Successive active phases observed in periodontal diseases may be explained either by a sudden activation of the pro-forms of tissue-stored degradative enzymes such as metalloproteinases (MMPs) or by an imbalance between metalloproteinases and their tissue inhibitors (TIMPs). To discriminate between these two hypotheses, we quantified the levels, the percentage of active form, and the activities of four metalloproteinases (MMPs -1, -2, -3, and -9), as well as the levels of two tissue inhibitors of metalloproteinases (TIMP-1 and -2) and the activity of cathepsin C in tissue extract supernatants and their corresponding gingival crevicular fluid samples collected from periodontitis-affected and healthy patients. Our results supported evidence that tissue destruction results from an imbalance of metalloproteinases over their tissue inhibitors rather than from a sudden activation of the pro-forms of these enzymes. A significant reduction in the activity of cathepsin C also contributed to the degradative process.

Our results did not reveal significant differences in the expression of mRNAs encoding for the MMPs between healthy and periodontitis-affected patients, reflecting the great heterogeneity in the periodontal status of individuals. However, they indicate that gingival fibroblasts are an active source of MMP-2 production in response to a periopathogen.

In order to examine the possible implication of human epithelial and endothelial cells in the pathogenesis of various diseases associated with oral viridans streptococci, we tested the immunomodulatory effects of 11 representative strains of oral viridans streptococci on human epithelial KB cells and endothelial cells. We then examined the possible role of two major adhesins from oral viridans streptococci, protein I/II and rhamnoseglucose polymers (RGPs), in this process. In this study we demonstrate that oral viridans streptococci are potent stimulators of interleukin-8 (IL-8) production from KB cells and of IL-6 and IL-8 production from endothelial cells. The ability of protein I/II and RGPs to contribute to these effects was then examined. Using biotinylated protein I/IIf and RGPs from Streptococcus mutans OMZ 175, we showed that these adhesins bind to KB and endothelial cells through specific interactions and that the binding of these molecules initiates the release of IL-8 from KB cells and of IL-6 and IL-8 from endothelial cells. These results suggest that protein I/IIf and RGPs play an important role in the interactions between bacteria and KB and endothelial cells in that similar cytokine profiles are obtained when cells are stimulated with bacteria or surface components. We also provide evidence that protein I/IIf binds to and stimulates KB and endothelial cells through lectin interactions and that N-acetyl neuraminic acid (NANA) and fucose present on cell surface glycoproteins may form the recognition site since binding and cytokine release can be inhibited by dispase and periodate treatment of cells and by NANA and fucose. These results demonstrate that oral viridans streptococci, probably by engaging two cell surface adhesins, exert immunomodulatory effects on human KB and endothelial cells.

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Successive active phases observed in periodontal diseases may be explained either by a sudden activation of the pro-forms of tissue-stored degradative enzymes such as metalloproteinases (MMPs) or by an imbalance between metalloproteinases and their tissue inhibitors (TIMPs). To discriminate between these two hypotheses, we quantified the levels, the percentage of active form, and the activities of four metalloproteinases (MMPs -1, -2, -3, and -9), as well as the levels of two tissue inhibitors of metalloproteinases (TIMP-1 and -2) and the activity of cathepsin C in tissue extract supernatants and their corresponding gingival crevicular fluid samples collected from periodontitis-affected and healthy patients. Our results supported evidence that tissue destruction results from an imbalance of metalloproteinases over their tissue inhibitors rather than from a sudden activation of the pro-forms of these enzymes. A significant reduction in the activity of cathepsin C also contributed to the degradative process.

To examine the possible implication of protein SR, an VII-related antigen from Streptococcus mutans OMZ 175 (serotype f), in inflammatory reactions, we tested the immunomodulatory effects of protein SR on human monocytes. Using biotinylated protein, we provide evidence that protein SR binds to human monocytes in dose-, time-, and calcium-dependent manners through specific interactions. These results were confirmed by competition experiments using either soluble human monocyte extract or anti-SR immunoglobulin G. Binding occurred through lectin-like interactions between SR and carbohydrate portions of monocyte membrane glycoproteins, since binding could be inhibited by several sugars, especially fucose and N-acetylneuraminic acid (NANA), which were confirmed by ligand blotting to be the primer ligands recognized by SR on human monocyte extracts. The ability of protein SR to stimulate the production of cytokines by human circulating monocytes was then examined. The release of tumor necrosis factor alpha (TNF-a), interleukin 1p, and interleukin 6 is time and dose dependent and not affected by the addition of polymyxin B. Activation of monocytes resulted from specific binding of SR to NANA and fucose present on cell surface glycoproteins since TNF-a release could be inhibited by sialidase and pronase treatment of monocytes and by NANA and fucose. These results confirm that sialic acid and fucose present on cell surface macromolecules and especially glycoproteins are needed for the binding of SR to monocytes and for the release of TNF-a.

Despite the availability of melanoma treatment at the primary site, the recurrence of local melanoma can metastasize to any distant organ. Currently, the available therapies for the treatment of metastatic melanoma are of limited benefit. Thus, the functional analysis of conventional therapies may help to improve their efficiency in the treatment of metastatic melanoma. In the present study, the exposure of melanoma cells to vinblastine was found to trigger apoptosis as evidenced by the loss of mitochondrial membrane potential, the release of both cytochrome c and apoptosis inducing factor, activation of caspase-9 and 3, and cleavage of Poly (ADP-ribose)-Polymerase. Also, vinblastine enhances the phosphorylation of Ras homologous protein A, the accumulation of reactive oxygen species, the release of intracellular Ca(2+), as well as the activation of apoptosis signal-regulating kinase 1, c-jun-N-terminal kinase, p38, inhibitor of kappaBα (IκBα) kinase, and inositol requiring enzyme 1α. In addition, vinblastine induces the DNA-binding activities of the transcription factor NF-κB, HSF1, AP-1, and ATF-2, together with the expression of HSP70 and Bax proteins. Moreover, inhibitory experiments addressed a central role for Rho A in the regulation of vinblastine-induced apoptosis of melanoma cells via mitochondrial and non-mitochondrial-dependent mechanisms. In conclusion, the present study addresses for the first time a central role for Rho A in the modulation of vinblastine-induced apoptosis of melanoma cells and thereby provides an insight into the molecular action of vinblastine in melanoma treatment.

In order to examine the possible implication of capsular polysaccharide (CP) types 5 and 8 (CP5 and CP8) from Staphylococcus aureus in the pathological mechanism associated with staphylococcal infections, we tested the immunomodulatory effects of CP5 and CP8 on human epithelial KB cells, endothelial cells, and monocytes. Using biotinylated CP5 and CP8, we provide evidence that both CPs bind to KB cells, endothelial cells, and monocytes in a dose-and calcium-dependent manner through specific interactions. These results were confirmed by competition experiments using soluble cell extracts. Furthermore, we show that CPs bind to identical cell membrane receptors on all three types of human cells and that human normal serum contains a factor(s) which inhibits the binding of both CPs to human KB cells, endothelial cells, and monocytes. The ability of CP5 and CP8 to stimulate the production of cytokines by the human cells was then examined. CP5 and CP8 trigger KB cells to produce interleukin-8 (IL-8); endothelial cells to produce IL-8 and IL-6; and monocytes to produce IL-8, IL-6, IL-1␤, and tumor necrosis factor alpha. The release of cytokines by all three types of cells is time dependent and dose dependent, and the tumor necrosis factor alpha production by monocytes is not affected by the addition of polymyxin B. We further confirm that human normal serum inhibits the immunomodulatory effects of both polysaccharides on each kind of cell. These results confirm that S. aureus CPs act as bacterial adhesins having immunomodulatory effects for human cells.