Induction of hepatitis was associated with a time-dependent up-regulation of hepatic PBEF messenger RNA (mRNA) expression. As shown in Fig. 2A, PBEF expression

was induced 4.4-fold after 2 hours, 15.5-fold after 4 hours, and peaked after 6 hours showing a 46.3-fold expression. mRNA selleck chemical data were confirmed by way of western blot analysis (Fig. 2B). In accordance with the mRNA data, protein expression peaked after 6 hours. Cellular origins were determined by way of immmunofluorescent microscopy. Apart from hepatocytes, PBEF colocalized with F4/80-positive Kupffer cells (Fig. 2C) and CD31-positive liver sinusoidal endothelial cells (Fig. 2D). To study the role of mouse PBEF in mediating ConA-induced hepatitis, we first introduced the PBEF gene into mouse livers using hydrodynamic delivery. Hydrodynamic perfusion represents an effective method for in vivo gene transfer into mouse livers.27 The mouse PBEF gene was cloned into a pCI-neo

mammalian expression vector constitutively expressing murine PBEF gene under control of the cytomegalovirus immediate-early enhancer/promoter region (pCI-Pbef1). The same vector containing a nonsense sequence was used as a control (pCI-Ctrl). Quantitative reverse-transcription GS-1101 in vivo polymerase chain reaction (RT-PCR) analysis showed that PBEF mRNA was efficiently overexpressed in mouse livers 24 hours after injection (Supporting Fig. 1A). RT-PCR results were further confirmed by way of western blot analysis (Supporting Fig. 1B). Of note, circulating PBEF concentrations in pCI-Pbef1–injected animals were increased eight-fold compared with pCI-control injected mice (Supporting Fig. 1C). Experiments included four groups: (1) mice receiving the PBEF1-overexpressing plasmid (pCI-Pbef1) and 12.5 mg/kg ConA (Fig. 3A), (2) mice receiving the control plasmid (pCI-Ctrl) and mafosfamide 12.5 mg/kg ConA (Fig. 3B), (3) mice injected with the pCI-Pbef1 and saline (Fig. 3C), and (4) mice injected with the pCI-Ctrl

and saline (Fig. 3D). With respect to liver pathology, we found more severe lesions in group 1 (pCI-Pbef1 + ConA) compared with group 2 (pCI-Ctrl + ConA) (Fig. 3A,B). No necrotic areas were present in saline-injected control mice (group 3 and group 4) (Fig. 3C,D). Correspondingly, liver damage as measured by the release of liver enzymes (AST and ALT) was significantly higher in group 1 (pCI-Pbef1 + ConA) compared with group 2 (pCI-Ctrl + ConA) (Fig. 3E). AST and ALT tended to be higher in group 3 (pCI-Pbef1 + saline) versus group 4 (pCI-Ctrl + saline), with P values of 0.108 and 0.124, respectively (Fig. 3E). Potential differences in hepatic inflammatory cell infiltration were determined by way of immunohistochemistry. A vivid, comparable T cell infiltration was found in group 1 (pCI-Pbef1 + ConA) and group 2 (pCI-Ctrl + ConA), but not in ConA-naïve groups 3 and 4 (Supporting Fig. 1D).