Xenotransplantation

Xenotransplantation ,Xeno ,transplantation,non human,human ,Xenotransplantation ,transplant,human,non human,

Overview
Xenotransplantation
involves the transplantation of nonhuman tissues or organs into human
recipients. The concept was pioneered a century ago, when transplanting
human organs was considered ethically controversial. Grafts were quickly
rejected, however, because of unknown forces later identified as immune
responses. Interest in xenotransplantation reemerged during the
1960s, when large advances were made in immunology. Chimpanzee kidneys
have been transplanted into patients with renal failure. In 1984, a baboon heart was transplanted into a newborn infant, Baby Fae, who had hypoplastic left heart syndrome and lived 20 days after heart surgery. A baboon liver was transplanted to a patient with hepatic failure. Porcine islet cells of Langerhans have been injected into patients with type 1 diabetes mellitus. Porcine skin has been grafted onto burn patients, and pig neuronal cells have been transplanted into patients with Parkinson (Parkinson’s) disease and Huntington (Huntington’s) disease. During
these advances, several obstacles to the success of xenotransplantation
have been identified. These include, but are not limited to, (1)
preventing hyperacute rejection, (2) preventing acute vascular
rejection, (3) facilitating immune accommodation, (4) inducing immune
tolerance, (5) preventing the transmission of viruses from xenografts
into humans, and (6) addressing the ethical issues surrounding animal
sources for xenografts and the appropriate selection of recipients
(given that xenotransplantation remains experimental). The purpose of
this review is to identify the obstacles and recent progress made in the
field of xenotransplantation.
The rationale of xenotransplantation

The
motivation for using animal sources for organ or tissue transplantation
is driven by supply and demand. According to the most current report
from the United Network for Organ Sharing (UNOS), more than 107,241
Americans were waiting for organ transplantation as of May 2010.In
2009, 28,464 patients had transplants, and approximately 40% of listed
candidates on waiting list were younger than 50 years. In light
of the lack of supply of human organs for transplantation, several
alternatives have been investigated and debated. Implantable mechanical
devices have been explored in the field of cardiac transplantation.
Recently, research has increased in the area of transplanting embryonic
cells across species and growing kidneys and endocrine pancreas cells in
situ. Organs
from pigs have been the focus of much of the research in
xenotransplantation, in part because of the public acceptance of killing
pigs and the physiologic similarities between pigs and human and
nonhuman primates. Xenotransplantation of organs from chimpanzees and
baboons has been avoided, however, because of ethical concerns and fear
of transmission of deadly viruses (see Biologic Barriers to
Xenotransplantation). Xenografts have been proposed as
appropriate for infants who are physically too small to accommodate
organs retrieved from adult or pediatric donors. Additionally, organs
from animal sources could be transplanted into patients currently
excluded from the human organ transplantation list. Finally, most
patients perceive xenotransplantation as an acceptable bridge to
transplantation of human organs in life-threatening situations.
History of the procedure

Alexis
Carrel is known as the founding father of experimental organ
transplantation because of his pioneering work with vascular techniques.
Carrel and Guthrie contributed substantially to the science of
transplantation from 1904-1906. They performed autogenous vein grafts,
performed leg replantation in dogs, and developed the famous patch-graft
technique for widening narrowed vessels. They also performed
heterotopic experimental transplantation. Parts of a small dog were
transplanted into the neck of a larger dog. They developed the
buttonhole technique for anastomosis of donor and recipient vessels in
kidney transplantation to prevent thrombus formation. In 1906,
Jaboulay transplanted kidneys from goats, sheep, and monkeys into
humans. These attempts at kidney xenografting were unsuccessful. In
1910, Unger transplanted a nonhuman kidney into a man dying of renal
failure, which caused death a little more than a day later. In 1932,
Neuhof transplanted a lamb kidney into a patient with mercury poisoning.
The patient survived for only 9 days. In 1946, Demikhov transplanted a
heterotopic heart and lung; the animal survived for 9 hours. The
clinical interest in xenotransplants waned following the series of
disappointing results and the realization that transplant failure was
attributable to powerful unknown forces that would eventually be
identified as the body's immune system. Scientific interest did not
revive until the 1950s, following successful allografting of kidneys
from identical twins. Michon and Hamburger successfully performed a
living related donor kidney transplantation in Paris in 1952; in 1954,
Merrill and Murray, using no immunosuppression, performed the first
kidney transplantation between monozygotic twins. The
advent of dialysis therapy during the 1940s and 1950s and its
widespread availability in 1970s eventually made a significant and
lasting impact on kidney transplantation. Hemodialysis established a
bridge to transplantation and significantly expanded the population of
patients that could benefit from kidney transplantation. Patients with
end-stage renal disease had 2 therapeutic options: (1) dialysis and/or
therapy or (2) an allograft (cadaveric or living related donor).At
that time, an understanding of transplant immunobiology and
immunosuppressive drugs had just started to develop. Knowledge of organ
procurement and preservation was extremely limited, severely curtailing
the widespread clinical use of allografting. During this time, Starzl
and colleagues (and other groups) were experimenting with
xenotransplantation using chimpanzees or baboons.The momentum of
xenotransplantation was derailed in the 1990s with discovery of the
porcine retrovirus. Concerns about the risk of cross-species infections
resulted in moratoriums on clinical trials on xenotransplantation.
Definition

Xenotransplantation
refers to any procedure that involves the transplantation,
implantation, or infusion into a human recipient of either (1) live
cells, tissues, or organs from a nonhuman animal source or (2) human
body fluids, cells, tissues, or organs that have had ex vivo contact
with live nonhuman animal cells, tissues, or organs. (US Food and Drug
Administration [FDA], 1999; FDA, 2001). Xenotransplantation
products must be alive, and circulation and return of patients' blood
must occur through live nonhuman cells. For example, human skin cells
grown outside the body on a layer of nonhuman cells and then used in
humans for skin reconstruction can also be considered a
xenotransplantation product. This latter category of procedures was
included in the definition because scientists believe that the potential
for transmission of an infectious disease with such a procedure may be
similar to that of implanting live nonhuman animal cells, tissues, or
organs directly into a human recipient. Xenotransplantation
products include those from transgenic or nontransgenic nonhuman animals
and composite products that contain xenotransplantation products in
combination with drugs or devices. These include, but are not limited
to, porcine fetal neuronal cells, encapsulated porcine islet cells,
encapsulated bovine adrenal chromaffin cells, baboon bone marrow, and
external liver-assist devices using porcine liver or porcine
hepatocytes. Nonliving biological products or materials from nonhuman
animals, such as porcine heart valves and porcine insulin, are not
classified as xenotransplantation products for the purposes of this
definition. Xenotransplantation products are subject to
regulation by the FDA under section 351 of the US Public Health Service
Act [42 U.S.C. 262] and the Federal Food, Drug and Cosmetic Act [21
U.S.C. 321 et seq]. In accordance with the statutory provisions
governing premarket development, xenotransplantation products are
subject to FDA review and approval. Depending on the relationship
between donor and recipient species, the xenotransplant can be
described as concordant or discordant. Concordant species are
phylogenetically closely related species. These species combinations
include mouse to rat, baboon to cynomolgus monkey, or, presumably,
nonhuman primate to human. Discordant species, on the other hand, are
not closely related (eg, pig to mouse, pig to human). A
concordant recipient takes several days to reject an organ, whereas a
discordant recipient mounts a violent, hyperacute response that leads to
xenograft loss within a few minutes to a few hours. This difference in
time to rejection is related to the presence or absence of preformed
natural antibodies in discordant species pairs (described in Immunologic
Barriers to Xenotransplantation).
Next Section: Choosing the Donor Species

Choosing the Donor Species

Chimpanzees
were generally thought to be the best nonhuman primate donor compared
with baboons or rhesus monkeys. However, their availability rapidly
dwindled in the 1960s, and they were later listed as endangered species.
Consequently, scientists sought other potential animal sources for
organs for xenotransplantation. Species such as baboons, although
existing in greater abundance compared with chimpanzees, fare poorly in
captivity, have a long gestation, and have few offspring. The
FDA's Biologic Response Modifiers Advisory Committee (BRMAC), after
conducting an in-depth investigation of this issue and convening public
hearings, noted its findings and concern on this matter in the federal
register dated January 2001.In
this document, the BRMAC raises concern regarding interspecies
transmission of xenogeneic infectious agents. It also notes the
potential for subsequent transmission of a xenogeneic infectious agent
from the recipient to the recipient's close contacts, and propagation
through the general human population, as an additional risk and a
recognized public health concern. The BRMAC has also identified
the potential risk of insertional mutagenesis associated with the
infection of xenotransplant recipients, their close contacts, and the
general population with xenogeneic retroviruses. In addition to
potential horizontal transmission of infectious agents from the
recipient of a xenotransplantation product to the recipient's contacts,
BRMAC is concerned regarding vertical transmission of infectious agents
from the recipient to progeny during gestation (ie, transmission from
mother to fetus of infectious agents across the placenta or during
parturition). Vertical transmission of xenogeneic infectious
agents could result in the development of infectious disease in progeny.
In addition, vertical transmission of xenogeneic viruses can result in
insertional mutagenesis with disruption of normal human development or
integration into the germline, resulting in transmission to future
generations. The BRMAC considered nonhuman primate donors to pose the
greatest threat of transmitting latent, intracellular, or unidentified
organisms, including retroviruses, and recommended that nonhuman
primates not be used as sources of xenotransplantation products until
more information is available to assess safety issues. This led
investigators to seek other animal sources. Monkeys are not considered
acceptable organ donors for both practical and ethical reasons. In
addition to being uncomfortably close to humans on the evolutionary
ladder, they also produce few offspring, are slow to mature, and would
be difficult to rear under the sterile conditions required to minimize
contamination by shared pathogens. Most investigators agree that
pigs have the potential to be the prime candidates for organ donation.
Pigs are plentiful, are quick to mature, breed well in captivity, have
large litters, and have vital organs roughly comparable in size to those
of humans. Because they are already being used in the consumer market,
their use in xenografting is less likely to elicit major objections from
society. Because humans have had prolonged and close contact with pigs,
their use for the purpose of xenotransplantation is believed to be less
likely to introduce any new infectious agents. However, use of pig
xenografts is associated with major immunologic barriers, resulting in
hyperacute rejection (HAR) when transplanted into a nonhuman primate or
human recipient.
Previous
Next Section: Choosing the Donor Species


Immunologic Barriers to Xenotransplantation

Immunology of Xenograft Rejection

Hyperacute rejectionTransplanting
solid organs from animal sources into humans results in rapid loss of
the xenograft because of hyperacute rejection. In this dramatic
immunologic reaction, preformed antibodies circulating in human blood
bind to the vascular epithelium of the animal organ and trigger a
cascade that quickly results in thrombosis of the graft. Humans
have preformed antibodies known as xenoreactive natural antibodies
(XNAs), which are directed against nonprimate species. XNAs appear in
the early neonatal period following colonization of the large bowel by
coliform bacteria. These antibodies primarily consist of immunoglobulin M
but also probably include the immunoglobulin G and immunoglobulin A
classes. Their binding is characterized by avidity and surprising
uniformity. Most XNAs recognize carbohydrate moieties associated
with bacterial cell walls. Most human XNAs are directed against terminal
carbohydrate, Gal1, and 3a-GalbGlcNAC-R, in which a galactosyl residue
is linked to another galactosyl residue. This process is controlled by
an enzyme galactosyl transferase. Humans lack this enzyme, and the
carbohydrate epitope is therefore perceived as a foreign antigen and
antibodies arise against it. This carbohydrate moiety is expressed on
pig cells. Thus, humans have naturally occurring antibodies (ie, XNAs)
against pig cells. XNAs recognize porcine glycoproteins of the
integrin family. Antibody binding initiates complement activation
through the classic pathway, triggering a number of effector mechanisms.
These mechanisms may include loss of heparan sulfate from endothelial
cells (EC) mediated by C5a and xenoreactive antibody, a change in
endothelial cell shape mediated by C5b-7 or the membrane attack complex,
procoagulant changes mediated by the membrane-attack complex, and
neutrophil adhesion mediated by iC3b. The
immunologic cascades triggered during hyperacute rejection destroy very
discordant xenografts within minutes to hours. This process is
characterized by immediate engorgement and discoloration of the organ.
Under light microscopy, interstitial hemorrhages and platelet
microthrombi are evident. Immunohistologically, dense deposition of
various immunoglobulins and multiple complement components is noted
throughout the vascular bed. The anaphylatoxins C3a and C5a generated in
the process stimulate basophils and mast cells to release histamine,
which, in turn, results in platelet degranulation. Binding of
histamine and serotonin to receptors on endothelial cells stimulate the
expression of platelet-activating factor and P-selectin.
Platelet-activating factor dramatically increases vascular permeability
and endothelial cell contraction, resulting in platelet and RBC sludging
within the microcirculation. This complex interplay of complement
components, platelets, and endothelial cells leads to platelet
aggregation, coagulation, fibrin deposition, and hemorrhage, typically
culminating in thrombosis and ischemic necrosis within minutes of
engraftment. Acute vascular rejectionIf the
transplanted organ is not rejected within minutes to hours, a more
delayed type of immunologic response ultimately leads to thrombosis of
the graft within hours to days. This process known as delayed xenograft
rejection or acute vascular rejection. Under light microscopy,
focal infarcts, interstitial hemorrhages, and widespread coagulation of
microvasculature are observed. DXR is characterized by progressive
infiltration of monocytes and natural killer cells (over several days),
endothelial cell activation, platelet and fibrin deposition, and
cytokine expression. Only very small numbers of T cells are noted (~5%).
The role of macrophages and natural killer cells in DXR has yet to be
determined; however, neither XNAs nor T cells are essential for DXR in
complement-depleted rats. Although endothelial cell activation is
believed to play a key role, factors that trigger it are not well
defined. Endothelial activation is type II in nature because it involves
gene induction and protein synthesis. This includes a shift to a
procoagulant state, secretion of chemokines such as membrane cofactor
protein-1, and induction of leukocyte adhesion molecules such as
E-selectin, intercellular adhesion molecule-1, and vascular cell
adhesion molecule-1.
Overcoming Xenograft Rejection

To
devise therapeutically effective strategies to defeat HAR and DXR, a
detailed understanding and identification of complex inflammatory
pathways and key events are indispensable. This involves in-depth study
of both donor and recipient factors that play critical roles in mounting
and sustaining a rejection response. This section focuses on approaches
proposed to circumvent xenograft rejection.

 Donor-based strategies: Transgenic organs

Genetically
engineered pigs have been designed to downregulate expression of
various immunogenic substances. Several groups have been successful at
developing a breed of knockout pigs that lack the 1,3
galactosyltransferase gene. This gene ordinarily codes for an enzyme
that is responsible for the expression of the immunogenic Galalpha1,3Gal
carbohydrate moiety on the vascular endothelium of pig organs. In 2005,
pig hearts from alpha 1,3 galactosyltransferase knockout (GT-KO) pigs
were transplanted into baboons.These grafts survived 6 months.Others
have bred pigs with increased expression of H-transferase (also known
as 1,2 fucosyltransferase). H-transferase is an enzyme that competes
with alpha 1,3 galactosyltransferase, the substrate that is a precursor
to the Galalpha1,3Gal moiety. The rationale is to decrease the amount of
Galalpha1,3Gal carbohydrate on pig vascular endothelium. Swine
islet cells that express N-acetylglucosaminyltransferase III (GnT-III)
have been developed and have been recently transplanted into monkeys. Unlike
other glycosyltransferases, GnT-III interrupts the biosynthesis of the
Galalpha1,3Gal xenoantigens by several mechanisms not fully understood,
including interruption of carbohydrate branching.Another
variation of transgenic pigs has been developed to interfere with the
mechanisms of graft rejection. For example, expression of human
ecto-5'-nucleotidase (E5'N) pig organs has protected grafts from natural
killer cell–mediated lysis.Transgenic
pigs have also been bred to express glycoproteins that inhibit the
human complement cascade. These include CD55 (human decay accelerating
factor [hDAF]), CD46 (membrane cofactor 1), and CD59 (protectin, which
blocks the membrane attack complex from binding to cells). Advances
in genetic engineering have made double transgenic pig organs
available. Xenotransplantation of double transgenic porcine skin to
cynomolgus monkey survived 31 days. The skin expressed both GnT-III and
hDAF.Double
transgenic pig kidneys with both human alpha-galactosidase and alpha
1,2-fucosyltransferase have been shown to decrease Gal expression and
resist human serum–mediated lysis in ex vivo experiments.Others have engineered double transgenic pig hearts expressing both hDAF and CD59.Triple
transgenic pigs have recently become available. Hyperacute rejection
was prevented in baboon recipients of orthotopically transplanted swine
livers that expressed hDAF, human CD59, and H-transferase.Japanese
scientists recently reported success with breeding alpha 1,3
galactosyltransferase knockout pigs that express hDAF and Gnt-III. Another variation of triple transgenic pigs has been bred to express human CD59, human membrane cofactor protein, and hDAF.
Experimental xenotransplantation

Several
experimental designs have been developed for xenotransplantation of
these genetically modified organs. In the ex vivo model, the genetically
transformed porcine organ is infused with human blood or serum to see
if hyperacute rejection occurs. Alternatively, in the life-sustaining
model, genetically modified pig organs have been transplanted into
baboons or monkeys undergoing immunosuppressive therapy.
Xenotransplantation may be performed orthotopically such that the native
organ is removed and the transplanted organ occupies the anatomic
location. Xenotransplantation of organs from transgenic pigs for
CD55 (hDAF) has been extensively studied over the last five years with
mixed results. Researchers recently showed that higher levels of CD55
expression in transgenic pigs increase survival of grafts in baboons
that have undergone xenotransplantation.CD55
transgenic porcine hearts have been successfully transplanted
orthotopically into baboons, with a median survival of 14.6 days.Organs transgenic for CD55 also appear resistant to cellular lysis by human serum.In
other ex vivo models involving xenotransplantation of transgenic pig
lungs into baboons, however, hyperacute rejection has been reported upon
perfusion of the graft.Many
groups have prolonged survival of CD55 (hDAF) transgenic pig organs
with infusion of various agents or soluble antibodies. For example,
reduced myocardial damage has been reported in ex vivo experiments in
which CD55 transgenic grafts were perfused with GPIIb/IIIa inhibitor
tirofiban.In
other experiments, CD55 transgenic pig hearts have been transplanted
into baboons that were also administered soluble Gal-glycoconjugates to
block baboon antibodies from binding to the Gal moiety on renal
endothelium. These grafts survived for 3 months.Anti–nonGal antibodies have been suggested to be involved in the ultimate acute humeral xenograft rejection of the kidneys.Other
groups have attempted life-supporting xenotransplantation of transgenic
CD55 pig kidneys in baboons with infusion of soluble complement
receptor type 1, TP 10. These grafts ultimately failed because of
chronic deposition of complement in the endothelium.

Recipient-based strategies

Acute
vascular rejection is thought to be triggered by antibodies to the
xenograft and complete activation of the complement cascade. In
theory, the complement cascade can be interrupted therapeutically by
using several inhibitory agents. Such complement inhibitory molecules
include cobra venom factor (to deplete C3), soluble complement receptor
type 1, anti-C5 antibodies, K76COOH, and FUT-125.Toxicity
associated with cobra venom factor is a major obstacle to its clinical
use. Moreover, recent studies show the administration of cobra venom
alone does not appear to prevent the deposition of C3 complement in
grafts.The
soluble complement receptor type 1 (TP10) does not appear to be clearly
effective in interrupting the complement cascade. Deposition of
complement was found in grafts of monkeys that were transplanted with
life-supporting hDAF transgenic pig kidneys and given cyclosporine,
mycophenolate, steroids, soluble glycoconjugates to Galalpha1,3Gal, and
TP10.Several
immune-modulating therapies have been developed to prolong xenograft
survival. Combinations of immunosuppressive agents, including
cyclosporine A, mycophenolate sodium, and steroids, have prolonged
survival of hDAF porcine renal xenografts in primates.Recently, soluble decay-accelerating factor has been used to prevent humeral rejection.Others
have experimented with various mechanisms to downregulate
co-stimulation and alter the immune response to interleukins. Swine
hearts have been successfully xenografted into baboons treated with
anti-CD154 antibodies and CD28/7 blockade.Murine
studies have suggested that acute vascular rejection can be attenuated
by CD8alpha+ dendritic cells that secrete IL-12 and induce a Th1 slow
cell-mediated response to the xenograft.Finally,
interrupting the initiation of acute humeral rejection by blocking IL-1
with receptor antagonists appears promising in models of guinea pig
hearts transplanted into rats treated with cobra venom.

Achieving accommodation

Accommodation
is defined as graft survival despite the circulation of xenoreactive
antibodies. This phenomenon was first identified in the late 1980s, when
blood group O recipients were able to tolerate transplantation of group
A or B kidneys.In
accommodation, the graft is given a break from attack when circulating
antibodies are removed from the system or when the complement cascade is
interrupted. During this break from acute vascular rejection, the graft
is able to up-regulate and express protective genes, like heme
oxygenase 1, which impart graft resistance to injury. Although still
unclear, other changes also appear to occur in the function of
circulating antibodies and the expression of surface antigens on the
graft. Studies have also shown that accommodation might be
achieved by interrupting the complement cascade with the administration
of heparin sulfate. This is because full activation of the complement
cascade characteristically lacks the presence of syndecan-4 phosphate.
One group showed, however, that warfarin or low molecular weight heparin
failed to induce accommodation in pig hearts xenografted into primates.
Coagulation dysregulation as a barrier to xenotransplantation

The
ability to generate pigs that express a human complement regulatory
protein (hCRP) and/or alpha 1,3 GT-KO pigs has largely overcome the
barrier of HAR of a pig organ transplanted into a primate. However,
acute humoral xenograft rejection (AHXR) presenting as microvascular
thrombosis, consumptive coagulopathy, or both remains a major hurdle to
successful xenotransplantation.Until now, thrombosis was believed
to result from antibody-mediated and complement-mediated EC activation,
initiating AHXR. Exposure of porcine ECs to xenoantibodies, complement,
and cells of the innate immune system results in EC activation and loss
of anticoagulant regulators on their surface, with a subsequent change
to a procoagulant phenotype. In xenotransplantation, distinct,
immune-independent factors contribute to the development of coagulation
disorders (eg, molecular incompatibilities between pigs and primates
that promote or fail to regulate pathological clotting). For example,
porcine von Willebrand factorand loss of porcine tissue factor inhibitor (TFPI)initiate coagulation cascade in primates. GT-KO pigs that express an hCRP gene (eg, CD46 or hDAF) have exhibited some protection against the humoral-mediated immune response but do not overcome the problems of coagulation.The
interactions between porcine ECs, platelets, and other blood cells are
at the nexus of a complex network that contributes to coagulation during
AHXR. According to the standard paradigm, the process is initiated by
the immune response against the graft, such that activation of porcine
ECs caused by antibodies with/without complement leads to expression of
TF that triggers the coagulation cascade.Loss
of anticoagulant regulators, such as TFPI, ectonucleotide triphosphate
diphosphohydrolase-1 (CD39), and ecto-5′-nucleotidases (CD73) activity
is associated with platelet activation and aggregation. Administration
of CD39 substitutes inhibits platelet activation and aggregation,
thereby significantly prolonging graft survival.Hearts from CD39-transgenic mice were resistant to thrombosis in a mouse-to-rat xenotransplantation model.
Future directions in coagulation dysregulation

The
resolution of coagulation dysregulation is critical to allow
xenotransplantation to advance sufficiently for clinical trials.
Thrombosis manifests in the graft, but systemic consumption coagulopathy
may have more than one cause. Therefore, in addition to genetic
modification of the organ-source pig, systemic medication may be
necessary. GT-KO pigs that express an hCRP and one or more
anticoagulant or antithrombotic genes are anticipated to inhibit the
generation of thrombosis and subsequent platelet activation. Withnew
cloning techniques (F2A system), mice transgenic for CD55/hTFPI/hCD39
have been generated. The same technology is expected to be successfully
applied to generate pigs with multiple gene modifications and will
enable these pigs to become available in a shorter period of time than
through conventional breeding.
Systemic medication

Statins,
inhibitors of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase, used
for hypercholesterolemia, also have other independent effects, such as
immunoregulatory, anti-inflammatory, and anticoagulant actions.Atorvastatin
reduces porcine EC activation induced by interferon gamma and inhibits
the proliferative response of primate peripheral blood mononuclear cells
(PBMC) and CD4+T cells when stimulated with porcine ECs. Simvastatin
was shown to prevent the induction of TF on human aortic ECs by
thrombin, at least in part through inhibition of rho-kinase-dependent
Akt dephosphorylation.Lovastatin
enhanced ecto-5′-nucleotidase activity and membrane expression in ECs,
consequently inhibiting platelet aggregation through the action of
adenosine.
Antiplatelet agents

Antiplatelet
agents, such as antagonists of P2Y12 or GPIIbIIIa receptors, are
expected to prevent aggregation when platelets are activated during
AHXR. A high dosage of a GPIIbIIIa antagonist prolonged graft survival
and decreased platelet aggregations in a rodent model.If
thrombosis in the graft is prevented by antithrombotic gene
modification, this systemic approach may offer survival benefits by the
prevention of systemic consumption coagulopathy.
Previous
Next Section: Choosing the Donor Species

Biologic Barriers to Xenotransplantation


The use of xenotransplantation products carries a natural and expected risk
of transmitting infectious pathogens. This risk is beyond the risk of
infections known to be associated with allotransplantation, which is a
consequence of immunosuppression. This serious risk originates from the
potential of xenotransplantation for transmitting infectious agents from
nonhuman animals to humans. The HIV pandemic, Creutzfeldt-Jakob
disease, Ebola virus outbreaks, and, more recently, the severe acute
respiratory syndrome scare have elicited serious concern about the
potential of inadvertent transmission of known and unknown infectious
agents that may spread to recipients and to their contacts and health
care workers, quickly becoming a public health issue. Thus, for
the following reasons, infectious risks associated with
xenotransplantation may be far greater than those noted with
allotransplantation:

• The
  level of immune suppression and/or rejection may be greater in
  xenograft recipients, enhancing the activation of latent pathogens,
  including viruses.
  
• Organisms
  carried by the graft may not be known human pathogens and/or may include
  xenotropic organisms, ie, organisms that are not pathogens in the
  native host species but which cause disease in other species, in this
  case, the human recipient.
  
• Microbiologic assays may not exist for some organisms derived from nonhuman species.
  
• Novel animal-derived organisms may cause novel and thus unrecognized clinical syndromes.
  
• Genetic
  modification of the donor animals (one xenotransplantation strategy) or
  treatment of the recipient with, for example, tolerance induction or
  antibody removal, may alter the host's susceptibility to organisms.
  

Xenosis

The term xenosis has been coined to describe the transmission of infections
by the transplantation of xenogeneic tissues or organs. Xenosis, or
xenozoonosis, potentially poses unique epidemiological hazards owing to
the efficiency of transmission of pathogens, particularly viruses, with
viable cellular grafts. When an infectious agent gains entry into a new
host species, its capacity to produce disease is unpredictable. For
example, in its natural host, the macaque monkey, cercopithecrine
herpesvirus 1 (B virus) infection has a clinical profile very similar to
that of herpes simplex virus infection in humans. However, B virus
infection of humans or other non–macaque primates results in rapidly
progressive myeloencephalitis with a mortality rate of approximately
70%.This
failure of the pathogenic potential of a microbe in its host species to
reliably predict the pathology that results when it is introduced into
another species is evident with many other zoonotic agents and diseases.
Organisms of serious concern include herpesviruses and
retroviruses, which can be screened for and eliminated from the donor
pool. Others include Toxoplasma gondii, Mycobacterium
tuberculosis, and encephalomyocarditis virus. Filoviruses (Marburg and
Ebola), monkeypox virus, and simian hemorrhagic virus are less likely to
be found in animals reared in captivity in the United States.

Retroviruses

Retroviruses,
by virtue of the enzyme reverse transcriptase, become inserted into
host chromosomal DNA. Compelling arguments suggest that the HIV pandemic
resulted from the adaptation of simian retroviruses introduced across
species lines into humans. Existing data suggest that the HIV-2 pandemic
in East Africa began with the horizontal transmission of simian
immunodeficiency virus from a sooty mangabey monkey into a human with
subsequent transmission through the human population. In Central Africa,
horizontal cross-species transmission of simian immunodeficiency virus
from a different primate species, probably a chimpanzee, resulted in the
HIV-1 pandemic. Initial human infections before 1970 resulted in more
than a decade of insidious human-to-human transmission before AIDS was
first recognized as a public health problem in the 1980s. Endogenous
retroviruses exist as part of the genomic material of most, if not all,
mammalian species, including humans. Endogenous retroviruses cause
equal concern and greater uncertainty than the exogenous retroviruses.
Endogenous retroviruses, presumably originating as exogenous viruses
that became permanently integrated into the host germ line, are
vertically transmitted through inheritance. In the host species, they
are benign. However, endogenous viruses are frequently xenotropic, ie,
although the original host is refractory to infection, the viruses can
infect related species. The increased phylogenetic distance
between swine and humans presumably makes pigs safer donors than
nonhuman primates. This presumption has not been completely explored.
The biology and pathogenicity of a type C retrovirus identified in the
blood of leukemic or irradiated swine are incompletely characterized.
Similarly, the discovery of porcine endogenous retroviruses (PERVs)
capable of infecting human cells in vitro has raised issues regarding
the safe clinical application of xenotransplantation.Phylogenetic
analysis reveals that PERVs are closely related to gibbon ape leukemia
virus, endogenous koala retrovirus, and inducible murine endogenous
retrovirus PERV
RNA is expressed in several porcine tissue types (eg, kidney, lung,
liver, heart, pancreatic islets); however, expression of virus mRNA does
not necessarily correlate with the release of infectious particles.
Many human cells clearly express receptors specific for PERV A and B,
whereas PERV C–specific receptors cannot be detected in most instances.Several
viral pathogens have been identified in the xenografts from pigs, which
are the most common animal source of xenografts. These include, but are
not limited to, porcine endogenous retrovirus (PERV), porcine
cytomegalovirus (PCMV), and porcine lymphotrophic herpes virus (PLHV),
and porcine circovirus type 2, (PCV). In New Zealand, pigs raised for
xenotransplantation were found to harbor encephalomyocarditis (EMCV) and
hepatitis E.^[65] Importantly,
pigs do not have the exogenous retroviral equivalent of the HTLV or HIV
virus. Two strains of the PERV virus (strain A and B) are present in
only a subset of swine and have the potential to infect human cells in
vitro. Importantly, the PCMV strains can be selected out of the pool of
potential xenografts by early weaning of piglets. Experimental
xenotransplantation of organs from swine to nonhuman primates has
demonstrated the absence of PERV transmission. One study suggested that
decreased risk of transmission of endogenous retroviruses from pigs to
baboons is correlated to decreased amounts of circulating anti–alpha Gal
antibody.^[66] Others have shown an absence of PERV infection in baboons receiving transgenic livers.^[67] Over
the past 10 years, many sensitive diagnostic assays have been developed
to detect most potential viruses associated with xenotransplantation of
organs into humans. For example, 1 long-term follow-up study on 18
human recipients of pig islet cell transplants showed no evidence of
PLHV, PCMV, PCV, or PERV infection in any of the patients 9 years after
xenotransplantation.^[68] No
in vivo infection of human cells by the PERV virus has been reported to
date. By contrast, the transplantation of baboon livers and chimpanzee
kidneys into humans had resulted in deaths due to illnesses not related
to organ failure.^[69] Japanese
researchers are attempting to engineer transgenic pigs that would be
genetically incapable of harboring endogenous retroviruses. Such a breed
of pigs would express the RNA interference silence genes.^[70]
Transmissible spongiform encephalopathy

Transmissible
spongiform encephalopathy is a uniformly fatal family of diseases of
humans and animals that causes irreversible cumulative brain damage.
These diseases, which include Creutzfeldt-Jakob disease, chronic wasting
disease, and bovine spongiform encephalopathy, are believed to be
caused by a novel class of agents termed prions. Various reports have
documented the prions jumping the species barrier from cattle,
squirrels, and rabbits to humans. Transmissible spongiform
encephalopathies have exhibited transmission into new hosts through
transplanted grafts and across species lines.^{[71, 72]} Of
these diseases, cows can be naturally infected with bovine spongiform
encephalopathy and pigs can be experimentally infected with
transmissible spongiform encephalopathy.^[73]
Strategies to avoid xenosis

Given
the complexity of xenotransplantation and the uncertainties of issues
surrounding it, strategies to address xenosis are being developed,
although a universally adopted guideline on xenosis remains to be
issued. The US Public Health Service agencies (ie, FDA, Centers for
Disease Control and Prevention, National Institutes of Health, and
Healthcare Resource Services Administration) and the Office of the
Associate Secretary for Planning and Evaluation of the Department of
Health and Human Services are collaborating to develop an integrated
approach to address infectious disease issues in xenotransplantation. Bach and colleagues have suggested a 3-tiered approach to policy development and decisions to address the issue of xenosis.^[57] This
method suggests approaching the issue at societal, institutional, and
individual (patient-physician) levels. Because the risk is societal and
not merely individual, the decision whether to undertake the procedure
involves more than ensuring the ability of the surgeon and the
transplant team, the capacity of the institution, and the willingness of
the patient.In situations in which the risks are collective, the
public must be educated about the risk and must be involved in the
decision-making process. Therefore, the first part of the
decision-making process must occur at the level of social policy; the
second part must occur at the level of the institutions performing the
xenografts, and the third part must occur at the level of individual
patients and physicians, especially affecting the processes of informed
consent and medical confidentiality. Because of the risk of
xenosis, all the major reports on xenotransplantation released to date
have recommended comprehensive monitoring and surveillance of xenograft
recipients. The legal and ethical problems associated with imposing such
surveillance on recipients (and perhaps their sexual partners) for what
would likely be many years or for life and the details about the nature
and frequency of monitoring require further discussion. That patients
manifesting signs of a possible xenosis after transplantation would have
to be quarantined is not inconceivable. The maintenance of
patient confidentiality, as in all areas of medicine, remains paramount
and further complicates the need for adequate monitoring of recipients.
The community must be educated about any risk that could arise from
xenotransplantation, regardless of whether the extent of that risk and
the degree to which that risk is controllable can be precisely defined.
Finally, it would be helpful for the public to have a better
understanding of the process by which decisions are made in situations
of uncertainty.^[57] At
the level of the hospital or research center, institutions must be
responsible for establishing and enforcing standards for quality of
care, management of risk, monitoring of patients and their contacts, and
evaluation of the effectiveness of the procedure in accordance with
public guidelines and regulations. Institutions should avoid situations
in which individuals proceed with xenotransplantation in advance of
adequate safeguards and should curtail clinical trials until societal
guidelines are available.^[57] A
new approach to informed consent as it relates to xenotransplantation
is necessary. A patient's agreement to participate in
xenotransplantation must be based on perceptions of individual risks, as
is the case with any experimental or extreme procedure, and on the risk
of new disease to family, friends, close contacts, and society at
large. Because of the need for monitoring for signs of infection, the
patient and others must commit to participate in such monitoring for a
period considered to be longer than the potential time necessary for an
infection to manifest. Thus, the xenotransplant recipient undertakes a
social obligation to submit to close and frequent follow-up monitoring,
even if this means relinquishing certain freedoms in order to gain the
potential benefits of participation.^[57]
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Ethical Issues

The
subject of ethics in relation to xenotransplantation has been widely
explored. This includes issues related to both humans and nonhuman
animals. The concept that certain ethical principles must be applied to
experimentation conducted in humans is widely accepted. Such principles
include respect for persons, beneficence, and justice. These ethical
issues are addressed in great length by the International
Xenotransplantation Association Ethics Committee's position paper.^[74]
Beneficence and risk-to-benefit analysis

Risk
assessment is based on the principle that the possible harm of the
research must be outweighed by its probable benefits. Preclinical data,
including nonhuman primate data, must adequately support the possibility
of a successful outcome.^[75] In addition, for any ethically conducted trials, risks to the patient and to society must be minimized. Animals
used for xenotransplantation should be bred in captive, closed colonies
in order to ensure the exclusion from the colony of known potential
pathogens to humans. The extensive human experience with short-term
exposure to porcine materials, including patients receiving porcine
insulin, clotting factors, and temporary skin grafts, is reassuring.
However, none of these situations involves the long-term presence of
large numbers of porcine cells or organs in an immunocompromised
individual.
Autonomy and informed consent

The potential
risk of xenotransplantation to society elicits unique challenges in
developing an appropriate informed consent process. In addition to the
research subject, the burden of risk is also carried by close contacts,
medical caregivers, and society, all of whom may reasonably insist that
the research subject agrees to life-long monitoring, avoids blood
donation, and informs close contacts about the xenotransplantation and
its potential risk for spreading zoonotic infections. Asking a
subject to agree to life-long monitoring effectively denies him or her
the right to withdraw from the study at any time. This is a denial of a
fundamental individual right as delineated in the Declaration of
Helsinki and the US Code of Federal Regulations.^[76] Some
authors have even gone so far as to suggest the need for mandatory
“Ulysses contracts” when obtaining informed consent to proceed with
xenotranplantation.^[77] These
contracts, which currently exist in some psychiatric practices, are an
advance directive from a patient to the primary care provider given at a
time when the patient is psychiatrically well. In this setting, the
patient essentially preauthorizes the physician to involuntarily commit
him or her during times of psychiatric relapse. In a similar fashion, a
Ulysses contract for xenotransplantation could allow the treating team
to enforce a commitment made prior to surgery. Therefore, should the
subject change their mind in the future regarding their previous
agreements, such as the right to withdraw from the trial or to inform
close contacts of the potential risks of their xenotransplantation,
there would be a binding contract enforceable by quarantine or
detention, thereby protecting society at large.^[78] From
a public health perspective, notification of close contacts and
caregivers about potential infectious risks surrounding a
xenotransplantation recipient could violate principles of
confidentiality. This raises questions regarding whether it is necessary
to obtain third party informed consent during patient selection for
transplant.^[78] Another
hurdle is that close contacts of xenotransplant recipients could be
expected to refrain from blood donation and agree to monitoring if this
becomes necessary. Enforcement of such rules could be deemed next to
impossible given that intimate contacts may change multiple times over a
person’s lifetime.^[78] Given
these difficult issues, societal input and governmental oversight
regarding the decision of whether a country will proceed with
xenotransplantation research are necessary.^[79] The
Department of Health and Human Services Secretary’s Advisory Committee
on Xenotransplantation suggested that raising public awareness about the
health concerns of xenotransplantation is the only adequate mechanism
to ensuring community-wide vigilance toward the potential health hazards
of xenosis.^[78]
Xenotourism

The
potential risks of xenotransplantation will not respect the geographic
borders of the country in which the procedure is undertaken. In the
absence of international regulations and monitoring procedures, the most
aggressive safety measures of any nation are likely to be unsuccessful.
This issue arises because of a constantly mobile population and the
wide use of intercontinental air travel, which can quickly spread an
infectious agent to geographically distant locations. The ethical
principle of justice requires all nations to bear responsibility
regarding the control of infectious disease risks. This problem is
highly complex and requires a globally respected international treaty
with a uniform immigration surveillance system to check for the entry of
potentially infectious pathogens.
Animal-related ethical issues

Xenotransplantation
might give rise to a variety of psychosocial problems pertaining to
emotional and personal identity issues associated with implantation of
organs from nonhuman animals. While not a reason to remove
xenotransplantation from consideration, these issues should be
thoroughly discussed with the potential recipient in advance. The
concept of rights for donor animals is controversial. Nonhuman primates
such as baboons have complex social behaviors, and various ethical
concerns exist regarding their use. The use of pigs is far less
controversial. In a response to the use of animal-derived xenogeneic
biologic meshes for soft tissue repairs, People for the Ethical
Treatment of Animals (PETA) stated that they were “opposed to the use of
animals and animal tissues for experimentation, medical training and
clinical treatments…including the use of biological meshes."^[76] Various
animal rights activists are opposed to the idea of xenotransplantation
because they maintain that humans do not have right to breed and use
other animals for their own needs. While these issues require
considerable debate, the accepted opinion is that animals used for
research or clinical xenotransplantation must be treated respectfully
and humanely, and they must not be used without institutional approval.
Religious views on xenotransplantation

Religion
plays a significant role in the everyday life of many individuals and
thus influences lifestyles, eating habits, and medical treatments. The 3
major monotheistic religions have certain aspects in common. All posit a
hierarchy in the order of creation, in which humanity has a unique
place.^[80] Furthermore, the Roman Catholic Church holds that humanity has a mandate to guide the life of creation toward the integral good.^[80] However,
both Judaism and Islam forbid the raising and consumption of pigs.
Nevertheless, using pig organs for xenotransplantation is not regarded
as eating pork. Moreover, both Judaism and Islam allow for exceptions to
dietary laws, particularly in situations in which a human life might be
saved.^{[76, 81]} Buddhism
regards organ donation as a matter that should be left to an
individual's conscience. A Hindu tenet is that the body must remain
whole in order to pass into the eternal life; therefore, transplantation
is not condoned. However, Hindu law does not prohibit individuals from
donating their organs or accepting an organ. With the exception of cows,
which are sacred in Hinduism, there are no prohibitions on using parts
of animals to alleviate human suffering. Interestingly, one of the
oldest mythological accounts of xenotransplantation is described in
Hinduism, wherein Lord Ganesha (Lord Shiva's son) received a xenograft
of an elephant head after Lord Shiva inadvertently severed his head.
Popular perception of xenotransplantation

In
light of the rapid advances in genetic engineering and immune
modulating therapies, the scientific and popular media have declared
xenotransplantation as becoming a clinical reality fraught with ethical
controversy. Television shows like "Frontline" and "60 Minutes" have
discussed many of the issues mentioned above. Similarly, the medical
literature reflects ethical debates. Scientists at Harvard have
questioned the ethical framework used to transplant pig islet cells into
Mexican children.^[82] Others
have interviewed patients to determine under which clinical
circumstances they would undergo xenotransplantation and identified a
"reluctance" to endorse the procedure.In 2008, controversial
issues surrounding xenotransplantation were explored at the Metropolitan
Museum of Modern Art by artist Elio Caccavale. In the Utility Pets
exhibit, the works My BioBoy and My BioPig depict a boy connected to a
pig by an umbilical cord and challenge the audience to contemplate
"transhuman" creatures. In the scenario, the boy harmoniously coexists
with the pig while waiting for the day of xenotransplantation. The art
work offers an alternative to the potentially uncomfortable idea of
organ farming and playfully displays inventions like The Smoke Eater
(which protects the pig from secondhand smoke) and The Toy Communicator
(which allows the boy and the pig to communicate when in different
rooms).^[83]
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Current Status and Future Directions

As
previously mentioned, xenotransplantation and xenotransplantation
products come under the regulatory authority of the FDA. In 1997, the
FDA formed the Xenotransplantation Subcommittee of the BRMAC as an
ongoing mechanism for open discussions of the scientific, medical,
social, ethical, and public health issues raised by xenotransplantation
and the specific ongoing and proposed protocols. The FDA has
developed a xenotransplantation action plan to provide a comprehensive
approach for the regulation of xenotransplantation that addresses the
potential public health and safety issues associated with
xenotransplantation and to provide guidance to sponsors, manufacturers,
and investigators regarding xenotransplantation product safety and
clinical trial design and monitoring. From time to time, the FDA
publishes guidance documents to assist sponsors and investigators
interested in conducting clinical trials in the field of
xenotransplantation. These documents provide reasonably detailed and
timely pragmatic guidance to sponsors regarding xenotransplantation
product safety and clinical trial development, including specific
recommendations for the procurement and screening qualification of
source animals, the manufacture and testing of xenotransplantation
products, preclinical testing, clinical trial design, and
posttransplantation monitoring and surveillance of recipients of
xenotransplantation products. The FDA provides notice of and invites
public comment on these draft documents. One such final guidance
document for the industry can be accessed on the Internet at http://www.fda.gov/cber/guidelines.htm.^[84] The
FDA has sponsored, planned, or participated in numerous open public
meetings and workshops (both domestic and international) that partially
or wholly focused on xenotransplantation. These activities are essential
for both sharing information and receiving public input on issues
relevant to xenotransplantation. Although clearly an experimental
procedure, investigators in clinical xenotransplantation have been
accused of using "the guise of bridge-to-transplantation" to appear
acceptable to institutional review or ethics boards.^[85] However,
the use of xenografts solely as bridges to allotransplantation does not
increase the donor pool; therefore, successful, permanent
xenotransplantation must itself be viewed as the target of future
clinical investigations.^[86] In
the future, clinical xenotransplantation may accomplish its intended
goal of achieving prolonged graft survival. Learning from the lessons of
allotransplantation, clinicians performing xenotransplantations must
persevere under justifiable scrutiny