The emergence of xenotransplantation

Transplant Immunology 1995; 3: 21-31

The emergence of xenotransplantation Jonathan P Fryer, Joseph R Leventhal and Arthur J Matas Department of Surgery, University of Minnesota, Minneapolis Revised manuscript accepted 18 May 1994.

Abstmctz The field of transplantation is faced with a growing shortage of human organs as the list of

potential recipients continues to increase. Those currently listed can already expect long waits; some die waiting. Xenotransplantation is a potential solution to this widening donor-recipient disparity. Consequently, in recent years, there have been several clinical attempts using organs from nonhuman primates and pigs. The results with nonhuman primates as donors have been encouraging, but it is unlikely that these species will provide a long-term solution to the organ shortage. Most recent xenotransplantation research has therefore shifted to more phylogenetically disparate species, such as pigs, as potential donors. The major barrier to transplantation between members of disparate species combinations has been hyperacute rejection (HAR). The elements of humoral immunity involved in this rejection process include (1) naturally occurring antibodies directed against carbohydrate and other antigens expressed on pig endothelium, and (2) the complement system, which is activated by binding of natural antibodies to their targets. Several elegant strategies to prevent HAR are being developed. The creation of transgenic pigs, whose cells express human regulators of complement activation, is one such strategy. Another promising approach has been to remove antidonor antibodies from the recipient by absorption with some recently characterized carbohydrate epitopes of porcine endothelial xenoantigens. Recent experimental work indicates that HAR can successfully be prevented by inhibition or depletion of complement. A delayed type of xenograft rejection, characterized by endothelial cell antibody deposition and cellular infiltration, occurs over the next three to four days. The likely mechanisms involved in delayed xenograft rejection include antibody-dependent cell-mediated cytotoxicity and the phenomenon of endothelial cell activation.

Introduction As the list of patients needing transplants grows, while the pool of available organs stagnates (Figure l), the search for new sources of donor organs becomes increasingly relevant. Efforts to enhance the yield from potential human donors have included campaigns to increase public and physician awareness of organ donation, greater emphasis on living donors, and relaxation of acceptance criteria for cadaver donors.‘-4 Despite these efforts, an allogeneic solution seems unlikely. As a result, xenotransplantation is undergoing serious consideration. Recently, an international group of xenotransplant researchers representing numerous specialized areas in medicine and the basic sciences met in Cambridge, UK, for the Second International Congress on Xenotransplantation. They discussed the most significant areas of research in this rapidly emerging discipline. This review highlights most of these areas. In xenotransplantation, species combinations have typically Address for correspondence: Jonathan P Fryer, Box 119 UMHC, 420 Delaware Street SE, Minneapolis, MN 55455, USA. 0 Edward Arnold 1995

been designated as either concordant or discordant, depending on whether or not hyperacute rejection (HAR) results.5 But over the past two decades, study of the pathobiology of xenograft rejection has demonstrated this classification system to be greatly oversimplified; it is not necessarily based on the pathogenesis of the rejection process observed in different species combinations.6 This review avoids broad categorization, and instead discusses xenografting in terms of the phylogenetic disparity of specific species pairs and the underlying mechanisms of xenograft loss. In addition, the process which lead to graft loss even if HAR is prevented (i.e. beyond HAR) are discussed.

Transplantation between closely related primate species Clinical application of xenotransplantation has been limited almost exclusively to the use of nonhuman primates as organ donors. Indeed, encouraging results have been obtained with the transplantation of primate kidneys,7 heartss and livers’ into humans.

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17939

18006 l6ooo __

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Persons on waiting list

- I- -

Number transplanted

12000 -Numbar in

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01. 1980

1982

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1986

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Year Figure 1 Demand for kidney transplantation. receiving one each year in the USA.

The growing disparity between the number of patients waiting for a kidney transplant and those

The first attempt at primate-to-human xenotransplantation was by Unger, who, in 1910, performed a chimpanzee-tohuman renal transplant. The graft clotted and the recipient died. Over 50 years later, primate-to-human renal xenotransplantation was again attempted, with markedly improved results. In 1964, Reemtsma and colleagues performed a series of chimpanzee-to-human renal transplants, achieving nine-month graft survival in one patient. Baboon-to-human renal transplants were attempted by Starzl, who noted poorer graft survival-despite an immunosuppressive protocol similar to the one used by Reemtsma. Although chimpanzee xenografts fared better than baboon xenografts, the rejection process was characterized in both by the infiltration of recipient cellular elements. Rejection of primate renal xenografts appeared to resemble an aggressive form of clinical allograft rejection. Clinical attempts at renal xenotransplantation were soon abandoned in favour of the use of cadaver and living related human donor organs. A limited number of unsuccessful nonhuman primate-to-human heart and liver transplants were also performed during the 1960s and 197Os’OJi In the 198Os, the introduction of cyclosporine (CsA) helped to revolutionize clinical solid organ transplantation. With this powerful new immunosuppressive agent, many felt the close species barrier between humans and apes could be overcome. In 1984, Bailey er al. performed a baboon-to-human h&t transplant into a newborn infant (Baby Fae) using an ABO incompatible donor.’ Imrnunosuppression of intravenous CsA begun 38 hours pretransplant, while methylprednisolone and antithymocyte globulin (ATG) were instituted when evidence of graft rejection was first detected. Baby Fae’s clinical course was characterized by progressive cardiac failure, beginning three days post-transplant and resulting in death 20 days post-transplant. Histological evaluation of the xenograft failed to demonstrate a prominent cellular rejection. Instead, graft loss appeared to be related to humoral rejection. Immunofluorescence studies revealed finely granular deposits of IgG, IgM, IgA, and complement (C3). It is important to note that Baby Fae was blood type G-rare in baboons-and that an ABO-compatible donor was not available. This donor-recipient blood group incompatibility complicates analysis of the cause of xenograft loss; it is unclear Transplant Immunology

1995; 3: 21-31

whether the depositing antibodies were directed against novel xenoantigens, or baboon blood group antigens expressed in the xenograft. Her case did indicate that CsA alone may not be sufficient to bridge interspecies barriers, and that future strategies would probably require intervention against antibody. The 1980s saw the advent of intensive scientific investigation in xenotransplantation using small and large animal models. Hamster organs placed into untreated rats are rejected over several days; this rodent model has been used as a small animal correlate of transplantation between closely related primate species. Using the hamster-to-rat species combination, several investigators reported extension of heart and liver xenograft survival with a variety of immunosuppressive regimens.6 In general, these studies indicate that protocols combining both antihumoral and anti-T cell immunosuppression are most effective. Importantly, extended hamster xenograft survival appears to require prolonged suppression of rat xenoreactive antibody synthesis (a phenomenon likely to require agents with B cell and T cell immunosuppressive action) or prevention of the deleterious consequences of antidonor antibody binding to the graft (i.e. complement activation), or both. Results from these rodent studies have been applied to nonhuman primate models with some success. Using a cynomolgus monkey-to-olive baboon combination, Michler et al.” prolonged cardiac xenograft survival with CsA and steroids from a mean of seven days in untreated controls to 77 days. The combination of CsA, steroids, azathioprine and ATG extended mean xenograft survival to 81 days. Although hyperacute rejection was not seen, an increase in cytotoxic antibodies correlated chronologically with graft loss. Cooper and co-workers,‘3,‘4 using vervet monkeys as donors and chacma baboons as recipients, found that cardiac xenograft survival was not prolonged with conventional immunosuppression; the incidence of humoral rejection was high. However, they extended graft survival with the addition of 15deoxyspergualin and rabbit ATG or with preoperative total lymphoid irradiation (TLI). Norin et all5 transplanted cynomologous monkey hearts into baboons and obtained graft survival of 2.5 to 18 months with TLI, CsA, and steroids. In this study, only baboons surviving more than 60 days had

The emergence ofxenotransplantation

increased serum levels of cytotoxic IgG, as well as IgG deposition in the xenograft. IgM xenograft deposition occurred early in the nonirradiated groups, but was eliminated by irradiation. *These fmdings suggest that IgM was the most significant constituent of preformed xenoreactive antibody, since marked reduction in serum levels after irradiation was associated with prolonged graft survival. On the other hand, the increase in xenoreactive IgG likely reflects an induced antibody response, since it is delayed with or without irradiation. These studies indicate that different primate species combinations vary in the aggressiveness of the immune response generated against the donor and that the intensity of the humoral response is a significant determinant of graft survival. Successful extension of liver xenograft survival in the hamster-to-rat combination with FK506 and cyclophosphamide”’ has served as the basis for two recent clinical attempts of baboon-to-human liver transplantation by Starzl et aL9 Patients received an aggressive immunosuppressive regimen, including FK506, cyclophosphamide, steroids, and perioperative prostaglandin E. Although extended graft survival was achieved in both cases, the patients died of infectious and neurological complications. Both liver xenografts displayed evidence of antibody-mediated injury post-transplant and vascular and sinusoidal deposition of antibody was also seen on later biopsies. At autopsy the baboon livers showed extensive steatosis and biliary tract changes, which resembled the pathological lesions observed in ABO-incompatible liver transplants. Both livers failed to show evidence of severe cellular rejection. Thus, antibody-dependent mechanisms of graft rejection appear to play a significant role in the baboonto-human species combination. Despite the promising results with nonhuman primate-tohuman xenotransplantation, species such as the chimpanzee and baboon are unliiely to be widely used as donors. Fit, chimpanzees and other anthropoid apes, the species which are most closely related to humans, are endangered. The few that are made available for biomedical research are used in AIDS or hepatitis research. Old world monkeys (baboons, cynomologous monkeys or macaques, and vervet monkeys) still exist in the wild in fairly large numbers so are now most wmmon in xenotransplant research. However, a growing segment of society is outwardly opposed to the use of primates in medical research. It is unclear whether an increase in the use of old world monkeys-an increase sufficient to solve the current organ shortage-would be acceptable to society. Secondly, there is a theoretical risk that nonhuman primate viruses will be transmitted when their organs are transplanted into humans. Some of these viruses, though harmless to nonhuman primates, can be deadly to humans.‘7*‘8 The risk involved is unclear, but the development of virus-free primate colonies necessary to address these wncems would be an arduous task. Thirdly, the limited size range of primate species such as the baboon may pose problems for appropriate donorrecipient size matching for certain organs, such as the heart and lungs. In light of the many real and potential obstacles to use of nonhuman primate organs, focus has shifted to the use of more phylogenetically disparate species as donors.

Transplant Immunology 1995; 3: 21-31

TrzAantation

23

between pigs and

There may always be objections to the transplantation of animal organs into humans. It may be more acceptable to obtain organs from a species that is already being bred and killed for human use. Pigs are easy and inexpensive to breed and have many physiological similarities to humans. Furthermore, they attain adult weights comparable to humans, so can provide large enough organs for any age of recipient. Unfortunately, immediately vascularized porcine organs are rapidly rejected by human and nonhuman primate recipients in a matter of minutes to hours. This process of HAR is mediated by naturally occurring, wmplement-lixing xenoreactive antibodies that bind to the pig vascular endothelium. HAR has become a major focus of xenotransplantation research, as investigators strive to overcome this formidable immunological barrier. Clinical experience with organ transplantation between pigs and humans has been very limited. In 1968, in separate instances occurring on the same day, pig hearts were incorporated into the circulation of two patients who could not be weaned from cardiopulmonary bypass support. In both cases, the grafts rejected within minutes.” There have been numerous attempts to support patients with fuhninant hepatic failure using ex vivo perfusion of liver xenografts. 20-U In most of these cases, pig livers were used. Routinely, antibodies to pig semm proteins developed and titres of antipig antibodies increased. These livers were functional, and demonstrated the ability to clear lactate and ammonia and to synthesize donor proteins for many hours. Porcine livers appear to be less susceptible to antibodymediated graft damage than other porcine organs perfused by human blood, suggesting an inherent resistance to antibodymediated graft damage. This is similar to observations made in clinical ABO-incompatible liver transplants. Recently, Makowka et aL” transplanted a pig liver into a young woman with fulminant hepatic failure, with the intent of using it as a ‘bridge’ until a human liver became available. Pretransplant, the recipient’s blood was perfused ex vivo through the pig’s kidneys to absorb antipig antibodies. Posttransplant, antipig antibodies rapidly rebounded, perhaps induced by the administration of unabsorbed blood products to the patient. Immunopathological evidence of graft rejection occurred in about three hours, and the patient died before successful bridging to an allograft could be achieved. Pathological analysis of the xenograft revealed diffuse vascular thrombosis, haemorrhagic necrosis, and extensive infiltration by neutrophils. Vascular deposits of IgG, IgM, and complement components were found. These results corroborate laboratory studies demonstrating the importance of antibody, complement, and elements of acute inflammatory reaction during hyperacute xenograft rejection.

The problem of hyperacute rejection Before porcine xenografting can become a reality, the first hurdle to overwme is HAR (Figure 2). Evidence now strongly suggests that this immunological barrier to xenotransplantation between disparate species combinations derives from one or more of three factors: (1) the specific

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JP Fryer et al

QUIESCENT EC

ACTIVATED

n

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LETHAL CELL INJURY LOSS OF BARRIER FUNCTION

UPREGULATION OF ADHESION MOLECULES: LEUCOCYTE ADHESION

NATURAL v ANTIBODY AND COMPLEMENT

PROCOAGULANT EFFECTS: FIBRIN DEPOSITION; PLATELET THROMBI

Figure 2 A model of discordant xenograft rejection. Xenogeneic natural antibody and complement xenoantigens from the recipient are the most important factors in discordant xenograft rejection.

interaction of recipient xenoreactive antibodies with antigens present on the endothelium of the donor organ, followed by activation of the complement cascade, (2) the direct activation of the recipient complement system by donor endothelial cells, (3) the relative failure of complement inhibitory proteins in the donor organ to impede activation of the recipient complement system. This improved understanding of the pathogenesis of HAR has provided a scientific foundation on which to devise and evaluate therapeutic approaches to extend porcine xenograft survival.

Preformed natural xenoantibody A critical role for preformed natural xenoantibody (PNXAb) in the pathogenesis of HAR follows from several lines of evidence: (1) the rejection process occurs extremely rapidly, indicating a role for circulating factors, rather than a cellmediated response, (2) the immunopathology of rejected xenografts in disparate species combinations reveals antibody deposition on donor endothelium,2” (3) removal of PNXAb is frequently associated with prolonged xenograft surviva1,26.27 and (4) infusion of PNXAb leads to rejection of organs previously engrafted in modified recipients? Recent studies have provided important information on the characteristics and porcine targets of PNXAb in humans and nonhuman primates.2”m34 Platt and Holznecht examined, in great detail, the endothelial cell antigens recognized by PNXAb in the pig-to-primate species combination.35 Evidently, primate PNXAb predominantly recognize three porcine endothelial cell glycoproteins of 115, 125, and 135 kDa; these antigens are also present on porcine platelets. The antigenic determinants of these membrane glycoproteins appeared to be located on N-linked carbohydrate substitutions. Examination of porcine red cells and lymphocytes failed to identify the glycoproteins expressed on endothelial cells and platelets. However, cross-absorption studies with red cells and lymphocytes reduced the levels of antiglycoprotein 115/135 antibodies, suggesting that primate PNXAb recognize the same or cross-reactive carbohydrate determinants expressed on the surface of a variety of cells. Isolated glycoprotein 115/135 has been shown to block about 90% of Transplant Immunology

1995; 3: 21-31

from the recipient and endothelial

cell

antiporcine PNXAb binding to pig endothelial cells, while removal of 115/135 from membrane extracts was shown to eliminate 88% of antibody binding. Amino terminal sequencing of these glycoproteins indicates they are members of the integrin family, with significant homology to human integrin membrane proteins. In addition, Platt and Holznecht have reported that human antipig PNXAb recognize a 250 kDa porcine glycoprotein homologous to human von Willebrand factor. Several investigators have reported that humans and old world primates possess circulating PNXAb with anti-cu-galactose (anti-gal) specificity. These antibodies are xenoreactive and bind to pig endothelial cells.“6.37 The presence of anti-gal specific antibodies in certain primates has been linked to their lack of expression of the enzyme (cY-galactosyltransferase) needed to produce nonreducing terminal a-gaIn vitro studies suggest that lactosyl sugar residues.38 membrane proteins and lipids bearing a-galactosyl structures are widely expressed on porcine cells, and that absorption of antibodies with anti-gal specificity prevents the in vitro lysis of porcine cells. Cooper et al. have described antibodies eluted from porcine organs perfused with human plasma to bind carbohydrates with terminal a-galactose residues.“’ Furthermore, in vivo administration of oligosaccharides capable of binding anti-gal antibodies has been shown to reduce antipig PNXAb titres in baboons and result in extended porcine xenograft survival.

Complement In the pig-to-human combination, HAR occurs within minutes to hours of engraftment. It is initiated when PNXAb binding to antigens on the endothelial cell activate the classical pathway of the complement cascade (Figure 3). Activation of the classical pathway in turn allows for subsequent recruitment of the complement alternative pathway. As a result, several biologically active complement fragments are produced culminating in the formation of the C5b9 complex (membrane attack complexes, MAC). These products of complement activation may play significant roles in vascular injury by inducing an increase in vascular permeabil-

The emergence

Antigen-Antibody Cl

f,

(IgG or IgM) Complex

25

PATHWAY ]

Activated Cl (Classical Pathway C3 conveftase)

c4 +c2-[C4b2a]-

C3b

t C3b 7-T Factor

(Classical Pathway C5 convertase)

[C4b2a3b)

i C3 --)

C3 --)

1CLASSICAL

ojxenotrcmsplantation

(CBbBb]-----+ (Alternative Pathway C3 convertase)

Factor / I3 D Microbial surfaces Polysaccharides

C6 C7 C8 C9

Complex)

[C3bBb3b] (Alternative Pathway C5 convertase)

1ALTERNATIVE

PATHWAY 1

Figure 3 The classical

and alternative pathways of complement activation have both been shown to play a role in xenograft rejection. Manipulation of these pathways has been the most successful strategy for preventing hyperacute rejection.

ity (C3a, CSa)+ontributing to development of a procoagulant state (MAC), promoting cell adhesion (C3bi), or inducing direct endothelial cell damage (MAC). A number of studies have supported the importance ot complement in xenografting between disparate species.4w3 These studies have demonstrated a decrease in recipient serum complement levels after xenoengraftment, in association with accumulation of complement proteins in the xenograft. Furthermore, deliberate depletion of complement by cobra venom factor, inhibition of complement with substances such as soluble complement receptor type 1, or engraftment into congenitally complement-deficient recipients is associated with dramatically prolonged xenograft survival. Studies in primates support a process wherein complement-fixing PNXAb of the IgM isotype are thought to initiate HAR of porcine organs.29 The severity of hyperacute rejection may result in part from the failure of membrane-associated regulators of complement in the donor organ to inhibit the activation of the recipient complement system. These regulators of complement activation (RCA) are known to be inhibitory only for homologous complement or for complement of a closely -probably not for complement of disrelated species-’ tantly related species. Thus, pig RCA would fail to inhibit activation of human complement. Incorporation of recipient RCA into the vascular endothelium of the xenogeneic donor organ could allow for marked inhibition of recipient complement activation, with an associated impact on HAR. In vitro studies where genes for human RCA have been introduced into xenogeneic cells have demonstrated the feasibility and cytoprotective effects of this approach.47,48 Attempts to achieve expression of human RCA in porcine organs through the development of transgenic human RCA pigs may help tackle the problem of HAR.

Transplant Immunology

1995; 3: 21-31

Prevention of hyperacute rejection Three basic strategies to prevent HAR are: (1) reduction or elimination of PNXAb; (2) prevention of complement activation; (3) alteration or elimination of endothelial cell xenoantigens.

Reduction or elimination of preformed natural xenoreactive antibody The problem of PNXAb has been approached through various selective and nonselective techniques. Plasmapheresis, a nonselective technique for antibody removal, has been employed in xenotransplantation with some success. It has proven useful for antibody removal in human ABO-incompatible kidney transplants49 and for renal transplants into with plasmahighly sensitized recipients. 5o Early experience pheresis for xenotransplantation involving nonprimate species during the 1960s and 1970s found that it extended xenotransplants, Alexxenograft survival. 5’ In pig-to-primate andre et al. used plasmapheresis and immunosuppression in splenectomized baboons to extend porcine renal xenograft survival, with one organ surviving 22 days.52 Recently, we have described the ability of plasmapheresis and immunosuppression to extend cardiac xenograft survival from one hour in untreated controls to greater than two days.53 When we combined plasmapheresis and immunosuppression with complement depletion, xenograft survival was extended to 17.5 days. Despite these encouraging results with plasmapheresis, it is hampered by the concomitant reduction of other plasma proteins-such as clotting factors and complement system proteins-that may be essential for the recipient. The effect of plasmapheresis on the coagulation system is likely to limit its application in the peritransplant period, since bleeding complications may result. Replacement of clotting factors with fresh frozen plasma would be hazardous, since it contains high levels of xenoreactive antibodies.“4

26 JPFryer et al

Another, more selective approach to remove xenoreactive antibodies is the use of immunosorbent columns. They avoid the consequences of wholesale plasma protein removal seen with plasmapheresis. Protein A and protein G effectively remove of antibodies of the IgG and IgM isotype, and have been used successfully in a variety of clinical situations:’ We have recently shown that columns containing polyclonal antihuman IgG or IgM antibodies conjugated to Sepharose are extremely effective for removing antipig antibody from human plasma.54 However, two potential disadvantages of column technologies are their nonspecific removal of all antibodies and the potential side effects of column-dependent complement activation. The optimal approach to selective xenoreactive antibody removal may be an extracorporeal source of target xenoantigens. The crudest application of this approach is the perfusion of a donor organ, most commonly the spleen,56 kidney,57 or liver.58 This approach, though effective, has several disadvantages. Organ perfusion results in the sequestration of blood volume and in the activation of the complement and coagulation cascades as the perfused organ is rapidly rejected. In addition, organ perfusion is cumbersome to perform, requiring sacrifice of a donor animal to obtain target organs. A more elegant approach to selective xenoreactive antibody removal would be to use purified or synthesized target antigens. Specific columns using human ABO blood group antigens have proved successful in removing anti-A and antiB antibodies.5g In addition, infusion of purified blood group antigens into transplant recipients has been associated with depression of antiblood group antibody titres and extension of allograft survival. The use of soluble antigen to achieve in viva blockade of circulating antibodies has recently been described by Cooper et al. Taking advantage of the anti-gal specificity of primate antipig PNXAbs, they showed that intravenous infusion of certain a-gal oligosaccharides reduced the cytotoxicity of baboon serum for pig cells and prevented HAR of porcine xenografts for up to 12 hours3’ In addition, in vitro work suggested that a similar result could be anticipated in humans. However, the rapid infusion of carbohydrates required to block antipig antibodies in baboons resulted in severe derangements in the serum osmolarity. A safer and equally effective use of xenoantigens for antibody removal might be an absorptive column for extracorporeal antibody depletion.a An attractive solution to the problem of xenoreactive antibodies is to selectively prevent antibody synthesis. Soares et al. have shown that administration of an anti-IgM monoclonal antibody (MARM-7) to rats may allow for depletion of IgM and depression of serum IgM levels for several weeks.61 This reduction in total IgM was associated with a reduction in xenoreactive antibody titres. The same group also described a successful reduction in total and antipig xenoreactive antibody titres in baboons after anti-IgM administration. However, despite these promising findings, Van der Werf et al. were unable to demonstrate improved graft survival using this approach in a guinea pig-to-rat model even though they successfully prevented endothelial IgM deposition.@ Although this may merely indicate that antibody deposition is not essential for rejection to occur in this particular model,6 it remains to be determined whether the anti-mu approach will result in safe and effective prolongation of porcine xenograft survival.

Transplant Immunology

1995; 3: 21-31

Prevention of complement activation Fluid phase inhibition

Manipulation of the normal pathway of complement activation has proven to be extremely effective for overcoming HAR. The most effective agent to date has been cobra venom factor (CVF).40A’.53”3Cobra venom contains, in addition to several toxins and enzymes, a C3b-like molecule, CVF, that is resistant to human and rat C3b inactivating factors. This C3b analogue combines with components of the alternative pathway of complement to form a highly stable enzyme complex that causes massive consumption of C3, factor B, and members of the MAC. The result is exhaustion of the complement cascade. Removal of phospholipade contaminants in cobra venom@ has greatly reduced its toxicity in rodents. To date, toxicity has not been a significant factor in primates.53.65 CVF has extended xenograft survival in several rodent models, and has prolonged pig-to-baboon xenograft survival. Importantly, the combination of complement depletion with CVF and antibody depletion by plasmapheresis has synergistically prolonged of pig-to-baboon cardiac xenograft survival.53 Clinical use may be limited by the potential toxicity of CVF as well as its immunogenicity. Repeated use may induce anti-CVF antibodies. A variety of complement-inhibiting agents have been evaluated in small and large animal models. Soluble human complement receptor type 1 (sCR1) is a truncated, recombinant form of the complement regulatory protein CRl. Investigators have shown that its infusion inhibits both the classical and alternative pathways of complement activation. It also inhibits complement-dependent tissue injury in a number of in vivo models.66*67Using both an ex vivo pig-tohuman heart perfusion model and an in vivo pig-to-primate heart transplant model, Pruitt et al. have shown sCR1 to significantly prolong xenograft survival.67 Miyagawa et al. recently reported on the effects of two novel anticomplement reagents, K-76 COOH and FUT175, which both exert inhibitory activity on the classical and alternative complement pathways. Their studies in the guinea pig-to-rat model indicate that these compounds prevented HAR for short periods.68 Dalmasso et al. have investigated the effect of purified human Cl inhibitor (Cl inh) on the cytotoxicity of porcine endothelial cells caused by human serum.“,69 Cl inh is a soluble serine protease that selectively blocks activation of the classical complement pathway. In Dalmasso’s study, it resulted in a dose-dependent reduction in cytotoxicity. Host-specific

regulators

Several biologically important regulators of complement activation (RCA) are located on the membranes of most cells that are in contact with blood or other body fluids containing complement system proteins. Decay accelerating factor (DAF or CD55), membrane inhibitor of reactive lysis (CD59 or protectin), homologous restriction factor (HRF), and membrane cofactor protein (CD46) interfere with the complement reaction only at the membrane sites where they are located. DAF inhibits formation of the classical and alternative pathway C3 and C5 convertases; if such convertases have been formed it promotes the dissociation of these enzymatic complexes. CD59 and HRF interfere with formation of the MAC at the C8 and C9 binding steps. CD46 acts as a co-factor for the inactivation of C3b and C4b by factor

The emergence ojxenotransplantation

I. DAF, CD59, and HRF are uniquely anchored to the cell membrane by a phosphatidylinositol tail, which affords these molecules great mobility in the plane of the cell membrane and allows purified forms of RCA to be inserted into cell membranes?0-73 Membrane-associated RCA display the phenomenon of homologous restriction, i.e. they are inhibitory to homologous complement but have little inhibitory capacity over xenogeneic complement. This phenomenon has led several investigators to explore whether the introduction of human RCA molecules into xenogeneic cells would afford protection against human complement-mediated damage. Dahnasso et al. were the first to show that purified human DAF could be incorporated into porcine endothelial cell membranes and that the incorporated human DAF protected these cells from the cytotoxic effects of human complement.74 They obtained similar results with purhied human CD59.” However, the protection afforded by inserted human RCA molecules is relatively short-lived, as they undergo rapid turnover. Long-term protection from human complement-dependent damage could be achieved through genetic expression of human RCA molecules in xenogeneic cells. Toward this end, experiments have been conducted with xenogeneic cell types that were transfected with cDNA for various human RCA molecules, resulting in expression of these proteins on cell membranes and protection from human complement.47*48 These findings served as the basis for several groups to develop animals transgenic for human RCA molecules. White et al. have demonstrated that cells from mice transgenic for human CD55 or CD46 are protected from lysis by human complement. 76 Subsequently, this group has developed a line of pigs transgenic for CD55 and have demonstrated mRNA expression in 65% of those expressing the gene, with downregulation of complement activation in these pigs.77*78Researchers at the DNX Corporation have developed transgenic mice and pigs expressing both human CD55 and CD59.79*80In an ex vivo perfusion system with human plasma, these proteins inhibited human complement activation. Interestingly, hearts from mice transfected with human CD59 alone did not survive longer than controls, despite immunohistochemical evidence of CD59 function. Effective protection of xenogeneic cells from complement in viva may therefore require higher levels of gene expression than have thus far been achieved; or they may require expression of multiple human RCA molecules in a transgenic animal. However, transfection studies have provided conlIicting data on the additive or synergistic effect of different human RCA molecules coexpressed on xenogeneic cells.81-83 Ultimately, transgenie organs must be evaluated in a preclinical in vivo model to see if they are indeed protected from hyperacute rejection. Valdivia er al. demonstrated that, if a liver xenograft recipient later receives a second organ from the same species, HAR is prevented. The homology of the recipient complement-now produced by the liver xenograft-and of the target cells of the second xenograft provide protection from rejection.&l The clinical applicability of this novel approach has yet to be determined. Alteration or elimination of endothelial xenoantigens As discussed above, the majority of naturally occurring human antipig antibodies appear to be directed to carbohyTransplant hmunology

1995; k 21-31

27

drate epitopes, in particular terminal galactose residues with a gal(a-1,3)gal linkage. The formation of these carbohydrate epitopes is controlled by the enzyme a-1,3-galactosyltransferase, whose gene has recently been cloned in pig cells.= Using the technique of homologous recombination in porcine embryonic stem cells, it may be possible to genetically engineer ‘knock-out* pigs deficient in the gal(cu-1,3)gal sequence. Lack of expression of the important xenogeneic terminal galactose epitope might therefore avoid xenoreactive antibody binding and prevent HAR. However, eliminating porcine o-gal expression may not completely abrogate the potential for HAR in a human recipient, since these carbohydrates do not represent the sole antigenic targets for the entire human antipig xenoantibody repertoire. Other proposed strategies to alter the antigenicity of the donor endothelium include enzymatic cleavage of the (a-1,3-galactose xenoantigens from the endothelial cells% and replacement of the entire endothelium with recipient endothelial cell~.~’

Beyond hyperacute rejection Several investigators in both small and large animal models have shown that HAR can be prevented through depletion or inhibition of the recipient complement system. However, graft loss still occurs after several days. We and others have begun to examine this process, referred to as delayed xenograft rejection or acute vascular xenograft rejection. We hope to identify pathogenetic mechanisms that may affect the longterm fate of a xenograft. The most extensive study of delayed xenograft rejection has been performed in the guinea pig-to-rat heart transplant model. Preventing HAR by complement depletion of recipients results in xenograft function for up to four days. Delayed xenograft rejection in this model has pathological similarities to HAR. Both types of rejection are characterized by vascular changes, including interstitial oedema, haemorrhage, and fibrin accumulation. However, acute vascular xenograft rejection differs from HAR in that a prominent infiltration of leucocytes-predominantly mononuclear cells and macrophages-and a widespread interstitial deposition of fibrin develops over the 72-96-hour engraftment period. Interestingly, the sequestration of rat effector cells in the xenograft occurs hours after evidence of diffuse deposition of Ig within blood vessels. We have hypothesized that the accumulation of rat leucocytes in the guinea pig xenograft is a antibody-mediated endothelial cell consequence of activation. Complement-depleted rats that receive guinea pig hearts develop marked elevations of antiguinea pig antibody titres within days post-transplant. Some, but not all, of this induced antidonor antibody is directed against cellular epitopes also bound by naturally occurring xenoreactive antibody.ss This induced xenoreactive antibody might result in guinea pig endothelial cell activation in the absence of complement, leading to upregulation or de nova expression of adhesion molecules, (such as E-selectin), capable of binding rat leucocytes. Other consequences of endothelial cell activation (synthesis of proinflammatory cytokines including IL-l, induction of endothelial cell procoagulant proteins such as tissue factor) would lead to the interstitial haemorrhage, oedema, and

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JP Fryer et al

widespread fibrin deposition observed in these xenografts. This process might be driven in complement-depleted animals by the induction of antidonor antibody synthesis, a process we have observed. 6s Recently, Hancock et al. examined the role of endothelial cell activation in the delayed rejection of guinea pig xenografts, showing that proinflammatory, proadhesive, and procoagulant protein synthesis occurs.89 They also suggested an important role for natural killer cells in delayed xenograft rejection. Our laboratory has recently examined the contribution of effector cells and antibody to the process of delayed xenograft rejection using a guinea pig-to-rat adoptive transfer model. We found that either sensitized cells or heat-inactivated sensitized serum could significantly increase the tempo of xenograft rejection in antibody- and complement-depleted recipients.= In addition, our analysis of sensitized rat leucocyte subsets determined that both macrophages and B cells were critical for inducing accelerated rejection by adoptively transferred cells. Depletion of either cell type was associated with rejection times similar to controls (manuscript in preparation). Our results with B cell depletion support the promise that antidonor antibodies play an important role in delayed xenograft rejection. Macrophages-a prominent feature of the cellular infiltrate in xenografts rejected by complement depleted ratss8*s9-may interact with xenoreactive antibodies through the Fc receptors and facilitate rejection by antibodydependent cell-mediated cytotoxicity (ADCC). In vitro work by Schaapherder et al. has shown that human leucocytes can indeed kill pig cells through ADCC mechanisms.gO

Endothelii cell activation It is increasingly apparent from in vitro and in vivo xenotransplant research that nonlethal metabolic and physiological changes in the endothelial cell (broadly termed endothelial cell activation) play an important role in the fate of the immediately vascularized xenograft. As reviewed above, procoagulant and proadhesive consequences of endothelial cell activation have been identified during delayed xenograft rejection in rodent models. Platt and co-workers have shown that incubation of porcine endothelial cells with human natural antibody and complement induces the loss of membrane-associated heparan sulphate. Loss of this membrane proteoglycan diminishes the endothelial cells’ ability to bind antithrombin III and thereby maintain a localized anticoagulant state. They also noted corresponding conformational changes 91,92in the endothelial cells, possibly due to alterations in the extracellular matrix93 or cytoskeleton. These changes occur in vitro without endothelial cell lysis if sublytic quantities of antibody and complement are used. They may account for the increase in vascular permeability and disruption of vascular integrity encountered in vivo. Recent work at our centre has shown that exposure of porcine endothelial cells to human natural antibody and complement results in additional procoagulant changes, including the loss of thrombomodulin and the induction of tissue factor (Vercelotti GM, unpublished observation). We have also demonstrated that exposure of porcine endothelial cells to human inflammatory cytokines, tumour necrosis factor-a, or prolonged incubation in complement-depleted human serum, results in the loss of thrombomodulin and the synthesis of tissue factor. 94In summary, these results suggest that significant procoagulant activity develops when the vasTransplant Immunology

1995; 3: 21-31

cular endothelium of a donor organ is activated by xenogeneic antibody, complement, or cytokines. The potential pathogeneic significance of procoagulant endothelial cell activation is further underscored by studies our group has conducted of pig-to-primate xenotransplants.53 Using a protocol of antibody and complement depletion, we extended porcine cardiac xenograft survival to 17.5 days in one case. Graft loss in this baboon correlated with the development of markedly elevated antipig IgG titres, and was characterized by widespread intravascular thrombosis without evidence of classical vascular or cellular rejection. In addition, functioning xenografts removed at one and eight days post-transplant revealed evidence of intravascular fibrin deposition. Thus, activation of coagulation in a xenograft may occur under conditions where HAR is avoided, and may be causally associated with graft loss. Endothelial cell activation is also associated with the novel or increased expression of adhesion molecules. Allograft infiltration by lymphocytes is an essential step in the rejection of a transplanted organ.9s As in other inflammatory responses, the diapedesis of circulating cells into an allograft requires adhesion molecule receptor-ligand interactions between leucocytes and graft vascular endothelium. In xenotransplantation between phylogenetically disparate species (e.g. pig-to-human), the process of HAR has prevented any detailed study of leucocyte+ndothelial cell interactions in vivo. Recently, we have shown that porcine xenografts rejected by complement-depleted baboons became infiltrated by baboon lymphocytes and macrophages.53 Thus, it appears that baboon (and possibly human) lymphocytes are capable of interacting with ligands on porcine endothelium. Using an in vifro adhesion system in an attempt to better understand the human antipig cellular immune response. Witson et al. at our institution showed that normal human lymphocytes possess transendothelial cell migratory capacity for porcine endothelium. Memory T lymphocytes (CD3+/ CD45RA-/CD45RO+/L-selectindimlCD29bti@’h’ cells) displayed an increased ability to adhere to and migrate through porcine endothelium, compared with phenotypically naive T cells (CD45RA +/CD45RO - /L-selectinti@“/CD29dim cells).” In addition, natural killer cells, macrophages, and B cells exhibit significant transendothelial cell migratory capacity. Importantly, endothelial cell activation with human cytokines or human serum quantitatively increased transendothelial cell migration by human leucocytes. These results indicate that human lymphocytes are capable of interacting with adhesion molecules on porcine endothelium and that endothelial activation promotes this interaction. However, it remains to be determined which receptor-ligand interactions must occur across a species barrier in order for cellular infiltration of a xenograft to occur and transplant rejection to proceed.97.98

Conclusion Recent advances in clinical and experimental xenotransplantation have heightened overall awareness of its potential to solve the organ shortage. It seems likely that any largescale implementation of xenotransplantation will involve phylogenetically disparate donors, such as pigs. Currently, the poor results of experimental transplantation between pigs and nonhuman primates preclude clinical application. However, research in xenotransplantation by representatives from

The emergence

clinical medicine, the basic sciences, and private industry has escalated in the last decade. Impressive progress has been

achieved. And most in the field believe that although the emergence of xenotransplantation into the clinical realm may not be imminent, it is inevitable.

References 1 Evans RW. Need, demand, and supply in kidney transplantation: a review of the data, and examination of the issues, and projections through the year 2000. Semin Nephrol1992,12: 234-35. 2 Alexander JW, Vaughn WK. The use of ‘marginal’ donors for organ transplantation. Tramplan~ation 1991,51: 13wl. 3 First MR. Transplantation in the nineties. Transplan~arian 1992; 53: l-11. 4 Randall T. Too few human organs for transplantation, too many in need. . . and the gap widens. JAMA 1991; 265: 1223-27. 5 Calne RY. Organ transplantation between widely disparate species. Transplant Proc 1970,2: 550-59. 6 Leventhal JR, Matas AJ. Xenotransplantation in rodents: a review and reclassification. Transplant Rev 1994; 8: 80-92. 7 Reemtsma K, McCracken BH, Schlegel JU et al. Renal heterotransplantation in man. Ann Surg 1%4; 16013~10. 8 Bailey LL, Nehlsen-Cannarella SL, Concepion W et al. Baboonto-human cardiac xenotransplantation in a neonate. JAMA 1985; 254: 3321-29. 9 Starzl TE, Fung J, Tzakis A et al. Baboon-to-human plantation. Lancer 1993; 341: 65-71.

liver trans-

10 Cooper DKC, Ye Y. Experience with clinical heart xenotransplantation. In: Cooper DKC, Kemp E, Reemtsma K, White DJG eds. Xenolransplantation. New York: Springer-Verlag. 1992: 541-57. 11 Cramer DV, Sher L, Makowka L. Liver xenotransplantation: clinical experience and future considerations. In: Cooper DKC, Kemp E, Reemtsma K, White DJG, eds. Xenotransplanration. New York: Springer-Verlag. 1992: 541-57. 12 Michler RE, McManus RP, Smith CR et al. Prolongation of primate cardiac xenograft survival with cyclosporine. Transplantation 1987; 44: 632-36. 13 Cooper DKC, Rose AG. Experience

with experimental xenografting in primates. In: Hardy M ed. Xenografi 25. Amsterdam: Elsevier, 1989: 95. 14 Rose AG, Cooper DKC, Human PA et al. Histopathology of hyperacute rejection of the heart: experimental and clinical observations in allografts and xenografts. J Heart Lung Transplant 1991; lo:223-34. 15 Norin AJ, Roslin MS, Panza A et al. TLI induces specific B-cell unresponsiveness and long-term monkey heart xenograft survival in cyclosporine-treated baboons. Transplant Proc 1992, 24: 508-10. 16 Murase N, Starzl TE, Demetris AJ et al. Hamster-to-rat

liver xenotransplantation

heart and with FK506 plus antiproliferative drugs.

Transplantation 1993; 53: 701. 17 Martini GA, Sliegert R. Marburg

18 19 20 21

22

virus disease. New York: Springer-Verlag, 1971. Centers for Disease Control. MMWR 1990,39: 22. Ross DN. In: Shapiro H ed. Experience with human heart framplanration. Durban: Butterworths, 1969: 227. Dubernaud JM, Bonneau M, Latour M. Heterografts in primates. Villeurbanne: Simep Editions, 1974. Norman MC, Saravis CA, Brown ME. Immunochemical observations in clinical heterologous (xenogeneic) liver perfusions. Surgery 1%; 60: 179-90. Fair J, Mattei P, Guo Y et al. Oligoclonal V-beta TCR usage in vitro after in vivo exposure to pig xentoantigen via extracorporeal pig liver perfusion. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK.

Transplant

Immunology

1995; 3: 21-31

ojxenotransplantation

29

23 Abouna GM, Serrou B, Boehmig HG et al. Long-term support by intermittent multi-species liver perfusions. Lancer 1970; 2: 391-99. 24 Makowka L, Cramer DV, Hoffman A et 01. pig liver xenografts as a temporary bridge for human allografting. Xeno 1993; 1: 27-29. 25 Mazes M, Gewurz H, Gunnarson A, Moberg A. Xenograft rejection by dog and man: isolated kidney perfusions with blood plasma. Transplant Proc 1971; 3: 531-33. 26 Moberg A, Shons A, Gewurz H et al. Prolongation of renal xenografts by the simultaneous sequestration of preformed antibody and the inhibition of complement, coagulation, and antibody synthesis. Transplant Proc 1971; 3: 538-41. 27 Giles GR, Boehming I-IJ, Lilly J et al. Mechanism and modification of rejection of heterografts between widely divergent species. Transplant Proc 1970; 2: 522-37. 28 Perper RJ, Najarian JS. Experimental renal heterotransplantation III: Passive transfer of transplantation immunity. Transplunrarion 1%7; 5: 514-33. 29 Platt JL, Vercelloti GM, Dalmasso AP et al. Transplantation of discordant xenografts: a review in progress. Zmmunol Today 1990, 11:456-57. 30 Dahnasso AP, Vercellotti GM, Fischel RJ et al. Mechanism of complement activation in the hyperacute rejection of porcine organs transplanted into primate recipient. Am / Path01 1992; 1401 1157-66. 31 Platt JL, Lindman BJ, Chen H et al. Endothelial cell antigens recognized by xenoreactive human natural antibodies. Transplantation 1990; 50: 817-22. 32 Koren E, Neethling FA, Ye Y et al. Heterogeneity of preformed human antipig xenogeneic antibodies. Transplant Proc 1992; 24: 598-601.

33 Calmus Y, Ayani E, Chereau C et al. Study of the target antigens of hyperacute xenogeneic rejection in discordant combinations. Transplanr Proc 1993; 25: 379-81.

34 Oriol R, Ye Y, Koren K, Cooper DK. Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for hyperacute vascular rejection in pig-to-man organ xenotransplantation. Transplanration 1992,s 1433-2. 35 Platt JL, Holznecht ZE. Porcine platelet antigens recognized by human xenoreactive natural antibodies. Transplantation 1994; 57: 327-35.

36 Galili U, Shohet SB, Kobrin E et al. Man, apes, and old world monkeys differ from other mammals in the expression of alphagalactosyl epitopes on nucleated cells. J Rio1 Chem 1988, 263: 1775562.

37 Galili U, Macher BA, Buehler J et al. Human natural anti alpha galactosyl IgG: II. The specific recognition of alpha (1-3)-linked galactose residues. J Exp Med 1985; 162: 573-82. 38 Gowda DC, Schultz M, Bredehorst R et al. Structure of the major oligosaccharide of cobra venom factor. Mol Zmmunol lm, 29: 33542. 39 Cooper DKC, Good AH, Koren E et al. Identification of alphagalactosyl and other carbohydrate epitopes that are bound by human anti-pig antibodies: relevance to discordant xenografting in man. Transplant Zmmunol1993; 1: 198-205. 40 Gewurz H, Clark D, Finstad J et al. Role of the complement system in graft rejections in experimental animals and man. Ann NYAcad Sci 1966,129: 673-713. 41 Adachi H, Rosengard BR, Hutchins GM et al. Effects of cyclospoke, aspirin, and cobra venom factor on discordant cardiac xenograft survival in rats. Transplant Proc 1987; 19: 1145-48. 42 Otte KE, Anderson N, Jorgenson KA et 01. Xenoperfusion of pig kidney with human AB or 0 whole blood. Transplanr Proc 19w, 22: 1091-92. 43 Chartrand C, O’Regan S, Robitaille P, Pinto-Blonde M. Delayed rejection of cardiac xenografts in C&deficient rabbits. Zmmunology 1979; 38: 245-48. 44 Medof ME, Kinoshita T, Nussenweig V. Inhibition of complement activation on the surface of cells after incorporation of

30 JP Fryer et al

decay accelerating factor (DAF) into their membranes. J Exp Med 1984; 160:1558-78.

45 Hourcade, D, Holers VM, Atkinson JP. The regulators of complement activation (RCA) gene cluster. Adv Zmmunol 1989; 45: 381-416. 46 Nicholson-Weller A, March JP, Rosenfeld SI, Austen KF. Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria are deficient in the complement regulatory protein, decay accelerating factor. Proc Nat1 Acad Sci USA 1983; 80: 5066-75. 47 Oglesby TJ, White DJ, Tedja I et al. Protection

of mammalian cells from complement-mediated lysis by transfection of human membrane cofactor protein (MCP) and decay accelerating factor (DAF). Tranr Assoc Am Physicians 1991; 104: 164-72. 48 Akami T, Sawada R, Naruto M et al. Cytoprotective effects of CD59 antigen on xenotransplantation immunity. Transplant Proc

1992; 24: 485-87. 49 Alexandre GPJ, Squifflet JP, DeBruyere M et al. Present experi-

ences in a series of 26 ABO-incompatible living donor renal allografts. Transplanl Proc 1987; 19:4538-42. 50 Taube DH, Williams DG, Cameron JS et al. Renal transplantation after removal and prevention of resynthesis of HLA antibodies. Lancet 1984; 1: 824-28.

51 Hammer C, Saumweber D, Krombach F. Xenotransplantation in canines. In: Hardy MA ed. Xenografi 25. New York: Elsevier, 1989: 67-68. 52 Alexandre GPJ, Gianello P, Latinne D et al. Plasmapheresis and splenectomy in experimental renal xenotransplantation. In: Hardy MA ed. Xenograft 25. New York: Elsevier, 1989: 259. 53 Leventhal JR, Sakiyalak P, Witson J et al. Synergistic effect of combined antibody (Ab) and complement depletion upon discordant cardiac xenograft survival in nonhuman primates. Transplantation

1994; 57: 974-83.

54 John R, Leventhal JR, Yang MQ et al. Removal of baboon and human anti-porcine IgG and IgM natural antibodies using absorptive columns: results of in vitro and in vivo studies. Abstract presented at the American Society of Transplant Surgeons ammal scientific meeting, May 18-20,1994, Chicago. 55 Palmer A, Welsh K, Gjorstrup P et al. Removal of anti-HLA antibodies by extracorporeal immunoadsorption to enable renal transplantation. Lancell989; 1: l&12. 56 Giles GR, Boehmig JH, Beavers CD et al. Selective plasmapheresis in dogs for delay of heterograft response. Trans Am Sot Artif Intern Organs 1970; 16:325-38. 57 Terman DS, Garcis-Rinaldi R, McCalmon R et al. Modification of hyperacute renal xenograft rejection after extracorporeal immunoadsorption of heterospecific antibody. Int J Artif Organs

cyclin and cobra venom factor. Transplant Proc 1987; 19: 4471-74. 64 Beukelman CJ, Aerts PC, van Dijk H et al. A one-step isolation procedure for phospholipase AZ-free cobra venom factor by fast protein liquid chromatography. J Immunol Methods 1987; 97: 119-22. 65 Leventhal JR, Dahnasso AP, Cromwell JW et al. Prolongation of cardiac xenograft survival by depletion of complement. Transplantation 1993; 55: 857-66. 66 Xia W, Fearon DT, Moore FD et al. Prolongation of guinea pig

cardiac xenograft survival in rats by soluble human complement receptor type 1. Transplant Proc 1992,24: 479-80. 67 Pruitt SK, Kirk AD, Bollinger RR er al. The effect of soluble complement receptor type 1 on hyperacute rejection of porcine xenografts. Transplanration 1994; 57: 363-70. 68 Miyagawa S, Shirakura R, Matsumiya F et al. Effect of anticomplement reagents, K-76 COOH and FUT175, on discordant xenograft survival. Transplant Proc 1992; 24: 483-84. 69 Dalmasso AP, Platt JL. Prevention of complement-mediated activation of xenogeneic endothelial cells in an in vitro model of xenograft hyperacute rejection, by Cl inhibitor. Transplantation 1993; 56: 1171-76. 70 Holguin MH, Fredrick

LR, Bershaw NJ et al. Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria. J Clin Invest 1989; 84:

7-17. 71 Medof ME, Walter EI, Roberts WL, Haas R, Rosenbeny

TL. Decay accelerating factor of complement is anchored to cells by a C-terminal glycolipid. Biochemistry 1986,25: 67U7. 72 Hansch GMN, Weller EI, Nicholson-Weller A. Release of CZ3 binding protein (C8bp) from the cell membrane by phosphatidylinositol-specific phospholipase C. Blood 1988; 72: 1089-92. 73 Lublin DM, Atkinson JP. Decay-accelerating factor: biochemistry, molecular biology, and function. Annu Rev Zmmunol 1989; 7: 35-38. 74 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: 530-33. 75 Dalmasso AP. The complement system in xenotransplantation. Immunopharmacology 1992; 24: 149-60. 76 White DJG, Oglesby T, Liszewski MK et al. Expression of human

decay accelerating factor or membrane cofactor protein genes on mouse cells inhibits lysis by human complement. Transplant Proc 1992; 24: 474-76. 77 Langford GA, Yannoutos

1979; 2: 3549. 58 Tanaka M, Latinne D, Gianello P et al. Xenotransplantation

from pig to cynomolgus monkey: the potential for overcoming xenograft rejection through induction of chimerism. Transplant Proc

78

1994; 26: 1326-27. 59 Bannett AD, McAlack RF, Raja R et al. Experiences with known ABO-mismatched renal transplants. Transplant Proc 1987; 19: 4543. 60 Good AH, Cooper DKC, Malcolm AJ et al. Identification of

79

carbohydrate structures that bind human antiporcine antibodies: implications for discordant xenografting in humans. Transplant Proc 1992; 24: 559-62. 61 Soares M, Nisol F, Bach FH et al. Use of anti-p monoclonal

80

antibody as a therapeutic approach to achieve depletion of xenoreactive natural antibodies. Transplant Proc 1994; 26: 1357-59. 62 Van der Werf WJ, Blakely ML, Hancock WW el al. Sustained

immunosuppression of xenoreactive natural antibodies: anti-&M monoclonal antibody and anti-B cell immunosuppressants. Transplant Proc 1994; 26: 1372-73. 63 Kemp E, Steinbruchel D, Starklint

H et al. Renal xenograft rejection: prolonging effect of captopril, ACE-inhibitors, prosta-

Transplant Immunology 1995; 3: 21-31

81

N, Cozzi E et al. Production and analysis of pigs transgenic for human decay accelerating factor. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK. Cozzi E, Langford GA, Richards A et al. Expression of human decay accelerating factor in transgenic pigs. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK. Kooyman D, Byrne GW, McClellan S et al. Erythroid specific expression of human CD59 and transfer to vascular endothelial cells. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK. Harland RC, Logan JS, Kooyman D et al. Ex-vivo perfusion of mouse hearts expressing the human complement regulatory protein CD59. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK. Hayashi S, Isobe K, Emi N et al. Inhibition of species-specific complement-mediated cytolysis in xenoendothelial cells transfected with GPI-anchoring complement regulatory factor (DAF, HmO) gene using retroviral vector comparison between single (DAF or HRF?20) and double (DAF and HFU?ZO)transfected xenoendothelial cells. Abstract presented at the International

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82

83

84

85

Congress on Xenotransplantation, September 1993, Cambridge, UK. McKenzie IF, Johnstone R, Szokolai K er al. The coordinate functions of multiple complement regulating molecules, CD46, CD55 and CD59. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK. Miyagawa S, Shirakura R, Matsuda H er al. Effects of transfectant molecules, MCP, DAF, and MCP/DAF hybrid on xenogeneic vascular endothehum. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK. Valdivia LA, Fung JJ, Demetris er al. Complement and target cells belong to the same species after liver xenotransplantation: protection from hyperacute rejection. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK. Dabkowski PL, Vaughn HA, McKenzie IFC, Sandrin MS. Characterization of a cDNA clone encoding the pig alpha(lZ)galactose transferase: implications for xenotransplantation.

Transplant Proc 1993; 25 2921. 86 Cairns T, Hammlemann W, Gray D et al. Enzymatic

removal from various tissues of the galactose (alpha 1,3)-galactose target antigens of human antispecies antibodies. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK. 87 Stevens LH, Axe-Graham P, Faulk WP. Endothelial reseeding prevents hyperacute rejection in guinea pig-to-rat femoral artery interposition grafts. Transplunt Proc 1994 (in press). 88 Fryer JP, Leventhal JR, Dalmasso AP et al. Cellular rejection in a discordant xenograft when hyperacute rejection is prevented: analysis using adoptive and passive transfer. Transplant Immunol 1994; 2: 87-93. 89 Hancock WW, Blakely ML, van der Werf WJ er al. Rejection of guinea pig cardiac xenografts post cobra venom factor therapy is associated with infiltration by mononuclear cells secreting interferon-gamma and diffuse endothelial activation. Trunsplant Proc

Transplant

Immunology

1995; 3t 21-31

of xenotransplantation

31

90 Schaapherder AFM, Daha MR, te Bulte MTJW et al. Antibodydependent cell-mediated cytotoxicity against porcine endothehum induced by a majority of human sera. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK. 91 Saadi S, Ihrcke NS, Platt JL.The pathogenesis of hyperacute rejection. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK. 92 Geller RL, Ihrcke NS, Platt JL. Release of endothelial cellassociated heparan sulfate proteoglycan by activated T cells. Transplantation 1994; 57~770-74. 93 De Sousa M, Tilney NL, Kupiec-Weglinski JW. Recognition of self within self; specific lymphocyte positioning and the extracellular matrix. Immunol To&y 1991; 12: 262-66. 94 Leventhal JR, Witson JC, Vercelotte GM et al. Endothelial cell activation in discordant xenografting; Induction of procoagulant effects. Abstract presented at the American Society of Transplant Surgeons--Annual Scientific Meeting, May lS20, 1994, Chicago. 95 Hall BM, De Saxe I, Dorsch SE. The cellular basis of ahograft rejection in vivo. III. Restoration of first-set rejection of heart grafts by T-helper cells in irradiated rats. Transplantation 1983; 36: 700-705. 96 Witson JC, Leventhal

JR, Matas AJ, Vercellotti GM, Bohnan RM. Adhesive interactions between human lymphocytes and porcine endothelium: a model for cellular infiltration of a xenograft. Abstract presented at American Society of Transplant Surgeons-Annual Scientific Meeting, May 18-20,1994, Chicago. 97 Fyfe AI, Harper CM, Stevenson LW et al. Human mononuclear cells do not exhibit enhanced adhesion of porcine arterial endothelium. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK. 98 Pleass HCC, Kirby JA, Forsythe JLR ef al. Adhesion molecule blockade in a porcine xenograft mode. Abstract presented at the Second International Congress on Xenotransplantation, September 1993, Cambridge, UK.