Robert Ian Lechler

Nitric oxide-mediated expression of Bcl-2 and Bcl-xl and protection from tumor necrosis factor-alpha-mediated apoptosis in porcine endothelial cells after exposure to low concentrations of xenoreactive natural antibody.

BACKGROUNDnCardiac and renal allo- and xenografts can acquire a natural resistance to vascular rejection. This accommodation involves endothelial cell (EC) expression of survival genes such as Bcl family members and hemoxygenase 1. Understanding what initiates this protective process would have profound implications; our hypothesis is that low concentrations of antigraft antibodies may mediate these changes.nnnMETHODSnIn vitro cultured primary and immortalized porcine EC were incubated with polyclonal human IgG for 6 days and then examined for phenotype changes.nnnRESULTSnThe cells acquired resistance to tumor necrosis factor-alpha-mediated apoptosis (50-100% reduction at 6 hr) and up-regulated expression of Bcl-2 and Bcl-xl; sustained expression was accompanied by inducible nitric oxide (NO) synthase expression and by enhanced production of NO by EC. Two observations suggested that NO was actively involved in the process of Bcl-2 and Bcl-xl induction. First, (z)-1-2-[2-aminoethyl)-N- (2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, an NO donor, was able to induce similar changes in porcine EC to those induced by anti-pig antibodies. Second, an NO synthase inhibitor NG-monomethyl-L-arginine.monoacetate was able to specifically inhibit the anti-pig antibody-mediated expression of Bcl-2 or Bcl-xl.nnnCONCLUSIONSnThese data strongly support the hypothesis that Bcl-2 and Bcl-xl expression and protection from apoptosis in EC may result from antibody-mediated NO production through the neoexpression of inducible NO synthase.

CREATION OF TOLEROGENIC ANTIGEN PRESENTING CELLS VIA INTRACELLULAR CTLA4: A NOVEL STRATEGY WITH POTENTIAL CLINICAL UTILITY IN TRANSPLANTATION.

Aims: Activation of T lymphocytes requires the recognition of peptide–MHC complexes and co-stimulatory signals provided by antigen-presenting cells (APCs). It has been shown that T cell activation without co-stimulation can lead to T cell anergy, a state of T cell hyporesponsiveness. In this study we developed a novel strategy to inhibit expression of B7 molecules by transfecting APCs with a gene construct encoding a modified CTLA4 molecule (CTLA4-KDEL) that is targeted to the endoplasmic reticulum (ER). Our approach is to express within the cells CTLA4-KDEL. This will bind to the newly synthesised B7 molecules in the ER and prevent them from reaching the cell surface. Methods: The CTLA4-KDEL gene was constructed using standard technology. It was then transfected into a range of APCs. In the case of DCs transfection was performed using a new non-viral gene therapy system. The APCs were then tested in their ability to activate peptide specific T cell clones and primary allospecific T cells. OVA TG model was used to assess in vivo tolerance. Results: Our in vitro and in vivo data using CTLA4-KDEL in various APCs show that this strategy does indeed block B7 expression by APC and that antigen-reactive (both allospecific and peptide-specific) T cells not only are hyporesponsive to antigen presented by these cells but also are rendered anergic. Conclusions: This gene-based strategy to inhibit expression of key surface receptors in order to generate ‘B7deficient’ APCs has significant advantages over the currently described approaches for treatment of transplant rejection. Transplant 6328