alpha-Methyldopa and the erythrocyte membrane.

To test the hypothesis that complement-mediated cell lysis and cell-mediated cytotoxicity operate by analogous mechanisms, cell membranes from two antibody-dependent cytotoxicity systems were examined by electron microscopy after negative staining. Ring-shaped membrane lesions generally similar to, but larger than, those previously described for complement lysis were observed. These findings are in agreement with recent measurements of larger functional pores for ADCC than complement.     

Saccharomyces boulardii interferes with enterohemorrhagic Escherichia coli-induced signaling pathways in T84 cells

Enterohemorrhagic Escherichia coli (EHEC) infections are associated with the modification of tight-junction permeability and synthesis of proinflammatory cytokine interleukin-8 (IL-8). In a previous study, it was demonstrated that EHEC-induced IL-8 secretion is due to the involvement of the mitogen-activated protein kinase (MAPK), AP-1, and NF-kappaB pathways. In this study, we investigated the effect of the yeast Saccharomyces boulardii on EHEC infection in T84 cells. For this purpose, cells were (i) incubated with bacteria and yeast at the same time or (ii) incubated overnight with yeast cells that were maintained during infection or eliminated by several washes before infection. Coincubation is sufficient to maintain the transmonolayer electrical resistance (TER) of EHEC-infected cells, whereas the preincubation of cells with the yeast without elimination of the yeast during infection is necessary to significantly decrease IL-8 secretion. We thus analyzed the mechanisms of S. boulardii action. We showed that S. boulardii has no effect on EHEC growth or on EHEC adhesion. Kinetics studies revealed that EHEC-induced myosin light chain (MLC) phosphorylation precedes the decrease of TER. ML-7, an MLC kinase inhibitor, abolishes the EHEC-induced MLC phosphorylation and decrease of TER. Studies show that S. boulardii also abolishes EHEC-induced MLC phosphorylation. We demonstrated that the preincubation of cells with S. boulardii without washes before EHEC infection inhibits NF-kappaB DNA binding activity, phosphorylation and degradation of IkappaB-alpha, and activation of the three members of a MAPK group (extracellular signal-regulated protein kinases 1 and 2, p38, and c-jun N-terminal kinase). These findings demonstrate that S. boulardii exerts a preventive effect on EHEC infection by (i) interfering with one of the transduction pathways implicated in the control of tight-junction structure and (ii) decreasing IL-8 proinflammatory secretion via inhibition of the NF-kappaB and MAPK signaling pathways in infected T84 cells.     

Effects of cyclic enteral nutrition on the immunological status of malnourished patients

The effects of 2 weeks of refeeding by cyclic enteral nutrition on chronically malnourished (mean global nutritional deficiency 19.9 +/- 1.1%) hospitalized patients were assessed in a prospective study with special attention paid to immunological status. All patients were immunodeficient, with cell-mediated immunity being more affected than humoral immunity. After 2 weeks of refeeding, nutritional status had improved by 29.8%. Initially abnormal parameters of humoral immunity (IgM, C3 and C4) improved significantly (P < 0.05) between day 0 and day 15. The following cell-mediated immunity parameters also improved significantly (P < 0.05): CD8, monocyte count, natural killer cell activity and skin tests. Short-term refeeding by cyclic enteral nutrition appears to be a safe and effective way of improving immunodeficiency in chronically malnourished patients, with predictable consequences on infection.     

Phosphorylation of factor Va and factor VIIIa by activated platelets

Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II- like kinase may downregulate the activity of the two cofactors.