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H.-J. to wild-type or hDAF+/+lungs. However, GalTKO lungs adsorbed cytotoxic anti-non-Gal antibody and elaborated high levels of thrombin; their demise was associated with increased PVR, capillary congestion, intravascular thrombi and strong CD41 deposition not seen at earlier time points. == Conclusions == In summary, GalTKO lungs are substantially guarded from injury but, in addition to anti-non-Gal antibody and complement, platelet adhesion Rabbit polyclonal to GAPDH.Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) is well known as one of the key enzymes involved in glycolysis. GAPDH is constitutively abundant expressed in almost cell types at high levels, therefore antibodies against GAPDH are useful as loading controls for Western Blotting. Some pathology factors, such as hypoxia and diabetes, increased or decreased GAPDH expression in certain cell types and non-physiologic intravascular coagulation contribute to Gal-independent lung injury mechanisms. Keywords:alphaGal, antibodies, ex vivo lung perfusion, genetically engineered, hyperacute rejection, lung, swine, Xenotransplantation == Introduction == The shortage of human organ donors remains a major limitation to the field of transplantation [1], and might be solved using xenografts. Pigs are favoured as a potential xenograft donor source because this use of pigs causes little ethical controversy and the actual risk of retrovirus transmission to humans is usually thought to be substantially lower than originally perceived [2]. The major initial immunological obstacle to discordant pig-to-human solid organ transplantation is usually hyper-acute rejection (HAR). Many breakthroughs in concept and practice have significantly advanced HDAC-IN-7 the understanding of HAR over the past several decades [24]. As organs from unmodified pigs were evaluated in animal models, HAR was found to be primarily a consequence of the recipients pre-formed anti-pig antibodies binding on porcine vascular endothelial cells leading to complement activation, thrombosis and graft HDAC-IN-7 failure [5]. This process occurs within minutes to hours of human blood perfusion in porcine organs. Consequently, conventional strategies to delay or prevent hyper-acute rejection include anti-xenograft antibody removal and complement inhibition [611]. As the carbohydrate structure Galactose-(1,3)-Galactose (Gal) is usually recognized by over 80% of anti-pig antibodies found in man, genetically altered galactosyl transferase knock-out (GalTKO) pig organs have been developed [12,13]. Endothelium and parenchymal cells from GalTKO animals lack the Gal1,3Gal epitope. As predicted, pilot studies using heart and kidney [1416] and orthotopic lung [17] transplants in baboons showed that this Gal-TKO phenotype is usually associated with decreased antibody binding and reduced activation of the complement cascade. Here, we report results from ex vivo perfusion of lungs with human blood, which allow us to describe for the first time the mechanisms of organ failure associated with GalTKO lungs, compared with historical wild-type, hDAF-expressing and autologous reference groups. == Materials and methods == == Animals == GalTKO pigs (n = 5, 20 to 60 kg) were supplied by Immerge Biotherapeutics, Inc. (Boston, MA, USA). The lungs were harvested at the Transplant Biology Research Center in Boston as described previously [18,19] and below, and transported to University of Maryland with a cold ischemic time of four to 6 h. Results using transgenic pigs homozygous for human decay-accelerating factor (hDAF+/+, Novartis Pharma, Basel, Switzerland, n = 13, 12 to 18 kg) [11] and some of the wild-type (WT) and autologous lungs tested using the same model [20] have previously been reported. All procedures were approved by the University of Maryland Animal Care and Use Committee, and in compliance with guidelines from NIH publication 86 to 23, theGuide for the Care and Use of Animals. == Lung harvest == Animals were anesthetized with ketamine (10 mg/kg) (Ketaset; Fort Dodge Animal Health, Fort Dodge, IA, USA) HDAC-IN-7 and xylazine (1 mg/kg) (Rompun; Bayer Pharmaceuticals, Shawnee Mission, KS, USA), and kept under general anesthesia (isoflurane 0.5 to 3%) throughout the surgical procedure. After intubation, median sternotomy, and heparinization (500 models/kg), 0.5 mg prostaglandin E1(PGE1) (ProstinVR Pediatric; Pfizer; New York, NY, USA) was administrated directly into the right heart. The main pulmonary artery was cannulated and lungs were flushed with 50 ml/kg HDAC-IN-7 Perfadex (Vitrolife, Goteburg, Sweden) made up of 0.5 mg PGE1/l at 4 C). The heart-lung block was removed and placed on iced normal saline. The lungs were transported on ice in a partially inflated state. == Ex vivo lung perfusion model == The ex vivo pig lung perfusion was performed as described in detail previously [11], with the modification that a single lung (right or left) was selected for ex vivo perfusion with human or pig perfusate (~1 l/lung) prepared as described below. As previously reported [11,18,19,21] blood flow, pulmonary artery and airway pressures were monitored constantly and PVR was calculated using the equation: PVR (mmHg/ml/min) = pulmonary artery pressure/measured flow. Blood gas samples were collected from the pulmonary veins to assess for oxygenation throughout the experiments. A pop-off valve was installed and set at 50 cm H2O.