Background Transgenic expression of human complement regulatory proteins (hCRPs) reduces the frequency of hyperacute rejection (HAR) in Gal-positive cardiac xenotransplantation. In this study we examine the impact of human CD55 (hCD55) expression on a Gal knock-out (GTKO) background using pig-to-primate heterotopic cardiac xenotransplantation. Methods Cardiac xenotransplantation was performed with GTKO (Group 1; n=6) and GTKO.hCD55 (Group 2; n=5) donor pigs using similar immunosuppression. Cardiac biopsies were obtained 30 minutes after organ reperfusion. Rejection was characterized by histology and immunohistology. Intragraft gene expression, serum non-Gal antibody and antibody recovered from rejected hearts were analyzed. Results HAR of a GTKO heart was observed. Remaining grafts developed delayed xenograft rejection. Median survival was 21 and 28 days for Groups 1 and 2 respectively. Vascular antibody deposition was uniformly detected 30 minutes after organ reperfusion and at explant. A higher frequency of vascular C5b deposition was seen in GTKO organs at explant. Serum non-Gal antibody, antibody recovered from the graft and intragraft gene expression were similar between the groups. Conclusion HAR of GTKO hearts without hCD55 may occur. Expression of hCD55 appeared to restrict local complement activation, but did not improve graft survival. Chronic vascular antibody deposition with evidence of protracted endothelial cell activation was seen. These observations suggest that non-Gal antibody-induced chronic endothelial cell activation coupled to possible haemostatic incompatibilities may be the primary stimulus for DXR of GTKO hearts. To avoid possible HAR, future clinical studies should employ donors expressing hCRPs in the GTKO background.

Background Xenograft rejection of pigs organs with an engineered mutation in the GGTA-1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non-Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig-to-primate cardiac xenotransplantation. One of these gene products was a glycosyltrasnferase with homology to the bovine β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. Methods The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK-B4T) was produced. Expression of porcine B4GALNT2 in HEK-B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK-B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK-B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non-Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. Results The porcine B4GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4GALNT2 genes respectively. The B4GALNT2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4GALNT2 in human HEK cells (HEK-B4T) results in increased binding of antibody to the B4GALNT2 enzyme, and increased reactivity with anti-Sda and DBA. HEK-B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO:CD55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non-Gal antibody, measured using GTKO PAECs, and the induction of antibodies which preferentially bind to HEK-B4T cells. Conclusion The functional isolation of the porcine B4GALNT2 gene from a PAEC expression library, the pattern of B4GALNT2 gene expression and its sensitization of HEK-B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4GALNT2 produces a new immunogenic non-Gal glycan which contributes in part to the non-Gal immune response d...

The identified proteins include key EC functions and suggest that non-Gal antibody responses may compromise EC functions and thereby contribute to DXR. Recovery of the porcine β1,4 N-acetylgalactosaminyl transferase 2 suggests that an antibody response to a SD-like carbohydrate may represent a new carbohydrate moiety involved in xenotransplantation. The identification of these porcine gene products may lead to further donor modification to enhance resistance to DXR and further reduce the level of xenograft antigenicity.

Stabilization of the levels of active oxygen species (AOS) is important to the survival of organisms. To clarify the system controlling levels of AOS in plants, this study used an electron spin resonance (ESR) method to directly measure superoxide radical (O(2)(.-)) scavenging activities in the wild-type Arabidopsis thaliana (Col and Ler ecotypes), two anthocyanin mutants (tt3 and ttg1), and an ascorbic acid mutant (vtc1). Under ordinary growth conditions, Arabidopsis contained superoxide-scavenging activity (SOSA) of approximately 300-500 SOD units/g of fresh weight. The ESR pattern indicated that most (40-50%) of this activity was due to ascorbic acid. For the analysis of SOSA under conditions of oxidative stress, synthesis of AOS was induced by gamma-irradiation. The radical scavenging activity in irradiated plants increased approximately 10-fold following an associated increase in the accumulation of ascorbic acid and anthocyanin. The accumulation of ascorbic acid and anthocyanin was suppressed by treatment with an antioxidant before irradiation and was induced by treatment with a radical-generating reagent. The contributions of ascorbic acid and anthocyanin to the total superoxide radical scavenging activity differed among ecotypes. In the Ler ecotype, ascorbic acid accumulated at twice the level of that in the Col ecotype, and induction of anthocyanin was half that in Col. To confirm the activity of ascorbic acid and anthocyanin against AOS stress, the viability of the wild type and mutants (tt2, tt3,tt5, ttg1, and vtc1) was examined after gamma-irradiation. Only the plants in which ascorbic acid and anthocyanin were induced had the ability to grow and flower.