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Dr. Ramachandran, my friend, philosopher and guide for over four decades had played a vital role in shaping my interests in veterinary public health. His training in Veterinary State Medicine in U.K., subsequent researches on canine erhlichiosis, clostridial infections and epidemiological investigations on several bacterial and viral diseases of large ruminants provided him with a deep insight into the practical implications of zoonotic diseases. He had remarkable technical skill, which he constantly endeavoured to update and improve upon. The newer biotechnologies had always interested him and in the last few years before his passing away he used to discuss eloquently on the modern concepts of cloning, application of stem cells, tissues and organs in allo and xeno transplantations, the concomitant immunological phenomena and xenozoonotic diseases. With this in the backdrop, I have made an attempt to present the following review.
Introduction
Millions of people are dying annually of heart, kidney, lung, and liver failure all over the world. Many of them are due to end stage organ failure, which can be averted by transplantation. However, it has not been found to be feasible for want of suitable organs from either healthy people (allotransplantation) or from animals (xenotransplantation) and for the various problems still to be addressed in such transplantations. Demand for organs, which far outstrips the supply, continues to grow. It has been estimated that approximately 45,000 Americans under 65 years of age could benefit each year from heart transplantation yet, only 2,000 human hearts are available annually (1). A multi-billion dollar market is anticipated from the sale of patented techniques and organs, as well as existing and new drugs to overcome organ-rejection (2). According to the United Network for Organ Sharing, the number of transplants increased from 12,000 to 20,000 between 1988 and 1996 while the number on the waiting list soared from 16,000 to 50,000 and the number of deaths rose from about 1000 to 3000 (3).
Historical
An isolated report on the use of a dog bone for skull repairs in humans was first made in the 17th century in Russia (4). Organized clinical cross-species transplantations were, however, started in the early twentieth century with kidney xenografts from rabbit, pig, goat, non-human primate and lamb, and they were largely unsuccessful (5). Among the different animal species, the chimpanzees are closely comparable to humans in the size of their organs and the presence of blood type 0 and the first description of transplantation of chimpanzee kidneys in humans was made in 1963 (6). The first cardiac xenotransplantation in humans was performed by Hardy in 1964 using again the chimpanzee as a donor (7). Since then there have been eight documented attempts at clinical heart xenotransplantation including transplants from 2 baboons, 3 chimpanzees, 1 sheep and 2 pigs (8-10).
Source animals
Baboons
The failure of baboon-to-human liver transplantation in the early 1990s, has been attributed to ethical issues related to their genetic closeness to humans, size discrepancy between baboons and adult humans, the infrequency of blood group 0 (universal donor) animals, the limited number of specially bred animals, their slow breeding and dwindling population and the expense of breeding them in a pathogen-free environment (1). Further, many zoonotic diseases, such as, those caused by the herpesviruses, retroviruses and Marburg, Ebola, monkeypox, encephalomyocarditis and Simian haemorrhagic fever viruses, Toxoplasma gondii and Mycobacterium tuberculosis, are potentially transmissible between baboons and humans through xenotransplantation. Lymphocytic choriomeningitis virus, gastrointestinal parasites and GI bacterial pathogens are some of the other organisms also to be screened (11,12). Evidence to the presence and persistence of the DNA of two retroviruses, namely, the simian foamy virus (SFV) and baboon endogenous virus (BaEV) in many tissues of two human patients transplanted with baboon livers was presented in 1998. The presence of baboon mitochondrial DNA in those tissues suggested that baboon leukocytes harbouring latent or active viral infections had migrated from the xenografts to distant sites in the human transplant recipients (13). Ho and Cummins, commenting upon the risks of spread of viral pandemics through xeno-transplantation stated that the potential for both exogenous and endogenous viruses to reside in human recipients of animal organs persisted for a significant period after transplantation and possibly the circulating xenogeneic cells also acted as sources for new human infections (14). The reports on the spread of retroviruses, such as, SFV and simian immunodeficiency viruses (SIV) to animal handlers with occupational exposures to baboons and rhesus monkeys provide further evidence to the potential transmission of these viruses in xenotransplantation (15).
Pigs
Sheep and pig hearts were first transplanted into human recipients in 1968 (8,16). Of these, the pig has been identified as the most suitable donor animal for cells, tissue and organs, in xenotransplantation for several reasons. Firstly, humans and pigs have existed together for centuries and the pork constitutes a common food for human consumption with obviously no ill effects; secondly, pigs breed rapidly and produce large litters and they are not an endangered species; and finally, some breeds of pigs have the organs which have the correct size for use in humans as xenografts (17-19).
Clinical consequences
The two important clinical consequences of transplantation are immunological rejection and infection. Both of these assume greater significance in xeno-transplantation as compared to allotransplantation.
Immunological rejection
There are several detailed reports on the phenomenon of immunological rejection particularly with the pig xenografts. Immediately following xenotransplantation, a severe hyperacute rejection (HAR) of the graft occurs in the recipient, which has been attributed to the presence of naturally occurring, pre-existing human anti-pig antibodies. These antibodies bind the carbohydrate antigen, galactose-alpha-1, 3-galactose attached to pig cell-surface proteins. The antigen-antibody complexes so formed activate the complement and cause immediate destruction of the donor organ and cells. Antibodies against the foreign graft induced in the recipient host are responsible for organ rejection in the longer term. The HAR can be blocked by depleting the pre-existing antibodies, by reducing antigen expression in the donor cells or by inhibiting complement activation (20,21). Of these, the inhibition of complement activation had found practical application and transgenic pigs containing the decay accelerating factor (DAF), which blocks an early step in complement activation, designated as hDAF, were developed (19,22-25). There was considerable variability in expression of hDAF in the transgenic animals, not only between animals, but also between organs from the same animal which indicated the unpredictable and uncontrollable nature of the transgenic process, the low rate of success, the stability of the transgenic inserts and lack of reproducibility of the experiments besides the potential for creation of new viruses and the resultant biosafety considerations (19).
Infection
Several documents have described the nature of infectious diseases especially with the porcine xenograft. It is known that pigs carry several bacteria, fungi, protozoa, helminths and viruses, which can be transmitted to human recipients during transplantation (26-28,29). The detailed listing of these pathogens has been provided in tables 1 to 3 (30).
Table 1
Bacterial pathogens of pigs capable of transmission to humans following xenotransplantation
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Actinobacillus, Actinomyces pyogenes, Brucella suis, Campylobacter coli, Campylobacter jejuni, Chlamydia psittaci, Clostridium perfringens, Clostridium septicum, Clostridium tetani, Corynebacterium suis, Enterobacteriaceae, Erysipelothrix rhusiopathiae, Haemophilius sp., Leptospira interrogans, Listeria monocytogenes, Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium fortuitum, Pasteurella multocida, Pseudomonas aeruginosa, Pseudomonas pseudomallei, Salmonella choleraesuis, Salmonella typhimurium, Shigella species, Staphylococcus aureus, Streptococcus species, Yersinia enterocolitica, Yersinia pseudotuberculosis.
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Table 2
Fungal, parasitic, and other microbial agents of pigs capable of transmission to humans followings xenotransplantation
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Fungi
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Parasites and Protozoa
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Others
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Aspergillus species
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Ascaris suum
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Prions
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Candida albicans
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Babesia species
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Unknown agents
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Coccidioides immitis
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Balantidium coli
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Cryptococcus neoformans
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Capillaria hepatica
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Histoplasma capsulatum
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Clonorchis sinensis
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Microsporum nanum
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Cryptosporidium species
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Petriellidium boydii
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Echinococcus granulosa
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Prototheca
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Entamoeba histolytica
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Sporothrix schenkii
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Entamoeba polecki
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Zygomycetes
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Fasciola hepatica
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Nocardia asteroides
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Isospora species
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Paragonimus westermani
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Pneumocystis carinii
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Sarcocystis species
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Schistosoma species
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Strongyloide ransomi
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Taenia species
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Toxoplasma gondii
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Trypanosoma cruzi
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Trichinella spiralis
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Table 3
Porcine viruses capable of transmission to humans following xenotransplantation
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Porcine adenovirus, Porcine cytomegalovirus, Porcine rotavirus, Porcine endogenous and exogenous retroviruses, Aujeszky’s disease virus, Japanese encephalitis virus, Encephalomyocarditis virus, Vesicular stomatitis virus, Swine vesicular disease virus, Foot-and-mouth disease virus, Rabies virus, Swine influenza virus, Swine parainfluenza-I virus (Malaysian “Nipah” virus, Australian Paramyxovirus)
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Pigs harbour endogenous retroviruses and it is estimated that hundreds of different endogenous retroviruses may be present in one animal (30). The demonstration that a pig endogenous retrovirus (PERV) can infect cultured human cells and then spread to a wide range of human cells was first made by Robin Weiss and his coworkers in 1997 (31). They drew attention to the fact that many copies of PERV existed in the pig genome and it would be extremely difficult to breed pigs free of PERV. Millions of people have become infected with the monkey SV40 virus through polio and adenovirus vaccines made in monkey kidney cells. Many viruses, such as, herpes and retro which are normally dormant in animals might become activated under immunocompromised conditions during xenotransplantation. All the natural barriers to infection are bypassed in xenotransplantation (32). Further, virus adaptation or recombination with other retroviruses could also occur in the new host (2,33,34).
Potentially, the porcine xenografts may be the source for several porcine viruses, such as, retrovirus, polyomavirus, parvovirus, circovirus, cytomegalovirus, reproductive and respiratory syndrome virus, influenza virus, hepatitis E virus and herpesvirus (35). In pigs, the retroviruses have been associated with the development of leukemia and lymphoma (36) while in humans, they cause chronic, life-long infections, long latency malignancies, neurological disorders, wasting diseases and immunodeficiencies, for which treatment is limited or unavailable (15).
Pigs harbour type C endogenous retroviruses and they are present in most cell lines derived from porcine tissues (33,36). Two infectious variants of porcine endogenous proviruses, namely, PERV-A and PERV-B are present in various organs, cells and tissues (spleen, heart, kidney, liver, lung, thymus) of different breeds of pigs and these are vertically transmitted posing problems even in xenotransplants from specific pathogen-free animals (30,34). Furthermore, the specific-pathogen-free pigs may be silent carriers of enteric organisms such as micrococci, streptococci D, and colibacillus and unrecognized pathogens (30,37). Studies have shown that PERVs can also infect human cells in test tubes (33,38-40). Their replication capacity increases when passaged in human kidney cells (41). The possible transmission of porcine viruses of diseases, such as, pseudorabies, vesicular exanthema, foot-and-mouth disease, swine vesicular disease, swine influenza, paramyxovirus infections and Nipah viral encephalitis have also been reported to occur in human recipients of porcine xenografts (30,42).
Retroviruses from pigs may recombine with human endogenous retroviruses, leading to recombinant “superviruses” with unknown, and possibly more virulent properties for human infection and subsequent human-to-human transmission (15,31,41). Some recombinant retroviruses have been shown to induce cancer (30). Accurate screening for such viruses may be difficult. In this context, some scientists are concerned that xenotransplants could alter the human gene pool by favouring the evolution of porcine-human chimeras with unknown susceptibility to infection (43,44). Finally, the genetic modification, or “humanization,” of pigs could provide an opportunity for animal viruses to fool the human immune system and “hide’ inside the human body (45).
Alternatives to xenotransplantation
Recent advances in the study of cell, tissue and organ engineering are providing safe and sustainable alternatives to xenotransplantation. The stromal cells from human tissue are used for developing blood vessels, bone, cartilage, nerve, oral mucosa, bone marrow, liver and pancreatic cells. Possibilities are also being explored for regenerating organs and tissues from the stem cells of the patients themselves requiring transplantation with a view to avoid immune rejection as well as viral epidemics. For example, bone-marrow stem cells, either from a donor or from the patient could be used to generate liver cells for replacing damaged tissue (46). Encapsulated cell therapy is another example of a technique under development, which employs biomaterials in order to augment circulating or local levels of the deficient molecules in the treatment of certain serious, chronic diseases, such as, insulin-producing cells in diabetics and dopamine-secreting cells in patients with Parkinson’s disease (14).
With a positive approach to encourage development of safe, effective, ethical, and accessible methods for improving human health worldwide, the World Health Organization has arrived at the followings conclusions:
- The practice of xenotransplantation carries with it an unquantifiable risk of xenozoonotic infection and disease and hence research and development in xenotransplantation should evolve measures to minimize risk and maximize safety in the potential use of this technology.
- The implications of this technology raise the issues of ethical, social, cultural and religious acceptability inviting the adoption of national policies and efforts for international cooperation and coordination.
- Consideration should be given to the relative costs and benefits of the technology to the recipients and to the health care system (47).
Conclusion
Organ, tissue and cell transplantations have become essential tools in the surgical correction of end organ failures in human patients. Although allotransplantation has a proven value, it poses the problem of availability of suitable transplants from human sources. The cost involved is also very high. An alternative to this exists in the application of xenotransplants. Among the animals studied so far, the pig has been found to be a potentially viable source. There are, however, several constraints in the practical application of pig xenografts, the major ones being the problems connected with immunological rejection and infection in the recipient human host.
The transgenic technology has been addressed to circumvent the hyperacute rejection. Xenozoonoses are reported not only in the xenotransplant recipient but also in other humans in the population. The risk for recognized zoonotic pathogens can be reduced, if not eliminated, by controlling the donor animal source and the individual donor animal by employing prescribed screening tests and strict sterile procedures during organ harvesting and donor autopsy for tissue and blood. The risk for unrecognized pathogens, however, is present but ill defined.
The detection of infection or contamination in the xenografts has been facilitated by the modern biotechnological techniques of PCR, hybridization and rDNA. Despite these advances, much remains to be done in identifying the plethora of infectious entities involved in xenotransplantation, especially, the unknown endogenous and exogenous viruses and prions significant in xenozoonoses. Scientific, veterinary and economic concerns apart, the ethical, religious, legal and psychological values pose serious challenges to the large-scale practice of this technology and are presently being debated. Religious leaders and the proponents of ethical and environmental care vociferously oppose the acceptance of the new technology and call for a total ban on its implementation. Vociferous reactions to such cutting-edge technologies are not unexpected and scientists should not be deterred by them, but, strive to generate convincing evidences to dispel them.
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