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Ann Thorac Surg 1997;64:1858-1865
© 1997 The Society of Thoracic Surgeons

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Current Reviews

Cardiac Xenotransplantation

Department of Cardiothoracic Surgery, Allegheny University Hospital, MCP, Philadelphia, Pennsylvania

	Abstract

Top Footnotes Abstract Introduction The Immunobiology of the... Zoonoses Ethical Issues Strategies to Achieve Successful... Genetic Engineering Donor-specific Graft Acceptance Predictions for the Future References

 
Heart failure is an important medical and public health problem. Although medical therapy is effective for many people, the only definitive therapy is heart transplantation, which is limited severely by the number of donors. Mechanical devices presently are used as "bridges" to transplantation. Their widespread use may solve the donor shortage problem, but at present, mechanical devices are limited by problems related to blood clotting, power supply, and foreign body infection. Cardiac xenotransplantation using animal donors is a potential biologic solution to the donor organ shortage. The immune response, consisting of hyperacute rejection, acute vascular rejection, and cellular rejection, currently prevents clinical xenotransplantation. Advances in the solution of these problems have been made using conventional immunosuppressive drugs and newer agents whose use is based on an understanding of important steps in xenoimmunity. The most exciting approaches use tools of molecular biology to create genetically engineered donors and to induce states of donor and recipient bone marrow chimerism and tolerance in xenogeneic organ recipients. The successful future strategy may use a combination of a genetically engineered donor and a chimeric recipient with or without nonspecific immunosuppressive drugs.

	Introduction

Top Footnotes Abstract Introduction The Immunobiology of the... Zoonoses Ethical Issues Strategies to Achieve Successful... Genetic Engineering Donor-specific Graft Acceptance Predictions for the Future References

 
Every year, heart failure requiring treatment develops in more than 400,000 Americans [1]. Although medical therapy is effective for many of these patients, heart transplantation using human donors (allotransplantation) remains the only definitive therapy for end-stage heart failure. However, heart allotransplantation has inherent limitations. Recipients must suffer long waiting times, usually in a hospital intensive care unit, before a suitable human donor can be found. In addition, after transplantation, all patients must take potentially toxic immunosuppressive drugs to control the immune response to the cardiac allograft. These medications make patients susceptible to potentially life-threatening opportunistic infections but do not eliminate the immune response completely, thereby also leaving them vulnerable to life-threatening rejection. In addition, all immunosuppressive drugs, including steroids, cyclosporine, and azathioprine (the most commonly used agents), have significant nonimmunologic side effects. Notwithstanding these imperfections of current immunosuppressive therapy and its complications and side effects, the fundamental problem with present-day heart transplantation is that the supply of donor hearts will never meet the demand for them.

The discrepancy between supply and demand is well illustrated by data from the United Network for Organ Sharing [2]. In calendar year 1995, 2,360 patients received heart transplants in the United States. At the end of that year, there remained 3,468 patients still waiting for a heart transplant, and during that year, 770 patients died waiting. The demand for heart transplants in calendar year 1995 can be estimated by adding these three patient groups. In total, 6,598 patients were deemed to need heart transplantation in 1995. The total number of hearts transplanted, which represents the supply, was 2,360. Subtracting this from the demand yields an actual deficit of 4,238 hearts needed for transplantation in the United States in 1995.

As daunting as this discrepancy between supply and demand is, the actual picture is much worse. A number of estimates have suggested that in the United States, as many as 40,000 to 60,000 patients per year might benefit from heart transplantation [3]. Obviously, the 2,360 hearts procured for transplantation could never meet this demand. Even the most optimistic estimates of the maximum donor supply fall far short. Reviews of charts of brain-injured patients who meet the criteria to be heart transplant donors suggest that the maximum number of transplantable hearts might approach 10,000 per year. Even this most optimistic estimate leaves a theoretic deficit of hearts for transplantation of 30,000 to 50,000 per year. Given the rate noted above at which new cases of heart failure are diagnosed, this number can only grow.

In part, this deficit is a reflection of the success of cardiac allotransplantation. Heart transplantation is an effective therapy, and this success has been part of the incentive to develop medical therapies for these patients with heart failure. As a result, medical therapy has advanced from digoxin and diuretics to sophisticated treatments such as angiotensin-converting enzyme inhibitors and ß-blockers [4]. These advances in therapy also have been an impetus to procedures such as cardiomyoplasty and to the development and improvement of mechanical devices. Mechanical ventricular assist devices and total artificial hearts presently are used as temporary support or "bridges" before transplantation [5]. Further development of these mechanical devices eventually might provide an alternative to allotransplantation of the heart. In fact, experimental protocols whereby patients supported by ventricular assist devices are discharged from the hospital and managed carefully at home already are under way in this country and overseas.

However, there are significant underlying problems with the mechanical device alternative to cardiac allotransplantation. Some of these are amenable to scientific study and someday may be overcome. Specifically, one of the primary problems is the interaction of the recipient's blood with the artificial surface of the device. As was well publicized in several clinical experiments involving mechanical hearts, the large artificial surface of the blood pump creates a tendency to blood clotting and thromboemboli. This is particularly devastating when the brain is the organ injured. Advances in the scientific understanding of the surface of normal blood vessels and the biology of the formed and protein elements of blood, coupled with advances in materials technology, someday may lead to a solution to this difficulty.

Another fundamental limitation of all present mechanical devices is the provision of an adequate energy supply to power the pumps in the devices. The current state of the art involves either a transcutaneous wire attached to a power supply or an externally worn power pack that transmits electrical energy transcutaneously through induction coils. A wire through the skin has the inherent limitation of breaching the integument and allowing for potential infection. Transcutaneous induction coils obviate this problem. However, the patient still is bound to bulky power packs. Again, advances in materials, technology, and energy storage will reduce, but never eliminate, these problems.

Mechanical devices, however, remain foreign bodies. As such, they are susceptible to infection from the innumerable episodes of bacteremia that occur during everyday life. Infection of a ventricular assist device or mechanical heart will remain a devastating complication because removal of the device threatens the life of the patient who is dependent on it. Treatment of infection, short of device removal, often is impossible.

The nonmechanical biologic alternative to cardiac allotransplantation is the use of nonhuman heart donors (xenotransplantation). A xenogeneic heart from an appropriately chosen species would solve the supply problem by offering an essentially inexhaustible number of usable hearts for transplantation. Nature has taken care of some of the problems that limit mechanical devices. The xenograft has mechanisms to prevent clotting of the formed elements of blood. Further, mammalian organs have developed the ability to replenish continuously their energy supply. Finally, a xenograft is not an inert foreign body made of artificial material and would not be subject to the infectious complications of mechanical devices.

Of course, there remain barriers to the use of xenografts. The complex immune response to xenogeneic organs has the capacity to destroy the graft in days or even hours. Molecular science is being applied to solving this problem using techniques that someday also may obviate the need for nonspecific immunosuppressive drugs. Should these immune barriers to xenotransplantation be overcome, there are other issues worthy of consideration. One is the possibility of transmitted infections (zoonoses). Organisms that reside in animals and do not cause disease in them may have the potential to cause disease in humans. Finally, as a society, we must deal with the ethical considerations of using animal organs for the benefit of humans. Although there is a great deal of precedent for the use of animals for human benefit throughout medicine and society, failure to acknowledge and deal with this issue will only hinder the development of xenotransplantation.

This review emphasizes the potential for xenotransplantation and molecular solutions to xenograft rejection to revolutionize the care of patients with heart failure. It does not consider other future, but potentially promising, molecular biologic approaches to improving the function of the failing heart, such as myocyte transplantation [6] or adrenergic receptor overexpression in transgenic animals [7]. The following paragraphs summarize the immunobiology of the xenotransplantation response and describe presently available and potential future strategies for achieving successful cardiac xenotransplantation.

	The Immunobiology of the Xenotransplantation Rejection Response

Top Footnotes Abstract Introduction The Immunobiology of the... Zoonoses Ethical Issues Strategies to Achieve Successful... Genetic Engineering Donor-specific Graft Acceptance Predictions for the Future References

 
Unlike the immune response to cardiac allotransplants that is mediated mainly by the cellular immune system and T lymphocytes, there are several complex components of the immune response to a xenograft that involve both humoral and cellular immunity. Lawson and Platt [8] have described the phases of xenorejection, which have been termed hyperacute rejection, acute vascular rejection, and cellular rejection.

Hyperacute Rejection
Hyperacute rejection is mediated by naturally occurring antibodies and complement and leads to graft destruction in a matter of minutes. In some species combinations, hyperacute rejection may be triggered without naturally occurring antibodies. In these settings, complement is activated by the alternate pathway. In earlier days of transplantation immunobiology, it was suggested that species combinations could be divided into concordant and discordant:

• Concordant
  • No preformed antibodies
    
  • No hyperacute rejection
    
  
  
• Discordant
  • Preformed antibodies
    
  • Hyperacute rejection
    
  
  

In concordant species combinations, a graft from one individual to another did not undergo hyperacute rejection. In contrast, in discordant combinations, a graft from one individual to another was destroyed rapidly by the hyperacute rejection response. Initially, concordance and discordance were believed to be related to the phylogenetic disparity between species. However, the present understanding is that phylogenetic disparity is less important than the presence or absence of naturally occurring xenoantibodies in the graft recipient.

This is illustrated by the hyperacute rejection response between pig organs and human blood. Parker and colleagues [9] have characterized the repertoire of xenoreactive antibodies that cause hyperacute rejection of pig organs by primates such as humans. More than 1% of the human antibody repertoire is capable of reacting with a carbohydrate antigen that is present on pig cells but missing on human cells. This antigen, called the galactose alpha 1-3 galactose (gal) antigen, was identified originally by Galili [10] and is not expressed on human cells. Similarly, expression of this antigen has been lost by old-world monkeys such as the baboon. New-world monkeys and lower, nonprimate mammals such as the pig continue to express the gal antigen. Mammals such as humans and old-world monkeys that do not express the gal antigen have naturally occurring antibodies that interact with epitopes on this antigen. Furthermore, these antibodies appear to play a primary role in the initiation of the hyperacute rejection response. It is against this antigen that most naturally occurring antibodies are directed. Collins and associates [11] and others [12] have provided evidence that suggests that the gal determinant is the primary trigger of the hyperacute rejection response in the combination of pig to human or pig to old-world monkey.

The process of hyperacute rejection when a pig organ is placed in contact with human blood or in a primate that does not express the gal antigen starts with the binding of naturally occurring immunoglobulin (Ig) M antibodies specific for the gal antigen [8]. This antigen is expressed on the surface of endothelial cells, and it is on the endothelium that hyperacute rejection begins. The binding of these IgM antibodies leads to the activation of complement, with the formation of complement terminal complexes (C5b67). The activation of complement leads to the release of heparan sulfate from endothelial cells and the generation of intercellular gaps. These gaps breach the endothelial barrier in the organ. The gaps also allow for the binding of platelets and the deposition of fibrin. This leads to the thrombosis of blood vessels and death of the graft within minutes.

Acute Vascular Rejection
Several maneuvers that are described later can be undertaken to limit the hyperacute rejection response. However, if hyperacute rejection is overcome, the xenogeneic graft remains subject to acute vascular rejection, which typically occurs 3 to 5 days after engraftment. Acute vascular rejection probably also is antibody-mediated, perhaps by the same naturally occurring antibodies that mediate hyperacute rejection. Acute vascular rejection also is characterized by changes in endothelial cells and local activation of cytokines. As noted, it leads to graft destruction in several days.

The sequence of events in acute vascular rejection begins with antibody binding to endothelial cells, followed by sublytic levels of complement activation [8]. These events cause changes in the endothelial cell and the induction of tissue factor. Other changes include the release of plasminogen activator, a decrease in the activity of plasminogen activator inhibitor, the expression of E-selectin, and the loss of thrombomodulin activity. Activation of these cytokines causes thrombosis in the graft and its relatively rapid destruction. Alternatively, the endothelial cells may be stimulated directly by binding of antibodies to cellular receptors plus the gal epitope. These receptors may be members of a group of glycoproteins called integrins.

Cellular Rejection
The cellular immune response to xenografts is the least well studied of the rejection responses. This is not the least because the formidable barriers of hyperacute rejection and acute vascular rejection provide relatively few opportunities to study events that occur after these virulent rejection episodes. Yamada and colleagues' [13] studies of the immune response to specially bred miniature swine suggest that the cellular rejection response to a xenograft is similar in strength and specificity to an allogeneic response. Their observations support the conclusion that the T-cell receptor repertoire, T-cell accessory molecule interactions, and cytokine production required for both direct and indirect recognition of xenogeneic antigens are present in the cellular immune response to a xenograft. If this is the case, it suggests that the cellular immune response may be amenable to the same sorts of pharmacologic and biologic control as presently are used clinically in allotransplantation [14].

Accommodation
Although the immune rejection response to a discordant xenogeneic organ is complex and virulent, it is not the only immune response that has been observed after experimental xenotransplantation. In rodents, temporary depletion of antibody or complement can prevent hyperacute rejection. If grafting is done during this interval, it can lead to a state of long-term graft survival even after antibody and complement levels return. This is called accommodation [15]. The mechanism of accommodation is unknown, and it has not been observed in primate recipients of organs from discordant species donors. However, the fact that accommodation can happen offers some hope that proper modulation of the immune response could lead to long-term acceptance of a cardiac xenograft.

	Zoonoses

Top Footnotes Abstract Introduction The Immunobiology of the... Zoonoses Ethical Issues Strategies to Achieve Successful... Genetic Engineering Donor-specific Graft Acceptance Predictions for the Future References

 
Another potential barrier to successful xenotransplantation is the simultaneous transplantation of potentially infectious agents that cause infections called zoonoses [16]. Animals may bear bacterial or viral organisms that do not cause disease in their natural hosts but have the potential to cause disease in humans. For example, viral transmission from primates to humans is thought by some to be the origin of the human immunodeficiency virus epidemic. Therefore, in any examination of the issues that must be addressed to make clinical xenotransplantation a reality, consideration has to be given to the identification and elimination of potentially pathogenic microorganisms. Thus far, relatively little attention has been paid to this important potential problem. Michaels and Simmons [17] have suggested potential strategies for the prevention of xenotransplant-associated zoonoses that incorporate meticulous microbiologic screening of donor animals. Because of the close relation between nonhuman primates and humans, the potential for disease transmission appears to be greatest when nonhuman primates are used as xenotransplant organ donors [18]. The potential for zoonotic infection is likely to be much lower with nonprimate donors, as suggested by Ye and coauthors [19].

	Ethical Issues

Top Footnotes Abstract Introduction The Immunobiology of the... Zoonoses Ethical Issues Strategies to Achieve Successful... Genetic Engineering Donor-specific Graft Acceptance Predictions for the Future References

 
The use of animal donors for human transplantation also raises important ethical issues. It is likely that members of our society would have legitimate moral, legal, ethical, and economic objections to the use of nonhuman primates as organ donors on a large scale. Primates phylogenetically are related closely to humans and have highly developed brains, emphasizing their close relation to humans. Significant objections to the use of a sentient being with this degree of intellectual capacity have been raised in the past. Further, not all primates breed well in captivity, and maintaining adequate donor colonies would be at least expensive, if not impossible.

These issues, like those of zoonoses, are likely to be less prohibitive with nonprimate donors. There certainly is precedent for the use of animals such as cows and pigs for food, clothing, and other purposes, including medical uses in humans. Porcine insulin has been in use for many years and bioprosthetic valves fashioned from pig heart valves or bovine pericardium also are in wide use. Further, the pig has the appropriate size and physiology to serve as an organ transplant donor. Therefore, most of the discussion and scientific work has been applied to consideration of the pig as a heart donor for human use. As noted previously, the pig expresses the gal antigen, and therefore the pig and humans are discordant species and the barrier of hyperacute rejection must be overcome.

	Strategies to Achieve Successful Xenotransplantation

Top Footnotes Abstract Introduction The Immunobiology of the... Zoonoses Ethical Issues Strategies to Achieve Successful... Genetic Engineering Donor-specific Graft Acceptance Predictions for the Future References

 
This section describes several proposed strategies for achieving successful xenotransplantation:

1. Conventional immunosuppressive drugs
   
2. Complement depletion/inactivation
   
3. Antibody depletion/inactivation
   
4. Genetic engineering of donors
   
5. Donor–recipient bone marrow chimerism
   

Many of these strategies have been tested experimentally. These include various combinations of conventional immunosuppressive drugs, including newer agents, in both rodent and primary experimental xenotransplantation. Because of the important role of antibody and complement, some investigators have studied antibody and complement depletion or inactivation as a component of therapy for xenotransplantation. Antigen-specific manipulations of the recipient immune system also have been tried. For example, intrathymic inoculation of donor antigen to respective recipients, which has demonstrated considerable success in experimental allotransplantation, also has been applied to models of xenotransplantation. More complex donor-specific recipient manipulations have included bone marrow transplants with the induction of xenogeneic bone marrow chimerism.

In other recent experiments, investigators have sought to engineer genetically a donor animal whose organs possess characteristics that would eliminate hyperacute rejection. Because complement activation plays such a crucial role in the hyperacute immune response to a discordant xenograft, genetically engineered transgenic animals that express human complement inhibitors have been created. Some investigators have produced transgenic mice and pigs that express human complement inhibitors. The engineering of animals that express other transgenes has been proposed. Another suggestion is to engineer a donor animal that does not express a key xenoantigen, such as the gal antigen. The manipulation of embryonal stem cells has produced such gene "knockouts" in mice.

Conventional Immunosuppressive Drugs
A number of conventional immunosuppressive drugs have been studied in experimental xenotransplantation, mostly in rodents. Some experiments have been done in primates. In rodents, both cyclosporine and FK506 (tacrolimus) have been shown to be effective in prolonging the survival of concordant hamster-to-rat heart xenotransplants [20–22]. Fujino and associates [23] demonstrated that combination therapy including cyclosporine, mycophenolate mofetil, and brequinar sodium was effective in delaying the rejection of hamster-to-rat xenografts. Prolongation of graft survival has been seen using other agents that are effective in allotransplantation, such as deoxymethylspergualin [24]. These observations have been confirmed by other investigators, including Murase and colleagues [25] and Hayashi and associates [26], who have suggested that combination therapy with conventional immunosuppressive drugs has a synergistic effect on the prolongation of graft survival in concordant rodent xenogeneic transplantation experiments. In some cases, permanent graft survival has been achieved. In discordant rodent xenografting experiments, Adachi and co-workers [27] have observed that combination chemotherapy with cyclosporine, aspirin, and cobra venom factor leads to significantly prolonged xenograft survival in the guinea pig-to-rat discordant species combination.

There have been a number of experimental attempts to use conventional immunosuppression to produce xenotransplant survival prolongation in primate models using nonhuman primate recipients. Sadeghi and colleagues [28] observed significant prolongation of cynomolgus monkey-to-baboon cardiac transplants using combination treatment with cyclosporine, methylprednisolone acetate, azathioprine, and antithymocyte globulin. Prolongation of discordant xenograft survival in a nonhuman primate recipient has not been observed using conventional immunosuppressive drugs.

Complement Depletion/Inactivation
Depletion or inactivation of complement has been tested in experimental settings of xenotransplantation [29, 30]. The administration of cobra venom factor leads to nearly total, but temporary, depletion of complement. When used as a pretreatment in experimental xenotransplantation, cobra venom factor can prevent the initiation of the hyperacute rejection response. In some settings, the use of cobra venom factor together with cyclosporine has induced a state of accommodation in rodent xenotransplants. Cobra venom factor, when used together with other pretreatment strategies such as intrathymic inoculation of donor antigen, has not been effective in producing long-term graft survival [31]. In contrast, Hayashi and co-workers [32] have evidence that combination therapy using cobra venom factor, splenectomy, and deoxymethylspergualin is effective in preventing xenotransplantation rejection in the guinea pig-to-rat discordant species combination.

Anticomplement reagents also have been tested experimentally. Miyagawa and co-workers [33] observed prolongation of rodent discordant xenograft survival using anticomplement drugs. Combination therapy with cobra venom factor and soluble complement receptor type I also has produced prolongation of xenograft survival experimentally [34]. Dalmasso and Platt [35], using human serum and porcine endothelial cells in an in vitro setting, showed that complement regulatory protein C1 inhibitor prevented complement-mediated activation of xenogeneic endothelial cells. Cobra venom factor also has been tested in a primate model, with some prolongation of graft survival [29, 30]. In addition, Pruitt and colleagues [36] showed that soluble complement receptor type I markedly inhibited total and alternative pathway serum complement activity after experimental porcine-to-cynomolgus monkey cardiac transplants.

Antibody Depletion/Inhibition
Just as strategies to deplete or inactivate complement, a primary activator of the hyperacute rejection response, have shown some success, similar attempts to deplete or inactivate naturally xenoreactive antibody have been studied experimentally. Tuso and associates [37] showed that removal of natural xenoantibodies to pig vascular endothelium by perfusion of blood through pig kidneys and livers led to significant reduction in the subsequent binding of IgM and IgG to pig kidney vascular endothelium. In conceptually similar experiments, Cooper and colleagues [38] observed that specific intravenous carbohydrate therapy coupled with triple pharmacologic immunosuppressive therapy prevented hyperacute rejection in ABO-incompatible cardiac allografting in baboons. This effect was thought to be due to binding and inactivation of antibody by the soluble carbohydrates. Cooper and colleagues proposed that this form of therapy might allow successful discordant organ xenotransplantation in man. Leventhal and associates [39] proposed immunoabsorption as a mechanism to remove the antibodies responsible for hyperacute rejection. In in vitro and in vivo studies, Leventhal and associates tested the efficacy of removing baboon and human antiporcine IgG and IgM natural antibodies. The use of an extracorporeal column to remove antibodies appeared to be safe and effective in these experimental settings.

Because the alpha-galactosyl epitopes of porcine endothelial cells are both primary triggers of the hyperacute rejection response and accessible surface antigens, Sandrin and associates [40] and LaVecchio and colleagues [41] proposed that enzymatic removal of the epitopes might diminish the cytotoxic effect of natural antibodies. Tests of this hypothesis showed that removal of the antigen reduced antibody binding and complement-mediated cytolysis.

	Genetic Engineering

Top Footnotes Abstract Introduction The Immunobiology of the... Zoonoses Ethical Issues Strategies to Achieve Successful... Genetic Engineering Donor-specific Graft Acceptance Predictions for the Future References

 
The most exciting recent work has been in the development and testing of genetically engineered xenogeneic donors [42, 43]. Two potential genetic engineering approaches are to add a gene that is not usually expressed in the donor animal (transgenic animals) and to remove or inactivate a gene that encodes a product important to the initiation of the immune response ("knockout"). Because control of human complement activation by porcine donor organs is considered paramount, the first experiments to produce transgenic genetically engineered donors have introduced transgenes for human complement inhibitors into the genetic material of the donor pig. In knockout experiments, because the gal epitope expressed on donor pig endothelium is so crucial to the initiation of the immune response, attention has been focused on producing animals that no longer express the gal antigen.

Transgenic Donors
Transgenic pigs that express human complement inhibitors have been produced. Experiments have been done using genes for three major human complement inhibitors, including decay-accelerating factor, membrane cofactor protein, and CD59. The genes for these human proteins have been isolated and incorporated in constructs that are introduced into a fertilized pig embryo at an early stage of development. Reintroduction of these manipulated embryos into pseudopregnant females leads to the production of a certain number of offspring that express the transgene in a heterozygous but stable fashion. One of these transgenic offspring that expresses a high level of the transgene product is selected as the "founder" pig. The transgenic founder expresses the gene for the complement inhibitor in all cells, including germ cells. Therefore, the gene is expressed in the progeny of this pig. Once a founder pig is selected, cross-breeding through several generations renders offspring homozygous for the transgene. Experiments by Cozzi and White [44] studying such transgenic pig hearts on a human blood perfusion apparatus have shown significant delay in destruction of the organ. Similar observations have been made by Kroshus and co-workers [45]. Preliminary experiments transplanting hearts from transgenic pigs into nonhuman primate recipients with anti-gal antibodies suggest promising improvements in long-term graft survival [46]. These results raise the possibility that the use of such a transgenic donor, together with conventional immune suppression, might lead to the clinical applicability of cardiac xenotransplantation. The experimental work using conventional immunosuppressive drugs in animal models and the observations that the cellular xenoresponse is similar to the response to an allograft may make this hypothesis plausible.

It is possible to imagine using this technology to introduce other potentially relevant genes, including major histocompatibility complex antigens and other regulators of the immune response. The advantage to the transgenic genetically engineered donor is the ease in producing copies of the original transgenic pig, because the porcine reproductive system takes care of the passage of the genetic information to subsequent generations.

Knockout Donors
Another genetic engineering strategy is to eliminate or inactivate a specific gene that produces a product critical to the initiation of the rejection response. This technique might be useful in xenotransplantation, for example, by knocking out the gene that encodes the enzyme responsible for attaching the gal carbohydrate antigen to the surface of endothelial cells. These knockout animals no longer would express the gal epitope. The production of such knockouts requires the availability of embryonal stem cells, which have been isolated for the mouse but not yet for the pig. The gal antigen murine knockout has been produced by Tearle and colleagues [47]. Studies using tissues from this mouse may be relevant to clinical xenotransplantation. In preliminary experiments, Tearle and colleagues have shown that substantially less xenoantibody from human serum binds to cells and tissues from knockout mice compared with normal mice. Similarly, there is less activation of human complement on cells from mice that lack the gal epitope.

	Donor-specific Graft Acceptance

Top Footnotes Abstract Introduction The Immunobiology of the... Zoonoses Ethical Issues Strategies to Achieve Successful... Genetic Engineering Donor-specific Graft Acceptance Predictions for the Future References

 
One of the primary limitations of present-day allotransplantation is the nonspecific nature of the immunosuppressive therapy given. Donor-specific recipient manipulations, including intrathymic inoculation and the induction of bone marrow chimerism, have been effective in settings of experimental allotransplantation. Several investigators have shown that pretreatment of rodent recipients with antilymphocyte serum and intrathymic inoculation with donor cells can lead to permanent acceptance of a rat cardiac allograft with normal immune reactivity to nondonor antigens. In related work, pretransplantation induction of bone marrow chimerism can lead to donor-specific permanent acceptance of solid organ allografts without long-term immunosuppression. Bone marrow chimerism is a state in which the individual expresses bone marrow–derived cells of both native and donor origin. Bone marrow chimerism in this setting is viewed as a preliminary state brought about to induce the acceptance of a subsequent donor solid organ graft. Kostecke and Ildstad [48] have identified a facilitator cell whose presence appears to promote the engraftment of donor bone marrow without the development of potentially lethal graft-versus-host disease. Graft-versus-host disease can be prevented by the depletion of mature T cells from donor bone marrow. However, engraftment rates are reduced when T cells are depleted. Specific attempts to preserve facilitator cells in T-cell–depleted marrow have led to increased rates of engraftment without concomitant increases in graft-versus-host disease. Both these strategies to produce donor-specific graft acceptance also have been applied to experimental xenotransplantation.

Intrathymic Inoculation
Attempts to translate directly current intrathymic treatment strategies that are effective in allotransplantation to models of xenotransplantation in rodents have not led consistently to significant prolongation of xenograft survival. For example, the administration of antilymphocyte serum followed by intrathymic inoculation of donor cells that are effective in allotransplants produces hyperacute rejection of hamster hearts by xenogeneic rat recipients [31]. Adding pretreatment with cobra venom factor eliminates hyperacute rejection without leading to further prolongation of graft survival. Combination therapy with antilymphocyte serum, intrathymic cells, and the antiproliferative agent cyclophosphamide, which prevents B-cell development and antibody formation, has led to some prolongation of xenograft survival in rodent recipients.

Bone Marrow Chimerism in Xenotransplantation
A potentially more fruitful approach to producing donor-specific acceptance of xenografts is the induction of multilineage mixed xenogeneic bone marrow chimerism using donor bone marrow cells:

1. Donor bone marrow
   1. T-cell–depleted
      
   2. Presence of facilitator cell
      
   
   
2. Recipient pretreatment
   1. Nonlethal irradiation
      
   2. Antibodies
      
   
   
3. Stable mixed xenogeneic bone marrow microchimerism
   
4. Tolerance to donor solid organs
   

As in allotransplantation, this involves bone marrow transplantation and subsequent establishment of stable expression of all blood elements of both the recipient and the xenogeneic donor. A number of studies from the laboratories of Ildstad and Sachs [49–52] have shown that the induction of xenogeneic bone marrow chimerism in rodents can produce long-term acceptance of skin and cardiac grafts. In early experiments, recipients were conditioned with multiple antibodies to formed elements of the blood plus lethal whole-body irradiation. It subsequently has been shown in rodents that the induction of mixed (donor plus recipient) xenogeneic bone marrow chimerism can be achieved using a preparative regimen that does not include lethal radiation. In these experiments, lower doses of whole-body irradiation are given in combination with antibodies and thymic irradiation. The animals achieve stable chimerism and demonstrate tolerance to skin grafts, but do not have graft-versus-host disease. Recently, Zhao and associates [53] from Sachs' laboratory have demonstrated skin graft tolerance across a discordant xenogeneic barrier. In these experiments, T-cell–depleted mice underwent thymectomy and grafting with xenogeneic fetal pig thymus and liver. The animals demonstrated recovery of functional immune cells. When tested for tolerance by skin grafting, donor-matched pig skin survived permanently, whereas allogeneic mouse skin was rejected rapidly. Control mice rejected pig skin within 26 days. Zhao and associates suggest that their study demonstrates that using the stringent criterion of skin grafting, tolerance can be induced across a widely disparate discordant species barrier.

In primates, stable xenogeneic chimerism has not been achieved. However, Latinne and associates [54] have advanced evidence that bone marrow microchimerism will produce tolerance to solid organ grafts in a pig-to-primate xenotransplantation setting. They pointed out that both humoral and cellular tolerance can be induced in rodents through pretransplantation induction of bone marrow microchimerism. They demonstrated that the human natural antibody target antigens expressed on swine endothelial cells also were expressed on cells derived from swine bone marrow. The presence of these key antigens on bone marrow cells suggests that successful bone marrow engraftment would lead to human immune tolerance to solid organ grafts that also express these antigens.

	Predictions for the Future

Top Footnotes Abstract Introduction The Immunobiology of the... Zoonoses Ethical Issues Strategies to Achieve Successful... Genetic Engineering Donor-specific Graft Acceptance Predictions for the Future References

 
These observations suggest a prescription for future success as proposed also by Fox and colleagues [55]. Long-term donor-specific tolerance to a discordant xenogeneic heart graft without toxic, long-term suppression will be achieved using a donor engineered to prevent rejection and to facilitate the induction of recipient multilineage mixed xenogeneic chimerism. The donor will be engineered by introducing relevant transgenes and knocking out critical native genes. Using a nonlethal preparative regimen, the human xenograft recipient will undergo pretransplantation induction of donor recipient xenogeneic mixed chimerism using cells from the engineered donor. After the induction of stable xenogeneic mixed bone marrow chimerism, the recipient will undergo solid organ transplantation using a graft from the same donor used to supply the bone marrow cells. The combination of genetically engineered donor and donor-specifically pretreated recipient will lead to permanent acceptance of the organ graft. The xenogeneically chimeric recipient will be tolerant to the replacement organ without the need for toxic and nonspecific long-term immunosuppression. Because the supply of genetically engineered donors will be essentially unlimited, and because each recipient will be pretreated with cells from his or her own donor, an inexhaustible supply of biologic replacement organs will be available for patients with cardiac and other organ failure.

How close is this goal? First, there can be no doubt that the need is great. Symptomatic congestive heart failure develops in hundreds of thousands of Americans annually. Tens of thousands of these individuals could benefit from replacement of their hearts if an appropriate biologic replacement were available. This is clearly an important public health problem. The loss of economic productivity and well-being that these patients suffer is enormous.

Second, the technology needed to develop this vision of cardiac xenotransplantation is available today. It is possible to produce transgenic animals and animals whose genes have been knocked out specifically. These animals have been demonstrated to be able to reproduce themselves. If primates are avoided and a lower mammal such as the pig is used, the ethical issues are manageable.

Third, the economics are right. Although it will be a feat of biomedical science to produce a genetically engineered donor pig, once the engineered animal is made, expenses decrease. Pigs breed readily in captivity and are relatively inexpensive to maintain. Of course, a medical-grade pig must be handled in a more careful manner than a food-grade animal, especially because care must be taken to avoid the risk of zoonotic infections. However, the cost of producing and maintaining these animals surely will be less than the cost of repeated hospitalizations and other presently available palliative therapy for patients with end-stage heart failure.

These observations suggest that experimental trials of cardiac xenotransplantation using genetically engineered animal donors will be undertaken in less than 5 years. It is conceivable, given the pace of advancement of biomedical science, that in less than 20 years, large numbers of cardiac xenotransplantations will be done in patients with heart failure.

	Footnotes

Top Footnotes Abstract Introduction The Immunobiology of the... Zoonoses Ethical Issues Strategies to Achieve Successful... Genetic Engineering Donor-specific Graft Acceptance Predictions for the Future References

 
Address reprint requests to Dr DiSesa, Department of Cardiothoracic Surgery, Allegheny University Hospital, MCP, 3300 Henry Ave, Philadelphia, PA 19129 (e-mail: disesa{at}auhs.edu).

	References

Top Footnotes Abstract Introduction The Immunobiology of the... Zoonoses Ethical Issues Strategies to Achieve Successful... Genetic Engineering Donor-specific Graft Acceptance Predictions for the Future References

 

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