Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes

Background Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes. Methods The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/β4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons. Results Cells from GGTA1/CMAH/β4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Nonhuman primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased nonhuman primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/β4GalNT2. Conclusions Inactivation of the β4GalNT2 gene reduces human and nonhuman primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of nonhuman primates towards GGTA1/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/β4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the nonhuman primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/β4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering

Hepatic mitochondrial oxidative metabolism and lipid peroxidation in iron-loaded rats fed ethanol

The aims of this study were to determine whether chronic ethanol consumption potentiates mitochondrial lipid peroxidation or impairment of mitochondrial oxidative metabolism in rats with chronic iron overload. Experimental iron overload was induced by feeding rats a chow diet supplemented with 2.5% carbonyl iron. After 8 to 12 weeks, half of the iron-loaded and control animals were changed to a liquid diet containing ethanol for 4 to 5 weeks. The remaining animals were fed an isocaloric amount of diet containing dextrin-maltose instead of ethanol for 4 to 5 weeks. Iron-supplemented animals had a 20-fold increase in hepatic iron concentration as compared with controls. Iron and ethanol independently increased plasma alanine aminotransferase (ALT) levels (p < 0.05) while the combination resulted in an additive increase in ALT levels (p < 0.01). Although iron overload increased the levels of mitochondrial conjugated dienes and significantly reduced the mitochondrial respiratory control ratio, ethanol administration did not affect these parameters in animals with or without iron overload. Livers from iron-loaded rats that received ethanol showed mild to moderate steatosis with scattered necroinflammatory foci. There was no significant increase in necroinflammatory foci in the livers of the iron plus ethanol group as compared with the iron group. In conclusion, we have demonstrated an additive increase in hepatocellular injury when ethanol is fed to iron-loaded rats, as evidenced by an increase in plasma ALT level. However, there were no additive or synergistic effects of iron and ethanol on either mitochondrial lipid peroxidation or mitochondrial oxidative metabolism

Simultaneous Liver and Pancreas Transplantation in Patients With Cystic Fibrosis

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. 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. 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. 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