Burcin Ekser

Clinical xenotransplantation: the next medical revolution?

The shortage of organs and cells from deceased individuals continues to restrict allotransplantation. Pigs could provide an alternative source of tissue and cells but the immunological challenges and other barriers associated with xenotransplantation need to be overcome. Transplantation of organs from genetically modified pigs into non-human primates is now not substantially limited by hyperacute, acute antibody-mediated, or cellular rejection, but other issues have become more prominent, such as development of thrombotic microangiopathy in the graft or systemic consumptive coagulopathy in the recipient. To address these problems, pigs that express one or more human thromboregulatory or anti-inflammatory genes are being developed. The results of preclinical transplantation of pig cells--eg, islets, neuronal cells, hepatocytes, or corneas--are much more encouraging than they are for organ transplantation, with survival times greater than 1 year in all cases. Risk of transfer of an infectious microorganism to the recipient is small.

Impact of Thrombocytopenia on Survival of Baboons with Genetically Modified Pig Liver Transplants: Clinical Relevance

A lack of deceased human donor livers leads to a significant mortality in patients with acute‐on‐chronic or acute (fulminant) liver failure or with primary nonfunction of an allograft. Genetically engineered pigs could provide livers that might bridge the patient to allotransplantation. Orthotopic liver transplantation in baboons using livers from α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs (n = 2) or from GTKO pigs transgenic for CD46 (n = 8) were carried out with a clinically acceptable immunosuppressive regimen. Six of 10 baboons survived for 4–7 days. In all cases, liver function was adequate, as evidenced by tests of detoxification, protein synthesis, complement activity and coagulation parameters. The major problem that prevented more prolonged survival beyond 7 days was a profound thrombocytopenia that developed within 1 h after reperfusion, ultimately resulting in spontaneous hemorrhage at various sites. We postulate that this is associated with the expression of tissue factor on platelets after contact with pig endothelium, resulting in platelet and platelet‐peripheral blood mononuclear cell(s) aggregation and deposition of aggregates in the liver graft, though we were unable to confirm this conclusively. If this problem can be resolved, we would anticipate that a pig liver could provide a period during which a patient in liver failure could be successfully bridged to allotransplantation.

Clinical islet xenotransplantation: how close are we?

Type 1 diabetes (T1D) is a major health problem throughout the world. In the U.S., it is estimated that about 1.5 million people suffer from T1D. Even when well controlled—by frequent monitoring of blood glucose and administration of insulin, the long-term complications of the disease are significant and include cardiovascular disease, nephropathy, retinopathy, and neuropathy (1). Here we review recent progress in preclinical models of pig islet xenotransplantation and discuss the remaining challenges that need to be addressed before the application of this form of therapy can be established in patients with T1D. During the past decade, islet allotransplantation alone (without previous kidney transplantation) using deceased human donor pancreata has been indicated mainly in patients who have had T1D for >5 years with life-threatening hypoglycemic episodes and wide fluctuations in blood glucose levels. Although the initial long-term results were rather disappointing (2), the results of islet allotransplantation have improved significantly in recent years, with 5-year insulin-independent normoglycemia achieved in >50% of patients at experienced centers (3). There is increasing evidence that successful islet allotransplantation greatly reduces the incidence of hypoglycemic episodes (2) and reduces or slows the incidence of late complications of T1D (4). This may extend the indications for islet transplantation to patients with progressive complications. For example, islet transplantation in a patient with preterminal renal failure may prevent disease progression, possibly avoiding the need for hemodialysis and kidney transplantation, provided that nonnephrotoxic immunosuppressive drug therapy is administered. Currently, in the U.S., the median waiting time for a kidney allograft from a deceased human donor is >4 years (5). However, islets from two deceased human donor pancreata are frequently required to achieve normoglycemia in a diabetic patient. Because of the limited number of suitable deceased donor pancreata, the overall number of …

Evaluation of Cytomegalovirus (CMV)-Specific T Cell Immune Reconstitution Revealed That Baseline Antiviral Immunity, Prophylaxis, or Preemptive Therapy but not Antithymocyte Globulin Treatment Contribute to CMV-Specific T Cell Reconstitution in Kidney Transplant Recipients

BACKGROUND The ultimate goal of organ transplantation is the reestablishment of organ function and the restoration of a solid immunity to prevent the assault of potentially deadly pathogens. T cell immunity is crucial in controlling cytomegalovirus (CMV) infection. It is still unknown how preexisting antiviral T cell levels, prophylaxis, or preemptive antiviral strategies and pharmacological conditioning affect immune reconstitution. METHODS Seventy preemptively treated CMV-seropositive recipients, 13 prophylaxis-treated CMV-seronegative recipients of seropositive donor transplants, 2 seropositive recipients of seronegative donor kidneys, and 27 pretransplant subjects were enrolled in a cross-sectional study and analyzed for CMV viremia (DNAemia) and CMV-specific T cell response (interferon-gamma enzyme-linked immunospot assay) before transplantation and at 30, 60, 90, 180, and 360 days after transplantation. RESULTS CMV-seropositive transplant recipients displayed a progressive but heterogeneous pattern of immune reconstitution starting from day 60 after transplantation. CMV-seronegative recipients did not mount a detectable T cell response throughout the prophylaxis regimen. A single episode of CMV viremia (CMV copy number, 7000-170,000 copies/mL) was sufficient to prime a protective T cell immune response in CMV-seronegative recipients. Antithymocyte globulin treatment did not significantly affect CMV-specific T cell response. CONCLUSIONS Baseline immunity, antiviral therapy but not antithymocyte globulin treatments profoundly influence T cell reconstitution in kidney transplant recipients.

Overcoming the barriers to xenotransplantation: prospects for the future

Cross-species transplantation (xenotransplantation) has immense potential to solve the critical need for organs, tissues and cells for clinical transplantation. The increasing availability of genetically engineered pigs is enabling progress to be made in pig-to-nonhuman primate experimental models. Potent pharmacologic immunosuppressive regimens have largely prevented T-cell rejection and a T-cell-dependent elicited antibody response. However, coagulation dysfunction between the pig and primate is proving to be a major problem, and this can result in life-threatening consumptive coagulopathy. This complication is unlikely to be overcome until pigs expressing a human ‘antithrombotic’ or ‘anticoagulant’ gene, such as thrombomodulin, tissue factor pathway inhibitor or CD39, become available. Progress in islet xenotransplantation has been more encouraging, and diabetes has been controlled in nonhuman primates for periods in excess of 6 months, although this has usually been achieved using immunosuppressive protocols that might not be clinically applicable. Further advances are required to overcome the remaining barriers.

Recipient tissue factor expression is associated with consumptive coagulopathy in pig-to-primate kidney xenotransplantation.

Consumptive coagulopathy (CC) remains a challenge in pig‐to‐primate organ xenotransplantation (Tx). This study investigated the role of tissue factor (TF) expression on circulating platelets and peripheral blood mononuclear cells (PBMCs). Baboons (n = 9) received a kidney graft from pigs that were either wild‐type (n = 2), α1,3‐galactosyltransferase gene‐knockout (GT‐KO; n = 1) or GT‐KO and transgenic for the complement‐regulatory protein, CD46 (GT‐KO/CD46, n = 6). In the baboon where the graft developed hyperacute rejection (n = 1), the platelets and PBMCs expressed TF within 4 h of Tx. In the remaining baboons, TF was detected on platelets on post‐Tx day 1. Subsequently, platelet‐leukocyte aggregation developed with formation of thrombin. In the six baboons with CC, TF was not detected on baboon PBMCs until CC was beginning to develop. Graft histopathology showed fibrin deposition and platelet aggregation (n = 6), but with only minor or no features indicating a humoral immune response (n = 3), and no macrophage, B or T cell infiltration (n = 6). Activation of platelets to express TF was associated with the initiation of CC, whereas TF expression on PBMCs was concomitant with the onset of CC, often in the relative absence of features of acute humoral xenograft rejection. Prevention of recipient platelet activation may be crucial for successful pig‐to‐primate kidney Tx.

The role of genetically engineered pigs in xenotransplantation research

There is a critical shortage in the number of deceased human organs that become available for the purposes of clinical transplantation. This problem might be resolved by the transplantation of organs from pigs genetically engineered to protect them from the human immune response. The pathobiological barriers to successful pig organ transplantation in primates include activation of the innate and adaptive immune systems, coagulation dysregulation and inflammation. Genetic engineering of the pig as an organ source has increased the survival of the transplanted pig heart, kidney, islet and corneal graft in non‐human primates (NHPs) from minutes to months or occasionally years. Genetic engineering may also contribute to any physiological barriers that might be identified, as well as to reducing the risks of transfer of a potentially infectious micro‐organism with the organ. There are now an estimated 40 or more genetic alterations that have been carried out in pigs, with some pigs expressing five or six manipulations. With the new technology now available, it will become increasingly common for a pig to express even more genetic manipulations, and these could be tested in the pig‐to‐NHP models to assess their efficacy and benefit. It is therefore likely that clinical trials of pig kidney, heart and islet transplantation will become feasible in the near future. Copyright

Technical Aspects of Unilateral Dual Kidney Transplantation from Expanded Criteria Donors: Experience of 100 Patients

One option for using organs from donors with a suboptimal nephron mass, e.g. expanded criteria donors (ECD) kidneys, is dual kidney transplantation (DKT). In adult recipients, DKT can be carried out by several techniques, but the unilateral placement of both kidneys (UDKT) offers the advantages of single surgical access and shorter operating time. One hundred UDKT were performed using kidneys from ECD donors with a mean age of 72 years (Group 1). The technique consists of transplanting both kidneys extraperitoneally in the same iliac fossa. The results were compared with a cohort of single kidney transplants (SKT) performed with the same selection criteria in the same study period (Group 2, n = 73). Ninety‐five percent of UDKTs were positioned in the right iliac fossa, lengthening the right renal vein with an inferior vena cava patch. In 69% of cases, all anastomoses were to the external iliac vessels end‐to‐side. Surgical complications were comparable in both groups. At 3‐year follow‐up, patient and graft survival rates were 95.6 and 90.9% in Group 1, respectively. UDKT can be carried out with comparable surgical complication rates as SKT, leaving the contralateral iliac fossa untouched and giving elderly recipients a better chance of receiving a transplant, with optimal results up to 3‐years follow‐up.

Endoscopic Gastric Submucosal Transplantation of Islets (ENDO‐STI): Technique and Initial Results in Diabetic Pigs

The results of transplantation of human donor islets into the portal vein (PV) in patients with diabetes are encouraging. However, there are complications, for example, hemorrhage, thrombosis and an immediate loss of islets through the ‘instant blood‐mediated inflammatory reaction’ (IBMIR). The gastric submucosal space (GSMS) offers potential advantages. Islets were isolated from adult pigs. Recipient pigs were made diabetic by streptozotocin. Donor islets were injected into the GSMS through a laparotomy (Group 1A, n = 4) or endoscopically (Group 1B, n = 8) or into the PV through a laparotomy (Group 2, n = 3). The pigs were followed for a maximum of 28 days. Monitoring of C‐peptide in Group 1 indicated that there was minimal immediate loss of islets whereas in Group 2 there was considerable loss from IBMIR. In Group 1, there were significant reductions in mean blood glucose and mean exogenous insulin requirement between pretransplantation and 20 days posttransplantation. In Group 2, there was no significant reduction in either parameter. Insulin‐positive cells were seen in the GSMS in Group 1, but not in the liver in Group 2. Endoscopic gastric submucosal transplantation of islets (ENDO‐STI) offers a minimally invasive and quick approach to islet transplantation, avoids IBMIR and warrants further exploration.

Hepatic Function After Genetically Engineered Pig Liver Transplantation in Baboons

Background. If “bridging” to allo-transplantation (Tx) is to be achieved by a pig liver xenograft, adequate hepatic function needs to be assured. Methods. We have studied hepatic function in baboons after Tx of livers from &agr;1,3-galactosyltransferase gene-knockout (GTKO, n=1) or GTKO pigs transgenic for CD46 (GTKO/CD46, n=5). Monitoring was by liver function tests and coagulation parameters. Pig-specific proteins in the baboon serum/plasma were identified by Western blot. In four baboons, coagulation factors were measured. The results were compared with values from healthy humans, baboons, and pigs. Results. Recipient baboons died or were euthanized after 4 to 7 days after internal bleeding associated with profound thrombocytopenia. However, parameters of liver function, including coagulation, remained in the near-normal range, except for some cholestasis. Western blot demonstrated that pig proteins (albumin, fibrinogen, haptoglobin, and plasminogen) were produced by the liver from day 1. Production of several pig coagulation factors was confirmed. Conclusions. After the Tx of genetically engineered pig livers into baboons (1) many parameters of hepatic function, including coagulation, were normal or near normal; (2) there was evidence for production of pig proteins, including coagulation factors; and (3) these appeared to function adequately in baboons although interspecies compatibility of such proteins remains to be confirmed.