First Part - Scientific Aspects
Rejection: Immunology of Organ Xenografting
Xenozoonoses: the Transmission of Infectious Agents from One Species to Another
Advances in Biotechnology and Molecular Genetics
Moving to the clinical phase
Part Two - Anthropological and Etichal Aspects
Human Intervention in the Created Order
The Use of Animals for the Good of Man
Xenotransplantation and the Identity of the Recipient
The Health Risk
Allocation of Health Care Resources
Patentability and Xenotransplantation
Transplantation represents a highly successful means of treating a variety of human illnesses. However, the number of transplants performed is limited by a shortage of human organs and tissues1. Xenotransplantation, the transplantation of organs, tissues or cells from one species to another, if applied to man, would offer the possibility of a huge supply of organs, tissues and cells for transplantation thereby relieving the "chronic" shortage of human donors.
However, before xenotransplantation becomes a clinical reality, there are practical challenges that must be overcome. One is rejection, the process by which the body of the transplant recipient attempts to rid itself of the transplant. Another is to ensure the correct functioning, across species barriers, of the transplant in its new host. Also, there is the need to minimize the likelihood of the introduction of new infectious agents into the human population via the transplant.
In addition there are concerns about xenotransplantation that require theological, anthropological, psychological and ethical considerations, as well as an examination of legal issues and procedural matters.
1. To date, there is only very limited experience in transplanting xenogenic organs or tissues to humans. The attempts made in the 1960s and early 1970s used immunosuppressive therapies on the recipient to prolong survival of the organ. The most striking success was the nine-month survival of a chimpanzee kidney transplanted into a human by Reemtsma and colleagues2. In the 1980s, a baboon heart was transplanted to a baby (Baby Fae) that survived briefly3; however, rejection occurred within a few weeks. In the 1990s, baboon livers were transplanted in two patients by Starzl and colleagues4. Those patients survived for 70 days in one case and 26 days in the other. The first patient was placed on an oral diet on the fifth post-transplant day and spent most of his time in a regular ward, leaving the hospital briefly on one occasion5. However, in one of the two cases, a baboon pathogen (cytomegalovirus) was apparently transferred to the patient, even though this did not result in a disease process6. However, in both patients there was evidence of an adequately functioning liver mass, sufficient to sustain life. The baboon livers led to the presence of baboon proteins synthesized by the liver; in some cases those proteins assumed the blood levels that are characteristic of the baboon and not of the human. Possible molecular incompatibility of those proteins poses a potential problem of functionality for humans.
Transplants have also been attempted using pig hearts (three cases) or livers (one case); in no case did the recipient survive more than 24 hours7. While non-human primates have been preferred in the past as source organs for humans, at present the scientific community and the regulatory agencies in those countries which are addressing the issue have ruled out the use of such source animals both because of the increased risk of transmission of infection and because of a variety of other ethical and practical concerns8. As a consequence many researchers have settled on the use of pigs as a potential source animals for xenotransplantation9. The use of genetic engineering has resulted in significant improvement in survival time for a pig organ in a non-human primate receiving immunosuppression10. However, the survival time of such organs does not yet approach that of human organs transplanted to other humans (allotransplantation). Therefore, certain barriers to xenotransplantation remain11.
Further genetic engineering of source animals and/or use of additional/new immunosuppressive agents are the two approaches that are considered most likely to prolong the survival of a xenotransplant12. Clearly more research in xenotransplantation is needed and should be done.
2. There are four immunological barriers that must be overcome for achieving successful organ xenotransplantation from pig to primate (human and non-human). First, hyperacute rejection, which is caused by xenoreactive natural antibodies and complement of the recipient acting against endothelial cells of the source animal organ. Second, acute vascular rejection caused by the combined effect of elicited xenoreactive antibodies and activated host natural killer cells and monocytes. In combination these stimuli (the anti-graft antibodies and the activated host cells) result in activation of the endothelial cells of the source organ. Endothelial cell activation leads to general inflammation with resultant thrombosis (platelet aggregation and activation of the coagulation cascade) resulting in organ rejection. Third, the xenograft counterpart of classical T cell mediated rejection of allografts (transplantation between individuals of the same species) will almost certainly occur. Finally, xenografts may also be subject to chronic rejection in a manner analogous to allografts.
Hyperacute Rejection. Recipient xenoreactive natural antibodies and complement are the two major factors that result in hyperacute rejection of an immediately-vascularized organ. Pre-existing xenoreactive natural antibodies bind with vascular endothelial cells of the pig organ13. These antibodies are directed primarily towards a sugar moiety, the Gal-∝ (1,3)-Gal-ß (1,4)-G1cNac antigens of the pig, also known as "∞-gal"14. The bound antibodies fix and activate complement, with the combination of antibodies and activated complement leading to endothelial activation which result in thrombosis, rapid graft ischemia and rejection. Elimination of xenoreactive natural antibodies provides one method to overcome hyperacute rejection15. Hyperacute rejection has also been overcome by methods that inhibit complement16.
Among the different approaches for achieving inhibition of complement, the one that has proven most effective is based on in vitro experiments in which a human protein that inhibits human complement activation is introduced into the membrane of pig endothelial cells. The molecule first tested was human decay accelerating factor, or hDAF. The presence of hDAF in the pig endothelial cells prevented lysis of those cells and would thus, presumably, prevent the activation of the cells17. These findings suggested that the production of transgenic pigs expressing hDAF might provide an approach for overcoming hyperacute rejection of pig organs transplanted into primates. Certain research groups have produced such transgenic pigs and have demonstrated that organs from these pigs usually do not undergo hyperacute rejection18. Based on these results with transgenic hDAF-expressing pigs, it appears that hyperacute rejection can be overcome, which is the first major triumph of gene therapy in the field of organ transplantation.
Another possible solution to hyperacute rejection is to eliminate, or greatly reduce the expression of "∞-gal" from pigs by knocking out the 1,3 galactosyl transferase gene, which is needed for the expression of "∞-gal"19. This has not yet been accomplished in pigs, although present-day cloning technology could make this possible.
Acute Vascular Rejection. Acute Vascular Rejection is precipitated by elicited xenoreactive antibodies and by the possible infiltration of host inflammatory cells, monocytes and natural killer cells, that invade the xenograft20. Endothelial cells are activated resulting in thrombosis, compromised blood flow and rejection21. Acute vascular rejection now represents the principle immunological barrier to successful xenotransplantation. Studies of acute vascular rejection in animals has shown that the use of immunosuppression leads to organ survival for a far greater length of time than is seen in untreated cases22. An alternative approach for overcoming acute vascular rejection is further genetic engineering animals/organs23. A number of genes may suppress the inflammatory response that appears to cause acute vascular rejection are now being studied.
T Cell Response. If acute vascular rejection can be overcome, it is expected that there will be a xenograft counterpart of the allogeneic T cell rejection response24. There are disagreements whether the xenogenic T cell response will be more difficult to overcome than the allogeneic one, which today is easily controlled. In addition to the use of immunosuppression, there is the possibility that in pig-to-primate transplants we might achieve tolerance (non-reactivity of the immune system of the recipient to pig antigens without immunosuppression)25. Such tolerance is the hope of transplantation in general and may be aided in the xenogenic arena by further genetic engineering of the source animal.
Chronic Xenograft Rejection. There is evidence that - as with allotransplants, - even when a transplant survives all the above rejection phases, there is the possibility that it will be rejected months or years later26. This is referred to as "chronic" rejection. The main pathology of chronic graft failure involves smooth muscle cell proliferation and obliteration of the lumens of blood vessels.
3. Xenotransplantation of organs has been studied primarily in small animal models and in pig-to-nonhuman-primate combinations.
Small animal models. The principal model used involves transplantation of hamster or mouse hearts to rats. For the most part, the rejection of a hamster heart by a rat is similar to the rejection of a mouse heart. However, the rat does not have sufficient preformed xenoreactive natural antibodies to reject a mouse or hamster heart hyperacutely; thus rejection is dependent on the synthesis of anti-graft antibodies that, together with recipient complement, lead to rejection of the organ27. Transplantation of mouse or hamster hearts to rats is therefore thought to be a good model of acute vascular rejection. The preliminary findings that have emerged from small animal transplants are the following: administration of immunosuppression to the rat can lead to long-term survival of hamster hearts28. In this sense, rejection of a hamster organ transplanted to a rat appear to differ from acute vascular rejection of a pig organ in a non-human primate in which hyperacute rejection has been overcome. In the pig-to-nonhuman-primate model, immunosuppression alone is currently unable to lead to long-term survival. The second finding in the hamster or mouse heart transplants to rats has been the achievement of "accommodation"29. Accommodation refers to the survival of an organ in the presence of anti-graft antibodies and complement. Short-term inhibition of complement coupled with continuing inhibition of T cells leads to long-term survival in these two situations. An interesting finding regarding accommodation is that the surviving organ expresses genes in its endothelium and smooth muscle cells that protect the organ from rejection30. To what extent these protective genes can be used therapeutically to aid pig organ survival in primates is not clear. Isolated cases of accommodation have been described in human allogeneic transplants as well31.
Large animal models. The principal model today remains transgenic pigs expressing hDAF32 and, in some cases, other human genes inhibiting complement cascade, coupled with immunosuppression in order to achieve survival. In most cases, normal pig organs are rejected hyperacutely by non-human primates, and thus, more rapidly than transgenic pig organs expressing hDAF33. Even when hyperacute rejection is avoided, the hDAF transgenic organs are rejected in non-human primates by a process that mimics acute vascular rejection, although rejection can be very much delayed34. Transgenic pig hearts have been shown to survive for up to 99 days when they are not asked to do life-supporting work (heterotopic transplant)35. When placed in the position of having to support life (orthotopic transplant), the longest survival periods have been a month for a cardiac xenograft36and 78 days for a renal xenograft37; most organs are rejected in a shorter period of time. Scientists propose two different approaches, which can be combined for achieving longer survival periods of pig organs in primates. The first is to try different immunosuppressive protocols, and the second is to produce pigs that express additional transgenes that might inhibit rejection factors associated with acute vascular rejection.
4. Over sixty porcine infectious agents with a potential to cause disease in humans have been identified38. Development of "clean" lines of source animals, with a certified health status, is under way39. Control measures include the birth of pigs by hysterotomy (caesarean derived), carefully controlled environments and routine monitoring of pigs and their handlers. These steps appear to have excluded almost all known infectious agents of concern. However, it cannot be ruled out that an unknown porcine virus might exist which causes no pathology in pigs but which may cause disease in humans.
As is true for all other mammalian species, pigs have sequences in their DNA that encode retroviruses (PERV - Porcine Endogenous RetroViruses)40. Weiss and colleagues showed that pig retroviruses could infect human cells in vitro41. There are no satisfactory animal models to test the pathogenicity of these agents. The blood of 160 patients exposed to living pig tissues was studied for the presence of PERV. In 135 patients exposure was for only one hour or a little more. In a few of the remaining patients exposure was for longer periods, in one case for 460 days. None of the patients showed evidence of PERV infection, although pig cells containing retroviral sequences were found even several years after exposure to the pig tissue42. It is a matter open to conjecture the extent to which one can take confort from negative results in persons exposed for such short period of time, except for a few cases, and in any event to very few pig cells, as compared with the years of exposure that would presumably occur in an organ were successfully transplanted into a human. Certainly, the elimination from pigs of all PERV, which represents a continuing concern and hinders the move to clinical trials, will remain a challenge for years to come.
5. The major advances in biotechnology that might favour further development of xenotransplantation relate to producing transgenic pigs that express human genes which inhibit rejection. Two break-throughs are especially important. First, recent studies have led to the cloning of pigs43, allowing for simple genetic manipulation compared with the methods currently available. With this procedure, at least in principle, new genes can easily be introduced into the DNA of the pig genome during the cloning process, and other genes "knocked out" so that they would no longer be functional. For instance, the gene that leads to expression of the "∞-gal" antigen on porcine endothelial cells could be knocked out so that at least one of the causes of rejection would presumably be reduced.
Second, although still at the experimental level, methods to regulate the expression of transgenes have been devised44. It may well be that a certain transgene would be highly desirable at a given moment after transplantation while it would be undesirable at a different moment. Therefore, being able to regulate the expression of a transgene would represent a great advance in the development of xenotransplantation.
6. Because transplanted cells and tissues are not immediately perfused with recipient blood after transplantation they are not hyperacutely rejected. Clinical trials using such transplants have therefore progressed further compared to clinical trials with solid organ transplants. Porcine pancreatic islets have been transplanted into a number of patients with diabetes45 and foetal porcine neural cells have been injected into a significant number of patients (more than 50) suffering from Parkinson's Disease, Huntington's Disease or strokes46. Only limited clinical benefit has been reported to date. A significant number of patients with acute liver failure has taken part in multicentre studies using pig hepatocytes in artificial devices (bioartificial liver) with promising initial results47.
There are considerable differences of opinion as to how long a pig organ should survive in a non-human primate before one proceeds to clinical trials involving the transplantation of pig organs into humans. Some suggest that clinical trials on humans could begin only after routine survival periods of ninety days or more are obtained for pig organs which are transplanted into nonhuman primates and which must perform life-supporting functions48. At present, survival periods for this type of xenotransplants vary from a few weeks to about three months, and three-month survival is certainly not routine49. Clearly, a significant improvement on current figures must be achieved before clinical trials using solid organ xenografts are warranted.
However, while survival of pig organs in non-human primates at present is not sufficiently long to consider transplanting such organs into humans as a permanent replacement organ, the option of using pig organs as 'bridge' transplants may well be possible in a shorter time.