Organ xenotransplantation

Xenotransplantation is the transplantation of cells, tissues, or organs occurring between species. This includes body fluids, cells, tissues or organs that have had ex vivo contact with the live cells, tissues or organs of a different species (FDA, 2006). In contrast, allotransplantation is the transplantation of cells, tissues, and organs between members of the same species.

Much scientific research is currently devoted to xenotransplantation driven by the worldwide shortage of organs available for transplantation. In the United States alone, roughly ten patients die each day while on the organ transplant waiting list (FDA, 2006). Advances in molecular techniques and immunosuppressants, which help make this prospect possible, have also increased interest in xenotransplantation.

As in most transplants, a variety of immune responses against the xenograft occur, in an attempt to reject the foreign organ. The strength of these reactions depends on how closely related the donor and recipient species are to each other. In phylogenetically close, or concordant combinations (for example nonhuman primates and humans) rejection occurs within several days, at a pace similar to that observed in allotransplantations. With discordant combinations (such as from pig to human), the two species are not closely related. A rapid, violent hyperacute response results due to preformed natural antibodies, known as XNAs (Dooldeniya and Warrens, 2003). Although xenotransplantation appears to present a solution to the organ shortage problem, by theoretically providing an almost endless supply of organs for transplantation, many issues currently stand in the way of allowing it to enter clinical use.

Potential animal organ donors
Since they are the closest relatives to humans, nonhuman primates were first considered as a potential organ source for xenotransplantation to humans. Chimpanzees were originally considered to be the best option since their organs are of similar size, and they have good blood type compatibility with humans. However, since chimpanzees are listed as an endangered species, other potential donors were sought out. Baboons are more readily available, however they are also not practical as potential donors. Problems include their smaller body size, the infrequency of blood group O (the universal donor), their long gestation period, and they typically produce few offspring. In addition, a major problem with the use of nonhuman primates is the increased risk of disease transmission, since they are so closely related to humans (Michler, 1996). Pigs are currently thought to be the best candidates for organ donation. The risk of cross-species disease transmission is decreased because of their increased phylogenetic distance from humans (Dooldeniya and Warrens, 2003). They are readily available, their organs are anatomically comparable in size, and new infectious agents are less likely since they have been in close contact with humans through domestication for many years (Taylor, 2007). Current experiments in xenotransplantation most often use pigs as the donor, and baboons as human models.

Immunologic Barriers
To date no xenotransplantation trials have been entirely successful due to the many obstacles arising from the response of the recipient’s immune system. This response, which is generally more extreme than in allotransplantations, ultimately results in rejection of the xenograft. There are several types of rejection organ xenografts are faced with, these include:

• Hyperacute rejection

• Acute vascular rejection

• Cellular rejection

• Chronic rejection

Hyperacute Rejection
This rapid and violent type of rejection occurs within minutes to hours from the time of the transplant. It is mediated by the binding of XNAs (xenoreactive natural antibodies) to the donor endothelium, causing activation of the human complement system which results in endothelial damage, inflammation, thrombosis and necrosis of the transplant. XNAs are first produced and begin circulating in the blood in neonates, after colonization of the bowel by bacteria which have galactose moieties on their cell walls. Most of these antibodies are the IgM class, but also include IgG, and IgA. (Taylor, 2007). The epitope XNAs target is an α-linked galactose moiety, Gal-α-1,3Gal (also called the α-Gal epitope), produced by the enzyme α-galactosyl transferase. (Candinas and Adams, 2000). Most non-primates contain this enzyme thus, this epitope is present on the organ epithelium and is perceived as a foreign antigen by primates, which lack the galactosyl transferase enzyme. In pig to primate xenotransplantation, XNAs recognize porcine glycoproteins of the integrin family (Taylor, 2007). The binding of XNAs initiate complement activation through the classical pathway. Complement activation causes a cascade of events leading to: destruction of endothelial cells, platlet degranulation, inflammation, coagulation, fibrin deposition, and hemorrhage. The end result is thrombosis and necrosis of the xenograft (Taylor, 2007).

Overcoming Hyperacute rejection
Since hyperacute rejection presents such a barrier to the success of xenografts several strategies to overcome it are under investigation:

Interruption of the complement cascade

• The recipient's complement cascade can be inhibited through the use of cobra venom factor (which depletes C3), soluble complement receptor type 1, anti-C5 antibodies, or C1 inhibitor (C1-INH). Disadvantages of this approach include the toxicity of cobra venom factor, and most importantly these treatments would deprive the individual of a functional complement system (Dooldeniya and Warrens, 2003).

Transgeneic organs (Genetically engineered pigs)

•1,3 galactosyl transferase gene knockouts - These pigs don’t contain the gene which codes for the enzyme responsible for expression of the immunogeneic gal-α-1,3Gal moiety (the α-Gal epitope) (LaTemple and Galili, 1998).

•Increased expression of H-transferase (α 1,2 fucosyltransferase), an enzyme that competes with galactosyl transferase. Experiments have shown this reduces α-Gal expression by 70% (Sharma et al., 1996).

•Expression of human complement regulators (CD55, CD46, and CD59) to inhibit the complement cascade (Huang et al., 2001).

Acute Vascular Rejection
Also known as delayed xenoactive rejection, this type of rejection occurs in discordant xenografts within 2 to 3 days, if hyperacute rejection is prevented. The process is much more complex than hyperacute rejection and is currently not completely understood. Acute vascular rejection requires de novo protein synthesis and is driven by interactions between the graft endothelial cells and host antibodies, macrophages, and platelets. The response is characterized by an inflammatory infiltrate of mostly macrophages and natural killer cells (with small numbers of T cells), intravascular thrombosis, and fibrinoid necrosis of vessel walls (Candinas and Adams, 2000). Binding of the previously mentioned XNAs to the donor endothelium leads to the activation of host macrophages as well as the endothelium itself. The endothelium activation is considered type II since gene induction and protein synthesis are involved. The binding of XNAs ultimately leads to the development of a procoagulant state, the secretion of inflammatory cytokines and chemokines, as well as expression of leukocyte adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) (Taylor, 2007). This response is further perpetuated as normally binding between regulatory proteins and their ligands aid in the control of coagulation and inflammatory responses. However, due to molecular incompatibilities between the molecules of the donor species and recipient (such as porcine major histocompatibility complex molecules and human natural killer cells), this may not occur (Candinas and Adams, 2000).

Overcoming Acute Vascular Rejection
Due to its complexity, with the use of immunosuppresive drugs along with a wide array of approaches are necessary to prevent acute vascular rejection, and include:

• Administering a synthetic thrombin inhibitor to modulate thrombogenesis

• Depletion of anti-galactose antibodies (XNAs) by techniques such as immunoadsorption, to prevent endothelial cell activation

• Inhibiting activation of macrophages (stimulated by CD4+ T cells) and NK cells (stimulated by the release of Il-2). Thus, the role of MHC molecules and T cell responses in activation would have to be reassessed for each species combo (Candinas and Adams, 2000).

Accommodation
If hyperacute and acute vascular rejection are avoided accommodation is possible, which is the survival of the xenograft despite the presence of circulating XNAs. The graft is given a break from humoral rejection (Takahashi et al., 1997) when the complement cascade is interrupted, circulating antibodies are removed, or their function is changed, or there is a change in the expression of surface antigens on the graft. This allows the xenograft to up-regulate and express protective genes, which aid in resistance to injury, such as heme oxygenase-1 (an enzyme that catalyzes the degradation of heme) (Taylor, 2007).

Cellular rejection
Rejection of the xenograft in hyperactute and acute vascular rejection is due to the response of the humoral immune system, since the response is elicited by the XNAs. Cellular rejection is based on cellular immunity, and is mediated by:

1. Natural killer cells, which accumulate in and damage the xenograft; and

2. T-lymphocytes - which are activated by MHC molecules through both direct and indirect xenorecognition.

In direct xenorecognition, antigen presenting cells from the xenograft present peptides to recipient CD4+ T cells via xenogeneic MHC class II molecules, resulting in the production of interleukin 2 (IL-2). Indirect xenorecognition involves the presentation of antigens from the xenograft by recipient antigen presenting cells to CD4+ T cells. Antigens of phagocytosed graft cells can also be presented by the host’s class I MHC molecules to CD8+ T cells. (Dooldeniya and Warrens, 2003; Abbas and Lichtman, 2005). The strength of cellular rejection in xenografts remains uncertain, however it is expected to be stronger than in allografts due to differences in peptides among different animals. This leads to more antigens potentially recognized as foreign, thus eliciting a greater indirect xenogenic response (Dooldeniya and Warrens, 2003).

Overcoming Cellular rejection
A proposed strategy to avoid cellular rejection is to induce donor non-responsiveness using haematopoietic chimerism. Donor stem cells are introduced into the bone marrow of the recipient, where they coexist with the recipient’s stem cells. The bone marrow stem cells give rise to cells of all haematopoietic lineages, through the process of hematopoiesis. Lymphoid progenitor cells are created by this process and move to the thymus where negative selection eliminates T cells found to be reactive to self. The existence of donor stem cells in the recipient’s bone marrow causes donor reactive T cells to be considered self and undergo apoptosis (Dooldeniya and Warrens, 2003).

Chronic rejection
This final type of rejection is slow and progressive, and is usually described in transplants which survive the initial rejection phases. Scientists are still unclear how chronic rejection exactly works, research in this area is difficult since xenografts rarely survive past the initial acute rejection phases. Nonetheless, it is known is that XNAs and the complement system are not primarily involved (Candinas and Adams, 2000). Fibrosis in the xenograft occurs as a result of immune reactions, cytokines (which stimulate fibroblasts), or healing (following cellular necrosis in acute rejection). Perhaps the major cause of chronic rejection is arteriosclerosis. Lymphocytes, which were previously activated by antigens in the vessel wall of the graft, activate macrophages to secrete smooth muscle growth factors. This results in a build up of smooth muscle cells on the vessel walls, causing the hardening and narrowing of vessels within the graft. Chronic rejection leads to pathologic changes of the organ, and is why transplants must be replaced after so many years (Abbas and Lichtman, 2005). It is also anticipated that chronic rejection will be more aggressive in xenotransplants as opposed to allotransplants (Vanderpool, 1999).

Physiology
Extensive research is required to determine whether animal organs can replace the physiological functions of human organs. Many issues include:

• Size - Differences in organ size limit the range of potential recipients of xenotransplants.

• Longevity - The lifespan of most pigs is roughly 15 years, currently it is unknown whether or not a xenograft may be able to last longer than that.

• Hormone and protein differences - Some proteins will be molecularly incompatible, which could cause malfunction of important regulatory processes. These differences also make the prospect of hepatic xenotransplantation less promising, since the liver plays an important role in the production of so many proteins (Dooldeniya and Warrens, 2003).

• Environment - For example, pig hearts work in a different anatomical site and under different hydrostatic pressure than in humans (Candinas and Adams, 2000).

• Temperature - The body temperature of pigs is 39°C (2°C above the average human body temperature). Implications of this difference, if any, on the activity of important enzymes are currently unknown. (Dooldeniya and Warrens, 2003).

Xenosis
The term xenosis was coined to describe the transmission of infectious agents between species (also known as zoonosis) via a xenograft. Animal to human infection is normally rare, but has occurred in the past. An example of such is the avian influenza, when an influenza A virus was passed from chickens to humans (WHO committee, 2005). Xenotransplantation may increase the chance of disease transmission for 3 reasons: 1. Implantation breaches the physical barrier that normally helps to prevent disease transmission, 2. The recipient of the transplant will be severely immunosuppressed; and 3. Human complement regulators (CD46, CD55, and CD59) expressed in transgenic pigs have been shown to serve as virus receptors, and may also help to protect viruses from attack by the compliment system (Takeuchi and Weiss, 2000).

Examples of viruses carried by pigs include porcine herpesvirus, rotavirus, parvovirus, and circovirus. Porcine herpesviruses and rotaviruses can be eliminated from the donor pool by screening, however others (such as parvovirus and circovirus) may contaminate food and footwear then re-infect the herd. Thus, pigs to be used as organ donors will have to be housed under strict regulations and screened regularly for microbes and pathogens. Unknown viruses, as well as those which aren’t harmful in the animal, may also pose risks (Takeuchi and Weiss, 2000). Of particular concern are PERVS (porcine endogenous retroviruses), vertically transmitted microbes which are imbedded in swine genomes. The risks with xenosis are twofold as not only could the individual become infected, but a novel infection could initiate an epidemic in the human population. Because of this risk, the FDA has suggested any recipients of xenotransplants shall be closely monitored for the remainder of their life, and quarantined if they show signs of xenosis (FDA, 2006).

Porcine endogenous retroviruses
Endogenous retroviruses are remnants of ancient viral infections, found in the genomes of most, if not all, mammalian species. Integrated into the chromosomal DNA, they are vertically transferred through inheritance (Vanderpool, 1999). Due to the many deletions and mutations they accumulate over time, they usually are not infectious in the host species, however the virus may become infectious in another species (Taylor, 2007). PERVS were originally discovered as retrovirus particles released from cultured porcine kidney cells (Armstrong et al., 1971). Most breeds of swine harbor approximately 50 PERV genomes in their DNA (Patience et al., 1997). Although it is likely that most of these are defective, some may be able to produce infectious viruses so every proviral genome must be sequenced to identify which ones pose a threat. In addition, through complementation and genetic recombination, two defective PERV genomes could give rise to an infectious virus (Rogel-Gaillard et al, 1999). There are three subgroups of infectious PERVs (PERV-A, PERV-B, and PERV-C). Experiments have shown that PERV-A and PERV-B can infect human cells in culture (Patience et al., 1997; Takeuchi et al, 1998). To date no experimental xenotransplantations have demonstrated PERV transmission, yet this does not mean PERV infections in humans are impossible (Takeuchi and Weiss, 2000).

Ethics
In addition to the infectious disease risks posed by xenotransplants, there are several other ethical issues with xenotransplantation that require consideration:

• The laboratory use of pigs, baboons and other animals

• Genetic alterations of animals

• Religious and individual beliefs

• Informed consent complexities for research subjects, as well as the selection of human subjects

• Public education (as many companies may go ahead with experiments without public awareness) (Vanderpool, 1999).