Brenner and Rector's The Kidney, 8th ed.

CHAPTER 67. Xenotransplantation

David H. Sachs   Jay A. Fishman

  

  

Historical Perspective, 2183

  

  

The Most Appropriate Donor, 2184

  

  

Nonimmunologic Compatibility Issues, 2185

  

  

Physiologic Compatibility, 2185

  

  

Coagulation and Endothelial Activation, 2185

  

  

Immunologic Considerations, 2185

  

  

The Humoral Response to Xenograft, 2185

  

  

The Cellular Response to Xenografts, 2187

  

  

Tolerance, 2189

  

  

Mixed Chimerism, 2189

  

  

Thymic Transplantation, 2189

  

  

The Risk of Infection in Xenotransplantation, 2190

  

  

Bacterial Infections, 2190

  

  

Viral Infections, 2190

  

  

Identifying Potential Human Pathogens, 2190

  

  

Retroviruses, 2191

  

  

Herpesviruses, 2191

  

  

Other Potential Pathogens, 2193

  

  

The Potential for New Pathogens, 2193

  

  

Infectious Risks, Surveillance, and the Search for Novel Pathogens, 2193

  

  

Infectious Benefits of Xenotransplantation, 2193

  

  

The Future of Xenotransplantation, 2193

  

  

Fear of New Viruses, 2194

  

  

Ethical Issues, 2194

As of June 2007, the Organ Procurement and Transplantation Network in the United States listed 72,192 individuals on “waiting lists” for renal transplantation. In 2006 in the United States, there were 10,659 deceased organ donors, with the number of living donors approaching that of deceased donors. If the U.S. waiting list were to grow at the current rate, it would include 76,000 individuals by 2010. Eurotransplant reports 12,251 people waiting for renal transplants in 2005, with 3981 kidneys procured for transplantation in that year. Thus, worldwide, there remains a great disparity between the availability of organs for transplantation and the patients awaiting this life saving therapy. This gap is expected to grow. The at-risk population for end-stage renal disease due to diabetes is expanding with the challenge of obesity among children and adults. Pre-emptive transplantation (pre-dialysis) has significant benefits for patients with types 1 and 2 diabetic nephropathy; however, this approach is limited by the organ shortage.[1] The lists might include many more individuals who could benefit from organ transplantation but are not yet deemed “sick enough” to be placed on official waiting lists or who are unaware of the potential benefits of transplantation.

The shortage of organs is also reflected in the use of possibly “less desirable” organs and in prolonged waiting times for transplantation. Such expanded criteria donor (ECD) kidneys are those from any brain dead donor older than 60 years of age, or from a donor older than the age of 50 years with two of the following: a history of hypertension, a terminal serum creatinine greater than or equal to 1.5 mg/dL, or death resulting from a cerebral vascular accident (stroke). The term “expanded” is used because an expansion of the donor pool increases transplantation and is preferred over the term “marginal donor” but nevertheless reflects the excessive demand for organs over supply. Increasing numbers of ECD organs are being used. Recipients of ECD kidneys are generally those for whom it is more difficult to obtain a donor, such as those who are more highly human leukocyte antigen (HLA) mismatched, older, non-Caucasian, or diabetic. After ECD transplantation, the death rate is approximately 100 per 1000 patient-years at risk compared with approximately 48 deaths per 1000 patient-years at risk for non-ECD organ recipients, although it is impossible to determine accurately how much of this difference reflects the quality of the organ versus the generally poorer health status of the recipient. The impact of pre-existing sensitization of recipients to donor antigens, as measured by the panel reactive antibodies (PRAs), is an added constraint on organ availability. Patients with a high PRA level wait twice as long for kidneys as those with low a PRA level. The percentage of patients with elevated levels (>10%) of PRA was approximately 33% in 2003. As a result, by 2003, 43% of patients had been wait-listed for more than 2 years and 11% for more than 5 years. For young adults (18 to 34 years of age) listed in 2001, the median waiting time was 987 days with a death rate while waiting of 100 deaths per 1000 patient-years at risk. Blacks wait twice as long as whites.

In 1907, Alexis Carrel suggested that the future of transplantation for the treatment of organ failure would lie in “heterotransplantation,” the clinical use of cells, tissues, or vascularized organs from nonhuman species, now termed xenotransplantation. Xenotransplantation carries the theoretical ad-vantage of providing an unlimited supply of transplantable organs as either permanent organ replacements or as “bridges” to the availability of human-derived organs. In addition, these animal-derived organs might be resistant to infection by common human pathogens such as hepatitis C virus or human immunodeficiency virus (HIV).

Multiple barriers exist to the broad clinical application of xenotransplantation. These include immunologically mediated graft rejection of xenogeneic tissues, metabolic or molecular incompatibilities between donor organs and humans, ethical concerns, and the risk of infections that might be transmitted from the donor species to the human recipient and to the general human population. The likelihood of clinical success of xenotransplantation has been enhanced over the past decade by genetic engineering. The first modification involved modified swine that expressed transgenes for human complement regulatory proteins[2] and more recently, pigs with disrupted genes for the enzyme alpha-1,3-galactosyltransferase have been produced. [3] [4] In the latter so-called Gal-T-Knockout (KO) swine, the enzyme producing the major endothelial antigen targeted by natural antibodies and causing hyperacute graft rejection has been disrupted. This chapter reviews the current status of renal xenotransplantation with a focus on the major advances and clinical applicability of this technology for patients with renal failure.

HISTORICAL PERSPECTIVE

The history of renal xenotransplantation dates anecdotally to ancient times, but it was not until the 1960s that major clinical efforts were begun by Reemtsma and colleagues,[5] who transplanted kidneys from chimpanzees into humans, with at least one remarkable 9-month survival, using only rudimentary immunosuppression. Slightly later, Hitchcock and Starzl transplanted baboon kidneys into six human patients, with variable survivals up to 98 days.[6]As allogeneic transplantation became more successful over the next 15 years, clinical xenotransplantation essentially disappeared. Paradoxically, however, the success of renal allografting brought with it a new limitation to the field—the severe shortage of available organs to meet the increasing demand described earlier, resulting in a rebirth of interest in xenotransplantation.

In the last 2 decades, research into xenotransplantation as a potential solution to the organ shortage problem has exploded. Investigators took advantage of the wealth of knowledge that had been gained in the field of allotransplantation with regard to new immunosuppressive drugs and T cell-specific monoclonal antibodies, coupled with the possibility for genetic engineering of the donor species. Most of the studies were carried out in nonhuman primates as surrogate recipients, although a few forays were made into the clinical realm—one using baboon livers to treat terminally ill patients in hepatic failure,[7] and another studying the effects of perfusing the blood of human volunteers through pig kidneys.[8] However, concerns regarding the potential that xenotransplantation might promote interspecies spread of novel, animal-derived pathogens into human populations (see later), resulted in a self-imposed wait-and-see approach to clinical xenotransplantation by most workers in the field.[9] The Food and Drug Administration (FDA) and National Academy of Sciences held meetings on the subject of microbiologic safety in xenotransplantation, and guidelines were prepared to ensure adequate attention to infectious agents if and when clinical trials of xenotransplantation might begin.[10] With the exception of a few small trials of porcine cells injected into the brains of patients with Parkinson disease,[11] there have been no further reports of attempts at clinical xenotransplantation since that time. However, as will be elaborated upon in the remainder of this chapter, preclinical studies of xenotransplantation into nonhuman primates have been numerous and have achieved results that would have been startling just a decade ago. Many of these preclinical studies have used renal transplants, largely because of the ease with which renal function can be followed. As detailed later, the fate of primates receiving transplantation of life-supporting porcine kidneys has gone from survivals measured in minutes just a few years ago to greater than 80 days, with functioning kidneys and normal histology at the time of death.[12] Living donation of kidneys has made the shortage of other organs, such as the heart and the liver, even more evident. However, there is little question that, if they were available and successful, xenogeneic kidneys would be used in preference to living donors and possibly in place of many ECD human donors.

THE MOST APPROPRIATE DONOR

Despite the remarkable success achieved by Reemstma using chimpanzees as organ donors or source species,[5] these animals, as well as the great apes, are endangered species, and therefore no longer under consideration as appropriate xenograft donors. The baboon, although relatively available, is rather small for potential use in adult humans, and also poses a risk for transmission of infectious agents to recipients, notably viruses adapted to primates, in addition to ethical considerations. For these reasons, most investigators have turned to pigs as more appropriate donors.

Among the advantages of swine as xenograft donors are virtually unlimited availability, physiologic similarity of many organ systems to those of humans,[13] and favorable breeding characteristics, making inbreeding and genetic engineering possible. One particular strain of swine that has been developed extensively for this purpose are inbred miniature swine,[14] several lines of which have been produced by a selective breeding program over the past 35 years. [15] [16] The size of these animals provides an additional advantage as a potential xenograft donor. When full grown, they achieve maximum adult weights similar to those of humans, approximately 100 to 140 kg, so that their organs would be appropriate in size for any potential human recipient, from a newborn baby to a large adult. In contrast, domestic strains of swine attain weights in excess of 400 kg, making them unfit as donors of xenograft organs to humans after the swine reach the age of about 1 year.

The favorable breeding characteristics of swine have enabled us to produce lines of miniature swine that are homozygous for swine leukocyte antigens (SLA), the major histocompatibility complex (MHC) of swine, and for recombinants within the MHC ( Fig. 67-1 ). [15] [16] In addition, intentional brother-sister matings from a single inbred line has recently resulted in a subline, the SLAdd subline, with a coefficient of inbreeding greater than 94%. This subline is sufficiently inbred that reciprocal skin grafts within this line are accepted indefinitely without immunosuppression.[17] These breeding characteristics have also made it possible to add or “knock out” genes of importance to the immunologic response to xenografts, [18] [19] [20] making these animals increasingly more appropriate as a donor species.

FIGURE 67-1  Origin of swine leukocyte antigen a (SLAa), SLAc, SLAd and recombinant haplotypes of inbred miniature swine.

 

Finally, the availability of inbred miniature swine in which the MHC antigens have been defined and characterized is of particular importance for strategies directed toward the induction of tolerance. For example, such animals make it possible to consider therapies in which the potential recipient is pretreated with thymic tissue, bone marrow stem cells, or even genes carried by expression vectors from inbred strains of pigs in order to induce tolerance to a subsequent tissue or organ graft from an identical donor. Some of these strategies are discussed further later.

NONIMMUNOLOGIC COMPATIBILITY ISSUES

In addition to immunologic interactions, which have been most widely studied, potential incompatibilities of function and of the coagulations system also deserve mention.

Physiologic Compatibility

The life-supporting function of xenografts requires that the tissues be physiologically compatible with the human host. Anatomically, the pig kidney is comparable in size to the human organ with similar concentrating capacity (approximately 1080 mOsm/L) and glomerular filtration rate (GFR; 126 to 175 mL/h).[21] There have been few technical problems with the implantation of kidneys from pigs into primates.

Porcine kidneys transplanted into nonhuman primates function well enough to sustain life [5] [22] [23] and, in the absence of rejection, can maintain a normal serum creatinine level and acid-base balance. However, subtle biochemical and structural differences may exist between human and porcine kidneys that could affect the clearance of metabolic byproducts (e.g., uric acid), the synthesis of essential hormones (e.g., erythropoietin, renin, calcitriol), and receptor binding by angiotensin II or parathyroid hormone. For example, the fact that porcine kidneys produce a higher urine volume with higher pH and excrete less uric acid than human organs could influence uric acid metabolism. Responses to pharmacologic intervention (e.g., diuretics, angiotensin-converting enzyme inhibitors) could also require attention. However, any such differences that may exist between human and pig kidneys remain to be defined, and from a functional viewpoint, the similarities are more striking. In addition, any problematic differences could be approached with either medication (e.g., for uric acid metabolism) or through genetic engineering to modify metabolic pathways so as to remove such differences.

Porcine xenografts in baboons subjected to bilateral nephrectomy were able to maintain serum creatinine levels (before graft rejection) less than 2.0 mg/dL.[24] Porcine xenografts had the capacity in cynomolgous monkeys to maintain fluid balance, and normal serum sodium and potassium levels. [25] [26] [27] It is not known whether blood pressure control will be normal in human recipients of porcine kidneys because some studies suggest that heterologous renin may be less active in the release of angiotensin than homologous renin.[27] The efficacy of porcine erythropoietin in humans is unknown, but primate renal xenograft recipients have often required transfusions to maintain red cell volume. Engraftment of porcine hematopoietic precursors in primate marrow often requires the use of porcine growth factors, suggesting that heterologous growth factors are less active in support of marrow differentiation. [28] [29] [30]

Coagulation and Endothelial Activation

An area of active investigation relates to the compatibility of porcine and human coagulation systems after xenotransplantation, notably in vascularized xenografts (as opposed to cells or tissues). Coagulation abnormalities including microangiopathy and thrombocytopenia have been observed in association with solid organ xenograft rejection. [31] [32] We and others have demonstrated that porcine endothelial cells (EC) of xenografts are activated by immune responses and by infection after transplantation, and cause vascular prothrombotic and inflammatory changes. [33] [34] [35] This is probably a reflection of poor compatibility of porcine tissue-factor-pathway inhibitor with human factor Xa, whereas porcine thrombomodulin interacts poorly with human tissue factor and human protein C. [36] [37] [38] These changes are associated with disordered regulation of blood clotting, fibrinolysis and platelet activation.[34] [39] Thus, the porcine xenograft may activate the coagulation cascade in the recipient with disturbances in coagulation, platelet activation, and vascular injury. Indeed, intravascular coagulation with microthrombus formation is a common component of xenograft rejection.[40]

Xenoreactive antibodies may directly induce endothelial activation with production of tissue factor procoagulant responses. [41] [42] [43] [44] Quiescent ECs express effective anticomplement, anticoagulant, and platelet antiaggregation mechanisms; it is not clear whether these factors are effective across species barriers. [45] [46] [47] Platelet sequestration within xenografts may be linked to the expression on EC of porcine von Willebrand factor that interacts with receptors on human and other primate platelets with high affinity, resulting in platelet aggregation. [39] [48] CD154 expressed on activated human platelets interacts with CD40 expressed on porcine ECs and might potentiate EC activation responses. There are also abnormalities in the regulation of human plasminogen activators by porcine plasminogen activator inhibitor type I that may compromise fibrinolytic pathways and promote vascular occlusion in xenografts.[49] Activation of porcine cytomegalovirus (PCMV) or other viral pathogens within the xenograft may cause endothelial activation and promote localized thrombosis. [35] [50] Reflecting the tendency to endothelial activation and clotting, anticoagulation has been shown to prolong xenograft survival.[51]

IMMUNOLOGIC CONSIDERATIONS

Both the humoral and the cellular arms of the immune response play important roles in xenograft rejection. A sequence of immune responses to vascularized organ xenografts has been characterized, involving (1) hyperacute rejection (HAR) occurring immediately owing to preformed antibodies, (2) delayed or acute humoral xenograft rejection (AHXR) occurring up to 2 weeks after transplantation and involving the recrudescence of preformed antibodies, as well as T cell-dependent humoral responses to new antigens, overlapping or followed by (3) cellular rejection, both innate and acquired. The initial targets for each of these responses are epitopes of xenograft vascular endothelial cells, and the details of these responses are considered in the next section.

The Humoral Response to Xenograft

It is useful to consider humoral immune responses in terms of two components: (1) “innate responses,” such as those due to preformed antibodies (without known prior antigenic exposure), and (2) “acquired responses,” resulting from antigenic exposure after transplantation.

Natural Antibodies and Concordant versus Discordant Species

Calne originally proposed the term “discordant species” to designate species between which organ grafts are rejected in an accelerated, or hyperacute manner, usually less than 24 hours, as opposed to “concordant species,” which reject in a manner similar to that of allotransplants.[52] The rapidity of the rejection process has been shown to depend largely upon the levels of preformed, or “natural” antibodies between the two species, these levels being higher the more disparate the species are phylogenetically, and being the major cause of HAR.[53] Natural antibodies (nAb), like ABO alloantibodies, probably develop in response to common environmental antigens. Consistent with this hypothesis, germ-free animals have low or absent titers of nAb. [54] [55]

Hyperacute Rejection

The role nAbs plays in rejection of discordant xenografts is probably the best-studied component of the xenogeneic immune response because of the devastating effects of HAR. HAR is caused by the binding of nAbs to antigens on the surface of the donor vascular endothelium, which, in turn, activates the complement cascade, damaging the endothelium and activating coagulation pathways with rapid destruction of the xenograft. [53] [56] Histologically, HAR is characterized by edema, interstitial hemorrhage, and vascular thrombi within the graft. Given the fact that all four components—antibodies, complement, the vascular endothelium, and the coagulation system—are integral to phenomenon of HAR, each has been the subject of attempts to down-modulate this reaction, as is discussed in more detail later.

In the case of pig-to-primate xenografts, the most abundant nAbs are directed to the single antigenic carbohydrate determinant Gala1-3Galb1-4GlcNAc (Gal), found as the terminal sugar on all cell-surface glycoproteins in all mammalian species except Old World primates and humans. [57] [58] [59] The reason for the absence of this determinant on glycoproteins of Old World primates and humans is that the enzyme responsible for adding this sugar moiety, α1,3 galactosyltransferase (GalT), was apparently lost during evolution, becoming a pseudogene at the level Old World primates.[60] Because these primates do not express the sugar, and because Gal is found ubiquitously in nature (e.g., in bacterial cell wall carbohydrates), there is ample opportunity for anti-Gal antibodies to form during the exposure of the primate's immune system to normal environmental antigens.

Anti-Gal nAbs in humans were originally described by Galili and colleagues,[61] who have estimated that pig endothelial cells express approximately 107 Gal epitopes per cell.[57] Anti-Gal antibodies account for approximately 4% to 8% of circulating IgM and about 2% of circulating human IgG,[62] but comprise more than 80% of human nAbs to pig.[63] With such a high level of expression on pig tissues, and with such high levels of nAbs to Gal in the serum of primates, it is no wonder that these antibodies turned out to be the major cause of HAR following pig-to-primate organ xenografts. This correlation was first described by Good and co-workers,[64] who demonstrated that the major antibodies binding to pig hearts and kidneys perfused ex vivo with normal human plasma, were directed to Gal. Confirming their major role in HAR, removal of these antibodies by perfusion enabled the phenomenon to be avoided,[23] [65] as described further later.

Avoiding Hyperacute Rejection Caused by Anti-Gal

Given the uniformly high levels of anti-Gal nAbs and high incidence of HAR, a prerequisite for prolongation of discordant xenografts beyond the hyperacute phase is removal of these antibodies or avoidance of their effects. Based on the mechanism by which nAbs cause injury (see earlier), a variety of approaches have been taken toward achieving this goal.

Plasmapheresis

Clinically, plasma exchange was reported by Alexandre and associates,[66] to be effective in avoiding HAR in cases of anti-A or anti-B sensitization before kidney allotransplantation. The procedure involved essentially complete replacement of the recipient's plasma with AB plasma. In Alexandre's patients, either the antibodies did not return, or if they did, they did not cause rejection, the phenomenon termed “accommodation”. Alexandre and colleagues[24]also attempted this procedure for pig-to-baboon kidney xenotransplantation, although replacement was not with antibody-free plasma but with other fluids. Prolonged renal xenograft survival was observed, up to 23 days, but there was no evidence of accommo-dation, and the grafts were lost when anti-Gal antibodies returned.[24]

Adsorption

As opposed to nonspecific removal of all immunoglobulins, including nAbs, through exhaustive plasmapheresis, adsorption techniques have sought to remove the specific nAbs by binding to antigen, leaving the remainder of the recipient's immunoglobulins intact. Early studies used perfusion of the recipient's blood through a pig liver intraoperatively, followed by transplantation of a pig kidney after the recipient's nAb level was sufficiently low so as to avoid HAR. [23] [65] Subsequently, after it was learned that the majority of nAbs in the pig-to-primate model were directed toward the Gal epitope, specific adsorption by plasmapheresis of the recipient baboon's plasma through columns bearing covalently bound α-1,3-Gal epitopes was implemented.[67] This technique was found to remove more than 97% of anti-aGal antibody,[68] and to eliminate HAR as effectively as did the previous liver perfusions. In our opinion, this result remains the most convincing evidence available that although there are preformed antibodies to other non-Gal determinants (see later), anti-Gal is overwhelmingly the most important nAb that needs to be dealt with to avoid HAR.

Inhibition of Complement

Because activation of complement is the major means by which antibodies damage the endothelium during HAR, a variety of means of avoiding complement activation have been employed, including both the administration of exogenous agents to the recipient and modification of the donor pig through genetic engineering. Among the agents that have been used to inhibit complement activity in vivo are cobra venom factor, [69] [70] [71] [72] [73] [74] [75] [76]soluble human complement receptor I (SCR1), [77] [78] [79] and peptide inhibitors of serine proteases.[80] Although all of these treatments have delayed HAR, none has provided long-term protection against antibody-mediated destruction of vascularized organ xenografts.

Genetic engineering has been used to prepare strains of transgenic pigs expressing human complement-inhibitory proteins, which are capable of protecting the transplanted organs from human complement. These regulatory proteins include human decay accelerating factor (hDAF or CD55), membrane cofactor protein (CD59), and human membrane cofactor protein (CD46). Experience with hDAF pig-to-baboon kidney or heart transplantation, coupled with intensive immunosuppressive therapy targeting acquired humoral and cellular immune responses, has produced survival times between 1 and 2 months. [81] [82] The protection against complement supplied by these transgenes seems to prevent HAR, but this protection can be overwhelmed by the humoral response, resulting in delayed xenograft rejection.

Production of Gal-T-Knockout Animals

Although all of the above-mentioned strategies have been capable of delaying HAR, they have been inadequate in preventing the eventual rejection of xenograft organs through humoral mechanisms. In order to completely eliminate anti-Gal antibody-mediated rejection, elimination of the Gal epitope from the surface of donor pig cells has recently been accomplished through genetic engineering involving homologous recombination, followed by nuclear transfer.[83] [84] [85] Two papers appeared in 2002, reporting elimination of the expression of one allele of the α-1,3-galactosyltransferase gene through this technology. [83] [86] In the laboratory of the authors, the nucleus used for nuclear transfer was from a fibroblast cell line derived from the most highly inbred SLAdd subline of miniature swine (described earlier, see Figs. 67-1 and 67-2 [1] [2]), so that the knockout animals are also inbred. As shown in Figure 67-2, production of full knockouts (i.e., GalT-KO) from these animals required two further generations of inbreeding, because only animals of a single sex are produced by nuclear transfer (dependent on the sex of the animal whose cells were used for the homologous recombination). Animals produced by this type of breeding are the most genetically pure, because both chromosomes bear the same mutation and have recently become available. However, both groups that had produced the single gene knockout animals, sped up the process of producing GalT-KO animals for experimentation by selection for a second-loss mutant, followed by another nuclear transfer, leading to pigs expressing no Gal ( Fig. 67-3 ). [3] [4]

FIGURE 67-2  Schematic for the breeding of a new genetic modification (Tg2) onto the current inbred (Tg1) line (in this case, the GalT-KO SLAd line). Theoretically the new inbred line, with both modifications homozygous, can be obtained in only two generations.

 

FIGURE 67-3  The first double GalT-KO double knockout produced from the swine leukocyte antigen dd (SLAdd) inbred miniature swine.

 

Initial results with organ transplants from the inbred GalT-KO miniature swine into baboons have shown the absence of HAR without the need for inhibition of anti-Gal antibodies or of complement. [12] [87] Heterotopic, non-life-supporting heart xenografts maintained with chronic immunosuppression survived up to a record 179 days,[87] but with eventual graft failure associated with thrombotic microangiopathy, most likely induced by acquired cellular and humoral rejection. More impressive, were results with life-supporting renal xenografts using a protocol designed to induce tolerance, which led to survival times of up to 83 days[12] (see later) with several of the longest survivors dying of other causes with functioning kidney grafts. Somewhat disappointing were subsequently published results for renal xenografts from the GalT-KO standard (i.e., nonminiature) swine using a chronic immunosuppressive treatment regimen, in which the longest survival time was 16 days, with kidneys showing marked evidence of rejection.[88] The contrast between these two sets of results for renal xenografts suggests that (1) chronic immunosuppression alone is unlikely to prevent rejection of renal xenografts, even using GalT-KO donors; and (2) that a combination of a tolerance-induction approach with the use of GalT-KO donors may be required for eventual success.[89]

The Cellular Response to Xenografts

Given the broad antigenic disparities between the organ donor and recipient in xenotransplantation, consideration of cellular immune responses includes a number of unique considerations in addition to the innate and acquired immune responses. These include the relative role of xenograft-derived versus host-derived cells in antigen presentation (i.e., direct versus indirect pathways); the role of T cells in propagating humoral immune responses, including those of acute vascular rejection; and the potential for diminished clearance of infection (notably viral) within the xenografts due to MHC disparities. Cellular immune responses are likely to be most prominent when examined in the absence of HAR and AHXR (e.g., in primate recipients of grafts from a GalT-KO pig).

Neutrophils

Neutrophils are most prominent in tissues damaged by hyperacute and acute humoral xenograft rejection. [90] [91] Neutrophils are activated during complement activation through the chemoattractant C5a. Human neutrophils have been observed to adhere to activated porcine endothlia after complement deposition and up-regulation of adhesion molecules including P-selectin and E-selectin. [92] [93] Neutrophils, like natural killer (NK) cells and macrophages, express inhibitory receptors that normally recognize class I MHC and other ligands, but these inhibitory signals may not function across xenogeneic barriers. [94] [95] After xenogeneic vascular endothelium are activated (e.g., through contact with human monocytes,[93] antibodies, or viruses), human neutrophils may be attracted by chemokines, such as interleukin-8 (IL-8), and adhesion molecules, such as E-selectin. Neutrophils may also activate porcine endothelial cells directly in the absence of antibody or complement.

Monocytes and Macrophages

The role of macrophages in xenograft rejection remains under investigation, although they are thought to be a major effector cell in both T cell-mediated and humoral xenograft rejection.[96] Like neutrophils, macrophages may be recruited into xenografts by inflammatory mediators or may directly attack porcine endothelial cells in a process mediated by cell surface lectins binding endothelial sugar moieties.[97] Activated macrophages may also produce tissue factor and directly activate systemic coagulation.

Macrophages have a central role in antigen presentation to T lymphocytes through both the direct and indirect pathways; both mechanisms have been implicated in rejection of discordant islet xenografts. Host-derived antigen-presenting cells and indirect antigen presentation appear to dominate in the process of rejection in concordant xenotransplantation models. [98] [99] Recent data suggest that primed and T cell-activated macrophages are also capable of recognizing and attacking xenografts independent of subsequent T cell help, possibly through xenoreactive antibodies bound to cell surfaces. [100] [101] CD4+ T cell-dependent cellular xenograft rejection is mediated by interferon-γ (IFN-γ) with recruitment of macrophages and NK cells. [102] [103] Blocking macrophage function enhances the engraftment of human hematopoietic cells in SCID mice, [104] [105] indicating the importance of macrophages in highly disparate combinations.

Natural Killer Cells

The roles of NK cells in xenograft rejection are initiated by multiple mechanisms including (1) response to stimulation by FcgRIII receptor (CD16) receptors by xenoreactive IgG antibodies bound to cell surfaces, resulting in ADCC[106]; (2) the absence of inhibitory signals from xenogeneic MHC class I molecules[107]; and (3) direct activation of NK cells by cell surface sugars.[108] NK cells have also been shown to provide indirect antigen presentation for T cell-mediated responses and release of cytokines such as IFN-γ and tumor necrosis factor-α (TNF-α) that amplify the host response through activation of macrophages, endothelial cells, and some T cells.

NK cells are cytotoxic for porcine targets through a number of mechanisms including ADCC, perforin-dependent cytolysis, and probably the Fas/FasL pathway. [109] [110] [111] As a result, NK cells are important mediators of antibody-mediated acute humoral xenograft rejection (described earlier). The cytotoxic role of NK cells is most apparent for nonvascularized grafts such as pancreatic islets and bone marrow.[112] Thus, NK cells have a greater role in resisting the engraftment of xenogeneic than allogeneic bone marrow in mice.[113] This observation is relevant to the requirement to avoid NK cells when using bone marrow transplantation (mixed chimerism) in the induction of xenograft tolerance (see later).

NK cell inhibitory receptors do not interact well across species barriers, in contrast to some MHC-recognizing NK cell activating receptors, which have been shown to function normally. [114] [115] [116] [117] This difference is likewise consistent with the apparently greater role of NK cells in xenograft than allograft rejection. NK activation may also be stimulated through non-MHC restricted “natural cytotoxicity receptors (NCR),” members of an Ig-superfamily that recognize xenogeneic targets and can, for example, induce killing by human NK cells of murine tumor target cells.[118] These activating signals are normally overridden by inhibitory signals received through class I inhibitory receptors that are absent in the xenogeneic combination enhancing the value for xenotransplantation of production of a transgenic pig expressing human class I molecules.

Based on limited studies, adhesion molecules (e.g., E-selectin) appear to function normally between species in terms of the modulation of interactions between NK cells and xenogeneic endothelial cells in AHXR. [119] [120]Interaction of human NK cells with porcine EC resulted in activation of the endothelium to express E-selectin as well as the chemokine IL-8 This activation would be expected to promote migration and infiltration of NK cells and other cells into xenografts.[121]

T Cells

The major role of T cells in xenograft rejection was first demonstrated by the ability of xenografts to grow in nude mice while being destroyed in wild-type mice. The degree to which T cells participate in xenograft rejection may be limited by poor xenogeneic stimulation through direct (i.e., donor) APCs or due to the lack of efficacy of cytokines and costimulatory molecules between species. [122] [123] However, in vitro studies of murine T cell responses to highly disparate xenogeneic species have not proven to be predictive of T cell function in other highly disparate species combinations.

Multiple studies have demonstrated effective xenogeneic T cell stimulation across species' lines and through indirect antigen presentation mechanisms. [40] [124] [125] Analysis of the T cell repertoire used in the rejection of vascularized hamster xenografts in rats revealed a gaussian distribution of T cell receptor usage, in contrast to the more restricted pattern observed in rejected allografts.[126] These results probably reflect the greater degree of protein polymorphism present between species than within species and are consistent with the interpretation that a more diverse array of T cell receptors is capable of recognizing xenoantigens than alloantigens.[40] As was noted earlier, both direct and indirect pathways for antigen presentation are used in generating the cellular responses to xenoantigens. [127] [128] In addition, porcine endothelial cells are capable of presenting MHC antigens to human T cells directly.[129] A possible role for gamma-delta T cells in xenograft rejection has been proposed based on the recognition of nonpeptide antigens, but confirmatory studies are not yet available.[40]

As in allotransplantation, T cells play a role in the development of AHXR through the development of acquired cellular and humoral immune responses to both MHC and non-MHC epitopes (including anti-Gal). Endothelial injury and activation are key components of this delayed form of rejection, with a variety of mechanisms implicated including (1) Infiltration by host NK cells and macrophages, with activation and cytokine production; (2) intrinsic (incompatibility) or extrinsic (e.g., antibody mediated including anti-Gal and anti-MHC or inflammatory) endothelial activation and coagulopathy; and (3) autocrine and paracrine EC activation. It is likely that induced high-affinity anti-Gal IgG as well as antibodies directed against non-Gal determinants are central to AHXR. These antibodies are thought to be produced and maintained by T cell-dependent mechanisms.

Suppression of Cellular Immune Responses to Xenografts

The full impact of acquired immune responses to xenografts can be studied only in the absence of HAR. Even in the setting of AHXR, cellular xenograft injury has been observed.[130] Many pharmacologic agents have been studied in the attempt to suppress acquired cellular and humoral immune responses to xenografts. Clinically relevant programs of immune suppression are under investigation. Although prolonged xenograft survival has been achieved, this has generally been in the setting of combinations of pharmacologic immune suppression with a degree of tolerance induction (discussed later).

TOLERANCE

Two approaches to the induction of T cell tolerance to pig organs in baboons have been investigated extensively in the laboratory of the authors and their colleagues at the Massachusetts General Hospital in Boston. These are (1) mixed mixed lymphohematopoietic chimerism [131] [132] [133] [134] [135] and (2) thymic transplantation. [12] [136] Although full tolerance has yet to be achieved, there has been considerable progress in both approaches:

Mixed Chimerism

Using either miniature swine or hDAF pigs as donors, the source of hematopoietic stem cells (HSC) infused into irradiated baboons has been either bone marrow [30] [137] or cytokine-mobilized peripheral blood progenitor cells (PBPCs). [138] [139] In the initial studies, administration of 2 to 3 × 108 pig bone marrow cells/kg along with swine recombinant growth factors porcine interleukin-3 (pIL-3) and porcine stem cell factor (pSCF) to immunosuppressed baboons led to transient and low levels of chimerism as detected by FACS analysis, although porcine colony-forming units (CFUs) were detectable in the recipients for over 6 months by polymerase chain reaction. [30] [140] [141]Further indication that pig stem cells were probably present at very low levels was the fact that sequential assays were variably positive over time.[30]

Subsequent studies attempted to increase engraftment by raising the dose of progenitor cells administered through the use of cytokine-mobilized PBPC, achieving doses as high as 2 to 4 × 1010 cells/kg following mobilization and pheresis. [142] [143] Even after the administration of these enormous doses of pig cells, they were generally detected in the baboon circulation for only 2 to 5 days, although one animal demonstrated a second appearance of pig cells in the circulation on days 16 to 21.[144] Specific hyporesponsiveness at the T cell level was observed in these animals by in vitro assays, suggesting a functional effect of the transient engraftment. It was reasoned that the failure of engrafted swine HSCs to achieve peripheral chimerism might be due to the appearance of Gal on progeny of these cells and elimination by natural anti-Gal antibodies (nAbs). If so, use of HSC from the new GalT-KO swine to baboons may achieve higher levels and longer duration of peripheral chimerism, and preliminary data appear to confirm this possibility (from reference 139 and unpublished data).

Thymic Transplantation

Yamada and colleagues [12] [136] have transplanted primarily vascularized thymic tissue from swine to baboons, following T cell depletion and thymectomy of the recipients, in an attempt to induce tolerance at the T cell level. The thymic tissue was either in the form of “thymokidneys”—that is, donor kidneys in which autologous thymic tissue had been allowed to engraft for 1 to 2 months under the kidney capsule before use as a renal transplant[145]; or vascularized thymic lobes.[146] As for mixed chimerism studies, the initial attempts were carried out using miniature swine or hDAF swine as donors. The vascularized xenografts survived for up to 30 days, with evidence of viable thymic epithelium and Hassall corpuscles in the thymic implants, and with evidence of donor-specific hyporesponsiveness by in vitro assays in some of the animals after immunosuppression had been stopped. However, all grafts were rejected, coincident with the return of anti-Gal antibodies. In addition, the rejected grafts showed evidence for humoral rather than cellular rejection. Therefore, it was concluded that vascularized thymic tissue grafts induced specific T cell hyporesponsiveness, with the consequent absence of new, T cell-dependent antibody responses. Again, recurrence of anti-Gal antibody appeared to be resistant to down-regulation, and there was no evidence for accommodation.

In order to avoid the effects of these resistant and recurrent anti-Gal responses, the most recent studies of this modality have used GalT-KO pigs as donors of vascularized thymic tissue and kidneys. This change appears to have achieved a major increase in survival of renal xenografts compared with the previous studies using the same protocol.[147] Thus, the survival of life-supporting kidneys was prolonged from a previous maximum of about 30 days to more 80 days by the use of GalT-KO donors. Most importantly, the longest surviving kidneys were still functioning and showed relatively normal gross appearance and histology ( Fig. 67-4 ) up to time that the animals expired or were sacrificed for other reasons. Although these results are early, they are very encouraging for the future use of tolerance induction to achieve long-term xenograft survival.

FIGURE 67-4  Biopsy of a life-supporting GalT-KO thymokidney in a baboon at day 60. Both gross appearance (left) and histology (right) were essentially normal.

 

THE RISK OF INFECTION IN XENOTRANSPLANTATION

Transplantation poses a unique epidemiologic hazard due to the efficiency of the transmission of pathogens, parti-cularly viruses, with transplantation of viable tissues into immunocompromised recipients. Experience with immunocompromised patients suggests that organisms of low native virulence may emerge as pathogens in transplant recipients, including organisms not commonly associated with human disease. [148] [149] [150] [151]

The terms xenosis, direct zoonosis, and xenozoonosis were coined to reflect the unique epidemiology of infection due to organisms carried by xenogeneic tissues. [150] [151] [152] [153] [154] Among the factors that may increase the risk of infection in xenotransplantation are (1) that the xenograft serves as a permissive reservoir in which donor organisms bypass host defenses without a need for a vector to achieve disease transmission; (2) that the virulence and kinetics of donor species-derived infection in immunosuppressed humans is not predictable; (3) that pathogens may produce novel clinical syndromes with atypical clinical features; for example, donor-derived organisms may not cause disease in the native species but may cause disease in a new host (xenotropic organisms), or may acquire new characteristics (genetic recombination or mutation) in the human host [150] [151] [155] [156] [157] [158] [159] [160] [161] [162] [163]; (4) that clinical laboratory assays may not exist for some organisms from nonhuman species; and (5) that donor-recipient incompatibility of MHC antigens may reduce the efficacy of the host's cellular immune responses to infection within the xenograft.

Bacterial Infections

Soon after swine became the most likely source species for xenografts, studies identified a broad array of potential bacterial or parasitic organisms of swine that could be infectious for humans. Most of these were treatable with available antimicrobial agents, although many would not ordinarily be diagnosed or speciated by clinical microbiology laboratories. In addition, given the extensive commercial development of pigs, most extracellular organisms including bacteria and parasites, although not commonly found in humans, have been well studied and can be excluded from closed herds of swine by combinations of breeding, sterilization of food, or through selective use of antibiotics. Furthermore, exposures of humans to products derived from pigs and other non-human species have had no demonstrable adverse effects on individuals or the general population. [162] [164] [165] Caesarian-derived porcine fetal tissues intended for human xenotransplantation carried antibodies to Leptospira interrogans and Aspergillus fumigatus, [160] [162] [166] [167] but no evidence of infections was reported after transplantation into humans. Transplantation of porcine fetal brain cells for the treatment of intractable neural disorders has likewise been achieved without infectious complications to date, although in limited trials.

Viral Infections

By contrast, less information is available regarding porcine viruses. Two classes of viruses are thought to be of special importance: (1) the herpesviruses, based on the central role of this group in causing infection after human allotransplantation, and (2) the porcine endogenous retroviruses (PERV), with the demonstration that this family of viruses is capable of infecting human cells in vitro. [168] [169] [170] [171] [172] [173] [174] In addition to the potential for cross-species infections under normal circumstances, modification of swine to avoid hyperacute graft rejection (described earlier) and post-transplantation coagulopathy may also have some impact on infectious risk. Thus, genetic engineering of swine to express human complement regulatory proteins on porcine xenografts to decrease complement deposition could lead to expression of complement regulatory proteins that also serve as cellular receptors for human pathogens (e.g., CD46 and measles), to which swine would not naturally be susceptible. In this situation, human measles might have the capacity to infect organs derived from CD46-transgenic swine. In addition, xenoreactive natural antibodies, the main mediator of HAR, may be advantageous in the prevention of human infection by retroviruses, parasites, and other common organisms carrying the α-1,3-Gal epitopes. Thus, depletion of anti-Gal antibodies could theoretically allow transmission of porcine viruses (carrying the Gal epitope) from a porcine xenograft to the host. [175] [176] [177] Similarly, GalT-KO swine,[86] which lack the Gal transferase and the Gal epitope on their cell membranes, will produce porcine viruses lacking this epitope, potentially diminishing human antiviral protection provided by natural antibodies. Finally, the infectious consequences of tolerance induction (antigen-specific immunologic unresponsiveness) and of the variety of immunosuppressive regimens used to prevent cellular rejection remain to be determined. At this point, these concerns remain theoretical, and no evidence for xenotic viral transmission to non-human primates has been reported to date.

Identifying Potential Human Pathogens

Only a subset of porcine pathogens is likely to pose a unique threat in immunocompromised human hosts ( Table 67-1 ). Lists have been generated to guide the breeding of source animals for xenotransplantation including organisms thought likely to cause disease in xenograft recipients based on known ability to infect humans (Toxoplasma gondii), similarity to organisms commonly causing infection in transplant recipients (e.g., PCMV), and high predilection for recombination (e.g. parvovirus). In addition, there may be organ-specific exclusion lists (e.g., Mycoplasma sp., adenovirus, or influenza in lung xenografts). Such lists, although inexact in the absence of clinical experience, serve a variety of purposes in the progress of xenotransplantation. These include the possibilities that (1) organisms thought to pose an unacceptable risk to the recipient can be bred out of a donor herd prospectively (designated pathogen-free) [152] [153]; (2) microbiologic assays for listed organisms can be developed for clinical use; (3) studies in preclinical xenograft models may clarify the biology of these organisms; and (4) prophylactic antimicrobial strategies can be developed for organisms not “removed” from donors.


TABLE 67-1   -- Organisms for Exclusion from Source Swine for Clinical Xenotransplantation

Bacteria

Actinomyces

Brucella suis

Leptospira

Listeria monocytogenes

Mycobacterium bovis

Mycobacterium tuberculosis

Mycobacterium avium-intracellulare complex

Mycoplasma hyopneumoniae (lung transplant)

Salmonella spp. (typhi, typhimurium, cholerasuis)

Shigella sp.

Parasites

Ascaris suum

Cryptosporidium parvum

Isospora sp.

Microsporidia sp.

Neospora

Strongyloides ransomi

Trichinella spiralis

Fungi

Cryptococcus neoformans

Aspergillus sp. (colonized or lesions)

Candida sp. (lesions)

Histoplasma capsulatum

Viruses

Circovirus

Porcine endogenous retrovirus (PERV)

Porcine cytomegalovirus (PCMV)

Encephalomyocarditis virus

Influenza viruses (porcine, avian, human)

Porcine γ-herpesvirus or porcine lymphotropic herpesvirus (PLHV-1)

Porcine reproductive and respiratory syndrome virus

Porcine parvovirus

Nipah (Hendra-like) and Menangle virus

Pseudorabies

Rabies

Rotavirus

Modified from Fishman JA: Xenosis and xenotransplantation: addressing the infectious risks posed by an emerging technology. Kidney Int 58(suppl):41–45, 1997.

 

 

 

Organism exclusion lists vary with the donor animal species and the use intended for the xenograft. Accordingly, such microbiologic standards must be dynamic, that is, they must be rigorously tested and subject to revision based on experimental and clinical data. Standards for testing must reflect the evolution of testing strategies (e.g., new quantitative molecular assays) and adjusted for differences in immunosuppressive regimens and epidemiology. Long-term “archiving” and testing of tissue and serum samples from donor animals and xenograft recipients have been mandated by FDA guidelines for clinical xenotransplantation. These items can also be used to track the epidemiology of unsuspected or novel pathogens in clinical trials. The mobility of human populations necessitates communication between public health authorities in the detection of unusual pathogens conveyed by xenotransplantation. Data are most easily shared if common international standards are developed for screening of donor animals and recipients, and the data are available in formats that are universally interpretable. Approaches to the sharing of data internationally have been addressed (see the “Consultation on Xenotransplantation Surveillance” sponsored by the OECD, WHO, and Health Canada, http://www.oecd.org).

Retroviruses

Exogenous retroviruses (human T cell lymphotrophic virus-1 [HTLV-1], HTLV-2, and HIV) have been transmitted with human tissues during organ transplantation. The course of accidentally transmitted infection due to HIV-1 is accelerated in transplant recipients not receiving effective antiretroviral therapy, generally manifesting disease (acquired immunodeficiency disease [AIDS]) within 6 months.[178] Concern about retroviral transmission in xenotransplantation relates to the potential for “silent” transmission, that is, asymptomatic infection that may subsequently cause altered gene regulation, oncogenesis, or recombination in the recipient. The activation of latent virus and the development of clinical manifestations might be delayed. Pigs do not possess exogenous viruses equivalent to HTLV or HIV. However, PERVs (part of the germ line DNA) have been demonstrated in swine. Three closely related C-type PERV (PERV A, B, C) have been identified in swine that possess infectious potential ( Fig. 67-5 ). [168] [173] [179] [180] [181] [182] [183] Two of these, PERV-A and PERV-B, can infect human and pig cells in vitro.[173]The third subgroup, PERV-C, infects porcine cells only. Infectious forms of the remaining PERV families have not been isolated and are unlikely to encode infectious virus due to disruptions in open reading frames (ORFs).[182]PERV mRNAs are expressed in all pig tissues and in all breeds of swine tested to date, and expression is amplified by stimulation of swine peripheral blood lymphocytes in vitro. There is variation between tissues in terms of the size and amount of PERV mRNA transcripts, consistent with in vivo recombination and processing.[168]

FIGURE 67-5  Three closely related C-type porcine endogenous retroviruses (PERV A, B, C) with infectious potential have been identified in swine. PERV-A and PERV-B can infect human and pig cells in vitro. The third subgroup, PERV-C, infects only porcine cells. PERV isolated from “transmitting” animals appear to be recombinants between PERV-A and PERV-C sequences. These are likely exogenous recombinants produced by homologous recombination between PERV-A and PERV-C. PERV-AC are derived from the recombination of PERV-A elements with the post-VRA (envelope) region of PERV-C. Autoinfection or horizontal infection of pigs by PERV-AC may occur. No evidence of productive infection due to PERV has been demonstrated in human cells in vivo, and no disease due to this family of viruses has been described in swine or humans to date.

 

PERV-A and PERV-B infect several human cell lines and primary cell cultures (see Fig. 67-5 ). [170] [173] [181] [184] [185] [186] [187] Swine can be classified according to whether their peripheral blood mononuclear cells either do or do not transmit PERV to human cells in vitro. Such animals are identified as either transmitters or nontransmitters. Productive infection by PERV-A is observed in human cells in vitro and in other cells and transgenic mice carrying the PERV-A receptor. [181] [188] However, high-titer human-tropic PERV isolated from “transmitting” animals appear to be recombinants between PERV-A and PERV-C sequences. [181] [188] Although the site of recombination varies, viral sequences are derived from the recombination of PERV-A elements with the post-VRA (envelope) region of PERV-C (see Fig. 67-5 ). Therefore, although PERV-C is not capable of infecting human cells, it appears to be an essential component of human-tropic PERV from swine and may be important in the assessment of infectious risk associated with xenotransplantation. The source of such recombinants in vivo is unknown; analysis of the germ line DNA of transmitting animals has recently been shown to include recombinant PERV A-C, although the significance of this observation and the infectivity of this provirus are unknown. Swine with incomplete genomic provirus (i.e., PERV A env sequence) might be able to generate infectious recombinant viruses in the presence of infectious PERV-C.

No evidence of productive infection due to PERV has been demonstrated in human cells in vivo and no disease due to this family of viruses has been described in swine, non-human primates, or humans to date. [8] [162] [164] [189] [190] [191] PERV appears to be susceptible to available antiretroviral agents. [192] [193] Some data suggest that primary cell lines of primates (baboons, gorilla, and macaques) can be infected by PERV-A, -B, and possibly -C, which enhances the value of preclinical studies in primates. However, other studies question the value of these models because the infection of non-human primate tissues is often abortive. Whether humans are equally nonpermissive remains to be determined.

Herpesviruses

Activation of latent herpesvirus infection during periods of intensified immune suppression or immune dysfunction and by immune reactivity to grafts (rejection) is an important problem in human allotransplantation. Comparable viruses exist in swine but tend to be species-specific, and would be expected to cause infection only in tissues derived from the usual host species for each viral strain.

Most importance is placed on cytomegalovirus (CMV), which causes invasive tissue disease, fever, neutropenia, and immune modulation that contributes to the risk for secondary infections, graft rejection, and lymphoma. Extensive molecular screening identified two families of herpesviruses in swine: PCMV and the porcine lymphotropic viruses (PLHV-1, -2, -3). Replication of PCMV is enhanced by intensive immune suppression in pig-to-primate models of xenotransplantation. [163] [194] [195] PCMV infection causes tissue-invasive infection in porcine xenografts in baboon hosts and contributes to endothelial injury and consumptive coagulopathy (CC) in some animals.[196] Based on molecular and histologic evaluations, PCMV does not appear to cause invasive disease in tissues of baboons that have received porcine xenografts.[195] It is possible to exclude PCMV from herds of swine by early weaning of newborns. [35] [197] Porcine CMV has reduced susceptibility to ganciclovir, foscarnet, and cidofovir compared with human strains.[198]

A novel family of γ-herpesviruses, PLHV-1, -2, -3, has been identified in swine by amplification of short DNA polymerase sequences from pigs. [199] [200] [201] PLHV-1 is associated with a syndrome of lymphoid proliferation in swine undergoing experimental allogeneic hematopoietic stem cell transplantation. This syndrome has characteristics similar to post-transplantation lymphoproliferative disease (PTLD). [201] [202] [203] Based on sequence analysis, this virus has some genetic homology with known sequences of lymphocryptovirus and the rhadinoviruses. [199] [201] The role of this virus in the pathogenesis of PTLD is under investigation. In porcine allogeneic hematopoietic stem cell transplantation, the risk of PTLD in swine is related to the overall intensity of immune suppression, the MHC disparity between donor and host, the degree of T cell depletion, and the PLHV activation that precedes B cell proliferation. The roles of PLHV-2 and PLHV-3 are unknown. Unlike PCMV, PLHV-1 is not removed from source animals by early weaning of newborns. [204] [205]

Other Potential Pathogens

A variety of potential human pathogens have been described in swine. These include porcine circovirus types 1 and 2 (PCV-1, -2), porcine reproductive and respiratory syndrome virus, porcine encephalomyocarditis virus, swine influenza viruses, African swine fever virus, hepatitis E-like virus, pseudorabies virus, parvovirus, and polyomaviruses of swine. None has yet been associated with human disease. PCV is highly prevalent in herds of swine and is exacerbated by immune suppression, causing pneumonitis. PCV-1 and -2 cause nonproductive infection of human cells in vitro, with only PCV-2 causing cytopathic effect.[206]

The Potential for New Pathogens

Recent human epidemics of viral infection have been traced to animal-derived viruses that have become adapted to human hosts. These include influenza (avian), hantavirus (mice), severe acute respiratory syndrome due to a new coronavirus possibly associated with civets (SARS), bovine spongiform encephalopathy, and HIV thought to evolve from primate viruses. In each case, the epidemiology has been defined after the recognition of a new clinical syndrome and the development of new, rapid, molecular assays for the causative agent. Pigs may serve as hosts for the adaptation of avian respiratory viruses to human hosts. Thus, human, porcine, and avian viruses could undergo genetic reassortment in swine to produce novel strains of pandemic potential. It is possible that infected lung xenografts might, for example, provide the “Petri dish” for recombination between porcine and human influenza viruses, particularly in the immunocompromised host. This theoretical concern can be addressed through the breeding of herds of source animals in isolation from human respiratory viruses and adventitious birds and rodents. Given highly sensitive molecular assays, it is feasible to ensure that pigs used as source animals for xenografts, particularly lungs, be screened for influenza viruses.

Hepatitis E virus is a major cause of viral hepatitis in the developing world and has recently been detected in swine in North America and Asia. Although spread of hepatitis E virus to humans from pigs has not been demonstrated, the viruses found in swine are closely related genetically to those causing human disease, suggesting that pigs may serve as a reservoir of infection and might merit screening of animals to be used as a source of porcine hepatic grafts.[207] [208]

Infectious Risks, Surveillance, and the Search for Novel Pathogens

In xenograft recipients, the risks of infection and rejection necessitate lifelong monitoring. In addition to the recipients, intimate contacts of the recipients might also be at increased risk for xenogeneic infection. Monitoring schemes have been proposed to detect known pathogens and archive specimens from source animals, and from patients, intimate contacts, and animal handlers on a routine basis for use in the event of unexplained infectious episode. These samples may be used as further microbiologic assays are developed against previously unrecognized pathogens.

The identification of PERV and other viruses have allowed the development of sensitive assays for these agents and strategies for exclusion of these pathogens from xenograft donors. Additional human pathogens may be identified in preclinical models, whereas others may appear only in clinical trials. Thus, investigators and public health authorities must share data that suggest the presence of unusual infectious events in xenotransplantation. These suggest the need for shared definitions for xenogeneic infectious disease events (case definition, laboratory assays, and specific organisms) and international cooperation in reporting, recording, and response to adverse events associated with xenotransplantation.

Infectious Benefits of Xenotransplantation

Infection is one of the major problems of human allotransplantation. Infection takes the form of a primary diagnosis underlying organ failure (hepatitis A, B, or C), as a cause of recurrent disease and graft failure, as an opportunistic infection in immunocompromised hosts (CMV, varicella zoster virus), or an underlying condition complicating the process of transplantation (HIV, recurrent pulmonary infections in cystic fibrosis, sepsis or aspiration pneumonia while awaiting liver transplantation). Clinical xenotransplantation may resolve some of these limitations.

Careful microbiologic screening of the animals used for xenotransplantation may provide a greater degree of safety than that which is presently available in the limited time for screening of organs from deceased human donors. Patients can receive their transplants at the time of greatest clinical need, reducing waiting times and the risks for nosocomial colonization or complications of long-term debility. Xenogeneic tissues also appear to be resistant to infection by some of the human pathogens commonly complicating allotransplantation including HIV (1, 2), HTLV, hepatitis viruses, and herpes viruses (including human CMV). Such species' specificity may reflect the absence of receptors or of host cellular machinery needed to support for viral replication in human cells.

THE FUTURE OF XENOTRANSPLANTATION

The remarkable success of allogeneic transplantation over the past three decades has led to an awareness of how many more lives could be saved if additional donor organs were available. This gap is such that, in the field of renal transplantation , the number of living donor transplants being performed annually in the United States (and in many other countries) now exceeds the number of deceased donor transplants.[209] Even so, there are far more patients waiting for transplants than the number performed each year, and only those patients who meet stringent criteria are able to be registered on a waiting list.

New sources of organs must be found if the field of transplantation is to continue to grow to meet this demand. Although tissue engineering[210] and stem cell research[211] have received much public attention recently, and although these technologies may be capable of providing functional cells or tissues for transplantation in the near term, it will undoubtedly be many years before they can produce an organ with all the functions of a normal kidney. Therefore, xenotransplantation is at present the best hope for overcoming the limitation that organ availability has imposed on the field of clinical transplantation, and it seems likely that research toward this goal is likely to continue. Among the issues that remain to be answered on the way toward clinical trials, some of the most important are fear of new viruses, and a series of important ethical issues.

Fear of New Viruses

As described in detail in the section on infectious diseases earlier, there has been much concern raised about the possibility that xenotransplantation could lead to the release of new viruses that the human immune system may be unprepared to handle. The best studied of these is PERV; the discovery that PERV could infect human cells had a chilling affect on the field for a few years while worst case scenarios were envisioned, especially in the news media.[212] However, as described earlier, no evidence of productive infection due to PERV has been described in vivo in either primates or swine despite vigorous attempts to detect such infections. [164] [213] In addition, PERV has been shown to be susceptible to available antiviral agents.[193] At this point, the FDA has encouraged further research in the area, with clear guidelines on the caution to be taken in initial studies.[214] Given the importance and attention that surveillance for potential viral pathogens has been given, it is likely that concerns about viruses will constitute a manageable risk that should not delay research or implementation of xenotransplantation in the future.

Ethical Issues

A full discussion of ethical concerns related to xenotransplantation would be beyond the scope of this chapter. Among the most apparent are animal rights, religious attitudes, impact on the right to die, the need for additional genetic engineering, and the decision on “who goes first”.

Animal Rights

There are well-organized groups both in the United States and in other countries with the expressed goal of stopping all research involving animals. As might be expected, these groups have been vocal in their opposition to xenotransplantation. However, given that the field of xenotransplantation has now settled predominantly on the pig as the most likely organ donor, and that use of pigs for food is accepted in most modern societies, it is generally thought that the potential saving of human lives through xenotransplantation would be an appropriate use of swine bred for this purpose.

Religious Attitudes

All of the major religious denominations have agreed that the use of pig organs to save lives appears justified. [215] [216] Indeed, during the International Transplantation Congress in Rome in 2000, the Pope endorsed the use of animal organs for xenotransplantation.[217] However, given the availability of dialysis therapies, orthodox Jewish and Muslim leaders have not yet endorsed the use of pig kidneys for renal replacement.

Impact on the Right To Die

The fear has been expressed that if organ availability were to become unlimited, no one would be allowed to die. However, as for all life-prolonging measures, the allocation of transplanted organs should be dependent upon the quality of life attainable by the patient following transplantation, and such decisions are in the province of the individual doctor-patient relationship. Although the availability of xenogeneic organs could eliminate waiting lists and avoid the need to subject living donors to unnecessary risks, consideration must always be given to the fact that transplants should be provided only to patients whose lives might be restored to a healthy physical and mental state by the procedure. This fear also appears to constitute a manageable ethical concern.

Need for Additional Genetic Engineering

The relative ease of genetic manipulation of swine has already made possible the production of transgenic animals and knockouts, both of which have facilitated pig-to-primate organ transplantation. However, as promising as the recent results with GalT-KO donors have been, it is likely that new problems will become apparent as organ survival times increase. This is particularly true for the liver, which produces a large number of enzymes and other critical proteins, some of which would likely be incompatible between species. However, as long as the number of such incompatibilities is limited, genetic engineering would also have the potential to overcome them. Thus, for example, it is possible to envision production of an ideal swine for donation of a liver, in which cross-species incompatible proteins would be replaced by their human counterparts, using a combination of transgenic and knockout technologies. Similarly, should porcine erythropoietin (EPO) not be an adequate replacement of its human counterpart, an ideal renal xenotransplant donor might be engineered to express human EPO. As indicated in Figure 67-2 , production of inbred animals with new genetic modifications should be greatly facilitated by the use of inbred miniature swine.

Decision on Who Goes First

There is general consensus among most scientists and clinicians that clinical organ xenotransplantation should not be attempted until there is a reasonable expectation of success, on the basis of successful pig to nonhuman primate experiments. However, debate remains concerning which organ should be attempted first, as well as concerning which patients would be most qualified as recipients. Advocates for heart and liver transplantation point out that there are no alternatives at present when a patient has reached the point at which life can be saved only by an immediate transplant. They argue further that a xenotransplant might be attempted in such situations even if only as a “bridge” until an allogeneic transplant becomes available. This argument makes sense for the individual desperate recipient, and might also be reasonable as a way to determine the safety and potential efficacy of xenotransplantation in early clinical trials. However, use of the xenograft as a bridge to allotransplantation would not only fail to overcome the problem of organ shortage but might even make it worse, because additional patients would accumulate on the waiting list for allogeneic donor organs.

The fact that there are other means available to support life in the face of renal failure provides arguments both for and against using kidneys as the first organs for clinical xenotransplantation. The positive argument is one of safety, because failure of the xenotransplant would not necessarily cause death of the recipient, who could receive life-supporting dialysis. On the negative side with regard to early trials is the fact that a “bridge” might not make sense for renal transplantation, because dialysis already provides this function. Thus, only if xenotransplantation could be viewed as destination therapy, at least as effective as allogeneic transplantation, would it be reasonable to offer this modality to a potential recipient. One important exception would be potential recipients who are highly sensitized to all potential donors, so-called high-PRA individuals, who might otherwise remain on a waiting list for many years, or die while on a waiting list.[218] A recent study tested the cross-reactivity of sera from high PRA individuals with GalT-KO cells and found no increase in reactivity over sera from non-PRA-positive individuals.[219] It was concluded that such patients might be excellent candidates for first trials of renal xenotransplantation. Another exception might be diabetic patients with end-stage diabetic nephropathy. Recent work from the authors' laboratory on the use of composite organs, so-called islet-kidneys, indicates that it may be possible to cure both diabetes and renal failure simultaneously with such transplants. [220] [221] In these experiments, the pig donor's islets were isolated and implanted under the autologous kidney capsule 2 to 3 months before transplanting the composite organ into a diabetic recipient. The islet-kidneys were effective in curing surgically induced diabetes in fully allogeneic pigs, and therefore, may have potential as xenografts if long-term survival of xenotransplanted kidneys can be accomplished.

These few examples illustrate some of the questions that will still require answers as the field of xenotransplantation progresses. Although some of these considerations may now be seen as obstacles to eventual clinical applications, none of these obstacles appear to be insurmountable. In the last analysis, the success of xenotransplantation as a clinical modality is more likely to depend on the functional and technical success of preclinical large animal experiments than on any of these remaining issues.

References

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