Mohamed Ezzelarab

Clinical Intestinal Transplantation: A Decade of Experience at a Single Center

ObjectiveTo assess the long-term efficacy of intestinal transplantation under tacrolimus-based immunosuppression and the therapeutic benefit of newly developed adjunct immunosuppressants and management strategies. Summary Background DataWith the advent of tacrolimus in 1990, transplantation of the intestine began to emerge as therapy for intestinal failure. However, a high risk of rejection, with the consequent need for acute and chronic high-dose immunosuppression, has inhibited its widespread application. MethodsDuring an 11-year period, divided into two segments by a 1-year moratorium in 1994, 155 patients received 165 intestinal allografts under immunosuppression based on tacrolimus and prednisone: 65 intestine alone, 75 liver and intestine, and 25 multivisceral. For the transplantations since the moratorium (n = 99), an adjunct immunosuppressant (cyclophos-phamide or daclizumab) was used for 74 transplantations, adjunct donor bone marrow was given in 39, and the intestine of 11 allografts was irradiated with a single dose of 750 cGy. ResultsThe actuarial survival rate for the total population was 75% at 1 year, 54% at 5 years, and 42% at 10 years. Recipients of liver plus intestine had the best long-term prognosis and the lowest risk of graft loss from rejection (P = .001). Since 1994, survival rates have improved. Techniques for early detection of Epstein-Barr and cytomegaloviral infections, bone marrow augmentation, the adjunct use of the interleukin-2 antagonist daclizumab, and most recently allograft irradiation may have contributed to the better results. ConclusionThe survival rates after intestinal transplantation have cumulatively improved during the past decade. With the management strategies currently under evaluation, intestinal transplant procedures have the potential to become the standard of care for patients with end-stage intestinal failure.

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.

Spermatogonial Stem Cell Transplantation into Rhesus Testes Regenerates Spermatogenesis Producing Functional Sperm

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a mans life and may have application for treating some cases of male infertility, including those caused by chemotherapy before puberty. We performed autologous and allogeneic SSC transplantations into the testes of 18 adult and 5 prepubertal recipient macaques that were rendered infertile with alkylating chemotherapy. After autologous transplant, the donor genotype from lentivirus-marked SSCs was evident in the ejaculated sperm of 9/12 adult and 3/5 prepubertal recipients after they reached maturity. Allogeneic transplant led to donor-recipient chimerism in sperm from 2/6 adult recipients. Ejaculated sperm from one recipient transplanted with allogeneic donor SSCs were injected into 85 rhesus oocytes via intracytoplasmic sperm injection. Eighty-one oocytes were fertilized, producing embryos ranging from four-cell to blastocyst with donor paternal origin confirmed in 7/81 embryos. This demonstration of functional donor spermatogenesis following SSC transplantation in primates is an important milestone for informed clinical translation.

Tolerogenic dendritic cells and their role in transplantation.

The pursuit of clinical transplant tolerance has led to enhanced understanding of mechanisms underlying immune regulation, including the characterization of immune regulatory cells, in particular antigen-presenting cells (APC) and regulatory T cells (Treg), that may play key roles in promoting operational tolerance. Dendritic cells (DC) are highly efficient APC that have been studied extensively in rodents and humans, and more recently in non-human primates. Owing to their ability to regulate both innate and adaptive immune responses, DC are considered to play crucial roles in directing the alloimmune response towards transplant tolerance or rejection. Mechanisms via which they can promote central and peripheral tolerance include clonal deletion, the induction of Treg, and inhibition of memory T cell responses. These properties have led to the use of tolerogenic DC as a therapeutic strategy to promote organ transplant tolerance. In rodents, infusion of donor- or recipient-derived tolerogenic DC can extensively prolong donor-specific allograft survival, in association with regulation of the host T cell response. In clinical transplantation, progress has been made in monitoring DC in relation to graft outcome, including studies in operational liver transplant tolerance. Although clinical trials involving immunotherapeutic DC for patients with cancer are ongoing, implementation of human DC therapy in clinical transplantation will require assessment of various critical issues. These include cell isolation and purification techniques, source, route and timing of administration, and combination immunosuppressive therapy. With ongoing non-human primate studies focused on DC therapy, these logistics can be investigated seeking the optimal approaches. The scientific rationale for implementation of tolerogenic DC therapy to promote clinical transplant tolerance is strong. Evaluation of technical and therapeutic logistic issues is an important next step prior to the application of tolerogenic DC in clinical organ transplantation.

THE INNATE IMMUNE RESPONSE AND ACTIVATION OF COAGULATION IN α1,3-GALACTOSYLTRANSFERASE GENE-KNOCKOUT XENOGRAFT RECIPIENTS

Background. The role of the innate immune system in the development of thrombotic microangiopathy (TM) after α1,3-galactosyltransferase gene-knockout (GTKO) pig organ transplantation in primates is uncertain. Methods. Twelve organs (nine hearts, three kidneys) from GTKO pigs were transplanted into baboons that received no immunosuppressive therapy, partial regimens, or a full regimen based on costimulation blockade. After graft failure, histologic and immunohistologic examinations were carried out. Results. Graft survival of less than 1 day was prolonged to 2 to 12 days with partial regimens (acute humoral xenograft rejection) and to 5 and 8 weeks with the full regimen (TM). Clinical or laboratory features of consumptive coagulopathy occurred in 7 of 12 baboons. Immunohistochemistry demonstrated IgM, IgG, and complement deposition in most cases. Histopathology demonstrated neutrophil and macrophage infiltrates, intravascular fibrin deposition, and platelet aggregation (TM). Grafts showed expression of primate tissue factor (TF), with increased mRNA levels, and TF was also expressed on baboon macrophages/monocytes infiltrating the graft. Conclusions. Our data suggest that (1) irrespective of the presence or absence of the adaptive immune response, early or late xenograft rejection is associated with activation of the innate immune system; and (2) porcine endothelial cell activation and primate TF expression by recipient innate immune cells may both contribute to the development of TM.

Carbohydrates in xenotransplantation

The success of allotransplantation has led to an increasing shortage of human organs from deceased donors. This crisis could be resolved by the use of organs from an anatomically suitable animal, such as the pig. The pig and human have, however, been evolving differently for approximately 80 million years, and numerous immunological and physiological barriers have developed that need to be overcome. Differences in carbohydrate epitopes on pig and human cells have been found to play a major role in some of the immunological barriers that have been identified to date. The rejection caused by the presence of galactose‐α1,3‐galactose (Gal) on the pig vascular endothelium and of natural anti‐Gal antibodies in humans has recently been prevented by the breeding of pigs that do not express Gal, achieved by knocking out the gene for the enzyme α1,3‐galactosyltransferase, which was made possible by the introduction of nuclear transfer/embryo transfer techniques. N‐glycolylneuraminic acid (the so‐called Hanganutziu‐Deicher antigen) has been identified as another carbohydrate antigen present in pigs that may need to be deleted if xenotransplantation is to be successful, although some doubt remains regarding its importance. There remain other antipig antibodies against hitherto unidentified antigenic targets that may well be involved in graft destruction; their possible carbohydrate target epitopes are discussed.

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 …

In vitro investigation of pig cells for resistance to human antibody-mediated rejection.

Although human complement‐dependent cytotoxicity (CDC) of α1,3‐galactosyltransferase gene‐knockout (GTKO) pig cells is significantly weaker than that of wild‐type (WT) cells, successful xenotransplantation will require pigs with multiple genetic modifications. Sera from healthy humans were tested by (i) flow cytometry for binding of IgM/IgG, and (ii) CDC assay against peripheral blood mononuclear cells and porcine aortic endothelial cells from five types of pig – WT, GTKO, GTKO transgenic for H‐transferase (GTKO/HT), WT transgenic for human complement regulatory protein CD46 (CD46) and GTKO/CD46. There was significantly higher mean IgM/IgG binding to WT and CD46 cells than to GTKO, GTKO/HT, and GTKO/CD46, but no difference between GTKO, GTKO/HT, and GTKO/CD46 cells. There was significantly higher mean CDC to WT than to GTKO, GTKO/HT, CD46, and GTKO/CD46 cells, but no difference between GTKO and GTKO/HT. Lysis of GTKO/CD46 cells was significantly lower than that of GTKO or CD46 cells. CD46 expression provided partial protection against serum from a baboon sensitized to a GTKO pig heart. GTKO/CD46 cells were significantly resistant to lysis by human serum and sensitized baboon serum. In conclusion, the greatest protection from CDC was obtained by the combination of an absence of Gal expression and the presence of CD46 expression, but the expression of HT appeared to offer no advantage over GTKO. Organs from GTKO/CD46 pigs are likely to be significantly less susceptible to CDC.

PIG-TO-MONKEY ISLET XENOTRANSPLANTATION USING MULTI-TRANSGENIC PIGS

The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an α1,3‐galactosyltransferase gene‐knockout background with ubiquitous expression of human CD46, with islet beta cell‐specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4‐lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin‐diabetic, immunosuppressed cynomolgus monkeys (n = 5). Immunosuppression was based on anti‐CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi‐transgenic islet grafts did not demonstrate consistency in regard to long‐term success, with only two of five demonstrating function beyond 5 months.