Search

PDF file 145K, Tables containing information about genes residing on the amplicon and compiled data

PDF file 1687K, The supplementary data contains immunostains, mRNA and DNA qPCR, ELISA analyses and descriptive cartoons including a model highlighting the manuscript

Mouse models of hepatocellular carcinoma (HCC) simulate specific subgroups of human HCC. We investigated hepatocarcinogenesis in Mdr2-knockout (Mdr2-KO) mice, a model of inflammation-associated HCC, using gene expression profiling and immunohistochemical analyses. Gene expression profiling showed that although Mdr2-KO mice differ from other published murine HCC models, they share several important deregulated pathways and many coordinately differentially expressed genes with human HCC data sets. Analysis of genome positions of differentially expressed genes in liver tumors revealed a prolonged region of down-regulated genes on murine chromosome 8 in three of the six analyzed tumor samples. This region is syntenic to human chromosomal regions that are frequently deleted in human HCC and harbor multiple tumor suppressor genes. Real-time reverse transcription-PCR analysis of 16 tumor samples confirmed down-regulation of several tumor suppressors in most tumors. We show that in the aged Mdr2-KO mice, cyclin D1 nuclear level is increased in dysplastic hepatocytes that do not form nodules; however, it is decreased in most dysplastic nodules and in liver tumors. We found that this decrease is mostly at the protein, rather than the mRNA, level. These findings raise the question on the role of cyclin D1 at early stages of hepatocarcinogenesis in the Mdr2-KO HCC model. Furthermore, we show that most liver tumors in Mdr2-KO mice were characterized by the absence of β-catenin activation. In conclusion, the Mdr2-KO mouse may serve as a model for β-catenin–negative subgroup of human HCCs characterized by low nuclear cyclin D1 levels in tumor cells and by down-regulation of multiple tumor suppressor genes. (Mol Cancer Res 2007;5(11):1159–70)

Supplementary Data from Molecular Mechanisms of Liver Carcinogenesis in the Mdr2-Knockout Mice

Supplemental Experimental Materials and Methods

Supplemental Figures and Tables

Hepatocellular carcinoma (HCC) typically develops on a background of chronic hepatitis for which the proinflammatory cytokine IL6 is conventionally considered a crucial driving factor. Paradoxically, IL6 also acts as a hepatoprotective factor in chronic liver injury. Here we used the multidrug-resistant gene 2 knockout (Mdr2−/−) mouse model to elucidate potential roles of IL6 in chronic hepatitis–associated liver cancer. Long-term analysis of three separate IL6/Stat3 signaling–deficient Mdr2−/− strains revealed aggravated liver injury with increased dysplastic nodule formation and significantly accelerated tumorigenesis in all strains. Tumorigenesis in the IL6/Stat3-perturbed models was strongly associated with enhanced macrophage accumulation and hepatosteatosis, phenotypes of nonalcoholic steatohepatitis (NASH), as well as with significant reductions in senescence and the senescence-associated secretory phenotype (SASP) accompanied by increased hepatocyte proliferation. These findings reveal a crucial suppressive role for IL6/Stat3 signaling in chronic hepatitis–associated hepatocarcinogenesis by impeding protumorigenic NASH-associated phenotypes and by reinforcing the antitumorigenic effects of the SASP.Significance:These findings describe a context-dependent role of IL6 signaling in hepatocarcinogenesis and predict that increased IL6-neutralizing sgp130 levels in some patients with NASH may herald early HCC development.See related commentary by Huynh and Ernst, p. 4671

Supplementary Figures 1-4 from Multiple Adaptive Mechanisms to Chronic Liver Disease Revealed at Early Stages of Liver Carcinogenesis in the Mdr2-Knockout Mice

Molecular events preceding the development of hepatocellular carcinoma were studied in the Mdr2-knockout (Mdr2-KO) mice. These mice lack the liver-specific P-glycoprotein responsible for phosphatidylcholine transport across the canalicular membrane. Portal inflammation ensues at an early age followed by hepatocellular carcinoma development after the age of 1 year. Liver tissue samples of Mdr2-KO mice in the early and late precancerous stages of liver disease were subjected to histologic, biochemical, and gene expression profiling analysis. In an early stage, multiple protective mechanisms were found, including induction of many anti-inflammatory and antioxidant genes and increase of total antioxidant capacity of liver tissue. Despite stimulation of hepatocyte DNA replication, their mitotic activity was blocked at this stage. In the late stage of the disease, although the total antioxidant capacity of liver tissue of Mdr2-KO mice was normal, and inflammation was less prominent, many protective genes remained overexpressed. Increased mitotic activity of hepatocytes resulted in multiple dysplastic nodules, some of them being steatotic. Expression of many genes regulating lipid and phospholipid metabolism was distorted, including up-regulation of choline kinase A, a known oncogene. Many other oncogenes, including cyclin D1, Jun, and some Ras homologues, were up-regulated in Mdr2-KO mice at both stages of liver disease. However, we found no increase of Ras activation. Our data suggest that some of the adaptive mechanisms induced in the early stages of hepatic disease, which protect the liver from injury, could have an effect in hepatocarcinogenesis at later stages of the disease in this hepatocellular carcinoma model. (Cancer Res 2006; 66(8): 4001-10)

Supplementary Tables 1-3 from Multiple Adaptive Mechanisms to Chronic Liver Disease Revealed at Early Stages of Liver Carcinogenesis in the Mdr2-Knockout Mice

Supplementary Methods from Multiple Adaptive Mechanisms to Chronic Liver Disease Revealed at Early Stages of Liver Carcinogenesis in the Mdr2-Knockout Mice

Hepatocellular carcinoma (HCC) typically develops on a background of chronic hepatitis for which the proinflammatory cytokine IL6 is conventionally considered a crucial driving factor. Paradoxically, IL6 also acts as a hepatoprotective factor in chronic liver injury. Here we used the multidrug-resistant gene 2 knockout (Mdr2−/−) mouse model to elucidate potential roles of IL6 in chronic hepatitis–associated liver cancer. Long-term analysis of three separate IL6/Stat3 signaling–deficient Mdr2−/− strains revealed aggravated liver injury with increased dysplastic nodule formation and significantly accelerated tumorigenesis in all strains. Tumorigenesis in the IL6/Stat3-perturbed models was strongly associated with enhanced macrophage accumulation and hepatosteatosis, phenotypes of nonalcoholic steatohepatitis (NASH), as well as with significant reductions in senescence and the senescence-associated secretory phenotype (SASP) accompanied by increased hepatocyte proliferation. These findings reveal a crucial suppressive role for IL6/Stat3 signaling in chronic hepatitis–associated hepatocarcinogenesis by impeding protumorigenic NASH-associated phenotypes and by reinforcing the antitumorigenic effects of the SASP. Significance: These findings describe a context-dependent role of IL6 signaling in hepatocarcinogenesis and predict that increased IL6-neutralizing sgp130 levels in some patients with NASH may herald early HCC development. See related commentary by Huynh and Ernst, p. 4671

Chronic liver inflammation (CLI) is a risk factor for development of hepatocellular carcinoma (HCC). Galectin-1 (Gal1) is involved in the regulation of inflammation, angiogenesis, and tumorigenesis, exhibiting multiple anti-inflammatory and protumorigenic activities. We aimed to explore its regulatory role in CLI and HCC progression using an established model of CLI-mediated HCC development, Abcb4 [multidrug-resistance 2 (Mdr2)]-knockout (KO) mice, which express high levels of Gal1 in the liver. We generated double-KO (dKO) Gal1- KO/Mdr2-KO mice on C57BL/6 and FVB/N genetic backgrounds and compared HCC development in the generated strains with their parentalMdr2-KO strains. Loss of Gal1 increased liver injury, inflammation, fibrosis, and ductular reaction in dKO mice of both strains starting from an early age. Aged dKO mutants displayed earlier hepatocarcinogenesis and increased tumor size compared with control Mdr2-KO mice. We found that osteopontin, a well-knownmodulator of HCC development, and oncogenic proteins Ntrk2 (TrkB) and S100A4 were overexpressed in dKO compared with Mdr2-KO livers. Our results demonstrate that in Mdr2-KO mice, a model of CLI-mediated HCC, Gal1-mediated protection from hepatitis, liver fibrosis, and HCC initiation dominates over its known procarcinogenic activities at later stages of HCC development. These findings suggest that anti-Gal1 treatmentsmay not be applicable at all stages of CLI-mediated HCC.—Potikha, T., Pappo, O., Mizrahi, L., Olam, D., Maller, S. M., Rabinovich, G. A., Galun, E., Goldenberg, D. S. Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model. Fil: Potikha, Tamara. Universidad Academica de Hadassa Jerusalem; Israel Fil: Pappo, Orit. Universidad Academica de Hadassa Jerusalem; Israel Fil: Mizrahi, Lina. Universidad Academica de Hadassa Jerusalem; Israel Fil: Olam, Devorah. Universidad Academica de Hadassa Jerusalem; Israel Fil: Maller, Sebastián M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Rabinovich, Gabriel Adrián. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Galun, Eithan. Universidad Academica de Hadassa Jerusalem; Israel Fil: Goldenberg, Daniel S.. Universidad Academica de Hadassa Jerusalem; Israel

The oncofetal long noncoding RNA (lncRNA) H19 is postnatally repressed in most tissues, and re-expressed in many cancers, including hepatocellular carcinoma (HCC). The role of H19 in carcinogenesis is a subject of controversy. We aimed to examine the role of H19 in chronic inflammation-mediated hepatocarcinogenesis using the Mdr2/Abcb4 knockout (Mdr2-KO) mouse, a well-established HCC model. For this goal, we have generated Mdr2-KO/H19-KO double knockout (dKO) mice and followed spontaneous tumor development in the dKO and control Mdr2-KO mice. Cellular localization of H19 and effects of H19 loss in the liver were determined in young and old Mdr2-KO mice. Tumor incidence and tumor load were both significantly decreased in the liver of dKO versus Mdr2-KO females. The expression levels of H19 and Igf2 were variable in nontumor liver tissues of Mdr2-KO females and were significantly downregulated in most matched tumors. In nontumor liver tissue of aged Mdr2-KO females, H19 was expressed mainly in hepatocytes, and hepatocyte proliferation was increased compared to dKO females. At an early age, dKO females displayed lower levels of liver injury and B-cell infiltration, with higher percentage of binuclear hepatocytes. In human samples, H19 expression was higher in females, positively correlated with cirrhosis (in nontumor liver samples) and negatively correlated with CTNNB1 (beta-catenin) mutations and patients' survival (in tumors). Our data demonstrate that the lncRNA H19 is pro-oncogenic during the development of chronic inflammation-mediated HCC in the Mdr2-KO mouse model, mainly by increasing liver injury and decreasing hepatocyte polyploidy in young mice.

ObjectiveBoth non-alcoholic fatty liver disease (NAFLD) and the multitarget complexity of microRNA (miR) suppression have recently raised much interest, but the in vivo impact and context-dependence of hepatic miR-target interactions are incompletely understood. Assessing the relative in vivo contributions of specific targets to miR-mediated phenotypes is pivotal for investigating metabolic processes.DesignWe quantified fatty liver parameters and the levels of miR-132 and its targets in novel transgenic mice overexpressing miR-132, in liver tissues from patients with NAFLD, and in diverse mouse models of hepatic steatosis. We tested the causal nature of miR-132 excess in these phenotypes by injecting diet-induced obese mice with antisense oligonucleotide suppressors of miR-132 or its target genes, and measured changes in metabolic parameters and transcripts.ResultsTransgenic mice overexpressing miR-132 showed a severe fatty liver phenotype and increased body weight, serum low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) and liver triglycerides, accompanied by decreases in validated miR-132 targets and increases in lipogenesis and lipid accumulation-related transcripts. Likewise, liver samples from both patients with NAFLD and mouse models of hepatic steatosis or non-alcoholic steatohepatitis (NASH) displayed dramatic increases in miR-132 and varying decreases in miR-132 targets compared with controls. Furthermore, injecting diet-induced obese mice with anti-miR-132 oligonucleotides, but not suppressing its individual targets, reversed the hepatic miR-132 excess and hyperlipidemic phenotype.ConclusionsOur findings identify miR-132 as a key regulator of hepatic lipid homeostasis, functioning in a context-dependent fashion via suppression of multiple targets and with cumulative synergistic effects. This indicates reduction of miR-132 levels as a possible treatment of hepatic steatosis.

Adenosine deaminase acting on RNA (ADAR) 1 binds and edits double-stranded (ds) RNA secondary structures found mainly within untranslated regions of many transcripts. In the current research, our aim was to study the role of ADAR1 in liver homeostasis. As previous studies show a conserved immunoregulatory function for ADAR1 in mammalians, we focused on its role in preventing chronic hepatic inflammation and the associated activation of hepatic stellate cells to produce extracellular matrix and promote fibrosis. We show that hepatocytes specific ADAR1 knock out (KO) mice display massive liver damage with multifocal inflammation and fibrogenesis. The bioinformatics analysis of the microarray gene-expression datasets of ADAR1 KO livers reveled a type-I interferons signature and an enrichment for immune response genes compared to control littermate livers. Furthermore, we found that in vitro silencing of ADAR1 expression in HepG2 cells leads to enhanced transcription of NFκB target genes, foremost of the pro-inflammatory cytokines IL6 and IL8. We also discovered immune cell-independent paracrine signaling among ADAR1-depleted HepG2 cells and hepatic stellate cells, leading to the activation of the latter cell type to adopt a profibrogenic phenotype. This paracrine communication dependent mainly on the production and secretion of the cytokine IL6 induced by ADAR1 silencing in hepatocytes. Thus, our findings shed a new light on the vital regulatory role of ADAR1 in hepatic immune homeostasis, chiefly its inhibitory function on the crosstalk between the NFκB and type-I interferons signaling cascades, restraining the development of liver inflammation and fibrosis.

Mutations in MCM9, which encodes DNA helicase, were recently shown to cause a clinical phenotype of primary ovarian failure and chromosomal instability. MCM9 plays an essential role in homologous recombination-mediated double-strand break repair. We describe a multiplex family with early colorectal carcinoma and mixed polyposis associated with primary hypergonadotropic hypogonadism. A combination of whole genome homozygosity mapping as well as exome sequencing and targeted gene sequencing identified a homozygous c.672_673delGGinsC mutation that predicts a truncated protein, p.Glu225Lysfs*4. Our data expand the phenotypic spectrum of MCM9 mutations and suggest a link between MCM9 and inherited predisposition to mixed polyposis and early-onset colorectal cancer.

Background and Aims: Rats are resistant to acetaminophen (APAP) hepatotoxicity. In this study, we evaluated whether by augmentation of the hepatic oxidative stress, through the induction of hepatic iron overload (IO), it will be feasible to overcome the resistance of rats to the toxic effects of APAP. Method: Rats with no or increased hepatic IO. Results: Providing iron by diet induced hepatocellular IO, while parenteral iron administration induced combined hepatocellular and sinusoidal cell IO. APAP administration to rats with no IO caused an increase in hepatic oxidative stress and a decrease in the hepatic antioxidative markers but no hepatic cell damage. APAP administration to rats with hepatocellular IO further amplified the hepatic oxidative stress but induced only hepatocyte feathery degeneration without any increase in serum aminotransaminases. APAP administration to rats with combined hepatocellular and sinusoidal cell IO caused an unexpected decrease in hepatic oxidative stress and increase in the hepatic antioxidative markers and no hepatic cell damage. No hepatic expression of activated c-jun-N-terminal kinase was detected in any of the rats. Conclusions: The hepatic distribution of iron may affect its oxidative/antioxidative milieu. Augmentation of hepatic oxidative stress did not increase the rats’ vulnerability to APAP.

Teratomas of the liver are exceedingly rare. Neuroblastoma is the most common, extracranial solid tumor of infancy. We describe the case of a 2-month-old, female infant who presented with an abdominal mass arising in the right lobe of the liver, and a severe coagulopathy, which necessitated cryoprecipitate infusion. Biopsy was interpreted as hepatoblastoma. Following resection, difficulty classifying the mass led to several consultations, and an eventual diagnosis of teratoma. During follow-up, the patient was diagnosed with right adrenal neuroblastoma, which, in retrospect, had been present before the hepatic resection. To our knowledge, these 2 tumors have never been reported together, or in combination with isolated hypofibrinogenemia.

// Ezra Ella 1 , Denise Heim 2 , Evgeniy Stoyanov 1 , Rona Harari-Steinfeld 1 , Israel Steinfeld 3 , Orit Pappo 4 , Temima Schnitzer Perlman 1 , Natalie Nachmansson 5 , Ludmila Rivkin 1 , Devorah Olam 1 , Rinat Abramovitch 1,5 , Henning Wege 2 , Eithan Galun 1 and Daniel Goldenberg 1 1 The Goldyne Savad Institute of Gene Therapy, Hadassah-Hebrew University Medical Center, Jerusalem, Israel 2 Department of Gastroenterology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 3 Computer Science Department, Technion-Israel Institute of Technology, Haifa, Israel 4 Department of Pathology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel 5 Magnetic Resonance Imaging/Magnetic Resonance Spectroscopy Laboratory, Human Biology Research Center, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Correspondence: Daniel Goldenberg, email: // Keywords : HCC, liver regeneration, chronic hepatitis, genomic instability, Crem Received : August 24, 2014 Accepted : September 24, 2014 Published : September 25, 2014 Abstract Resection of hepatocellular carcinoma (HCC) tumors by partial hepatectomy (PHx) is associated with promoting hepatocarcinogenesis. We have previously reported that PHx promotes hepatocarcinogenesis in the Mdr2-knockout (Mdr2-KO) mouse, a model for inflammation-mediated HCC. Now, to explore the molecular mechanisms underlying the tumor-promoting effect of PHx, we compared genomic and transcriptomic profiles of HCC tumors developing in the Mdr2-KO mice either spontaneously or following PHx. PHx accelerated HCC development in these mice by four months. PHx-induced tumors had major chromosomal aberrations: all were amplifications affecting multiple chromosomes. Most of these amplifications were located near the acrocentric centromeres of murine chromosomes. Four different chromosomal regions were amplified each in at least three tumors. The human orthologs of these common amplified regions are known to be amplified in HCC. All tumors of untreated mice had chromosomal aberrations, including both deletions and amplifications. Amplifications in spontaneous tumors affected fewer chromosomes and were not located preferentially at the chromosomal edges. Comparison of gene expression profiles revealed a significantly enriched expression of oncogenes, chromosomal instability markers and E2F1 targets in the post-PHx compared to spontaneous tumors. Both tumor groups shared the same frequent amplification at chromosome 18. Here, we revealed that one of the regulatory genes encoded by this amplified region, Crem, was over-expressed in the nuclei of murine and human HCC cells in vivo , and that it stimulated proliferation of human HCC cells in vitro . Our results demonstrate that PHx of a chronically inflamed liver directed tumor development to a discrete pathway characterized by amplification of specific chromosomal regions and expression of specific tumor-promoting genes. Crem is a new candidate HCC oncogene frequently amplified in this model and frequently over-expressed in human HCC.

An increase in hepatic iron concentration might exacerbate liver injury. However, it is unknown whether hepatic iron overload may exacerbate acute liver injury from various toxins. Therefore, we evaluated how manipulations to increase hepatic iron concentration affected the extent of acute liver injury from thioacetamide. In this study, we used rats with either "normal" or increased hepatic iron concentration. Iron overload was induced by either providing excess iron in the diet or by injecting iron subcutaneously. Both routes of providing excess iron induced an increase in hepatic iron overload. Meanwhile, the subcutaneous route induced both hepatocellular and sinusoidal cell iron deposition; the oral route induced lesser degree of hepatic iron concentration and only hepatocellular iron overload. Thioacetamide administration to the rats with "normal" hepatic iron concentration induced hepatic cell necrosis and apoptosis associated with a remarkable increase in serum aminotransaminases and depletion of hepatic glutathione and other antioxidative indices. Thioacetamide administration to the iron-overloaded rats exacerbated the extent of liver injury only in the rats orally induced with iron overload. In the rats subcutaneously induced with iron overload, the extent of liver injury from thioacetamide was not different from that observed in the rats with "normal" iron overload. It was concluded that the outcome of thioacetamide-induced acute liver injury may depend on both the level of hepatic iron concentration and on the cellular distribution of iron. While isolated hepatocellular iron overload may exacerbate thioacetamide-induced acute liver injury, a combined hepatocellular and sinusoidal cell iron deposition, even at high hepatic iron concentration, had no such an effect.

Extended partial hepatectomy may be needed in cases of large hepatic mass, and can lead to fulminant hepatic failure. Macroporous alginate scaffold is a biocompatible matrix which promotes the growth, differentiation and long-term hepatocellular function of primary hepatocytes in vitro. Our aim was to explore the ability of implanted macroporous alginate scaffolds to protect liver remnants from acute hepatic failure after extended partial hepatectomy. An 87% partial hepatectomy (PH) was performed on C57BL/6 mice to compare non-treated mice to mice in which alginate or collagen scaffolds were implanted after PH. Mice were scarified 3, 6, 24 and 48 h and 6 days following scaffold implantation and the extent of liver injury and repair was examined. Alginate scaffolds significantly increased animal survival to 60% vs. 10% in non-treated and collagen-treated mice (log rank = 0.001). Mice with implanted alginate scaffolds manifested normal and prolonged aspartate aminotransferases and alanine aminotransferases serum levels as compared with the 2- to 20-fold increase in control groups (P < 0.0001) accompanied with improved liver histology. Sustained normal serum albumin levels were observed in alginate-scaffold-treated mice 48 h after hepatectomy. Incorporation of BrdU-positive cells was 30% higher in the alginate-scaffold-treated group, compared with non-treated mice. Serum IL-6 levels were significantly decreased 3 h post PH. Biotin-alginate scaffolds were quickly well integrated within the liver tissue. Collectively, implanted alginate scaffolds support liver remnants after extended partial hepatectomy, thus eliminating liver injury and leading to enhanced animal survival after extended partial hepatectomy.

We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7–treated animals had lower body weight (−9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (−51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (−40%) and serum aldosterone (−48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7–treated rats. WAT from Ang 1-7–treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7–treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7–treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.

Objective: Blood loss and transfusion are frequent among patients undergoing liver surgery. Concerns have been raised about the safety and efficacy of transfusing stored blood. The influence of transfusing fresh vs. stored blood on the liver has not been studied to date. We tested the hypothesis that transfusion of stored, but not fresh blood, adversely affects liver outcome in vivo following acute hemorrhage. Additionally, possible mechanisms linking adverse liver outcome with increased storage duration were evaluated. Design: Prospective, controlled, animal study. Setting: University research laboratory. Subjects: Adult male Sprague-Dawley rats Interventions: Anesthetized rats were randomized to control, hemorrhagic and shock group (acute bleeding; HSG), or hemorrhagic and blood resuscitation groups (BR) (with fresh blood [BRd0], blood stored for 4 [BR-d4] or 7 [BR-d7] days, or packed RBCs stored for 7 days [packed RBC-d7]). Measurements and Main Results: Administration of blood or packed RBC stored for 7 days exacerbated liver injury as reflected by liver necrosis and enhanced apoptosis (p < 0.001). Functional MRI analysis of the liver demonstrated significant improvement in liver perfusion with fresh blood (% change in functional MRI signal intensity due to hyperoxia was 16% ± 3% in BR-d0 vs. 4% ± 3% in hemorrhagic group, p < 0.001) but not with stored blood (12% ± 2% and 9% ± 5% for BR-d4 and BR-d7, respectively). Analysis of stored blood showed reduction in RBC deformability at 7 days of storage, reflecting a five-fold increase in the number of undeformable cells. Conclusion: Liver injury is exacerbated by the transfusion of stored blood, primarily due to the change in the rheological properties of RBC. This data call for clinical studies in patients undergoing liver resection or transplantation. (Crit Care Med 2013; 41:842–849)

BACKGROUND AND AIM Distinguishing biliary atresia (BA) from other causes of neonatal cholestasis (NC) is challenging. Continuous BreathID C-methacetin breath test (MBT) is a novel method that determines liver function. Methacetin is metabolized uniquely by the liver and CO2 is measured passively, through a nasal cannula in the exhaled breath. The aim of this study was to assess the ability of MBT to differentiate BA from other causes of NC. METHODS MBT was performed in infants with NC before any invasive procedure. Percent dose recovered (PDR) peak and time to peak (TTPP) of C recovered were correlated with blood test results and degree of fibrosis on liver biopsy. RESULTS Fifteen infants were enrolled in the study. Eight were eventually diagnosed as having BA. MBT showed that infants with NC from various causes reached the PDR peak after 44.5 ± 6.7 minutes, whereas infants with BA reached the PDR peak value after 54.7 ± 4.3 minutes (P < 0.005). This suggested low cytochrome P450 1A2 activity in the BA group. The area under the curve (AUC) was 0.95 (95% confidence interval [CI] 0.83-1), sensitivity of 88%, and specificity of 100%. CONCLUSIONS This pilot study shows that MBT can differentiate between BA and other causes of NC by time to peak of methacetin metabolism. The results suggest that MBT may be used as part of the diagnostic algorithm in infants with liver disease. Larger-scale studies should be conducted to confirm these initial observations.

We recently observed patients with chronic liver disease (CLD) or chronic reflux symptoms (CRS) who developed gastric polyps (GPs) while undergoing surveillance gastroscopies for the detection of esophageal varices or Barrett's esophagus, respectively.To identify risk factors for GP growth and estimate its growth rate.GP growth rate was defined as the number of days since the first gastroscopy (without polyps) in the surveillance program, until the gastroscopy when a GP was discovered.Gastric polyp growth rates in CLD and CRS patients were similar. However, hyperplastic gastric polyps (HGPs) were detected more often (87.5% vs. 60.5%, P = 0.051) and at a higher number (2.57 ± 1.33 vs. 1.65 ± 0.93, P = 0.021) in the CLD patients. Subgroup analysis revealed the following findings only in CLD patients with HGPs: (i) a positive correlation between the GP growth rate and the patient's age; the older the patient, the higher the GP growth rate (r = 0.7, P = 0.004). (ii) A negative correlation between the patient's age and the Ki-67 proliferation index value; the older the patient, the lower the Ki-67 value (r = -0.64, P = 0.02). No correlation was detected between Ki-67 values of HGPs in CLD patients and the presence of portal hypertension, infection with Helicobacter pylori, or proton pump inhibitor use.In comparison with CRS patients, CLD patients developed HGPs more often and at a greater number. Young CLD patients may have a tendency to develop HGPs at a faster rate than elderly CLD patients.

Galectin-1 (Gal1) is a known immune/inflammatory regulator which actsboth extracellularly and intracellularly, modulating innate and adaptive immuneresponses. Here, we explored the role of Gal1 in liver regeneration using 70% partial hepatectomy (PHx) of C57BL/6 wild type and Gal1-knockout (Gal1-KO, Lgals1-/-) mice. Gene or protein expression, in liver samples collected at time intervals from 2 to 168 hours post-operation, was tested by either RT-PCR or by immunoblotting and immunohistochemistry, respectively. We demonstrated that Gal1 transcript and protein expression was induced in the liver tissue of wild type mice upon PHx. Liver regeneration following PHx was significantly delayed in the Gal1-KO compared to the control liver. This delay was accompanied by a decreased Akt phosphorylation, and accumulation of the hepatocyte nuclear p21 protein in the Gal1-KO versus control livers at 24 and 48 hours following PHx. Transcripts of several known regulators of inflammation, cell cycle and cell signaling, including some known PHx-induced genes, were aberrantly expressed (mainly down-regulated) in Gal1-KO compared to control livers at 2, 6 and 24 hours post-PHx. Transient steatosis, which is imperative for liver regeneration following PHx, was significantly delayed and decreased in the Gal1- KO compared to the control liver and was accompanied by a significantly decreased expression in the mutant liver of several genes encoding lipid metabolism regulators.Our results demonstrate that Gal1 protein is essential for efficient liver regeneration following PHx through the regulation of liver inflammation, hepatic cell proliferation, and the control of lipid storage in the regenerating liver. Fil: Potikha, Tamara. Hadassah Hebrew University Medical Center; Israel Fil: Ella, Ezra. Hadassah Hebrew University Medical Center; Israel Fil: Cerliani, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Mizrahi, Lina. Hadassah Hebrew University Medical Center; Israel Fil: Pappo, Orit. Hadassah Hebrew University Medical Center; Israel Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Galun, Eithan. Hadassah Hebrew University Medical Center; Israel Fil: Goldenberg, Daniel S.. Hadassah Hebrew University Medical Center; Israel

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease, typically diagnosed by elevated cholestatic liver enzymes and a positive antimitochondrial antibody (AMA) test. The clinical significance of AMA positivity in patients with normal cholestatic liver enzymes is uncertain.Charts of patients with normal cholestatic liver enzymes and AMA positivity who underwent liver biopsy between 2012 and 2015 were retrospectively analyzed.Six AMA-positive patients with normal cholestatic liver enzymes who underwent a liver biopsy were identified. Four (67%) showed florid bile duct lesion compatible with early-stage PBC, whereas the other two showed mild and non-specific histological findings. The patients with histological findings compatible with PBC had higher enzyme-linked immunosorbent assay-determined AMA titers and significantly elevated immunoglobulin M (IgM) level. Patients with non-specific histological findings (33%) had low-titer AMA and a borderline elevated IgM level.Antimitochondrial antibody-positive patients with normal cholestatic liver enzymes should be meticulously evaluated for PBC including a liver biopsy, mainly in patients with high-titer seropositivity for AMA and a significantly elevated IgM level. More studies are required to clarify the role of liver biopsy in these patients and further follow-up may elucidate the relationship of these patients to those with more classical forms of PBC.

Variation in the prevalence of various types of gastric polyps worldwide may reflect different etiologies. Here, the authors report the dynamic changes in histological distribution of gastric polyps over time and by ethnicity for individuals who underwent gastroscopies between 1994 and 2009 at two hospitals in Jerusalem, Israel. During this time period, the proportion of patients receiving proton pump inhibitors (PPIs) increased while the proportion of patients infected with Helicobacter pylori (H. pylori) decreased.Pathological reports of biopsies from 50,071 consecutive gastroscopies were reviewed.Gastric polyps were detected in 727 individuals. The yearly prevalence of gastric polyps was ≤ 1% between 1994 and 2001 and ≥ 2% from 2004 to 2009, of which overall 66% were hyperplastic polyps and 23% fundic gland polyps (FGPs). FGPs were diagnosed exclusively in the Jewish population. From 2001 to 2004, an increase in the absolute number of newly discovered hyperplastic and FGPs per year was observed. However from 2005, a divergent trend of changes was observed: While the proportion of patients with hyperplastic polyps dropped from 0.72 during the 2001-2004 period to 0.62 during the 2005-2009 period (p = 0.02), the proportion of patients with FGPs at these time periods increased from 0.16 to 0.33 (p = 0.0001).The yearly prevalence of gastric polyps in Jerusalem has recently doubled. This occurred mainly due to the increasing prevalence of FGPs. The changing epidemiology of gastric polyps is probably related to the interaction between genetic factors and fluctuating environmental factors like H. pylori infection rates and exposure to PPIs.

Background/Aims: Toll-like receptor 4 (TLR4) is expressed on hepatic non-parenchymal cells and hepatocytes. Hepatic signaling through TLR4 is critical in the pathogenesis of ischemia reperfusion injury (IRI) and leads to the release of cytokines. The role of bone marrow-derived TLR4 in the early reperfusion stage is unclear. Methods: We used wild type mice (WT), TLR4deficient (TLR4ko) mice and chimeras to dissociate between the role of TLR4 expression in the liver (TLR4ko/WT) and in the immuno-hematopoietic system (WT/TLR4ko) in mouse hepatic IR injury model. Mice were subjected to in vivo partial IRI (70% for 60 min). Results: Compared with WT IR livers, TLR4ko IRI mice (4 hours) showed a significant reduction in serum liver enzyme, hepatic TNF-α and interleukin-1β levels. Fewer apoptotic hepatocytes cells were identified by morphological criteria and immunohistochemistry for caspase-3. In TLR4ko mice, decreased hepatic CJUN and NF-ĸB expression during IRI was noted compared with WT mice. Chimeric mice having either TLR4 bone-marrow or non-bone marrow derived cells following IRI exhibited almost similar hepatic injury as WT mice in the immediate reperfusion stage. Conclusion: Both TLR4 bone marrow-derived and non-bone marrow-derived cells are necessary in the initial process of hepatic injury. Activating TLR4-dependent signaling is required for IRI. The absence of the TLR4 gene plays a pivotal role in reducing hepatic IR injury.

Ectopic lymphoid-like structures (ELSs) are often observed in cancer, yet their function is obscure. Although ELSs signify good prognosis in certain malignancies, we found that hepatic ELSs indicated poor prognosis for hepatocellular carcinoma (HCC). We studied an HCC mouse model that displayed abundant ELSs and found that they constituted immunopathological microniches wherein malignant hepatocyte progenitor cells appeared and thrived in a complex cellular and cytokine milieu until gaining self-sufficiency. The egress of progenitor cells and tumor formation were associated with the autocrine production of cytokines previously provided by the niche. ELSs developed via cooperation between the innate immune system and adaptive immune system, an event facilitated by activation of the transcription factor NF-κB and abolished by depletion of T cells. Such aberrant immunological foci might represent new targets for cancer therapy.

We present a novel method for the automatic classification and grading of liver fibrosis based on hepatic hemodynamic changes measured noninvasively from functional MRI (fMRI) scans combined with hypercapnia and hyperoxia. The supervised learning method automatically creates a classification and grading model for liver fibrosis grade from training datasets. It constructs a statistical model of liver fibrosis by evaluating the signal intensity time course and local variance in T2(*)-W fMRI scans acquired during the breathing of air, air-carbon dioxide, and carbogen with a hierarchical multiclass binary-based support vector machine (SVM) classifier. Two experimental studies on 162 slices from 34 mice with the hierarchical multiclass binary-based SVM classifier yield 96.9% separation accuracy between healthy and histological-based fibrosis graded subjects, and an overall accuracy of 75.3% for healthy, fibrotic, and cirrhotic subjects. These results outperform existing image-based methods that can discriminate between healthy and mild-grade fibrosis subjects.

An increase in serum ferritin levels during combined interferon–ribavirin treatment in chronic patients infected with hepatitis C virus (HCV) can occur. A study was conducted to determine whether observing the kinetics of serum ferritin levels during antiviral therapy, may assist in predicting the rate of sustained virological response. The kinetics of serum ferritin levels during antiviral therapy in treatment-naive, adherent patients with chronic HCV who had early virological response were characterized. Thirteen patients achieved sustained virological response (group 1) while eight patients did not (group 2). Pre-treatment serum ferritin levels were higher in group 2 patients. During antiviral therapy, serum ferritin levels increased in both groups. On treatment, the median increase (compared to baseline) and the calculated rate of the increase in serum ferritin levels was higher in group 1 patients (874% vs. 272%, P

Background & Aims Interleukin-6 (IL-6) is a crucial factor in liver regeneration following partial hepatectomy (PH); however, the role of IL-6 and IL-6 trans -signaling in particular, in hepatocyte mitosis remains controversial. IL-6 trans -signaling relies upon the release of the soluble IL-6R (sIL-6R), which binds IL-6 to form an agonistic IL-6/sIL-6R complex. Herein we have examined the hypothesis that IL-6 trans -signaling plays a crucial and distinct role in liver regeneration following PH. Methods The specific IL-6/sIL-6R antagonist, sgp130Fc, was expressed in mice and analyzed for its effect on hepatocyte mitosis following PH. Alternatively, we examined the effect of the IL-6/sIL-6R super-agonist, Hyper-IL-6, or IL-6 expressed either alone or in combination with hepatocyte growth factor (HGF) on hepatocyte mitosis in the absence of PH. Results Following PH, the dramatic rise of circulating IL-6 levels is accompanied by a concurrent ∼2-fold increase in circulating sIL-6R levels. Ectopic expression of sgp130Fc reduced hepatocyte mitosis by about 40% at early times following PH, while substantially reducing AKT, but not STAT3, activation. But, ectopic Hyper-IL-6 expression in mice without PH was not mitogenic to hepatocytes in vivo . Rather, Hyper-IL-6, but not IL-6, markedly increased HGF-induced hepatocyte mitosis. This cooperative effect correlated with greater resistance of HIL-6 than IL-6 to HGF-mediated reduction of AKT activation, rather than changes in STAT3 or MAPK signaling, and was completely blocked by PI3K inhibition. Conclusions Following PH, IL-6/sIL-6R cooperates with growth factors, through a PI3K/AKT-dependent mechanism to promote entry of hepatocytes into the cell cycle.