Xenotransplantation-the current status and prospects.

Introduction: There is a continuing worldwide shortage of organs from deceased human donors for transplantation into patients with end-stage organ failure. Genetically engineered pigs could resolve this problem, and could also provide tissues and cells for the treatment of conditions such as diabetes, Parkinson's disease and corneal blindness. Sources of data: The current literature has been reviewed. Areas of agreement: The pathobiologic barriers are now largely defined. Research progress has advanced through the increasing availability of genetically engineered pigs and novel immunosuppressive agents. Life-supporting pig kidneys and islets have functioned for months or years in nonhuman primates. Areas of controversy: The potential risk of transfer of a pig infectious microorganism to the recipient continues to be debated. Growing points: Increased attention is being paid to selection of patients for initial clinical trials. Areas timely for developing research: Most of the advances required to justify a clinical trial have now been met.

The Resurgence of Xenotransplantation.

There has been an upsurge of interest in xenotransplantation in recent years. This resurgence can attributed to a combination of factors. First, there has been a dramatic improvement in efficacy in several preclinical models, with maximum xenograft survival times increasing to 950 days for islets, 945 days for hearts, and 310 days for kidneys. Second, the rapid development of genome editing technology (particularly the advent of clustered regularly interspaced short palindromic repeats/Cas9) has revolutionized the capacity to generate new donor pigs with multiple protective genetic modifications; what once took many years to achieve can now be performed in months, with much greater precision and scope. Third, the specter of porcine endogenous retrovirus (PERV) has receded significantly. There has been no evidence of PERV transmission in clinical trials and preclinical models, and improved screening methods and new options for the treatment or even elimination of PERV are now available. Balancing these positive developments are several remaining challenges, notably the heavy and often clinically inapplicable immunosuppression required to prevent xenograft rejection. Nonetheless, the potential for xenotransplantation as a solution to the shortage of human organs and tissues for transplantation continues to grow.

Characterization of swine leucocyte antigen alleles in a crossbred pig to be used in xenotransplant studies.

We have characterized swine leucocyte antigen (SLA) classes I and II molecules of a domestic pig as a model for use in our xenotransplant program. Molecular characterization of the SLA classes I and II genes is critical to understanding the adaptive immune responses between swine and humans in the event of xenotransplantation. Seven swine leucocyte antigen genes (SLA-1, SLA-2, SLA-3, DQB1, DRB1, DQA and DRA) were analyzed and 15 alleles were identified. A novel DRA*w04re01 is reported for this limited polymorphic class II gene. The heterozygous haplotypes, Hp-32.0/35.0 and Hp-0.13/0.23 were deduced for our IU-pig model, for SLA classes I and II regions, respectively.

Reduced binding of human antibodies to cells from GGTA1/CMAH KO pigs.

Xenotransplantation using genetically modified pig organs could solve the donor organ shortage problem. Two inactivated genes that make humans unique from pigs are GGTA1 and CMAH, the products of which produce the carbohydrate epitopes, aGal and Neu5Gc that attract preformed human antibody. When the GGTA1 and CMAH genes were deleted in pigs, human antibody binding was reduced in preliminary analysis. We analyzed the binding of human IgM and IgG from 121 healthy human serum samples for binding to GGTA1 KO and GGTA1/CMAH KO peripheral blood mononuclear cells (PBMCs). We analyzed a sub population for reactivity toward genetically modified pig PBMCs as compared to chimpanzee and human PBMCs. Deletion of the GGTA1 and CMAH genes in pigs improved the crossmatch results beyond those observed with chimpanzees. Sorting the 121 human samples tested against the GGTA1/CMAH KO pig PBMCs did not reveal a distinguishing feature such as blood group, age or gender. Modification of genes to make pig carbohydrates more similar to humans has improved the crossmatch with human serum significantly.

Aminocaproic Acid (amicar) as an alternative to aprotinin (trasylol) in liver transplantation.

INTRODUCTION: This study compared clinical outcomes for a large number of liver transplant patients receiving intraoperative epsilon-aminocaproic acid (EACA), aprotinin, or no antifibrinolytic agent over an 8-year period. PATIENTS AND METHODS: Records for deceased donor liver transplants were reviewed. Data included antifibrinolytic agent, blood loss, early graft function, and postoperative complications. Study groups included low-dose aprotinin, high-dose aprotinin, EACA (25 mg/kg, 1-hour infusion), or no antifibrinolytic agent. RESULTS: Data were included for 1170 consecutive transplants. Groups included low-dose aprotinin (n = 324 [28%]), high-dose aprotinin (n = 308 [26%]), EACA (n = 216 [18%]), or no antifibrinolytic (n = 322 [28%]). EACA had the lowest intraoperative blood loss and required the fewest transfusions of plasma. Patients receiving no agent required the most blood transfusions. Early graft loss was lowest in the EACA group, and 90-day and 1-year patient survival rates were significantly higher for the low-dose aprotinin and EACA groups according to Cox regression. Complications were similar, but there were more episodes of deep vein thrombosis in patients receiving EACA. CONCLUSIONS: These results suggest that transitioning from aprotinin to EACA did not result in worse outcomes. In addition to decreased intraoperative blood loss, a trend toward improved graft and patient survival was seen in patients receiving EACA.

Closure of the abdominal wall with acellular dermal allograft in intestinal transplantation.

Loss of abdominal domain is a common problem in intestinal transplantation. Several surgical options are available perioperatively for abdominal wall reconstruction. This study reports the management and complications for intestinal transplant patients with abdominal wall closure either primarily or with foreign material. This single center study reviews the records of intestinal transplant patients between 2004 and 2010. Study outcomes included reoperation for dehiscence, hernia or enterocutaneous fistula. There were 37 of 146 patients (25%) who required implantation of foreign material at transplant. Of these 37, 30 (81%) had implantation of acellular dermal allograft (ADA) and 7 (19%) implantation of another mesh. Perioperative dehiscence was rare with 2/109 (2%) for primary closure, 0/30 (0%) for ADA and 1/7 (14%) for other mesh. There were 12/146 (8%) patients who underwent ventral hernia repair: primary closure 7/109 (6%), ADA 3/30 (10%) and other mesh 2/7 (28%). There were 4/146 (3%) patients who required surgery for enterocutaneous fistulas: 2/109 (2%) primary closure, 1/30 (3%) ADA and 1/7 (14%) synthetic mesh. Abdominal wall reconstruction with ADA biologic mesh provides an expeditious means of performing a tension-free closure of the fascial layer after intestinal transplantation with complications similar to those seen for primary closure.

Clinical experience with histidine-tryptophan-ketoglutarate solution in abdominal organ preservation: a review of recent literature.

Histidine-tryptophan-ketoglutarate (HTK) solution was introduced by Bretschneider as a cardioplegia solution in the early 1980s and has subsequently been applied to organ preservation for transplantation in Europe during the 1980s and in North America during the last six yr. With the increasing use of HTK for primary preservation, it is important that the transplant community be aware of the properties of this solution and the advantages and disadvantages of its use. Even if a center uses an alternative preservation solution, it is likely that import offers will be made for allografts that have been preserved in HTK. In this review article, recent literature describing the clinical use of HTK in abdominal transplantation will be summarized with references to earlier reports when indicated.

Predictors of blood product use in orthotopic liver transplantation using the piggyback hepatectomy technique.

UNLABELLED: Orthotopic liver transplantation (OLT) has historically been associated with massive blood loss and hemodynamic instability related to the coexistence of varices, coagulopathy, thrombocytopenia, and portal hypertension. Piggyback hepatectomy (PGB) is a technique increasingly utilized in OLT to avoid veno-venous bypass and vena cava clamping. This study evaluated the factors associated with blood loss and blood product requirement in PGB. METHODS: This study is a retrospective review of the anesthesia preoperative and operative notes and computerized lab values for all adult cadaveric liver transplants over a 42-month period. These data were combined with the liver transplant database for analysis. Approximately 98% of the transplants were performed using a standard piggyback approach with no use of veno-venous bypass. RESULTS: Data were included for all 526 transplants performed during this time period. Estimated blood loss (EBL) was 1000 cc. Median transfusion requirement was 3 units packed red blood cells, 7 units fresh frozen plasma, and 6 units platelets. Multivariate linear regression demonstrated that predictors of EBL were age, MELD score, preoperative hemoglobin, initial fibrinogen, initial central venous pressure, and total anesthesia time. Predictors of PRBC useage were age, MELD score, preoperative hemoglobin, initial fibrinogen, and anesthesia time. Postoperatively increased transfusion requirement was associated with increased length of hospital stay and lower 90-day and 1-year graft and patient survivals. CONCLUSION: These results demonstrate that PGB can be safely accomplished in nearly all liver transplant patients without venovenous bypass or vena cava clamping and with less warm ischemia, which may ultimately be associated with less perioperative morbidity and improved outcomes.

In vitro monitoring of in vivo development of human anti-thymoglobulin antibodies by ELISA.

Thymoglobulin (rATG), polyclonal immunoglobulin, is prepared from rabbits immunized with human thymocytes. It is effective in prevention and treatment of renal allograft rejection. Human antibodies against antilymphocyte preparations can reduce efficacy by accelerating drug clearance or by inducing serum sickness. We developed an enzyme-linked immunosorbent assay (ELISA) to study posttreatment development of anti-rATG. In an Institutional Review Board-approved trial, we tested 101 allograft recipients for anti-rATG antibodies. Patients received rATG intravenously at 1.25 to 2.0 mg/kg/d for 2 to 14 days. Serum samples were obtained pretreatment and at weeks 1, 2, 4, 6, and months 3 and 6 post-rATG. ELISA plates were coated with rATG (10 microg/mL). Samples were diluted 1:100 and tested in quadruplicate. Positive samples were titrated. Horseradish peroxidase-conjugated (HRPO) affinity-purified goat anti-human immunoglobulin G (H&L) antibody reacted with bound human antibody. A chromagenic substrate for HRPO was added and optical density (OD, 490 nm) was read. An OD of twice the negative control was considered positive. Mean ODs of negative and positive controls were 0.113 +/- 0.030 and 1.042 +/- 0.196, respectively. Ten patients had detectable anti-rATG before rATG administration (1:100). Thirty-five of 101 patients (35%) developed anti-rATG antibody. Patients showed an initial positive anti-rATG antibody from days 8 to 59 after infusion and titers from 1:100 to 1:4000. In spite of rATG's postulated anti-B-cell activity, this study confirms that rATG induces sensitization at a frequency and titer seen with other xenogeneic antilymphocyte antibodies. Formation of such antixenoantibodies can have a negative impact on treatment response and hence warrant monitoring.

Simultaneous liver and pancreas transplantation in patients with cystic fibrosis.

BACKGROUND: Improved survival in patients with cystic fibrosis (CF) has led to an increased incidence of extrapulmonary complications of this disease. Of these, cirrhosis and pancreatic insufficiency, including CF-related diabetes (CFRD) and exocrine insufficiency, are significant causes of morbidity and mortality. Liver transplantation is the treatment of choice for cirrhosis in this setting, but the addition of an isolated simultaneous pancreas transplant in patients with CFRD has not been reported. METHODS: Two female patients with CF underwent simultaneous pancreas and liver transplantation. Both had pancreatic insufficiency, CFRD, cirrhosis, and preserved renal function. In each case, the liver and pancreas were procured from a single cadaveric donor. The liver transplant was performed first. A lower midline extension was added for improved exposure of the iliac vessels. The donor pancreas transplant was performed with systemic venous drainage and enteric exocrine drainage. Immunosuppression included rabbit anti-thymocyte globulin, tacrolimus, mycophenolate mofetil, and early steroid withdrawal. RESULTS: Both patients recovered well with normal liver function, resolution of portal hypertension, and normal blood glucoses independent of insulin. As a result of the enteric exocrine drainage of the pancreas, they are now independent of supplemental pancreatic enzymes. CONCLUSIONS: Simultaneous liver and pancreas transplantation in CF patients provides the advantages of normalization of glucose and improved nutrition for patients requiring liver transplantation and should be considered in CF patients with CFRD who require liver transplants.

False-positive Histoplasma antigenemia caused by antithymocyte globulin antibodies.

False-positive Histoplasma antigen results were identified in two patients who received rabbit antithymocyte globulin (ATG, Thymoglobulin(R)) to prevent allograft rejection. To determine the prevalence of false-positive results following the administration of Thymoglobulin, sequential specimens were tested from a cohort of transplant recipients. Of 107 such patients, 17 (15.9%) demonstrated false-positive tests for Histoplasma antigenemia. False antigenemia peaked at 2-4 weeks after ATG administration and cleared over the next few months. Physicians should be aware of the potential for false-positive results in specimens from patients who have received ATG.

HeartMate VE LVAS design enhancements and its impact on device reliability.

OBJECTIVE: The HeartMate VE left ventricular assist system (LVAS) has supported more than 2300 patients and has been shown to be effective for bridge to cardiac transplantation and has demonstrated improved outcomes in survival as a destination therapy. Improvements in device durability are needed as bridge to transplant times increase and as we move into the era of LVAD as destination therapy. The purpose of this study is to determine if design enhancements to the HeartMate LVAS have improved device reliability and durability. METHODS: A retrospective analysis of serious mechanical failures was performed in 1865 devices (1458 VE, 407 XVE). The analysis of data included devices used to support patients from September 1998 for bridge to transplantation and destination therapy. Serious mechanical failures were defined as inflow valve dysfunction, percutaneous lead breaks, diaphragm fractures or punctures, bearing failures, outflow graft erosion and pump disconnects. RESULTS: Median device duration for the VE was 97 days (max 1206 days), and 85 days (max 517 days) for the XVE. A total of 134 serious mechanical failures occurred and included inflow valve dysfunction (5.3% VE, 2.4% XVE) (P = 0.853) percutaneous lead breaks (1.9% VE, 0% XVE) (P < 0.001) diaphragm fractures (0.1% VE, 0% XVE) (P = 0.134) outflow graft erosion (0.2% VE, 0% XVE) (P = 0.1096), pump disconnects (0.1% VE, 0% XVE) (P = 0.1336) and bearing failures (0.6% VE, 0.2% XVE) (P = 0.5538). Of the XVEs 97% were free of serious mechanical failures at 6 months and 82% at 1 year compared to 92 and 73% for the VE, respectively. The 6-month difference between the devices was statistically significant (P = 0.0063) and there was no statistically significant difference at 1 year (P = 0.1492). CONCLUSIONS: Preliminary experience with the HeartMate XVE LVAS demonstrated a significant reduction in percutaneous lead breaks. Early trends indicate positive impact of recent design modifications on XVE performance. These design modifications may improve device durability and reliability, which is crucial as we enter the era of LVADs as an alternative to medical therapy.

Purely internal thoracic artery grafts: outcomes.

BACKGROUND: Most of our patients with coronary artery disease have undergone bypass exclusively with purely internal thoracic artery grafts (PITA). Our goal has been to lengthen the time a patient benefits from coronary bypass operations. The present report describes an 8.5-year study of outcomes including mortality and the need for reintervention in patients who have undergone bypass with PITA. METHODS: We studied 897 patients who underwent PITA with a total of 3,784 internal thoracic artery (ITA) grafts (4.2 grafts per patient). Connecting ITA to ITA along with sequential anastomosis made the procedure possible. RESULTS: Early mortality for the group was 2.3%. Freedom from death was 86% and freedom from reintervention was 94% at 5 years after the operation. CONCLUSIONS: The acceptable early and late mortality and the 94% freedom from reintervention as long as 8.5 years after operation in this group of patients inspire us to continue choosing PITA for patients with three-vessel coronary artery disease.

Rejection of pig liver xenografts in patients with liver failure: implications for xenotransplantation.

The pathophysiological state of rejection in liver xenotransplantation is poorly understood. Data from clinical pig liver perfusion suggest that pig livers might be rejected less vigorously than pig hearts or kidneys. Pig livers used in clinical xenoperfusions were exposed to blood from patients with liver failure. We have shown in an animal model that transplant recipients with liver failure are less capable of initiating hyperacute rejection of a xenografted liver than a healthy transplant recipient. The goal of this report is to examine the pathological characteristics of pig livers used in 2 clinical pig liver perfusions and combine this information with in vitro studies of pig-to-human liver xenotransplantation to determine whether the findings in the perfused pig livers could be explained in part by the diminished capacity of the patient with liver failure to respond to xenogeneic tissue. Pathological analysis of the perfused pig livers showed immunoglobulin M deposition in the sinusoids with little evidence of complement activation. Our in vitro studies showed that serum from patients with liver failure caused less injury to pig liver endothelium than serum from healthy subjects. Serum from patients with liver failure had similar levels of xenoreactive antibodies as serum from healthy humans. Incubation of serum from patients with liver failure with pig hepatic endothelial cells generated less iC3b, Bb fragment, and C5b-9 than serum from healthy subjects. We conclude that the altered injury in the perfused pig livers can be attributed to the relative complement deficiency that accompanies liver failure.

Experimental discordant hepatic xenotransplantation in the recipient with liver failure: implications for clinical bridging trials.

BACKGROUND: Clinical xenotransplantation might start with bridge-to-bridge trials. Situations where hyperacute rejection is avoided would provide opportunities for the initiation of bridging trials. Patients with liver failure have a diminished capacity to initiate antibody and complement-induced injury of xenogeneic endothelium. Hyperacute rejection of a liver xenograft manifests as a coagulopathy. We examined the ability of a recipient with liver failure to hyperacutely reject a liver xenograft in the dog-to-pig model in the immediate postoperative period. STUDY DESIGN: Liver failure in pigs was induced with galactosamine. Canine livers were transplanted into pigs with liver failure and into healthy pigs. The postoperative course was monitored for 1 hour for histologic changes in the xenograft, changes in platelet counts, and whole blood clotting with Sonoclot analysis. In vitro assays with pig serum and canine hepatic sinusoidal endothelial cells were used to assess the effect of liver failure on serum cytotoxicity and xenoreactive antibody levels. RESULTS: All untreated pig recipients of liver xenografts died from a coagulopathy. Recipients with liver failure manifested no signs of coagulopathy, and had minimal change in platelet counts or Sonoclot (Sienco Inc., Morrison, CO) tracings. Liver xenograft biopsies from recipients with liver failure showed no evidence of the tissue injury that characterized the biopsies of control recipients. Serum from pigs was less cytotoxic to the canine hepatic sinusoidal endothelium after induction of liver failure. The xenoreactive antibody levels and repertoire were similar in the pig serum before and after liver failure was induced. CH50 (total complement) levels were diminished in pigs after the induction of liver failure. CONCLUSIONS: Liver xenotransplantation used in bridging trials in recipients with liver failure might not face the barrier of hyperacute rejection.

A native soluble form of CTLA-4.

CTLA-4 is an immunoregulatory receptor expressed on the surface of activated T and B lymphocytes. The counterreceptors for CTLA-4 are the B7 family molecules. We describe alternatively spliced mRNAs expressed in hematolymphoid tissues of humans, mice, and rats that lack the transmembrane domain coded by exon 3 of the CTLA-4 gene. These alternate transcripts were detected by RT-PCR in B cells and resting T cells of both the CD4 and the CD8 phenotype. Activation of human blood mononuclear cells with PHA or anti-CD3 + anti-CD28 monoclonal antibodies appears to effect a decrease in the amount of the alternative transcript relative to the full-length transcript. Recombinant sCTLA-4 is a B7-binding protein and has immunomodulatory effects as measured by inhibition of the mixed leukocyte response. Human serum contains immunoreactive material consistent with a native soluble form of CTLA-4.

Pulmonary injury in recipients of discordant hepatic and renal xenografts in the dog-to-pig model.

Work in our lab demonstrated that the early post-operative course of discordant hepatic and renal xenotransplantation is complicated by a pulmonary injury. The aim of this study was to characterize the nature of this injury, as well as to determine whether endothelin-1 (ET-1) and inducible-nitric oxide synthase (iNOS) are present in this form of pulmonary injury. Dog-to-pig orthotopic liver and kidney xenografts were performed. Pulmonary artery pressures were monitored throughout all procedures. The lungs were stained with monoclonal antibodies for ET-1, endothelin converting enzyme-1, and iNOS. The lungs from pig recipients of hepatic or renal xenografts were compared to lungs from untreated pigs. Pulmonary artery pressures were elevated in recipients of liver xenografts when the suprahepatic caval cross clamp was placed and continued to rise to systolic levels following unclamping. The mean pulmonary artery pressures in recipients of renal and hepatic xenografts rose significantly following revascularization. Pathology in lungs from kidney and liver recipients was similar, showing congestion with peribronchial and septal edema, with diffuse adhesion of PMN to alveolar endothelium. ET-1, endothelin converting enzyme-1 (ECE-1), and iNOS staining was widespread and intense in alveolar and pulmonary arterial endothelium. Discordant xenotransplantation of livers and kidneys is associated with a significant early pulmonary injury that is associated with early PMN infiltration and expression of ET-1 and iNOS.