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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: Curr Opin Organ Transplant. 2012 Apr;17(2):148–154. doi: 10.1097/MOT.0b013e3283509120

Cardiac Xenotransplantation: Progress and Challenges

Guerard W Byrne 1,2, Christopher GA McGregor 1,2
PMCID: PMC3326642  NIHMSID: NIHMS362492  PMID: 22327911

Abstract

Purpose of Review

Cardiac xenotransplantation (CXTx) remains a promising approach to alleviate the chronic shortage of donor hearts. This review summarizes recent results of heterotopic and orthotopic CXTx, highlights the role of non-Gal antibody in xenograft rejection, and discusses challenges to clinical orthotopic CXTx.

Recent Findings

Pigs mutated in the α 1,3 galactosyltransferase gene (GTKO pigs) are devoid of the galactose α 1,3 galactose (αGal) carbohydrate antigen. This effectively eliminated any role for anti-Gal antibody in GTKO cardiac xenograft rejection. Survival of heterotopic GTKO cardiac xenografts in nonhuman primates continues to increase. GTKO graft rejection commonly involves vascular antibody deposition and variable complement deposition. Non-Gal antibody responses to porcine antigens associated with inflammation, complement, and hemostatic regulation and to new carbohydrate antigens have been identified. Their contribution to rejection remains under investigation. Orthotopic CXTx is limited by early perioperative cardiac xenograft dysfunction (PCXD). However, hearts affected by PCXD recover full cardiac function and orthotopic survival up to 2 months without rejection has been reported.

Summary

CXTx remains a promising technology for treating end-stage cardiac failure. Genetic modification of the donor and refinement of immunosuppressive regimens have extended heterotopic cardiac xenograft survival from minutes to in excess of 8 months.

Keywords: Organ transplantation; xenotransplantation; antibody-mediated rejection; α1,3 galactosyltransferase gene-knockout; complement regulatory proteins; transgenic

Introduction

The chronic shortage of donor organs limits the full benefit of organ transplantation as a therapy for end-stage organ failure. This has spurred interest in alternative therapies including mechanical devices, regenerative medical approaches, and xenotransplantation. Left ventricular assist devices, used as a bridge to transplant or as destination therapy, are effective for patients with end-stage heart failure (1). Changes in device design and patient selection have improved survival, although infections and bleeding complications remain. As destination therapy, mechanical devices may not provide an equal quality of life compared to organ transplantation. Regenerative medicine promises immunologically compatible organs produced from recipient progenitor cells. This approach has had good success for simple tissues (2), but regenerative medicine for heart transplantation remains at a nascent stage (3). Genetic modification of the porcine genome has transformed xenotransplantation (4). Survival of pig-to-primate cardiac xenotransplantation (CXTx) has increased from a few hours to a median of 3 months (5). If replicated using life-supporting orthotopic transplants, CXTx may warrant clinical testing. This review summarizes recent developments in heterotopic and orthotopic CXTx and highlight the role of non-Gal antibody-mediated endothelial cell (EC) activation as a primary effector of delayed xenograft rejection (DXR).

Elimination of the αGal antigen

It has been nearly 10 years since the development of pigs with a targeted mutation in the α-galactosyltransferase gene (6, 7) (GGTA-1; GTKO pigs). This gene encodes the enzymatic function to produce the galactose α1,3, galactose glycan (αGal). Initially there was concern that residual levels of αGal might persist in GTKO pigs. Residual αGal expression was detected on cultured GTKO porcine fetal fibroblasts isolated after in vitro selection of GT+/− cells (8) and a second α-galactosyltransferase, encoding the production of the rat isogloboside iGb3, was shown to induce αGal expression in human cells (9). This suggested that GTKO pigs might still produce αGal glycolipids. This is not the case. Cells from GTKO pigs fail to bind the αGal specific lectin GSIB-4 or any of a series of anti-Gal monoclonal antibodies. ((10) and GWB personal comm.) Comparisons of neutral and acidic glycolipids from Gal-positive and GTKO pig organs found no evidence of αGal in GTKO tissues (11) and molecular analysis failed to observe expression of the iGb3 α-galactosyltransferase in GTKO pig cells (12). Xenotransplantation of GTKO hearts has no effect on preformed anti-Gal antibody and does not stimulate an anti-Gal antibody response (13, **14). These findings indicate that the GTKO mutation has eliminated the αGal antigen from pig tissues and marginalized any role of anti-Gal antibody in rejection of GTKO donor organs.

Heterotopic GTKO cardiac transplants

The initial pig-to-primate GTKO cardiac xenografts observed no hyperacute rejection and obtained a median cardiac xenograft survival of 78 days (13). Recipients in this study were strongly immunosuppressed and showed hyporesponsive cellular reactivity and no notable induction of non-Gal antibody. Despite effective immune regulation and use of recipients with minimal pretransplant non-Gal antibody, DXR of GTKO hearts showed vascular antibody and complement deposition along with platelet and fibrin rich microvascular thrombosis (MT). The extent of MT paralleled increases in antibody and complement deposition, EC expression of tissue factor and von Willebrand's factor and decreased expression of CD39 (15). We recently compared the histopathology of GT+;CD46 and GTKO;CD55 cardiac xenograft rejection.(14) Despite depletion of anti-Gal antibody for GT+ graft recipients prior to transplant, both donor types show vascular antibody deposition within 30 minutes of reperfusion and in all subsequent interim biopsies. Complement activation occurred, as evidenced by C3d deposition. Vascular deposition of C5b and C5b-9 were minimal, consistent with the intrinsic regulatory function of CD46 or CD55. Early histopathology showed myocyte vacuolization, generally prior to significant levels of MT, followed by increasing levels of MT, ischemic injury, and myocardial coagulative necrosis. (Figure 1) These later histologic features paralleled decreased cardiac contractility and increased levels of serum troponin. Elevated expression of porcine ICAM-1, VCAM and tissue factor, consistent with EC activation, was observed at explant. This study and others using GT+ donors suggests that when early effects of anti-Gal antibody are blocked the histopathology of DXR is the same in both GT+ and GTKO cardiac xenografts (16, 17). Together these studies strongly indicate that rejection of GTKO organs remains primarily an antibody-mediated process resultant to non-Gal antibody-mediated injury or activation of vascular ECs.

Figure 1.

Figure 1

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Common histopathologic changes observed in DXR of GTKO organs. The illustration depicts the relative intensity and timing of major histologic features based on analysis of interim biopsies from Gal-positive and GTKO heterotopic cardiac xenografts (14). IgM, vascular antibody binding; MV, myocyte vacuolization; MT, microvascular thrombosis; CN, coagulative necrosis

Non-Gal Antigens and Cardiac Xenograft Rejection

The natural antibody repertoire consists of germline encoded polyreactive antibody (IgM) produced in the absence of clear immune challenge (18, 19). Natural antibody also includes reactivity to αGal and to blood groups A and B glycans which develop in response to intestinal microflora (20). Naïve human and nonhuman primate sera includes antibody reactivity to GTKO pig cells. In primates this in mainly IgM and in humans it is a mixture of IgG and IgM. Preformed non-Gal antibody is cytotoxic to GTKO peripheral blood mononuclear cells in most human sera and about 64% of baboon sera (21, 22). The level of non-Gal antibody and cytotoxicity is generally reduced compared to wild type pig cells and there is a wide range of individual variation.

The impact of preformed non-Gal antibody in CXTx is not evident using recipients with minimal antibody reactivity to GTKO pig cells. Recent studies using recipients with demonstrable preformed non-Gal antibody report early immune injury to GTKO hearts, including an instance of hyperacute rejection (23–*25). GTKO cardiac xenograft survival of less than 1 day was observed in the absence of immunosuppression and early graft failure was reported in recipients with variant sub-therapeutic levels of immunosuppression (24, 25). Rejection was characterized by vascular antibody and complement (C4d) deposition, MT, and graft infiltration by neutrophils and macrophages. We reported hyperacute rejection of a GTKO cardiac xenograft in a recipient treated with a full regimen of immunosuppression (23). At explant, 90 minutes after reperfusion, the histology showed extensive intramyocardial hemorrhage, vascular IgM and C5b deposition. (Figure 2A–C) Despite elimination of αGal antigen, these studies show that preformed non-Gal antibody is commonly present and can induce early graft injury. This suggests that methods to deplete or block preformed non-Gal antibodies may be needed to prevent early graft injury and GTKO xenograft rejection.

Figure 2.

Figure 2

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Immunohistopathology comparison of GTKO hyperacute rejection after heterotopic cardiac xenotransplantation and perioperative cardiac xenograft dysfunction (PCXD) after orthotopic cardiac xenotransplantation. Hyperacute rejection (A – C). PCXD (D – F). Panels show hematoxylin and eosin staining (A and D), vascular IgM deposition (B and E) and vascular C5b deposition (C and F).

Panels A – C (Byrne GW, McGregor CGA, et al, 2012 Transplantation, accepted pending revision)

Human preformed non-Gal antibody includes antibody reactivity to terminally linked N-glycolylneuraminic acid (anti-Neu5Gc) (26). The Neu5Gc sialic acid modification is produced by hydroxylation of N-acetylneuraminic acid (Neu5Ac) through CMP-Neu5Ac hydroxylase (CMAH) (27). A mutation of the CMAH locus eliminates this pathway in humans, but not nonhuman primates. Anti-Neu5Gc antibody is estimated to constitute 7–13% of the preformed non-Gal human antibody repertoire (28). The potential for anti-Neu5Gc antibody to affect cardiac xenograft rejection remains unclear since they are not present in nonhuman primates, and binding of anti-Neu5Gc may be highly context dependent (27). The development of a targeted CMAH mutation in mice will be useful to define the potential of anti-Neu5Gc antibody to mediated organ rejection (29).

Several studies have attempted to identify non-Gal antigenic targets detected after CXTx. Yeh and colleagues (30) used sensitized baboon sera to screen a panel of 15 prospective glycan polyacrylamide conjugates. These neoconjugates included blood group A and B glycans, αGal, α- and β-lactoasamine, Forssman disaccharide, sulphated derivatives of β-lactosamine, Neu5Gc, Neu5Ac, P1, Pk, and Lewis A, B and C glycans. GTKO sensitized baboon sera showed no specific induced immune response to any of these glycans. In contrast Diswall and colleagues (11) detected an induced antibody response to an undefined acidic glycolipid present in GTKO tissue. We have examined the specificity and diversity of induced non-Gal IgG using proteomic and expression library screening methods (23, **31). These studies identified both protein and putative carbohydrate non-Gal antigens. (Table 1) Expression library screening identified a porcine glycosyl transferase homologous to human and murine β1–4 N-acetylgalactossminyl transferase 2 (B4GALNT2) (32, 33). Lectin and antibody binding suggests that human cells expressing the porcine B4GALNT2 homologue produces a CAD-like glycan(32), which may be consistent with the acidic glycolipid response reported by Diswall et al (11).

Table 1.

Candidate Non-Gal antigens involved in Cardiac Xenograft rejection

Antigen Function
Fibronectin Extracellular matrix adhesion
MG-160 Golgi membrane
ORP150 Stress response
HSP70RY Stress response
HSP90B1 Stress response
GRP75 Stress response
GRP78 Stress response
HSC70 Stress response
HSP60 Stress response
TCP1 Stress response
Annexin A6 Inflammation
Annexin A2 Inflammation
Annexin A1 Inflammation
Vimentin Cytoskeleton
CD9 Platelet and EC activation
CD46 Complement regulation
CD59 Complement regulation
PROCR Haemostasis
B4GALNT2 Glycosylation
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Our analysis suggests that the induced non-Gal antibody response is directed to a limited number of non-Gal antigens composed of members of the heat shock and annexin protein families, porcine complement and thromboregulatory proteins. Induced antibody responses to porcine fibronectin, the endothelial cell protein C receptor, CD46 and the B4GALNT2 produced glycan are frequently observed (23, **31). Antibody responses to these non-Gal antigens may contribute to xenograft rejection by blocking key EC functions which directly or indirectly contribute to MT. This may occur if non-Gal antibody blocks the function of annexin A2 (ANAX2) which regulates fibrin deposition (34), porcine CD46 and CD59 which control complement amplification, porcine GRP78 and ECPCR involved in regulating thrombosis and inflammation (35, 36) and CD9 involved in platelet activation (37). If this is the case then substitution of the human gene for these non-Gal targets could eliminate the induced antibody response and maintain key EC functions to promote graft survival. Likewise elimination of the glycan produced by the porcine B4GALNT2 homologue could further reduce the antigenicity of pig organs.

Orthotopic Cardiac Xenotransplantation

Pig-to-primate CXTx has mainly been studied using the abdominal heterotopic transplantation model. This is an immunological model as the xenograft is perfused and beating but does not contribute to circulation. Median heterotopic cardiac xenograft survival of 3 months has been achieved (5) with individual survival beyond 8 months (25). Replicating these results with life supporting orthotopic transplantation might justify future clinical applications (38). The number of reported orthotopic pig-to-primate cardiac transplants is limited (39–**46) with survival ranging from 1 to 57 days. In this difficult model recipient death was mainly from postoperative complications with several cases explanted between 9 and 57 days showing little histologic evidence of DXR. This suggests that the efficacy of life sustaining CXTx is limited by postoperative complications and not cardiac function.

These studies also identify a problem in orthotopic CXTx which was not apparent in heterotopic CXTx. Each research group reported variable perioperative mortality ranging from 40 to 60% within the first 48 hours. (Figure 3) Xenograft failure was not due to hyperacute rejection as the explanted hearts showed vascular antibody deposition but otherwise mainly normal myocardial histology. (Figure 2D–F) Early graft failure was mainly due to primary organ dysfunction. This primary organ dysfunction which we have called perioperative cardiac xenograft dysfunction (PCXD) is a significant barrier to the clinical application of CXTx.

Figure 3.

Figure 3

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Orthotopic cardiac xenograft survival. The graph summarizes the published results for pig-to-primate cardiac xenotransplantation based on recipient survival. Values are based on the data reported in references (39–46).

PCXD may occur due to xenotransplantation-specific factors, such as the use of young donor organs, the inflammatory effects of preformed non-Gal antibody, and incompatibility between porcine and primate plasma including known dysfunctions in thromboregulation and may be further effected by pig-specific factors, most notably the sensitivity of the pig heart to cardiopulmonary bypass and ischemia-reperfusion (I/R) injury (47, 48). If this is the case then a combination of pretransplant immunoabsorption, cardiac preconditioning (49) or pharmacological treatments to improve cardiac resistance to I/R injury (50, 51) may modulate PCXD. Genetic modification to increase adenosine receptor (52) and CD39 expression (53) in the heart or to increase the number of collateral arteries (54) might also alleviate PCXD. It is important to note that PCXD is not an insurmountable barrier as 20 – 50% of current orthotopic xenografts recover from the initial effect. Over a period of 1 – 2 weeks these hearts recover full cardiac function and can go on to support the life of the primate for nearly 2 months.(43)

Recently there was the first report of pig-to-baboon heterotopic intrathoracic cardiac xenotransplantation (HTCXTx) (*55). The HTCXTx model is a load-bearing heterotopic transplant in which both the donor and recipient heart contribute to circulation. Reichart and colleagues demonstrated the feasibility of this complex surgical procedure and show good GTKO;CD46 porcine cardiac function for 45 days using clinical immunosuppression. Since circulation of the recipient is not fully dependent on xenograft function, the HTCXTx model may be an important model to study PCXD and to define diagnostic criteria and methods to treat and reverse DXR. HTCXTx may also be an alternative clinical application.

Gene Expression during Orthotopic Cardiac Xenotransplantation

We recently reported a genome wide scan of changes in porcine cardiac gene expression after orthotopic CXTx (**56). In cardiac xenografts subject to PCXD, DXR, or recovered from recipients which died of transplant complications in the absence of strong histologic evidence of rejection (Surviving grafts) we identified a set of 260 genes with widely variant changes in gene expression compared to control not transplanted pig hearts. This analysis showed that all orthotopic samples, including those from hearts not subject to DXR, showed evidence of inflammation and myocardial injury consistent with the effects of cytokines and antibody-mediated inflammation. There was also evidence of altered gene expression indicative of a cardioprotective response. Since surviving hearts were fully supporting the circulation of the recipient and not rejected by DXR despite vascular antibody deposition, this analysis suggests that for long periods of time there is a dynamic EC response which balances haemostasis against the prothrombotic effects of EC activation. DXR with MT and coagulative necrosis may be induced when this balance is pushed towards thrombosis. This might occur after an induced antibody response, when key EC functions are blocked, or as a result of species specific dysfunctions in coagulation.

Coagulation and Xenograft Rejection

Fibrin and platelet rich MT and myocardial coagulative necrosis are the prominent histologic features of GTKO cardiac xenograft rejection. Similar pathology is seen only in severe manifestations of antibody-mediated allograft rejection (57). It is unclear if this difference is the consequence of higher concentrations and greater diversity of non-Gal antibody, or due to known disparities in haemostatic regulation between pigs and humans (*58). Dysfunction of porcine thrombomodulin, and von Willebrand factor, spontaneous porcine EC dependent aggregation of human platelets, and loss of CD39 expression from activated ECs would all promote intragraft thrombosis (59–62). These disparities may also contribute to systemic derangement of coagulation parameters sometimes reported after xenotransplantation (63). Systemic therapies using clinical antiplatelet and antithrombotic drugs or using activated protein C fail to prevent MT, fibrin deposition, or prolong xenograft survival (64–68). This result may be due to the level of redundancy in haemostatic regulation, the intensity of non-Gal antibody-mediated rejection, or a failure to durably control haemostasis at the surface of the vascular EC. Numerous transgenic donor pigs designed to effect coagulation have recently been reported (69–75) but their ability to prevent MT, systemic coagulopathies or prolong graft survival remains to be tested.

Conclusion

GTKO organs have eliminated the role of anti-Gal in DXR but DXR remains an aggressive form of antibody-mediated rejection involving chronic non-Gal antibody induced EC activation and injury and possible species restricted dysfunctions in haemostasis. New methods to block the effects of preformed and induced non-Gal antibody may be required for durable xenograft survival. Orthotopic cardiac xenotransplantation has demonstrated the ability of the porcine heart to support life in the nonhuman primate model for up to 2 months; however the efficacy of this approach is limited by early xenograft dysfunction. If improvements in early cardiac xenograft function can be achieved then cardiac xenotransplantation has the potential to make a significant impact in future cardiovascular care.

Key Points.

  1. GTKO donor organs eliminate the role of anti-Gal antibody in xenograft rejection.

  2. Cytotoxic non-Gal antibody is commonly present in nonhuman primates. Preformed non-Gal antibody contributes to early cardiac xenograft injury and is sufficient to induced hyperacute rejection at low frequency.

  3. Survival of GTKO and GTKO;hCRP organs has improved, but organ rejection remains an antibody-mediated process with chronic vascular antibody deposition and variable complement activation.

  4. Orthotopic cardiac xenotransplantation is limited by early perioperative cardiac xenograft dysfunction (PCXD), not observable in heterotopic xenotransplantation. The origin of PCXD may be related to porcine cardiac ischemic injury and the effects of preformed non-Gal antibody.

  5. Further progress towards clinical use of cardiac xenotransplantation will require techniques to mitigate PCXD and new therapies to control both preformed and induced non-Gal antibody.

Acknowledgements

This work was supported by NIH Grant AI66310, CBRC Grant from University College London, and supplemental funding from the Mayo Clinic. The authors wish to acknowledge the expert technical contributions of Karen Schumacher in preparation of this manuscript.

Funding: NIAID Immunobiology of Xenotransplantation Cooperative Research Grant (AI66310) CBRC Grant, University College London

Footnotes

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Conflicts of interest

There are no conflicts of interest to report.

References

  • 1.Garbade J, Bittner HB, Barten MJ, Mohr FW. Current trends in implantable left ventricular assist devices. Cardiol Res Pract. 2011;2011:290561. doi: 10.4061/2011/290561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Atala A, Bauer SB, Soker S, Yoo JJ, Retik AB. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet. 2006 Apr 15;367(9518):1241–1246. doi: 10.1016/S0140-6736(06)68438-9. [DOI] [PubMed] [Google Scholar]
  • 3.Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Nat Med. 2008 Feb;14(2):213–221. doi: 10.1038/nm1684. [DOI] [PubMed] [Google Scholar]
  • 4.d'Apice AJ, Cowan PJ. Xenotransplantation: the next generation of engineered animals. Transpl Immunol. 2009 Jun;21(2):111–115. doi: 10.1016/j.trim.2008.10.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.McGregor CG, Davies WR, Oi K, et al. Cardiac xenotransplantation: recent preclinical progress with 3-month median survival. J Thorac Cardiovasc Surg. 2005 Sep;130(3):844–851. doi: 10.1016/j.jtcvs.2005.04.017. [DOI] [PubMed] [Google Scholar]
  • 6.Phelps CJ, Koike C, Vaught TD, et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science. 2003;299(5605):411–414. doi: 10.1126/science.1078942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Dai Y, Vaught TD, Boone J, et al. Targeted disruption of the α1,3-galactosyltransferase gene in cloned pigs. Nat Biotech. 2002;20(3):251–255. doi: 10.1038/nbt0302-251. [DOI] [PubMed] [Google Scholar]
  • 8.Sharma A, Naziruddin B, Cui C, et al. Pig cells that lack the gene for alpha1–3 galactosyltransferase express low levels of the gal antigen. Transplantation. 2003;75(4):430–436. doi: 10.1097/01.TP.0000053615.98201.77. [DOI] [PubMed] [Google Scholar]
  • 9.Sandrin MS, Milland JA, Taylor S, et al. A second galacytosyltransferase which synthesizes the major xenoepitope Galα(1,3)Gal. Xenotransplantation. 2001;8:27. [Google Scholar]
  • 10.Nottle MB, Beebe LF, Harrison SJ, et al. Production of homozygous alpha-1,3-galactosyltransferase knockout pigs by breeding and somatic cell nuclear transfer. Xenotransplantation. 2007 Jul;14(4):339–344. doi: 10.1111/j.1399-3089.2007.00417.x. [DOI] [PubMed] [Google Scholar]
  • 11.Diswall M, Angstrom J, Karlsson H, et al. Structural characterization of alpha1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies. Xenotransplantation. 2010 Jan;17(1):48–60. doi: 10.1111/j.1399-3089.2009.00564.x. [DOI] [PubMed] [Google Scholar]
  • 12.Kiernan K, Harnden I, Gunthart M, et al. The anti-non-gal xenoantibody response to xenoantigens on gal knockout pig cells is encoded by a restricted number of germline progenitors. Am J Transplant. 2008 Sep;8(9):1829–1839. doi: 10.1111/j.1600-6143.2008.02337.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Kuwaki K, Tseng YL, Dor FJ, et al. Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat Med. 2005 Jan;11(1):29–31. doi: 10.1038/nm1171. [DOI] [PubMed] [Google Scholar]
  • 14.Tazelaar HD, Byrne GW, McGregor CG. Comparison of Gal and Non-Gal-Mediated Cardiac Xenograft Rejection. Transplantation. 2011 Mar 11;91(9):968–975. doi: 10.1097/TP.0b013e318212c7fe. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Shimizu A, Hisashi Y, Kuwaki K, et al. Thrombotic microangiopathy associated with humoral rejection of cardiac xenografts from alpha1,3-galactosyltransferase gene-knockout pigs in baboons. Am J Pathol. 2008 Jun;172(6):1471–1481. doi: 10.2353/ajpath.2008.070672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Houser SL, Kuwaki K, Knosalla C, et al. Thrombotic microangiopathy and graft arteriopathy in pig hearts following transplantation into baboons. Xenotransplantation. 2004 Sep;11(5):416–425. doi: 10.1111/j.1399-3089.2004.00155.x. [DOI] [PubMed] [Google Scholar]
  • 17.McGregor CGA, Teotia SS, Byrne GW, et al. Cardiac xenotransplantation: Progress toward the clinic. Transplantation. 2004 Dec;78(11):1569–1575. doi: 10.1097/01.tp.0000147302.64947.43. [DOI] [PubMed] [Google Scholar]
  • 18.Manson JJ, Mauri C, Ehrenstein MR. Natural serum IgM maintains immunological homeostasis and prevents autoimmunity. Springer Semin Immunopathol. 2005 Mar;26(4):425–432. doi: 10.1007/s00281-004-0187-x. [DOI] [PubMed] [Google Scholar]
  • 19.Merbl Y, Zucker-Toledano M, Quintana FJ, Cohen IR. Newborn humans manifest autoantibodies to defined self molecules detected by antigen microarray informatics. J Clin Invest. 2007 Mar;117(3):712–718. doi: 10.1172/JCI29943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Galili U, Mandrell RE, Hamadeh RM, et al. Interaction between human natural anti-α-Galactosyl immunoglobulin G and bacteria of the human flora. Infect Immun. 1988;56(7):1730–1737. doi: 10.1128/iai.56.7.1730-1737.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Ezzelarab M, Hara H, Busch J, et al. Antibodies directed to pig non-Gal antigens in naive and sensitized baboons. Xenotransplantation. 2006 Sep;13(5):400–407. doi: 10.1111/j.1399-3089.2006.00320.x. [DOI] [PubMed] [Google Scholar]
  • 22.Rood PP, Hara H, Ezzelarab M, et al. Preformed antibodies to alpha1,3-galactosyltransferase gene-knockout (GT-KO) pig cells in humans, baboons, and monkeys: implications for xenotransplantation. Transplant Proc. 2005;37(8):3514–3515. doi: 10.1016/j.transproceed.2005.09.082. [DOI] [PubMed] [Google Scholar]
  • 23.Byrne GW, Stalboerger PG, Davila E, et al. Proteomic identification of non-Gal antibody targets after pig-to-primate cardiac xenotransplantation. Xenotransplantation. 2008 Jul;15(4):268–276. doi: 10.1111/j.1399-3089.2008.00480.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ezzelarab M, Garcia B, Azimzadeh A, et al. The innate immune response and activation of coagulation in alpha1,3-galactosyltransferase gene-knockout xenograft recipients. Transplantation. 2009 Mar 27;87(6):805–812. doi: 10.1097/TP.0b013e318199c34f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Mohiuddin MM, Corcoran PC, Singh AK, et al. Pig to Baboon Cardiac Xenotransplantation: Essential Role of B Cell Depletion in Prolonging Cardiac Xenograft Survival. Am J Transplant. 2010;10 Supplement:186. Describes the longest individual survival of a pig-to-primate abdominal heterotopic cardiac transplant.
  • 26.Zhu A, Hurst R. Anti-N-glycolylneuraminic acid antibodies identified in healthy human serum. Xenotransplantation. 2002 Nov;9(6):376–381. doi: 10.1034/j.1399-3089.2002.02138.x. [DOI] [PubMed] [Google Scholar]
  • 27.Padler-Karavani V, Yu H, Cao H, et al. Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: potential implications for disease. Glycobiology. 2008 Oct;18(10):818–830. doi: 10.1093/glycob/cwn072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Saethre M, Baumann BC, Fung M, et al. Characterization of natural human anti-non-gal antibodies and their effect on activation of porcine gal-deficient endothelial cells. Transplantation. 2007 Jul 27;84(2):244–250. doi: 10.1097/01.tp.0000268815.90675.d5. [DOI] [PubMed] [Google Scholar]
  • 29.Hedlund M, Tangvoranuntakul P, Takematsu H, et al. N-glycolylneuraminic acid deficiency in mice: implications for human biology and evolution. Mol Cell Biol. 2007 Jun;27(12):4340–4346. doi: 10.1128/MCB.00379-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Yeh P, Ezzelarab M, Bovin N, et al. Investigation of potential carbohydrate antigen targets for human and baboon antibodies. Xenotransplantation. 2010 May–Jun;17(3):197–206. doi: 10.1111/j.1399-3089.2010.00579.x. [DOI] [PubMed] [Google Scholar]
  • 31. Byrne GW, Stalboerger PG, Du Z, et al. Identification of new carbohydrate and membrane protein antigens in cardiac xenotransplantation. Transplantation. 2011 Feb 15;91(3):287–292. doi: 10.1097/TP.0b013e318203c27d. Screens porcine PAEC expression libraries to identify induced antibody responses to non-Gal antigens. This paper isolates a porcine B4GALNT2 like glycosyltransferase and gives the strongest evidence to date for an induced antibody response in nonhuman primates to a new non-Gal glycan.
  • 32.Renton PH, Howell P, Ikin EW, Giles CM, Goldsmith KLG. Anti-Sda, a New Blood Group Antibody. Vox Sang. 1967;13:493–501. [Google Scholar]
  • 33.Montiel MD, Krzewinski-Recchi MA, Delannoy P, Harduin-Lepers A. Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acalpha2–3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen: evidence for an unusual extended cytoplasmic domain. Biochem. J. 2003 Jul 15;373(Pt 2):369–379. doi: 10.1042/BJ20021892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Cesarman-Maus G, Rios-Luna NP, Deora AB, et al. Autoantibodies against the fibrinolytic receptor, annexin 2, in antiphospholipid syndrome. Blood. 2006 Jun 1;107(11):4375–4382. doi: 10.1182/blood-2005-07-2636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Al-Hashimi AA, Caldwell J, Gonzalez-Gronow M, et al. Binding of anti-GRP78 autoantibodies to cell surface GRP78 increases tissue factor procoagulant activity via the release of calcium from endoplasmic reticulum stores. J Biol Chem. 2010 Sep 10;285(37):28912–28923. doi: 10.1074/jbc.M110.119107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Van de Wouwer M, Collen D, Conway EM. Thrombomodulin-protein C-EPCR system: integrated to regulate coagulation and inflammation. Arterioscler Thromb Vasc Biol. 2004 Aug;24(8):1374–1383. doi: 10.1161/01.ATV.0000134298.25489.92. [DOI] [PubMed] [Google Scholar]
  • 37.Kawakatsu T, Suzuki M, Kido H, et al. Antithrombotic effect of an anti-glycoprotein IIB/IIIA antibody in primate lethal thrombosis. Thromb Res. 1993 May 1;70(3):245–254. doi: 10.1016/0049-3848(93)90131-7. [DOI] [PubMed] [Google Scholar]
  • 38.Cooper DKC, Keogh AM, Brink J, et al. Report of the Xenotransplantation Advisory Committee of the International Society for Heart and Lung Transplantation: the present status of xenotransplantation and its potential role in the treatment of end-stage cardiac and pulmonary diseases. J Heart Lung Transplant. 2000;19(12):1125–1165. doi: 10.1016/s1053-2498(00)00224-2. [DOI] [PubMed] [Google Scholar]
  • 39.Waterworth PD, Dunning J, Tolan M, et al. Life-supporting pig-to-baboon heart xenotransplantation. J Heart Lung Transplant. 1998;17(12):1201–1207. [PubMed] [Google Scholar]
  • 40.Vial CM, Ostlie DJ, Bhatti FNK, et al. Life supporting function for over one month of a transgenic porcine heart in a baboon. J Heart Lung Transplant. 2000;19(2):224–229. doi: 10.1016/s1053-2498(99)00099-6. [DOI] [PubMed] [Google Scholar]
  • 41.Schmoeckel M, Bhatti FNK, Zaidi A, et al. Orthotopic heart transplantation in a transgenic pig-to-primate model. Transplantation. 1998;65(12):1570–1577. doi: 10.1097/00007890-199806270-00006. [DOI] [PubMed] [Google Scholar]
  • 42.Xu H, Gundry SR, Hancock WW, et al. Prolonged discordant xenograft survival and delayed xenograft rejection in a pig-to-baboon orthotopic cardiac xenograft model. J Thorac Cardiovasc Surg. 1998;115(6):1342–1349. doi: 10.1016/S0022-5223(98)70218-1. [DOI] [PubMed] [Google Scholar]
  • 43.McGregor CGA, Davies WR, Oi K, et al. Recovery of cardiac function after pig-to-primate orthotopic heart transplant. (Abstr 98) Am J Transplant. 2008;8(s2):205–206. [Google Scholar]
  • 44.Brandl U, Michel S, Erhardt M, et al. Transgenic animals in experimental xenotransplantation models: orthotopic heart transplantation in the pig-to-baboon model. Transplant Proc. 2007 Mar;39(2):577–578. doi: 10.1016/j.transproceed.2006.12.021. [DOI] [PubMed] [Google Scholar]
  • 45.Brandl U, Michel S, Erhardt M, et al. Administration of GAS914 in an orthotopic pig-to-baboon heart transplantation model. Xenotransplantation. 2005 Mar;12(2):134–141. doi: 10.1111/j.1399-3089.2005.00208.x. [DOI] [PubMed] [Google Scholar]
  • 46. McGregor CGA, Byrne GW, Vlasin M, et al. Cardiac function after preclinical orthotopic cardiac xenotransplanation. Am J Transplant. 2009;9(S2):380. This abstract describes the full recovery of cardiac function after PCXD in pig-to-primate orthotopic cardiac xenotransplantation.
  • 47.Calne RY, Bitter-Suermann H, Davis DR, et al. Orthotopic heart transplantation in the pig. Nature. 1974;247(437):140–142. doi: 10.1038/247140b0. [DOI] [PubMed] [Google Scholar]
  • 48.Ryan JB, Hicks M, Cropper JR, et al. Cariporide (HOE-642) improves cardiac allograft preservation in a porcine model of orthotopic heart transplantation. Transplantation. 2003 Mar 15;75(5):625–631. doi: 10.1097/01.TP.0000054619.13962.30. [DOI] [PubMed] [Google Scholar]
  • 49.Candilio L, Hausenloy DJ, Yellon DM. Remote ischemic conditioning: a clinical trial's update. J Cardiovasc Pharmacol Ther. 2011 Sep–Dec;16(3–4):304–312. doi: 10.1177/1074248411411711. [DOI] [PubMed] [Google Scholar]
  • 50.Hing AJ, Watson A, Hicks M, et al. Combining cariporide with glyceryl trinitrate optimizes cardiac preservation during porcine heart transplantation. Am J Transplant. 2009 Sep;9(9):2048–2056. doi: 10.1111/j.1600-6143.2009.02736.x. [DOI] [PubMed] [Google Scholar]
  • 51.Bhamra GS, Hausenloy DJ, Davidson SM, et al. Metformin protects the ischemic heart by the Akt-mediated inhibition of mitochondrial permeability transition pore opening. Basic Res Cardiol. 2008 May;103(3):274–284. doi: 10.1007/s00395-007-0691-y. [DOI] [PubMed] [Google Scholar]
  • 52.Ashton KJ, Peart JN, Morrison RR, et al. Genetic modulation of adenosine receptor function and adenosine handling in murine hearts: insights and issues. J Mol Cell Cardiol. 2007 Apr;42(4):693–705. doi: 10.1016/j.yjmcc.2006.12.012. [DOI] [PubMed] [Google Scholar]
  • 53.Crikis S, Lu B, Murray-Segal LM, et al. Transgenic overexpression of CD39 protects against renal ischemia-reperfusion and transplant vascular injury. Am J Transplant. 2010 Dec;10(12):2586–2595. doi: 10.1111/j.1600-6143.2010.03257.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Bollini S, Smart N, Riley PR. Resident cardiac progenitor cells: at the heart of regeneration. J Mol Cell Biol. 2011 Feb;50(2):296–303. doi: 10.1016/j.yjmcc.2010.07.006. [DOI] [PubMed] [Google Scholar]
  • 55. Bauer A, Postrach J, Thormann M, et al. First experience with heterotopic thoracic pig-to-baboon cardiac xenotransplantation. Xenotransplantation. 2010 May–Jun;17(3):243–249. doi: 10.1111/j.1399-3089.2010.00587.x. First description of pig-to-primate intrathoracic cardiac xenotransplantation.
  • 56. Byrne GW, Du Z, Sun Z, et al. Changes in cardiac gene expression after pig-to-primate orthotopic xenotransplantation. Xenotransplantation. 2011 Jan;18(1):14–27. doi: 10.1111/j.1399-3089.2010.00620.x. First genome-wide assessment of changes in porcine cardiac gene expression after orthotopic cardiac xenotransplantation.
  • 57.Berry GJ, Angelini A, Burke MM, et al. The ISHLT working formulation for pathologic diagnosis of antibody-mediated rejection in heart transplantation: evolution and current status (2005–2011) J Heart Lung Transplant. 2011 Jun;30(6):601–611. doi: 10.1016/j.healun.2011.02.015. [DOI] [PubMed] [Google Scholar]
  • 58.Cowan PJ, Roussel JC, d'Apice AJ. The vascular and coagulation issues in xenotransplantation. Curr Opin Organ Transplant. 2009 Apr;14(2):161–167. doi: 10.1097/mot.0b013e3283279591. [DOI] [PubMed] [Google Scholar]
  • 59.Siegel JB, Grey ST, Lesnikoski BA, et al. Xenogeneic endothelial cells activate human prothrombin. Transplantation. 1997 Sep 27;64(6):888–896. doi: 10.1097/00007890-199709270-00017. [DOI] [PubMed] [Google Scholar]
  • 60.Robson SC, Siegel JB, Lesnikoski BA, et al. Aggregation of human platelets induced by porcine endothelial cells is dependent upon both activation of complement and thrombin generation. Xenotransplantation. 1996;3:24–34. [Google Scholar]
  • 61.Lin CC, Chen D, McVey JH, et al. Expression of tissue factor and initiation of clotting by human platelets and monocytes after incubation with porcine endothelial cells. Transplantation. 2008 Sep 15;86(5):702–709. doi: 10.1097/TP.0b013e31818410a3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Deaglio S, Robson SC. Ectonucleotidases as regulators of purinergic signaling in thrombosis, inflammation, and immunity. Adv Pharmacol. 2011;61:301–332. doi: 10.1016/B978-0-12-385526-8.00010-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Lin CC, Cooper DK, Dorling A. Coagulation dysregulation as a barrier to xenotransplantation in the primate. Transplant Immunol. 2009 Jun;21(2):75–80. doi: 10.1016/j.trim.2008.10.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Schirmer JM, Fass DN, Byrne GW, et al. Effective antiplatelet therapy does not prolong transgenic pig to baboon cardiac xenograft survival. Xenotransplantation. 2004 Sep;11(5):436–443. doi: 10.1111/j.1399-3089.2004.00159.x. [DOI] [PubMed] [Google Scholar]
  • 65.Byrne GW, Schirmer JM, Fass DN, et al. Warfarin or low-molecular-weight heparin therapy does not prolong pig-to-primate cardiac xenograft function. Am J Transplant. 2005;5(5):1011–1020. doi: 10.1111/j.1600-6143.2005.00792.x. [DOI] [PubMed] [Google Scholar]
  • 66.Byrne GW, Davies WR, Oi K, et al. Increased immunosuppression, not anticoagulation, extends cardiac xenograft survival. Transplantation. 2006 Dec 27;82(12):1787–1791. doi: 10.1097/01.tp.0000251387.40499.0f. [DOI] [PubMed] [Google Scholar]
  • 67.Cozzi E, Simioni P, Boldrin M, et al. Effects of long-term administration of high-dose recombinant human antithrombin in immunosuppressed primate recipients of porcine xenografts. Transplantation. 2005;80(10):1501–1510. doi: 10.1097/01.tp.0000178377.55615.8b. [DOI] [PubMed] [Google Scholar]
  • 68.Simioni P, Boldrin M, Gavasso S, et al. Effects of long-term administration of recombinant human protein C in xenografted primates. Transplantation. 2011 Jan 27;91(2):161–168. doi: 10.1097/TP.0b013e318200ba0e. [DOI] [PubMed] [Google Scholar]
  • 69.Petersen B, Ramackers W, Tiede A, et al. Pigs transgenic for human thrombomodulin have elevated production of activated protein C. Xenotransplantation. 2009 Nov–Dec;16(6):486–495. doi: 10.1111/j.1399-3089.2009.00537.x. [DOI] [PubMed] [Google Scholar]
  • 70.Klymiuk N, Wuensch A, Kurome M, et al. GalT-KO/CD46/hTM triple-transgenic donor animals for pig-to-baboon heart transplantation. Xenotransplantation. 2011;18(5):271. [Google Scholar]
  • 71.Iwamoto M, Yazaki S, Onishi A, et al. Successful production and breeding of cloned pigs expressing human thrombomodulin in endothelial cells. Xenotransplantation. 2011;18(5):270. doi: 10.1111/j.1399-3089.2012.00696.x. [DOI] [PubMed] [Google Scholar]
  • 72.Ayares D, Phelps C, Vaught T, et al. Multi-transgenic pigs for vascularized pig organ xenografts. Xenotransplantation. 2011;18(5):269. [Google Scholar]
  • 73.Ahrens H, Petersen B, Cunha A, et al. Production and characterization of TF knock-down pigs and hTFPI transgenic pigs. Xenotransplantation. 2011;18(5):296. [Google Scholar]
  • 74.Salvaris E, Fisicaro N, Harrison S, et al. Expression of human thrombomodulin in GTKO/hCD55-hCD59-HTF pigs. Xenotransplantation. 2011;18(5):296. [Google Scholar]
  • 75.Perota A, Lagutina I, Colleoni S, et al. High ubiquitous hCD55 and hCD39 co-expression in live transgenic alpha 1,3-Gal Knock-out piglet. Xenotransplantation. 2011;18(5):297. [Google Scholar]

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