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• INTRODUCTION
• MAJOR OBSTACLES IN XENOTRANSPLANTATION
• Immunological
• Hyper Acute Rejection and Anti-Gal antibodies
• Anti-nonGal antibodies
• Coagulationand Anticoagulation system
• Microbiological
• TRANSGENIC TECHNOLOGY AND HISTORICAL PERSPECTIVE
• Transgenesis by Viral transfection
• Sperm-mediated DNA transfer
• Transgenesis by embryonic stem cells
• Transgenesis by pronuclear injection
• Transgenesis by SCNT
• SUS SCROFAGENOME MODIFICATION
• CONSIDERATIONS FOR ANIMAL MODELS FOR HEART TRANSPLANTATION
• SURGICAL CONSIDERATIONS
• FUTURE DIRECTIONS
• CONCLUSION

INTRODUCTION

 The first successful human-to-human heart transplantation in the human history was done in the December, 1967 by Christian Barnard. However, in Korea, the first human-to-human heart transplantation was done in the November 11^th 1992. And since then almost 227 heart transplantations have been reported to be performed uptill January 2004. Recently, due to acute shortage in the donated human organs, patients have been waiting in the list to receive the organs for transplantation for more than 4-5 years. However, Mulligan et al. (2008) have recently reported that the mortality of patients awaiting heart alloTx has declined over the past 10 years. These declines owe to the increasing usage of ventricular assist devices which helps the patients in sustaining the condition. Although mechanical devices have proven valuable in the treatment of heart failure, the insertion of a foreign body is not ideal, as the device is susceptible to infection and leading to complications such as thrombo-embolism (Tseng et al., 2005). Therefore, it was assumed and realized that only a readily available organs from animal source only could resolve the increasing discrepancy between the availability of donated human organs and the demand for xeno-transplantation.

 Transgenic pigs are promising donor organisms for xenotransplantation (transfer of organs from genetically different animals of different species) as they share many anatomical and physiological characteristics with humans. The most profound barrier in pig-to-primate xenotransplantation is the rejection of the grafted organ by a cascade of immune mechanisms commonly referred to as hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), immune cell-mediated rejection and chronic rejection. Various strategies for the genetic modification of pigs facilitate tailoring them to be donors for organ transplantation. Genetically modified pigs lacking α-1,3- Gal epitopes, the major xenoantigens triggering HAR of pig-to-primate xenografts, are considered to be the basis for further genetic modifications that can address other rejection mechanisms and incompatibilities between the porcine and primate blood coagulation systems (Chen et al., 2005).

 In view of the steadily improving results of heart Tx in the pig-to nonhuman primate model (Struber et al., 2007), particularly of hearts from pigs homozygous for α1,3- galactosyltransferase gene-knockout (GT-KO), where graft survival has reached almost 6 months, cardiac xenoTx is likely to be a valid option for the treatment of end-stage heart failure. Although the introduction of geneticallymodified pigs for xenoTx has increased the resistance of the organs to the xenoreactive immune response, there remain immunological and other barriers that currently prevent the clinical application of xenoTx.

 This update provides an overview of the transgenic approaches that have been used to generate donor pigs and also discuss in detail immunological hurdles for xenotransplantation.

MAJOR OBSTACLES IN XENOTRANSPLANTATION

Immunological

Hyper Acute Rejection and Anti-Gal antibodies

 Transplantation of an unmodified pig heart into a non-immunosuppressed (or standard pharmacologicallyimmunosuppressed) human or higher non-human primate results in destruction of the graft within minutes or hours by a process known as hyperacute rejection (HAR). Rejection of xenografts entails participation of the innate immune system with natural antibody-producing B cells, natural killer (NK) cells, macrophages, and complement, as well as adaptive immune responses from T and B cells (Fig. 1). In addition, it is becoming increasingly clear that physiological incompatibilities, such as incompatibilities between human and pig coagulation pathways, lead to xenograft injury and contribute significantly to rejection. The established nomenclature of the different types of xenograft rejection includes HAR, AVR (acute vascular rejection) and its major component acute humoral xenograft rejection (AHXR), and chronic rejection, and refers to the time course and predominant mechanism of rejection. HAR develops within 24 h and it results from the binding of pre-existing xenoreactive antibodies to endothelial cells and activation of the complement system, resulting in vascular deposition of immunoglobulins and complement, endothelial swelling, and microvascular thrombosis (Schuurman et al., 2003). In HAR, the recognition of pig antigens, predominantly Galα-1,3-Gal (Gal), by primate preformed (natural) antibodies leads to complement activation, resulting in extensive intravascular coagulation and thrombosis, endothelial injury, interstitial hemorrhage and edema, and infiltration of polymorphonuclear leukocytes into the tissues (Shimizu and Yamada, 2006). The relatively recent introduction of GT-KO pigs (Kolber-Simonds et al., 2004), that do not express the major antigenic target for primate anti-pig antibodies (Gal), has brought clinical xenoTx one step closer by avoiding HAR (Tseng et al., 2005). GT-KO hearts transplanted heterotopically into immunosuppressed baboons have survived for up to 6 months (Tseng et al., 2005). Graft failure did not exhibit the typical features of humoral rejection caused byantibody-mediated complement activation, but was due to the development of a thrombotic microangiopathy that resulted in vascular occlusion and surrounding ischemic injury.

Fig. 1. Principal mechanisms involved in rejection of porcine xenografts in nonhuman primate. HAR is initiated when preexisting ‘natural’xenoantibodies, which are primarily directed toward α Gal, bind to donor endothelium. Endothelial cells are activated, complement and coagulation systems are involved, and endothelial damage and thrombosis occur.

 The key obstacle to using pig organs and cells for transplantation in humans has been the strong immune response elicited by porcine antigens. The most immunogenic epitope, the gal-epitope, is generated by the enzyme α-1,3 galactosyl transferase (Sandrin et al., 1994). This epitope is responsible for the hyperacute rejection phenomena. The epitope isexpressed on the tissues of all mammals except humans and subhuman primates, which have antibodies against the epitope. If a pig kidney is perfused with human blood, the preformed antibodies react with the gal-epitope. This antibody-antigen reaction elicits an activation of the complement and clotting systems, with subsequent injury to the vascular endothelium and intravascular clotting, a chain of events that results in hyperacute rejection.

 In the early 1990s, pigs that were transgenic for a human complement inhibitor, h-DAF, were developed therefore due to this the hyperacute rejection was usually avoided when the kidneys and hearts from such pigs were transplanted into primates. If the primates were treated with immunosuppressive agents in high doses, then the pig organs could, in some animals, function for several weeks or even months (Cozzi et al., 2003). However, many of the pig organs were lost early after transplantation due to vascular rejection, while many of the primate recipients died from adverse reactions related to the immunosuppressive treatment. It was concluded that the transgenic pigs constituted a significant advance regarding the avoidance of hyperacute rejection; however, the technology was not ready for clinical trials. Moreover, there was also the potential risk of transmitting porcine endogens retroviruses. Due tothese reasons, interest in xenotransplantation research abated in the late 1990s.

 Recently, however, research groups in Boston and Pittsburg succeeded in cloning pigs and in this context, they able to eliminate the gene that encodes α-galactocyltransferase. These "gal-knockout pigs" do not express the gal-epitope. When kidneys and hearts from such pigs were transplanted into baboons, hyperacute rejection was not encountered and in some immunosuppressed recipients the pig organs survived and functioned for a few months. However, long-term function was prevented by graft rejection, histological examinations revealing thrombotic microangiopathy (Chen et al., 2005). The next step is to combine the two technologies and currently laboratories in the USA and Australia are aiming at making gal-knock out pigs transgenic for h-DAF and for a human gene encoding for an inhibitor of coagulation, (h-TFPI), thereby hopefully alleviating the thrombotic microangiopathy (Table. 1)

Table 1. Different approach to overcome xenograft rejection

Anti-nonGal antibodies

 These studies highlighted the remaining major immunologic problems that need to be overcome (Cooper et al., 2008). Although the GT-KO pig organs overcame the presence of anti-Gal antibodies in the nonhuman primates, and thus prevented HAR, there are clearly antibodies directed toward non-Gal targets that can result in early humoral rejection (Rood et al., 2007; Ezzelarab et al., 2006). The exact targets for these anti-non-Gal antibodies remain uncertain (Ezzelarab et al., 2005). Asthe thrombotic microangiopathy seen in the GT-KO pig-to baboon experiments (Houser et al., 2004) that may be the form of delayed antibody-mediated rejection. The focal deposition of IgM and IgG and C4d on the grafted tissue suggests that a component of this process may involve the binding of anti-non-Gal antibodies to the pig vascular endothelial cells (Ezzelarab et al., 2005). This may be secondary to vascular endothelial cell activation from anti-non-Gal antibodies and complement. GT-KO pigs transgenic for one or more human complement-regulatory proteins (CRP), such as CD46 (membrane cofactor protein, MCP) or CD55 (decay-accelerating factor, DAF) and may inhibit the development of this complication (Cooper et al., 2008).

Coagulationand Anticoagulation system

 Incompatibilities between the coagulation anti-coagulation systems of pig and primate that may predispose to the development of thrombosis in the transplanted pig vessels has been observed. This dysfunction might be overcome by the introduction into the pig of a human ‘anticoagulant’ or ‘anti-thrombotic’ gene (Cowan et al., 2008), such as tissue factor pathway inhibitor, hirudin or CD39 (Dwyer et al., 2004). Wu et al. (2007) studied the coagulation cascade in the pathogenesis of HAR and in the early failure of pig hearts transgenic for a human CRP in the pig-to-baboon heterotopic heart Tx model. The need for genetically-modified pigs is recognized as being essential, as is the need for improved immunosuppressive regimens, possibly with the addition of anticoagulant or antithrombotic medication, such as acetylsalicylic acid [aspirin], clopidogrel bisulfate [plavix], enoxiparin sodium  [lovenox], warfarin [coumadin], or heparin. Advances in immunosuppressive medication, such as co-stimulatory blockade, have allowed prevention of a T cell-dependent elicited antibody response (Zhu et al., 2007). However, Byrne et al. (2006) have recently demonstrated that significant prolongation of xenograft survival after cardiac transplantation in a pig-to-nonhuman primate model can be achieved by improved immunosuppression, rather than through an increase in anticoagulation (Wu et al., 2007).

Microbiological

 With xenotransplantation, there is a risk of transmitting infectious agents from animal to man. With regard to most microbiological agents, the risk can be minimized by using animals from strictly controlled herds. However, such measures will not affect the porcine endogenous retrovirus (PERV). These viruses are a permanent part of the genome in all mammalian species (Blusch et al., 2002) and so all recipients of porcine transplant will inevitably be exposed to PERV. However, the endogenous retroviruses do not replicate or cause disease under physiological conditions. However, in the late 1990s it was found that when pig cells were co-cultured with human cells, the transmission of PERV could occur  (Patience et al., 1997). Furthermore, the transmission of PERV was found when immune- incompetent mice were injected with porcine cells (Vander Laan et al., 2000). The question was then raised whether the transplantation of porcine tissue into humans might transmit PERV and if the dormant virus in this context might recombine or otherwise be activated in the new environment and thus become pathogenic. If so, the recipient would become infected, and mighthis/her relatives and other people attending to the patient. In a worst case scenario, the activated viruses would cause an epidemic. The transplantation of organs or cells from pig to man would then be a highly hazardous under taking. In the meantime, a number of new technologies for the monitoring of PERV were developed and the application of these tools greatly increased the understanding of the behavior of the virus (Blusch et al., 2002).

TRANSGENIC TECHNOLOGY AND HISTORICAL PERSPECTIVE

Transgenesis by Viral transfection

 The first successfully reported foreign DNA transfer to a mammal with germ-line transmission was obtained by using a retrovirus (Jaenisch et al., 1975). Retrovirus is an RNA virus that can infect mammals, causing several diseases (like herpes, cancers and immunodeficiency syndromes). During infection the virus fuses with the cell membrane and when the viral RNA is in the cytoplasm, it is converted into a DNA molecule that integrates into the host-cell genome. Therefore the retrovirus is an efficient transgene-delivery vehicle and it has been used to infect the bovine embryo when injected into the perivitelline space, between the surface of the fertilized egg and the zona pellucid (Chan et al., 1998). However, DNA transfer by a retrovirus has some limitations, such as: (1) preferential integration of retroviruses into dividing cells; (2) recognition of specific target cells; and (3) high probability of chimeric animal production, which is incapable of transferring the transgene to the next generation. Actually, studies are continuously been going on in this aspect to overcome the limitation of retrovirus and also for lentivirus to which some infectious diseases are generally been associated such equine infectious anemia virus (EIAV) and the immunodeficiency viruses of cattle and man (BIV and HIV, respectively) that is still being considered as the biggest obstacles in the production of transgenic livestock by this technology (Clark and Whitelaw, 2003).

Sperm-mediated DNA transfer

 The sperm-mediated DNA transfer in rabbits, during in vitro fertilization, was the pioneer experiment to produce transgenic animals. However, the generated animals were generally mosaic for the transgenes, so the genes were not always expressed in the second generation. The transformed sperm have been used for in vitro fertilization (Maione et al.,1998). Nevertheless this technique has a limited capability to incorporate the DNA into the host genome. In order to improve the DNA incorporation it is possible to expose the sperm to electrical field (electroporation), which has brought good results in cattle (Rieth et al., 2000). An alternative technique of sperm-mediated DNA transfer is through male germinal stem (GS) cell transfection and transplantation into the recipient seminiferous tubules (Nagano et al., 2001). This technique was first utilized in mice and recently has been successfully introduced in goats and pigs (Honaramooz et al., 2002). Almost 5% of the male progeny from mice transplanted with transformed GS cells were transgenic and transmitted the gene to subsequent generations (Nagano et al., 2001), making this methodology very promising.

Transgenesis by embryonic stem cells

 The production of transgenic animals by embryonic stem (ES) cells, generates chimeric embryos composed of
 2 distinct cell lines, and one of them carries the desired gene(s). ES cell lines are pluripotent cells derived from the inner cell mass of the blastocysts, and are capable of producing all tissues of an individual, including the germinative tissue. This gene transfer method involves the injection of transgenic ES cells into expanded blastocysts, and was first utilized in mice. This method is feasible in all animals in which ES cells can be manipulated and transfected in vitro. Mice were the first mammals from which ES cells were isolated and cultured (Martin,1981). Some of their advantages for the production of transgenic animals include the fact that ES cell lines can be targeted through homologous DNA recombination, which can be screened and selected for the incorporation of the foreign DNA before the transgenic embryo production. Moreover, the site of transgenic integration in the genome can be controlled to replace existing genes. Hence human gene function can be studied by knockout or knockin of candidate genes (Capecchi, 1989). One of the limitations ofthis technique to produce transgenic livestock is that chimeric products must be crossbred to carry the transgene in all tissues, which can take a long time in animals like cattle. Additionally, the isolation and establishment of the livestock ES cell culture in vitro are still in fancy in some species, like swine and cattle, and further studies are warranted to fully establish the proper in vitro manipulation of ES cells from such species (Saito et al., 2003).

Transgenesis by pronuclear injection

 Another method for introducing foreign genes into animals is by direct pronuclear injection, where the gene of interest is directly injected into one of the pronuclei of a zygote. This technique was first developed in mice (Palmiter et al., 1982) and is still the main choice in production of transgenic rodents. Subsequently, the pronuclear injection was responsible for the first successful attempt to produce transgenic livestock species (including rabbits, sheep and swine) with the rat and human growth hormone gene (Hammer et al.,1985), as well as the first transgenic bovine (Krimpenfort et al.,1991). However, this technique has serious limitations, such as: (1) impossibility to produce knockouts by homologous recombination; (2) inefficiency in generating embryos in which the injected DNA was stably integrated into the host genome (Nottle et al. 2001); and (3) production of chimeric transgenic embryos resulting in a mosaic animal with some cells containing the transgene and others not (Keefer, 2004); (4) unpredictability of the site of transgene integration in the host genome and the resulting variation in transgene expression because of the position effect (Clark et al., 1994). Due to the low efficiency of this technique, around 1000 bovine, and 300 ovine, and 200 goat zygotes must be microinjected to produce one founder transgenic animal (Seidel, 1993), so this increases the costs of producing large transgenic animals.

Transgenesis by SCNT

 Recently, the first successful nuclear transfer (NT) using somaticcells from the mammary gland has lead to the birth of the famed clone Dolly (Wilmut et al., 1997). In this method, DNA from the MII oocytes is removed (enucleation), leaving only the cytoplasm (the cytoplast). Following enucleation a donor nucleus, which can be almost any cell of the body, is injected into the perivitelline space and fused to the cytoplast by electrofusion. After fusion, the zygote clone is activated by either chemical or mechanical stimulation in order to initiate embryo development (Wilmut et al.,1997). This process is known as cloning, since a series of identical individuals can be generated from a single DNA donor cell line (Fig. 2). The great impact of NT on transgenic livestock production is due to the possibility to cultivate primary cells in vitro during a long lifespan without loss of viability (Kasinathan et al., 2001), and to execute the genetic manipulation before NT in order to produce transgenic embryos with the same rate of non-transgenic ones (Iguma et al., 2005). However, a good cell transfection system and the isolation of a transgenic cell line derived from a unique transfection event (clonal origin) must be efficiently produced before actual transfer of the nucleus (Melo et al., 2005). A variety of methods for transfection into mammalian cells have been reported such as microinjection, particle bombardment, calcium phosphate, viral infection, and liposomes (Caplen et al., 1995). However, the easy cell manipulation and the uniform transfection efficiency make the cationic liposome-mediated gene transfer the most appropriate method for many livestock species (Oliveira et al., 2005). A great advantage of producing transgenic animals by NT, in comparison to pronuclear microinjection, is the possibility of gene targeting through homologous recombination between the exogenous and the host DNA producing knockouts and knockins (McCreath et al., 2000). Although being a recent technique, with low efficiency, the NT associated with transgenesis enables the production of transgenic embryos without chimeric hazards. Then, it is expected that all generated embryos will be transgenic, reducing the time and the cost of transgenic animal production, compared to other methods (Melo et al., 2005).

Fig. 2. Applications of transposition to pig transgenesis.

SUS SCROFAGENOME MODIFICATION

 With the emergence of technologies for animal transgenesis and genetic engineering, scientists have also sought to improve the performance or change the phenotype of pigs based on directed genetic modification. So far pigs were considered as the primary animals for studying enhanced growth and nutrient partitioning, pork composition, pig resistance to pathogens, and impact of environment on pig waste (Golovan et al., 2001). Researchers have further utilized these animalsand turned them into bio-reactors for the pharmaceutical production of therapeutic proteins in milk, blood, urine and semen (Lee et al., 2005).

 Xenotransplantation i.e. the transplantation of cells, tissue, and organs from one species to another-may bethe most important application of pig genetic engineering. Targets for the genetic modification of pigs for xenotransplantation have thus far emphasized reducingthe immunogenicity of pig cells and tissues, and preventing the hyperacute rejection (HAR) andacute vascular rejection responses that are observed within minutes and days, respectively, after transplantation of pig organs to nonhuman primates (NHPs). HAR of porcine organs by old world primate recipients is mediated through preformed antibodies against galactosyl-α-1,3-galactose epitopes expressed on the surface of pig cells. Antigen recognition leads to complement activation and assembly of membrane attack complexes on the surface of donor tissue endothelium, causing cell lysis, hemorrhage, and clotting that occludes the donor tissue blood supply. Transgenic pigs have been developed that express regulators of the complement cascade, including CD55 (decay accelerating factor), CD59, and CD46 (membrane co-factor protein), which are intended to suppress the assembly of membrane attack complexes on donor tissues (Houdebine, 2005). Xenogenic transplants of organs from these pigs into NHPs have indeed exhibited significant improvement in terms of controlling HAR. A complementary approach has focused on eliminating the galactosyl-α-1,3-galactose antigen from the surface of donor cells. Some research groups have generated pigs without the gene encoding α-1,3-galactosyltransferase, which is the enzyme that is required for this sugar modification (Zhong, 2007). This was accomplished by the serial ‘knockout’of the gene in cultured pig fibroblasts, followed by somatic cell nuclear transfer (SCNT) to generate pigs. This revolutionary accomplishment marks the beginning of a new era in pig genetic engineering, providing a path to the generation of pigs based on both gene supplementation and ablation. Pig cells are also a promising resource to counter the limited supply of human tissues for cell-based therapy, particularlyneurologic disorders and diabetes. Recent clinical and preclinical trials of islet cell transplantation and xenotransplantation, respectively, suggested that xenogeneic cellular therapy may indeed provide a viable option for the treatment of diabetes. Serendipitously, adult pig islets do not express the galactosyl-α-1,3-galactose epitope. Instead, rejection (Rayat et al., 2003) of xenogeneic islets in NHPs results from direct or indirect activation of T cells by donor pig xenopeptides. Targeted prevention of T cell co-stimulation has led to great strides in pig islet xenotransplantation to NHPs (Hering et al., 2006). However, maintenance of immunosuppression puts patients at risk for opportunistic infections, and can cause significant cardiovascular, renal, hematologic, gastrointestinal, and (in female patients) reproductive toxicity (Rother and Harlan, 2004).

 Pig transgenesis could provide an alternative approach to systemic T-cell co-stimulation blockade, instead relying on the local provision of immunotherapeutic proteins by the xenograft (Sutherland et al., 2000). Prevention of zoonotic transmission of pathogens from donor pigs to patients is also crucial for clinical application of porcine xenotransplantation.

CONSIDERATIONS FOR ANIMAL MODELS FOR HEART TRANSPLANTATION

 Kinetics of anti-pig and anti-Gal IgM and IgG antibodies after perfusing human blood containing GAS914, a Gal trisaccharide conjugated to poly-L-lysine, through hDAF pig hearts using a working ex vivo model has been demonstrated by Brandl et al. (2007). Their study showed improved survival and function of cardiac xenograft. When hDAF pig hearts were perfused with human blood containing GAS914, there was an immediate and extensive reduction in both anti-Gal IgM and IgG. Their study indicated that, by causing an immediate and profound reduction in Gal-specific antibodies, soluble Gal conjugates not only prolonged pig graft survival, but also improved the hemodynamic performance of the heart of hDAF pigs. Charniot et al. (2007) perfused seven small pig hearts with human blood using a Langendorff blood perfusion model. Reactive oxygen species were generated, probably promoting arrhythmias and impairment of left ventricular pressure. This group concluded that xenoTx was associated with a significant increase in ischemic injury and oxidative stress, factors that might play a role in the development of HAR. Smolenski et al. (2007) perfused hearts from five hDAF transgenic pigs (generated by spermmediated gene transfer) ex vivo with human blood. The hearts were protected from HAR, were relatively metabolically stable, and maintained mechanical function above the threshold level for life-support.

 Porter et al. (2007) have recently studied CD4+ CD25+ regulatory T cells (Treg) in baboons to see whether Treg can modulate the xenogeneic immune response. The characterization of baboon Treg will be beneficial in experiments relating to tolerance induction. Treg were isolated from baboon lymph nodes, spleens, and blood. Porcine antigen-specific baboon CD4+ CD25+ high cells were purified and expanded in vitro, and their effect on the baboon antipig xenogeneic response was studied. Baboon Treg suppressed the response to xenogeneic stimulation. This study suggests that adoptive transfer of expanded Treg into xenotransplant recipients may prevent cell-mediated rejection of grafts and potentially induce tolerance in the pig-to-baboon xenoTx preclinical model.

 Accommodation occurs in ABO-incompatible organ alloTx and in rodent models of xenoTx, but has not yet been conclusively described in large animal models of xenoTx. In accommodation, a graft becomes resistant to destruction despite the presence of specific antidonor antibodies and normal levels of complement (Holgersson, 2007). An understanding of the mechanism of accommodation might provide clues to potential therapeutic manipulations of the donor or recipient that could improve the survival of xenografts. Recently, Komori et al. (2008)have shown that host accommodation could be more important than graft accommodation. The study was performed in a hamster-to-rat cardiac Tx model. Accommodated grafts expressing protective genes were rejected with an increase of both IgM and B-1 cells. However, in accommodated hosts, both IgM and B-1 cells decreased. Their results suggest that sufficient suppression of recipient B-1 cells, resulting in decreased titers of antibodies, may play an important role in the development of accommodation.

SURGICAL CONSIDERATIONS

 The majority of experimental studies have utilized the heterotopic (auxiliary) heart Tx approach. Because a heterotopic heart graft does not contribute to support of the circulation, orthotopically-placed grafts would be likely to fail earlier. Siepe et al. (2007) investigated the anatomical differences between human and pig hearts that would require special care in cardiac xenoTx. They transplanted pig hearts into deceased human recipients. They drew attention to the following surgical points: 1) special care must be paid to the anastomosis of the donor recipientpulmonary arteries because of different outflow angles; 2) the biatrial technique should be used rather than performing caval anastomoses as this avoids potential stenoses at the anastomoses of the SVC and IVC related to the differing angles at which these structures enter the heart; 3) the porcine left azygous vein that drains into the coronary sinus needs to be ligated to avoid bleeding. These technical considerations could not present any problems to trained cardiac surgeons. Ricci et al. (2007) explored the value of right ventricular endomyocardial biopsy for the diagnosis of rejection after pig-to-baboon heterotopic cardiac xenoTx. Their results indicated that delayed xenograft rejection is a widespread process involving both right and left ventricles similarly. They concluded that histological assessment of right ventricular endomyocardial biopsy specimens is an effective method for monitoring acute humoral xenograft rejection after cardiac xenoTx. Additionally, Human beings are, in contrast to pigs, a species of upright position. Therefore, gravity affects the heart differently to allow a more vertical position in the human thorax. The four-legged walk of pigs and, in consequence, the transversal position of their hearts leads to a different outflow- and inflow-angle of the great vessels as compared to human hearts (Hammer, 1998). In particular, the thin-walled pulmonary artery tends to kink and narrow. Moreover, because the porcine pulmonary artery is friable and tears easily (Swindle, 1998), its anastomosis should be carried out using pericardial pledges.

 Although, the bicaval technique combines some advantages in the clinical heart transplantation such as lesser distorsion of the tricuspid valve,we recommend using the biatrial technique for pig-to-human xenotransplantation. The biatrial technique is preferred to avoid stenoses of the caval veins. While using bicaval technique by correcting the different inflow-angles of the superior caval vein ofpigs and human proved to be problematical. As the left azygos vein in pigs is a species particularity it must be ligated near to the coronary sinus to avoid retrograde bleeding (Fig. 3).

Fig. 3. Porcine heart in the human thorax after (Courtsey; Seipe et al., 2007) 1. Biatrial implantation; 2. Anastomosis of the pulmonary artery; 3. Porcine heart in human thorax after bicaval implantation.

 To estimate the proportions of the porcine heart beforehand, the published correlations between porcine cardiac morphology and parameters which are easy to measure from the outside view will help in donor selection. Allan et al. (2001) demonstrated that pigs’body weight strongly correlates with the heart weight, length, width, and depth. The diameters of the aortic root and the main pulmonary artery correlate with the pigs’ length. Moreover, additive magnetic resonance imaging of the potential donor organs and the recipient will allow finding a proper organ for every recipient.

FUTURE DIRECTIONS

 Although newer approaches to immunosuppression may contribute to prolonged graft survival, it seems most likely that further genetic modification of the organ-source pig will be most efficacious. Identification of pig non-Gal antigens that are targets for natural or elicited antibodies, natural killer cells, and/or macrophages might allow further genetic modification, either by knock-out those antigens or by masking them by the introduction of competitive genes, e.g., α-1,2-fructosyltransferase (H transferase) (Sandrin et al.,1995). Genetically-modified pigs have overcome the problem of HAR. However, coagulation dysregulation between species remains an important challenge. Potential contributing factors toward the development of the thrombotic microangiopathy currently being seen in pig grafts include: 1) the presence of preformed anti-non-Gal antibodies, 2) the development of very low levels of elicited antibodies to non-Gal antigens, 3) natural killer cell or macrophageactivity, and 4) inherent coagulation dysregulation between pigs and primates. The breeding of pigs transgenic for an ‘anticoagulant’ or ‘anti-thrombotic’ gene, such as human TFPI, hirudin, or CD39, or lacking the gene for the prothrombinase, fibrinogen-like protein-2 (fgl-2), is anticipated to inhibit the change in the endothelium to a procoagulant state that takes place in the pig organ after Tx (Spranger et al., 2008). Some investigators have tested several anticoagulant/antiplatelet transgenes in small animal cardiac Tx models and demonstrated efficacy for human CD39 (Dwyer et al., 2004) and trans membrane-anchored forms of human TFPI and hirudin b (Chen et al., 2005). Deletion of Fgl2 was also effective at reducing intravascular thrombosis (Ghanekar et al., 2004), although the high rate of neonatal and cardiac dysfunction associated with its deletion rules it out as a practical approach in pigs.

CONCLUSION

 Increased consideration has been given towards clinical trials for potential candidates using pig organs or cells. Ibrahim et al. (2005) considered the case for cardiac bridging as the first clinical trial of xenoTx .The ideal adult patients might be those with severe biventricular dysfunction from ischemic cardiomyopathy with ventricular dysrhythmias, but who do not have any significant co morbidities, such as hemodynamic shock, hepato-renal failure with dysregulatedcoagulation, sepsis, or multi-organ failure. An assessment of the experimental results that might justify the initiation of a clinical trial was made some years ago by the International Society for Heart and Lung Transplantation (Cooper et al., 2000). A prerequisite of a minimum of 60% graft survival for 3 months in a series of consecutive lifesupporting
 (orthotopic) heart transplants in the pig-to-nonhuman primate model was considered necessary, with at least 10 recipients surviving for this period of time on a clinically applicable immunosuppressive regimen. With the GT-KO and newer genetically-modified pigs that are becoming availableand the advances in immunosuppresive agents that prevent an elicited antibody response, the immunological problems of xenoTx will steadily be reduced (Zhong, 2007). Furthermore, there is now less concern with regard to the transfer of porcine endogenous retroviruses to the recipient than previously (Sprangers et al., 2008). ‘Physiological’ problems, however, particularly those relating to coagulation pathways, remain an important challenge that is being addressed aggressively by several groups. In this context, it is noteworthy that pigs transgenic for human TFPI and human CD39 have recently been produced and the use of organs from these engineered animals could result in improved survival of porcine xenografts transplanted into primates (Sprangers et al., 2008). The prospect of pig organs resolving the critical shortage of human organs is getting closer. As the longest survival of a pig organ in a nonhuman primate to date has been the heart, cardiac xenoTx might provide the first clinical trial of solid organ xenoTx.

Note: CRP, complement-regulatory protein; DAF, decay-accelerating factor; ESC, embryonic stem cell; Gal, Galα1,3Gal; GT-KO, α-1,3-galactosyltranferase geneknockout; HAR, hyperacute rejection; MCP, membrane cofactor protein; Treg, regulatory T cells; TFPI, tissue factor pathway inhibitor; Tx, transplantation.