATERIALS & METHODS. Representative results from 3 independent experiments are presented in B-D.

View larger version (23K): [in a new window]   Figure 3. Correlations between CD14/LPS-binding protein and activation of human gingival fibroblasts induced by phenol-water extracts. Cells were stimulated with the phenol-water extracts or with E. coli LPS with (closed bar) or without (open bar) T. medium glycoconjugate in the presence or absence of CD14- and LPS-binding protein. The results are presented as the mean ± SEM. Data were analyzed by a one-way analysis of variance (ANOVA) with the Bonferroni or Dunn method, and an asterisk indicates statistically significant inhibition by T. medium glycoconjugate (p < 0.01). Representative results from 3 independent experiments are presented.

View larger version (31K): [in a new window]   Figure 4. Inhibition of phenol-water extract binding to immobilized CD14 by T. medium glycoconjugate. Biotinylated phenol-water extract or E. coli LPS with or without T. medium glycoconjugate were added to CD14- or BSA-coated wells in the presence or absence of LPS-binding protein. The binding of biotinylated phenol-water extract or E. coli LPS was detected with the use of HRP-conjugated streptavidin. Representative results from 3 independent experiments are presented.