Specifity of staining was verified via isotope control carried along for the antibody used. and mutational mechanisms of bacterial resistance [19,20]. Environmental persistence is usually further increased by the ability of to form biofilms [21]. In addition, the microorganism has been reported DDIT4 to synchronize gene expression by an intercellular communication mechanism, the quorum sensing [21,22]. This mechanism enables the bacterial populace to act as a single organism and to modulate Butabindide oxalate a number of virulence factors, including biofilm formation as well as the production of numerous toxins [21,22]. Feeding studies concerning the impact of PUFA supplementation on immune defense mechanisms yielded conflicting findings, so far. This is aggravated by variations in experimental settings leading to a lack in comparability of gained results. Moreover, virtually no data regarding the relevance of PUFA in case of macrophage contamination with or exist. Hence, in this and respectively induced an increase in the concentration of pro-inflammatory cytokines in cell supernatants (Physique 1). Significant differences depending on the stimulator added could be assessed. Treatment of the cells with LPS resulted in a significant increase in the concentration of IL-1, IL-6 as well as TNF- (Physique 1). In contrast, after stimulation of the macrophages with PMA a significant increase could only be seen for TNF- (Physique 1). Addition of the quorum sensing molecule N3-oxododecanoyl-l-homoserine lactone (OdDHL) to the culture medium did not affect the concentration of pro-inflammatory cytokines in cell supernatants (Physique 1). The combination of LPS and OdDHL abrogated the stimulating effect of LPS on IL-1, IL-6 and TNF- synthesis (Physique 1). Culturing of RAW264.7 in presence of the viable pathogens and boosted proinflammatory cytokine synthesis as well (Determine 1). The virulent strain ATCC 33701 was found to act more effectively in increasing the production of IL-1, IL-6 and TNF- by the macrophages than the non-virulent strain ATCC 6939 (Physique 1). Butabindide oxalate Open in a separate window Physique 1 Concentration of IL-1, IL-6 and TNF- in supernatants of RAW264.7 macrophages, cultured in basic medium after 24 h of activation with lipopolysaccharide (LPS), ATCC 10145, ATCC 6939 and ATCC 33701 respectively. Data are mean SD (= 6). Bars denoted by different letters are significantly different. Enrichment of the culture medium with fatty acids diminished the stimulatory effects of LPS, and was decreased significantly following feeding of cells with the (Physique 3). For RAW264.7 stimulated with ATCC 6939 or ATCC 33701 no effect of PUFA supplementation on IL-6 production was seen (Determine 3). PUFA that experienced a decreasing effect on the secretion of TNF- by the macrophages were LNA, EPA and DHA for LPS stimulated cells, LNA, EPA, DHA and LA for cells treated with ATCC 6939 as well as LNA, EPA, DHA, LA and AA for cells treated with the virulent strain ATCC 33701 (Physique 4). For un-stimulated cells as well as for cells treated with PMA no effects of PUFA supplementation around the production of the pro-inflammatory cytokines IL-1, IL-6 and TNF- could be seen (data not shown). Furthermore, treatment of the cells with LPS in combination with the quorum sensing molecule OdDHL abolished the PUFA effects observed for LPS stimulated RAW264.7 (data not shown). Open in a separate window Physique 2 Concentration of IL-1 in supernatants of RAW264.7 macrophages cultured in basic medium as well as in medium Butabindide oxalate supplemented with 15 mol/L alpha-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or arachidonic acid (AA) after 24 h of activation with LPS, ATCC 10145, ATCC 6939 and ATCC 33701 respectively. Data are mean SD (= 6). Bars denoted by different letters are significantly different. Open in a separate window Open in a separate window Physique 3 Concentration of IL-6 in supernatants of RAW264.7 macrophages cultured in basic medium as well as in medium supplemented with Butabindide oxalate 15 mol/L alpha-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or arachidonic acid (AA) after 24 h of activation with LPS, ATCC 10145, ATCC 6939 and ATCC 33701 respectively. Data are mean SD (= 6). Bars denoted by different letters are significantly different. Open in a separate window Physique 4 Concentration of TNF- in supernatants of RAW264.7 macrophages cultured.

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