Serum alanine transaminase (ALT) concentration was measured using a Chemistry Analyser AU2700 (Olympus, Tokyo, Japan). Serum IL-6 concentration was measured by flow cytometry using the mouse inflammation selleck cytometric bead array (BD Biosciences) following the manufacturer’s instructions. Liver grafts were immersed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Whole sections were analyzed and scored following Suzuki’s method26 by a histopathologist blinded to treatment groups. Apoptosis was assessed using rabbit anti-cleaved caspase-3 antibody [Asp175] (Cell Signaling Technology, Danvers, MA). Sections were analyzed and apoptosis scored

according to percentage of central vein involvement by a histopathologist blinded to treatment groups. The computer software GraphPad Prism (v. 5, La Jolla, CA) was used for all statistical analyses and graphical representations, except for FACS plots and histograms, which were generated using FlowJo software. For statistical significance analyses, the unpaired Student t

test was used to give the stated P values. P < 0.05 was considered significant. To assess the impact of CD39 overexpression on hepatic IRI, donor livers (CD39tg and WT littermates) were subjected MAPK Inhibitor Library clinical trial to 18 hours of cold ischemia and transplanted into WT recipient mice. At 6 hours post reperfusion, CD39tg livers were significantly protected against IRI compared to WT donor livers, as reflected by serum ALT concentrations of 10,018 U/L (±653) this website and 15,638 U/L (±1,513), respectively (Fig. 1A). The level of systemic proinflammatory cytokine IL-6 was also significantly reduced in recipients of CD39tg donor livers (2,396 pg/mL ± 438) compared to WT livers (4,336 pg/mL ± 647) (Fig. 1B). Histologically, large necrotic areas were evident in WT donor livers, whereas the hepatic architecture of CD39tg donor livers was better preserved with well-defined, smaller, and nonconfluent necrotic areas. Hepatocellular damage was graded according to Suzuki’s histological score26 (Supporting

Fig. 1A): five out of six WT donor livers showed mild to severe congestion and vacuolization associated with more than 30% hepatocyte necrosis (score 2 or greater) compared to only two out of six CD39tg livers (Fig. 1C). Further, there were significantly fewer central veins surrounded by cleaved caspase-3 positive cells in donor livers from CD39tg mice (Fig. 1D; Supporting Fig. 1B). CD39 transgene expression in donor livers was confirmed by real-time polymerase chain reaction (PCR) (Supporting Fig. 2A) and flow cytometric analysis on donor liver lymphocytes (Supporting Fig. 2B). To determine whether the protection observed was due to the expression of CD39 transgene on the liver parenchyma or on the resident hepatic immune cells, bone marrow adoptive transfer experiments were performed.