Q J Med 2000; 93: 63-66
© 2000 Association of Physicians
Editorial |
Xenotransplantation: postponed by a millennium?
Department of Surgery and Liver Research Laboratories, MRC Centre for Immune Regulation, Liver Research Laboratories, University of Birmingham Institute for Clinical Science, Queen Elizabeth Hospital, Birmingham
In the late 1980s, a persistent shortage of human donor organs and the availability of new molecular techniques sparked a renewed interest in transplantation of organs and tissues across the species barrier. In spite of the considerable phylogenetic distance to humans, the pig was chosen for its size, breeding pattern and general availability as the most suitable xenogeneic donor species. The following years saw substantial financial and intellectual investments in xenotransplantation, which resulted in the development of genetically modified animals that were thought to be suitable for use in clinical trials. Although these clinical trials are yet to be implemented, the issues that drove the initial enthusiasm, notably the shortage of human donors, have become even more acute in recent years, strengthening the need for alternative sources of organs for transplantation. However, serious reservations remain about the use of xenotransplantation. If we assume, and this is a major assumption in the present climate, that the ethical concerns related to the use of animals as a source of donor organs will be met by society, three major problems remain to be overcome before xenotransplantation can become a viable treatment: (i) immunological acceptance of the xenograft; (ii) functional compatibility of organs between species; and (iii) concerns that transmission of novel infectious agents will occur between species.1
The transplantation of vascularized xenografts including heart, kidney or liver into unmodified hosts invariably leads to hyperacute xenograft rejection.2 This process occurs rapidly and does not require de novo protein synthesis, because it is mediated by pre-existing xenoreactive natural antibodies (XNA) that bind to specific terminal carbohydrate epitopes termed Gal-
-1,3Gal that are synthesized by a specific form of the enzyme galactosyltransferase. Gal--1,3Gal is expressed on pig cells but not on human cells, as man (in common with other higher primates) lacks this enzyme and uses another disaccharide for terminal glycosylation of proteins.3 One of the explanations for the existence in humans of anti-porcine antibodies is that Gal--1,3Gal is also found in many enteric bacteria. Thus most people have been exposed to Gal--1,3Gal and mounted protective immune responses against it. In addition, there is evidence that a T-cell-independent subset of B cells produces antibodies encoded by germline immunoglobulin genes which serve as a form of primitive humoral defense.4 Furthermore, some XNA may be crossreactive antibodies derived from neonatal B cells reacting with self antigens that persist in adult life.
Deposits of XNA on graft endothelium fix and activate the human complement system, leading to platelet activation, the generation of procoaculant and proinflammatory factors such as thrombin, histamine and leukotriens, culminating in intravascular thrombosis, interstitial haemorrhage and graft loss. It has long been recognised that hyperacute xenograft rejection is a major barrier to successful xenotransplantation, and several strategies have been advocated to overcome it.5 These include lowering circulating XNA, depleting host complement and altering the expression of the porcine-specific carbohydrate epitopes on the cell surface. An elegant approach was suggested recently in which retroviral gene transfer was used to express the pig epitope on bone marrow cells of a xenograft recipient. The recipient's immune system then saw the pig antigen as self, resulting in tolerance and a failure to mount an antibody response.6 However, this approach requires genetic modification of the host, which has ethical and logistic problems. A more practical approach is to modify the xenograft itself. Endothelium expresses proteins that prevent the deposition of complement, and fortunately these proteins are highly species-specific. Thus it was proposed over a decade ago that expression of regulators of human complement activation on pig endothelium would prevent activation of the host (human) complement by the xenograft.7 This theory was proven when pig organs that had been modified to express human decay accelerating factor, a crucial human regulator of complement activation, were transplanted into primates and hyperacute rejection was greatly diminished. Subsequently several transgenic pig lines were raised which expressed one or a combination of several human regulators of complement activation. When these modified donor organs were transplanted in the presence of standard immunosuppression in preclinical models, graft survival of several months was observed, demonstrating the feasibility of the approach.8
Despite these successes, xenograft rejection has not been solved. Not surprisingly, a more complex process of delayed acute vascular xenograft rejection still occurs and determines the long-term fate of the graft. This form of rejection does require de novo protein synthesis, and is different to vascular rejection in allogeneic transplants. It is characterized by extensive thrombotic changes within the graft and a dense mixed inflammatory infiltrate of predominantly macrophages, with lesser numbers of T cells and NK cells.9 This process does not require T-cells, and is driven by complex interactions between graft endothelial cells and host antibodies, macrophages and platelets.10 Host macrophages are activated by XNA bound on the donor endothelium, and the endothelium itself is activated directly by XNA, resulting in a procoagulant and inflammatory microenvironment characterized by expression of chemokines and inflammatory cytokines. This response is perpetuated and exacerbated, because molecular incompatibilities between regulatory proteins and their ligands may result in a failure of the physiological homeostasis that controls inflammatory and coagulatory responses.11 Human NK cells, for example, are inhibited by human MHC class I expressed on the endothelial cell surface, whereas porcine MHC may be less efficient in controlling human NK cell activation.12
Given the complex nature of delayed xenograft rejection, it is likely that its prevention will require the use of a wide array of strategies to modulate thrombogenesis, prevent endothelial cell activation and inhibit activation of macrophages and NK cells. Used alone, conventional immunosuppressive drugs do not significantly alter the course of delayed xenograft rejection. The role of the classical MHC/T-cell responses that drive alloactivation in conventional transplantation have to be reassessed for each species combination. In pig-to-human transplantation, there are sufficient similarities between the two systems for T-cell-mediated xenograft responses to be important. Porcine MHC antigens are recognized by human T-cells by both the direct (antigen presentation via xenogeneic MHC expressed on xenogeneic cells) and indirect (presentation of processed pig MHC by human antigen-presenting cells) pathways leading to activation of effector cells and cytotoxicity. Thus effective anti-rejection regimes in xenotransplantation will need to combine genetic engineering, in which protective genes are induced in donor tissue in order to prevent xenorejection, with more conventional strategies to induce immunosuppression or immunological tolerance and prevent rejection.
While most work so far has focused on the prevention of hyperacute or delayed xenograft rejection, attention also needs to be paid to chronic xenograft rejection. This slow but progressive destructive process has been described in concordant xenografts, where XNA and complement are not involved primarily. Of historical interest is the observation that many features of chronic xenograft rejection were described in the early 1960s following an initially successful chimpanzee-to-human kidney transplant,13 a practise abandoned later in view of animal ethics and issues of transmission of simian disease.
Even if the formidable immunological challenges can be overcome, other problems remain. One particular concern is whether the xenograft will function physiologically in the very different host environment. Numerous examples of possible graft dysfunction have been mentioned, including effects on the homeostasis of organ function. For instance, the porcine heart is designed to work in a different anatomical site under different hydrostatic pressures than found in man. Another obvious problem relates to the fact that some proteins are molecularly incompatible between species, leading to a malfunction of crucial regulatory processes.14 For example, the inability of porcine tissue factor pathway inhibitor to regulate human factor Xa adequately, contributes to the disordered thromboregulation observed following xenografting.15 Yet another concern derives from the observation that some porcine proteins produced by the xenograft, such as porcine erythopoietin, elicit an antibody response, which may crossreact and neutralize the human protein.
At the present time, the potential for compatible organ function cannot be predicted by theoretical modelling alone and will need further careful assessment in suitable in vivo models that take into account organ-specific characteristics. These problems are likely to be greatest for liver transplantation, because of the liver's role in the production of thousands of highly specialized proteins. The complexity of the systems that regulate protein synthesis and secretion in addition to concerns about how compatible many porcine proteins are in human systems make it impossible to predict how a xenogeneic liver will function in a human host. Furthermore, it is unclear whether porcine complement will damage human vessels leading to xenograft versus host reaction.
The problems above relate to feasibility and compatibility of xenotransplants but there is another less predictable and potentially more serious concern. ‘Xenosis’ is a novel term introduced a few years ago to describe the interspecies transmission of infectious agents via a xenograft. Whereas rejection and physiological compatibility only affect the xenograft recipient, xenozoonotic infections have the potential to introduce infectious agents into the wider community, leading to pandemic infections with unusual or novel agents.16 The risk of infections caused by bacteria, fungi, parasites or exogenous viruses could be minimized by keeping the donor animals pathogen-free. The main worry, however, relates to the transmission of porcine endogenous retroviruses (PERV) via the xenograft. PERVs are not only integrated into the pig genome and transmitted via the germline but may also play a physiological role in placental function. It has been shown in vitro with various cell types that PERV can be activated to produce virions that are infectious for a variety of human cells. 17,18 Against this, thousands of patients have come into contact with xenogeneic material through exposure to pig cells and tissue in, for instance cardiac surgery, and so far no evidence of viral infection has been detected.19,20 However, this situation is very different from the transplantation of a vascularized organ in the face of potent immunosuppression, which might promote viral transmission. At the moment the lack of clear evidence either way should not be used to overrule the substantial concerns. Policy makers in the USA have issued a de facto ban on clinical trials with xenotransplants from non-human primates to humans21 and in the UK have declared a moratorium on xenotransplantation. However, the climate is moving toward permitting limited closely monitored clinical trials with porcine tissue in the future.22
While issues such as cross-species infection apply to most forms of xenotransplantation, they may be less of a problem in the context of cellular xenografts. Cellular transplants are an easier target for complex genetic engineering than an entire organ and may, in some circumstances, be protected by semi-permeable polymer encapsulation.23 In fact (pre)clinical trials involving a variety of genetically engineered cell types for treatment of refractory pain, neurological disorders and malignant tumours are under way.24 In other words: the potential for cellular xenotransplants in the next future is considerable.
At the end of the millennium, xenotransplantation, with its potential benefits and intrinsic hazards, still has a long way to go to become a successful treatment. From the public's perspective, the risks will have to be continuously addressed by sound regulations that are derived from facts and do not end at national borders. From the researcher's perspective, enough space should be provided for new developments to be explored, and from the patient's point of view, we have to make sure that hopes for a better future do not detract from the dire need for human organs for today's patients.
References
1. Bach FH, Fishman JA, Daniels N, Proimos J, Anderson B, Carpenter CB, et al. Uncertainty in xenotransplantation: individual benefit versus collective risk. Nat Med 1998; 4:141–4.[Web of Science][Medline]
2. Platt JL. The immunological barriers to xenotransplantation. Crit Rev Immunol 1996; 16:331–58.[Web of Science][Medline]
3.
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.
4. Borie DC, Cramer DV, Shirwan H, Wu GD, Rodriguez O, Chapman FA, et al. Genetic control of the humoral immune response to xenografts. II. Monoclonal antibodies that cause rejection of heart xenografts are encoded by germline immunoglobulin genes. Transplantation 1995; 60:1504–10.[Web of Science][Medline]
5. Bach FH, Robson SC, Winkler H, Ferran C, Stuhlmeier KM, Wrighton CJ, et al. Barriers to xenotransplantation. Nat Med 1995; 1:869–73.[Web of Science][Medline]
6.
Bracy JL, Sachs DH, Iacomini J. Inhibition of xenoreactive natural antibody production by retroviral gene therapy. Science 1998; 281:1845–7.
7. Dalmasso AP, Vercellotti GM, Platt JL, Bach FH. Inhibition of complement-mediated endothelial cell cytotoxicity by decay-accelerating factor. Potential for prevention of xenograft hyperacute rejection. Transplantation 1991; 52:530–3.[Web of Science][Medline]
8. Bhatti FN, Schmoeckel M, Zaidi A, Cozzi E, Chavez G, Goddard M, et al. Three-month survival of HDAFF transgenic pig hearts transplanted into primates. Transplant Proc 1999; 31:958.[Web of Science][Medline]
9. Blakely ML, Van der Werf WJ, Berndt MC, Dalmasso AP, Bach FH, Hancock WW. Activation of intragraft endothelial and mononuclear cells during discordant xenograft rejection. Transplantation 1994; 58:1059–66.[Web of Science][Medline]
10. Candinas D, Belliveau S, Koyamada N, Miyatake T, Hechenleitner P, Mark W, et al. T cell independence of macrophage and natural killer cell infiltration, cytokine production, and endothelial activation during delayed xenograft rejection. Transplantation 1996; 62:1920–7.[Web of Science][Medline]
11. Robson SC, Schulte am Esch J, Bach FH. Factors in xenograft rejection. Ann N Y Acad Sci 1999; 875:261–76.[Web of Science][Medline]
12. Manilay JO, Sykes M. Natural killer cells and their role in graft rejection. Curr Opin Immunol 1998; 10:532–8.[Web of Science][Medline]
13. Reemtsma K. Renal heterotransplantation. Adv Surg 1966; 2:285–93.[Medline]
14. Hammer C, Dommer S, Allmeling A. Cross Species Interaction of xenogeneic interleukins. Transplant Proc 1996; 28:858–9.[Web of Science][Medline]
15. Kopp CW, Siegel JB, Hancock WW, Anrather J, Winkler H, Geczy CL, et al. Effect of porcine endothelial tissue factor pathway inhibitor on human coagulation factors. Transplantation 1997; 63:749–58.[Web of Science][Medline]
16. Fishman JA. Xenosis and xenotransplantation: addressing the infectious risks posed by an emerging technology. Kidney Int Suppl 1997; 58:S41–5.[Medline]
17. Martin U, Kiessig V, Blusch JH, Haverich A, von der Helm K, Herden T, et al. Expression of pig endogenous retrovirus by primary porcine endothelial cells and infection of human cells. Lancet 1998; 352:692–4.[Web of Science][Medline]
18. Patience C, Takeuchi Y, Weiss RA. Infection of human cells by an endogenous retrovirus of pigs. Nat Med 1997; 3:282–6.[Web of Science][Medline]
19. Heneine W, Tibell A, Switzer WM, Sandstrom P, Rosales GV, Mathews A, et al. No evidence of infection with porcine endogenous retrovirus in recipients of porcine islet-cell xenografts. Lancet 1998; 352:695–9.[Web of Science][Medline]
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. Science 1999; 285:1236–41.
21. Butler D. FDA warns on primate xenotransplants. Nature 1999; 398:549.[Medline]
22. Stoye J. No clear answers on safety of pigs as tissue donor source. Lancet 1998; 352:666–7.[Web of Science][Medline]
23. Lysaght MJ, Aebischer P. Encapsulated cells as therapy. Sci Am 1999; 280:76–82.
24. Aebischer P, Kato AC. Treatment of amyotrophic lateral sclerosis using a gene therapy approach. Eur Neurol 1995; 35:65–8.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||