Xenotransplantation Biological Barrier

Jeffrey L. Platt

Mayo Clinic, Rochester, Minnesota, U.S.A.

INTRODUCTION

Xenotransplantation, the transplantation of cells, tissues, or organs between individuals of different species, is a subject of interest because it might be used to address a shortage of human organs for transplantation and for other purposes. While the potential applications of xenotrans-plantation are widely appreciated, the biological hurdles have prevented all but a small number of experimental trials.

BIOLOGICAL HURDLES TO XENOTRANSPLANTATION

The biological hurdles to xenotransplantation are summarized subsequently and in recent reviews.[1,2] With certain notable exceptions, the hurdles are a direct function of the phylogenetic distance between the graft and the recipient. Because of this consideration, some have advocated using closely related species, for example, primates in the case of human recipients, as a source of xenografts. However, nonhuman primates are not sufficiently plentiful to provide organs needed to treat human disease and may harbor viruses potentially lethal to humans. Use of nonhuman primates also raises social and ethical controversy. For these reasons, many in the field of transplantation have turned to the pig as a potential source of xenografts. Swine are plentiful, and some strains, such as the mini-pig, have organs of appropriate size for use in humans. Further, the pig can be genetically engineered. This article will therefore focus on the pig, although what is mentioned for the pig would also apply to other nonprimate mammals.

IMMUNOLOGICAL HURDLES TO XENOTRANSPLANTATION

Many components of the immune system may target a xenograft (Table 1). The most important component may be xenoreactive natural antibodies. Xenoreactive natural antibodies are made by all immunocompetent humans and recognize predominantly Gala1-3Gal, a saccharide synthesized by lower mammals and New World monkeys, which have a functional a1,3galactosyltrans-ferase. Humans and Old World monkeys lack a functional a1,3galactosyltransferase and make anti-Gala1-3Gal. Binding of anti-Gala1-3Gal antibodies activates complement. Complement activation on porcine cells is amplified because porcine complement regulatory proteins, such as decay-accelerating factor, control human complement poorly.[3] Human natural killer cells and macrophages, bearing receptors specific for porcine cell surface molecules, such as Gala1-3Gal, also react with porcine cells. Finally, xenografts provoke elicited immune responses against porcine major histocompati-bility proteins.

XENOGRAFT REJECTION: SUSCEPTIBILITY AND TREATMENT

Whereas all xenografts may provoke immune responses, all xenografts are not equally susceptible to destruction by those immune responses. The critical factor determining the susceptibility of a xenograft to injury by the recipient's immune system is the nature of the blood vessels that serve the graft, and that is determined by whether the graft consists of an intact organ, free tissue, or isolated cells (Fig. 1).[1] Organ xenografts are subject to severe vascular injury (Fig. 1). This injury arises from the interaction of antibodies and complement of the recipient with the endothelial lining of blood vessels of the donor.

Hyperacute rejection is caused by the rapid activation of complement on donor blood vessels, and it typically destroys a xenograft in minutes to hours. Although hyperacute rejection is dramatic and severe, it can be prevented by depleting antidonor (Gala1-3Gal) antibodies with immunoabsorbant columns, or by inhibiting complement activation. The best way to inhibit complement is genetic engineering of swine to express human complement regulatory proteins, such as decay-accelerating factor. Some have suggested that hyperacute rejection might be prevented by knocking out a1,3GT to eradicate

Table 1 Immune and inflammatory components contributing to the barrier to xenotransplantation

Component

Target recognized

Natural antibodies

Galal 3Gal

Elicited antibodies

Porcine MHC, other proteins,

and Galal 3Gal

Complement

Complement fixing antibodies

Platelets

von Willebrand factor

Macrophages

Saccharides

T cells

Porcine MHC and other proteins

"Components organized by pig to human xenografts.

"Components organized by pig to human xenografts.

expression of Gala1-3Gal, but this application has yet to be tested.

When hyperacute rejection is prevented, an organ xenograft becomes subject to acute vascular rejection (AVR). AVR arises over a period of days to weeks and is caused by the interaction of host antibodies and a small amount of complement with donor blood vessels. AVR may also be caused by natural killer cells, platelets, and/or macrophages. AVR is a greater challenge than hyperacute rejection because it can be caused by very small amounts of immune reactants and may be promoted by incompat ibilities between complement and coagulation systems of the recipient and control proteins expressed in the donor

AVR has been prevented in some model systems by temporary depletion of antidonor antibodies from the recipient. Depletion of antidonor antibodies and their gradual return may bring about accommodation, a condition in which the organ acquires resistance to antibody-mediated injury.[3]

Unfortunately, accommodation has not been achieved reproducibly in pig-to-nonhuman primate organ transplants, and hence other approaches to preventing acute vascular rejection are sought. One such approach may be the induction of immunological tolerance, that is, specific immune nonresponsiveness with porcine cells. Unfortunately, tolerance to swine has yet to be achieved in primates.

Another approach to preventing acute rejection may involve eradicating donor antigen, particularly Galal-3Gal. This end is sought by targeting the a1-3galacto-syltransferase gene.[4,5] Gene knockout in swine involves targeting by homologous recombination, followed by transfer of targeted nuclei to targeting of enucleated eggs. This process, called cloning, was recently achieved and full knockout accomplished by pairing one allele of a1-3galactosyltranserase with

Type of Graft

Source of Blood Vessels

Organ

Donor

Tissue

Donor and Recipient

Cell

Recipient

B. Organ Xenografts

Hyperacute Rejection

Acute Vascular Rejection

Accommodation

Cellular _ Rejection

Cellular _ Rejection

_ Chronic Rejection

C. Cell and Tissue Primary Nonfunction

Xenografts -^ Failure of Neovascularization -►

Microenvironment Incompatibility Rejection

Fig. 1 Relationship between type of graft and source of blood vessels. (A) Cell and tissue transplants do not undergo humoral rejection because some or all blood vessels are of recipient origin. (B) Organ xenograft. The action of the immune system particularly humoral immunity on the blood vessels in organ xenografts gives rise to various types of vascular disease, including hyperacute rejection, acute vascular rejection, or accommodation and chronic rejection. Organ xenografts are also subject to cellular rejection. The figure depicts the various types of rejection in approximate temporal sequence. (C) Tissue or cell xenografts. Cell or tissue xenografts are subject to primary nonfunction and to cellular rejection, but not to the types of vascular disease shown in A and B.

spontaneous mutation in the other allele. Whether a1-3galactosyltransferase knockout pigs will truly resist AVR remains to be seen.

Xenografts are also susceptible to cellular rejection. Rejection results from immune responses to many foreign proteins, especially major histocompatibility proteins. Because all of the proteins of the pig differ to a certain extent from the proteins of humans, cell-mediated immunity might be very strong stronger than the immune reaction against an allograft. Still, cell-mediated immunity against xenografts can apparently be controlled by immunosuppressive therapies in common use.

Organ xenografts may also be susceptible to chronic rejection. Chronic rejection arises over a period of months to years and is characterized by thickening of blood vessel walls, interstitial fibrosis, and loss of epithelial ducts. Chronic rejection is the major cause of the loss of human allografts, and some believe the occurrence would be at least as frequent and at least as severe in organ xenografts. Whether this view is correct is unknown. However, because xenografts can potentially be replaced, the implications of chronic rejection for the well-being of the recipient are not nearly so great in the case of xenotransplantation as in the case of allotransplantation.

In contrast to organ grafts, cell and tissue xenografts are mainly subject to cellular rejection.[1] Cell and tissue xenografts do not undergo hyperacute, acute vascular, or chronic rejection because they derive their blood supply mainly by ingrowth of the recipient. Because cellular rejection can be controlled by immunosuppressive therapy, cell and tissue xenografts have achieved long-term survival and have been applied clinically.

PHYSIOLOGIC BARRIERS TO XENOTRANSPLANTATION

Some xenografts may function poorly because of physiologic incompatibility with the recipient. This problem may especially plague hepatic xenografts because of the complex metabolic pathways of the liver. A related concern is that the complement, coagulation, or other proteins secreted by the xenogeneic liver might disrupt physiologic systems in the recipient. Whether or not physiology of the graft will pose a significant hurdle to xenotransplantation of the liver is unclear.

XENOTRANSPLANTATION AND ZOONOSIS

A potential hurdle to xenotransplantation that has received much attention in recent years is zoonosis, the conveying of an infectious agent, especially a virus, from the donor to the recipient.[6] Zoonosis poses a significant hurdle to use of nonhuman primates as a source of xenografts because some viruses of nonhuman primates are lethal in humans. In the case of the pig, all of the known infectious agents can potentially be eliminated, so the problem of zoonosis may not be so severe. Indeed, because infection is a regular complication of allotransplantation, the problem of infection may be less severe in the case of xenografts than it is in the case of allografts.

One exception to the relatively low risk of zoonosis is porcine endogenous retrovirus (PERV). PERV is present in the genome of all swine and may infect human cells.[7] However, while PERV can infect human cells in vitro, studies of several hundred human recipients of porcine xenografts have failed to reveal evidence that PERV can infect human cells in vivo.[8] Still, the theoretical possibility of infection has raised concerns, especially that recombination or mutation might make the virus more infectious or pathogenic. Hence, the possibility of infection and spread of PERV among humans will be intensely studied in all clinical trials.

CONCLUSION

Although xenotransplantation has been seen as a potential approach to treating organ failure for more than a century, it has not been regularly applied in human subjects. The main barrier to application is the immunological reactions of the recipient against the graft. New technologies, such as genetic engineering and cloning, offer promise for overcoming this hurdle, and thus making xenotransplan-tation a potential treatment for many human diseases. How xenotransplantation will weigh against other technologies, such as implantable devices and tissue engineering, for the treatment of disease remains to be determined. Regardless, genetic engineering, cloning, and other technologies developed to enable xenotrans-plantation to be applied may find broader use in animal science and biotechnology.

ARTICLE OF FURTHER INTEREST

Biotechnology: Xenotransplantation, p. 152

REFERENCES

1. Cascalho, M.; Platt, J.L. The immunological barrier to xenotransplantation. Immunity 2001, 14, 437 446.

2. Platt, J.L. Immunology of Xenotransplantation. In Samter's

Immunologic Diseases, 6; Lippincott Williams & Wilkins: Philadelphia, 2001; 1132 1146.

3. Platt, J.L.; Vercellotti, G.M.; Dalmasso, A.P.; Matas, A.J.; Bolman, R.M.; Najarian, J.S.; Bach, F.H. Transplantation of discordant xenografts: A review of progress. Immunol. Today 1990, 11, 450 456.

4. Lai, L.; Kolber Simonds, D.; Park, K.W.; Cheong, H.T.; Greenstein, J.L.; Im, G.S.; Samuel, M.; Bonk, A.; Rieke, A.; Day, B.N.; Murphy, C.N.; Carter, D.B.; Hawley, R.J.; Prather, R.S. Production of a 1,3 galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002, 295, 1089 1092.

5. Phelps, C.J.; Koike, C.; Vaught, T.D.; Boone, J.; Wells, K.D.; Chen, S.H.; Ball, S.; Specht, S.M.; Polejaeva, I.A.; Monahan, J.A.; Jobst, P.M.; Sharma, S.B.; Lamborn, A.E.;

Garst, A.S.; Moore, M.; Demetris, A.J.; Rudert, W.A.; Bottino, R.; Bertera, S.; Trucco, M.; Starzl, T.E.; Dai, Y.; Ayares, D.L. Production of alpha 1,3 galactosyltransfer ase deficient pigs. Science 2003, 299, 411 414.

6. National Research Council of the National Academies. Animal Biotechnology: Science Based Concerns; The Na tional Academies Press: Washington, DC, 2002; vol.

7. Patience, C.; Takeuchi, Y.; Weiss, R.A. Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 1997, 3, 282 286.

8. Paradis, K.; Langford, G.; Long, Z.; Heneine, W.; Sandstrom, P.; Switzer, W.M.; Chapman, L.E.; Lockey, C.; Onions, D. Search for cross species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. Science 1999, 285, 1236 1241.

Gerald Wiener

Roslin Institute, Edinburgh, U.K.

Han Jianlin

International Livestock Research Institute, Nairobi, Kenya

INTRODUCTION

Yak, a species of bovidae, are the mainstay of livelihood for the nomads on the vast Qinghai-Tibetan Plateau of western China and in other countries bordering the Himalayas and to the north into Mongolia and Russia. These areas have harsh climate, short growing seasons, and high elevations. Yak withstand these extreme conditions and still remain productive.

HISTORY AND DISTRIBUTION

The yak, classified by Linnaeus in 1766 as Bos grunniens (on account of its grunting noises), was listed later as Poephagus grunniens, which recent evidence supports more strongly.[1] Both classifications remain in use.

The domestic yak is descended from wild yak, which may have been tamed by the ancient Qiang people in the Changtang area of Tibet, starting perhaps 10,000 years ago. Domestic yak herding, perhaps not too different from that practiced until recently, dates back about 4500 years.[1] From those times, the yak spread outward, but always at high altitudes, generally between 2000 m and 5000 m.

China is the main country for yak, with about 13 million animals around 4 million each in Tibet, Qinghai, and Sichuan provinces, 900,000 in Gansu province, and relatively few in Yunnan and Xinjiang provinces. Mongolia has about 600,000 yak, and smaller populations, but of great local importance, exist in Bhutan, Nepal, northeastern India, and in some of the climatically inhospitable parts of Commonwealth of Independent States (CIS) countries.

During the late 20th century, yak were introduced into parts of the northern and western United States and Canada. There are a few very small herds in some European countries and in New Zealand. In addition, there are collections in many zoos, but few of them are viable, self-reproducing herds.

BREEDS AND BREEDING Wild Yak

Fewer than 15,000 wild yak survive, principally in the Changtang area of Tibet and parts of the Kunlun mountains, from among former millions.[2] They have been driven to exist at elevations mostly above 4500 m by excessive hunting, albeit mostly for food. The wild yak is now a protected species, but protection is difficult and leaves them in danger of extinction.

Domestic Yak

The Chinese authorities have officially recognized 12 breeds of domestic yak, of which the Jiulong, Maiwa, Tianzhu White, Plateau, and Huanhu are best known and numerically most important. Yak in other countries are normally referred to by the name of the area in which they are found. The classification of breeds in China was based on characteristics of color, conformation, local history, distribution, and other factors, but most breeds of yak live in different parts of a vast territory (more than 2.5 million square km) and rarely intermingle or interbreed. While some of the breeds differ from each other in color and conformation, it is more difficult to say whether there is any significant genetic difference among the breeds in reproduction, survival, or performance traits. However, techniques of molecular genetics have recently started to show some degree of genetic distance between the yak populations.

Crosses and Hybrids

Crosses among yak breeds are relatively rare, but crossing with wild yak by using the semen of captured wild yak bulls is practiced at some breeding centers. Such crosses are larger and more vigorous than domestic yak. In times past, wild yak bulls on the perimeter of the domestic yak population mated with the latter, and herders liked the crosses. Attempts are now being made, by selection, to create a new breed of yak (the Datong yak) from such crosses.

Hybridization of domestic yak with local cattle, at intermediate elevations, has been practiced for generations. The hybrids inherit some of the good characteristics from each species, but lack the adaptation of the yak to the harsh conditions at higher elevations.

Over the past 50 years or so, hybridization of yak with exotic cattle breeds, such as the Holstein or the Simmental, has been achieved on a limited scale by using artificial insemination. These hybrids are larger and more productive than the yak, but need extra feed and better management at lower altitudes than pure yak.

The male hybrids of yak and cattle are sterile and are used for draft purposes and meat. The females can be mated to either yak or cattle and are especially liked for their milk production, but their calves are not usually kept for further breeding.

MANAGEMENT

Traditionally, management follows a transhumance system dictated by the seasons, climate, topography, and sociocultural factors. During summer and early autumn, the herds are kept at higher elevations. The herders live in campsites and move as often as necessary, depending on the availability of grazing. During winter and early spring, yak are kept at lower elevations nearer the permanent homes of the herders. Some shelters are provided, especially for calves.

Formerly, the animals of several families were herded together, and milking females and their calves were kept separate from younger females and from males. Now, some of the traditional and communal systems of transhumance management are breaking down under a policy of Household Responsibility in China, in which there is individual ownership of animals and rights (though not ownership) to parcels of rangeland, some of which are fenced. It has yet to be shown whether the new system is as effective as the old in utilizing the natural resources of the rangeland.

The natural vegetation is almost the only feed available for the yak in both summer and winter. Summer is a time of plenty. The animals gain weight rapidly after severe weight loss (up to 25 30% of their liveweight) over winter and early spring, when animals can be close to starvation and deaths are common, especially in years of heavy snow. Supplementary feeds, such as hay or crop byproducts, are not generally available except in very small quantities, mostly for weak animals.

ADAPTATION

The yak has adapted to high altitude (low oxygen), cold (almost no frost-free days and an annual mean temperature of 5°C), shortage of feed for up to 7 months of the year, precipitous terrain, and danger from predators. Yak conserve heat through a compact body; thick fleece of long outer hair and, in winter, an undercoat of fine down; thick skin; nonfunctioning sweat glands; and, by autumn, a layer of subcutaneous fat. Uptake of oxygen is aided by a large lung and heart, rapid breathing, and hemoglobin with a high affinity for oxygen. Pigmentation of skin and fleece (black color predominates) counteracts solar radiation. The yak's grazing habits allow use of diverse vegetation, from shrubs to the shortest grass. A special hoof shape and the yak's temperament help them in often treacherous terrain, and grouping into tight herds provides protection, especially from wolves.

PERFORMANCE

Compared to specialized dairy and beef cattle, the yak's productivity is low. There is no widespread recording of performance in yak herds, and nearly all available data stem from experimental stations and a few surveys. The values presented in this section[1] are therefore only approximate.

Reproduction

Mating takes place between June and November, with August and September being the peak months. Traditionally, the bulls run in groups and fight for possession of the females. The strongest, generally older bulls get the most mates. More controlled breeding practices, including the use of artificial insemination, have been introduced, especially for producing hybrids with exotic cattle breeds and for crossing with wild yak.

Calves are born in the spring and early summer, but their mothers are often in very poor condition at that time. Calves thus have a hard time, and those that have not put on enough weight by autumn may not survive the following winter.

In China, the majority of yak females do not show estrus until two years old and they calve first at four years old. Occurrence of estrus is greatly influenced by seasonal and environmental factors, and by the body condition of the cow. One estrus period per year is common. Cows that have not calved in the current year and others under good conditions (and in some other countries) may be polyestrous. One calf every two years is the norm, although two calves in three years is not unusual. Four to five calves, on average, are produced in a lifetime. Gestation length averages 258 days.

Body Weight

Average birth weights for calves of different breeds vary from 10 to 16 kg (with a higher range for individual animals). For one breed (Maiwa yak), weights (kg) of female yak at different ages during the warm season are reported as: birth, 11.9; 1 year, 67; 2 years, 120; 3 years, 155; 4 years, 182; 5 years, 189; and 6 years, 222. From about 3 years onward, males are significantly heavier than females, and up to twice the weight of females at maturity.

Castrated males are not usually slaughtered for meat until 4 years old in September or October, when at their fattest. Surplus females are also slaughtered then. Carcass weights vary widely as a proportion of liveweight, from around 30 to 60%.

Milk Production

Milk production is seasonal and averages among breeds from 150 to 500 kg, with fat content from 5.4 to 7.5%. Cows do not dry off completely during winter, and lactation will resume in a second year without calving again, but the cow will produce only one-half to two-thirds the quantity of milk, although it has a higher fat percentage.

Fleece

Fiber yields vary among breeds and locations, from 0.5 to 2.9 kg. The valuable down component represents 60 70% of the total fleece in calves, but can decline to as little as 20% in adults.

Other Produce

Hides, feces, blood, viscera, horn, and, to some extent, bones are all harvested.

PRODUCTS

Milk is made into butter (the main product) and various soft cheeses, mostly locally. Milk is also used skimmed or soured and made into yogurt. In Mongolia, yak milk is also fermented into an alcoholic drink. Dried milk powder is produced in factories, and in Nepal, a Swiss-style hard cheese is also produced in local factories. Milk is mostly brewed up with tea, to which butter may be added. Butter is used in cooking, as ointment, in lamps, and for sculptures in religious contexts.

Meat is eaten fresh or kept air-dried, smoked, or frozen (by nature). Meat products include a variety of sausages, often with blood added. The hair is used for ropes, blankets, and tents, and in mixtures for clothing. The hides, when tanned, are used for leather goods and to make coracles, while pelts can be used as coats. The feces are dried, and on the Qinghai-Tibetan Plateau they are the main source of fuel for heating and cooking. Yak are widely used for carrying loads and people and, in agricultural areas, for plowing.

CONCLUSIONS

Yak live on the high plateau and mountain ranges of western China and adjacent countries. The animals are adapted to withstand cold, low oxygen, often-treacherous terrain, and long periods of semistarvation. Milk yield is rarely more than needed to sustain a calf, but most of it is used for human consumption. In addition, meat and other products help to sustain the life of the nomads. Sheep and goats provide an extra source of livelihood in some areas, and horses are common for riding.

Yak are an integral part of the culture and social fabric of these regions, and they even have religious significance.

The relatively small numbers of yak kept in North America demonstrate that, contrary to received wisdom, yak can adapt to better environments and can reproduce and perform significantly better than is traditionally believed. Yak may, because of their resistance to hardship, find a role in other parts of the world.

REFERENCES

1. Cai Li; Wiener, G. The Yak, 2nd Ed; Regional Office for Asia and the Pacific of the Food and Agriculture Organization (FAO) of the United Nations: Bangkok, Thailand, 2003; 476. (revised and enlarge by Wiener G.; Han Jianlin; Long Ruijun) xviii.

2. Schaller, G.B. Wild Yak. In Wildlife of the Tibetan Steppe; University of Chicago Press: Chicago, USA, 1998; 125 142.

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