The enzyme α1,3-galactosyltransferase (α1,3GT or GCTA1) synthesizes α1,3-galactose (α1,3Gal) epitopes (Galα1,3Galβ1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of α1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the α1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the α1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the α1,3GT protein. Four healthy α1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the α1,3GT gene hold significant value, as they would allow production of α1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.The enzyme α1,3-galactosyltransferase (α1,3GT or GGTA1) synthesizes α1,3Gal epitopes (Galα1,3Galβ1,4GlcNAc-R) on the cell surface of almost all mammals with the exception of humans, apes, and Old World monkeys (1). α1,3Gal epitopes are the major xenoantigens causing hyperacute rejection (HAR) in pig-to-human xenotransplantation (2-4). Many reports have also indicated that α1,3Gal epitopes are involved in acute vascular rejection (AVR) of xenografts (4-6). Piglets with α1,3GT heterozygous knockout have been cloned Copyright © 2003 by our group (7) and another team (8) in the last year. To produce homozygous α1,3GT knockout piglets by natural breeding, assuming both male and female heterozygous knockout pigs are available at the same time and are fertile, is feasible but takes up to 12 months. However, by using a second-round knockout and cloning strategy, we could save up to 6 months and all cloned piglets would be α1,3GT double knockout (DKO). We have selected and enriched for α1,3GT DKO cells by using a bacterial toxin, toxin A from Clostridium difficile, which binds with high affinity to α1,3Gal epitopes and produces a cytotoxic effect on cells that are α1,3Gal-positive (9). Toxin A uses α1,3Gal epitopes as a cell surface receptor and causes "rounding" and lifting of the α1,3Gal-positive cells from the surface of the growth vessel (10, 11).Heterozygous α1,3GT knockout fetal fibroblasts, 657A-I11 1-6 cells, were isolated from a day-32 pregnancy as described in (7). To avoid using a second antibiotic-resistance gene as a selection marker, we constructed an ATG (start codon)-targeting α1,3GT knockout vector, pPL680 (12), which also contains a neo gene, to knock out the second allele of the α1,3GT gene. 657A-I11 1-6 cells were transfected by electroporation with pPL680 and selected for the α1,3Gal-negative phenotype with purified C. difficile toxin A (13). One colony (680B1) was isolated and expanded af...

The chimeric nature of the transplanted liver was first shown in our long-surviving human recipients of orthotopic hepatic allografts in 1969. 1 When liver grafts were obtained from cadaveric donors of the opposite sex, karyotyping studies showed that hepatocytes and endothelium of major blood vessels retained their donor specificity, whereas the entire macrophage system, including Kuppfer cells, was replaced with recipient cells. 2 Where donor cells that had left the liver had gone was unknown, but their continued presence was confirmed by the acquisition and maintenance in recipient blood of new donor-specific immunoglobulin (Gm) types 1,3 and red-blood-cell alloantibodies, if donors with ABO non-identity were used. 4 Davies et al 5 attributed the secretion of new soluble HLA class I antigens of donor type to transplanted hepatocytes. However, these HLA molecules come from bone-marrow-derived macrophages and/or dendritic cells, 6 and probably have the same origin from migrated donor cells as the additional Gm types and red-cell antibodies.Although this early evidence of systemic mixed allogeneic chimerism was circumstantial, we have recently shown with both anatomical and molecular techniques the presence, in clinically stable patients, of peripherally located donor cells many years after liver replacement. For instance, in patients with type IV glycogen storage disease, a disorder in which an insoluble amylopectin-like polysaccharide accumulates throughout the body because of a deficiency in a branching enzyme, we found resorption of extrahepatic amylopectin after liver replacement. 7 This process could not be explained until the migrated donor cells, which had acted as enzyme couriers, were identified by both HLA monoclonal antibodies (fig 1) and polymerase chain reaction (PCR) studies (fig 2) in the biopsied myocardium and skin of 2 patients, 33 and 91 months after hepatic transplantation.Recent experiments in rats have shown the timing and extent of seeding from the hepatic allograft to both non-lymphoid and lymphoid organs (fig 3). 8 A similar pattern of distribution was found after successful rat-to-mouse bone-marrow transplantation. 9 This similarity between liver transplantation and bone-marrow transplantation has not been reported before. The prompt development, and then the persistence, of this systemic chimerism may help to explain the resistance of the liver to cellular 10 and humoral 11 rejection, as well as its tolerogenicity to other organs from the same donor. 12 The chimeric structure of the transplanted liver was thought to be a unique feature of this organ for many years until we identified lymphoid and dendritic cell replacement under FK 506 immunosuppression in rat 13 and human 14 intestinal allografts; a similar finding has been reported in swine. 15 In our experiments with rats, the two-way traffic was the same, irrespective of whether bowel was transplanted alone or as a part of a multivisceral graft that also contained Correspondence to

The feasibility of hepatic homotransplantation has been clearly established in principle inasmuch as several animals are still alive almost 3 years after complete hepatectomy and liver replacement. Both orthotopic and auxiliary operations are complicated surgical techniques. Nevertheless, the results in dogs are comparable to those which can be obtained with homotransplantation of the kidney.In man the problem is more difficult. In patients who have a need for such operations, there is invariably a metabolic disorder more complex than that caused by renal failure. In addition, the new organ must function efficiently from the beginning since its complete functional failure leads to death within a few hours. There is no recourse to an artificial liver to maintain life until the reversal of an injury which is caused by either ischemia or rejection.Nevertheless, research of several kinds may soon make possible the successful use of hepatic transplantation procedures for the definitive treatment of human liver disease as exemplified by the reports in this symposium concerning new techniques of organ preservation, histocompatibility analysis, and immunosuppression.

Tacrolimus, a novel macrocyclic lactone with potent immunosuppressive properties, is currently available as an intravenous formulation and as a capsule for oral use, although other formulations are under investigation. Tacrolimus concentrations in biological fluids have been measured using a number of methods, which are reviewed and compared in the present article. The development of a simple, specific and sensitive assay method for measuring concentrations of tacrolimus is limited by the low absorptivity of the drug, low plasma and blood concentrations, and the presence of metabolites and other drugs which may interfere with the determination of tacrolimus concentrations. Currently, most of the pharmacokinetic data available for tacrolimus are based on an enzyme-linked immunosorbent assay method, which does not distinguish tacrolimus from its metabolites. The rate of absorption of tacrolimus is variable with peak blood or plasma concentrations being reached in 0.5 to 6 hours; approximately 25% of the oral dose is bioavailable. Tacrolimus is extensively bound to red blood cells, with a mean blood to plasma ratio of about 15; albumin and alpha 1-acid glycoprotein appear to primarily bind tacrolimus in plasma. Tacrolimus is completely metabolised prior to elimination. The mean disposition half-life is 12 hours and the total body clearance based on blood concentration is approximately 0.06 L/h/kg. The elimination of tacrolimus is decreased in the presence of liver impairment and in the presence of several drugs. Various factors that contribute to the large inter- and interindividual variability in the pharmacokinetics of tacrolimus are reviewed here. Because of this variability, the narrow therapeutic index of tacrolimus, and the potential for several drug interactions, monitoring of tacrolimus blood concentrations is useful for optimisation of therapy and dosage regimen design.

SummaryPost-transplant lymphomas or other lymphoproliferative lesions, which were usually associated with Epstein-Barr virus infections, developed in 8, 4, 3, and 2 recipients, respectively, of cadaveric kidney, liver, heart, and heart-lung homografts. Reduction or discontinuance of immunosuppression caused regression of the lesions, often without subsequent rejection of the grafts. Chemotherapy and irradiation were not valuable. The findings may influence policies about treating other kinds of posttransplantation neoplasms.

The treatment of diabetes mellitus by transplantation of isolated pancreatic islets is an approach that remains the subject of research by a large number of investigators throughout the world. A crucial requirement for the success of this enterprise is the ability to prepare viable isolated islets in adequate quantity. Over the years numerous descriptions of procedures for islet isolation from the pancreas of experimental animals and of man have been advanced; each claiming to be an improvement on previous methods. Indeed, there certainly have been advances, although few techniques live up to the claims that are made in their support Part of the problem is the generally poor methodology

The blood coagulation system of 66 consecutive patients undergoing consecutive liver transplantations was monitored by thrombelastograph and analytic coagulation profile. A poor preoperative coagulation state, decrease in levels of coagulation factors, progressive fibrinolysis, and whole blood clot lysis were observed during the preanhepatic and anhepatic stages of surgery. A further general decrease in coagulation factors and platelets, activation of fibrinolysis, and abrupt decrease in levels of factors V and VIII occurred before and with reperfusion of the homograft. Recovery of blood coagulability began 30-60 min after reperfusion of the graft liver, and coagulability had returned toward baseline values 2 hr after reperfusion. A positive correlation was shown between the variables of thrombelastography and those of the coagulation profile. Thrombelastography was shown to be a reliable and rapid monitoring system. Its use was associated with a 33% reduction of blood and fluid infusion volume, whereas blood coagulability was maintained without an increase in the number of blood product donors. Keywords BLOOD-coagulation; LIVER-transplantationLiver transplantation, which began as highly experimental surgery 20 yr ago, is now recognized as a major means of therapy for patients with end-stage liver disease (1). However, massive blood loss during liver transplantation is still a major concern. The liver produces most of the blood coagulation factors, so it is not surprising that we see very low levels of these factors and prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT) in many patients receiving liver transplants (2). Frequently we have seen thrombocytopenia, which may result from gastrointestinal bleeding, splenomegaly, or malnutrition (3). At the same time, numerous collateral channels and portal hypertension, together with increased capillary fragility, make maintenance of surgical hemostasis very difficult.Previous studies on the blood coagulation system in humans and in animals undergoing liver transplantation have demonstrated dilutional coagulopathy associated with massive blood transfusion, decreased fibrinogen levels, and thrombocytopenia (4,5). Fibrinolysis, beginning during the anhepatic stage of surgery and becoming "explosive" on reperfusion of the homograft, has been reported (6). A consumption coagulopathy accompanied by an increased level of fibrin degradation products appeared to play a major role (7). However, these observations were limited to a few patients in the early era of liver transplantation, and comprehensive information is still lacking.Another difficulty in the intraoperative management of liver transplantation has been actively monitoring the coagulation system and determining the appropriate treatment during dynamic blood volume changes. The use of the thrombelastograph (TEG) was suggested by von Kaulla et al. (4) and Howland et al. (8). However, the efficacy of thrombelastographic monitoring of blood coagulation during liver transplantatio...

ObjectiveThis study analyzed the incidence and timing of biliary tract complications after orthotopIc liver transplantation (OL Tx) in 1792 consecutive patients. These results were then compared with those of previously reported series. Finally, recommendations were made on appropriate management strategies. Summary Background DataTechnical complications after OL Tx have a significant Impact on patient and graft survival. One of the principle technical advances has been the standardization of techniques for biliary reconstruction. Nonetheless. biliary complications stili occur. A 1983 report from the University of Pittsburgh reported biliary complications in 19% of all transplants, and an update in 1987 reported biliary complications In 13.2% of transplants. MethodsThe medical records of all patients who underwent liver transplantation and were hospitalized between January 1,1988 and July 31.1991 were revieWed. The case matenal conSisted of the medical records of 217 patients treated for 245 biliary complICations. Results Primary biliary continuity was established by either choledochocholedochostomy over a T·tube(C-C, n = 129) or a Roux-en-Y choledochoJeJunostomy With an internal stent (C-RY, n = 85). The overall Incidence for biliary complication in this large senes was 11.5%. Strictures (n = 93) and bile leak (n = 58) were the most common complications (69.6%). Most biliary complications (n = 143, 66%) occurred Within the first 3 months after surgery. In general, leaks occurred early, and strictures developed later. Bile leaks were equally frequent In both C-C and CoRY (27.1 % and 25.9%, respectively): strictures were more common after a CoRY type of reconstruction (36.4% and 52.9%, respectively). Twenty-one patients died, an InCidence of 9.6%. Fifteen of the 21 biliary-related deaths were among patients treated for rejectIOn before the recognition of biliary tract pathologiC findings. 40