Xenotransplantation literature update, March–April 2015 (2015)

The minuscule molecular variation between pig, non-human primates (NHP), and human glycan-coated cells has a profound impact resulting in xenograft rejection. A literature review by Salama et al. 1 discussed the absence of cytidine monophosphate-N-acetyl-neuraminic acid hydroxylase (CMAH), an enzyme involved in sialic acid synthesis. Such deficiency results in a lack of the glycolyl form of neuraminic acid, Neu5Gc. A diet including milk and red meat creates a metabolic incorporation of Neu5Gc with expression at the surface of normal human epithelial and endothelial cells inducing immunization, with some individuals producing substantial amounts of anti-Neu5Gc IgG and IgM antibodies. The authors raised concern that the pig-to-NHP model of xenotransplantation is not suitable for studying anti-Neu5Gc antibodies' effect on vascularized or tissular xenotransplants and suggested that speculation on the final outcome of xenotransplantation from alpha-1,3-galactosyltransferase-1 knockout pigs to humans is hazardous. Pertinent to xenotransplantation, Neu5Gc has been identified on the vascular endothelium cell surface of pigs with the kidney, liver, heart, and pancreas being included, as well as all layers of the cornea. Devitalized animal tissue, such as porcine biological prosthetic heart valves (BPHVs), is currently under study for the expression of Neu5Gc epitopes. Although structural damage of the valves appears as the problem leading to valve failure, it could in part be caused by a modified immune response to carbohydrate antigens. Moreover, CMAH enzyme knockout pig serum tests were positive to some Neu5Gc. The authors gave rise to the need for reliable quantitative assays for measuring levels of antibodies of different isotypes binding Neu5Gc epitopes including methods to study functional abilities to activate or destroy target cells. With this understanding of strong molecular diversity of Neu5Gc epitopes, new studies have shown an increased antibody reactivity to Neu5Gc-terminated structures in immunosuppression-treated graft patients and pig kidney perfusion. The estimated percentage of normal individual sera with detectable anti-Neu5Gc antibodies is now at least 85%, with levels close to that of anti-Gal antibodies. Virus infections in either donor tissues or the recipient may create a significant barrier to xenotransplantation. Infections with latent viruses in transplanted patients have the potential to impact the recipient immune responses to the transplanted tissue, potentially leading to tissue rejection. Porcine endogenous retrovirus (PERV) is established in porcine germ lines and poses a significant safety concern to the use of pig tissues and cells for human transplantation and thus understanding the risk of cross-species infections with PERV and how human cells react to infections is paramount. Porcine endogenous retrovirus infection in human cells may be inhibited by intracellular restriction factors (RFs), including those in the tripartite motif (TRIM) family. Many of the candidate RFs are upregulated during PERV infection of human cells 2, 3. PERV isolates capable of overcoming host restriction more readily infect and productively replicate in human cells. A Letter to the Editor, Denner et al. 4 discuss research leading to conclusions that only certain isolates are capable of infecting human cells. The author explains that differences in experimental results may be the result of the PERV strains examined. Interestingly, only highly infectious PERV isolates that contain numerous repeats in the long terminal repeat (LTR) region can infect human peripheral blood monocytes. The potential impact of PERV infection on xenotransplantation success highlights the need for effective assays to detect PERV infection. A review in Xenotransplantation provided an update on the various methods of PERV detection and analysis of gene expression 5. In addition to the inherent risk of transplantation of non-sterile tissues, latent human infections may complicate matters. Ubiquitous herpesviruses, such as human cytomegalovirus (CMV) and human herpesvirus 1 (HHV-1), also known as herpes simplex virus, while normally latent, can reactivate following therapeutic immunosuppression during transplantation. CMV has been shown to infect and modulate pig cells 6, 7. If the cells contain PERV, then a herpesvirus infection can also result in the expression of PERV proteins 8. This ultimately may facilitate tissue targeting by immune cells and the subsequent tissue rejection. And finally, reactivation of latent viruses may have unintended pathological complications. Cavicchioli et al. 9 recently reported that active CMV infection in an immunosuppressed and xenotransplanted primate model results in severe gastrointestinal lesions. Thus, there is a need to understand latent infections in both donor and recipient. In a letter to the editor of Xenotransplantation, Lee et al. 10 praised Barone and team 11 for the identification of potential target antigens related to bioprosthetic heart valve (BHV) xenograft rejection. Through the use of liquid chromatography–mass spectrometry and monoclonal antibodies and lectins, they characterized acid and non-acid glycosphingolipids from aortic and pulmonary valve cusps and did not find N-glycolylneuraminic acid (NeuGc). It should be emphasized Barone et al. analyzed glycolipids and not glycoproteins, which could be the root of discrepancy between previous studies. NeuGc is believed to exist in the form of glycolipid or glycoprotein depending upon the cell type examined. Using immunofluorescence techniques, the authors believe NeuGc is in fact present on pig heart valves with high levels of expression. Although high levels of expression are becoming more definite, immunogenicity remains not yet confirmed. Going forward, they believe it is imperative to determine NeuGc expression in blood samples from porcine-derived tissue graft patients and whether or not there is correlation to long-term graft success. Further, they suggest human anti-NeuGc antibodies could recognize a certain structural form of NeuGc, and identifying the predominant structure on different tissue types would be beneficial for further advancement. Jiminez-Vera et al. 12 suggest that neonatal pig islets could be a suitable source for clinically transplantable islets if we better understood the relationship of time in culture to suitable insulin expression for rescue from diabetes. Kang et al. 13 have analyzed the specificity of antibodies generated after pig-to-non-human primate islet xenotransplantation. In the presence of anti-CD154 and sirolimus immune suppression, only one NHP analyzed produced significant non-Gal antibody, whereas 7 of 15 increased the production of anti-Gal antibody. The authors performed proteomic analysis to determine porcine albumin as the target of non-Gal antibody. The authors suggest that the anti-non-Gal antibody response is unique and not susceptible to immune suppression in sensitized NHP. Harris et al. 14 studied the compound factors influencing graft survival during porcine lung xenotransplantation from genetically modified pigs. The study used 157 lung xenoperfusion experiments ranging from wild type, one genetic modification, two genetic modifications, and three or more modifications for functional lung survival to 4 h of perfusion and markers of known xenograft injury. Increases in genetic modifications coincided with prolonged lung xenograft survival. Modifications associated with no significant effect on xenograft survival included hTBM, HLA-E, and hCD39, while improved survival occurred with GalTKO and the expression of hCD46, HO-1, hCD55, or hEPCR. Although survival improved with these increased modifications, elaboration of thrombin, histamine, and thromboxane remains abundant including neutrophil and platelet sequestration. The authors concluded the relationship between gene expression and pathway-specific injury is necessary in future studies before a solid conclusion that the new “platform lung” should be GalTKO.hCD46.hCD55.hEPCR to progress toward the clinic. Bone marrow (BM) transplant from α-1,3-galactosyltransferase knockout (GalTKO) pig to baboon has been largely unsuccessful due to loss of macrochimerism within 24 h. Taski et al. 15 developed a new strategy of intrabone BM (IBBM) transfer with improved engraftment and persistence of peripheral chimerism. Their procedure included baboons receiving half of the GalTKO BM cells transplanted directly into the bilateral tibiae and the remaining half injected intravenously with a conditioning immunosuppressive regimen. Half of the recipients received matched donor swine leukocyte antigen GalTKO kidneys in the IBBM/BM-Tx perioperative period, while the other half received kidneys 2 months later. The study observed an increased peripheral macrochimerism, detectable continuously for a mean of 7.7 days post-IBBM/Bm transfer. To date, their procedure provides the highest incidence of BM engraftment.