Xenotransplantation in Pediatrics

Updated: Sep 10, 2014
  • Author: Bryan A Mitton, MD, PhD; Chief Editor: Stuart M Greenstein, MD  more...
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Overview

Overview

The Art and Science of Organ Transplantation

The most efficacious treatment for patients with end-stage, life-threatening organ failure remains replacement of the failing organ with a functional one. Human-to-human transplantation dates back to the late 19th century; Theodor Kocher, an Austrian surgeon and Nobel laureate, first successfully transplanted a human thyroid in 1883. In the second half of the 20th century, discoveries in immunogenetics and immunosuppression paved the way for major organ transplantation with excellent outcomes.

Despite tremendous advances in our understanding of the pathophysiologic processes that lead to organ failure as well as the continual evolution of supportive care approaches that can sustain patients’ lives, organ transplant remains the definitive therapy of choice for severe organ dysfunction. Transplant affords individuals the chance of cure in a way that no other modern medical technology can.

To put the phenomenal success of transplantation in human terms, patients who require heart transplantation and are designated as 1A priority candidate on waiting lists in the United States have a life expectancy of less than a week. If they undergo transplantation, they typically experience more than 10 additional years of active life. Approximately 2000 heart transplants are performed annually in the United States. [1] Together, 27958 organ and tissue transplants were performed in the United States in 2008.

Fewer than one in 20 individuals who would be considered candidates for transplantation are able to undergo the procedure, due to a shortage of human organ donors. For this reason, organ transplantation has been called a victim of its own success. [2] The Organ Procurement and Transplantation Network/United Network of Organ Sharing (OPTN/UNOS) waiting list has nearly 3000 heart transplant candidates; this does not, however, estimate the full number of patients who would potentially benefit from a heart transplant, as this figure represents only those deemed to be in greatest need.

Indeed, for those patients in need of a transplant, rigorous criteria exist to determine their position on the transplant wait-list for nearly every transplantable organ. For instance, the International Heart and Lung Transplant society has estimated that more than 50,000 Americans annually could benefit from heart transplantation if donors were available. [3] For hearts, kidneys, livers, and pancreatic islets, approximately 500,000 transplants or more could be performed annually in the United States and more than 1.3 million transplants could be performed annually in the developed world if organs were available.

Xenotransplantation as a Solution

Although stem cell technology and tissue engineering are potential solutions to the organ shortage, xenotransplantation (transplanting organs and tissues from a different species to humans) has generated considerable interest as a potential solution. If this technology could be fully developed, it would provide a renewable, reliable source of organs for patients in need. The ideal species for these organs would be one that has the following characteristics:

  • Is, or could be made to be, immunologically similar to humans
  • Has a short generation time, with high number of offspring
  • Shares few viral, bacterial or multicellular pathogens in common with humans
  • Has organs of similar size and function

Xenogeneic organs would have several advantages over organs derived from human donors. For unpaired organs, donors are sometimes identified only a short time before or after brain death, and their organs are procured under urgent or emergent conditions; many times, the organs are transported from center to center, delaying implantation. These factors are critical, because both these situations can lead to non-viability of the organs or blood coagulation that disrupts full organ reperfusion following implantation.

The source of the organ may also not be ideal, even under controlled conditions. The transplant organ may come from a suboptimal donor with advanced age and chronic medical conditions or from a carrier with undetected infectious agents or malignant cells. In contrast, donor animals may be raised under controlled conditions, specifically for use as an organ donor, and selected for size and age. Potential pathogens can also be eliminated if these animals are bred in pathogen-free conditions.

For those reasons, pigs are often considered the optimal source of xenotransplant organs, and the potential for their use in organ generation is discussed in detail below. Advantages are as follows:

  • Many pig organs are similar to the human counterparts in size, anatomy, and physiology
  • Pigs can be bred quickly under standardized, clean conditions
  • Pigs can be readily genetically modified
  • Unlike primates, pigs share relatively few pathogens with humans

Xenografts may provide medical advantages as well. These grafts can be resistant to human pathogens specific to human tissues, such as HIV, hepatitis, and human cytomegalovirus. Furthermore, though immune and non-immune organ rejection remains a major hurdle in xenotransplantation, the disparities between the donor’s and recipient’s immune systems could be exploited. For example, xenogeneic pancreatic transplants may be able to escape the cellular and humoral autoimmune causes of Type 1 diabetes by virtue of its foreign nature.

The State of the Art

To date, successful whole-organ xenotransplantation is limited to animal models; no successful animal-to-human whole-organ transplants have been performed. The field faces four major challenges. First, xenogeneic transplants are subject to severe rejection (hyperacute, acute and chronic), due to large antigen disparities between humans and other species that elicit multiple mechanisms of immune rejection.

Second, for some tissues such as the liver, the physiological function of the xenogeneic organ is insufficiently close to the human to provide long-term support. Thus, xenotransplantation may not be an option unless significant tissue engineering is first undertaken.

Third, infectious agents arising from the donor species are a major concern. Two infectious agents known to arise from animal sources (xenozoonoses) include the influenza A pandemic virus and the human immunodeficiency virus (HIV). The influenza A virus periodically cross-infects humans following genomic rearrangement in birds or livestock with deadly effect, such as during the ‘avian flu’ pandemic of 1918 and the feared but ultimately mild ‘swine flu’ of 2010. HIV arose during the 20th century from the simian immunodeficiency virus and continues to be a major public health issue.

Infectious agents transmitted by transplantation have the theoretical potential to infect humans who are immunosuppressed, or to evolve into a novel infectious agent capable of being transmitted from human to human.

Fourth, a variety of ethical, social, and cultural issues surround this topic. As the technology of this field advances, society must give thorough consideration to these issues. This conversation should also be extended to the regulation of this technology, the economics of xenotransplantation and the medical threshold for the use of xenogeneic organs versus human organs.

In spite of these issues, xenotransplantation offers the hope of an adequate supply of organs for those in need. At present, it is estimated that fewer than 5% of patients deemed transplant candidates undergo transplantation. To reiterate, this number is unfortunately an understatement of the true medical need for organ transplantation. Should the obstacles to the xenotransplantation be overcome, the health and lives of countless patients could be improved.

With regard to the specifics of xenotransplantation, this article will discuss the following five major concept areas:

  • Xenograft rejection
  • Xenograft function
  • Xenozoonosis
  • Clinical xenotransplantation
  • The future of xenotransplantation
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Xenograft Rejection and Prevention of Rejection

The primary obstacle to successful xenotransplantation is graft rejection. In any transplant, the likelihood and severity of rejection is related to the extent to which the host immune system recognizes the transplanted tissue as foreign.

In the case of allogeneic (human to unrelated/non-identical human) transplantation, Human Leukocyte Antigen (HLA) molecules play a major role in determining immune tolerance of the foreign organ. Though a host of other cell surface and intracellular markers have been identified that can predict immune tolerance in a variety of organs, the degree of acute and chronic rejection can be predicted by how well-matched the donor and recipient HLA molecules are.

The HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ and HLA-DP genes encode the human Major Histocompatibility Complex (MHC) molecules. HLA-A, -B and -DR are the most genetically diverse HLA molecules in the human genome, and have roughly 1000, 1600, and 870 known alleles, respectively. [4]

Minor differences in these molecules can lead to acute or chronic organ dysfunction, failure, and rejection. This effect is thought to be mediated at least in part due to differences in intracellular processing of antigens and their subsequent presentation on MHC molecules. [5] This influences the extent to which the host immune system tolerates the donor HLA molecules. A number of other antigens, such as Kir, are coming to the fore as important co-determinants in transplantation success. [6]

While HLA matching is a major determinant of transplant success, the antigens that affect xenogeneic (animal to human) transplants remain undefined. Xenogeneic transplants would present a number of challenges to the human immune system; HLA disparity could be chief among these.

The chimpanzee, the contemporary animal species most genetically similar to humans, is reported to share 98.77% nucleotide and >99% amino acid identity with humans. [7] However, comparing the genetic sequence of the 3.7-Mb human chromosome MHC gene complex with the corresponding genomic region of the chimpanzee has revealed only 86.7% identity. [8] In contrast, a recent major study of human HLA variability, taken from 23500 individuals worldwide, showed that most HLA alleles differ by fewer than 30 nucleotides in the genes’ coding regions. [9]

Our best current evidence tells us that our closest living phylogenetic relatives are quite different from us immunologically. Indeed, there remains a great deal of work to be done to first define and characterize the unique features of HLA alleles that make humans different from other humans; examining how a human immune system might tolerate a foreign HLA molecule remains a critically important question.

A second significant challenge presented by xenogeneic transplants concerns the presence of antigens on animal tissue with no analogous gene product in humans and vice versa. Specifically, humans differ from each other only in allelic variation; humans differ from other species by the sets of genes expressed.

Tissue mismatches and humoral incompatibilities known to cause rejection

Perhaps the best-studied example of antigens causing difficulty in xenotransplantation is the Galα1-3Gal epitope, which is a post-translational modification of the extracellular domain of a variety of cell surface proteins. [10] Though virtually all mammalian species express this sugar moiety on proteins of the luminal surface of endothelial cells, humans and Old World primates do not. [11] Humans develop preformed antibodies against this epitope that can constitute up to 1% of the total circulating immunoglobulin in human serum. Consequently, implantation of organs rich in vasculature has a high chance of resulting in hyperacute or acute humoral-based rejection with thrombosis, ischemia, and hemorrhage of the organ.

Porcine knockout models of the galactosyltransferase enzyme have been developed, in which the absence of this enzyme prevents the placement of the Galα1-3Gal sugar moiety on key proteins on the endothelial surface. Transplant experiments show that this genetic manipulation alone reduces hyperacute rejection in pig-to-primate xenotransplantation, though significant rejection still frequently occurs in these experiments. [12] Specifically, kidney xenotransplants from galactosyltransferase (GT) knockout pigs into nonhuman primates eliminated hyperacute rejection but not overall acute xenograft rejection.

Lung xenografts and heart xenografts similarly were not hyperacutely rejected, but were ultimately rejected in this animal model. [13, 14] Cytotoxic antibodies to non-Gal antigens were observed pretransplant, offering an explanation for this phenomenon. [15, 16]

To examine whether additional endothelial cell antigens may contribute to rejection, or whether the Galα1-3Gal epitope is alternatively generated in the GT knockout pigs, Yung et al performed a series of elegant experiments. [17] Their research addressed whether carbohydrate epitopes similar to to Galα1-3Gal can be generated by alternative pathways in the GT knockout pigs.

The authors examined a large panel of available monoclonal anti-Gal antibodies and lectins and used ion trap mass spectroscopy to evaluate the expression of the Galα1-3Gal epitope as well as that of epitopes that may immunologically resemble this epitope. Aortic endothelial cells derived from GT knockout pigs did not express the Galα1-3Gal epitope at detectable levels. However, lectin staining showed increased presence of blood group H-type sugar structures in GT-knockout endothelial cells compared with wild-type endothelial cells. While these data confirm the absence of the antigenic Galα1-3Gal epitope, higher expression of other potentially immunogenic carbohydrate antigens may mediate rejection of these organs.

Exciting studies are under way to circumvent the antigenicity of the Galα1-3Gal epitope. GAS914 (Novartis) is a soluble, polymeric form of α-gal trisaccharide (Galα1-3Galβ1-4GlcNAc) and functions as an antigenic decoy. Intravenous administration of GAS914 (GAS) reduced xenoreactive antibodies in nonhuman primates, suggesting that this and similar agents could be developed to “reverse chelate” xenoreactive antibodies. [18]

Another potential problem of the xenografted endothelium is that components of human blood may alter porcine endothelial cells (pECs) and promote intravascular coagulation. After contact with human/primate blood, clots quickly develop in pECs. The increased clot formation may be immunologically mediated, or may be the result of specific hormone regulators.

Lee et al. recently examined this question. [19] Incubation of pECs with human tumor necrosis factor alpha (TNF-α) or interleukin-1α (IL-1α) caused an increase in porcine tissue factor (TF) activity and mRNA; however, those changes were not seen when these cells were exposed to 20% human serum or the complement protein C5a. The upregulation of TF expression by human TNF-α and IL-1α reduced coagulation time, while small interfering RNA (siRNA)-mediated knockdown of TF expression prolonged coagulation time.

Interestingly, incubation of porcine endothelial cells with human whole blood increased human TNF-α levels in serum, suggesting human TNF-α is released by human white cells following exposure to pECs, which in turn causes coagulation. Thus, TNF-α may be an important mediator of procoagulant changes in the porcine endothelium.

In spite of these innate immunologic barriers, prolonged function of vascularized pig xenografts in nonhuman primates is possible if the correct combination of immunosuppression is provided. Heterotopic pig heart xenografts have survived for up to 6 months in baboons. [20] However, it should be noted that these baboons were tremendously immune suppressed with anti-CD20 antibody (rituximab) and anti-thymocyte globulin (ATG) at induction, splenectomy, apheretic removal of anti-Gal antibodies, plus tacrolimus, sirolimus, and steroids. The donor pigs were transgenic, expressing the CD46 human complement inhibitor. This trial did at least demonstrate the possibility of sustained successful xenotransplantation, though these animals were arguably immune-ablated.

These examples illustrate some of the difficulties inherent to whole organ allografts; there is a large immunologic barrier to successful xenotransplantation. This barrier can be potentially overcome with the use of heavy immune suppression. Are there other ways to restore organ function to patients without the risks inherent to heavy immune suppression?

Cellular xenotransplantation circumvents the circulation

To circumvent some of these issues, attempts have been made to simply deliver xenogeneic cells rather than whole organs. This represents a novel solution to overcoming host rejection; only one cell type is presented to the host immune system, rather than the several cell types that may be contained within a whole organ (vascular endothelium, fibroblasts, parenchymal cells, and/or immune cells). The purified cells can be contained within a synthetic capsule that can be perfused directly or indirectly, and thus would be less likely to suffer hyperacute rejection. Alternatively, these cells can be infused directly via the portal vein, and allowed to “land” wherever these cells find a viable environment.

The Edmonton protocol, as an example, involves infusion of pancreatic beta cells in this manner, and there has been limited clinical success with this method. [21] Unfortunately, some 70% of the infused islet cell mass is lost within 24 hours in allogeneic human transplants. Nonetheless, encouraging progress has been made in the xenotransplantation field in this regard.

Initial experiments demonstrated that xenogeneic islet cells were also lost in the immediate post-infusion period through a combination of complement and intravascular coagulation. The process of procuring the islets from the pancreas induces proinflammatory factors in the islets, such as monocyte chemotactic protein (MCP)-1, IL-1β, TNF-α, interferon (IFN)-γ, IL-6, and IL-8, which may mediate some of the cell mass loss early after infusion.

On the other hand, prolonged engraftment of porcine islets and prolonged insulin-free survival in cynomolgus monkeys has been achieved using wild-type pigs rather than α-Gal knockout with T-cell–directed immunosuppression. [22]

In a remarkable set of experiments, islet cell clusters isolated from neonatal pigs (aged 1-2 d) were used to achieve prolonged insulin-free survival in rhesus macaques that had undergone total pancreatectomy. [23] These islet clusters contained endocrine precursor cells able to proliferate, differentiate, and reverse hyperglycemia in mice and pigs.

The key to these experiments was adequate immune suppression; the macaques were given antibodies to IL-2 receptor and CD154, plus maintained on immune suppression that included belatocept and sirolimus or mycophenolate. This methodology circumvented hyperacute rejection and provided good long-term functional results.

As alluded to earlier, encapsulation of porcine islets has also been pursued as a method to prevent acute rejection by protecting the graft from invading lymphocytes and antibodies. The initial formulations of polyalginate encapsulation led to capsule fibrosis and ultimately breakdown of the capsule. Recently, success has been achieved with the use of newer alginate macroencapsulation devices. [24] Adult pig islet cells were encapsulated in microcapsules implanted in the kidney or in a subcutaneous macrodevice in streptozocin-treated cynomolgus monkeys; in the latter group, diabetes was controlled for 6 months without immune suppression. This remarkable advance could pave the way for a variety of future human trials.

The regulation of T-regulatory cells

Still another approach involves inducing host immune tolerance and tissue accommodation; if the host immune system were ‘taught’ to tolerate specific antigens of the xenotransplant, perhaps rejection issues could be minimized.

Immune tolerance to ‘self’ is centrally maintained through clonal deletion of self-reactive lymphocytes in the thymus and peripherally in suppressor or regulatory cells that block self-reactive lymphocytes that escape thymic censure. [25] Specific immune tolerance is very difficult to achieve in adult recipients. One approach would be to induce mixed hematopoietic chimerism, wherein the recipient of an organ transplant also receives a simultaneous bone marrow transplant. In this way, the transplanted immune system would theoretically be able to tolerate the transplanted organ. [26, 27] However, this could obviously also result in graft-versus-host disease.

Thus, some work has been done looking at the potential of using T-regulatory cells to modulate the host immune response. T-regulatory cells are circulating CD4+/CD25+ T cells that facilitate the maintenance of tolerance to self. [28] These cells express the transcription factor foxP3, which is integral to the function of inhibiting self-reactive T cells. They mature within the thymus and are positively selected for cells with a T-cell receptor reactive to class II antigen expressed on thymic dendritic cells, and they are known to play a role in graft versus host disease after allogeneic transplants of hematopoietic stem cells. [29]

The broad idea is that one could use T-regulatory cells to mediate tolerance of the transplanted tissue. Ideally, regulatory cells would lead to specific inhibition of both adaptive and innate immunity protecting the xenograft but leaving the recipient sufficiently immune competent to defend against pathogens.{{Ef27} [30]

Initial work has shown that T-regulatory cells do have the potential to suppress host immune responses to xenogeneic organs. For example, expanded CD4+/CD25+ regulatory T cells cultured from baboon peripheral blood block the reaction of baboon responder lymphocytes to pig stimulator cells. [31] Indeed, transplantation of pig thymic tissue into nonhuman primates leads to tolerance of porcine pancreatic beta cells. [32] Previous work with pigs that were chimeric with human lymphocytes demonstrated that the chimeric lymphocytes specifically inhibited the human versus pig mixed lymphocyte reaction. [32]

Generating T-regulatory cells from chimeric pigs can provide specific immune tolerance to the pig xenograft. The tolerance is induced prior to organ transplantation and outside of the recipient, effectively avoiding the risk and complications often associated with the induction of tolerance. Using this approach, in two preclinical trials of diabetic monkeys, five of six transplants demonstrated prolonged survival and function of the islet xenografts without any posttransplant immune suppression. One monkey was insulin free 31 weeks post-transplant when he died of unrelated causes. [33]

Accommodation is the term applied to the phenomenon whereby cells, principally vascular endothelial cells, adapt to resist injury by complement. It is typically seen in allograft recipients with antigraft antibodies, such as with an AB mismatch. [34] If rejection can be prevented for 2 or more weeks, such as with plasmapheresis, the graft then becomes resistant to injury, even in the presence of antigraft antibodies.

Multiple protective proteins, such as heme oxygenase-1, protein inhibitor of apoptosis, and A20, are induced that protect the cells from apoptosis. [35] The protection is antigen nonspecific, providing protection to multiple antibodies.

Other new technologies are being actively explored to reduce the immune suppression required for prolonged xenograft function. The need for immune suppression could be reduced by reducing the overall antigenicity of the xenograft. Fetal pig tissues have a reduced density of antigens. These could be transplanted into the recipient, where they later mature. [35] Another approach is to induce specific immune tolerance to the pig tissue by reconstituting the immune system through a pig thymus within the recipient.

Still another approach is to block co-stimulation of the mature T-effector cells by inhibiting the second signal with monoclonal antibody, such as anti-CD154. Co-stimulator blockade causes the T-effector cells to undergo apoptosis. When combined with donor specific transfusions, prolonged acceptance of allografts has been achieved; unfortunately, anti-CD154 antibodies appear to be associated with thromboembolic complications. Alternatives to anti-CD154 are being developed to disrupt costimulation, such as antibodies to CD40. [36]

Designing the donor

Another strategy for using pigs as organ donors is to genetically modify the donor population. The overall thrust of this idea is to eliminate donor tissue antigens that are especially immunogenic, and to perhaps engineer the donor tissue to express antigens that are recognized as ‘self’ by the recipient’s immune system. The balancing act of altering the donor tissue would involve creating an organ tolerated in vivo by both donor and host immune systems.

Some intriguing work has been done to avert vascular rejection through thrombotic microangiopathy by engineering donor tissue. It is known that porcine thrombomodulin fails to activate primate protein C, an important anticoagulant. A rodent model of tissue engineering has been successfully used to demonstrate proof-of-principle. [37]

Recently, another group generated transgenic pigs that express the human heme oxygenase I (HO-1), an anti-apoptotic protein. Perfusion of these animals’ kidneys with human blood demonstrated that while control organs developed microthrombi and survived only about 60 minutes, transgenic organs survived for 240 minutes and did not develop microthrombi. [38]

Finally, CD8+ cytotoxic T lymphocytes are known to damage transplanted islet cells through a Fas ligand–mediated pathway. Transgenic expression of ‘decoy’ Fas ligand greatly improved survival of transplanted cells, and represented an interesting tissue engineering approach to evasion of immune response. [39, 40]

Overall, the immunologic barrier across species is the greatest barrier to overcome; all the efforts described above represent major advances in our understanding of cross-species antigen tolerance. As efforts continue, we begin to understand the interplay begin the immunologic challenge presented by a foreign organ and the immune system’s ability to tolerate this challenge.

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Xenograft Function

Naturally, the ability of any xenograft to replace the function of a failed human organ depends on multiple factors. To date, the technology has not yet advanced to the point where we can carefully examine the functionality, longevity or long-term physiological compatibility of xenotransplanted tissues; animal recipients of xenotransplants remain very immunosuppressed during experimentation, and studies of long-term successful transplants in humans or animals are lacking. Nonetheless, pig organs in particular have been studied and offer clues as to whether xenogeneic organs could provide appropriate function for a human patient.

Comparison of Human and Pig Organs

The data suggest that pig hearts, kidneys, and pancreases could serve as adequate substitutes for human organs.

Interestingly, the pig heart maintains systolic and diastolic pressures similar to the human heart; it is approximately the same size, has similar architecture, and beats at the same rate. [41] Pig hearts orthotopically transplanted into nonhuman primates provide adequate circulation with good blood pressures until the graft is rejected, suggesting its feasibility as a pump. [42] It is unclear whether cardiac endocrine function, such as its responsiveness to epinephrine or acetylcholine or its ability to elaborate atrial natriuretic factor, would remain similar following transplant.

Pig and human kidneys are similar with respect to physiologic parameters such as renal blood flow, glomerular filtration rate, and creatinine clearance. [43] However, little information is available on the endocrine functionality of a transplanted porcine kidney. For instance, porcine erythropoietin is about 80% homologous with human erythropoietin, though it is unclear whether this would function to fully stimulate the human erythropoietin receptor. [44]

As for the pancreas, porcine insulin differs from human insulin by just a single amino acid, and provides excellent control of glucose metabolism. However, the connecting C-peptide of humans and pigs differs at 11 of 31 amino acids, which may affect microvascular blood flow and the development of chronic vascular pathology. [45, 46]

There is a limited clinical track record, however, for the success of these organs in humans. In the 1960s, Dr. Keith Reemtsma performed 13 chimpanzee-to-human kidney transplants; though most failed within 1-2 months from immune rejection or infectious, one patient lived 9 months with the transplanted organs, indicating reasonable functionality in this patient. [47] Dr. James Hardy was the first to perform a baboon-to-human heart transplant in 1964, though the heart was not large enough to sustain function and failed in a few hours. [48]

However, perhaps the most well-known cases of xenotransplantation was that of “Baby Fae”. Dr. Leonard Bailey transplanted a baboon heart into an infant in 1983; this patient survived only 20 days, as the heart failed of rejection. [49] Thus, there is little available clinical data as to how well a transplanted organ would truly survive, especially in the context of modern-era immunosuppressive and supportive care techniques.

On the other hand, we may predict prima facie that some xenogeneic organs such as the liver might be inadequate for long-term support of humans. [50] The liver is arguably the organ most critical for regulating overall human metabolism; it stores fat and glucose and metabolizes medicines and toxins. Hepatocytes produce many proteins such as albumin, α1-antitrypsin, and critical clotting factors. Thus, a great deal of manipulation of the porcine liver would need to occur before it could be expected to fully replace a human liver over a long period.

Nevertheless, a number of attempts have been made to transplant this organ for patients in dire need. As discussed below, Dr. Tom Starzl performed a number of liver transplants between primates and humans in the 1960s, but had little success. Two other liver transplants were performed in the 1990s, and one patient survived 70 days. [51] The clinical results did not stimulate further clinical attempts.

Thus, in our current state, it is difficult to estimate the ability of xenogeneic organs to replace human organs with longstanding benefit; the graft is lost to immune rejection too often and too soon. It should be noted, however, that in its infancy, allogeneic organ transplants suffered a similar fate. Tremendous advances in immune suppression and supportive measures permitted extended survival of human-to-human transplants. Perhaps our experience with allogeneic transplants will extend to xenogeneic organ transplants and lead to future success.

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Xenozoonoses

One of the most-discussed and feared risks of xenotransplantation is the potential for infectious agents to be transmitted from the donor animal to the patient. History teaches us that rarely, infectious agents that infect only one set of species are able to ‘jump’ to other species, essentially creating a new pathogen with sometimes devastating results.

As alluded to earlier, the influenza A virus periodically cross-infects humans following genomic rearrangement in birds or livestock with deadly effect, such as during the ‘avian flu’ pandemic of 1918 and the feared but ultimately mild ‘swine flu’ of 2010. The human immunodeficiency virus (HIV) arose during the 20th century from the simian immunodeficiency virus (SIV) and continues to be a major public health issue; this arguably represents the most catastrophic example of animal-to-human adaptation of an infectious agent in modern history.

At present, it is difficult to estimate the risk of a novel pathogen that may arise from xenotransplantation; however, the risk is not ‘zero’. It should be noted that some evidence suggests that SIV itself may have originally arisen from a mouse retrovirus; through the course of history and species interaction, the virus moved to humans, with deadly results. Human T-lymphocyte virus (HTLV) 1 and 2 also both have origins in non-human primates, and these are well-known causes of disease in immune-compromised humans. [52]

The issue of the possible genesis of a new infectious agent may be especially important in organ recipients who are immunosuppressed, as the immunosuppressed state may facilitate the survival and dissemination of the novel virus within the host. During the 1990s, a moratorium against xenotransplantation was actively discussed in Europe and North America, in part for these reasons. Although a formal moratorium never took place, the US Food and Drug Administration (FDA) has issued a series of guidelines for xenotransplant experimentation, the latest in 2003. [53]

Infectious Agents

In general, bacterial pathogens that may be transmitted from animal to human can be effectively managed with current antibiotics. Transmission of bacterial pathogens is extremely rare in human-to-human transplants, and these species do not lie ‘dormant’ in their hosts; bacteria’s strategy is rapid expansion without latency, with few exceptions. Many infectious species of bacteria have no specific animal host; some bacterial species are only opportunistic, infecting the young, the old and the immunosuppressed, and these are naturally of concern. However, most antibiotics remain broad-spectrum enough to cover most organisms.

Thus, concerns focus on viral zoonotic agents, especially lysogenic viruses that are able to stably infect a eukaryotic cell. These include a wide variety of retroviruses and DNA viruses.

Retroviruses, by nature of their ability to stably integrate into the host’s genome, are difficult to eliminate from animal stock and pose the theoretical risk of infecting human cells and integrating into human DNA. Depending on where this virus integrates and its ability to replicate following integration, serious risks include organic dysfunction, as well as the development of neoplasm. There is also the possibility that a novel human-directed infectious agent may be generated through recombination with other already-present human-directed viruses in immunosuppressed patients, though the likelihood of this occurring is difficult to ascertain.

Porcine endogenous retroviruses (PERVs)

Important work has been done to identify infectious agents present in pigs that pose a potential threat to human cells. The porcine endogenous retroviridae (PERV) have received considerable attention. Careful studies have shown that of the three related viruses included in this family, PERV-A, -B and -C, only PERV-A and -B can infect a variety of mammalian cells; PERV-C is restricted to pigs. [54]

In 1997, it was first reported that co-culture of human and porcine cells could produce active infection of human cells in culture; this offered the first suggestion for these viruses as potential xenozoonotic agents. [55] Careful examination revealed that the PERVs can infect and replicate in human endothelial kidney (HEK) cells, they can only infect primary bone marrow cells.

PERVs can infect cells from the rhesus monkey, baboon, gorilla and chimpanzee. [56] They are unable to detectably infect any rodent species. Strain analyses of a variety of pig herds has found that the porcine genome contains between 10 and 100 proviral integration sites, though it is proposed that the majority of these are inactive/non-productive coding regions. [56]

Despite considerable research and the fact that a major portion of the world’s population consumes pork products, no infectious symptoms, syndromes or recognizable pathologies have ever been directly attributable to PERV infection. In a study of 14 pig-breeding farmers undergoing liver transplant, none had detectable anti-PERV antibodies, PERV DNA, or PERV RNA. [57] Individuals in the control group (n=82) were similarly negative. Moreover, in a number of pig-to-primate studies involving a variety of organs and immunosuppressive regimens, PERV has not once been detectably transmitted to the host species. [56] Would these data apply to human trials?

Again, in many contexts, PERV has never been detected in patients exposed to porcine cells or blood products. Studies of individuals who received pig-derived blood products such as plasma or factor VIII, islet cell transplantations, fetal pig mesencephalon/lateral ganglionic eminence cells, or extracorporeal kidney or liver perfusions have revealed no evidence of transmission of PERV. [56] Thus, though it is important to consider the potential risks of viral xenozoonoses, none have been identified to date in pig-to-primate or pig-to-human xenotransplantation procedures.

Pathogen-Free Animals  

Investigations are under way to examine whether the potential of PERV infection, or infections from as-yet unidentified pathogens, could be mitigated by raising and maintaining pigs in pathogen-free conditions. Lines of transgenic pigs have been produced that synthesize silencing interference RNA for PERV. [58, 59] These pigs have substantially reduced PERV expression (>20%). While this is an attractive idea, the nature of retroviruses may make complete silencing of PERV gene expression difficult.

Another approach to creating PERV-free pigs is selectively breeding pigs based on PERV genomic load, with the intent of selecting against the viral “allele”. [60] In this study, boars had an average of about 15 copies of PERV per cell, and sows averaged about 10 copies of PERV; through generations of breeding, two piglets were obtained that were PERV-C negative. This suggests that a strain of pigs could be bred to have no detectable copies of PERVs, so long as they were kept in a controlled environment without exposure to PERV.

Other stock in North America has been found to harbor little to no detectable PERV-A, PERV-B, or PERV-C. [61] One sow was found to be totally free of all three types of PERV. Most of the herd was free of PERV-C, and half of the herd was free of PERV-A, as well. In quantifying the PERV types, five pigs (one boar, four sows) were identified as PERV-A-negative and PERV-C negative, with one copy of PERV-B per cell. PERV-free breeders could potentially be realized within one generation.

While PERVs have received the most attention, based on their ability to infect and replicate in certain human and primate cells, other ubiquitous exogenous viruses may pose danger to xenograft recipients. [62, 63] Examples of viruses that potentially could be passed from swine to humans include the following:

  • Influenza viruses
  • Nipah virus
  • Menangle virus
  • Hepatitis E virus
  • Encephalomyocarditis virus
  • Japanese encephalitis

While some viruses may have a low potential to infect recipient tissues, they could adversely affect the graft, depending on tropism. Porcine lymphotropic herpes viruses, for example, may lead to post-transplant lymphoproliferative syndrome in recipients receiving porcine lymphocytes. [64, 65] Porcine cytomegalovirus (CMV) and porcine encephalomyocarditis virus may adversely affect vascular xenografts such as hearts. [66]

Under strict conditions, exogenous viruses could be eliminated from a selected pig donor herd, through careful husbandry and persistent monitoring of the herd. To produce a ‘clean’ herd, some have proposed deriving colonies from hysterotomy-delivered, colostrum-deprived piglets. This eliminates the colonization of normal bacterial and fungal pathogens, which naturally occurs following transvaginal delivery and subsequent nursing. This would not, however, eliminate pathogens that pass the placental barrier, such as circovirus, arterivirus, and parvoviruses. [60, 67] Porcine lymphotropic herpes viruses and encephalomyocarditis virus may also escape this procedure. [68, 69]

The development of a clean herd of donor animals does actually offer an advantage over allogeneic transplants; human donors are certainly not raised under pathogen-free conditions. Human CMV, Epstein-Barr virus (EBV), varicella-zoster virus (VZV), HIV, HTLV-1 and HTLV-2, and herpes simplex virus 1 (HSV-1) and HSV-2 are all potential threats to the immunocompromised patient that can be transmitted through allogeneic transplant.

Obviously, all donors are rigorously screened for many of these viruses, depending on the context, but the recipient’s need may sometimes trump the donor’s immune status to some of these agents. In this sense, xenotransplantation offers one advantage over allogeneic transplantation regarding infectious agents contracted through transplantation.

Interestingly, the reverse question can also be asked: could human viruses infect and harm the xenotransplant? It is known that human CMV, human adenovirus, and hepatitis C virus can infect pigs. [70] Thus, human xenotransplant recipients will still need to be screened for these viruses, just as they are prior to allogeneic transplants.

In summary, there are certainly numerous infectious risks involved with any organ transplant. Currently, infections pose an enormous risk to immune-compromised hosts in the post-transplant state. Xenotransplantation poses novel risks, however. It is difficult to estimate the risks posed to xenogeneic organ recipients by infectious agents restricted to pigs or primates.

However, the societal risks must also be considered; there is a non-zero risk that a novel infectious agent could arise someday from the transplantation of a xenogeneic organ into an immune-compromised human host. There is plenty of evidence that over the span of history, numerous infectious agents have jumped across species, often with disastrous results. A very careful consideration of these risks will be required before xenotransplantation could be applied in any significant way to clinical practice.

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Evolution of Clinical Xenotransplantation

The use of animals as organ and tissue donors extends back to the 17th century. At a time when medical technology and understanding of immunology and physiology were primitive, animals were the preferred source.

Indeed, the first formal transplants from animals to humans pre-date human to human transplants by hundreds of years. Jean Baptiste Denys performed the first blood transfusion into a patient in 1667, using blood taken from a sheep. [71] Numerous reports exist of the use of skin grafts, taken from sheep, dogs, cats, rats, chickens, pigeons or rabbits, to aid in human wound healing. [72] Interestingly, the first corneal transplant was a xenotransplantation, and pre-dates the first human-to-human corneal transplant by 65 years. [73]

In 1906, Jaboulay performed the first vascular xenotransplants, transplanting kidneys from a pig and a dog into patients with renal insufficiency. [74] In 1963, Hitchcock transplanted a kidney from a baboon into 65-year-old woman; it functioned for 4 days. [75] Reemtsma and Starzl achieved a measure of clinical success transplanting kidneys from nonhuman primates into human recipients, as mentioned above. [47, 76]

In the early 1990s, a number of remarkable successes were achieved for a variety of organs and cells. Porcine islets prepared from fetal pigs were transplanted into diabetic patients with modest immune suppression. Porcine C-peptide was monitored in the urine and was maintained until the grafts were eventually rejected. [77]

In 1992 and 1993, two orthotopic xenotransplants were performed in which baboon livers were placed into patients with liver failure related to hepatitis B virus infection. [51] Multidrug immunosuppressive therapy was administered to prevent cellular and antibody-mediated rejection. The patients survived 70 and 26 days, respectively, and the grafts provided at least partial function. Although the grafts did not undergo hyperacute or acute rejection, one of the patients developed aspergillosis related to the immune suppression.

It is known that baboon CD4 cells lack the receptor for the HIV virus; thus, a bone marrow transplant was performed for a patient with HIV. The patient was given non-myeloablative conditioning therapy; ultimately, the patient rejected the baboon cells, but there was a noted reduction in HIV viral load for 11 months. No xenozoonotic infections were noted in this immunocompromised patient. [78]

Dopaminergic neurons from a fetal pig were transplanted into the brain of a patient with Parkinson disease. [79] The transplant significantly improved the patient’s clinical course. Seven months later, the fetal pig neurons were identified as still present. The implantation of pig neural tissue into an ‘immune privileged’ environment of the brain reduced the risk of rejection. Unfortunately, a subsequent controlled trial failed to demonstrate a statistically significant difference with the control group. [80, 81]

Patients in acute liver failure have been supported for a few hours to days with extracorporeal liver perfusion (ECLP) while a human liver donor is sought. [82, 83] Blood from the patient is perfused through the pig liver and returned. These procedures indicate that the pig liver is functional on a short-term basis. Patients typically show clinical improvement, with reduction of blood ammonia and lactic acid levels, conjugation and excretion of bilirubin, and stabilization of prothrombin time. [84]

Devices that incorporate cells or tissue from animals or incorporate human cells or tissues that have been cocultured with animal cells are considered xenografts. One promising device provides short-term support for patients with acute liver failure. [85] Initial clinical trials were promising, providing patients time to bridge to a human liver transplant. Other patients showed spontaneous recovery during the support period.

These initial groundbreaking trials offer hope that xenotransplantation could play an important role in the health and well-being for generations to come. The lessons gained from each experiment builds our experience and helps to establish the safety and efficacy of these transplants.

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The Future of Xenotransplantation

For patients in greatest need, organ transplantation represents the only chance of survival. For many, a suitable organ donor will never become available, and they will die as a result. This large and unmet need causes the medical community to seek alternatives to human-to-human transplants; xenotransplantation offers a potential solution to this problem. A number of issues still restrict the use of this technology to animal models. However, tremendous advances have been made, bringing this technology closer to potential clinical use.

Though the technical and scientific issues surrounding xenotransplantation remain formidable, a variety of additional questions remain. As a society, we must consider the important ethical and cultural questions inherent in the use of animals as a source of medical tissues, as well as the regulatory and financial questions that this technology raises. If the technology evolves to the point that it could be used clinically on a large-scale basis, it remains an open question as to whether these organs should be used. How will their production and distribution be regulated? What are the cultural or religious issues to consider? What are the risks involved with the use of xenogeneic tissue?

The ethical concerns of xenotransplantation have been well-reviewed. [86, 87, 88] Here, we pose a few important questions with the intent of provoking a thoughtful discussion of this ethically challenging issue.

You Are What You Eat  

For millennia, humans have bred and raised animals for a wide variety of uses. Do we draw a line at raising animals for organ transplantation?

For example, is it acceptable to use the pig as a source of organs for human patients? Consider the fact that the pig is a world-wide food source. We feed these animals and maintain large herds to support our population; when a pig reaches a certain age and size, we slaughter the animal for our consumption. If we consider it acceptable to breed and raise these animals solely to consume it, why could they not be bred and raised for a medical need?

Naturally, this argument would lose a great deal of traction were we to apply it to animals we do not typically eat, such as primates or companion animals. However, humans have bred and raised horses for transportation and farm work for thousands of years. We breed and raise cats and dogs for companionship; we train dogs to lead the blind and assist the disabled. We breed numerous rodents and reptiles, fish and amphibians, even some insects and invertebrates for pets and food.

Perhaps more germane to the question, a large precedent already exists for the use of animals in biomedical research, spanning from invertebrates to rodents to primates. The point of this discussion is that we raise and breed animals for very specific purposes—sometimes for food, sometimes for a task, sometimes for pleasure. But would it be acceptable to breed and raise any animal species specifically for medical organ transplantation? Would we judge some species to be more acceptable to breed and raise for a medical purpose than others? What criteria should we apply to make these decisions? These are difficult questions without clear answers.

The Heart and the Soul  

The psychological impact of receiving a transplant is profound. The knowledge that one’s own heart has been replaced by another’s would certainly be expected to have a major effect on one’s emotional and spiritual outlook. How might that same person feel to know that a pig’s heart has replaced their own? Or that a chimpanzee was euthanized to sustain their own life?

Naturally, individuals may have very different responses. However, it will be important to consider the psychological well-being of potential recipients; some might find the very concept of xenotransplantation abhorrent. Certain cultures and religions do not consume pork or pork products, and many individuals from these cultures refuse to accept pig heart valve implantations. Such considerations must be taken into account as well when considering transplant candidates.

The use of animals for biomedical research in general has raised ethical questions for many years. There are those in our society who have serious concerns regarding the use of animals in any biomedical research; some individuals feel that no purpose justifies the use of these animals in pursuit of science. We must consider that animals cannot give informed consent for any procedure or experiment; neither can they withdraw from a study. They can, however, feel pain, and clearly possess many ‘human’ traits such as fear. The ethical treatment of animals is of the utmost importance, and this issue is of the highest importance in medical research. No matter one’s individual convictions, all would agree that a thoughtful dialog about these issues should continue as the field of xenotransplantation advances.

In the United States, federal guidelines designed to regulate xenotransplantation were formulated in 2003. These comprehensive guidelines provide recommendations for the procurement and use of animal subjects, the requirements for demonstrating xenogeneic transplant efficacy in animal models prior to human trials, and clear guidelines for human trials. These landmark guidelines help to establish standards for experimentation and help address a number of these ethical issues. [89]

The Beginning  

In summary, xenotransplantation stands as potential solution to a major problem in modern medicine; though this field is still in its infancy, its promise as a life-saving alternative to allogeneic transplant could save innumerable people’s lives in the future. We must be careful to appreciate not only the benefits, but also the risks and ethical constraints presented by this new technology.

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