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Introduction: After orthotopic cardiac xenotransplantation, the combination of both the inflammatory responses to the exposure of a recipient to the xenogeneic organ and the use of cardiopulmonary bypass has been assumed to cause detrimental side effects. These have been described not only to affect the transplanted organ (heart) itself, but also the recipient's lungs. In this article, we summarize how these possible detrimental processes can be minimized or even avoided. Methods: Data from eight pig‐to‐baboon orthotopic cardiac xenotransplantation experiments were analyzed with a special focus on early (within the first week) postoperative organ dysfunction and systemic inflammatory responses. Non‐ischemic heart preservation and the careful management of the heart‐lung machine were deemed essential to guarantee not only the immediate function of the transplanted xenogeneic organ but also the prompt recovery of the recipient. Results: After weaning from cardiopulmonary bypass, very low catecholamine amounts were needed to ensure an adequate pump function and cardiac output. Central venous oxygen saturation and serum lactate levels remained within normal ranges. All animals were successfully weaned from ventilation within the first postoperative hours. Serum parameters of the transplants and native kidneys and livers were initially slightly elevated or always normal, as were hemoglobin, LDH, and platelet measurements. Markers of systemic inflammation, C‐reactive protein, and IL‐6 were slightly elevated, but the reactions caused no lasting damage. Conclusion: Consistent short‐term and long‐term results were achieved after orthotopic cardiac pig‐to‐baboon transplantation without detrimental inflammatory responses or signs of multiorgan failure. In comparison to allogeneic procedures, non‐ischemic heart preservation was important for successful immediate organ function, as was the management of the heart‐lung machine. Thus, we believe that genetically modified porcine hearts are ready for use in the clinical setting. [ABSTRACT FROM AUTHOR]

The article focuses on the manipulation of the xenotransplantation method to pigs wherein unmatched, untested and immunosuppressing possibilities in the operation are overcome. It notes the potential genetic modification of the pig using the employed technology which include removing particular pig characteristics and adding human characteristics to the pig tissue. It mentions the manipulation of the power of genetic engineering making the pig donor tissue to look like human allograft.

Background: Despite overcoming xenograft hyperacute rejection (HAR), Gal (galactose-α1,3-galactose) expression may not be completely eliminated from the α1,3-galactosyltransferase gene knockout (Gal KO) pig because of alternative galactosyltransferases. Whether low levels of “residual” Gal are still susceptible to either complement fixing or non-complement fixing antibody beyond the HAR barrier remains unknown. Furthermore, it would be impossible to analyze the immune response specific to low-level Gal in a xenograft setting given the multitude of xenoantigens that could induce a recipient response. To investigate this question, we therefore used a skin graft model in BALB/c mice where the sole difference between donor and recipient was the expression of Gal, where rejection is caused by passively administered anti-Gal monoclonal antibody and where HAR does not occur. Methods: Gal expression over time was examined by immunohistochemistry in wildtype-to-Gal KO skin grafts. Graft rejection in response to passively administered anti-Gal monoclonal antibody at early and late time points was studied to determine changes in susceptibility to antibody. To independently test the effect of reduced Gal expression on antibody-mediated rejection, we used two separate lines of α1,2-fucosyltransferase transgenic mice as skin donors in the model. These mice have known reduced but different levels of Gal as determined by flow cytometry on peripheral blood leukocytes. Results: Gal expression on skin grafts diminished with time with a corresponding reduction in susceptibility to antibody-mediated rejection. Skin grafts at day 30 (n = 7) and 150 (n = 11) had a rejection rate of 100% and 45% respectively in response to non-complement fixing anti-Gal antibody administered to the recipient. Similar results were demonstrated with a complement fixing anti-Gal antibody. When α1,2-fucosyltransferase transgenic mice skin was used in the model, the line with lowest level of Gal expression was resistant to antibody-induced rejection with a rate 0% (n = 9) vs. 60% (n = 5) in the alternative line with relatively more Gal expressed but still much less than normal mice. Conclusions: Resistance to anti-Gal antibody-mediated damage in the model was observed in skin grafts 100 to 150 days post-grafting but not earlier and was associated with a reduction in Gal expression. It is possible that below a threshold level of Gal expression, the grafts were not susceptible to anti-Gal antibody. [ABSTRACT FROM AUTHOR]

Acute humoral rejection remains the major barrier to long-term pig-to-primate xenograft survival, and microvascular thrombosis is a critical element of the rejection process. It appears that persistent endothelial cell activation and injury, by even low levels of anti-graft antibodies, eventually overwhelm the cellular anticoagulant defences and promote the development of thrombotic microangiopathy. Porcine endothelium may be particularly vulnerable because of cross-species molecular incompatibilities affecting the function of thrombomodulin and possibly TFPI. Recent data from small animal models suggest that transgenic overexpression of anti-thrombotic molecules on xenograft endothelium is capable of inhibiting intravascular thrombosis and preventing acute humoral rejection. In conjunction with existing genetic modifications (e.g. Gal KO, hDAF), this is a promising strategy to move xenotransplantation to the clinic. [ABSTRACT FROM AUTHOR]

Abstract: Transplantation exposes vascularized grafts to several potential injuries, including ischemia-reperfusion injury and rejection. These processes are associated with, at least in part, extracellular nucleotide-triggered (type 2 purinergic receptor) inflammatory responses that if left unchecked might compromise the long-term function and survival of the graft. Modulation of type 2 purinergic receptor–mediated signaling occurs by ectoenzymes that hydrolyze nucleotides to adenosine, which in turn activate type-1 purinergic receptors, and play a critical role in limiting inflammation and thrombosis. CD39 is the prototype nucleoside triphosphate diphosphohydrolase and is highly expressed on the endothelium. Different levels of CD39 expression by the vasculature influence the fate of the transplanted organ in ischemia-reperfusion injury and transplant rejection models. Additional roles for extracellular nucleotides/adenosine have also been defined within the immune system. Moreover, CD39 is a phenotypic marker of regulatory T cells, and through the generation of adenosine, contributes to the suppressive capabilities of these cells in vitro and in vivo. Our experimental models suggest that purinergic mechanisms may provide an integration point for the vascular and immunological responses in transplantation. CD39, the dominant ectonucleotidase, receptors for nucleotide mediators and adenosine are expressed in a regulated manner on both vascular and immune cells. We predict that administration of soluble CD39 and adenosine agonists or the targeted expression of CD39 through genetically modified organs or cells may have future therapeutic application in transplant-associated and other vascular diseases. [Copyright &y& Elsevier]

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 wild-type mice were then transplanted into streptozotocin-induced 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. [ABSTRACT FROM AUTHOR]

Important phylogenetic differences between pig and human tissues prevent xenotransplantation from becoming a clinically feasible option. Humans lack the galactose-α1,3-galactose (αGal) epitope on endothelial cell surfaces and therefore have preformed anti-αGal antibodies. The role of these antibodies in rejection of non-vascular xenografts remains controversial. This study investigared the role of anti-αGal antibodies in rejection of non-vascularized αGal+/+ grafts in αGal -/- mice. αGal +/+ and αGal -/- pancreatic islets were transplanted under the renal capsule of streptozotocin-induced diabetic (1) αGal -/- mice and (2) αGal +/+ mice. αGal -/- recepients were immunized with rabbit red blood cell membranes (RRBCs) to produce elevated anti-αGal antibody levels. Six of the 18 αGal -/- mice rejected the αGal +/+ grafts within 68 days whereas indefinite graft survival was achieved in the control groups. Animals with surviving islet grafts were challenged with αGal +/+ skin grafts. Although all αGal +/+ skin grafts were rejected within 58 days, the islet grafts remained intact. This observation correlated with the level of αGal expression (which was very low on islets compared to skin) rather than the actual titre of anti-αGal antibody. The results suggest that the level of αGal expression plays an important role in graft survival. Therefore, its removal is important in the development of a pig islet donor for future clinical therapy. [ABSTRACT FROM AUTHOR]

Focuses on transgenic strategies for overcoming complement-mediated hyperacute rejection of a xenotransplant. Shortage of human donor organs, tissues and cells; Use of pronuclear microinjection to produce pigs containing up to three transgenes.

Xenotransplantation offers a potential solution to the shortfall in donor organs for human transplantation. This review describes the barriers to xenotransplantation and the progress that has been made towards making it a clinical reality. Data from preclinical pig-to-primate cardiac and pulmonary xenografts are highlighted. (Heart, Lung and Circulation 2002; 11: 32-41). [ABSTRACT FROM AUTHOR]

In this article, the authors discuss the study on the production of galactosyltransferase knockout (Gal KO) pigs expressing multiple transgenes in one generation using porcine embryonic stem cells (pESCs) to advance xenotransplantation research. The authors say that they employed new approach in isolating pESCs by using expanded blastocysts and added growth factors and additives in place of foetal calf serum. Benefits of pESCs including removal of antibiotic resistance genes are also discussed.

Extracellular nucleotides play an important role in thrombosis and inf lammation,triggering a range of effects such as platelet activation and recruitment,endothelial cell activation,and vasoconstriction.CD39, the major vascular nucleoside triphosphate diphosphohydrolase (NTPDase),converts ATP and ADP to AMP, which is further degraded to the antithrombotic and anti-inf lammatory mediator adenosine.Deletion of CD39 renders mice exquisitely sensitive to vascular injury,and CD39-null cardiac xenografts show reduced survival.Conversely,upregulation of CD39 by somatic gene transfer or administration of soluble NTPDases has major benefits in models of transplantation and inf lammation.In this study we examined the conse- quences of transgenic expression of human CD39 (hCD39)in mice.Importantly,these mice displayed no overt spontaneous bleeding tendency under normal circumstances.The hCD39 transgenic mice did,however, exhibit impaired platelet aggregation,prolonged bleeding times,and resistance to systemic thromboembo- lism.Donor hearts transgenic for hCD39 were substantially protected from thrombosis and survived lon- ger in a mouse cardiac transplant model of vascular rejection.These thromboregulatory manifestations in hCD39 transgenic mice suggest important therapeutic potential in clinical vascular disease and in the control of serious thrombotic events that compromise the survival of porcine xenografts in primates. [ABSTRACT FROM AUTHOR]