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normalities in the malignant hyperthermiasusceptible pig ryanodine receptor. Am J Physiol 264:C125–C135. Shou W, Aghdasi B, Armstrong DL, et al.: 1998. Cardiac defects and altered ryanodine receptor function in mice lacking FKBP12. Nature 391:489–492. Simon BJ, Hill DA: 1992. Charge movement and SR calcium release in frog skeletal muscle can be related by a Hodgkin-Huxley Model with four gating particles. Biophys J 61:1109–1116. Sobieszek A, Strobl A, Ortner B, Babiychuk EB: 1993. Ca(2⫹)-calmodulin-dependent modification of smooth-muscle myosin light-chain kinase leading to its co-operative activation by calmodulin. Biochem J 295(Pt 2):405–411. Takeshima H, Nishimura S, Matsumoto T, et al.: 1989. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 339:439–445. Tanabe T, Beam K, Adams B, Niidome T, Numa S: 1990. Regions of the skeletal muscle dihydropridine receptor critical for excitation–contraction coupling. Nature 346: 567–569. Timerman AP, Ogunbumni E, Freund E, Wiederrecht G, Marks AR, Fleischer S: 1993. The calcium release channel of sarcoplasmic reticulum is modulated by FK506-binding protein. J Biol Chem 268: 22,992–22,999. Timerman AP, Onoue H, Xin H-B, et al.: 1996. Selective binding of FKBP12.6 by the cardiac ryanodine receptor. J Biol Chem 271:20,385–20,391. Tong J, Oyamada H, Demaurex N, Grinstein S, McCarthy TV, MacLennan DH: 1997. Caffeine and halothane sensitivity of intracellular Ca2⫹ release is altered by 15 calcium release channel (ryanodine receptors) mutations associated with malignant hyperthermia and/or central core disease. J Biol Chem 272:26,332–26,339. Tripathy A, Xu L, Mann G, Meissner G: 1995. Calmodulin activation and inhibition of skeletal muscle Ca2⫹ release channel (ryanodine receptor). Biophys J 69:106–119. Van de Van PF, Jap PH, terLaak HJ, et al.: 1995. Immunophenotyping of congenital myopathies: disorganization of sarcomeric, cytoskeletal and extracellular matrix proteins. J Neurol Sci 129:199–213. Wagenknecht T, Grassucci R, Frank J, Saito A, Inui M, Fleischer S: 1989. 3-dimensional architecture of the calcium release channel/foot structure of sarcoplasmic reticulum. Nature 338:167–170. Wagenknecht T, Radermacher M, Grassucci R, Berkowitz J, Xin H-B, Fleischer S: 1997. Locations of calmodulin and FK506-binding protein on the three-dimensional archi-

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tecture of the skeletal muscle ryanodine receptor. J Biol Chem 272:32,463–32,471. Wu Y, Aghdasi B, Dou SJ, Zhang JZ, Liu SQ, Hamilton SL: 1997. Functional interactions between cytoplasmic domains of the skeletal muscle Ca2⫹ release channel. J Biol Chem 272:25,051–25,061. Wu Y, Zhang J-Z, Mandel F, et al.: 1998. Calmodulin binding to RYR1 is altered by modification of RYR1 sulfhydryls. Biophys J 74:A61. Yamazawa T, Takeshima H, Shimata M, Iino M: 1997. A region of the ryanodine receptor critical for excitation–contraction coupling in skeletal muscle. J Biol Chem 272:8161–8164. Yang H-C, Reedy MM, Burke CL, Strasburg GM: 1994. Calmodulin interaction with the skeletal muscle sarcoplasmic reticulum calcium channel protein. Biochemistry 33: 518–525.

Yano M, E-Hayek R, Antoniu B, Ikemoto N: 1994. Neomycin: a novel potent blocker of communication between t-tubule and sarcoplasmic reticulum. FEBS Lett 351:349–352. Zhang Y, Chen HS, Khanna VK, et al.: 1993. A mutation in the human ryanodine receptor gene associated with central core disease. Nat Genet 5:46–50. Zorzato F, Fujii J, Otsu K, et al.: 1990. Molecular cloning of cDNA encoding human and rabbit forms of the Ca2⫹ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. J Biol Chem 265: 2244–2256. Zorzato F, Menegazzi P, Treves S, Ronjat, M: 1996. Role of malignant hyperthermia domain in the regulation of Ca2⫹ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. J Biol Chem 271:22,759–22,763. PII S1050-1738(98)00023-1

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Xenotransplantation: An Update Anthony J. F. d’Apice,* David J. Goodman, and Martin J. Pearse

Over the past 20 years, the mortality and morbidity associated with cardiac allotransplantation has fallen significantly, providing a viable treatment for patients with terminal cardiac failure. Unfortunately, the increase in the number of patients who could benefit from cardiac transplantation has not been matched with an increase in the number of organs available for transplantation. Thus, many patients with cardiac failure die waiting for a suitable organ, unlike patients with renal failure, who can be maintained on dialysis while waiting for a kidney. Although the development of artificial hearts may provide a lifesustaining bridging therapy until a donor organ becomes available, the quality of life associated with cardiac prostheses is currently less than that following successful cardiac allotransplantation. (Trends Cardiovasc Med 1998;8:319–325) © 1998, Elsevier Science Inc.

Anthony J. F. d’Apice is at the Immunology Research Centre, St. Vincent’s Hospital, Fitzroy, Victoria, Australia. * Address correspondence to: Anthony J. F. d’Apice, Immunology Research Centre, St. Vincent’s Hospital, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia. © 1998, Elsevier Science Inc. All rights reserved. 1050-1738/98/$-see front matter

The increasing shortfall of donor organs compared with the increasing number of patients requiring transplantation has led to a reevaluation of animals as potential organ donors. The transplantation of primate kidneys to humans performed by Reemstma et al. (1964) and Starzl et al. (1964) in the 1960s confirmed that

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animal organs could sustain human life and demonstrated organ survival of up to 9 months (Reemstma 1989). Most organs were rejected or patients died from infectious complications resulting from high doses of nonspecific immunosuppressive agents. The introduction of cyclosporin A resulted in a prolongation in renal allograft survival and renewed interest in cardiac allotransplantation. With the hope that cyclosporin would overcome the rejection of xenogeneic organs, “baby Fae” received a baboon heart but died 20 days following surgery (Bailey et al. 1985). More recently, two patients with hepatic failure as a consequence of hepatitis B infection received baboon livers. The rationale for transplanting a primate liver was that human hepatitis B will readily infect human allografts but will not infect baboon cells. Again, both patients died (Starzl et al. 1993). The choice of the donor animal is influenced by physiological, immunological, microbiological, and ethical factors. Pigs and primates have emerged as the preferred species for xenotransplantation, with the former being the clear favorite (Table 1). Among the primate species, only baboons can be adapted to captivity and bred sufficiently readily to represent a practical possibility. Nevertheless, there is continuing interest in their future use as cardiac donors for pediatric patients (Bailey et al. 1985) and as liver donors (Starzl et al. 1993). In contrast, pigs breed readily in captivity, and

porcine reproductive biology and methods to genetically manipulate the expression of genes contributing to the pathogenesis of xenograft rejection is well understood. The recent demonstration of cross-species infection of human cells by porcine endogenous retrovirus (Patience et al. 1997) suggests that retroviral transmission is not limited to primate–human transplants and has delayed human xenotransplantation until the risks have been clearly defined. • The Difference Between Xenografts and Allografts The difference between xenografts and allografts resides in the physiological and immunological differences between organs. Size matching is critical for heart or lung transplants but is of lesser importance for renal transplants. The long-term consequences of transplanting organs from four-legged animals to upright animals has yet to be defined, but porcine heart valves are structurally different than human or kangaroo heart valves owing to posturally related hemodynamic requirements, and they may fail as a result of physiological rather than immunological incompatibility. The molecular interactions that form the basis of physiological and immunological function will determine the ultimate feasibility of xenotransplantation. Thus, as the complexity and multiplicity of those interactions increases, the feasibility of overcoming the molecular incompatibili-

ties decreases. For example, porcine insulin is fully functional in humans, but multiple liver–derived factors such as porcine complement, coagulation factors, and angiotensinogen are not. For these reasons, it is unlikely that pig-to-human liver grafts will be successful even if the immunological barriers can be overcome. In contrast, factors generated by the heart such as atrial naturetic factor and those that regulate cardiac function including adrenaline and noradrenaline are generally well conserved, even between phylogenetically distant species. Thus cardiac xenotransplantation will probably be among the earliest clinically attempted xenografts of vascularized organs and is the main preclinical model. The immunological consequences of xenotransplantation enable the categorization of donor recipient combinations into two broad groups, concordant or discordant combinations (Calne 1970). Discordant species combinations such as pig-to-primate transplants are rapidly rejected owing to the presence of preformed xenoreactive natural antibody (XNA) in the recipient’s serum. In contrast, concordant combinations such as primate-to-human transplants lack XNA and are rejected by cell-mediated rejection similar to allograft rejection. • Pathogenesis of Xenograft Rejection The pathogenic steps in rejection of a concordant vascularized organ xenograft

Table 1. Comparison of pigs and primates for some attributes relevant to favored donor status Attribute

Pigs

Primates

Litter size Gestation time Time to maturity/appropriate size Adaptation to captivity and ease of management Community acceptability Organ size Organ physiology

Large 120 days About 6 months

Single 184 days (baboons) Approximately 5 years

High Variable but generally good All sizes Variable between organs but very good for hearts Well understood and technology developed Low

Low Generally unacceptable Small sizes only Very good Physiology understood but manipulation not required High

Possible

Nearly impossible

Discordant ⫽ Hyperacute rejection

Concordant ⫽ no hyperacute rejection

Reproductive physiology and genetic manipulation Risk of transmitting infections to humans Ability to maintain in a specific pathogen free environment Immunological compatibility with human recipient

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are similar or identical to their counterparts in allograft rejection, but additional mechanisms as a consequence of qualitative and quantitative differences in the molecular incompatibilities contribute to the rate and tempo of the rejection response. The pig is currently the favored organ donor, and the greatest advances in xenobiology have been in our understanding of the mechanisms of discordant xenograft rejection. For this reason, the following discussion focuses on the pig-to-primate combination. Hyperacute Rejection Transplantation of discordant vascularized organs results in rejection within 15 minutes to 1 hour. Hyperacute rejection (HAR) is the result of the binding of preformed XNA to the donor endothelium, complement activation, and endothelial cell dysfunction leading to intravascular thrombosis and ischemic necrosis (Figure 1). Xenoantibody. In the pig-to-human combination, the major porcine antigen targeted by human XNA is a single carbohydrate galactose-␣1,3-galactose-␤1,4-Nacetylglucosamine-R antigen frequently abbreviated to ␣Gal (Good et al. 1992, Galili et al. 1988b, Sandrin et al. 1993, Cooper et al. 1993, Oriol et al. 1994). ␣Gal has significant homology to human blood group B (Figure 2) and is formed by the addition of galactose to terminal

Figure 1. A simplified pathogenesis of hyperacute rejection of discordant vascularized organ xenografts.

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Figure 2. Formation of the ␣Gal antigen and its structural relationship to the human ABO blood group antigens.

N-acetyl lactosamine residues on glycolipids and glycoproteins by the enzyme ␣1,3 galactosyltransferase. This glycosyltransferase is present in all mammals up to New World monkeys but is absent from Old World monkeys and humans owing to inactivation of the gene (Galili and Swanson 1991). In a similar way to the development of the isoagglutinins in humans, those species that lack the antigen develop antibodies against it after birth owing to exposure to cross-reacting antigens on normal enteric flora (Galili et al. 1987, 1988a). Humans have immunoglobulin (Ig)M, IgG, and IgA anti-␣Gal antibodies (Koren et al. 1992). The IgM anti-␣Gal is responsible for HAR and binds to the graft endothelium and activate complement (Platt et al. 1991, Sandrin et al. 1996). Complement. In the pig-to-human (primate) combination, the complement cascade is activated predominantly via the classic pathway [see d’Apice and Pearse (1996), Platt (1996) for reviews] and, in some models, via the alternate pathway (Romanella et al. 1997). This cascade is in a state of permanent lowlevel activity, “ticking over” but restrained by a series of soluble and cell

surface complement regulatory factors (CRFs) (Figure 3). As far as xenotransplantation of vascularized organs is concerned, the CRFs of relevance are those present on the xenograft endothelium, as this bears the brunt of the humoral response. They are CD55 [decay accelerating factor (DAF)], CD46 [membrane cofactor protein (MCP)], and CD59, which has many synonyms (Lachmann 1991). DAF and MCP act to accelerate the decay of the C3 and C5 convertases. In addition, MCP also serves as a cofactor for factor I–mediated cleavage of the C3 convertase. CD59 acts to prevent assembly of the membrane attack complex at the level of C8 and C9 [for a review, see Mathieson et al. (1996)]. DAF and CD59 are species-specific “homologously restricted,” explaining why porcine cells that express CD55 and CD59 cannot inhibit human complement. MCP, on the other hand, functions relatively efficiently across species. It is probable but not formally proved that the inability to limit the extent of complement activation induced by anti-␣Gal antibodies contributes to the pathogenesis of HAR because expression of human CRF molecules on the endothelium of pig organs prevents HAR (see later discussion).

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Figure 3. The complement cascade and its regulators on the endothelial cell surface.

Damage to the Graft Endothelium and Intravascular Coagulation. Activation of complement on the endothelial cell (EC) surface results in damage to the endothelium. The consequences of this injury are a loss of EC function, with retraction of the cells resulting in gaps with exposure of the basement membrane, leakage of blood components causing graft edema, and interstitial hemorrhage. An important feature of the normal intact endothelium is the maintenance of an anticoagulant surface, which is achieved by several mechanisms including heparan sulfate, a heavily N-sulfated proteoglycan akin to heparin with similar anticoagulant activity; thrombomodulin, which exerts an anticoagulant effect through the protein C/protein S system; tissue factor pathway inhibitor (TFPI); ecto-ADPase (CD39); and anti-thrombin III. Damage to the endothelium results in loss of heparan sulfate (Platt et al. 1990), as well as exposure of the basement membrane and matrix proteins, including collagen, gpIb, and von Willebrand factor, causing platelet adhesion and activation with secretion of platelet activating factor (PAF) and other inflammatory mediators, with further platelet recruitment and activation. The damaged endothelium also secretes PAF and translocates P-selectin from Weibel-Palade bodies to the cell surface. The latter, together with deposited complement components (iC3b and C5b7) and the anaphylotoxins C3a and C5a, adhere to and

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activate polymorphonuclear leukocytes, another feature of HAR. This series of events leads to the well-known appearances of HAR and, in particular, to intravascular coagulation with resulting ischemic necrosis of the graft [see Bach et al. (1995) for a review]. Delayed Xenograft Rejection Inhibition of complement activation by cobra venom factor has inhibited HAR and prolonged xenograft survival from minutes to several days. These organs are rejected by a novel form of rejection that has no counterpart in rejecting allografts. This delayed form of rejection has two major components, EC activation and a mononuclear cell infiltrate comprising predominantly monocytes/ macrophages and natural killer (NK) cells (Hancock et al. 1993). This has been termed delayed xenograft rejection (DXR) (Bach et al. 1994) and acute vascular rejection (AVR) (Leventhal et al. 1993), emphasizing different aspects of the phenomenon. The first and probably most intractable is the vascular component with features suggesting an attenuated form of HAR. It is not clear to what extent this component of DXR is simply due to incomplete control of the pathogenic mechanisms of HAR leading to a slower tempo, enabling more active EC responses. The vascular component is recognized in allograft recipients who appear to be presensitized to their do-

nors, albeit with negative direct crossmatch. Like HAR, it is characterized by varying degrees of EC damage and activation, often with graft edema and interstitial hemorrhage. EC activation may be classified into type I responses that do not require new protein synthesis such as the release of heparan sulfate from the cell surface of the translocation of P-selectin to the cell membrane (Bach et al. 1994) and type II responses that are characterized by new protein synthesis. Many inflammatory mediators including the cytokines interleukin (IL)-8, MCP-1, IL-1, and IL6 (Hancock et al. 1993) and leukocyte adhesion molecules, ICAM-1, VCAM-1, E-selectin, and P-selectin (Hancock et al. 1993) are generated by type II activation. These proteins promote local accumulation, adhesion and tissue infiltration of NK cells, and monocyte/ macrophages. The deposition of IgG anti-␣Gal antibodies can also lead to endothelial damage by ADCC mediated by NK cells (Inverardi et al. 1992). Once again, these changes, together with EC retraction, platelet adhesion, and expression of tissue factor on infiltrating monocyte/macrophages, lead to intravascular coagulation. Many of the factors associated with type II EC activation are linked by a final common pathway involving the activation of a family of transcription factors called the NF-␬B. NF-␬B was first described as an essential transcription factor; DXR presents the latest of what is probably a succession of so far unsurmounted hurdles. The fact that the NF␬B system represents a single final intracellular signaling pathway for most of the events that characterize type II EC activation offers another target for genetic manipulation, which, if successful, may prevent DXR (Ferran et al. 1995, Goodman et al. 1996). This would represent an important advance, as the alternative, immunosuppression of the innate immune response, would almost certainly be lethal. T Cell–Mediated Rejection The initial assumption that recognition of class I and II antigens across species would not occur owing to molecular incompatibilities has been rejected by recent data that confirm that human cells can recognize SLA class I and II and TCM Vol. 8, No. 7, 1998

also present antigen to T cells. Mixed lymphocyte reactions have demonstrated higher reactivity when human cells are incubated with porcine cells. Furthermore, the activity can be enhanced by the addition of xenogeneic endothelial cells. The major difference between the T-cell responses in allograft and discordant xenograft rejection is once again a function of the type and number of molecular incompatibilities encountered by the host’s immune system. Allografts present a relative small number of differences [the polymorphic portions of the major histocompatability complex (MHC) molecules and their associated antigen peptides] on an otherwise relatively invariant background. There is predominantly direct recognition of these differences by host T cells, and a predominantly Th1 type response ensues. In contrast, phylogenetically distant xenografts present the recipient with a vast array of molecular differences. Of these, only a relatively small number, the porcine SLA antigens, have the potential of being directly recognized by the host’s T-cell antigen receptors and, provided they are presented by a porcine antigen presenting cell (APC) that is able to provide effective costimulation, of stimulating direct immune activation. Although there are potential molecular incompatibilities that may reduce “direct” activation, such as between human T-cell reactivity (TCR) and porcine MHC molecules, human CD4/8 and porcine MHC class I/II, adhesion receptors and ligands, costimulatory molecules and receptors, and cytokines and receptors [see Moses and Auchincloss (1997) for review], it is evident that it does occur, although to a lesser extent than in allorecognition (Dorling et al. 1996a). This is, however, more than compensated for by the remaining very large number of differences (xenoantigens) that are processed and presented effectively by host antigen presenting cells to host CD4⫹ T cells (“indirect” recognition) (Dorling et al. 1996b). Overall, the effect is to generate very powerful T cell responses to porcine xenografts. The resulting immune response is dominated by indirect antigen presentation and a Th2-type response that favors humoral responses (Moses and Auchincloss 1997). The current immunosuppressive agents were developed to deal effectively with direct antigen recognition and the reTCM Vol. 8, No. 7, 1998

sulting Th1 type response. Thus experience gained in the treatment of autoimmune diseases characterized by indirect antigen recognition and preliminary pig-to-primate xenotransplantation data suggests that current immunosuppressive agents may be less effective in preventing xenograft rejection. • Safety The issue of safety of xenotransplants had received relatively little attention until the demonstration that porcine retrovirus could infect human cells in vitro. Porcine retrovirus is endemic in pigs, and there are variable numbers of copies of the virus in the genome of pigs. The finding caused serious concern that such viruses could be transmitted from the porcine donor to the human recipient and then be transmitted laterally among humans. This precipitated the establishment of various inquiries, the results of which have varied from a temporary ban on clinical studies pending further research in the United Kingdom (The UK Government 1997) to agreement that researchers may proceed with caution in the United States (Nasto 1997). • Therapeutic Approaches Genetic Modification The fundamental conceptual difference between the attempts at xenotransplantation in previous eras and that of today is that the pig, a discordant species, is now a potential donor as a result of recent advances in molecular biology and in the understanding of porcine reproductive physiology, which have allowed its genetic manipulation. Thus, HAR of pig-to-primate heart grafts has been prevented by transgenic expression in the donor animal of human DAF, MCP, and CD59 in various combinations (Lawson et al. 1997, Kroshus et al. 1997, Bhatti et al. 1997). This represents an enormous advance because it limits the therapeutic attack that must be directed at the human recipient. It is probable that these are only the first of many genetic modifications that will be made to pigs to correct the crucial molecular incompatibilities, which will vary depending on the organ. Current technology probably limits the introduction of transgenes to about six at present, and even this

number assumes simultaneous introduction of three genes by coinjection. This usually results in their insertion into a single site and cotransmission to offspring. Candidate genes (in addition to the CRFs) include native or suitably modified human genes for H-transferase and ␣-galactosidase to suppress production of the ␣Gal antigen, MHC Class I including HLA-G to inhibit NK cell activation, and thrombomodulin, TFPI-I and hirudin to restore the anticoagulant environment, and inhibitors of NF-␬B to prevent the many manifestations of EC activation. Targeted gene inactivation by homologous recombination (gene knock-out) is the obverse of positive transgenesis. We have used knock-out technology to inactivate the ␣1,3 galactosyltransferase gene and eliminate the ␣-Gal xenoantigen in the mouse (Tearle et al. 1996). At present, this technology is not available in the pig and is limited to a few mouse strains. If knock-out technology becomes feasible in the pig, there are two immediate candidates, the ␣1,3 galactosyltransferase gene and the endogenous porcine retroviral sequences. The recent reports of cloning of a sheep, “Dolly,” by transfer of a nucleus of a differentiated adult cell (Wilmut et al. 1997), is of direct relevance because if this could be replicated in pigs, it would provide an alternative technology to generate Galdeficient pigs without the need for embryonic stem cells. Recipient Modification Treatment of the recipient imposes a substantial morbidity and mortality burden, even in cardiac allotransplantation. Many cardiac transplant recipients surviving more than 10 years have hypertension and renal impairment and a significant number have progressed to endstage renal failure requiring dialysis support. The spectrum and intensity of therapies that may be required to prevent rejection of a discordant xenograft, if the only manipulations were those directed at the recipient, may be lethal. Thus it appears that many treatments that are known to be effective against various pathogenetic steps in experimental models, for example, plasmapheresis and immunoabsorption and various anticomplementary agents, may never see clinical application.

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At the recent Congress for Xenotransplantation, held September 7 through 11, 1997, in Nantes, France, three groups (Lawson et al. 1997, Kroshus et al. 1997, Bhatti et al. 1997) reported their experience with preclinical models (pig-tobaboon heart and kidney grafts) in which HAR was effectively prevented (as already discussed). There were three important observations. First, despite massive and frequently lethal immunosuppression, which included cyclosporin, corticosteroids, and cyclophosphamide 10 mg/kg, the grafts were ultimately lost. Second, the cause of the graft loss was unclear, as there appeared to be little cellular infiltrate. Finally, although heterotopic grafts survived for many weeks, orthotopic grafts were able to support life only for about a week. Steady progress is being made toward clinical application in allografts and xenografts of techniques to induce immunological tolerance by mixed hemopoetic chimerism (Kawai et al. 1995). In xenografts, molecular incompatibility of host hemopoetic growth factors and the corresponding donor receptors is a limiting factor in achieving sustained mixed chimerism.

• Prospects for Clinical Application The wave of interest and enthusiasm for xenotransplantation of the 1990s is fueled by an increasing demand for organs that could not be satisfied by allografts, together with new technological capabilities that encouraged renewed hubris. This was based on the incorrect assumption that once the problem of HAR was overcome, the new and more powerful immunosuppressive agents would readily cope with the ensuing cellular rejection. It was also implicit that we could draw readily on experience with allografts and our knowledge would be directly transferable to xenografts. Again, this was incorrect. HAR has been overcome, but a new and still incompletely understood entity, DXR, which currently is not preventable and is untreatable, blocks progress. Just as DXR was unexpected, so too may be the next barrier. The results of preclinical studies presented at the Congress for Xenotransplantation gave no encouragement to those looking for endorsement to progress to clinical trials.

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