Background Cholestatic liver diseases can be caused by genetic defects, drug toxicities, hepatobiliary malignancies or obstruction of the biliary tract. Cholestasis leads to accumulation of bile acids (BAs) in hepatocytes. Direct toxicity of BAs is currently the most accepted hypothesis for cholestatic liver injury. However, information on which bile acids are actually accumulating during cholestasis is limited. Aims Assess BA composition in liver and serum after bile duct ligation (BDL) in male C57Bl/6 mice between 6 h and 14 days and evaluate toxicity of most abundant BAs. Results BA concentrations increased in liver (27-fold) and serum (1400-fold) within 6 h after surgery and remained elevated up to 14 days. BAs in livers of BDL mice became more hydrophilic than sham controls, mainly due to increased 6β-hydroxylation and taurine conjugation. Among the 8 unconjugated and 16 conjugated BAs identified in serum and liver, only taurocholic acid (TCA), β-muricholic acid (βMCA) and TβMCA were substantially elevated representing >95% of these BAs over the entire time course. Although glycochenodeoxycholic acid and other conjugated BAs increased in BDL animals, the changes were several orders of magnitude lower compared to TCA, βMCA and TβMCA. A mixture of these BAs did not cause apoptosis or necrosis but induced inflammatory gene expression in cultured murine hepatocytes. Conclusion The concentrations of cytotoxic BAs are insufficient to cause hepatocellular injury. In contrast, TCA, βMCA and TβMCA are able to induce pro-inflammatory mediators in hepatocytes. Thus, BAs act as inflammagens and not as cytotoxic mediators after BDL in mice.

Angioblasts are multipotent progenitor cells that give rise to arteries or veins . Genetic disruption of the gridlock gene perturbs the artery/vein balance, resulting in generation of insufficient numbers of arterial cells . However, within angioblasts the precise biochemical signals that determine the artery/vein cell-fate decision are poorly understood. We have identified by chemical screening two classes of compounds that compensate for a mutation in the gridlock gene . Both target the VEGF signaling pathway and reveal two downstream branches emanating from the VEGF receptor with opposing effects on arterial specification. We show that activation of ERK (p42/44 MAP kinase) is a specific marker of early arterial progenitors and is among the earliest known determinants of arterial specification. In embryos, cells fated to contribute to arteries express high levels of activated ERK, whereas cells fated to contribute to veins do not. Inhibiting the phosphatidylinositol-3 kinase (PI3K) branch with GS4898 or known PI3K inhibitors, or by expression of a dominant-negative form of AKT promotes arterial specification. Conversely, inhibition of the ERK branch blocks arterial specification, and expression of constitutively active AKT promotes venous specification. In summary, chemical genetic analysis has uncovered unanticipated opposing roles of PI3K and ERK in artery/vein specification.

Compound EGFR mutations, defined as double or multiple mutations in the EGFR tyrosine kinase domain, are frequently detected with advances in sequencing technology but its clinical significance is unclear. This study analyzed 61 cases of EGFR mutation positive lung adenocarcinoma using next-generation sequencing (NGS) based repeated deep sequencing panel of 16 genes that contain actionable mutations and investigated clinical implication of compound EGFR mutations. Compound EGFR mutation was detected in 15 (24.6%) of 61 cases of EGFR mutation-positive lung adenocarcinoma. The majority (12/15) of compound mutations are combination of the atypical mutation and typical mutations such as exon19 deletion, L858R or G719X substitutions, or exon 20 insertion whereas 3 were combinations of rare atypical mutations. The patients with compound mutation showed shorter overall survival than those with simple mutations (83.7 vs. 72.8 mo; P = 0.020, Breslow test). Among the 115 missense mutations discovered in the tested genes, a few number of actionable mutations were detected irrelevant to the subtype of EGFR mutations, including ALK rearrangement, BCL2L11 intron 2 deletion, KRAS c.35G>A, PIK3CA c.1633G>A which are possible target of crizotinib, BH3 mimetics, MEK inhibitors, and PI3K-tyrosine kinase inhibitors, respectively. 31 missense mutations were detected in the cases with simple mutations whereas 84 in those with compound mutation, showing that the cases with compound missense mutation have higher burden of missense mutations (P = 0.001, independent sample t-test). Compound EGFR mutations are detected at a high frequency using NGS-based repeated deep sequencing. Because patients with compound EGFR mutations showed poor clinical outcomes, they should be closely monitored during follow-up.

Hong JY, Lebofsky M, Farhood A, Jaeschke H. Oxidant stressinduced liver injury in vivo: role of apoptosis, oncotic necrosis, and c-Jun NH2-terminal kinase activation. Am J Physiol Gastrointest Liver Physiol 296: G572-G581, 2009. First published January 8, 2009 doi:10.1152/ajpgi.90435.2008.-Oxidant stress is critically involved in various liver diseases. Superoxide formation causes c-Jun NH2-terminal kinase (JNK)-and caspase-dependent apoptosis in cultured hepatocytes. To verify these findings in vivo, male Fisher rats were treated with diquat and menadione. The oxidant stress induced by both compounds was confirmed by increased formation of glutathione disulfide and 4-hydroxynonenal protein adducts. Plasma alanine aminotransferase activities increased from 46 Ϯ 4 U/l in controls to 955 Ϯ 90 U/l at 6 h after diquat treatment. Hematoxylin and eosin staining of liver sections revealed large areas of necrotic cells at 3 and 6 h. DNA strandbreaks, evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, showed clusters of TUNEL-positive cells, where the staining was predominantly cytosolic and the cells were swollen, indicating oncotic necrosis. There was no significant increase in caspase-3 activities or relevant release of DNA fragments into the cytosol at any time between 0 and 6 h after diquat treatment. Despite the activation of JNK after high doses of diquat, the JNK inhibitor SP-600125 did not protect against diquat-induced necrosis. Menadione alone did not cause liver injury, but, in combination with phorone and FeSO4, induced moderate oncotic necrosis. On the other hand, if animals were treated with galactosamine/endotoxin as positive control for apoptosis, caspase-3 activities were increased by 259%, the number of TUNEL-positive cells with apoptotic morphology was increased 103-fold, and DNA fragmentation was enhanced 6-fold. The data indicate that liver cell death initiated by diquat-induced superoxide formation in vivo is mediated predominantly by oncotic necrosis and is independent of JNK activation.

Patients with TB had a higher prevalence of vitamin D deficiency than control subjects in a Korean population. The median 25(OH)D level increased after TB treatment. Further studies are needed to establish a causal relationship.