Salmonella typhimurium SH5014 porins induce the release of tumor necrosis factor alpha (TNF-), interleukin lee (IL-la), and IL-6 by human monocytes and of gamma interferon (IFN-y) and ILH4 by human lymphocytes. Porins at 1 ,ug/ml induce the greatest release of TNF-a, IL-let, and IL-6 by monocytes and of I]LA by lymphocytes, while porins at 5 ,ug/ml induce the greatest release of IFN-y by lymphocytes. The R form of lipopolysaccharide (LPS-R) induces the greatest release of TNF-a and IL-lee by monocytes when used at a low concentration (1 jLg/mI). At higher concentrations (5 and 10 ,ug/ml, respectively), LPS-R induces the maximal release of IL-6 from monocytes and the maximal release of IL-4 from lymphocytes. The S form of LPS (LPS-S) induces the greatest release of TNF-e, IL-le, and IL-6 by monocytes and that of IL-4 by lymphocytes when used at a concentration of 1 ;ag/ml. After stimulation with LPS-S, the largest quantity of TNF-a and IL-let released was less than that obtained after stimulation with LPS-R at the same concentration, while the quantity of IL-6 released was found to be slightly higher than that obtained after stimulation with porins or LPS-R. LPS-S (1 ,ug/ml) induces IFN-y release from lymphocytes in notably smaller quantities than that obtained with LPS-R and slightly larger quantities than that obtained with porins. The preparation of porins used was found to be contaminated with 10 pg of LPS per 10 jLg of porins, a quantity which was found to have no biological effect; furthermore, porin preparations with the addition of polymyxin B gave the same results. Many of the pathophysiologic mechanisms of gram-negative bacterial infections are due to endotoxins acting on tissue directly or via mediators such as cytokines (40). Mediators of these reactions include tumor necrosis factor (TNF), interleukin la (IL-la), and IL-6 (2, 8, 9) released by monocytes. In addition, endotoxins appear to potentiate antigen-specific proliferation of T helper cell lines (4); among the different mediators released by lymphocytes, IL-4 and gamma interferon (IFN-y) are of particular interest. There

Bacterial heat shock proteins (HSPs) from Escherichia coli (GroES, GroEL, and DNAk) were tested for their ability to induce by themselves the expression and release of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-␣), and granulocyte-monocyte colony-stimulating factor (GM-CSF) by human monocytes and GM-CSF, IL-6, E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) by human umbilical vein endothelial cells (HUVEC). Our study demonstrated that treatment of monocytes with DNAk increased IL-6, TNF-␣, and GM-CSF release in a dose-dependent manner. The same effect was elicited by GroEL but at a lower rate. Treatment of HUVEC cultures with DNAk and GroEL also increased GM-CSF, IL-6, E-selectin, ICAM-1, and VCAM-1 release in a dose-dependent fashion. In any case, the greatest release was obtained by using DNAk and GroEL at a concentration of 1 g/ml. DNAk and GroEL were also able to up-regulate the surface expression of E-selectin, ICAM-1, and VCAM-1. As detected by reverse transcription-PCR analysis, DNAk and GroEL also increased the steady-state levels of cytokines and adhesion molecules in human monocytes and endothelial cells. In our study GroES showed a significant activity only on the release, surface expression, and mRNA transcription of E-selectin. Adhesion molecule expression seems to be a direct effect of HSPs and not via cytokines. Furthermore, these effects are due to HSPs properties because they are inhibited by specific monoclonal antibodies. These findings support the potential role of HSPs in modulating cell interactions during immunological and inflammatory responses. MATERIALS AND METHODS E. coli HSPs. E. coli HSPs GroES, the 10-kDa HSP (HSP 10), GroEL, the 60-kDa HSP (HSP 60), and DNAk, the 70-kDa HSP (HSP 70), were purchased from Boehringer Mannheim (Mannheim, Germany). Cell preparation and culture. (i) Monocyte isolation. To exclude variability, leukocyte populations were obtained for each test series from a buffy coat of a

Studies were carried out on the ability of some anesthetic agents (Propofol, Dormicum, Ketalar and Penthotal) to induce the release of cytokines by human monocytes and lymphocytes in vitro. All anesthetic agents tested at hematic concentrations reached during anesthetic administration cause an increase in the production of Tumor necrosis factor (TNF) from human monocytes; the increase is 4-5 times greater than controls. The greatest Interleukin -1 alpha (IL-1 alpha) production increase was induced by Propofol. The release of Interleukin -6 (IL-6) is notably increased by Ketalar (about 10 times greater than controls). In the presence of different anesthetic agents, human lymphocytes release Interleukin -4 (IL-4) and Interferon gamma- (IFN-gamma). Penthotal and Ketalar increase IL-4 production which appears quite high compared to that obtained with Con A used as standard challenge. Propofol induce IL-4 release which is about the same as that seen with Con A. IFN-gamma is released in high quantities by lymphocytes treated with Propofol. Dormicum, Ketalar and Penthotal induce non-significant increase of IFN-gamma release. The results concern the choice of anesthetic, in relation to its action on host immune response. This aspect is particularly interesting in immunocompromised host.

In understanding the regulation of the specific immune response to Salmonella typhimurium, the role of a surface major component (porins) was studied. In this study we demonstrate that purified porins are able to induce a different response to that induced by the porins present on the S. typhimurium cell surface. Porin-treated or orally infected mice show anti-porin antibodies with bactericidal activity. The complete adoptive transfer of resistance to S. typhimurium is achieved only using splenic T cells from survivor mice after experimental infection. After stimulation with specific antigen in vitro CD4+ cells from porin-immunized mice released large amounts of interleukin-4 (IL-4), at a time when CD4+ cells from S. typhimurium-infected mice predominantly secreted interferon-gamma (IFN-gamma). Limiting dilution analysis showed that infection resulted in a higher precursor frequency of IFN-gamma-producing CD4+ T cells and a lower precursor frequency of IL-4-producing CD4+ T cells, while immunization with porins resulted in a higher precursor frequency of IL-4-producing cells and a low frequency of IFN-gamma-producing cells. Analysis of polymerase chain reaction-amplified cDNA from the spleens of infected mice revealed that IFN-gamma, IL-2 and IL-12 p40 mRNA were found 5 days after in vitro challenge and increased after 15 days; IL-10 expression was barely present after both 5 and 15 days, while IL-4 mRNA expression was not detected. In immunized mice, the IL-4 mRNA expression increased after 15 days, IFN-gamma mRNA expression disappeared entirely after 15 days, while IL-2, IL-10 and IL-12 mRNA remained relatively unchanged.

The response of Salmonella typhimurium to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 32-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Lipid and carbohydrate content of bacterial cells rapidly declined within the first 16 days, while DNA and proteins exhibited a more gradual decline over the 32 days of starvation. In contrast, RNA content did not decrease appreciably upon nutrient starvation. Structural damage occurred especially after 16 days of starvation. After 32 days of nutrient deprivation, we recorded degenerative cellular forms, a coccoidal cell shape, a decrease in cellular volume, and the loss of the three-layered outer membrane. The morphological and structural alterations correlated with virulence in infected animals. We observed a decrease in virulence of S. typhimurium after 9, 16, and 32 days of starvation, reaching a maximal decrease after 32 days of nutrient deprivation. The decrease in virulence correlated to surface hydrophobicity alterations, adherence to eukaryotic cells, and phagocytosis.