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The production of proinflammatory cytokines is likely to play a major pathophysiological role in meningitis and other infections caused by Haemophilus influenzae type b (Hib). Previous studies have shown that Hib porin contributes to signaling of the inflammatory cascade. We examined here the role of Toll-like receptors (TLRs) and the TLR-associated adaptor protein MyD88 in Hib porin-induced production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Hib porin-induced TNF-alpha and IL-6 production was virtually eliminated in macrophages from TLR2- or MyD88-deficient mice. In contrast, macrophages from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, which are defective in TLR4 function, responded normally to Hib porin. Moreover anti-TLR2 antibodies but not anti-TLR4 antibodies significantly reduced Hib porin-stimulated TNF-alpha and IL-6 release from the human monocytic cell line THP-1. These data indicate that the TLR2/MyD88 pathway plays an essential role in Hib porin-mediated cytokine production. These findings may be useful in the development of alternative therapies aimed at reducing excessive inflammatory responses during Hib infections.

ABSTRACTThe production of proinflammatory cytokines is likely to play a major pathophysiological role in meningitis and other infections caused by Haemophilus influenzaetype b (Hib). Previous studies have shown that Hib porin contributes to signaling of the inflammatory cascade. We examined here the role of Toll-like receptors (TLRs) and the TLR-associated adaptor protein MyD88 in Hib porin-induced production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Hib porin-induced TNF-α and IL-6 production was virtually eliminated in macrophages from TLR2- or MyD88-deficient mice. In contrast, macrophages from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, which are defective in TLR4 function, responded normally to Hib porin. Moreover anti-TLR2 antibodies but not anti-TLR4 antibodies significantly reduced Hib porin-stimulated TNF-α and IL-6 release from the human monocytic cell line THP-1. These data indicate that the TLR2/MyD88 pathway plays an essential role in Hib porin-mediated cytokine production. These findings may be useful in the development of alternative therapies aimed at reducing excessive inflammatory responses during Hib infections.

ABSTRACTMicromolar concentrations of porin, purified from the outer membranes of Pseudomonas aeruginosa, induced in vitro the classic morphological and biochemical signs of apoptosis in an epithelial cell line (SVC1) derived from the rat seminal vesicle secretory epithelium. The programmed cell death (PCD) was p53 independent and associated with significant decrease ofbcl-2expression, a marked increase of c-myctranscriptional activity, and an absence of the mRNA coding for tissue transglutaminase. The Ca2+influx, caused by the porin treatment of SVC1 cells, appears to play an important role in the triggering of apoptosis in our biological model. The possibility that the porin property of inducing PCD plays a role in the infertility of individuals chronically infected by gram-negative bacteria is discussed.

Human osteoblasts were obtained by migration and proliferation of cells from embryonic membranous bone on glass fragments. Light and electron microscopy analyses revealed a typical osteoblast-like appearance with high protein synthesis activity. The cells showed high alkaline phosphatase activity that was associated with plasma membranes and matrix vesicles and was 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] responsive. In contrast to the adult osteoblasts, embryonic cells could not produce detectable levels of osteocalcin, not even in the presence of 1,25(OH)2D3. Osteoblasts grown in multilayers produced a thick extracellular matrix, mainly composed of type I collagen, that mineralized in the presence of 10 mM ß-glycerophosphate. Because of their intrinsic osteogenic capacity, embryonic osteoblasts represent a valuable model for studying the mineralization process in vitro. In addition, the embryonic origin of these cells renders them a precious experimental system for the elucidation of mechanisms at the basis of differentiation of osteoblastic lineage.

Micromolar amounts of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, inducein vitroa marked release of a variety of cytokines (interferon-γ, tumor necrosis factor-α, interleukin 6, and granulocyte–monocyte colony-stimulating factor) from human resting peripheral blood mononuclear cells as well as from isolated resting lymphocytes and monocytes. This effect was found to be significantly higher when the spermidine adduct of SV-IV (Spd2-SV-IV), synthesizedin vitroby the enzyme transglutaminase, was used instead of the native protein. Furthermore, the pretreatment of monocytes with transglutaminase caused an increase of the inducing effect of both native and modified SV-IV on the release of interleukin 6 from these cells. The inducing effect of these proteins on the cytokine release was markedly inhibited by actinomycin D and cycloheximide.

A decreased concentration or total disappearance of Lactobacillus acidophilusin the vagina constitutes a frequent observation in bacterial vaginosis.

Summary: In this study we provide evidence that structural and soluble components of periodontopathogenic bacteria, such asPrevotella melaninogenica andFusobacterium nucleatum, induce the release of cytokines in vitro known to cause in vivo necrotic inflammatory phenomena and bone resorption (tumor necrosis factor-α, interleukin-1α and interleukin-6). Human monocytes and gingival fibroblasts were cultivated in vitro in the presence of both particulate and soluble bacterial fractions. A dose-dependent production of tumor necrosis factor-α by monocytes and gingival fibroblasts was observed in the presence of fractions ofP. melaninogenica andF. nucleatum. Interleukin-1α was produced in approximately the same quantities in the presence of soluble fractions of eitherP. melaninogenica orF. nucleatum, but in greater quantities in response to particulate fractions ofP. melaninogenica. Monocytes release larger amounts of interleukin-1α (about 3000 pg/ml) than gingival fibroblasts (about 1500 pg/ml). Interleukin-6 was released in greater quantities by monocytes in the presence of the pellet fraction ofP. melaninogenica (about 5.5 ng/ml), but gingival fibroblasts released larger amounts of interleukin-6, especially in the presence of particulate and soluble components ofF. nucleatum (about 12 ng/ml). The ability to induce the release of these cytokines notably increases the pathogenic potential of the bacteria involved in the damage of periodontal tissue.