INDUCTION OF CYTOKINE MRNA EXPRESSION IN U937 CELLS BY SALMONELLA TYPHIMURIUM PORINS IS REGULATED BY DIFFERENT PHOSPHORYLATION PATHWAYS.

Lipopolysaccharide (LPS) and porins of Gram-negative outer membranes are the main pathogenic factors implicated in the clinical syndrome of septic shock. The biological activity of porins and LPS are similar, but they occur by different mechanisms. It seems that porins act through different intracellular pathways with respect to LPS. In this study we analyzed the role of several inhibitors of the MEK/ERK signal pathway on the induction of proinflammatory and immunological cytokines in U937 cell line stimulated by Salmonella typhimurium porins and compared it to the cytokine induction after LPS stimulation. We investigated the effects of p38 MAP kinase inhibitor SB-203580, MEK/ERK kinase inhibitor PD-098059 and Raf-1 inhibitor forskolin, and demonstrated that they modulate cytokine mRNA expression in a different manner as a consequence of the use of porins or LPS as stimuli. TNF-alpha and IL-1beta mRNA expression is decreased by PD-098059 after stimulation with LPS but not with porins in differentiated U937 cells. IL-10 mRNA expression is inhibited by SB-203580 and PD-098059 after stimulation with porins in U937 cells. IL-6 and IL-8 mRNA expression is not changed by PD-098059 or SB-203580, after stimulation either with porins or LPS. Furthermore, mRNA expression of the studied cytokines, except for GM-CSF, is not changed using forskolin

Porins from Salmonella enterica serovar Typhimurium activate the transcription factors AP-1 and NF-kB through the Raf-1-mitogen activated protein kinase cascade

In this study we examined the ability of Salmonella enterica serovar Typhimurium porins to activate activating protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) through the mitogen-activated protein kinase (MAPK) cascade, and we identified the AP-1-induced protein subunits. Our results demonstrate that these enzymes may participate in cell signaling pathways leading to AP-1 and NF-kappaB activation following porin stimulation of cells. Raf-1 was phosphorylated in response to the treatment of U937 cells with porins; moreover, the porin-mediated increase in Raf-1 phosphorylation is accompanied by the phosphorylation of MAPK kinase 1/2 (MEK1/2), p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase. We used three different inhibitors of phosphorylation pathways: 2'-amino-3'-methoxyflavone (PD-098059), a selective inhibitor of MEK1 activator and the MAPK cascade; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of the p38 pathway; and 7beta-acetoxy-1alpha,6beta,9alpha-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), an inhibitor at the level of Raf-1 kinase. PD-098059 pretreatment of cells decreases AP-1 and NF-kappaB activation by lipopolysaccharide (LPS) but not by porins, and SB203580 pretreatment of cells decreases mainly AP-1 and NF-kappaB activation by porins; in contrast, forskolin pretreatment of cells does not affect AP-1 and NF-kappaB activation following either porin or LPS stimulation. Our data suggest that the p38 signaling pathway mainly regulates AP-1 and NF-kappaB activation in cells treated with S. enterica serovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only after porin stimulation. These data suggest different molecular mechanisms of activation induced by porins or by LPS

IL RUOLO DEGLI EVENTI SENTINELLA NELLA MISURAZIONE DELLO STRESS DA LAVORO CORRELATO. UNA PROPOSTA METODOLOGICA

La letteratura ha rilevato che lo stress-lavoro correlato rappresenta uno dei maggiori problemi di salute nei luoghi di lavoro le cui conseguenze negative si ripercuotono sul benessere psico-fisico e sociale dei lavoratori e sulla produttività e la competitività delle imprese. Recependo quanto stabilito dall’Accordo Europeo sullo Stress nei luoghi di lavoro (8/10/2004), il D. Lgs. 81/2008 e la successiva circolare del Ministero del Lavoro e delle Politiche Sociali del 18/11/2010 hanno reso obbligatoria anche in Italia la valutazione dello stress lavoro-correlato, proponendo linee guida che lasciano liberi gli operatori di utilizzare le metodologie ritenute più appropriat