Extracellular factors and immunosuppressive drugs influencing insulin secretion of murine islets.

Approximately 60% of transplanted islets undergo apoptosis within the first week post-transplantation into the liver attributed to poor engraftment, immune rejection and toxicity of immunosuppressive drugs. Understanding how extracellular matrix (ECM) components, immunosuppressive drugs and proinflammatory cytokines affect insulin secretion will contribute to an improved clinical outcome of islet transplantations. In this study, functional activity of isolated murine islets was measured by glucose-stimulated insulin secretion (GSIS) and by electrophysiological measurements using patch-clamp. Cultivating islets with soluble fibronectin or laminin, as opposed to with coated laminin, markedly increased GSIS. Addition of cyclosporin A reduced GSIS and suppressed glucose-induced spike activity. Tacrolimus affected neither GSIS nor spike activity, indicating a different mechanism. To evaluate the influence of proinflammatory cytokines, islets were incubated with interleukin (IL)-1ß, tumour necrosis factor (TNF)-α or with supernatants from cultured Kupffer cells, the main mediators of inflammation in the hepatic sinusoids. IL-1ß exerted a bimodal effect on insulin secretion, stimulating below 2 ng/ml and suppressing above 10 ng/ml. Soluble laminin in combination with a stimulatory IL-1ß concentration further increased insulin secretion by 20% compared to IL-1ß alone, while with high IL-1ß concentrations soluble laminin slightly attenuated GSIS inhibition. TNF-α alone did not affect GSIS, but with stimulatory IL-1ß concentrations completely abolished it. Similarly, supernatants derived from Kupffer cells exerted a bimodal effect on GSIS. Our data suggest that improved insulin secretion of transplanted islets could be achieved by including soluble laminin and low IL-1ß concentrations in the islet cultivation medium, and by a simultaneous inhibition of cytokine secretion from Kupffer cells.

Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis.

Rituxan, a chimeric anti-CD20 antibody, is the first antibody approved for immunotherapy in non-Hodgkin's B-cell lymphoma and other B-cell lymphoproliferative disorders. Additionally, efficacy of Rituxan treatment has been reported in nonmalignant autoimmune diseases such as rheumatoid arthritis. Crosslinking of CD20 molecules by Rituxan induces therapeutic B-cell depletion. CD20 is a B-lymphocyte specific integral membrane protein, proposed to function as a store-operated calcium channel, which is activated upon receptor-stimulated calcium depletion of intracellular stores. Crosslinking of CD20 by antibodies has been reported to induce a redistribution of CD20 molecules to specialized microdomains at the plasma membrane known as lipid rafts. Here, we report that in the absence of Rituxan, CD20 exhibits a low affinity to lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca(2+) levels and downstream apoptotic signalling.

Role of penicillin-binding proteins in the initiation of the AmpC beta-lactamase expression in Enterobacter cloacae.

Penicillin-binding proteins (PBPs) are involved in the regulation of beta-lactamase expression by determining the level of anhydromuramylpeptides in the periplasmatic space. It was hypothesized that one or more PBPs act as a sensor in the beta-lactamase induction pathway. We have performed induction studies with Escherichia coli mutants lacking one to four PBPs with DD-carboxypeptidase activity. Therefore, we conclude that a strong beta-lactamase inducer must inhibit all DD-carboxypeptidases as well as the essential PBPs 1a, 1b, and/or 2.

beta-Lactamase induction and cell wall recycling in gram-negative bacteria.

beta-Lactams with the ability to induce beta-lactamase in gram-negative bacteria bind to essential penicillin-binding proteins (PBPs) after entering the periplasmic space. This leads to inactivation of transpeptidase activities and thereby a decrease in the number of peptide cross-links, allowing further degradation of murein by soluble lytic transglycosylases. If all DD-carboxypeptidases (PBP 4, 5, 6a and 6b) are inhibited as well, the degradation product aD-pentapeptide (N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic-acid-D-alanyl-D- alanine) accumulates, which is the case with inducing beta-lactams such as imipenem. These molecules in addition to tri- and tetrapeptides (N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic-acid-[D-alanine]) which are the usual degradation products of peptidoglycan, are released into the cytoplasm and displace the UDP-pentapeptide (UDP-N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic-acid-D-alanyl-D-alanine) from the DNA-binding protein AmpR, converting it into an activator of AmpC beta-lactamase expression.