Antonella Marcatili

ROLE OF PASTEURELLA MULTOCIDA PORIN ON CYTOKINE EXPRESSION AND RELEASE BY MURINE SPLENOCYTES

The aim of this study was to verify whether Pasteurella multocida porin can affect the expression and release of IL-1alpha, IL-6, TNF-alpha, IL-4, IFN-gamma, IL-10 and IL-12 by murine splenocytes in vitro. P. multocida porin and lipopolysaccharide (LPS) were able to induce the release of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 in a dose-dependent fashion. The greatest release of these cytokines was obtained using P. multocida porin at a concentration of 5 microg ml(-1) and LPS at a concentration of 1 microg ml(-1). The time-courses of release showed that P. multocida LPS was able to stimulate the production of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 earlier than porin and at a greater rate. No effect was observed on IL-4 and IL-10 release under the same experimental conditions. P. multocida porin and LPS were also able to up-regulate the mRNA expression of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 p40. Our findings suggest that P. multocida porin is able to modulate inflammatory and immunological responses by affecting the release of several cytokines and the expression of their genes.

Effect of low-nutrient seawater on morphology, chemical composition, and virulence of Salmonella typhimurium

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.

Growth hormone modulates IL-α and IFN-γ release by murine splenocytes activated by LPS or porins of Salmonella Typhimurium

The effect of growth hormone (GH) on the release of IL-1α and IFN-γ from murine splenocytes was investigated. Their release from splenocytes activated by Salmonella enterica serovar Typhimurium lipopolysaccharide (LPS) 0.5γg/ml was increased by c. 65% in the presence of GH 100 pg/ml. With splenocytes activated by S. Typhimurium porins 5 μg/ml, GH increased the production of both IL-1α and IFN-γ by c. 56%. Polymyxin treatment abolished the cytokine-releasing activity of LPS but had no effect on the activity of the porin preparation.

Release of GM-CSF, sE-Selectin, and SICAM-1 by Human Vascular Endothelium Stimulated with Gram-Negative and Gram-Positive Bacterial Components

Gram-negative (Salmonella typhimurium porins and lipopolysaccharide-R), and Gram-positive bacterial components (lipoteichoic acid, muramic acid, muramyl-dipeptide, adjuvant peptide, protein A, toxic shock syndrome toxin-1, α-hemolysin) were tested for their ability to stimulate both the release of Granulocyte-Monocyte Colony Stimulating Factor (GM-CSF), soluble Intercellular Adhesion Molecule-1 (ICAM-1) and soluble E-selectin from human endothelial cells, and the surface expression of the adhesion molecules. Salmonella typhimurium porins and lipopolysaccharide-R (LPS-R) were able to induce the release of GM-CSF, sE-selectin and sICAM-1 in a dose dependent fashion. The greatest release of these factors was obtained using pork at a concentration of 5μg/ml and LPS-R at a concentration of 1μg/ml. The kinetics of release showed that LPS-R was able to stimulate the production of these factors earlier than porins but at a lower rate. Porins and LPS-R was also able to up-regulate the surface expression of E-selec...

Correlation between changes in surface hydrophobicity and interaction of Streptococcus pyogenes with human polymorphonuclear leukocytes after prolonged starvation in sea water.

The aim of this research was to evaluate the persistence of virulence characteristics of Streptococcus pyogenes cells after prolonged starvation in sea water. Studies were carried out on changes in viability, alterations in the chemical composition and surface hydrophobicity and the interaction of S. pyogenes with human polymorphonuclear leukocytes (PMN) after starvation. Results showed that surface hydrophobicity decreased progressively starting after three days of starvation and was correlated with the decrease in total carbohydrate, lipid and protein content. These values correlated with a better interaction of S. pyogenes cells with the PMN, as shown by a chemiluminescence increase that reached a peak after 32 days of starvation. Furthermore, bacterial cells became more easily phagocytized and killed by human PMN.

Role of Pasteurella multocida, Pasteurella haemolytica and Salmonella typhimurium porins on inducible nitric oxide release by murine macrophages.

The aim of this study was to verify whether Pasteurella haemolytica, P. multocida and Salmonella typhimurium porins could affect the inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release by murine resident peritoneal macrophages in vitro. We also compared their effect with that elicited by P. haemolytica, P. multocida and S. typhimurium lipopolysaccharide (LPS) whose biological activity is well known. Variations in NO release and iNOS mRNA expression due to variable concentrations of porins were recorded and compared. We also investigated the synergism between bacterial products and interferon gamma (IFN-gamma). With this aim cells were incubated with porins together with murine rIFN-gamma prior to assessing the presence of NO in the supernatant and mRNA analysis. Porins in themselves were not able to induce NO release by resident peritoneal macrophages. Incubation of macrophages with IFN-gamma in the presence of porins increased NO release, whereas incubation in the presence of the arginine analog N(G)-monomethyl-L-arginine (NMA) inhibited NO release. The greatest NO release was obtained using porins at a concentration of 5 microg/mL. Porins, together with IFN-gamma, were also able to upregulate the mRNA expression of iNOS. Our findings suggest that gram-negative porins are able to modulate inflammatory and immunological responses by affecting the release of NO and the expression of iNOS gene in activated macrophages.

Biological activities of cell envelope fragments of the archaeobacterium Sulfolobus solfataricus: lethal toxicity, local hypersensitivity, pyrogenicity and spleen lymphocyte mitogenicity

The sensitizing effect and the local and general toxicity related to membrane components of the archaeobacterium Sulfolobus solfataricus was studied. Cell envelope fragments were biologically active but this activity was lost upon separation of the lipid and protein components. The envelope fragments exerted lethal effects on mice sensitized with D-galactosamine that were prevented by pretreatment with anti-TNF-alpha serum. This lethal activity occurred in both LPS-responder (BALB/cByJ) and LPS-nonresponder (C3H/HeJ) mouse strains. In addition, Sulfolobus envelope fragments tested in rabbits caused a local Schwartzman reaction, and showed pyrogenic activity. In vitro, the envelope fragments that act on spleen lymphocytes of the LPS-responder (BALB/cByJ) and LPS-nonresponder (C3H/HeJ) mice caused an uptake of [3H]thymidine similar to that caused by concanavalin A. A similar toxic activity to that exerted by eubacteria is therefore exerted by this non-pathogenic archaeobacterium despite the difference in surface chemistry.