Context: Liver cancers (including hepatocellular carcinoma [HCC] and cholangiocarcinoma) are the fifth most common cause of cancer death. The most powerful independent histologic predictor of overall survival after transplantation for HCC is the presence of microscopic vascular invasion (VI). Aims: Given that VI is known to have somewhat high interobserver variability in both HCC and other tumors, we hypothesized that pathologists with special interest and training in liver pathology would be more likely to identify and report VI in HCC than would general surgical pathologists. Settings and Design: We searched our departmental surgical pathology archives for transplant hepatectomies performed for HCC. Subjects and Methods: We identified 143 such cases with available sign-out reports and hematoxylin and eosin-stained slides. Statistical Analysis Used: Kappa results (level of agreement) were calculated. Results: Before surgical pathology subspecialty sign-out (SSSO) implementation, 49 of 88 HCC cases were reported as negative for VI; on rereview, 20 of these had VI. After SSSO implementation, 39 of 55 cases were reported as negative for VI; on our review, 8 of these had VI. Kappa (agreement) between general SO and subspecialty rereview was 0.562 (95% confidence interval [CI] = 0.411-0.714) "weak agreement." Kappa (agreement) between SSSO and rereview by select liver pathologists was 0.693 (95% CI = 0.505-0.880) "moderate agreement." Conclusions: Our study is one of only a few so far that have suggested improved accuracy of certain parameters under SSSO.

BACKGROUND:Liver cancer, of which hepatocellular carcinoma (HCC) is by far the most common type, is the second most deadly cancer (746,000 deaths in 2012). Currently, the only curative treatment for HCC is surgery to remove the malignancy (resection) or to remove the entire diseased liver followed by transplantation of healthy liver tissue. Given the shortage of healthy livers, it is crucial to provide transplants to patients that have the best chance of long-term survival. Currently, transplantation is determined via the Milan criteria-patients within Milan (single tumor < 5 cm or 2-3 tumors < 3 cm with no extrahepatic spread nor intrahepatic vascular invasion) are typically eligible for transplantation. However, combining microRNA expression profiling with the Milan criteria can improve prediction of recurrence. HCC often presents with multiple distinct tumor foci arising from local spread of a primary tumor or from the oncogenic predisposition of the diseased liver. Substantial genomic heterogeneity between tumor foci within a single patient has been reported; therefore, biomarker development must account for the possibility of highly heterogeneous genomic profiles from the same individual.METHODS:MicroRNA profiling was performed on 180 HCC tumor samples from 89 patients who underwent liver transplantation at the University of Rochester Medical Center. The primary outcome was recurrence-free survival time, and patients were observed for 3 years post-transplantation.RESULTS:MicroRNA expression profiles were used to develop a biomarker that distinguishes HCC patients at greater risk of recurrence post-transplantation. Unsupervised clustering uncovered two distinct subgroups with vast differences in standard transplantation selection criteria and recurrence-free survival times. These subgroups were subsequently used to identify microRNAs strongly associated with HCC recurrence. Our results show that reduced expression of five specific microRNAs is significantly associated with HCC recurrence post-transplantation.CONCLUSIONS:MicroRNA profiling of distinct tumor foci, coupled with methods that address within-subject tumor heterogeneity, has the potential to significantly improve prediction of HCC recurrence post-transplantation. The development of a clinically applicable HCC biomarker would inform treatment options for patients and contribute to liver transplant selection criteria for practitioners.

OBJECTIVES:The precise measurement of fat accumulation in the liver, or steatosis, is an important clinical goal. Our previous studies in phantoms and mouse livers support the hypothesis that, starting with a normal liver, increasing accumulations of microsteatosis and macrosteatosis will increase the lossy viscoelastic properties of shear waves in a medium. This increase results in an increased dispersion (or slope) of the shear wave speed in the steatotic livers. METHODS:In this study, we moved to a larger animal model, lean versus obese rat livers ex vivo, and a higher-frequency imaging system to estimate the shear wave speed from crawling waves. RESULTS:The results showed elevated dispersion in the obese rats and a separation of the lean versus obese liver parameters in a 2-dimensional parameter space of the dispersion (slope) and shear wave speed at a reference frequency of 150 Hz. CONCLUSIONS:We have confirmed in 3 separate studies the validity of our dispersion hypothesis in animal models.

It has been considered that epigenetic modulation can affect a diverse array of cellular activities, in which ten eleven translocation (TET) methylcytosine dioxygenase family members refer to a group of fundamental components involved in catalyzation of 5-hydroxymethylcytosine and modification of gene expression. Even though the function of TET proteins has been gradually revealed, their roles in immune regulation are still largely unknown. Recent studies provided clues that TET2 could regulate several innate immune-related inflammatory mediators in mammals. This study sought to explore the function of TET family members in potential T-helper (Th) cell differentiation involved in adaptive immunity by utilizing a zebrafish model. As shown by results, soluble antigens could induce expression of zebrafish IL-4/13A (i.e. a pivotal Th2-type cytokine essential in Th2 cell differentiation and functions), and further trigger the expression of Th1- and Th2-related genes. It is noteworthy that this response was accompanied by the up-regulation of two TET family members (TET1 and TET3) both in immune organs (spleen and kidney) and cells (peripheral lymphocytes). Knocking-down of TET1 and TET3 will give rise to the decreased responses of IL-4/13A induction against exogenous soluble antigen stimulation, and further restrain the expression of Th2-related genes, which indicates a restrained Th2 cell differentiation. Nonetheless, TET2 did not exhibit effect on the modification of Th1/Th2 related gene expression. Hence, these data showed that TET1 and TET3 might be two significant epigenetic regulators involved in Th2 differentiation through regulation of IL-4/13A expression. This is the first report to show that TET family members play indispensable roles in Th2-type immunity, indicating an epigenetic modulation manner involved in adaptive immune regulations and responses.

Vascular invasion provides a direct route for tumor metastasis. The degree to which microRNA (miRNA) expression plays a role in tumor vascular invasion is unclear. Here, we report that miR‐494 is up‐regulated in human hepatocellular carcinoma (HCC) tumors with vascular invasion and can promote HCC cell invasiveness by gene inactivation of multiple invasion‐suppressor miRNAs. Our results show that ten eleven translocation (TET) methylcytosine dioxygenase, predominantly TET1 in HCC cells, is a direct target of miR‐494. The reduced 5′‐hydroxymethylcytosine levels observed in the proximal cytosine‐phosphate‐guanine (CpG) regions of multiple invasion‐suppressor miRNA genes are strongly associated with their transcriptional repression upon miR‐494 overexpression, whereas enforced DNA demethylation can abolish the repression. Furthermore, TET1 knockdown shows a similar effect as miR‐494 overexpression. Conversely, miR‐494 inhibition or enforced TET1 expression is able to restore invasion‐suppressor miRNAs and inhibit miR‐494‐mediated HCC cell invasion. Conclusions: miR‐494 can trigger gene silencing of multiple invasion‐suppressor miRNAs by inhibiting genomic DNA demethylation by direct targeting of TET1, thereby leading to tumor vascular invasion. (Hepatology 2015;62:466–480

The accumulation of fat droplets within the liver is an important marker of liver disease. This study assesses gradations of steatosis in mouse livers using crawling waves, which are interfering patterns of shear waves introduced into the liver by external sources. The crawling waves are detected by Doppler ultrasound imaging techniques, and these are analyzed to estimate the shear wave speed as a function of frequency between 200 and 360 Hz. In a study of 70 mice with progressive increases in steatosis from 0% to >60%, increases in steatosis are found to increase the dispersion, or frequency dependence, of shear wave speed. This finding confirms an earlier, smaller study and points to the potential of a scoring system for steatosis based on shear wave dispersion.

We describe a surface-based approach to the generation of shear wave interference patterns, called crawling waves (CrW), within a medium and derive local estimates of biomechanical properties of tissue. In previous experiments, elongated bars operating as vibration sources were used to generate CrW propagation in samples. In the present study, however, a pair of miniature circular vibration sources was applied to the overlying skin to generate the CrW within the medium. The shape and position of the miniature sources make this configuration more applicable for in vivo implementation. A modified ultrasound imaging system is used to display the CrW propagation. A shear speed mapping algorithm is developed using a detailed analysis of the CrW. The proposed setup is applied to several biomaterials including a homogeneous phantom, an inhomogeneous phantom and an ex vivo human liver. The data are analyzed using the mapping algorithm to reveal the biomechanical properties of the biomaterials.

pared by t-test ( p<0.001). Polyunsaturated fatty acids in hepatic total lipids were measured by gas chromatography. Results are given in % of total fatty acids. Spearman correla-tions were used to identify potential associations. Results: Twenty-six miRNAs were differentially expressed between HC, SS and NASH, including miR10b which was upregulated in HC vs NASH (p=0.00001). Total omega-3 PUFA were lower in NASH (mean ± SD) (2.35 ± 0.65 %) and SS (3.28 ± 1.23 %) compared with HC (4.44 ± 1.61 %) (p<0.05). Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the biologically active long-chain omega-3 PUFA, were also lower in NASH and SS than in HC (p<0.05). Twenty of the differentially expressed miRNAs were significantly correlated with at least one omega-3 PUFA, including miR10b which was positively correlated with DHA (r=0.417, p=0.001) and total omega-3 PUFA (r=0.343, p=0.008). Conclusion: The expression of miR10b was higher in HC than NASH and positively correlated with hepatic omega-3 PUFA. A potential target of miR10b is peroxisome proliferator-activated receptor- α , which can contribute to steatogenesis and inflammation in NAFLD. These results support the concept of associations between PUFA, epigenetic mechanisms, and NAFLD-related gene expression. Further studies are required to establish cause-effect relationships and examine the potential of omega-3 PUFA supplementation to regulate miRNA in NAFLD. Background: Hepatitis C (HCV) is the most frequent indication for liver transplantation. Several donor factors have been identified influencing post-transplant outcomes; however the impact of donor graft steatosis is debated. The aim of this study is to assess the impact of donor graft steatosis on patient and graft survival in HCV+ recipients after transplantation Methods: We reviewed the clinical course of all adult primary liver transplants from 2002 – 2010. 448 patients were included in the final analysis. Patients were grouped according to their HCV status (+/-) and level of donor steatosis (>30% or ≤ 30%); group 1: HCV-/DSteatosis ≤ 30%; 2: HCV+/DSteatosis ≤ 30%; 3: HCV-/DSteatosis>30%; 4: HCV+/DSteatosis>30%. Survival was analyzed with univariate statistics and regression models and correlated with donor and recipient characteristics; associations were included in the final multivariate model. Results: Patients were followed up for a median of 60 months. Overall patient and graft survival was significantly different across the 4 groups: graft 78.7%, 70.3%, 71.8%, 36% (p=0.01); patient: 87.2, 79.7, 79.7, 45.6% for group 1, 2, 3 and 4 respectively (p=0.02). HCV positive patients who received a graft with more than 30% steatosis demonstrated the worst overall graft and those with non-HCV diagnosis and ≤ 30% steatosis had superior outcomes to all other groups. This held true after multivariate adjustment graft (p=0.02) Background: Recent studies have suggested that microRNAs (miRs) play an important role in the pathogenesis of NAFLD and NASH but human studies especially those that integrate miRs with mRNAs are lacking. Aim: To identify miRs, mRNAs as well as the miR-mRNA regulatory network involved in hepatic fat accumulation and human NAFLD. Materials and Methods: This study consisted of 206 human liver samples obtained from organ donors on which transcriptome has been profiled and total hepatic fat content (HFC, mg fat/mg total protein) was measured. In a subset (n=73) we also measured genome-wide miRs; out of which 50 samples were characterized into normal (n=34) and NAFLD (n=16) based on their histology. Spearman correlation was calculated between miR/mRNA expression and HFC to identify miRs and mRNA significantly associated with HFC (p < 0.05 for miR and p< 0.001 for mRNA). Further, miR-mRNA association network was built separately for both NAFLD and normal groups based on miRs and mRNA exhibiting high level correlation (p<0.001). To build a post-transcrip-tional regulatory network associated with HFC, miRs and mRNAs strongly correlated with HFC were further integrated based on the potential miR-mRNA targeting by employing two miR target prediction tools (TargetScan and MiRanda). mRNAs targeted by miRNAs in this network (significantly correlated with HFC) were analyzed by pathway enrichment analysis tools, DAVID and Panther. Results: We identified 67 miRs significantly correlated with HFC (p<0.05), among which 16 miRs were strongly associated with NAFLD phenotype (p<0.05). Background and Aim: Elevated serum free fatty acid and hepatocyte apoptosis are features of nonalcoholic steatohepatitis (NASH). We have previously shown that saturated free fatty acids (FFAs) induce hepatocyte apoptosis by translational upregulation of death receptor (DR) 5 (Cazanave et al, JBC, 2010). However, details regarding DR5-mediated lipoapoptosis are unexplored. Cellular inhibitor of apoptosis 1 protein is a potent inhibitor of DR5 mediated cell death (Guicciardi et al, Exp Cell Res. 2011). Previous studies have also demonstrated that proteasomal degradation of cIAP-1 occurs through their auto-ubiquitination, which is mediated by the E3 ubiquitin ligase activity of its RING domain. Aim of the current study was to determine the detailed role of cIAP-1 degradation during hepatocyte lipoapoptosis. Methods: Huh-7 cells, Hep3B cells, primary hepatocytes from wild type and DR5 -/- mice were employed for the study. Cells were treated with unsaturated free fatty acid, palmitate (200-800 μ M). Huh-7 cells were tran-siently transfected with constructs expressing cIAP-1 mutant deficient in their E3 ligase activity due to specific replacement of critical histidine residues in their RING domains. cIAPs protein was assessed by immunoblotting. Apoptosis was assessed by characteristic nuclear staining with DAPI and cleavage of PARP by immunoblotting. Results: cIAP-1 protein underwent cellular elimination following treatment with the 800 μ M palmitate (PA) in Huh7, Hep3B, and HepG2 cells. Huh-7 cells transfected with RING-mutant cIAP-1 did not degrade with PA treatment, indicating that cIAP-1 undergoes proteasomal degradation by PA. Furthermore, transfection of cells with RING-mutant cIAP-1 decreased PA-induced apoptosis. Incubation with the SMAC mimetic JP1584 (500 nM), which induces degradation of cIAPs, significantly enhanced PA- mediated apoptosis in Hep3B cells and Mouse primary hepatocytes. In contrast, JP 1584 failed to sensitize primary hepatocytes from DR5 -/- mouse to apoptosis, suggesting that degradation of cIAP-1 by PA enhances death receptor mediated pathway of lipoapoptosis. Conclusions: Collectively, these results implicate RING domain dependent degradation of cIAP-1 by FFA regulates lipoapoptosis via DR5-induced hepatocyte lipoapoptosis. cose intolerance in diabetes. Collectively, these new drug targets of metformin may sever to mediate autophagy and metabolic homeostasis with important implications for obesity-related fatty liver disease, insulin resistance, and cancer. Background: There is considerable evidence that intestinal microbiota are involved in the development of metabolic syn-dromes and consequently with that of nonalcoholic fatty liver disease (NAFLD). Toll-like receptors (TLRs) are essential for the recognition of microbiota. However, the induction mechanism of TLR signals through the gut-liver axis for triggering the development of nonalcoholic steatohepatitis (NASH) or NAFLD remains unclear. In this study, we investigated the role of fatty acids on the formation of pro-inflammatory state of NAFLD. Methods: C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks. Antibiotics treatment was followed by feeding a HFD for further 8 weeks. The mice were sacrificed and histopathological evaluation was performed. The expressions of TLRs, tumor necrosis factor (TNF), interleukin 1 β (IL-1 β ), and phospho-inter-leukin-1 receptor-associated kinase 1 (pIRAK1) in the liver and small intestine were assessed. In addition, Huh7 and THP-1 cells, both of which are representatives of hepatocytes and Kupffer cells, respectively, were treated with fatty acids, and the direct effects of fatty acids on TLR induction by these cells were evaluated. Results: Histopathological evaluation showed intracellular fat droplets and ballooning degeneration of hepatocytes, but no obvious infiltration of inflammatory cells was noted. The expressions of inflammatory cytokines such as TNF, IL-1 β , and TLR2, TLR4, TLR5, and TLR9 and pIRAK1 were increased in the liver, but decreased in the small intestine of HFD mice in vivo. Antibiotics treatment improved steatosis, serum ALT, serum free fatty acids, and TLR expressions in the liver. In addition, the expression of carbohydrate response element binding protein (ChREBP) and sterol response element binding protein 1 (SREBP1) were decreased in the liver. The expressions of TLRs in Huh7 and THP-1 cells were increased by treatment with fatty acids. Conclusion: Our data suggests that dietary fatty acids trigger the expressions of TLRs, which contribute to the pro-inflammatory state of NAFLD. Furthermore intestinal microbiota of HFD develops it via induction of hepatic de novo lipogenesis. CD18, or β 2-integrin, is a leukocyte adhesion molecule that promotes neutrophil and macrophage invasion into sites of tissue injury. Mice lacking CD18 have impaired neutrophil migration and activation. Studies have shown that CD18-deficient mice are protected from some BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) can progress to more severe lesions including nonalcoholic steatohepatitis (NASH) and cirrhosis. A diet rich in fat along with host environmental factors, including gut-flora, play a central role in NASH pathogenesis. Investigation for the molecular basis of gut-permeability in NAFLD progression has not been previously evaluated. We obtained knockout mice for Junctional Adhesion Molecule A (JAM-A -/- ) which have a 10 fold increase in intestinal permeability as previously assessed by de