Xenotransplantation

No area of medicine stimulates as much excitement or as much controversy as transplantation. The rationale for xenotransplantation is the shortage of human organs and tissues. For several reasons, however, most in the field of xenotransplantation have abandoned the use of nonhuman primates. Therefore, instead of using primates, most in the field of transplantation focus on the use of pigs or other non-primate species. If opportunities offered by xenotransplantation are great, the hurdles, at present, appear equally so. These hurdles include (i) the immune response of the host against the graft leading to rejection of the graft, (ii) the inherent physiologic limitations of the animal tissue or organ in a human system or induced disruption of the normal functions of the recipient, and (iii) the possibility of transferring infectious agents from the transplant to the recipient and, potentially, more broadly to the general population. This chapter focuses on the hurdles to transplanting porcine organs and cells into humans. The past few years have brought significant progress in defining the hurdles to xenotransplantation and progress in overcoming the immunologic and physiologic hurdles in this field.

Transplantation is considered to be established therapy for a variety of end-stage diseases. Despite some obvious parallels, transplantation stands in stark contrast to many other health care services. First, while cost is an extremely critical issue, donor organ supply has always been the foremost concern. Second, the gap between need and demand is narrowing. Third, this situation has been the source of highly controversial disputes. Finally, it is conceivable that if we were to resolve the supply issue, the financial one would become even more significant than it is today. Too often, naive analysts and misleading analyses give the impression that the future will be unlike the past, since both xenotransplantation and mechanical devices will enable to overcome the supply constraint. This chapter focuses on actual and potential cadaveric donors and donor organs. It presents data in tables to enable to put xenotransplantation into an appropriate perspective. Despite the data reported here, futile health policy efforts will be directed toward eliminating the disparity between the need for solid organ transplantation and the supply of human donor organs. The pace of progress may be exceedingly slow, as nonclinical issues take center stage as resource constraints become as apparent as they are real.

This chapter discusses the role of complement in the pathogenesis of tissue injury in xenograft rejection and reviews methods to inhibit complement activation as part of strategies to achieve xenograft survival. It begins with a brief overview of the complement system. The complement system is composed of 35 plasma and membrane-associated proteins, which include control proteins and receptors on cell membranes that recognize various fragments that result from complement activation. The membrane-associated complement regulators are of great interest to xenotransplantation. Although soluble complement inhibitors that can be used in vivo are beginning to be developed, small nontoxic molecules that inhibit complement efficiently are not yet available. Some of the large molecules that have been successful to abrogate hyperacute rejection (HAR) of the xenograft in experimental animals may be useful, at least temporarily, to reduce reperfusion injury and prevent HAR. A recent report indicates that a hexadecemeric multiple peptide of the C1q-binding site from human IgGi inhibits lysis of pig red cells by human serum, with an I50 value of 1 μM. This study suggests that peptides derived from the C1q binding region of immunoglobulins could hold promise to inhibit activation of the classical complement pathway.

The use of heterologous species for clinical transplantation has attracted increasing interest in recent years due to the shortage of human donors, resulting in significant progress toward the understanding of the mechanisms leading to recognition and rejection of xenogeneic grafts. Analysis of the leukocyte infiltrates found in tissues undergoing delayed xenograft rejection (DXR) has led to the identification of natural killer (NK) lymphocytes as an important effector cell subset. This paper focuses on the role of NK cells in xenogeneic graft rejection, on the biology of NK cells, and on NK-cell receptors in particular. NK cells contribute to the natural host defense mechanisms, both by exerting cytotoxicity on certain virally infected or tumor cells and by releasing cytokines that promote an inflammatory response, such as γ-interferon (IFN), tumor necrosis factor (TNF), granulocyte/macrophage-colony stimulating factor, and macrophage colony-stimulating factor. During NK-cell ontogeny, the CD34 surface antigen is lost and cells acquire the expression of membrane markers characteristic of mature NK cells (CD2, CD 16, CD56) . Chimerism induced by donor-specific bone marrow transplantation has been shown to produce alio- and xenotolerance, even across a discordant barrier, with evidence of NK as well as T- and B-cell tolerance. However, it seems likely that more than one of the approaches briefly outlined above might be eventually necessary to efficiently prevent NK-cell-mediated xenograft damage.

This chapter centers on adaptive or antigen-specific cellular responses to xenografts. It emphasizes the nature of T-lymphocyte-dependent immunity to xenografts. Most of the concepts concerning T-cell responses to xenografts developed in the chapter are derived from rodent studies. When considering the T-lymphocyte response to non-self antigens, including xenografts, it is imperative to emphasize the two fundamental properties of T-cell reactions. Although transferred human cells can mediate the rejection of human allografts or porcine xenografts in these animals, they generally fail to initiate vigorous GVHD against the scid mouse host itself. This model of donor MHC-restricted (direct) and host MHC-restricted (indirect) pathways of graft antigen presentation has important implications for the nature of T-cell-dependent immune responses to both allografts and xenografts. Two differing pathways of graft antigen presentation can be envisioned that would fulfill the two-signal requirement for T-cell activation, each involving APC-dependent processes: (i) donor MHC-restricted responses, and (ii) host MHC-restricted responses. CD4-dependent xenograft rejection depends on host and not on donor MHC class II expression. We find that CD4 T cells trigger rapid rejection of rat islet xenografts established in immunodeficient recombinase-activating gene (rag)-deficient hosts. Immunodeficient scid and rag-deficient mice accept tissue and organ xenografts despite retaining innate immune reactivity, including NK-cell function. The precise molecular mechanisms of cellular xenograft rejection remain to be identified, especially regarding the role of particular Th1 and Th2 cytokines in triggering tissue injury.

To achieve successful xenotransplantation, it is necessary to overcome the immunological barriers that evolution has built up between different species. In contrast to allotransplantation, where the cellular response is the main hurdle, in xenotransplantation both humoral and cellular responses have to be overcome. In attempts to achieve successful pig-to-human xenotransplantation, several approaches are currently being evaluated. Genetic engineering techniques are being applied to the problems of xenotransplantation. To achieve successful xenotransplantation, it will probably be necessary to combine several therapeutic techniques and/or agents, as is the case with allotransplantation today. Xenotransplantation offers the first opportunity for modifying the donor as opposed to the recipient, which opens up new possibilities in this era of rapidly developing techniques such as genetic engineering, gene transfer, and cloning. The breeding of a pig with a vascular endothelial structure against which humans have no preformed antibodies would be a major advance. In the recipient, however, it will still be necessary to inhibit the production of induced antibodies, as well as the strong cellular response, either by some form of immunosuppressive therapy or by the induction of tolerance.

The humoral immune system comprises elements that, in part, provide protection against invading organisms. This chapter discusses the effects of manipulating the humoral immune system on the risk of developing microbial infections. Plasmapheresis (also called plasma exchange) has been utilized as a therapy for a limited number of immunological diseases that are caused by autoantibodies. Plasmapheresis has several possible benefits. First, plasma exchange may remove a portion of pathogenic autoantibodies. Second, removal of some circulating immune complexes may be achieved by plasmapheresis, although it has almost no effect on tissue-bound immune complexes that may be formed in situ. Specific methods, such as specific immunoadsorption methods, have been developed to remove specific pathogenic antibodies. Immunoadsorbents have been developed that remove immunoglobulins nonspecifically or that remove specific antibodies selectively. Elements of innate immunity such as natural antibodies and complement have clearly been shown to play an important role in host resistance against microorganisms. Experimental and clinical studies are needed to fully appreciate the beneficial and harmful effects of methods that interfere with unwanted pathologic effects of elements of the humoral immune system.

The complement (C) system comprises some 15 soluble plasma proteins that interact with one another in three distinct enzymatic activation cascades (the classical, alternative, and lectin pathways) and in the nonenzymatic membrane attack pathway. C is an important component of the host innate immune system. This chapter reviews briefly the main situations in vivo where C may cause disease, discusses recent strategies for regulating C activation in order to reduce C-mediated pathology, and addresses the potential problems related to therapies that inhibit the physiological effects of C activation. Harm will result when C activation occurs in an uncontrolled manner and/or at an inappropriate site; the end result of this will be inflammation and tissue destruction. "Iatrogenic" (treatment-precipitated) C activation is a common consequence of the many modern therapies that involve contact of blood with a foreign surface. The naturally occurring C regulators are excellent inhibitors of C and have potential as therapeutic agents. Inhibitors acting in the terminal pathway are less likely to predispose to bacterial infections and immune complex disease, particularly when used for long-term therapy. With the realization that hyperacute rejection was mediated by C attack came the suggestion that the human C regulators might be utilized to overcome this hurdle. The simplest approach to protecting the xenograft would be to utilize the fluid-phase C regulators. A xenograft hyperexpressing human membrane cofactor protein (MCP) might be rendered highly susceptible to infection with measles virus with consequences for the graft that are not predictable from current knowledge.

This chapter discusses human anti-pig T-cell responses as this system is particularly relevant to clinical transplantation. T-cell recognition of foreign major histocompatibility complex (MHC) antigens has represented a challenging enigma for cellular immunologists over more than three decades. First, T-cell reactivity to nominal antigens can be detected only if the individual had been primed in vivo with the respective antigen, or after in vitro immunization by multiple antigenic stimulations. The second major difference between T-cell responses to allogeneic or xenogeneic cells and reactivity to nominal antigens resides in the lack of self-MHC restriction and processing requirements for recognition of foreign cell surface MHC antigens. Elucidation of the three-dimensional structure of class 1(11) and class II MHC (17) molecules, together with major advances in our understanding of antigen processing and presentation, had clarified considerably the molecular basis of direct T-cell recognition. The emerging picture is that both the allogeneic MHC molecule itself and the bound peptides may each independently contribute to reactivity and, furthermore, that peptide binding to MHC may induce conformational changes in the MHC molecule itself that affect recognition. Indirect recognition of xenogeneic antigens is likely to induce more powerful immune responses than human allogeneic MHC antigens, because of the larger number of foreign antigenic peptides that result from the processing of xenogeneic proteins. In conclusion, xenografts, like allografts, elicit T-cell-mediated immune responses against foreign antigens present in the graft, via the direct and indirect recognition pathways.

This chapter reviews the issue of xenotransplantation as a vector for infection to humans and considers potential mechanisms for disease, screening systems, and evaluations that need to be carried out to identify these risks as the field moves forward. Knowledge of donor-associated infections after allotransplantation and of zoonotic infections is used to help estimate the potential risk of xenotransplantation. Bacterial or fungal infections in the airway of the donor can lead to disease after lung or heart-lung transplantation, and histoplasmosis has been transmitted with cadaveric kidneys. These types of acute donor-associated infections can theoretically be less of a risk after xenotransplantation as source animals can be maintained under optimal healthy conditions and surgery would be performed on an elective schedule rather than under the current time pressures of allotransplantation. In an attempt to minimize the risk of xenozoonoses and to learn how to evaluate animal-associated infections after xenotransplantation, more extensive protocols began to be developed in the early and mid-1990s that continue to be expanded upon today. Some microbes, particularly viruses, are considered to be "species specific." If this is the case, it is possible that xenotransplantation carries less risk of donor-transmitted infections than does allotransplantation. Acute viremia can lead to donor-associated infections after allotransplantation and would likewise be expected to have the same risk after xenotransplantation. Xenotransplantation may have positive implications for decreasing the risk of some infections after transplantation.

Scientists have proffered widely varying opinions on the extent to which xenotransplantation clinical trials threaten to introduce new infections into the human community. Observers at one extreme conclude that the risk of introducing new infections to the human community is too great, and the potential for benefit is too unclear. These observers argue that the only responsible stance is to impose a complete moratorium on clinical xenotransplantation until there is sufficient knowledge to assess these risks. Observers on the other extreme argue that in the absence of data to support these fears, restrictions that slow or halt the progress of xenotransplantation research would unnecessarily impede progress in an area that promises unmeasurable relief to human suffering. Other observers note that both the risks and the benefits associated with clinical xenotransplantation remain theoretical at present. Advocates of this approach argue that these trials can be accompanied by safeguards stringent enough to adequately protect the public. Laboratory-based studies of xenograft survivors will also increase one's ability to quantify xenotransplant-associated risks and thereby expand one's capacity to make science-based assessments of appropriate public policy. The risk that any xenograft recipient may become infected with porcine endogenous retrovirus (PERV) is likely a function of multiple factors associated with the source animal, the xenotransplantation technique, the characteristics of the human recipient, and the level of PERV expression by the transplanted cells. Reviews of historic developments in xenotransplantation coupled with critiques of developing public policy and procedures have furthered efforts at consensus development.

This chapter talks about infectious agents that have the potential to be transmitted via swine xenografts. These infectious agents fall into three categories: traditional zoonotic organisms, swine-specific organisms and undiscovered swine-specific organisms. Risk of disease transmission via xenografts and the public health aspects of porcine xenotransplantation are discussed. The chapter addresses the history, occurrence, etiology, epidemiology, mode of transmission, animal infection, diagnosis, prevention and control, human infection, and public health aspects of each specific disease. The intention is to narrow the scope of the review to describe the occurrence, the epidemiology, and the clinical signs of the traditional zoonotic diseases in the pig. The knowledge of the epidemiology, especially mode of transmission, in the pig itself is essential for the design of facilities and the establishment of biosafety and disease control programs that can minimize the risk of transmitting infectious disease agents via swine xenografts. The majority of affected herds may have no recognizable signs. Bratislava is now recognized as the most common swine-maintained serovar. Carrier pigs are probably the most common route of introduction, with infected replacement gilts or boars being one of the most important means of introducing infection. For those swine-specific organisms that are sequestered in tissues such as gastrointestinal tract, tonsil, and upper respiratory tract of clinically healthy donors, the risk of being transmitted via xenografts is probably minimal.

Most vertebrate species harbor multiple inherited proviruses, which are called endogenous retroviruses (ERV) to distinguish them from infectiously transmitted, exogenous retroviruses. These inherited retroviruses in potential source animals raise questions about their potential transmission via xenotransplantation and are discussed in this chapter. It is noteworthy that xenotropic retroviruses have been found to infect foreign cells in the xenotransplantation setting. The possibility of recombination between animal retroviruses and endogenous or infectious human retroviral genomes needs to be borne in mind in xenotransplantation. As pigs are a favored species for xenotransplantation to humans, there has been renewed interest in porcine retroviruses. Unlike ruminants such as sheep, pigs appear to carry only one group of infectious retrovirus, the C-type retroviruses, related to murine leukemia virus (MLV) and gibbon ape leukemia virus (GALV). The foregoing discussion of natural and experimental infection across large phylogenetic distances and other factors, show that retroviruses are able to infect and cause disease in hosts wholly unrelated to those from which they emerge. Indeed, the zoonoses discussed in this chapter illustrate that animal retroviruses have found other means of infiltrating humans. Xenotransplantation can offer the extremely rare event of zoonosis much more opportunity to occur, for many reasons. First, the physical barrier to cross-species infection is breached by implanting animal tissues in humans. Second, the immunosuppression necessary to prevent graft rejection may allow the virus to take and propagate in the human body. More research is required on retroviral and other infections in relation to xenotransplantation.

The recent in vitro observation that porcine endogenous retroviruses (PERV) can infect human cells has ignited tremendous fear among investigators in the xenotransplantation field and health officials. This chapter reviews the available experience with other endogenous retroviruses in different animal situations, with the aim of better understanding the potential impact of such organisms in the field of xenotransplantation. All mammals that have been examined so far, including pigs and baboons, have endogenous retrovirus DNA sequences integrated in their genomes. Although transmitted in the germ line, they exhibit a very significant sequence homology to exogenous retroviruses. The true source of endogenous retroviruses remains to be elucidated. One hypothesis is that they are viral remnants of remote infections with exogenous forms of the virus. Alternative explanations include that retroviruses may be derived from retrotransposons or that endogenous retroviruses are the precursors of exogenous retroviral agents. The majority of endogenous retroviruses act as colonizers in the germ line of the host and are noninfectious. The exact functions of the remaining "competent" or partially defective endogenous retroviruses remain enigmatic. It is speculated that, among a number of possible functions, they may enhance the pathogenicity of exogenous retroviruses by the recombination and production of a hybrid virus with altered receptor specificity and species tropism, now capable of infecting a new range of hosts. New animal viruses are being discovered on a regular basis, as detection methods are becoming more sophisticated. Similarly, many more animal endogenous retroviruses are yet to be discovered and fully characterized.

The central hypothesis for this chapter is that some of the infectious risks associated with xenotransplantation can be assessed before the broad application of this emerging technology. Epidemiologic exposures in the transplant recipient take two forms: those occurring within the hospital or the community, and those exposures carried with the transplanted organ. On the basis of experience with human allograft recipients and with immunosuppressed miniature swine, common infections in the first month after xenotransplantation are likely to be due to bacteria and fungi common to swine and to primates: staphylococci, streptococci, Candida spp., Aspergillus spp., Salmonella spp., and Actinomyces spp., which are routinely isolated from swine and from non-human primates. With prolonged xenograft survival, the susceptibility of the xenograft recipient to infection reflects the individual's epidemiologic exposures and the immune suppression and manipulations of donor and host needed to prevent graft rejection. While breeding strategies may enhance the safety of xenotransplantation, the greatest potential risk to the recipient and to the general community by xenograft-derived organisms may be due to infection by unknown pathogens that cause minimal or novel clinical syndromes and for which clinical laboratory testing is not available. Chimerism and False Positive Assays for Infection of the Host and Routine Monitoring for Xenogeneic Infection are among the issues discussed for maintaining safety in clinical trials of xenotransplantation. Further research is essential regarding the behavior of organisms known to be present in prospective donor species in xenograft recipients and on the detection of novel potential pathogens.