Xenotransplantation involves the transplantation of nonhuman tissues or organs into human recipients. The concept was pioneered a century ago, when transplanting human organs was considered ethically controversial. Grafts were quickly rejected, however, because of unknown forces later identified as immune responses.
Interest in xenotransplantation reemerged during the 1960s, when large advances were made in immunology. Chimpanzee kidneys have been transplanted into patients with renal failure.  In 1984, a baboon heart was transplanted into a newborn infant, Baby Fae, who had hypoplastic left heart syndrome and lived 20 days after heart surgery.  A baboon liver was transplanted to a patient with hepatic failure.  Porcine islet cells of Langerhans have been injected into patients with type 1 diabetes mellitus.  Porcine skin has been grafted onto burn patients,  and pig neuronal cells have been transplanted into patients with Parkinson (Parkinson’s) disease and Huntington (Huntington’s) disease. 
During these advances, several obstacles to the success of xenotransplantation have been identified. These include, but are not limited to, (1) preventing hyperacute rejection, (2) preventing acute vascular rejection, (3) facilitating immune accommodation, (4) inducing immune tolerance, (5) preventing the transmission of viruses from xenografts into humans, and (6) addressing the ethical issues surrounding animal sources for xenografts and the appropriate selection of recipients (given that xenotransplantation remains experimental).  The purpose of this review is to identify the obstacles and recent progress made in the field of xenotransplantation.
The rationale of xenotransplantation
The motivation for using animal sources for organ or tissue transplantation is driven by supply and demand. According to the most current report from the United Network for Organ Sharing (UNOS), more than 107,241 Americans were waiting for organ transplantation as of May 2010.  In 2009, 28,464 patients had transplants, and approximately 40% of listed candidates on waiting list were younger than 50 years.
In light of the lack of supply of human organs for transplantation, several alternatives have been investigated and debated. Implantable mechanical devices have been explored in the field of cardiac transplantation. Recently, research has increased in the area of transplanting embryonic cells across species and growing kidneys and endocrine pancreas cells in situ. [9, 10] Organs from pigs have been the focus of much of the research in xenotransplantation, in part because of the public acceptance of killing pigs and the physiologic similarities between pigs and human and nonhuman primates. Xenotransplantation of organs from chimpanzees and baboons has been avoided, however, because of ethical concerns and fear of transmission of deadly viruses (see Biologic Barriers to Xenotransplantation).
Xenografts have been proposed as appropriate for infants who are physically too small to accommodate organs retrieved from adult or pediatric donors. Additionally, organs from animal sources could be transplanted into patients currently excluded from the human organ transplantation list. Finally, most patients perceive xenotransplantation as an acceptable bridge to transplantation of human organs in life-threatening situations. 
History of the procedure
Alexis Carrel is known as the founding father of experimental organ transplantation because of his pioneering work with vascular techniques. Carrel and Guthrie contributed substantially to the science of transplantation from 1904-1906. They performed autogenous vein grafts, performed leg replantation in dogs, and developed the famous patch-graft technique for widening narrowed vessels. They also performed heterotopic experimental transplantation. Parts of a small dog were transplanted into the neck of a larger dog. They developed the buttonhole technique for anastomosis of donor and recipient vessels in kidney transplantation to prevent thrombus formation.
In 1906, Jaboulay transplanted kidneys from goats, sheep, and monkeys into humans. These attempts at kidney xenografting were unsuccessful. In 1910, Unger transplanted a nonhuman kidney into a man dying of renal failure, which caused death a little more than a day later. In 1932, Neuhof transplanted a lamb kidney into a patient with mercury poisoning. The patient survived for only 9 days. In 1946, Demikhov transplanted a heterotopic heart and lung; the animal survived for 9 hours.
The clinical interest in xenotransplants waned following the series of disappointing results and the realization that transplant failure was attributable to powerful unknown forces that would eventually be identified as the body's immune system. Scientific interest did not revive until the 1950s, following successful allografting of kidneys from identical twins. Michon and Hamburger successfully performed a living related donor kidney transplantation in Paris in 1952; in 1954, Merrill and Murray, using no immunosuppression, performed the first kidney transplantation between monozygotic twins. [12, 13, 14]
The advent of dialysis therapy during the 1940s and 1950s and its widespread availability in 1970s eventually made a significant and lasting impact on kidney transplantation. Hemodialysis established a bridge to transplantation and significantly expanded the population of patients that could benefit from kidney transplantation. Patients with end-stage renal disease had 2 therapeutic options: (1) dialysis and/or therapy or (2) an allograft (cadaveric or living related donor).
At that time, an understanding of transplant immunobiology and immunosuppressive drugs had just started to develop. Knowledge of organ procurement and preservation was extremely limited, severely curtailing the widespread clinical use of allografting. During this time, Starzl and colleagues (and other groups) were experimenting with xenotransplantation using chimpanzees or baboons.
The momentum of xenotransplantation was derailed in the 1990s with discovery of the porcine retrovirus. Concerns about the risk of cross-species infections resulted in moratoriums on clinical trials on xenotransplantation.
Xenotransplantation refers to any procedure that involves the transplantation, implantation, or infusion into a human recipient of either (1) live cells, tissues, or organs from a nonhuman animal source or (2) human body fluids, cells, tissues, or organs that have had ex vivo contact with live nonhuman animal cells, tissues, or organs. (US Food and Drug Administration [FDA], 1999; FDA, 2001).
Xenotransplantation products must be alive, and circulation and return of patients' blood must occur through live nonhuman cells. For example, human skin cells grown outside the body on a layer of nonhuman cells and then used in humans for skin reconstruction can also be considered a xenotransplantation product. This latter category of procedures was included in the definition because scientists believe that the potential for transmission of an infectious disease with such a procedure may be similar to that of implanting live nonhuman animal cells, tissues, or organs directly into a human recipient.
Xenotransplantation products include those from transgenic or nontransgenic nonhuman animals and composite products that contain xenotransplantation products in combination with drugs or devices. These include, but are not limited to, porcine fetal neuronal cells, encapsulated porcine islet cells, encapsulated bovine adrenal chromaffin cells, baboon bone marrow, and external liver-assist devices using porcine liver or porcine hepatocytes. Nonliving biological products or materials from nonhuman animals, such as porcine heart valves and porcine insulin, are not classified as xenotransplantation products for the purposes of this definition.
Xenotransplantation products are subject to regulation by the FDA under section 351 of the US Public Health Service Act [42 U.S.C. 262] and the Federal Food, Drug and Cosmetic Act [21 U.S.C. 321 et seq]. In accordance with the statutory provisions governing premarket development, xenotransplantation products are subject to FDA review and approval.
Depending on the relationship between donor and recipient species, the xenotransplant can be described as concordant or discordant. Concordant species are phylogenetically closely related species. These species combinations include mouse to rat, baboon to cynomolgus monkey, or, presumably, nonhuman primate to human. Discordant species, on the other hand, are not closely related (eg, pig to mouse, pig to human).
A concordant recipient takes several days to reject an organ, whereas a discordant recipient mounts a violent, hyperacute response that leads to xenograft loss within a few minutes to a few hours. This difference in time to rejection is related to the presence or absence of preformed natural antibodies in discordant species pairs (described in Immunologic Barriers to Xenotransplantation).
Choosing the Donor Species
Chimpanzees were generally thought to be the best nonhuman primate donor compared with baboons or rhesus monkeys. However, their availability rapidly dwindled in the 1960s, and they were later listed as endangered species. Consequently, scientists sought other potential animal sources for organs for xenotransplantation. Species such as baboons, although existing in greater abundance compared with chimpanzees, fare poorly in captivity, have a long gestation, and have few offspring.
The FDA's Biologic Response Modifiers Advisory Committee (BRMAC), after conducting an in-depth investigation of this issue and convening public hearings, noted its findings and concern on this matter in the federal register dated January 2001.  In this document, the BRMAC raises concern regarding interspecies transmission of xenogeneic infectious agents. It also notes the potential for subsequent transmission of a xenogeneic infectious agent from the recipient to the recipient's close contacts, and propagation through the general human population, as an additional risk and a recognized public health concern.
The BRMAC has also identified the potential risk of insertional mutagenesis associated with the infection of xenotransplant recipients, their close contacts, and the general population with xenogeneic retroviruses. In addition to potential horizontal transmission of infectious agents from the recipient of a xenotransplantation product to the recipient's contacts, BRMAC is concerned regarding vertical transmission of infectious agents from the recipient to progeny during gestation (ie, transmission from mother to fetus of infectious agents across the placenta or during parturition).
Vertical transmission of xenogeneic infectious agents could result in the development of infectious disease in progeny. In addition, vertical transmission of xenogeneic viruses can result in insertional mutagenesis with disruption of normal human development or integration into the germline, resulting in transmission to future generations. The BRMAC considered nonhuman primate donors to pose the greatest threat of transmitting latent, intracellular, or unidentified organisms, including retroviruses, and recommended that nonhuman primates not be used as sources of xenotransplantation products until more information is available to assess safety issues.
This led investigators to seek other animal sources. Monkeys are not considered acceptable organ donors for both practical and ethical reasons. In addition to being uncomfortably close to humans on the evolutionary ladder, they also produce few offspring, are slow to mature, and would be difficult to rear under the sterile conditions required to minimize contamination by shared pathogens.
Most investigators agree that pigs have the potential to be the prime candidates for organ donation. Pigs are plentiful, are quick to mature, breed well in captivity, have large litters, and have vital organs roughly comparable in size to those of humans. Because they are already being used in the consumer market, their use in xenografting is less likely to elicit major objections from society. Because humans have had prolonged and close contact with pigs, their use for the purpose of xenotransplantation is believed to be less likely to introduce any new infectious agents. However, use of pig xenografts is associated with major immunologic barriers, resulting in hyperacute rejection (HAR) when transplanted into a nonhuman primate or human recipient.
Immunologic Barriers to Xenotransplantation
Immunology of Xenograft Rejection
Transplanting solid organs from animal sources into humans results in rapid loss of the xenograft because of hyperacute rejection. In this dramatic immunologic reaction, preformed antibodies circulating in human blood bind to the vascular epithelium of the animal organ and trigger a cascade that quickly results in thrombosis of the graft.
Humans have preformed antibodies known as xenoreactive natural antibodies (XNAs), which are directed against nonprimate species. XNAs appear in the early neonatal period following colonization of the large bowel by coliform bacteria. These antibodies primarily consist of immunoglobulin M but also probably include the immunoglobulin G and immunoglobulin A classes. Their binding is characterized by avidity and surprising uniformity.
Most XNAs recognize carbohydrate moieties associated with bacterial cell walls. Most human XNAs are directed against terminal carbohydrate, Gal1, and 3a-GalbGlcNAC-R, in which a galactosyl residue is linked to another galactosyl residue. This process is controlled by an enzyme galactosyl transferase. Humans lack this enzyme, and the carbohydrate epitope is therefore perceived as a foreign antigen and antibodies arise against it. This carbohydrate moiety is expressed on pig cells. Thus, humans have naturally occurring antibodies (ie, XNAs) against pig cells.
XNAs recognize porcine glycoproteins of the integrin family. Antibody binding initiates complement activation through the classic pathway, triggering a number of effector mechanisms. These mechanisms may include loss of heparan sulfate from endothelial cells (EC) mediated by C5a and xenoreactive antibody, a change in endothelial cell shape mediated by C5b-7 or the membrane attack complex, procoagulant changes mediated by the membrane-attack complex, and neutrophil adhesion mediated by iC3b. 
The immunologic cascades triggered during hyperacute rejection destroy very discordant xenografts within minutes to hours. This process is characterized by immediate engorgement and discoloration of the organ. Under light microscopy, interstitial hemorrhages and platelet microthrombi are evident. Immunohistologically, dense deposition of various immunoglobulins and multiple complement components is noted throughout the vascular bed. The anaphylatoxins C3a and C5a generated in the process stimulate basophils and mast cells to release histamine, which, in turn, results in platelet degranulation.
Binding of histamine and serotonin to receptors on endothelial cells stimulate the expression of platelet-activating factor and P-selectin. Platelet-activating factor dramatically increases vascular permeability and endothelial cell contraction, resulting in platelet and RBC sludging within the microcirculation. This complex interplay of complement components, platelets, and endothelial cells leads to platelet aggregation, coagulation, fibrin deposition, and hemorrhage, typically culminating in thrombosis and ischemic necrosis within minutes of engraftment.
Acute vascular rejection
If the transplanted organ is not rejected within minutes to hours, a more delayed type of immunologic response ultimately leads to thrombosis of the graft within hours to days. This process known as delayed xenograft rejection or acute vascular rejection.
Under light microscopy, focal infarcts, interstitial hemorrhages, and widespread coagulation of microvasculature are observed. DXR is characterized by progressive infiltration of monocytes and natural killer cells (over several days), endothelial cell activation, platelet and fibrin deposition, and cytokine expression. Only very small numbers of T cells are noted (~5%). The role of macrophages and natural killer cells in DXR has yet to be determined; however, neither XNAs nor T cells are essential for DXR in complement-depleted rats.
Although endothelial cell activation is believed to play a key role, factors that trigger it are not well defined. Endothelial activation is type II in nature because it involves gene induction and protein synthesis. This includes a shift to a procoagulant state, secretion of chemokines such as membrane cofactor protein-1, and induction of leukocyte adhesion molecules such as E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1.
Overcoming Xenograft Rejection
To devise therapeutically effective strategies to defeat HAR and DXR, a detailed understanding and identification of complex inflammatory pathways and key events are indispensable. This involves in-depth study of both donor and recipient factors that play critical roles in mounting and sustaining a rejection response. This section focuses on approaches proposed to circumvent xenograft rejection.
Donor-based strategies: Transgenic organs
Genetically engineered pigs have been designed to downregulate expression of various immunogenic substances. Several groups have been successful at developing a breed of knockout pigs that lack the 1,3 galactosyltransferase gene. This gene ordinarily codes for an enzyme that is responsible for the expression of the immunogenic Galalpha1,3Gal carbohydrate moiety on the vascular endothelium of pig organs. In 2005, pig hearts from alpha 1,3 galactosyltransferase knockout (GT-KO) pigs were transplanted into baboons.  These grafts survived 6 months. 
Others have bred pigs with increased expression of H-transferase (also known as 1,2 fucosyltransferase). H-transferase is an enzyme that competes with alpha 1,3 galactosyltransferase, the substrate that is a precursor to the Galalpha1,3Gal moiety. The rationale is to decrease the amount of Galalpha1,3Gal carbohydrate on pig vascular endothelium.  Swine islet cells that express N-acetylglucosaminyltransferase III (GnT-III) have been developed and have been recently transplanted into monkeys.
Unlike other glycosyltransferases, GnT-III interrupts the biosynthesis of the Galalpha1,3Gal xenoantigens by several mechanisms not fully understood, including interruption of carbohydrate branching. 
Another variation of transgenic pigs has been developed to interfere with the mechanisms of graft rejection. For example, expression of human ecto-5'-nucleotidase (E5'N) pig organs has protected grafts from natural killer cell–mediated lysis.  Transgenic pigs have also been bred to express glycoproteins that inhibit the human complement cascade. These include CD55 (human decay accelerating factor [hDAF]), CD46 (membrane cofactor 1), and CD59 (protectin, which blocks the membrane attack complex from binding to cells).
Advances in genetic engineering have made double transgenic pig organs available. Xenotransplantation of double transgenic porcine skin to cynomolgus monkey survived 31 days. The skin expressed both GnT-III and hDAF.  Double transgenic pig kidneys with both human alpha-galactosidase and alpha 1,2-fucosyltransferase have been shown to decrease Gal expression and resist human serum–mediated lysis in ex vivo experiments.  Others have engineered double transgenic pig hearts expressing both hDAF and CD59. 
Triple transgenic pigs have recently become available. Hyperacute rejection was prevented in baboon recipients of orthotopically transplanted swine livers that expressed hDAF, human CD59, and H-transferase.  Japanese scientists recently reported success with breeding alpha 1,3 galactosyltransferase knockout pigs that express hDAF and Gnt-III.  Another variation of triple transgenic pigs has been bred to express human CD59, human membrane cofactor protein, and hDAF. 
Several experimental designs have been developed for xenotransplantation of these genetically modified organs. In the ex vivo model, the genetically transformed porcine organ is infused with human blood or serum to see if hyperacute rejection occurs. Alternatively, in the life-sustaining model, genetically modified pig organs have been transplanted into baboons or monkeys undergoing immunosuppressive therapy. Xenotransplantation may be performed orthotopically such that the native organ is removed and the transplanted organ occupies the anatomic location.
Xenotransplantation of organs from transgenic pigs for CD55 (hDAF) has been extensively studied over the last five years with mixed results. Researchers recently showed that higher levels of CD55 expression in transgenic pigs increase survival of grafts in baboons that have undergone xenotransplantation.  CD55 transgenic porcine hearts have been successfully transplanted orthotopically into baboons, with a median survival of 14.6 days.  Organs transgenic for CD55 also appear resistant to cellular lysis by human serum.  In other ex vivo models involving xenotransplantation of transgenic pig lungs into baboons, however, hyperacute rejection has been reported upon perfusion of the graft. 
Many groups have prolonged survival of CD55 (hDAF) transgenic pig organs with infusion of various agents or soluble antibodies. For example, reduced myocardial damage has been reported in ex vivo experiments in which CD55 transgenic grafts were perfused with GPIIb/IIIa inhibitor tirofiban.  In other experiments, CD55 transgenic pig hearts have been transplanted into baboons that were also administered soluble Gal-glycoconjugates to block baboon antibodies from binding to the Gal moiety on renal endothelium. These grafts survived for 3 months.  Anti–nonGal antibodies have been suggested to be involved in the ultimate acute humeral xenograft rejection of the kidneys.  Other groups have attempted life-supporting xenotransplantation of transgenic CD55 pig kidneys in baboons with infusion of soluble complement receptor type 1, TP 10. These grafts ultimately failed because of chronic deposition of complement in the endothelium. 
Acute vascular rejection is thought to be triggered by antibodies to the xenograft and complete activation of the complement cascade.
In theory, the complement cascade can be interrupted therapeutically by using several inhibitory agents. Such complement inhibitory molecules include cobra venom factor (to deplete C3), soluble complement receptor type 1, anti-C5 antibodies, K76COOH, and FUT-125. [36, 37] Toxicity associated with cobra venom factor is a major obstacle to its clinical use. Moreover, recent studies show the administration of cobra venom alone does not appear to prevent the deposition of C3 complement in grafts.  The soluble complement receptor type 1 (TP10) does not appear to be clearly effective in interrupting the complement cascade. Deposition of complement was found in grafts of monkeys that were transplanted with life-supporting hDAF transgenic pig kidneys and given cyclosporine, mycophenolate, steroids, soluble glycoconjugates to Galalpha1,3Gal, and TP10. 
Several immune-modulating therapies have been developed to prolong xenograft survival. Combinations of immunosuppressive agents, including cyclosporine A, mycophenolate sodium, and steroids, have prolonged survival of hDAF porcine renal xenografts in primates. [39, 40] Recently, soluble decay-accelerating factor has been used to prevent humeral rejection. 
Others have experimented with various mechanisms to downregulate co-stimulation and alter the immune response to interleukins. Swine hearts have been successfully xenografted into baboons treated with anti-CD154 antibodies and CD28/7 blockade. [42, 43] Murine studies have suggested that acute vascular rejection can be attenuated by CD8alpha+ dendritic cells that secrete IL-12 and induce a Th1 slow cell-mediated response to the xenograft.  Finally, interrupting the initiation of acute humeral rejection by blocking IL-1 with receptor antagonists appears promising in models of guinea pig hearts transplanted into rats treated with cobra venom. 
Accommodation is defined as graft survival despite the circulation of xenoreactive antibodies. This phenomenon was first identified in the late 1980s, when blood group O recipients were able to tolerate transplantation of group A or B kidneys. 
In accommodation, the graft is given a break from attack when circulating antibodies are removed from the system or when the complement cascade is interrupted. During this break from acute vascular rejection, the graft is able to up-regulate and express protective genes, like heme oxygenase 1, which impart graft resistance to injury. Although still unclear, other changes also appear to occur in the function of circulating antibodies and the expression of surface antigens on the graft.
Studies have also shown that accommodation might be achieved by interrupting the complement cascade with the administration of heparin sulfate. This is because full activation of the complement cascade characteristically lacks the presence of syndecan-4 phosphate. One group showed, however, that warfarin or low molecular weight heparin failed to induce accommodation in pig hearts xenografted into primates.  .
Coagulation dysregulation as a barrier to xenotransplantation
The ability to generate pigs that express a human complement regulatory protein (hCRP) and/or alpha 1,3 GT-KO pigs has largely overcome the barrier of HAR of a pig organ transplanted into a primate. However, acute humoral xenograft rejection (AHXR) presenting as microvascular thrombosis, consumptive coagulopathy, or both remains a major hurdle to successful xenotransplantation.
Until now, thrombosis was believed to result from antibody-mediated and complement-mediated EC activation, initiating AHXR. Exposure of porcine ECs to xenoantibodies, complement, and cells of the innate immune system results in EC activation and loss of anticoagulant regulators on their surface, with a subsequent change to a procoagulant phenotype. In xenotransplantation, distinct, immune-independent factors contribute to the development of coagulation disorders (eg, molecular incompatibilities between pigs and primates that promote or fail to regulate pathological clotting). For example, porcine von Willebrand factor  and loss of porcine tissue factor inhibitor (TFPI)  initiate coagulation cascade in primates.
GT-KO pigs that express an hCRP gene (eg, CD46 or hDAF) have exhibited some protection against the humoral-mediated immune response but do not overcome the problems of coagulation.
The interactions between porcine ECs, platelets, and other blood cells are at the nexus of a complex network that contributes to coagulation during AHXR. According to the standard paradigm, the process is initiated by the immune response against the graft, such that activation of porcine ECs caused by antibodies with/without complement leads to expression of TF that triggers the coagulation cascade. [50, 51] Loss of anticoagulant regulators, such as TFPI, ectonucleotide triphosphate diphosphohydrolase-1 (CD39), and ecto-5′-nucleotidases (CD73) activity is associated with platelet activation and aggregation. Administration of CD39 substitutes inhibits platelet activation and aggregation, thereby significantly prolonging graft survival.  Hearts from CD39-transgenic mice were resistant to thrombosis in a mouse-to-rat xenotransplantation model. 
Future directions in coagulation dysregulation
The resolution of coagulation dysregulation is critical to allow xenotransplantation to advance sufficiently for clinical trials. Thrombosis manifests in the graft, but systemic consumption coagulopathy may have more than one cause. Therefore, in addition to genetic modification of the organ-source pig, systemic medication may be necessary.
GT-KO pigs that express an hCRP and one or more anticoagulant or antithrombotic genes are anticipated to inhibit the generation of thrombosis and subsequent platelet activation. With
new cloning techniques (F2A system), mice transgenic for CD55/hTFPI/hCD39 have been generated. The same technology is expected to be successfully applied to generate pigs with multiple gene modifications and will enable these pigs to become available in a shorter period of time than through conventional breeding. 
Statins, inhibitors of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase, used for hypercholesterolemia, also have other independent effects, such as immunoregulatory, anti-inflammatory, and anticoagulant actions. [54, 55]
Atorvastatin reduces porcine EC activation induced by interferon gamma and inhibits the proliferative response of primate peripheral blood mononuclear cells (PBMC) and CD4+T cells when stimulated with porcine ECs. Simvastatin was shown to prevent the induction of TF on human aortic ECs by thrombin, at least in part through inhibition of rho-kinase-dependent Akt dephosphorylation.  Lovastatin enhanced ecto-5′-nucleotidase activity and membrane expression in ECs, consequently inhibiting platelet aggregation through the action of adenosine. 
Antiplatelet agents, such as antagonists of P2Y12 or GPIIbIIIa receptors, are expected to prevent aggregation when platelets are activated during AHXR. A high dosage of a GPIIbIIIa antagonist prolonged graft survival and decreased platelet aggregations in a rodent model.  If thrombosis in the graft is prevented by antithrombotic gene modification, this systemic approach may offer survival benefits by the prevention of systemic consumption coagulopathy.
Biologic Barriers to Xenotransplantation
The use of xenotransplantation products carries a natural and expected risk of transmitting infectious pathogens. This risk is beyond the risk of infections known to be associated with allotransplantation, which is a consequence of immunosuppression. This serious risk originates from the potential of xenotransplantation for transmitting infectious agents from nonhuman animals to humans. The HIV pandemic, Creutzfeldt-Jakob disease, Ebola virus outbreaks, and, more recently, the severe acute respiratory syndrome scare have elicited serious concern about the potential of inadvertent transmission of known and unknown infectious agents that may spread to recipients and to their contacts and health care workers, quickly becoming a public health issue.
Thus, for the following reasons, infectious risks associated with xenotransplantation may be far greater than those noted with allotransplantation: 
The level of immune suppression and/or rejection may be greater in xenograft recipients, enhancing the activation of latent pathogens, including viruses.
Organisms carried by the graft may not be known human pathogens and/or may include xenotropic organisms, ie, organisms that are not pathogens in the native host species but which cause disease in other species, in this case, the human recipient.
Microbiologic assays may not exist for some organisms derived from nonhuman species.
Novel animal-derived organisms may cause novel and thus unrecognized clinical syndromes.
Genetic modification of the donor animals (one xenotransplantation strategy) or treatment of the recipient with, for example, tolerance induction or antibody removal, may alter the host's susceptibility to organisms.
The term xenosis has been coined to describe the transmission of infections by the transplantation of xenogeneic tissues or organs. Xenosis, or xenozoonosis, potentially poses unique epidemiological hazards owing to the efficiency of transmission of pathogens, particularly viruses, with viable cellular grafts. When an infectious agent gains entry into a new host species, its capacity to produce disease is unpredictable.
For example, in its natural host, the macaque monkey, cercopithecrine herpesvirus 1 (B virus) infection has a clinical profile very similar to that of herpes simplex virus infection in humans. However, B virus infection of humans or other non–macaque primates results in rapidly progressive myeloencephalitis with a mortality rate of approximately 70%.  This failure of the pathogenic potential of a microbe in its host species to reliably predict the pathology that results when it is introduced into another species is evident with many other zoonotic agents and diseases.
Organisms of serious concern include herpesviruses and retroviruses, which can be screened for and eliminated from the donor pool. Others include Toxoplasma gondii, Mycobacterium tuberculosis, and encephalomyocarditis virus. Filoviruses (Marburg and Ebola), monkeypox virus, and simian hemorrhagic virus are less likely to be found in animals reared in captivity in the United States.
Retroviruses, by virtue of the enzyme reverse transcriptase, become inserted into host chromosomal DNA. Compelling arguments suggest that the HIV pandemic resulted from the adaptation of simian retroviruses introduced across species lines into humans. Existing data suggest that the HIV-2 pandemic in East Africa began with the horizontal transmission of simian immunodeficiency virus from a sooty mangabey monkey into a human with subsequent transmission through the human population. In Central Africa, horizontal cross-species transmission of simian immunodeficiency virus from a different primate species, probably a chimpanzee, resulted in the HIV-1 pandemic. Initial human infections before 1970 resulted in more than a decade of insidious human-to-human transmission before AIDS was first recognized as a public health problem in the 1980s.
Endogenous retroviruses exist as part of the genomic material of most, if not all, mammalian species, including humans. Endogenous retroviruses cause equal concern and greater uncertainty than the exogenous retroviruses. Endogenous retroviruses, presumably originating as exogenous viruses that became permanently integrated into the host germ line, are vertically transmitted through inheritance. In the host species, they are benign. However, endogenous viruses are frequently xenotropic, ie, although the original host is refractory to infection, the viruses can infect related species.
The increased phylogenetic distance between swine and humans presumably makes pigs safer donors than nonhuman primates. This presumption has not been completely explored. The biology and pathogenicity of a type C retrovirus identified in the blood of leukemic or irradiated swine are incompletely characterized. Similarly, the discovery of porcine endogenous retroviruses (PERVs) capable of infecting human cells in vitro has raised issues regarding the safe clinical application of xenotransplantation. [61, 62, 63]
Phylogenetic analysis reveals that PERVs are closely related to gibbon ape leukemia virus, endogenous koala retrovirus, and inducible murine endogenous retrovirus. [64, 65] PERV RNA is expressed in several porcine tissue types (eg, kidney, lung, liver, heart, pancreatic islets); however, expression of virus mRNA does not necessarily correlate with the release of infectious particles. Many human cells clearly express receptors specific for PERV A and B, whereas PERV C–specific receptors cannot be detected in most instances. [66, 67]
Several viral pathogens have been identified in the xenografts from pigs, which are the most common animal source of xenografts. These include, but are not limited to, porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotrophic herpes virus (PLHV), and porcine circovirus type 2, (PCV). In New Zealand, pigs raised for xenotransplantation were found to harbor encephalomyocarditis (EMCV) and hepatitis E.  Importantly, pigs do not have the exogenous retroviral equivalent of the HTLV or HIV virus. Two strains of the PERV virus (strain A and B) are present in only a subset of swine and have the potential to infect human cells in vitro. Importantly, the PCMV strains can be selected out of the pool of potential xenografts by early weaning of piglets.
Experimental xenotransplantation of organs from swine to nonhuman primates has demonstrated the absence of PERV transmission. One study suggested that decreased risk of transmission of endogenous retroviruses from pigs to baboons is correlated to decreased amounts of circulating anti–alpha Gal antibody.  Others have shown an absence of PERV infection in baboons receiving transgenic livers. 
Over the past 10 years, many sensitive diagnostic assays have been developed to detect most potential viruses associated with xenotransplantation of organs into humans. For example, 1 long-term follow-up study on 18 human recipients of pig islet cell transplants showed no evidence of PLHV, PCMV, PCV, or PERV infection in any of the patients 9 years after xenotransplantation.  No in vivo infection of human cells by the PERV virus has been reported to date. By contrast, the transplantation of baboon livers and chimpanzee kidneys into humans had resulted in deaths due to illnesses not related to organ failure. 
Japanese researchers are attempting to engineer transgenic pigs that would be genetically incapable of harboring endogenous retroviruses. Such a breed of pigs would express the RNA interference silence genes. 
Transmissible spongiform encephalopathy
Transmissible spongiform encephalopathy is a uniformly fatal family of diseases of humans and animals that causes irreversible cumulative brain damage. These diseases, which include Creutzfeldt-Jakob disease, chronic wasting disease, and bovine spongiform encephalopathy, are believed to be caused by a novel class of agents termed prions. Various reports have documented the prions jumping the species barrier from cattle, squirrels, and rabbits to humans. Transmissible spongiform encephalopathies have exhibited transmission into new hosts through transplanted grafts and across species lines. [74, 75] Of these diseases, cows can be naturally infected with bovine spongiform encephalopathy and pigs can be experimentally infected with transmissible spongiform encephalopathy. 
Strategies to avoid xenosis
Given the complexity of xenotransplantation and the uncertainties of issues surrounding it, strategies to address xenosis are being developed, although a universally adopted guideline on xenosis remains to be issued. The US Public Health Service agencies (ie, FDA, Centers for Disease Control and Prevention, National Institutes of Health, and Healthcare Resource Services Administration) and the Office of the Associate Secretary for Planning and Evaluation of the Department of Health and Human Services are collaborating to develop an integrated approach to address infectious disease issues in xenotransplantation.
Bach and colleagues have suggested a 3-tiered approach to policy development and decisions to address the issue of xenosis.  This method suggests approaching the issue at societal, institutional, and individual (patient-physician) levels. Because the risk is societal and not merely individual, the decision whether to undertake the procedure involves more than ensuring the ability of the surgeon and the transplant team, the capacity of the institution, and the willingness of the patient.
In situations in which the risks are collective, the public must be educated about the risk and must be involved in the decision-making process. Therefore, the first part of the decision-making process must occur at the level of social policy; the second part must occur at the level of the institutions performing the xenografts, and the third part must occur at the level of individual patients and physicians, especially affecting the processes of informed consent and medical confidentiality.
Because of the risk of xenosis, all the major reports on xenotransplantation released to date have recommended comprehensive monitoring and surveillance of xenograft recipients. The legal and ethical problems associated with imposing such surveillance on recipients (and perhaps their sexual partners) for what would likely be many years or for life and the details about the nature and frequency of monitoring require further discussion. That patients manifesting signs of a possible xenosis after transplantation would have to be quarantined is not inconceivable.
The maintenance of patient confidentiality, as in all areas of medicine, remains paramount and further complicates the need for adequate monitoring of recipients. The community must be educated about any risk that could arise from xenotransplantation, regardless of whether the extent of that risk and the degree to which that risk is controllable can be precisely defined. Finally, it would be helpful for the public to have a better understanding of the process by which decisions are made in situations of uncertainty. 
At the level of the hospital or research center, institutions must be responsible for establishing and enforcing standards for quality of care, management of risk, monitoring of patients and their contacts, and evaluation of the effectiveness of the procedure in accordance with public guidelines and regulations. Institutions should avoid situations in which individuals proceed with xenotransplantation in advance of adequate safeguards and should curtail clinical trials until societal guidelines are available. 
A new approach to informed consent as it relates to xenotransplantation is necessary. A patient's agreement to participate in xenotransplantation must be based on perceptions of individual risks, as is the case with any experimental or extreme procedure, and on the risk of new disease to family, friends, close contacts, and society at large. Because of the need for monitoring for signs of infection, the patient and others must commit to participate in such monitoring for a period considered to be longer than the potential time necessary for an infection to manifest. Thus, the xenotransplant recipient undertakes a social obligation to submit to close and frequent follow-up monitoring, even if this means relinquishing certain freedoms in order to gain the potential benefits of participation. 
The subject of ethics in relation to xenotransplantation has been widely explored. This includes issues related to both humans and nonhuman animals. The concept that certain ethical principles must be applied to experimentation conducted in humans is widely accepted. Such principles include respect for persons, beneficence, and justice. These ethical issues are addressed in great length by the International Xenotransplantation Association Ethics Committee's position paper. 
Beneficence and risk-to-benefit analysis
Risk assessment is based on the principle that the possible harm of the research must be outweighed by its probable benefits. Preclinical data, including nonhuman primate data, must adequately support the possibility of a successful outcome.  In addition, for any ethically conducted trials, risks to the patient and to society must be minimized.
Animals used for xenotransplantation should be bred in captive, closed colonies in order to ensure the exclusion from the colony of known potential pathogens to humans. The extensive human experience with short-term exposure to porcine materials, including patients receiving porcine insulin, clotting factors, and temporary skin grafts, is reassuring. However, none of these situations involves the long-term presence of large numbers of porcine cells or organs in an immunocompromised individual.
Autonomy and informed consent
The potential risk of xenotransplantation to society elicits unique challenges in developing an appropriate informed consent process. In addition to the research subject, the burden of risk is also carried by close contacts, medical caregivers, and society, all of whom may reasonably insist that the research subject agrees to life-long monitoring, avoids blood donation, and informs close contacts about the xenotransplantation and its potential risk for spreading zoonotic infections.
Asking a subject to agree to life-long monitoring effectively denies him or her the right to withdraw from the study at any time. This is a denial of a fundamental individual right as delineated in the Declaration of Helsinki and the US Code of Federal Regulations. 
Some authors have even gone so far as to suggest the need for mandatory “Ulysses contracts” when obtaining informed consent to proceed with xenotranplantation.  These contracts, which currently exist in some psychiatric practices, are an advance directive from a patient to the primary care provider given at a time when the patient is psychiatrically well. In this setting, the patient essentially preauthorizes the physician to involuntarily commit him or her during times of psychiatric relapse. In a similar fashion, a Ulysses contract for xenotransplantation could allow the treating team to enforce a commitment made prior to surgery. Therefore, should the subject change their mind in the future regarding their previous agreements, such as the right to withdraw from the trial or to inform close contacts of the potential risks of their xenotransplantation, there would be a binding contract enforceable by quarantine or detention, thereby protecting society at large. 
From a public health perspective, notification of close contacts and caregivers about potential infectious risks surrounding a xenotransplantation recipient could violate principles of confidentiality. This raises questions regarding whether it is necessary to obtain third party informed consent during patient selection for transplant. 
Another hurdle is that close contacts of xenotransplant recipients could be expected to refrain from blood donation and agree to monitoring if this becomes necessary. Enforcement of such rules could be deemed next to impossible given that intimate contacts may change multiple times over a person’s lifetime. 
Given these difficult issues, societal input and governmental oversight regarding the decision of whether a country will proceed with xenotransplantation research are necessary.  The Department of Health and Human Services Secretary’s Advisory Committee on Xenotransplantation suggested that raising public awareness about the health concerns of xenotransplantation is the only adequate mechanism to ensuring community-wide vigilance toward the potential health hazards of xenosis. 
The potential risks of xenotransplantation will not respect the geographic borders of the country in which the procedure is undertaken. In the absence of international regulations and monitoring procedures, the most aggressive safety measures of any nation are likely to be unsuccessful. This issue arises because of a constantly mobile population and the wide use of intercontinental air travel, which can quickly spread an infectious agent to geographically distant locations. The ethical principle of justice requires all nations to bear responsibility regarding the control of infectious disease risks. This problem is highly complex and requires a globally respected international treaty with a uniform immigration surveillance system to check for the entry of potentially infectious pathogens.
Animal-related ethical issues
Xenotransplantation might give rise to a variety of psychosocial problems pertaining to emotional and personal identity issues associated with implantation of organs from nonhuman animals. While not a reason to remove xenotransplantation from consideration, these issues should be thoroughly discussed with the potential recipient in advance.
The concept of rights for donor animals is controversial. Nonhuman primates such as baboons have complex social behaviors, and various ethical concerns exist regarding their use. The use of pigs is far less controversial. In a response to the use of animal-derived xenogeneic biologic meshes for soft tissue repairs, People for the Ethical Treatment of Animals (PETA) stated that they were “opposed to the use of animals and animal tissues for experimentation, medical training and clinical treatments…including the use of biological meshes."  Various animal rights activists are opposed to the idea of xenotransplantation because they maintain that humans do not have right to breed and use other animals for their own needs. While these issues require considerable debate, the accepted opinion is that animals used for research or clinical xenotransplantation must be treated respectfully and humanely, and they must not be used without institutional approval.
Religious views on xenotransplantation
Religion plays a significant role in the everyday life of many individuals and thus influences lifestyles, eating habits, and medical treatments. The 3 major monotheistic religions have certain aspects in common. All posit a hierarchy in the order of creation, in which humanity has a unique place. 
Furthermore, the Roman Catholic Church holds that humanity has a mandate to guide the life of creation toward the integral good.  However, both Judaism and Islam forbid the raising and consumption of pigs. Nevertheless, using pig organs for xenotransplantation is not regarded as eating pork. Moreover, both Judaism and Islam allow for exceptions to dietary laws, particularly in situations in which a human life might be saved. [79, 84]
Buddhism regards organ donation as a matter that should be left to an individual's conscience. A Hindu tenet is that the body must remain whole in order to pass into the eternal life; therefore, transplantation is not condoned. However, Hindu law does not prohibit individuals from donating their organs or accepting an organ. With the exception of cows, which are sacred in Hinduism, there are no prohibitions on using parts of animals to alleviate human suffering. Interestingly, one of the oldest mythological accounts of xenotransplantation is described in Hinduism, wherein Lord Ganesha (Lord Shiva's son) received a xenograft of an elephant head after Lord Shiva inadvertently severed his head.
Popular perception of xenotransplantation
In light of the rapid advances in genetic engineering and immune modulating therapies, the scientific and popular media have declared xenotransplantation as becoming a clinical reality fraught with ethical controversy. Television shows like "Frontline" and "60 Minutes" have discussed many of the issues mentioned above. Similarly, the medical literature reflects ethical debates. Scientists at Harvard have questioned the ethical framework used to transplant pig islet cells into Mexican children.  Others have interviewed patients to determine under which clinical circumstances they would undergo xenotransplantation and identified a "reluctance" to endorse the procedure.
In 2008, controversial issues surrounding xenotransplantation were explored at the Metropolitan Museum of Modern Art by artist Elio Caccavale. In the Utility Pets exhibit, the works My BioBoy and My BioPig depict a boy connected to a pig by an umbilical cord and challenge the audience to contemplate "transhuman" creatures. In the scenario, the boy harmoniously coexists with the pig while waiting for the day of xenotransplantation. The art work offers an alternative to the potentially uncomfortable idea of organ farming and playfully displays inventions like The Smoke Eater (which protects the pig from secondhand smoke) and The Toy Communicator (which allows the boy and the pig to communicate when in different rooms). 
Current Status and Future Directions
As previously mentioned, xenotransplantation and xenotransplantation products come under the regulatory authority of the FDA. In 1997, the FDA formed the Xenotransplantation Subcommittee of the BRMAC as an ongoing mechanism for open discussions of the scientific, medical, social, ethical, and public health issues raised by xenotransplantation and the specific ongoing and proposed protocols.
The FDA has developed a xenotransplantation action plan to provide a comprehensive approach for the regulation of xenotransplantation that addresses the potential public health and safety issues associated with xenotransplantation and to provide guidance to sponsors, manufacturers, and investigators regarding xenotransplantation product safety and clinical trial design and monitoring.
From time to time, the FDA publishes guidance documents to assist sponsors and investigators interested in conducting clinical trials in the field of xenotransplantation. These documents provide reasonably detailed and timely pragmatic guidance to sponsors regarding xenotransplantation product safety and clinical trial development, including specific recommendations for the procurement and screening qualification of source animals, the manufacture and testing of xenotransplantation products, preclinical testing, clinical trial design, and posttransplantation monitoring and surveillance of recipients of xenotransplantation products. The FDA provides notice of and invites public comment on these draft documents. One such final guidance document for the industry can be accessed on the Internet at http://www.fda.gov/cber/guidelines.htm. 
The FDA has sponsored, planned, or participated in numerous open public meetings and workshops (both domestic and international) that partially or wholly focused on xenotransplantation. These activities are essential for both sharing information and receiving public input on issues relevant to xenotransplantation.
Although clearly an experimental procedure, investigators in clinical xenotransplantation have been accused of using "the guise of bridge-to-transplantation" to appear acceptable to institutional review or ethics boards.  However, the use of xenografts solely as bridges to allotransplantation does not increase the donor pool; therefore, successful, permanent xenotransplantation must itself be viewed as the target of future clinical investigations.  In the future, clinical xenotransplantation may accomplish its intended goal of achieving prolonged graft survival. Learning from the lessons of allotransplantation, clinicians performing xenotransplantations must persevere under justifiable scrutiny.