Strong expression of CD55 and CD59 completely protected porcine kidneys from hyperacute rejection and allowed a detailed analysis of xenograft rejection in the absence of immunosuppression. Coagulopathy appears to be a common feature of pig-to-baboon renal transplantation and represents yet another major barrier to its clinical application.

Primary nonfunction of transplanted islets results in part from their sensitivity to reactive oxygen species (ROS) generated during the isolation and transplantation process. Our aim was to examine whether coexpression of antioxidant enzymes to detoxify multiple ROS increased the resistance of mouse islets to oxidative stress and improved the initial function of islet grafts. Islets from transgenic mice expressing combinations of human copper/zinc superoxide dismutase (SOD), extracellular SOD, and cellular glutathione peroxidase (Gpx-1) were subjected to oxidative stress in vitro. Relative viability after hypoxanthine/xanthine oxidase treatment was as follows: extracellular SOD ؉ Gpx-1 ؉ Cu/Zn SOD > extracellular SOD ؉ Gpx-1 > extracellular SOD > wild type. Expression of all three enzymes was the only combination protective against hypoxia/ reoxygenation. Islets from transgenic or control wildtype mice were then transplanted into streptozotocininduced diabetic recipients in a syngeneic marginal islet mass model, and blood glucose levels were monitored for 7 days. In contrast to single-and double-transgenic grafts, triple-transgenic grafts significantly improved control of blood glucose compared with wild type. Our results indicate that coexpression of antioxidant enzymes has a complementary beneficial effect and may be a useful approach to reduce primary nonfunction of islet grafts. Diabetes 54:2109 -2116, 2005

Organ xenografts in discordant combinations such as pig-to-man undergo hyperacute rejection due to the presence of naturally occurring human anti-pig xenoantibodies. The galactose alpha(1,3)-galactose epitope on glycolipids and glycoproteins is the major porcine xenoantigen recognized by these xenoantibodies. This epitope is formed by alpha(1,3)-galactosyltransferase, which is present in all mammals except man, apes, and Old World monkeys. We have generated mice lacking this major xenoantigen by inactivating the alpha(1,3)-galactosyltransferase gene. These mice are viable and have normal organs but develop cataracts. Substantially less xenoantibody from human serum binds to cells and tissues of these mice compared with normal mice. Similarly, there is less activation of human complement on cells from mice lacking the galactose alpha(1,3)-galactose epitope. These mice confirm the importance of the galactose alpha(1,3)-galactose epitope in human xenoreactivity and the logic of continuing efforts to generate pigs that lack this epitope as a source of donor organs.

The expression of intercellular adhesion molecule 2 (ICAM-2) in adult tissues is restricted to vascular endothelial cells and megakaryocytes. We have previously shown that the endothelial-specific in vivo activity of the human ICAM-2 promoter is contained within a small (0.33-kilobase (kbp)) 5-flanking region of the gene. Here we describe the in vitro characterization of this region. The ICAM-2 promoter is TATA-less, and transcription in endothelial cells initiates at four sites. Reporter gene expression directed by the promoter was 125-fold greater than vector alone in bovine aortic endothelial cells but less than 2-fold vector alone in non-endothelial (COS) cells, confirming that specificity in vivo was paralleled in vitro. The addition of 2.7 kbp of 5-flanking region to the 0.33-kbp fragment had no effect on promoter activity or specificity. The mutation of an Sp1 motif centered at base pair ؊194 or an eight-base pair palindrome at ؊268 each reduced promoter activity by 70%. Mutation of GATA motifs at ؊145 and ؊53 reduced promoter activity by 78 and 61%, respectively. Specific binding of bovine aortic endothelial cells nuclear proteins to the Sp1 and GATA sites was demonstrated by gel shift analysis. Promoter activity in COS cells was transactivated 3-4-fold by overexpression of GATA-2. The results presented here suggest that transcription from the ICAM-2 promoter in endothelial cells is regulated by the interplay of several positive-acting factors and provide the basis for further analysis of endothelial-specific gene expression.

Delayed rejection of pig kidney xenografts by primates is associated with vascular injury that may be accompanied by a form of consumptive coagulopathy in recipients. Using a life-supporting pig-to-baboon renal xenotransplantation model, we have tested the hypothesis that treatment with recombinant human antithrombin III would prevent or at least delay the onset of rejection and coagulopathy. Non-immunosuppressed baboons were transplanted with transgenic pig kidneys expressing the human complement regulators CD55 and CD59. Recipients were treated with an intravenous infusion of antithrombin III eight hourly (250 units per kg body weight), with or without low molecular weight heparin. Antithrombin-treated recipients had preservation of normal renal function for 4-5 days, which was twice as long as untreated animals, and developed neither thrombocytopenia nor significant coagulopathy during this period. Thus, recombinant antithrombin III may be a useful therapeutic agent to ameliorate both early graft damage and the development of systemic coagulation disorders in pig-to-human xenotransplantation.

Lectins were able to bind underlying carbohydrate structures (sialylated Tn and Forssman antigens) that are normally cryptic antigens on H-transferase transgenic mouse spleen and cardiac endothelial cells, probably as a consequence of the reduction in the electronegativity of the cell surface due to reduced sialylation. As humans have preformed anti-Tn and anti-Forssman antibodies, it is possible that these structures may become targets of the xenograft rejection process, including hyperacute rejection.

Genetic engineering of donor animals in xenotransplantation research has been directed largely toward obtaining expression of various immunoregulatory molecules on vascular endothelium, the initial target of recipient antibody and complement. However, specific high-level expression of transgenes throughout the vascular tree in adult animals has proved difficult to achieve, perhaps because of the inherent heterogeneity of endothelium. Using the promoter of the gene for intercellular adhesion molecule 2 (ICAM-2), which is constitutively expressed on all vascular endothelium, we have developed a system for endothelial cell gene targeting in vivo. A 334-basepair fragment from the 5' flanking region of the human ICAM-2 gene was used to drive the expression of human CD59 in transgenic mice. Strong and uniform expression of CD59 was observed on the endothelial cells of all blood vessels in the heart, kidney, lung, liver, and pancreas in the three lines of mice examined. Little or no expression was seen in other cell types, with the exception of neutrophils and monocytes. These results suggest that this small promoter region contains most of the signals necessary to endow it with endothelial cell specificity, making it a potentially valuable tool in areas ranging from xenotransplantation to gene therapy.

There is now considerable evidence implicating anti‐Gal xenoantibodies as a key instigator in the hyperacute rejection of discordant xenografts. As a consequence it is generally held that elimination or reduction of the Gal/anti‐Gal component is critical to overcoming hyperacute rejection. We have recently shown that in mice inactivation of the GalT gene by homologous recombination completely eliminates the expression of the Gal‐epitope and that hearts from these mice demonstrate prolonged survival when perfused ex vivo with human plasma. Unfortunately this strategy is currently not feasible in pigs because the technology to isolate porcine embryonic stem cells, which are critical for homologous recombination, is not yet available. This study investigates an alternative competition‐based transgenic strategy to suppress the level of the Gal epitope by expression of H‐transferase (α1,2‐fucosyltransferase) an enzyme which has the same substrate specificity (lactobiose) as α1,3‐galactosyltransferase. In vitro transfection of murine cells with H‐transferase reduced Gal‐epitope expression by 80–90%. A similar reduction in Gal expression was observed on PBL and thymocytes from H‐transferase transgenic mice. This reduction in Gal epitope expression resulted in a marked reduction in the reactivity of these cells with human serum. In tissues from these mice the reduction in Gal expression was inversely proportional to the endogenous level of Gal. The results of this study support pursuing this strategy as a means to reduce the xenoantigenicity of porcine tissues.