An in vivo model of hyperacute rejection: characterization and evaluation of the effect of transgenic human complement inhibitors

Hyperacute rejection (HAR) occurring after transplantation within phylogenetically distant species is a severe reaction triggered by preexisting xenoreactive antibodies and complement activation, leading to the destruction of the donor organ. Expression of human complement inhibitors in transgenic pig organs prolongs the survival of xenograft in experimental models. Moreover, the extent of protection from hyperacute rejection is dependent on the level and site of expression of the transgenic molecules and, probably, on the combination of different molecules. In this regard a small animal model to test the efficacy of expression vectors and different human molecules could be very advantageous. A murine model developed in our laboratory was characterized by measurement of several parameters characteristic of HAR in the livers of control and transgenic mice expressing transgenic human DAF (CD55) or MCP (CD46) at the end of 2h of perfusion with human plasma and after 1 day. The parameters studied were heamatological values of hepatic functions (GOT and GPT), induction of pro-inflammatory molecules and histopathological evaluation. Cytokines (IL-1, IL-1, IL-6) induction and exposure of P-selectin on the endothelial cell surface, was only observed in control animals after 2h of perfusion, as an early event. GOT and GPT values increase drammatically after 2h perfusion and 1 day after the treatment according to the histopathological observation of liver damage. On the contrary, the livers of hDAF or hMCP transgenic mice, under the same treatment were significantly protected although the extent of this protection is dependent on the level of expression of transgenic human molecules.     

Functional studies of new GLA gene mutations leading to conformational fabry disease

Fabry Disease (FD) is an X-linked multisystemic lysosomal disorder caused by mutations of α-galactosidase (GLA) gene. Only a few of the 450 genetic lesions identified so far have been characterised by in vitro expression studies. Thus the significance of newly identified GLA nucleotide variants in FD patients which lead to α-galactosidase (GAL-A) amino acid substitutions or intronic changes can be uncertain. We identified three GLA mutations, c.155G > A (p.C52Y), c.548G > C (p.G183A), c.647A > G (p.Y216C) in as many individuals (two male; one female) and performed in vitro expression studies and Western blot analysis in order to clarify their functional effects. Reduced GAL-A activity and normal or partially reduced mutant proteins were present in all overexpressed mutant systems in which three-dimensional structural analysis showed that the active site was not directly involved. We hypothesize that the three new mutations affect the GAL-A protein, leading to conformational FD. When mutant proteins overexpressed in COS-1 cells and in patients' lymphocytes were tested in the presence of the 1-deoxygalactonojirimicin (DGJ) chaperone, the p.G183A and p.Y216C systems showed increased GAL-A enzyme activities and protein stabilisation while p.C52Y was not responsive. We underline that genetic, biochemical and functional studies are helpful in clarifying the consequences of the missense genetic lesions detected in FD. ERT is the elective therapy for Fabry patients, but it is not always possible to issue the enzyme's active form in all involved organs. Our study endorses the hypothesis that an active site-specific chemical chaperone, which could be administered orally, might be effective in treating GAL-A conformational defects.