B cell phenotypes in baboons with pig artery patch grafts receiving conventional immunosuppressive therapy.

BACKGROUND: In the pig-to-baboon artery patch model with no immunosuppressive therapy, a graft from an α1,3-galactosyltransferase gene-knockout (GTKO) pig elicits a significant anti-nonGal IgG response, indicating sensitization to the graft. A costimulation blockade-based regimen, e.g., anti-CD154mAb or anti-CD40mAb, prevents sensitization. However, neither of these agents is currently FDA-approved. The aim of the present study was to determine the efficacy of FDA-approved agents on the T and B cell responses. METHODS: Artery patch xenotransplantation in baboons was carried out using GTKO/CD46 pigs with (n = 2) or without (n = 1) the mutant transgene for CIITA-knockdown. Immunosuppressive therapy consisted of induction with ATG and anti-CD20mAb, and maintenance with different combinations of CTLA4-Ig, tacrolimus, and rapamycin. In addition, all 3 baboons received daily corticosteroids, the IL-6R blocker, tocilizumab, at regular intervals, and the TNF-α blocker, etanercept, for the first 2 weeks. Recipient blood was monitored for anti-nonGal antibody levels by flow cytometry (using GTKO/CD46 pig aortic endothelial cells), and mixed lymphocyte reaction (MLR). CD22+B cell profiles (naïve [IgD+/CD27-], non-switched memory [IgD+/CD27+], and switched memory [IgD-/CD27+] B cell subsets) were measured by flow cytometry. At 6 months, the baboons were euthanized and the grafts were examined histologically. RESULTS: No elicited anti-pig antibodies developed in any baboon. The frequency of naïve memory B cells increased significantly (from 34% to 90%, p = 0.0015), but there was a significant decrease in switched memory B cells (from 17% to 0.5%, p = 0.015). MLR showed no increase in the proliferative T cell response in those baboons that had received CTLA4-Ig (n = 2). Histological examination showed few or no features of rejection in any graft. CONCLUSIONS: The data suggest that immunosuppressive therapy with only FDA-approved agents may be adequate to prevent an adaptive immune response to a genetically-engineered pig graft, particularly if CTLA4-Ig is included in the regimen, in part because the development of donor-specific memory B cells is inhibited.

Silencing porcine genes significantly reduces human-anti-pig cytotoxicity profiles: an alternative to direct complement regulation.

UNLABELLED: The future of solid organ transplantation is challenged by an increasing shortage of available allografts. Xenotransplantation of genetically modified porcine organs offers an answer to this problem. Strategies of genetic modification have 'humanized' the porcine model towards clinical relevance. Most notably, these approaches have aimed at either antigen reduction or human transgene expression. The object of this study was to evaluate the relative effects of both antigen reduction and direct complement regulation on the human-anti-porcine complement dependent cytotoxicity response. Genetically modified animals were created through CRISPR/Cas9-directed mutation and human transgene delivery. Pigs doubly deficient in GGTA1 and CMAH genes were compared to pigs of the same background that expressed a human complement regulatory protein (hCRP). A third animal was made deficient in GGTA1, CMAH and B4GalNT2 gene expression. Cells from these animals were subjected to measures of human antibody binding and antibody-mediated complement-dependent cytotoxicity by flow cytometry. Human IgG and IgM antibody binding was unchanged between the double knockout and the transgenic hCRP double knockout pig. IgG and IgM binding was reduced by 49.1 and 43.2 % respectively by silencing the B4GalNT2 gene. Compared to the double knockout, human anti-porcine cytotoxicity was reduced by 8 % with the addition of a hCRP (p = .032); It was reduced by 21 % with silencing the B4GalNT2 gene (p = .012). CONCLUSIONS: Silencing the GGTA1, CMAH and B4GalNT2 genes in pigs achieved a significant antigen reduction. Changing the porcine carbohydrate profile effectively mediates human antibody-mediated complement dependent cytoxicity.

Alcohol abuse in deceased liver donors: impact on post-transplant outcomes.

BACKGROUND: Many deceased liver donors with a history of alcohol abuse are excluded based upon medical history alone. This paper summarizes the transplant outcomes for a large number of deceased liver donors with a documented history of alcohol abuse. METHODS: The records for 1478 consecutive deceased liver donors were reviewed (2001-2012). As per the United Network for Organ Sharing criteria, heavy alcohol use by an organ donor is defined as chronic intake of two or more drinks per day. Donors with a documented history of alcohol abuse were divided into three groups according to duration of abuse (<10 years, 10-24 years and 25 + years). Reperfusion biopsies are reported. Outcomes include biopsy appearance, early graft function and early and late graft survival. RESULTS: There were 161 donors with alcohol abuse: <10 years (29%); 10-24 years (42%); and ≥25 years (29%). Risk of 90-day graft loss for these three groups was: 0%, 3% and 2%, compared to 3% for all other donors (P = 0.62). Graft survival at 1 year for donor grafts with and without alcohol abuse was 89% and 87% (P = 0.52). There was no difference in early graft function. Cox proportional hazards modelling for graft survival demonstrates no statistically significant difference in survival up to 10 years post-transplant. CONCLUSIONS: This study demonstrates successful transplantation of a large number of deceased donor liver grafts from donors with a documented history of alcohol abuse (n = 161; 11% of all grafts). These extended criteria donor allografts may, therefore, be utilized successfully with similar outcomes.

Role of cardiac catheterization and percutaneous coronary intervention in the preoperative assessment and management of patients before orthotopic liver transplantation.

Limited data regarding the optimal risk assessment strategy for evaluating candidates for orthotopic liver transplantation (OLT) exist. Our center has adopted a policy of performing cardiac catheterization (CATH) in patients with predefined risk factors, and this is followed by percutaneous coronary intervention (PCI) when it is indicated, even in the presence of negative stress test findings. The aim of this single-center, retrospective study of all patients who underwent OLT between 2000 and 2010 was to assess the effect of our policy on cardiovascular (CV) complications and survival rates after OLT. Data, including 1-year all-cause and CV mortality, postoperative myocardial infarctions (MIs), and frequencies of CATH and PCI, were abstracted. The study was divided into 3 subperiods to reflect the changes in policy over this period: (A) 2000-2004, (B) 2005-2008, and (C) 2009-2010. One thousand two hundred twenty-one patients underwent OLT between 2000 and 2010. The rate of catheterization increased during the 3 time periods (P < 0.001), as did the rate of PCI (P < 0.05). All-cause mortality decreased over the periods (P < 0.001), as did the MI rate (P < 0.001). Thirty-five of the 57 patients requiring PCI had normal stress tests. The mortality rate associated with postoperative MIs was significantly higher than the overall all-cause mortality rate. In conclusion, a significant improvement in the overall survival rate over the 3 analyzed time periods was noted. Increases in the frequencies of CATH and PCI corresponded to significant reductions in postoperative MIs and 1-year all-cause mortality rates. The increased use of CATH and PCI was associated with reduced overall all-cause mortality through reductions in the incidence of both fatal and nonfatal MIs. Further analyses of the role of stress testing and CATH in evaluating and treating patients before OLT are required to optimize this process.

A new vascular approach to the modified multivisceral graft procurement.

Modified multivisceral (MMV) transplantation including the stomach, pancreaticoduodenal complex and intestine requires preservation of the left gastric and splenic arteries. The previously described techniques require division of the hepatic artery proximal to the gastroduodenal artery leaving the liver transplant team with a very short and small caliber vessel. To increase MMV graft availability and provide the liver transplant team with an appropriate quality vessel, we developed the following technique. We also describe two cases where we used this method to support the technical feasibility of this procedure.

Addition of a total pancreatectomy and pancreas transplantation in a liver transplant recipient with intraductal papillary mucinous neoplasm of the pancreas.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMNs) of the pancreas are increasingly recognized in clinical practice. There are no published data suggesting a management approach for the potential organ transplant recipient with the incidental finding of this pre-malignant lesion. METHODS: The recipient was a 65-yr-old man with hepatocellular carcinoma in a background of Laennec's cirrhosis. During evaluation for transplant, he was found to have diffuse IPMN. This patient underwent liver transplantation with a planned simultaneous total native pancreatectomy and pancreas transplant from the same donor. RESULTS: The patient has had normal liver function and has been free of diabetes and exocrine insufficiency at 18 months post-transplant. CONCLUSION: IPMN in the potential transplant recipient should be managed more aggressively than in the general population because these patients will be committed to life-long immunosuppression. If a total pancreatectomy is contemplated, this is a unique opportunity to replace the pancreas with an allograft at the time of transplantation.