Vikas Satyananda

Progress in pig‐to‐non‐human primate transplantation models (1998–2013): a comprehensive review of the literature

The pig‐to‐non‐human primate model is the standard choice for in vivo studies of organ and cell xenotransplantation. In 1998, Lambrigts and his colleagues surveyed the entire world literature and reported all experimental studies in this model. With the increasing number of genetically engineered pigs that have become available during the past few years, this model is being utilized ever more frequently.

Pig‐to‐baboon heterotopic heart transplantation – exploratory preliminary experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade‐based regimens

Three costimulation blockade‐based regimens have been explored after transplantation of hearts from pigs of varying genetic backgrounds to determine whether CTLA4‐Ig (abatacept) or anti‐CD40mAb+CTLA4‐Ig (belatacept) can successfully replace anti‐CD154mAb.

New concepts of immune modulation in xenotransplantation.

Abstract The shortage of human organs for transplantation has focused research on the possibility of transplanting pig organs into humans. Many factors contribute to the failure of a pig organ graft in a primate. A rapid innate immune response (natural anti-pig antibody, complement activation, and an innate cellular response; e.g., neutrophils, monocytes, macrophages, and natural killer cells) is followed by an adaptive immune response, although T-cell infiltration of the graft has rarely been reported. Other factors (e.g., coagulation dysregulation and inflammation) appear to play a significantly greater role than in allotransplantation. The immune responses to a pig xenograft cannot therefore be controlled simply by suppression of T-cell activity. Before xenotransplantation can be introduced successfully into the clinic, the problems of the innate, coagulopathic, and inflammatory responses will have to be overcome, most likely by the transplantation of organs from genetically engineered pigs. Many of the genetic manipulations aimed at protecting against these responses also reduce the adaptive response. The T-cell and elicited antibody responses can be prevented by the biological and/or pharmacologic agents currently available, in particular, by costimulation blockade-based regimens. The exogenous immunosuppressive regimen may be significantly reduced by the presence of a graft from a pig transgenic for a mutant (human) class II transactivator gene, resulting in down-regulation of swine leukocyte antigen class II expression, or from a pig with “local” vascular endothelial cell expression of an immunosuppressive gene (e.g., CTLA4-Ig). The immunomodulatory efficacy of regulatory T cells or mesenchymal stromal cells has been demonstrated in vitro but not yet in vivo.

Initial in vivo experience of pig artery patch transplantation in baboons using mutant MHC (CIITA-DN) pigs.

BACKGROUND In the pig-to-nonimmunosuppressed baboon artery patch model, a graft from an α1,3-galactosyltransferase gene-knockout pig transgenic for human CD46 (GTKO/CD46) induces a significant adaptive immune response (elicited anti-pig antibody response, increase in T cell proliferation on MLR, cellular infiltration of the graft), which is effectively prevented by anti-CD154mAb-based therapy. METHODS As anti-CD154mAb is currently not clinically applicable, we evaluated whether it could be replaced by CD28/B7 pathway blockade or by blockade of both pathways (using belatacept + anti-CD40mAb [2C10R4]). We further investigated whether a patch from a GTKO/CD46 pig with a mutant human MHC class II transactivator (CIITA-DN) gene would allow reduction in the immunosuppressive therapy administered. RESULTS When grafts from GTKO/CD46 pigs were transplanted with blockade of both pathways, a minimal or insignificant adaptive response was documented. When a GTKO/CD46/CIITA-DN graft was transplanted, but no immunosuppressive therapy was administered, a marked adaptive response was documented. In the presence of CD28/B7 pathway blockade (abatacept or belatacept), there was a weak adaptive response that was diminished when compared with that to a GTKO/CD46 graft. Blockade of both pathways prevented an adaptive response. CONCLUSION Although expression of the mutant MHC CIITA-DN gene was associated with a reduced adaptive immune response when immunosuppressive therapy was inadequate, when blockade of both the CD40/CD154 and CD28/B7 pathways was present, the response even to a GTKO/CD46 graft was suppressed. This was confirmed after GTKO/CD46 heart transplantation in baboons.

Toward clinical islet xenotransplantation – are revisions to the IXA guidelines warranted?

The health problems related to diabetes – both to the individual patient and to the population – are immense, with an estimated 20% of all deaths occurring in people between the ages of 50 and 60 being the consequence of the complications of diabetes [1,2]. Glycemia can be controlled in many patients by intensive insulin therapy, including insulin pumps, but over a prolonged period of time this approach is often not completely successful [1]. In many countries, the healthcare costs are also considerable, with > 10% of annual expenditure on healthcare devoted to patients with diabetic complications. The high costs of treating patients with diabetes must be taken into consideration when providing the resources to develop pancreatic islet transplantation as a potential cure. The results of clinical islet allotransplantation in patients with type 1 diabetes (T1D) are steadily improving, suggesting that eventually this form of therapy will become routine [3–6]. The success of islet autotransplantation in patients with chronic pancreatitis who undergo total pancreatectomy suggests that failure of allotransplantation is related to the requirement for a strong immunosuppressive regimen to protect the graft, rather than from islet loss associated with the isolation procedure or from transplantation into an ‘unphysiological’ site [7]. However, there will never be sufficient deceased human donors to provide islets to impact the immense problem of diabetes mellitus, not only in the Western world, but also in the populous countries of Asia where the problem of diabetes is steadily increasing, as elsewhere (Table 1). For numerous reasons that have been discussed previously [8,9], the pig may prove a source of islets for transplantation in patients with diabetes. Progress in this experimental field is also advancing.

Further evidence for sustained systemic inflammation in xenograft recipients (SIXR)

In pig‐to‐baboon heart/artery patch transplantation models, adequate costimulation blockade prevents a T‐cell response. After heart transplantation, coagulation dysfunction (thrombocytopenia, reduced fibrinogen, increased D‐dimer) and inflammation (increased C‐reactive protein [CRP]) develop. We evaluated whether coagulation dysfunction and/or inflammation can be detected following pig artery patch transplantation.

Is there a correlation between anti-pig antibody levels in humans and geographic location during childhood?

Background An initial observation suggested high levels of anti-pig antibodies in healthy humans who had spent their childhood in the Middle East. We tested larger cohorts to determine whether anti-pig antibody levels correlated with the geographic location in which the subject spent his/her childhood, because this might have implications for clinical trials of xenotransplantation. Methods Anti-pig IgM and IgG levels (by flow cytometry using peripheral blood mononuclear cells from wild-type and &agr;1,3-galactosyltransferase gene-knockout pigs) and anti-Gal IgM and IgG levels (by enzyme-linked immunosorbent assay) were measured in 75 volunteers. Comparisons of antibody levels were also made based on subject age, gender, ABO blood group, diet, and history of vaccination. Results Antibody binding to &agr;1,3-galactosyltransferase gene-knockout pig cells was less than to wild-type cells. There was a reduction in anti-pig IgM and anti-Gal IgM, but a slight increase in anti-nonGal IgG, with age. Women had higher levels of anti-Gal IgM than men. Blood group A subjects had higher levels of anti-pig IgM and IgG than those of group AB. Diet had no influence on antibody levels. Typhoid or measles-mumps-rubella vaccination was associated with lower anti-nonGal IgG or anti-Gal IgG, respectively, whereas influenza vaccination was associated with higher anti-nonGal IgG. There were some significant variations in antibody levels associated with location during childhood, with subjects from the Middle East demonstrating higher anti-nonGal IgG and anti-Gal IgG. Conclusion Clinical trials of xenotransplantation may be influenced by various factors, including the geographic location of the recipient during childhood, possibly associated with exposure to different microorganisms.

In vitro testing of an anti-CD40 monoclonal antibody, clone 2C10, in primates and pigs

BACKGROUND The CD40/CD154 and CD28/B7 pathways are important in allo- and xeno-transplantation. Owing to the thrombotic complications of anti-CD154mAb, anti-CD40mAb has emerged as a promising inhibitor of costimulation. Various clones of anti-CD40mAb have been developed against primate species, e.g., clone 2C10 against rhesus monkeys. We have compared the in vitro efficacy of 2C10 to prevent a T cell response in primates and pigs. METHODS The binding of 2C10 to antigen-presenting cells (PBMCs [B cells]) of humans, rhesus and cynomolgus monkeys, baboons, and pigs was measured by flow cytometry, and was also tested indirectly by a blocking assay. The functional capacity of 2C10 was tested by mixed lymphocyte reaction (MLR) with polyclonal stimulation by phytohemagglutinin (PHA) and also with wild-type pig aortic endothelial cells (pAECs) as stimulators. RESULTS There was a significant reduction in binding of 2C10 to baboon PBMCs compared to rhesus, cynomolgus, and human PBMCs, and minimal binding to pig PBMCs. The blocking assay confirmed that the binding of 2C10 was significantly lower to baboon PBMCs when compared to the other primate species tested. The functional assay with PHA showed significantly reduced inhibition of PBMC proliferation in humans, cynomolgus monkeys, and baboons compared to rhesus monkeys, which was confirmed on MLR with pAECs. CONCLUSIONS Since both the binding and functional activity of 2C10 in the baboon is lower than in rhesus monkeys, in vivo treatment using 2C10 in the baboon might require a higher dose or more frequent administration in comparison to rhesus monkeys. It may also be beneficial to develop species-specific clones of anti-CD40mAb.

Plasma free triiodothyronine (fT3) levels in baboons undergoing pig organ transplantation: relevance to early recovery of organ function

It has been well-documented that the level of plasma free triiodothyronine (fT3) falls rapidly following brain death (1, 2, reviewed in 3) or during certain surgical procedures, e.g., heart surgery carried out on cardiopulmonary bypass (4-8). The level in patients following cardiopulmonary bypass usually recovers within 48 hours. We have measured plasma fT3 in healthy baboons (n=19) and healthy monkeys (n=5), in two groups of baboons undergoing transplantation (Tx) with pig organs (heterotopic heart Tx [n=9] or orthotopic liver Tx [n=10]), and in one group of monkeys undergoing heterotopic heart alloTx (n=5). The levels in healthy baboons ranged from 1.9-3.6pg/mL (mean 2.7pg/mL) and in healthy monkeys from 2.3-2.9pg/mL (mean 2.6pg/mL). Following heterotopic pig heart xenoTx in baboons, there was a rapid fall from a mean pre-Tx level of 2.3pg/mL to 0.9pg/mL at 4 hours (a reduction of 62% from pre-Tx) and 0.7pg/mL at 24 hours (a reduction of 69% from pre-Tx). Four days after the surgical procedure, the level had recovered to the low normal range (approximately 2.0pg/mL), where it remained for the 28 days of follow-up. Following orthotopic pig liver xenoTx in baboons, there was a rapid fall from a mean pre-Tx level of 3.0pg/mL to 0.4pg/mL at 4 hours (a reduction of 90% from pre-Tx) and to 1.0pg/mL at 24 hours (a reduction of 66% from pre-Tx). Thereafter, the level remained in the subnormal range (approximately 1.0pg/mL, or 33% of pre-Tx) for the next 4-7 days, at which time the baboons were euthanized. Following heterotopic heart alloTx in monkeys, the level fell from a mean pre-Tx level of 2.6pg/mL to 1.1pg/mL at 24 hours (a reduction of 60% from pre-Tx), recovering to 2.6 on day 4 where it remained for the 14 days of follow-up. The cause of the rapid fall in plasma fT3 (resulting in a euthyroid sick syndrome) in brain-dead potential organ donors and after major surgical procedures is associated with a generalized inhibition of mitochondrial function, which results in the loss of energy stores and diminished organ function, particularly of the heart (9; reviewed in 3). In the present study in the monkeys with cardiac allografts, recovery of fT3 to normal levels was more rapid and better sustained than in baboons with pig cardiac xenografts. Following xenotransplantation, the persisting low fT3, particularly in the liver recipients, may be associated with an ongoing inflammatory state (Ezzelarab M et al, manuscript submitted). We have documented high levels of C-reactive protein for several weeks after pig organ and artery patch transplantation in baboons (Iwase H et al, manuscript submitted). Deterioration of organ function after brain death or after open heart surgery can be reversed by hormonal replacement therapy in which T3 plays a critical role (2, 3, 6-8, 10, 11). This results in rapid replenishment of myocardial energy stores and improvement in cardiac output (2, 3, 6-8, 10, 11). There have been reports of a high early mortality after pig orthotopic heart Tx in baboons, particularly within the first 48 hours (12; Mohiuddin M, personal communication). If the baboon survives for 48 hours, then there is recovery of the myocardial function and longer-term survival is likely (12). We suggest that the early high morbidity and mortality may be associated with a reduced level of plasma fT3, and that consideration should be given to T3 replacement therapy.

Potential for clinical pancreatic islet xenotransplantation

© 2014 Bottino et al. Diabetes mellitus is increasing worldwide. Type 1 diabetes can be treated successfully by islet allotransplantation, the results of which are steadily improving. However, the number of islets that can be obtained from deceased human donors will never be sufficient to cure more than a very small percentage of patients who might benefit from transplantation. Although there are some differences in glucose metabolism between pigs and humans, the use of pigs could provide an unlimited supply of islets, and the insulin produced would undoubtedly control glucose levels. Transplantation of islets into the portal vein results in islets residing in the liver; however, an early inflammatory response and rejection remain problematic, even when the recipient is receiving immunosuppressive therapy. In the long term, immunosuppressive drugs may exhibit toxicities to patients and specifically harm the islet cells. In contrast, encapsulation techniques provide islets with a physical barrier that prevents antibodies binding to the islet graft while still allowing insulin to be released into the recipient’s circulation; in theory, patients receiving encapsulated grafts might not require exogenous immunosuppressive therapy. Nonhuman primates with encapsulated pig islet transplants have remained insulin-independent for several weeks, but long-term efficacy remains uncertain. Furthermore, techniques are now available to knock out genes from the pig and/or insert human genes, thus rendering the antigenic structure of pigs closer to that of humans, and providing protection from the human immune response. Islet transplantation from genetically engineered pigs has been followed by insulin independence in a small number of nonhuman primates for greater than 1 year. Neonatal islets have some advantages over adult islets in that they are easier to isolate and culture, and have the ability to proliferate during the first few months after transplantation. In 2009, the International Xenotransplantation Association set up a group to encourage and advise on clinical trials of pig islet xenotransplantation; this group’s guidelines are discussed. Clinical trials of encapsulated pig islets are already under way.