_Medically Speaking_ The Good_ the Bad_ and the Ugly in

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					KEYWORDS: xenotransplantation, transgenic pigs, immunology

(Medically Speaking) The Good, the Bad,
and the Ugly in Xenotransplantation
Sara Wilcox
Microbiology and Immunology, University of British Columbia
Submitted April 2003

 Introduction                                                        litter, and have been extensively farmed so the supply
      A large percentage of the deaths in Canada, Eu-                of potential organs for donation is unlimited1. Pigs can
 rope, and the U.S are caused by chronic illnesses such              be genetically engineered to render them more com-
 as cardiovascular disease, diabetes, cancer and liver               patible for organ donation and the histocompatibility
 disease1. Many of these individuals would potentially               is well established1. Finally, there is much less social
 benefit from an organ transplant but unfortunately the              opposition to killing a pig than killing a primate1. The
 demand for organs greatly supersedes the supply1. It                reasons are that they have been used for food for many
 is estimated that there are more than 400 individuals               years and lack the human-like intelligence and social
 awaiting organ transplant in B.C. alone and many of                 skills of primates1. Zoonotic infection from pigs to
 the individuals waiting for an organ die while on the               humans is thought to be limited1. The reason is that
 waiting list19.                                                     pigs have been captively bred for food for many years
      The supply of organs is from healthy donors                    with reasonable quality control1. One problem is that
 who have died in a trauma, which means that the                     pigs are distant from humans both in terms of their im-
 donor supply is small2. Another problem is that the                 mune and coagulation systems and potentially require
 families must consent to the donation (which occurs                 substantial genetic manipulation in order to provide
 only approximately 20% of cases)2. Since the supply                 useful donors 4.
 of organs is so small, other solutions to the organ                       So why is xenotransplant not a current option? The
 shortage problem are being investigated3.                           answer is that there are many technical problems with
      One solution is to xenotransplant which is                     xenotransplant including rejection, the possibility of a
 defined as the transplantation of organs from non-                  transfer of disease, and physical compatibility issues.
 human animals to humans4. From an immunological
 perspective, primates would be the preferred source                 History
 of organs for transplant but almost all of the species                   In general, transplantation has been around for a
 are endangered or too small to provide organs capable               long time. In ancient Egypt, 8000BC, surgeons were
 of sustaining a human5. There are also concerns about               making cosmetic operations on the nose, face and ears2.
 the ability of primates to transmit infectious agents to            In ancient India, doctors were transplanting skin2. In
 the human population as many primates are caught                    1682 a Russian physician reportedly repaired the skull
 from the wild or have been housed in colonies for                   of a wounded nobleman using a bone from a dog2.
 many generations5. Also, primates are difficult and                 The church however frowned upon the surgery and
 time consuming to keep and breed and there is a                     the bone was subsequently removed2. The first docu-
 lack of experience genetically engineering them in                  mented animal to human transplant occurred in 1906
 the scientific community3. The pig is believed by                   when a physician named Jaboulay transplanted a pig’s
 many investigators to be the preferred donor as the                 kidney into a woman and a goat’s liver into another3.
 organs are of similar size and physiology1. Another                 Both transplants however, were rejected in one hour3.
 benefit to using pigs is that they proliferate quickly1.            In 1963 one patient survived 9 months after receiving a
 Pigs reach sexual maturity at nine months, take three               chimpanzee kidney3. The kidney was excised post mor-
 to five months to gestate, have up to 16 piglets in a               tem and showed no sign of rejection3. One of the more

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publicized cases of xenotransplant was in the case of                 however there may be less common antibodies to other
Baby Fae6. At birth, Baby Fae was diagnosed with hy-                  epitopes which could still function in hyperacute re-
poplastic left heart syndrome6. She received a baboon                 jection12. Alternatively, soluble synthetic Gala-3Gal
heart and three weeks later died of organ rejection6.                 can be continuously infused into the blood in order
Another interesting case was that of Tom Getty6. Tom                  to bind anti-Gala-3Gal antibodies5. The problem with
was an AIDS patient who was given a baboon bone                       this approach is that at high levels the sugars can be
marrow transplantation in hope that it would combat                   toxic and the results of experiments done have not
the AIDS virus6. Baboons normally are not susceptible                 shown prolonged xenograft survival5. Another pos-
to infection by AIDS and it was hypothesized as be-                   sible solution is to use complement inhibitors4. These
ing due to the inability of the AIDS virus to infect the              inhibitors are designed to inhibit the cleavage of the
immune cells6. In terms of Tom Getty, the transplant                  pro-enzymes involved4. The problem with inhibitors
ended up being unsuccessful as the transplanted cells                 is that complement normally acts as a first line defense
failed to engraft however, mysteriously his white cell                against bacterial infection so the recipient becomes
count and condition seemed to improve6. The results                   more susceptible to bacterial infection4. Now, with the
were inconclusive, the improvement may have been                      availability of transgenics, it is possible to engineer
due to the transplantation or due to the irradiation used             animals that present complement regulatory proteins
to deplete his current immune system6.                                like human decay accelerating factor or membrane
                                                                      cofactor protein on their cell surfaces4. These proteins
Rejection                                                             regulate the activation of complement by dissociating
    There are four recognized phases of xenotrans-                    and degrading the complement enzymes involved in
plant rejection:                                                      the formation of the membrane attack complex13. When
    ·     Hyperacute rejection                                        these procedures are combined with immunosuppres-
    ·     Acute vascular rejection                                    sive therapy, xenografts can continue to function for
    ·     Cellular rejection                                          several weeks experimentally3.
    ·     Chronic rejection
    Each phase is discussed in detail below.                          Acute vascular rejection
                                                                           Once hyperacute rejection has been successfully
Hyperacute rejection                                                  prevented, the xenograft can become subject to acute
     One of the major hurdles in xenotransplantation                  vascular rejection4. Acute rejection begins after 24
is hyperacute rejection. Hyperacute rejection oc-                     hours of reperfusion and results in the failure of the
curs immediately and is characterized by interstitial                 graft within days to weeks 4. It is characterized by en-
hemorrhage, congestion, disruption of the vascular                    dothelial swelling, ischemia and vascular thrombosis
endothelium, edema within the graft and the forma-                    with blood extravasation 3. Acute rejection is generally
tion of platelet thrombi resulting in the rapid loss of               thought to be due to an antibody (both Gala-3Gal and
graft function7. Leakage of fluid and blood through the               non-Gala-3Gal) mediated activation of the graft endo-
capillary walls is closely followed by necrosis of the                thelium5. Endothelial activation induces the expression
graft endothelial cells5. It is caused by the interaction             of many inflammatory and prothrombotic molecules
of xenoreactive antibodies with the graft endothelium4.               that lead to the changes in the tissues of the graft4. For
Ninety-percent of the xenoreactive IgM antibodies are                 example, endothelial activation by antibody deposits
specific for the sugar Gala-3Gal and make up 1-4%                     on the vascular endothelium results in the transcription
of the circulating immunoglobulins in humans 8.                       of paracrine factors like interleukin-1a, which activates
Gala-3Gal sugars are normally present on the porcine                  other endothelial cells4. Also, the binding of antibodies
endothelium9. The binding of the antibodies causes the                to the endothelial cell epitopes may direct killer cells
activation of the complement regulatory proteins of the               through interactions with the Fc receptors of the anti-
recipient through the classical pathway, which results in             body to release their cytotoxic compounds on the endo-
the destruction of the foreign tissues 10. The presence of            thelial cell surface5. Therapeutic strategies for combat-
xenoreactive antibodies is not the only reason for rapid              ing acute rejection are similar for those of hyperacute
activation of the complement system4. Rapid activation                rejection including the depletion of xenoreactive
also reflects the lack of complement regulatory proteins              antibodies along with the expression of complement
expressed on the donor epithelium11.                                  regulatory proteins and immuno-suppressant drugs4.
     One solution to the problem of xenoreactive                      Recent studies have shown that co-stimulatory block-
antibodies is to deplete them from the serum4. Since                  ade with anti-CD154 monoclonal antibodies inhibits
most of the antibodies are involved are specific for                  the induction of an antibody response although it does
Gala-3Gal it is reasonably easy to isolate the xeno-                  not suppress the production of anti-Gal antibodies14.
reactive antibodies using columns bearing Gala-3Gal                   Another approach is to genetically engineer the donors

                                           Xenotransplantation – pros and cons
                                                             - 50 -
to inhibit donor endothelium activation4.                            of the piglets, which would theoretically make them
                                                                     safer organ donors than other humans18. The reason,
Cellular rejection                                                   however, that xenozoonosis has become such a major
      Cellular rejection occurs after 7 days due to                  concern is due to the discovery of endogenous retro-
the infiltration of tissue specific T cells, B cells and             viruses like porcine endogenous retrovirus (PERVs)3.
antibodies15. Natural killer (NK) cells may also be a                PERVs make up 1% of the genome in pig cells and are
potential problem in terms of cellular rejection15. There            similar to human endogenous retroviruses3. Although
is evidence that NK cells may fail to receive inhibitory             there is no evidence showing that PERVs can lead to
signals from the xenograft tissue15 . Not much is known              health problems in pigs or humans, the fear is that
about cellular rejection in xenografts as the hurdles of             these retroviruses could mutate and become harmful
hyperacute and acute rejection still stand in the way4.              to the human recipient and potentially spread through
Cellular rejection is a major problem in the tolerance               the population3.
of allografts (same species transplants) and so the same
approaches to solutions may be applicable2. One ap-                  Physical compatibility
proach is the formation of mixed chimeras where the                       If the problems with rejection and zoonosis can
bone marrow of the donor is simultaneously injected                  become overcome, there is still the question of the
into the irradiated host resulting in the deletion of xe-            physical compatibility of the xenograft with the human
noreactive cells16. Another strategy is to transplant the            system. Is the donor’s organ going to be sufficient to
donors thymus in order to ensure that the T cell rep-                fulfill the requirements of a human organ? Since hu-
ertoire is not reactive to the donor17. Both approaches              mans and pigs are anatomically and physiologically
are being experimentally investigated with good results              different, the transplanted organs may not function
however the current treatments are composed of cock-                 adequately in order to sustain the human recipient5.
tails of immunosuppressant drugs4.                                   For example, pigs normal body temperature is 103o,
                                                                     while the average human body temperature is 98.6o
 Chronic rejection                                                   indicating that the pigs cell metabolism may not be op-
     As for cellular rejection, not much is known about              timal at human body temperatures5. Also, it is unclear
chronic rejection in xenografts; however, as in all forms            whether porcine organs can generate the factors that
of rejection, it probably occurs in an accelerated form5.            would be necessary to sustain a human and whether
In allografts, chronic rejection affects long term graft             human cytokines and hormones are able to support
survival2. The average half life for kidney grafts is                the organs5. Another potential problem is the physi-
approximately 8 years2. Chronic rejection is character-              ological function of many of the organs for example
ized by concentric arteriosclerosis of the graft blood               human hearts must perfuse a high blood flow organ
vessels, along with fibrosis and atrophy of the tissue5.             (brain) against gravity where as a pig’s brain is a low
Chronic rejection is due to ischemia-reperfusion injury              blood flow organ5.
which occurs at the time of grafting along with chronic                   Pig insulin has been used for years to treat diabetes
inflammation and associated scarring5. It can also be                and pig aortic valves that have been rendered inactive
caused by infection and cyclosporin toxicity5. The only              have been used successfully in transplantation so there
solution thus far to chronic rejection is the transplan-             is optimism that the organs will function adequately
tation of a new tissue or organ2. As the availability of             in humans5.
organs from animals would greatly exceed the available
organs from humans, chronic rejection may not be such                Conclusion
a problem in terms of xenotransplantation.                                Xenotransplantation may provide an unlimited
                                                                     supply of organs to patients suffering from chronic
Transfer of disease                                                  diseases, however, there are still many barriers to
     One of the more recent concerns of xenotransplan-               overcome before xenotransplantation can become
tation is xenozoonosis1. Zoonosis is defined as a disease            common practice. As rejection is still a problem in
that can be transmitted from animals to humans1. Well                the transplantation of allografts, it will probably be a
known zoonosis include HIV, hauntevirus, Ebola, and                  while before xenotransplantation becomes a realistic
Bovine spongiform encephalopathy (mad cow dis-                       alternative. With the advent of genetic engineering and
ease)1. Xenozoonosis is defined as the transmission                  new drug regimens, however, it seems as though the
of pathogens from animal organs and blood products                   problems associated with the initial forms of rejec-
to human recipients of animal organ transplants 1. It                tion in xenotransplantation are being overcome. Once
may be possible to avoid transfer of microorganisms                  a major barrier, hyperacute rejection has become less
with xenotransplantation by the use of gnotobiotic tech-             of a determinant in the success of a transplanted organ.
niques in the delivery, weaning, housing and handling                This is important in terms of using animal organs tem-

                          BioTeach Online Journal | Vol. 1 | Fall 2003 |
                                                            - 51 -
porarily as bridge organs while the patient is waiting                   350-3 (1997).
for an allotransplant. Likewise, our understanding of
                                                                    9.   Oriol, R., Ye, Y., Koren, E. & Cooper, D. K.
the mechanisms of acute rejection has increased greatly
                                                                         Carbohydrate antigens of pig tissues reacting
in recent years extending the amount of time a foreign
                                                                         with human natural antibodies as potential
organ can survive in a host. In terms of cellular and
                                                                         targets for hyperacute vascular rejection
acute rejection, not much is known. There have been
                                                                         in pig-to-man organ xenotransplantation.
many advances in the field of allotransplantation but
                                                                         Transplantation 56, 1433-42 (1993).
our ability to conquer acute and chronic rejection is
still beyond our reach. In time there is no doubt that              10. Platt, J. L. et al. Immunopathology of
the problems will eventually get worked out and with                    hyperacute xenograft rejection in a swine-to-
luck, the solutions will apply to xenotransplantation as                primate model. Transplantation 52, 214-20
well. In terms of the problem of xenozoonosis, hope-                    (1991).
fully with time, research and genetic engineering we
                                                                    11. Dalmasso, A. P., Vercellotti, G. M., Platt, J.
can eradicate the threats of endogenous viruses in the
                                                                        L. & Bach, F. H. Inhibition of complement-
porcine genome and with better technology, gnoto-
                                                                        mediated endothelial cell cytotoxicity by decay-
biotic conditions for the housing and care of the donor
                                                                        accelerating factor. Potential for prevention of
animals should also reduce xenozoonosis. As we get
                                                                        xenograft hyperacute rejection. Transplantation
closer to unlocking the secrets of the immune system,
                                                                        52, 530-3 (1991).
xenotransplantation will undoubtedly become a major
achievement in the field of science; unfortunately, we              12. Lin, S. S. et al. The role of natural anti-Gal
still have a long way to go before xenotransplantation                  alpha 1-3Gal antibodies in hyperacute rejection
becomes a reality.                                                      of pig-to-baboon cardiac xenotransplants.
                                                                        Transpl Immunol 5, 212-8 (1997).
References                                                          13. Atkinson, J. P., Oglesby, T. J., White, D.,
1.   Halperin, E. C. Non-human to human organ                           Adams, E. A. & Liszewski, M. K. Separation of
     transplantation: its biologic basis and a potential                self from non-self in the complement system: a
     role for radiation therapy. Int J Cancer 96, 76-                   role for membrane cofactor protein and decay
     89 (2001).                                                         accelerating factor. Clin Exp Immunol 86 Suppl
2.   Hakim, N. S. Introduction to organ                                 1, 27-30 (1991).
     transplantation (Imperial College Press,                       14. Buhler, L. et al. High-dose porcine
     London, 1997).                                                     hematopoietic cell transplantation combined
3.   Platt, J. L. Xenotransplantation (ASM Press,                       with CD40 ligand blockade in baboons
     Washington, D.C., 2001).                                           prevents an induced anti-pig humoral response.
                                                                        Transplantation 69, 2296-304 (2000).
4.   Platt, J. L. & Nagayasu, T. Current status of
     xenotransplantation. Clin Exp Pharmacol                        15. Inverardi, L. et al. Early recognition of a
     Physiol 26, 1026-32 (1999).                                        discordant xenogeneic organ by human
                                                                        circulating lymphocytes. J Immunol 149, 1416-
5.   Cooper, D. K., Gollackner, B. & Sachs, D. H.                       23 (1992).
     Will the pig solve the transplantation backlog?
     Annu Rev Med 53, 133-47 (2002).                                16. Sachs, D. H., Sykes, M., Greenstein, J. L. &
                                                                        Cosimi, A. B. Tolerance and xenograft survival.
6.   Institute of Medicine (U.S.). Committee on                         Nat Med 1, 969 (1995).
     Xenograft Transplantation: Ethical Issues and
     Public Policy. Xenotransplantation : science,                  17. Zhao, Y. et al. Skin graft tolerance across a
     ethics, and public policy (National Academy                        discordant xenogeneic barrier. Nat Med 2, 1211-
     Press, Washington, D.C., 1996).                                    6 (1996).
7.   Rose, A. G. & Cooper, D. K. Venular thrombosis                 18. Onions, D. et al. An approach to the control
     is the key event in the pathogenesis of antibody-                  of disease transmission in pig-to-human
     mediated cardiac rejection. Xenotransplantation                    xenotransplantation. Xenotransplantation 7,
     7, 31-41 (2000).                                                   143-55 (2000).
8.   Deacon, T. et al. Histological evidence of fetal               19. British Columbia Transplant Society. http:
     pig neural cell survival after transplantation into                //
     a patient with Parkinson’s disease. Nat Med 3,

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