Indian Journal of Medical Sciences Medknow Publications on behalf of Indian Journal of Medical Sciences Trust ISSN: 0019-5359 EISSN: 1998-3654 Vol. 57, Num. 7, 2003, pp. 311-318

Indian Journal of Medical Sciences, Volume 57, Number 7, July 2003, pp. 311-318

Xenotransplantation Ethics and Immunological Hurdles!

U Shankarkumar

HLA department, Institute of Immunohaematology, 13^th Floor, K.E.M. Hospital, Parel, Mumbai 400012. India. E-mail: shankarkumar16@hotmail.com

Accepted on 6-3-2003.

Code Number: ms03006

Exiting new technologies, such as cellular transplantation, organogenesis and Xenotransplantation are thought to be promising approaches for the treatment of human disease.¹ Remarkable results have been achieves in the field of organ transplantation over the past 40 years, perhaps inconceivable in the pioneering days of the 1950's. Factors, which have contributed to these results, include better immuno-suppression, matching for HLA, better preservation, and resolution of most of the technical problems associated with organ transplantation. Scientists and transplant surgeons are considering the use of animals as a source of organs and tissues for transplantation into humans. This procedure is known as Xenotransplantation. This is not a new idea. In 1682 doctors have repaired the damaged skull of an injured Russian nobleman using bone from the skull of a dog. In 1905 a surgeon from France transplanted slices of rabbit kidney into a 16-yr. old boy suffering from end stage kidney failures, unfortunately patient died two weeks later.¹

The growing demand of organs for transplantation has made scientists to think for an alternative to allotransplantation especially in heart failures. It has been estimated that approximately 45,000 Americans under the age of 65 could be benefited each year from heart transplantation, yet only 2,000 human hearts are available annually.² Currently 3,500 Canadians are waiting for donor organ.¹ Hardy attempted the first cardiac xenotransplant in humans in 1964 with chimpanzee as a donor. Bailey in 1984 did the first human neonatal cardiac xenotransplantion with mismatched baboon heart that functioned for 20 days. The case is well known a "Baby Fae".² Recent advances in our understanding of organ rejection and in animal genetic modification and cloning made it possible for scientists and doctors to consider non-human organs as a viable source of organs for transplant into humans.¹

Early Xenografts failed because the animal organs used were too different from the recipients tissues. Now the pigs because of its physiological similarities with humans, the relative simplicity with which it can be bred and the large litter with ability to reproduce faster⁴ is considered for Xenotransplantation.³ The size of organ, the blood vessels and its plumbing is similar to that of humans. This facilitates easy reconnection of organ during surgery.⁴ The cloning of a genetically modified piglet brings scientists closer to their goal of xenotranplantation. However the substantial risks and ethical issues involved need to be considered in the light of its clinical application as a treatment for end stage organ diseases.

OVERVIEW

Xenotransplantation would offer an unlimited supply of organs and avert infections or disease that would occur in human transplant. It will also provide opportunity to gain expression of extrinsic genes already present or deliberately introduced. However one potential problem in its clinical application is dysfunction of transplanted tissue and physiological limitations.⁵

Though limited data is available, the porcine hearts can provide physiologic support for days to weeks in non-human primates. The significant challenge exists in minimizing the immunological response to the organ transplant.

A: IMMUNOLOGICAL BARRIERS

Two major immunological barriers have impeded the survival of porcine organs in transplanted in to primates.⁶

Hyperacute rejection: Which is a consequence of the recipient's preformed antibodies binding specifically to the carbohydrate structure of porcine endothelial cells.

Acute vascular rejection (AVR): It can also be termed as Delayed xenograft rejection. It is known to occur several days in animal in which HRA has been prevented.⁷

T-cell-mediated rejection: It cannot be precisely defined, as T-cell responses to xenograft have been difficult in most discordant models because of the problem non-survival of grafts. However experiments on murine skin and pancreatic islet grafts show that T cell mediated xenograft rejection is often vigorous.

Mechanism of HRA, AVR and T cell mediated rejection Hyperacute rejection (HRA)

The phenomenon of HRA depends on the binding of natural antibody to the vascular endothelium, fixation of complement by that antibody and finally, activation of the endothelial and initiation of coagulation.⁸ All human beings have naturally occurring antibodies XNA (Xenoreactive natural antibodies) in their serum, which are IgM in nature and react with the carbohydrate xenoantigens (Gal-alpha 1-3-Gal) expressed on the endothelial cells.⁹ This lead to complement activation.The XNA found in human serum shares many characteristics with naturally occurring blood group antibodies and are thought to arise due to the exposure to gastrointestinal bacteria expressing similar carbohydrate structure.⁷ Complement factors bind with the antibodies resulting in the classical pathway activation of the complement cascade. The increased levels of C3a and sC5b-9 found during this time has diagnostic value.¹¹ As a result, complement factors C3 and C5 are activated, ultimately leading to the formation of C5b-9.¹⁰ It is observed that levels of C3a and sC5b-9 are found increased during this time and is of diagnostic value.¹¹

Acute Vascular Rejection (AVR)

The pathophysiology of delayed xenograft rejection (DXR) is still poorly understood. The endothelial cell of the graft microvasculature undergoes activation accompanied by gene up regulation and protein synthesis. The genes that are up regulated include tissue factor, E-selctin, VCAM-1, ICAM-1 and certain cytokines such as IL-1, IL-6, IL-8 and MCP-1. As a result of increased expression of these proteins, EC undergo phenotypic changes which make them loose their antithrombotic properties and attract leucocytes, monocytes stimulate EC and thus accelerate the ongoing AVR.⁶

On left are factors that elicit EC and on right are factors that may modulate or play a role in rejection (Figure 1).¹²

T-cell Mediated rejection

The crucial molecular interaction required for human T cell action is efficiently supplied by porcine antigen presenting cells, resulting in an efficient "direct" xenoresponse. Also the enormous number of pig antigens, recognized as foreign by host MHC-class-II restricted T cells, provoke a strong "indirect" xenoresponse. The implication is that, in vivo, pig xenografts are almost certain to provoke vigorous direct and indirect T cell xenoresponses with in the first week after xenotransplantation.⁷

B: MICROBIOLOGICAL BARRIERS

In March 1997, British researchers reported that pig retroviruses (PERVs) infected human kidney cells in vitro and replicated themselves until the viral particles "were no longer susceptible to destruction by the human immune system.¹² Retrovirus infection is life long. Two sets of pig retrovirus, PERV-A and PERV-B are found. They are widely distributed in different pig breeds and expressed in different tissues, including spleen, kidney and heart, aortic endothelial cells, hepatocytes, skin and lung.¹³ The pigs may have many unknown retro and herpes viruses in addition to bacteria, fungi and parasites capable of xenozoonoses.¹⁴

Strategies to overcome immunological and microbiological barriers:^6,7,13

• Prevention of interaction between XNA and epitopes on xenografts
• Interferes with the activation of complement.
• Preserving antithrombotic properties of EC
• Role of platelet inhibitors or antibodies against adhesion molecules.
• Immunosupression.
• Induction of tolerance.
• Haemopoietic chimerism
• Genetic modification of the pigs.
• Screening the pigs for known viral risks.

Scientific developments to overcome barriers

To prevent the XNA interaction between XNA and xenografts following developments have been achieved. (a) Immunoadsorption through columns containing gala- alpha (1-3) gal linkages⁷ or protein-A immunoadsorpiton.¹⁸ (b) Removal of xenoreactive antibodies via perfusion of the recipients, blood through pig organs.¹⁵ (c) Intravenous infusion of carbohydrates, to saturate XNA binding sites before transplantation.⁷ (d) Decreased expression of the gal alpha (1-3) gal epitope on porcine cells. (e) The expression of the human fucosyltransferase. in transgenic mice and pigs resulted in a high expression of the H antigen, together with a strong reduction of the H antigen, together with a strong reduction in the expression of the Gal alpha 1,3 Gal structure.¹⁵

Inhibition of complement activation is successful by using variety of compounds like soluble complement agents such as FUT-175, cobra venom,¹⁶ drug like sesquiterpene also called K76¹⁶ and monoclonal antibodies directed against complement.⁶ In vitro studies have shown that human RCAs such as CD55, CD46 and CD59 can prevent complement mediated lysis when expressed on the surface of transfected xenogeneic cells. Transgenic pigs expressing DAF, CD59, or both molecules have been generated by several different groups.⁷ Preventing EC activation in graft by inhibition of NF-kB, a transcription factor has been successfully achieved by transfecting porcine EC with anti-apoptotic genes, such as A 20, bcl-2, and bcl-xL-genes. EC have been shown to be resistant to activation when exposed, in vitro to activating stimuli like TNF alpha. Chicken IgY antibodies have shown to block the complement mediated lysis of PAECs by human serum and inhibit antibody dependent cell mediated lysis of PACEs by heat inactivated human serum plus peripheral blood leukocytes.¹⁹ Combination of 1-BI and PGI2 results in moderate inhibition of platelet aggregation, without changes in coagulation. Heparin leads to good inhibition thrombin-induced platelet aggregation at doses that cause only minor prolongation of PTT.¹⁷ Use of conventional of conventional immunosuppression and hDAf transgenic pigs as organ donors have demonstrated greater survival in primates.⁶ Induction of mixed hematopietic chimerism has been demonstrated to be an effective method of inducing permanent tolerance of T and B cells in closely related concordant. Experiments on mice have shown improved porcine hematopoietic engraftment.²⁰ Controversies exist about the PERV transmission, studies involving cellular porcine material- islet cells, fetal neuronal cells, extracorporeal liver cells show no evidence of PERV transmission in any of the animals or human recipients.²¹

However, an ELISA test for detection of PERV has been developed by immunization of rabbits with a peptide corresponding to the C-terminal 19 a of the 10 kDa (p10) nucleocapsid (Nc) portion of the Gag polyprotein.²² To minimize the spread of PERV, Prater's team has used a line of pig- miniature swine, which are unable to spread PERV.⁴ Bio Transplant and Novartis AG, has also bred pigs it says are incapable of passing viruses to humans.¹⁴ Retroviruses produced in non-primate species frequently carry glycoproteins on their envelope with carbohydrate moieties that reflect the biosynthesis capacities of the host organism. Once introduced into the human blood steam, these virus particles will be opsonised by the anti-Gal alpha1, 3-Gal antibody, and be inactivated. A similar mechanism would protect humans against pig endogenous retroviruses that can be mobilized from the pig genome, and be secreted from a transplanted pig organ into the recipient.¹⁵

C: ETHICAL ISSUES

Ethics is broadly defined referred as a system of moral, scruples, principles or values that in itself defines what is right or good behavior: thus, it is appropriate on this level to deal with such philosophical topics as religion, political, liberty, human rights and animal rights.²⁴ Ethics and economics are conjoins, as they are very much interrelated. Economics is the science of human behavior that generally deals with the production, distribution and consumption of commodities, and the applicability of such market mechanisms as the well-known law of supply and demand. Although the animals that may become sources of organ often are thought of as commodities, human organs generally are not so regarded.²⁴

Some of the important ethical issues are briefly described below:

Emotion and Euphemism: Raising the animals for the purpose of "viciously" killing them and "selfishly" taking their organs raises different issues. Animal right activists almost ghoulish delight in showing pictures of mistreated laboratory animals. On the other hand, those who favor Xenotransplantation loosely use the word "donor" to refer the animal that supplies the organ. Its is euphemism bordering on mockery, however to refer to an animal which is killed solely so that its heart, for example, may be put into another creature's body- as a "donor". In genesis, God declares: "Let us make men in our image and likeness to rule the fish of the sea, the birds of heaven, the cattle, all wild animals on earth, and all reptiles that crawl upon the earth". Aristotle argued that the animals were inferior to people, and that people therefore had a right to own and use animals as they saw fit. Both Judeo-Christian teaching and Greek philosophy agreed that cruelty to animals was wicked in itself, and furthermore was demeaning to humans. The Islam and Orthodox Judaism are similar and well known with respect to pigs. It is fair to say that these views express a social consensus that exists to this day.²⁴

Xenozonooses and Public Health Risks posed: It is well known that microbial traffic has emerged on the planet. The diseases like AIDS and vCJD have emerged in human species, which is traced back to animal origin. These potential viruses do not cause any disease pathology in animals but have been found to endanger the human race. Pigs may have hundreds of unknown viruses and by transplanting organs we are welcoming the entry of such viruses, which are usually kept at bay by natural barriers. Once these viruses enter the human body are likely to cause newer diseases in transplant recipients. Also it is possible that the virus on entering human body may take its route to care takers of recipients and individuals closely associated. This may lead to epidemics of viral diseases of unknown pathology in the society. Measures to institute life long surveillance programs for organ recipients may inflict workload on the Public Health machinery and involve profound cost.¹² However some authors feel that xenotransplantion will reduce the risk of disease transmission. The deadly viruses such as HIV, HBV, HCV, EBV, CMV would not be transmitted as the donor is animal origin.¹⁴

Allocation of organs: Need and not finances should dictate organ allocation. The wealth and powerful now get first access to transplants. Therefore some framework is must for equity in the allocation of organs as per top priorities. Legal concerns include the patenting. It is likely that some corporations will develop monopolies over the artificially engineered and created species.²⁴

Consent and Psychological trauma: Patients who have been told they will surely die without receiving a transplant are likely to consent to virtually any treatment at all, which may not be their true consent. On receiving the xenograft, both pre and post operation counseling is necessary to prevent the individual from emotional crippling, as they try to accept images of themselves as "part animals". Equally important is that they are accepted by society.²⁴

The issues such as compensation to the xenogenic infected recipient; cost involved in the long-term treatment of such infected persons, establishment of tissue blood banks for storage of organs cannot be neglected. The xenotransplants are much more costly than allotransplantion. The cost for one xenograft in year 1995 was estimated to be 2,50,000 dollars. Considering these issues, a huge cost burden is likely to be imposed of society.¹²

CONCLUSION

The clinical Xenotransplantation may achieve its targeted goal of extended graft survival and remain 3 to 5 years from clinical trails but must persevere under the consideration of and often is spite of scrutiny by its most demanding critics. Success has a hundred fathers; failure is an orphan.² An article mentioned below reflects the present consensus of human race. Considering all the possible risks and ethical issues involved people are eager to receive xenografts. A study was conducted in Sweden with an aim to study the attitude of patients waiting for transplantation versus the general public. And the results were as follows: 60% expressed a positive attitude towards receiving an animal kidney graft with the same degree of risk as a human kidney graft, compared with 66% for the patients. The proportion in favor of receiving a heart remained 60% for the public, but rose to 70% for the patients, If a human heart was not available, 61% of public were for the use of an animal heart, compared with 73% in the patient group. A majority of the respondents would accept a transplant from an animal, provide the result and risk of infection were the same as with a human transplant. Xenotransplantation in India is still at its infancy eventhough in our Hindu Mythology Lord Ganesha, that reveals a success story of xenotransplant. Considering the number of patients added and waiting for solid organ transplantation every year in India it is mandatory to initiate research in this field for the betterment of the ailing patient population.

REFERENCES

1. Tucker A, Belcher C, Moloo B, Bell J, Mazzulli T, et al. The production of transgeneic pigs for potential use in clinical transplantation: baseline clinical pathology and organ size studies. Xenotranaplantation 2002;3: 203-8.
2. Michler RE. Xenotransplantation : Risks, clinical potential and future prospects. Emerg Infect Dis 1996;2:64-70.
3. Cozzi E, Whitle DJ. The generation of transgeneic pigs a potential organ donors for humans. Nat Med 1995;1:964-6.
4. Bijal PT. Cloned pigs modified for use in human transplants. National Geographic today. 2002;3:http://news.nationalgeographic.com/news.html
5. Platt JL. Physiologic barriers to xenotransplantation. Trans Proc 2000;32:1547-8.
6. Cozzi E, Masroor S, Soin B, Vial C, White DJ. Progress in Xenotransplantation. Clin Nephrol 2000;53:13-8.
7. Dorling A, Riesbeck K, Warrens A, Lechler R. Clinical xenotransplantation of solid organs. Lancet 1997;349:867-71.
8. Auchincloss H. Xenogeneic transplantation. A review. Transplantation. 1988;46:1-20.
9. Sandrin MS, Vaughan HA, Dabkowski PL, Mckenzie IF. Anti-pig IgM antibodies in human serum react predominantly with Gal (alpha 1-3) Gal epitopes. Proc Natl Acad Sci USA 1993;90:11391-5.
10. Platt JL, Fishel RJ, Matas AJ, Reif SA, Bolman RM, Bach FH. Immunopathology of hyperacute xenograft rejection in a swing to primate model. Transplantation 1991;52:214-20.
11. Loss M, Vangerow B, Schmidtho J, Kanz R, Jalahi A, et al. Acute vascular rejection is associated with systemic complement activation in a pig to primate kidney xenograft model. Xenotransplantation 2000;7:186-96.
12. Alix Fano MA, Cohen MJ, Cramer M, Greek R, Kaufmann SR. Pigs, Primates and Plagues: A lay persons guide to the problems with animal to animal organ transplants. Medical Research Council Publications (MRMC) UK. http://www.mrmcmed.org/pigs.html
13. Stoye J. No clear answers on safety of pigs as tissue donor source. Lancet 1998;352:666-7.
14. Parkins K. Animal to human transplants a creation of Frankenstein's monster. http://www.heureka.clara.net/gaia/x-trans.html
15. Joziasse DH, Oriol R. Xenotransplantation: the importance of the Gal alpha 1,3 Gal epitope in hyperacute vascular rejection. Biochem. Et Biopysica Acta 1999;1455: 403-18.
16. Starzl TE, Tzakis A, Fung JJ, Todo S, et al. Prospects of clinical xenotransplantation. Transplant Proc 1994;26:1082-8.
17. Alwayn IP, Appel JZ, Goepfert C, Bahler L, Cooper DK, Robson SC. Inhibition of platelet aggregation in baboons: therapeutic implications for xenotransplantation. Xenotransplantation 2000;7:247-57.
18. Ramos A, Reiez JC, de Francisco AL, Gomez-Fleitas M, Arias M. Removal of xenoreactive antibodies by protien A immunoadsorption: experience in 22 patients. Xenotransplantion 2000;7:14-20.
19. Fryer J, Firca J, Leventhal J, Blondin B, Malcom A, et al. IgG antiporcine endothelial cell antibodies effectively block human antiporcine xenoantibody binding. Xenotransplantation 1999;6:98-109.
20. Yang YG, Chen AM, Garret LJ, Sergio JJ, et al. Development and analysis of transgeneic mice expressing porcine hematopoietic cytokines: a model for achieving durable porcine hematopoietic chimerism across an extensive xenogeneic barrier. Xenotransplantation 2000;7:58-64.
21. Birmingham K. FDA subcommitte finds no evidence of PERV transmission. Nat Med 1999;5:855.
22. Krach U, Fisher N, Czanderna F, Kurth R, Tonjes RR. Generation and testing of a highly specific anti-serum directed against porcine endogenous retrovirus nucleocapsid. Xenotransplantation 2000;7:221-9.
23. Persson OM, Persson NH, Ranstam J, Hermeren G. Attitudes toward xenotransplantation patients waiting for transplantation versus the general public. Transpl Int 2001;14:334-42.
24. Kress JM. Xenotransplantation ethics and economics. Food Drug Law Journal 1998;53:353-84.
25. Salomon DR. The US public health service guidelines for xenotransplantation advances and limitations. Xenotransplantation 2001;8:86-9.

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