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Xenotransplantation: Just around the corner?
Volume 129
Number 3
SURGERY MARCH
2001
Surgical research reviews Xenotransplantation: Just around the corner? David Grant,* MD, FRCSC, Michael Mendicino, and Gary Levy,*MD, FRCPC, Toronto, Ontario, Canada
From the Departments of Immunology, Medicine, and Surgery, University of Toronto, Toronto, Ontario, Canada
XENOGRAFTS ARE ORGANS or tissues that have been transplanted between different species. Xenografting—the transplantation of animal organs into humans—offers a potential solution to the critical shortage of human donor organs for transplantation. One skeptic has stated: “Xenotransplantation is just around the corner... and it always will be.” However, recent advances in immunology and genetics have clearly moved this therapy closer to the bedside than ever before.1-4 No one has successfully performed a pig-to-human organ transplant yet, but clinical trials are contemplated in the near future. This article reviews the progress in xenotransplantation and discusses the problems that remain. Accepted for publication April 29, 2000. Reprint requests: David Grant, Toronto General Hospital, 621 University Ave, NU-10-114, Toronto, ON M5G-2c4, Canada. Surgery 2001;129:243-7. *Dr Levy and Dr Grant are executive officers in Transplantation Technologies, Inc, Toronto, Canada, a private biotechnology company that is studying the problems associated with xenografting. Copyright © 2001 by Mosby, Inc. 0039-6060/2001/$35.00 + 0 doi:10.1067/msy.2001.108380
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WHY XENOTRANSPLANTATION? Transplantation of human cadaveric and livingrelated organs allows patients with organ failure to resume a normal lifestyle. The long-term results of heart, kidney, and liver grafting are steadily improving, with 5-year survival rates of approximately 75%. More than 68,000 patients in the United States are waiting for a transplant. A new name is added to the waiting list every 16 minutes. The demand for transplantation would be even greater if the current stringent restrictions on candidacy were to be relaxed. (United Network for Organ Sharing [UNOS]; http://www.unos.org). Donor organ availability is currently the ratelimiting step for transplantation. Approximately 15 patients on the US waiting list die every day without ever getting the chance for a transplant. Attempts to meet this need have included the use of marginal-quality cadaveric donor organs; the use of organs from living-related and unrelated donors; financial or other incentives for the donor families; and even the re-use of transplanted organs. Despite these initiatives, the supply of organs for transplantation still falls far short of the demand, as evidenced by growing waiting times for transplantation. Moreover, even if the United States doubled its organ donor rate to that of SURGERY 243
244 Grant, Mendicino, and Levy
Spain (30-40 per million), where organ donation is governed by “presumed consent” legislation, there would still be a shortage of donor organs. Hence, the impetus to consider xenografts, which can potentially provide an unlimited supply of healthy donor organs. PIGS VERSUS PRIMATES AS A DONOR SOURCE At first glance, nonhuman primates would seem to be an ideal donor source for xenografts because of their close phylogenetic relationship with human beings, but there are many drawbacks. First, there are the ethical concerns about using highly sentient animals for this purpose. Second, there is a high risk of infectious disease transmission because of the physiologic similarities between nonhuman primates and human beings. Third, it is expensive to breed primates because of their long gestation and maturity times. Fourth, for technical and ethical reasons, primates are poor candidates for genetic manipulation.3 Pigs are currently the most promising source of donor organs.5 The use of pigs for human purposes is widely accepted. Pigs have large litters with a short maturation period, they are easy to breed in a pathogen-free environment, and their organ size and physiology are remarkably similar to humans. THE IMMUNOLOGY OF XENOTRANSPLANTATION Graft rejection has been the major barrier to xenotransplantation. Whole-organ xenografts are classified as either concordant (transplants between closely related species that do not reject immediately) or discordant (transplants between distantly related species that are rejected within minutes to hours). Pig-to-primate transplants are discordant xenografts. They incite an immediate, vigorous immune response, called hyperacute rejection, which leads to graft loss within minutes to hours. If this response is prevented, strong cellular immune responses and secondary antibody production result in acute humoral xenograft rejection; cellular rejection; or chronic rejection.3 STRATEGIES TO PREVENT XENOGRAFT REJECTION Xenoreactive antibodies (XRA) are naturally occurring IgG and IgM antibodies produced in response to gut bacterial flora. XRA bind to α 1,3 galactose residues that are expressed on all mammals, with the exception of humans and Old World monkeys who lack the α 1,3 galactosyltransferase gene required to produce the gal antigen.6 The
Surgery March 2001
XRA bind to the pig’s vascular endothelium where they activate serum complement resulting in hyperacute rejection.7 C1q binds to the Fc portion of the IgM molecule, thereby inducing the formation of the membrane attack complex causing endothelial cell lysis, massive hemorrhage, thrombosis with vessel occlusion, and abrupt graft failure. The creation of transgenic donor pigs that express human complement regulatory genes has recently solved the problem of hyperacute rejection.1 Insertion of the gene for the human decay accelerating factor, for example, modifies the response to α 1,3 gal by blocking the complement cascade at the level of the initial C3 convertase.8,9 Orthotopic kidney grafts and heterotopic cardiac grafts from these transgenic animals survive for up to 100 days in nonhuman primates when treated with clinically relevant immune suppression (cyclophosphamide, cyclosporine, mycophenolate mofetil, and steroids). All of these grafts eventually fail, however, because of acute humoral xenograft rejection. The problem of acute humoral xenograft rejection must be solved before it is ethical to start clinical trials of xenotransplantation. Acute humoral xenograft rejection is characterized by the localization of XRA within the graft vasculature: the perturbation and activation of endothelial cells; host natural killer cell and monocyte recruitment and activation; platelet sequestration and activation; and vascular thrombosis by implementing the coagulation cascade through molecules such as tissue factor and the prothrombinase complex.1,10-12 Acute humoral xenograft rejection is resistant to the standard immune suppressive agents, possibly because it is only partially a T-cell–dependent response.1,2 Research is ongoing to determine if acute humoral xenograft rejection can be ameliorated by (1) treatment with antithrombotic agents; (2) “knocking-out” the gal antigen by using nuclear transfer technology; (3) using novel pharmacologic agents directed against B cells, T cells (eg, blocking B7 interactions between swine leukocyte antigens and major histocompatibility complex 1); or (4) developing techniques to make the vascular endothelium resistant to the adverse effects of antibody binding and immune activation (accommodation).1,2,13-15 If hyperacute rejection and acute humoral xenograft rejection can be prevented, it is anticipated that the problems of cellular rejection and chronic rejection of xenografts will be similar to the problems seen with allografts and controllable by the same agents that are currently in clinical use. Time will tell if this hypothesis is correct.
Surgery Volume 129, Number 3
Ultimately, the goal of allotransplantation and xenotransplantation is to induce a state of tolerance in which the recipient’s immune system accepts the graft as “self” without the need for maintenance immune suppression. The opportunity to genetically manipulate pig donors and time the transplant surgery will provide new opportunities to induce transplantation tolerance. Potential approaches include: (1) blocking the second signals for xenoimmune stimulation (anti-CD40 or CTLA4-Ig); (2) inserting additional transgenes to further ameliorate the immune response to pigs tissues; and (3) concurrent donor bone marrow transplantation to create a tolerant state through microchimerism.13,16-18 PHYSIOLOGY There are limited data on the physiology of pig xenografts in the nonhuman primate. Nonhuman primates with porcine kidney grafts require supplemental erythropoietin to maintain normal hemoglobin levels. They have low serum phosphate and albumin levels. These biochemical changes are well-tolerated for the short term, but the long-term consequences are unknown (Peter Friend, Oxford, Presented at the 5th Congress on Xenotransplantation, Nagoya, Japan, in October 1999). Other incompatibilities will undoubtedly emerge. For example porcine heart valves may suffer from shear damage caused by the different pressure gradients in an erect, biped human recipient. Liver xenotransplantation may never become a reality because of the large (> 2000) number of species-specific proteins produced by this organ. Finally, it is unknown whether pig organs will work for a human lifetime given the fact that the normal lifespan of a pig is only 15 years. XENOZOONOSIS Human allografts are frequently a source of cytomegalovirus and herpes virus (including the Epstein-Barr virus) infections. Xenotransplantation will also potentially expose the recipient and their contacts to graft-derived pathogens. It is difficult to quantitate this risk. Pigs have been developed that are free of most known bacterial and viral pathogens except for genomic retroviruses. Porcine endogenous retroviruses can infect human cells in vitro.19 However, they have never been shown to cause disease in humans. A recent study of more than 159 people who were intimately exposed to living pig tissues found no evidence of retroviral infection.20 Furthermore, there has been no evidence of disease transmission in the nonhuman primate xenotransplant studies performed to date.21
Grant, Mendicino, and Levy 245
Until the risks are more clearly defined, xenotransplant recipients, their families, and their health care providers will require long-term surveillance for infections.22-24 It may be difficult to recognize xenozoonotic infections because of their unusual presentations, altered pathogen behavior in immune compromised recipients, and lack of laboratory tests to diagnose novel pathogens. In the United States, the Food and Drug Administration has a mandate to evaluate the safety and efficacy of xenografting. Proposed guidelines for monitoring the infectious disease risk in the first phase I clinical trials of xenotransplanation are posted at the Food and Drug Administration web site (http://www.fda.gov/cber/gdlns/zooxeno.pdf). CLINICAL XENOTRANSPLANTATION Tissue xenografting. Xenotransplantation that uses wild-type pig neuroendocrine cells has shown promising preliminary results as a treatment for Parkinson’s disease.25 Phase II trials are currently under way. Pancreatic islet xenotransplantation offers the potential to cure insulin-dependent diabetes.26 Pigs are a good source of donor tissue because (1) porcine and human insulin are structurally similar, (2) pigs and human beings have similar glucose metabolism, and (3) porcine insulin has been used for many years to treat diabetes. One clinical trial has reported the presence of viable pig islet cells after clinical xenotransplantation27; but to date, no one has been able to achieve long-term pig islet function in humans. Extracorporeal liver perfusion. Extracorporeal perfusion of pig livers has been used to treat patients with fulminant liver failure until transplantation or recovery of the native liver.28 A perfusion circuit is used to carry blood from the patient through the hepatic artery and portal vein of the ex vivo organ and then return the deoxified blood to the patient. The results with this therapy have been inconsistent, possibly because of hyperacute rejection; using livers from transgenic pigs may improve its effectiveness.29,30 Clinical whole organ xenografting. There is no experience yet with the transplantation of tissues or organs from transgenic pigs to human beings, but there is a limited experience with the transplantation of organs from nonhuman primates into human beings.31 In the early 1960s, Reemtsma32 transplanted chimpanzee kidneys into human recipients before dialysis was widely available. Some of these grafts had adequate early function, but all of the recipients eventually died of uncontrollable rejection or infection. In 1985, Bailey et al33 trans-
246 Grant, Mendicino, and Levy
planted a baboon heart into a newborn infant who survived for 3 weeks post-transplant until the graft was lost to antibody-mediated damage. In the early 1990s, Starzl et al34 reported about 2 patients who underwent baboon-to-human liver xenotransplantation for end-stage liver disease secondary to chronic active hepatitis B (one was also HIV positive), which was a contraindication to transplantation at the time. These patients cleared serum lactate and ammonia. They maintained a normal coagulation profile, but had low serum albumin levels. The first patient lived for 70 days, while the second patient died at 26 days. Both died of sepsis caused by profound immune suppression. ETHICS It is difficult to know when it is appropriate to start clinical trials with transgenic pig organs and which patients should be the first test candidates. Patients with renal failure who are highly sensitized and must wait for years (or indefinitely) until a suitably matched human kidney becomes available are one group to consider. Another group to consider are neonates with heart failure who currently face a severe shortage of size-matched donors. If an unlimited supply of donor xenografts becomes available, many patients who are currently denied transplantation because they are too well or because they have extra risk factors may become xenotransplant candidates. This therapy will present many ethical challenges related to informed consent, the minimal listing requirements for transplantation, the potential psychologic responses to living with tissues from a nonhuman source, animal rights, and the clinical outcomes required to justify the use of a donor animal. There will also be economic issues. Human organs are generous gifts from donor families. The expenses of xenotransplantation, on the other hand, include not only the costs of developing, breeding, and maintaining donor animals, but also the costs associated with lifetime surveillance for infectious diseases. FUTURE DIRECTIONS Xenotransplantation offers the potential to save lives and alleviate human suffering. However, this new technology will require thorough scrutiny at every step, with sound scientific analysis and broad societal input, to ensure that its clinical application proceeds in a safe and timely fashion.3 CONCLUSIONS The success of allotransplantation as a treatment for end-stage organ failure has resulted in an
Surgery March 2001
increasing need for organ donors. Unfortunately, even if current donation rates doubled, there would still be insufficient human organs to meet the clinical need. Xenografting—the transplantation of animal organs into humans—offers a potential way of addressing this shortage. As the immunologic barriers become more clearly defined, the barriers to xenografting are slowly being solved through the genetic engineering of donor animals and the development of new drug therapies. No one has successfully performed a pigto-human organ transplant yet, but clinical trials are contemplated in the near future. Before xenotransplantation is fully implemented, the scientificmedical communities and the general public must carefully consider and respond to the complex ethical, social, and economic challenges that this technology presents.
REFERENCES 1. Soin B, Vial CM, Friend PJ. Xenotransplantation. Br J Surg 2000;87:138-48. 2. Buhler L, Friedman T, Iacomini J, Cooper DK. Xenotransplantation—state of the art—update 1999. Front Biosci 1999;4:D416-32. 3. Bigam D, Zhong R, Levy G, Grant D. Xenotransplantation [see comments]. Can J Surg 1999;42:12-6. 4. Rogers NJ, Dorling A, Moore M. Xenotransplantation: steps towards a clinical reality. Immunol Today 1998;19:206-8. 5. Caplan A. The case for using pigs. Bull World Health Organ 1999;77:67-8. 6. Platt JL. Xenotransplantation: recent progress and current perspectives. Curr Opin Immunol 1996;8:721-8. 7. Cramer DV, Wu GD, Kearns-Jonker M, Gochi E, Wakiyama S, Shirwan H, et al. The humoral response to xenografts is controlled by a restricted repertoire of immunoglobulin VH genes. Transplantation 1998;66:1375-83. 8. Platt JL. Genetic engineering for xenotransplantation. Transplant Proc 1999;31:1488-90. 9. Ascher NL. Progress in transgenic pigs for xenotransplantation. Liver Transpl Surg 1998;4:150-1. 10. Robson SC, Schulteam EJ, Bach FH. Factors in xenograft rejection. Ann N Y Acad Sci 1999;875:261-76. 11. Friedman T, Shimizu A, Smith RN, Colvin RB, Seebach JD, Sachs DH, et al. Human CD4+ T cells mediate rejection of porcine xenografts. J Immunol 1999;162:5256-62. 12. Fukushima N, Ohtake S, Yamaguchi T, Kobayashi Y, Yoshitatsu M, Ahmet I, et al. Role of endothelial apoptosis in delayed xenograft rejection in pig-to-baboon cardiac transplantation. Transplant Proc 1999;31:2731-2. 13. Chen D, Riesbeck K, Kemball-Cook G, McVey JH, Tuddenham EG, Lechler RI, et al. Inhibition of tissue factor-dependent and -independent coagulation by cell surface expression of novel anticoagulant fusion proteins. Transplantation 1999;67:467-74. 14. Badrichani AZ, Stroka DM, Bilbao G, Curiel DT, Bach FH, Ferran C. Bel-2 and Bcl-XL serve an anti-inflammatory function in endothelial cells through inhibition of NF-kappaB. J Clin Invest 1999;103:543-53.
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Surgery Volume 129, Number 3 15. Ferran C, Stroka DM, Badrichani AZ, Cooper JT, Wrighton CJ, Soares M, et al. A20 inhibits NF-kappaB activation in endothelial cells without sensitizing to tumor necrosis factor-mediated apoptosis. Blood 1998;91:2249-58. 16. Cooke DT, Nikolic B, Sykes M. The role of atypical T cells and NK cells in inhibiting primary engraftment of xenogeneic donor rat bone marrow cells. Transplant Proc 1999;31:683. 17. Nikolic B, Gardner JP, Scadden DT, Arn JS, Sachs DH, Sykes M. Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC. J Immunol 1999; 162:3402-7. 18. Nikolic B, Lei H, Pearson DA, Sergio JJ, Swenson KG, Sykes M. Role of intrathymic rat class II+ cells in maintaining deletional tolerance in xenogeneic rat—>mouse bone marrow chimeras. Transplantation 1998;65:1216-24. 19. Patience C, Takeuchi Y, Weiss RA. Zoonosis in xenotransplantation. Curr Opin Immunol 1998;10:539-42. 20. Paradis K, Langford G, Long Z, Heneine W, Sandstrom P, Switzer WM, et al. Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue [see comments]. Science 1999;285:1236-41. 21. Martin U, Steinhoff G, Kiessig V, Chikobava M, Anssar M, Morschheuser T, et al. Porcine endogenous retrovirus is transmitted neither in vivo nor in vitro from porcine endothelial cells to baboons. Transplant Proc 1999;31:913-4. 22. US guidelines on xenotransplantafion [editorial]. Nat Med 1999;5:465. 23. Xenotransplant caution continues [editorial]. Nature 1999;398:543. 24. Pig in the middle [editorial]. Nature 1999;397:279. 25. Deacon T, Schumacher J, Dinsmore J, Thomas C, Palmer P, Kott S, et al. Histological evidence of fetal pig neural cell
26. 27.
28.
29.
30.
31. 32. 33.
34.
survival after transplantation into a patient with Parkinson’s disease. Nat Med 1997;3:350-3. Groth CG, Breimer ME. Xenotransplantatian in Sweden. Bull World Health Organ 1999;77:75-6. Groth CG, Korsgren O, Wennbeag L, Song Z, Wu G, Reinholt F, et al. Pig-to-human islet transplantation. Transplant Proc 1998;30:3809-10. Fox IJ, Langnas AN, Fristoe LW, Shaefer MS, Vogel JE, Antonson DL, et al. Successful application of extracorporeal liver perfusion: a technology whose time has come. Am J Gastroenterol 1993;88:1876-81. Luo Y, Levy G, Ding JW, et al. Transgenic hDAF pigs livers perfused ex-vivo with human blood have superior function and no evidence of hyperacute rejection compared to wild-type livers [abstract]. The 5th International Congress of Xenotransplantation, October 1999, Kyoto, 1999. Levy MF, Crippin J, Sutton S, Netto G, McCormack J, Curiel T, et al. Liver allotransplantation after extracorporeal hepatic support with transgenic (hCD55/hCD59) porcine livers: clinical results and lack of pig-to-human transmission of the porcine endogenous retrovirus. Transplantation 2000;69:272-80. Steele DJ, Auchincloss H Jr. Xenotransplantation. Annu Rev Med 1995;46:345-60. Reemtsma K. Renal heterotransplantation from nonhuman primates to man. Ann N Y Acad Sci 1969;162:412-8. Bailey LL, Nehlsen-Cannarella SL, Concepcion W, Jolley WB. Baboon-to-human cardiac xenotransplantation in a neonate. JAMA 1985;254:3321-9. Starzl TE, Fung J, Tzakis A, Todo S, Demetris AJ, Marino IR, et al. Baboon-to-human liver transplantation [see comments]. Lancet 1993;341:65-71.
Number 3
SURGERY MARCH
2001
Surgical research reviews Xenotransplantation: Just around the corner? David Grant,* MD, FRCSC, Michael Mendicino, and Gary Levy,*MD, FRCPC, Toronto, Ontario, Canada
From the Departments of Immunology, Medicine, and Surgery, University of Toronto, Toronto, Ontario, Canada
XENOGRAFTS ARE ORGANS or tissues that have been transplanted between different species. Xenografting—the transplantation of animal organs into humans—offers a potential solution to the critical shortage of human donor organs for transplantation. One skeptic has stated: “Xenotransplantation is just around the corner... and it always will be.” However, recent advances in immunology and genetics have clearly moved this therapy closer to the bedside than ever before.1-4 No one has successfully performed a pig-to-human organ transplant yet, but clinical trials are contemplated in the near future. This article reviews the progress in xenotransplantation and discusses the problems that remain. Accepted for publication April 29, 2000. Reprint requests: David Grant, Toronto General Hospital, 621 University Ave, NU-10-114, Toronto, ON M5G-2c4, Canada. Surgery 2001;129:243-7. *Dr Levy and Dr Grant are executive officers in Transplantation Technologies, Inc, Toronto, Canada, a private biotechnology company that is studying the problems associated with xenografting. Copyright © 2001 by Mosby, Inc. 0039-6060/2001/$35.00 + 0 doi:10.1067/msy.2001.108380
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WHY XENOTRANSPLANTATION? Transplantation of human cadaveric and livingrelated organs allows patients with organ failure to resume a normal lifestyle. The long-term results of heart, kidney, and liver grafting are steadily improving, with 5-year survival rates of approximately 75%. More than 68,000 patients in the United States are waiting for a transplant. A new name is added to the waiting list every 16 minutes. The demand for transplantation would be even greater if the current stringent restrictions on candidacy were to be relaxed. (United Network for Organ Sharing [UNOS]; http://www.unos.org). Donor organ availability is currently the ratelimiting step for transplantation. Approximately 15 patients on the US waiting list die every day without ever getting the chance for a transplant. Attempts to meet this need have included the use of marginal-quality cadaveric donor organs; the use of organs from living-related and unrelated donors; financial or other incentives for the donor families; and even the re-use of transplanted organs. Despite these initiatives, the supply of organs for transplantation still falls far short of the demand, as evidenced by growing waiting times for transplantation. Moreover, even if the United States doubled its organ donor rate to that of SURGERY 243
244 Grant, Mendicino, and Levy
Spain (30-40 per million), where organ donation is governed by “presumed consent” legislation, there would still be a shortage of donor organs. Hence, the impetus to consider xenografts, which can potentially provide an unlimited supply of healthy donor organs. PIGS VERSUS PRIMATES AS A DONOR SOURCE At first glance, nonhuman primates would seem to be an ideal donor source for xenografts because of their close phylogenetic relationship with human beings, but there are many drawbacks. First, there are the ethical concerns about using highly sentient animals for this purpose. Second, there is a high risk of infectious disease transmission because of the physiologic similarities between nonhuman primates and human beings. Third, it is expensive to breed primates because of their long gestation and maturity times. Fourth, for technical and ethical reasons, primates are poor candidates for genetic manipulation.3 Pigs are currently the most promising source of donor organs.5 The use of pigs for human purposes is widely accepted. Pigs have large litters with a short maturation period, they are easy to breed in a pathogen-free environment, and their organ size and physiology are remarkably similar to humans. THE IMMUNOLOGY OF XENOTRANSPLANTATION Graft rejection has been the major barrier to xenotransplantation. Whole-organ xenografts are classified as either concordant (transplants between closely related species that do not reject immediately) or discordant (transplants between distantly related species that are rejected within minutes to hours). Pig-to-primate transplants are discordant xenografts. They incite an immediate, vigorous immune response, called hyperacute rejection, which leads to graft loss within minutes to hours. If this response is prevented, strong cellular immune responses and secondary antibody production result in acute humoral xenograft rejection; cellular rejection; or chronic rejection.3 STRATEGIES TO PREVENT XENOGRAFT REJECTION Xenoreactive antibodies (XRA) are naturally occurring IgG and IgM antibodies produced in response to gut bacterial flora. XRA bind to α 1,3 galactose residues that are expressed on all mammals, with the exception of humans and Old World monkeys who lack the α 1,3 galactosyltransferase gene required to produce the gal antigen.6 The
Surgery March 2001
XRA bind to the pig’s vascular endothelium where they activate serum complement resulting in hyperacute rejection.7 C1q binds to the Fc portion of the IgM molecule, thereby inducing the formation of the membrane attack complex causing endothelial cell lysis, massive hemorrhage, thrombosis with vessel occlusion, and abrupt graft failure. The creation of transgenic donor pigs that express human complement regulatory genes has recently solved the problem of hyperacute rejection.1 Insertion of the gene for the human decay accelerating factor, for example, modifies the response to α 1,3 gal by blocking the complement cascade at the level of the initial C3 convertase.8,9 Orthotopic kidney grafts and heterotopic cardiac grafts from these transgenic animals survive for up to 100 days in nonhuman primates when treated with clinically relevant immune suppression (cyclophosphamide, cyclosporine, mycophenolate mofetil, and steroids). All of these grafts eventually fail, however, because of acute humoral xenograft rejection. The problem of acute humoral xenograft rejection must be solved before it is ethical to start clinical trials of xenotransplantation. Acute humoral xenograft rejection is characterized by the localization of XRA within the graft vasculature: the perturbation and activation of endothelial cells; host natural killer cell and monocyte recruitment and activation; platelet sequestration and activation; and vascular thrombosis by implementing the coagulation cascade through molecules such as tissue factor and the prothrombinase complex.1,10-12 Acute humoral xenograft rejection is resistant to the standard immune suppressive agents, possibly because it is only partially a T-cell–dependent response.1,2 Research is ongoing to determine if acute humoral xenograft rejection can be ameliorated by (1) treatment with antithrombotic agents; (2) “knocking-out” the gal antigen by using nuclear transfer technology; (3) using novel pharmacologic agents directed against B cells, T cells (eg, blocking B7 interactions between swine leukocyte antigens and major histocompatibility complex 1); or (4) developing techniques to make the vascular endothelium resistant to the adverse effects of antibody binding and immune activation (accommodation).1,2,13-15 If hyperacute rejection and acute humoral xenograft rejection can be prevented, it is anticipated that the problems of cellular rejection and chronic rejection of xenografts will be similar to the problems seen with allografts and controllable by the same agents that are currently in clinical use. Time will tell if this hypothesis is correct.
Surgery Volume 129, Number 3
Ultimately, the goal of allotransplantation and xenotransplantation is to induce a state of tolerance in which the recipient’s immune system accepts the graft as “self” without the need for maintenance immune suppression. The opportunity to genetically manipulate pig donors and time the transplant surgery will provide new opportunities to induce transplantation tolerance. Potential approaches include: (1) blocking the second signals for xenoimmune stimulation (anti-CD40 or CTLA4-Ig); (2) inserting additional transgenes to further ameliorate the immune response to pigs tissues; and (3) concurrent donor bone marrow transplantation to create a tolerant state through microchimerism.13,16-18 PHYSIOLOGY There are limited data on the physiology of pig xenografts in the nonhuman primate. Nonhuman primates with porcine kidney grafts require supplemental erythropoietin to maintain normal hemoglobin levels. They have low serum phosphate and albumin levels. These biochemical changes are well-tolerated for the short term, but the long-term consequences are unknown (Peter Friend, Oxford, Presented at the 5th Congress on Xenotransplantation, Nagoya, Japan, in October 1999). Other incompatibilities will undoubtedly emerge. For example porcine heart valves may suffer from shear damage caused by the different pressure gradients in an erect, biped human recipient. Liver xenotransplantation may never become a reality because of the large (> 2000) number of species-specific proteins produced by this organ. Finally, it is unknown whether pig organs will work for a human lifetime given the fact that the normal lifespan of a pig is only 15 years. XENOZOONOSIS Human allografts are frequently a source of cytomegalovirus and herpes virus (including the Epstein-Barr virus) infections. Xenotransplantation will also potentially expose the recipient and their contacts to graft-derived pathogens. It is difficult to quantitate this risk. Pigs have been developed that are free of most known bacterial and viral pathogens except for genomic retroviruses. Porcine endogenous retroviruses can infect human cells in vitro.19 However, they have never been shown to cause disease in humans. A recent study of more than 159 people who were intimately exposed to living pig tissues found no evidence of retroviral infection.20 Furthermore, there has been no evidence of disease transmission in the nonhuman primate xenotransplant studies performed to date.21
Grant, Mendicino, and Levy 245
Until the risks are more clearly defined, xenotransplant recipients, their families, and their health care providers will require long-term surveillance for infections.22-24 It may be difficult to recognize xenozoonotic infections because of their unusual presentations, altered pathogen behavior in immune compromised recipients, and lack of laboratory tests to diagnose novel pathogens. In the United States, the Food and Drug Administration has a mandate to evaluate the safety and efficacy of xenografting. Proposed guidelines for monitoring the infectious disease risk in the first phase I clinical trials of xenotransplanation are posted at the Food and Drug Administration web site (http://www.fda.gov/cber/gdlns/zooxeno.pdf). CLINICAL XENOTRANSPLANTATION Tissue xenografting. Xenotransplantation that uses wild-type pig neuroendocrine cells has shown promising preliminary results as a treatment for Parkinson’s disease.25 Phase II trials are currently under way. Pancreatic islet xenotransplantation offers the potential to cure insulin-dependent diabetes.26 Pigs are a good source of donor tissue because (1) porcine and human insulin are structurally similar, (2) pigs and human beings have similar glucose metabolism, and (3) porcine insulin has been used for many years to treat diabetes. One clinical trial has reported the presence of viable pig islet cells after clinical xenotransplantation27; but to date, no one has been able to achieve long-term pig islet function in humans. Extracorporeal liver perfusion. Extracorporeal perfusion of pig livers has been used to treat patients with fulminant liver failure until transplantation or recovery of the native liver.28 A perfusion circuit is used to carry blood from the patient through the hepatic artery and portal vein of the ex vivo organ and then return the deoxified blood to the patient. The results with this therapy have been inconsistent, possibly because of hyperacute rejection; using livers from transgenic pigs may improve its effectiveness.29,30 Clinical whole organ xenografting. There is no experience yet with the transplantation of tissues or organs from transgenic pigs to human beings, but there is a limited experience with the transplantation of organs from nonhuman primates into human beings.31 In the early 1960s, Reemtsma32 transplanted chimpanzee kidneys into human recipients before dialysis was widely available. Some of these grafts had adequate early function, but all of the recipients eventually died of uncontrollable rejection or infection. In 1985, Bailey et al33 trans-
246 Grant, Mendicino, and Levy
planted a baboon heart into a newborn infant who survived for 3 weeks post-transplant until the graft was lost to antibody-mediated damage. In the early 1990s, Starzl et al34 reported about 2 patients who underwent baboon-to-human liver xenotransplantation for end-stage liver disease secondary to chronic active hepatitis B (one was also HIV positive), which was a contraindication to transplantation at the time. These patients cleared serum lactate and ammonia. They maintained a normal coagulation profile, but had low serum albumin levels. The first patient lived for 70 days, while the second patient died at 26 days. Both died of sepsis caused by profound immune suppression. ETHICS It is difficult to know when it is appropriate to start clinical trials with transgenic pig organs and which patients should be the first test candidates. Patients with renal failure who are highly sensitized and must wait for years (or indefinitely) until a suitably matched human kidney becomes available are one group to consider. Another group to consider are neonates with heart failure who currently face a severe shortage of size-matched donors. If an unlimited supply of donor xenografts becomes available, many patients who are currently denied transplantation because they are too well or because they have extra risk factors may become xenotransplant candidates. This therapy will present many ethical challenges related to informed consent, the minimal listing requirements for transplantation, the potential psychologic responses to living with tissues from a nonhuman source, animal rights, and the clinical outcomes required to justify the use of a donor animal. There will also be economic issues. Human organs are generous gifts from donor families. The expenses of xenotransplantation, on the other hand, include not only the costs of developing, breeding, and maintaining donor animals, but also the costs associated with lifetime surveillance for infectious diseases. FUTURE DIRECTIONS Xenotransplantation offers the potential to save lives and alleviate human suffering. However, this new technology will require thorough scrutiny at every step, with sound scientific analysis and broad societal input, to ensure that its clinical application proceeds in a safe and timely fashion.3 CONCLUSIONS The success of allotransplantation as a treatment for end-stage organ failure has resulted in an
Surgery March 2001
increasing need for organ donors. Unfortunately, even if current donation rates doubled, there would still be insufficient human organs to meet the clinical need. Xenografting—the transplantation of animal organs into humans—offers a potential way of addressing this shortage. As the immunologic barriers become more clearly defined, the barriers to xenografting are slowly being solved through the genetic engineering of donor animals and the development of new drug therapies. No one has successfully performed a pigto-human organ transplant yet, but clinical trials are contemplated in the near future. Before xenotransplantation is fully implemented, the scientificmedical communities and the general public must carefully consider and respond to the complex ethical, social, and economic challenges that this technology presents.
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