Hepatitis B and C virus coinfection: a novel model system reveals the absence of direct viral interference.

Coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) has been associated with severe liver disease and frequent progression to cirrhosis and hepatocellular carcinoma. Clinical evidence suggests reciprocal replicative suppression of the two viruses, or viral interference. However, interactions between HBV and HCV have been difficult to study due to the lack of appropriate model systems. We have established a novel model system to investigate interactions between HBV and HCV. Stable Huh-7 cell lines inducibly replicating HBV were transfected with selectable HCV replicons or infected with cell culture-derived HCV. In this system, both viruses were found to replicate in the same cell without overt interference. Specific inhibition of one virus did not affect the replication and gene expression of the other. Furthermore, cells harboring replicating HBV could be infected with cell culture-derived HCV, arguing against superinfection exclusion. Finally, cells harboring replicating HBV supported efficient production of infectious HCV. Conclusion: HBV and HCV can replicate in the same cell without evidence for direct interference in vitro. Therefore, the viral interference observed in coinfected patients is probably due to indirect mechanisms mediated by innate and/or adaptive host immune responses. These findings provide new insights into the pathogenesis of HBV-HCV coinfection and may contribute to its clinical management in the future.

Traitement des tumeurs maligne du foie par radiologie interventionnelle: techniques intra-artérielles [Interventional radiology procedures for malignancies of the liver treatment: Intraarterial procedures].

Intraarterial procedures such as chemoembolization and radioembolization aim for the palliative treatment of advanced hepatocellular carcinoma (stage BCLC B and C with tumoral portal thrombosis). The combination of hepatic intraarterial chemotherapy and systemic chemotherapy can increase the probability of curing colorectal cancer with hepatic metastases not immediately accessible to surgical treatment or percutaneous ablation.

Bridging basic science and clinical research - The EASL Monothematic Conference on Translational Research in Viral Hepatitis.

The EASL Monothematic Conference on Translational Research in Viral Hepatitis brought together a group of leading scientists and clinicians working on both, basic and clinical aspects of viral hepatitis, thereby building bridges from bench to bedside. This report recapitulates the presentations and discussions at the conference held in Lyon, France on November 29-30, 2013. In recent years, great advances have been made in the field of viral hepatitis, particularly in hepatitis C virus (HCV) infection. The identification of IL28B genetic polymorphisms as a major determinant for spontaneous and treatment-induced HCV clearance was a seminal discovery. Currently, hepatologists are at the doorstep of even greater advances, with the advent of a wealth of directly acting antivirals (DAAs) against HCV. Indeed, promising results have accumulated over the last months and few years, showing sustained virological response (SVR) rates of up to 100% with interferon-free DAA combination therapies. Thus, less than 25years after its identification, HCV infection may soon be curable in the vast majority of patients, highlighting the great success of HCV research over the last decades. However, viral hepatitis and its clinical complications such as liver cirrhosis and hepatocellular carcinoma (HCC) remain major global challenges. New therapeutic strategies to tackle hepatitis B virus (HBV) and hepatitis D virus (HDV) infection are needed, as current therapies have undeniable limitations. Nucleoside/nucleotide analogues (NUC) can efficiently control HBV replication and reduce or even reverse liver damage. However, these drugs have to be given for indefinite periods in most patients to maintain virological and biochemical responses. Although sustained responses off treatment can be achieved by treatment with (pegylated) interferon-α, only about 10-30% of patients effectively resolve chronic hepatitis B. It was the goal of this conference to review the progress made over the last years in chronic viral hepatitis research and to identify key questions that need to be addressed in order to close the gap between basic and clinical research and to develop novel preventive and treatment approaches for this most common cause of liver cirrhosis and HCC.

Transcriptional dissection of pancreatic tumors engrafted in mice.

BACKGROUND: Engraftment of primary pancreas ductal adenocarcinomas (PDAC) in mice to generate patient-derived xenograft (PDX) models is a promising platform for biological and therapeutic studies in this disease. However, these models are still incompletely characterized. Here, we measured the impact of the murine tumor environment on the gene expression of the engrafted human tumoral cells. METHODS: We have analyzed gene expression profiles from 35 new PDX models and compared them with previously published microarray data of 18 PDX models, 53 primary tumors and 41 cell lines from PDAC. The results obtained in the PDAC system were further compared with public available microarray data from 42 PDX models, 108 primary tumors and 32 cell lines from hepatocellular carcinoma (HCC). We developed a robust analysis protocol to explore the gene expression space. In addition, we completed the analysis with a functional characterization of PDX models, including if changes were caused by murine environment or by serial passing. RESULTS: Our results showed that PDX models derived from PDAC, or HCC, were clearly different to the cell lines derived from the same cancer tissues. Indeed, PDAC- and HCC-derived cell lines are indistinguishable from each other based on their gene expression profiles. In contrast, the transcriptomes of PDAC and HCC PDX models can be separated into two different groups that share some partial similarity with their corresponding original primary tumors. Our results point to the lack of human stromal involvement in PDXs as a major factor contributing to their differences from the original primary tumors. The main functional differences between pancreatic PDX models and human PDAC are the lower expression of genes involved in pathways related to extracellular matrix and hemostasis and the up- regulation of cell cycle genes. Importantly, most of these differences are detected in the first passages after the tumor engraftment. CONCLUSIONS: Our results suggest that PDX models of PDAC and HCC retain, to some extent, a gene expression memory of the original primary tumors, while this pattern is not detected in conventional cancer cell lines. Expression changes in PDXs are mainly related to pathways reflecting the lack of human infiltrating cells and the adaptation to a new environment. We also provide evidence of the stability of gene expression patterns over subsequent passages, indicating early phases of the adaptation process.

Unusual association of diseases/symptoms: Encephalitis with herpes simplex-2 in the cerebrospinal fluid and anti-RI (ANNA-2) antibodies: an infectious or a paraneoplastic syndrome?

We report on a 70-year-old woman with partial complex status epilepticus who was initially diagnosed with herpes simplex-2 (HSV-2) encephalitis, based on brain magnetic resonance imaging (MRI) findings, cerebrospinal fluid (CSF) lymphocytic pleocytosis and HSV-2 DNA detection by polymerase chain reaction (PCR) in the CSF, but without improvement on intravenous acyclovir. Anti-Ri antibodies were positive and computed tomography (CT) investigations revealed a small cell carcinoma at biopsy suggesting paraneoplastic encephalitis. The outcome was unfavourable and the autopsy showed typical features of paraneoplastic encephalitis but no evidence of viral inclusions. This case report is interesting because: (1) it is the first report of an autopsy proven paraneoplastic widespread encephalitis with anti-Ri antibodies; (2) despite a positive HSV-2 PCR in the CSF, there was no sign of herpetic infections of the nervous system; and (3) it illustrates the fact that if paraneoplastic antibodies are usually good markers of the underlying tumour, they are not always predictive of neurological deficits.

Management of hepatitis C virus (HCV) infection in drug substitution programs.

BACKGROUND: In Switzerland, intravenous drug use (IDU) accounts for 80% of newly acquired hepatitis C virus (HCV) infections. Early HCV treatment has the potential to interrupt the transmission chain and reduce morbidity/mortality due to decompensated liver cirrhosis and hepatocellular carcinoma. Nevertheless, patients in drug substitution programs are often insufficiently screened and treated. OBJECTIVE/METHODS: With the aim to improve HCV management in IDUs, we conducted a cross sectional chart review in three opioid substitution programs in St. Gallen (125 methadone and 71 heroin recipients). Results were compared with another heroin substitution program in Bern (202 patients) and SCCS/SHCS data. RESULTS: Among the methadone/heroin recipients in St. Gallen, diagnostic workup of HCV was better than expected: HCV/HIV-status was unknown in only 1% (2/196), HCV RNA was not performed in 9% (13/146) of anti-HCV-positives and the genotype missing in 15% (12/78) of HCV RNA-positives. In those without spontaneous clearance (two thirds), HCV treatment uptake was 23% (21/91) (HIV-: 29% (20/68), HIV+: 4% (1/23)), which was lower than in methadone/heroin recipients and particularly non-IDUs within the SCCS/SHCS, but higher than in the, mainly psychiatrically focussed, heroin substitution program in Bern (8%). Sustained virological response (SVR) rates were comparable in all settings (overall: 50%, genotype 1: 35-40%, genotype 3: two thirds). In St. Gallen, the median delay from the estimated date of infection (IDU start) to first diagnosis was 10 years and to treatment was another 7.5 years. CONCLUSIONS: Future efforts need to focus on earlier HCV diagnosis and improvement of treatment uptake among patients in drug substitution programs, particularly if patients are HIV-co-infected. New potent drugs might facilitate the decision to initiate treatment.

Cardiovascular drug interactions with tyrosine kinase inhibitors

Imatinib mesylate, a selective inhibitor of tyrosine kinases, has excellent efficacy in the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal tumour (GIST). Inducing durable responses and achieving prolonged survival, it has become the standard of care for the treatment of these diseases. It has opened the way to the development of additional tyrosine kinase inhibitors (TKIs), including sunitinib, nilotinib, dasatinib and sorafenib, all indicated for the treatment of various haematological malignancies and solid tumours. TKIs are prescribed for prolonged periods and are often taken by patients with - notably cardiovascular - comorbidities. Hence TKIs are regularly co-administered with cardiovascular drugs, with a considerable risk of potentially harmful drug-drug interactions due to the large number of agents used in combination. However, this aspect has received limited attention so far, and a comprehensive review of the published data on this important topic has been lacking. We review here the available data and pharmacological mechanisms of interactions between commonly prescribed cardiovascular drugs and the TKIs marketed at present. Regular updating of the literature on this topic will be mandatory, as will the prospective reporting of unexpected clinical observations, given the fact that these drugs have been only recently marketed.

Mapping the Membrane Topology of Hepatitis C Virus Nonstructural Protein 4B

BACKGROUND: Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is an integral membrane protein that has only poorly been characterized to date. In particular, a precise membrane topology is thus far elusive. Here, we explored a novel strategy to map the membrane topology of HCV NS4B. METHODS: Selective permeabilization of the plasma membrane, maleimide-polyethyleneglycol (mPEG) labeling of natural or engineered cysteine residues and immunoblot analyses were combined to map the membrane topology of NS4B. Cysteine substitutions were introduced at carefully selected positions within NS4B and their impact on HCV RNA replication and infectious virus production analyzed in cell culture. RESULTS: We established a panel of viable HCV mutants with cysteine substitutions at strategic positions within NS4B. These mutants are infectious and replicate to high levels in cell culture. In parallel, we adapted and optimized the selective permeabilization and mPEG labeling techniques to Huh-7 human hepatocellular carcinoma cells which can support HCV infection and replication. CONCLUSIONS: The newly established experimental tools and techniques should allow us to refine the membrane topology of HCV NS4B in a physiological context. The expected results should enhance our understanding of the functional architecture of the HCV replication complex and may provide new opportunities for antiviral intervention in the future.

Enrichment of human CD4+ V(alpha)24/Vbeta11 invariant NKT cells in intrahepatic malignant tumors.

Invariant NKT cells (iNKT cells) recognize glycolipid Ags via an invariant TCR alpha-chain and play a central role in various immune responses. Although human CD4(+) and CD4(-) iNKT cell subsets both produce Th1 cytokines, the CD4(+) subset displays an enhanced ability to secrete Th2 cytokines and shows regulatory activity. We performed an ex vivo analysis of blood, liver, and tumor iNKT cells from patients with hepatocellular carcinoma and metastases from uveal melanoma or colon carcinoma. Frequencies of Valpha24/Vbeta11 iNKT cells were increased in tumors, especially in patients with hepatocellular carcinoma. The proportions of CD4(+), double negative, and CD8alpha(+) iNKT cell subsets in the blood of patients were similar to those of healthy donors. However, we consistently found that the proportion of CD4(+) iNKT cells increased gradually from blood to liver to tumor. Furthermore, CD4(+) iNKT cell clones generated from healthy donors were functionally distinct from their CD4(-) counterparts, exhibiting higher Th2 cytokine production and lower cytolytic activity. Thus, in the tumor microenvironment the iNKT cell repertoire is modified by the enrichment of CD4(+) iNKT cells, a subset able to generate Th2 cytokines that can inhibit the expansion of tumor Ag-specific CD8(+) T cells. Because CD4(+) iNKT cells appear inefficient in tumor defense and may even favor tumor growth and recurrence, novel iNKT-targeted therapies should restore CD4(-) iNKT cells at the tumor site and specifically induce Th1 cytokine production from all iNKT cell subsets.

Is magnetic resonance imaging of hepatic hemangioma any different in liver fibrosis and cirrhosis compared to normal liver?

PURPOSE: To compare qualitative and quantitative magnetic resonance (MR) imaging characteristics of hepatic hemangiomas in patients with normal, fibrotic and cirrhotic livers. MATERIALS AND METHODS: Retrospective, institutional review board approved study (waiver of informed consent). Eighty-nine consecutive patients with 231 hepatic hemangiomas who underwent liver MR imaging for lesion characterization were included. Lesions were classified into three groups according to the patients' liver condition: no underlying liver disease (group 1), fibrosis (group 2) and cirrhosis (group 3). Qualitative and quantitative characteristics (number, size, signal intensities on T1-, T2-, and DW MR images, T2 shine-through effect, enhancement patterns (classical, rapidly filling, delayed filling), and ADC values) were compared. RESULTS: There were 160 (69%), 45 (20%), and 26 (11%) hemangiomas in groups 1, 2 and 3, respectively. Lesions were larger in patients with normal liver (group 1 vs. groups 2 and 3; P=.009). No difference was found between the groups on T2-weighted images (fat-suppressed fast spin-echo (P=.82) and single-shot (P=.25)) and in enhancement patterns (P=.56). Mean ADC values of hemangiomas were similar between groups 1, 2 and 3 (2.11±.52×10(-3)mm(2)/s, 2.1±.53×10(-3)mm(2)/s and 2.14±.44×10(-3)mm(2)/s, P=87, respectively). T2 shine-through effect was less frequently observed in cirrhosis (P=.02). CONCLUSION: MR imaging characteristics of hepatic hemangioma were similar in patients with normal compared to fibrotic and cirrhotic livers. Smaller lesion size was observed with liver disease and less T2 shine-through effect was seen in hemangiomas developed on cirrhosis, the latter being an important finding to highlight in these patients at risk of developing hepatocellular carcinoma.

Characterization of fortuitously discovered focal liver lesions: additional information provided by shearwave elastography.

OBJECTIVES: To prospectively assess the stiffness of incidentally discovered focal liver lesions (FLL) with no history of chronic liver disease or extrahepatic cancer using shearwave elastography (SWE). METHODS: Between June 2011 and May 2012, all FLL fortuitously discovered on ultrasound examination were prospectively included. For each lesion, stiffness was measured (kPa). Characterization of the lesion relied on magnetic resonance imaging (MRI) and/or contrast-enhanced ultrasound, or biopsy. Tumour stiffness was analysed using ANOVA and non-parametric Mann-Whitney tests. RESULTS: 105 lesions were successfully evaluated in 73 patients (61 women, 84%) with a mean age of 44.8 (range: 20‒75). The mean stiffness was 33.3 ± 12.7 kPa for the 60 focal nodular hyperplasia (FNH), 19.7 ± 9.8 k Pa for the 17 hepatocellular adenomas (HCA), 17.1 ± 7 kPa for the 20 haemangiomas, 11.3 ± 4.3 kPa for the five focal fatty sparing, 34.1 ± 7.3 kPa for the two cholangiocarcinomas, and 19.6 kPa for one hepatocellular carcinoma (p < 0.0001). There was no difference between the benign and the malignant groups (p = 0.64). FNHs were significantly stiffer than HCAs (p < 0.0001). Telangiectatic/inflammatory HCAs were significantly stiffer than the steatotic HCAs (p = 0.014). The area under the ROC curve (AUROC) for differentiating FNH from other lesions was 0.86 ± 0.04. CONCLUSION: SWE may provide additional information for the characterization of FFL, and may help in differentiating FNH from HCAs, and in subtyping HCAs. KEY POINTS: ? SWE might be helpful for the characterization of solid focal liver lesions ? SWE cannot differentiate benign from malignant liver lesions ? FNHs are significantly stiffer than other benign lesions ? Telangiectatic/inflammatory HCA are significantly stiffer than steatotic ones.

GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

Abstract Background: Hypoxia-mediated HIF-1a stabilization and NF-kB activation play a key role in carcinogenesis by fostering cancer cell survival, angiogenesis and tumor invasion. Gangliosides are integral components of biological membranes with an increasingly recognized role as signaling intermediates. In particular, ganglioside GD3 has been characterized as a proapoptotic lipid effector by promoting cell death signaling and suppression of survival pathways. Thus, our aim was to analyze the role of GD3 in hypoxia susceptibility of hepatocarcinoma cells and in vivo tumor growth. Methodology/Principal Findings: We generated and characterized a human hepatocarcinoma cell line stably expressing GD3 synthase (Hep3B-GD3), which catalyzes the synthesis of GD3 from GM3. Despite increased GD3 levels (2-3 fold), no significant changes in cell morphology or growth were observed in Hep3B-GD3 cells compared to wild type Hep3B cells under normoxia. However, exposure of Hep3B-GD3 cells to hypoxia (2% O2) enhanced reactive oxygen species (ROS) generation, resulting in decreased cell survival, with similar findings observed in Hep3B cells exposed to increasing doses of exogenous GD3. In addition, hypoxia-induced c-Src phosphorylation at tyrosine residues, NF-kB activation and subsequent expression of Mn-SOD were observed in Hep3B cells but not in Hep3B-GD3 cells. Moreover, MnTBAP, an antioxidant with predominant SOD mimetic activity, reduced ROS generation, protecting Hep3B-GD3 cells from hypoxia-induced death. Finally, lower tumor growth, higher cell death and reduced Mn-SOD expression were observed in Hep3B-GD3 compared to Hep3B tumor xenografts. Conclusion: These findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-kB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy.

Hépatite C: épidémiologie, histoire naturelle et diagnostic [Hepatitis C: epidemiology natural course and diagnosis].

L'infection par le virus de l'hépatite C (HCV) représente une des causes les plus fréquentes d'hépatite chronique, de cirrhose et de carcinome hépatocellulaire au niveau mondial. D'énormes progrès ont été réalisés durant ces 25 dernières années depuis la découverte du HCV, notamment dans la compréhension de la virologie moléculaire, de la pathogenèse et de l'histoire naturelle ainsi que dans la prévention, le diagnostic et le traitement de l'hépatite C. Ces avancées seront résumées dans cet article et discutées à la lumière de nouveaux défis. Hepatitis C virus (HCV) infection represents a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. Great progress in the understanding of the molecular virology, pathogenesis and natural course as well as the prevention, diagnosis and treatment of hepatitis C have been made in over the last 25 years since the discovery of HCV. Here, we review recent advances and discuss them in the light of new challenges.

Metabolic syndrome and nonalcoholic fatty liver disease: Is insulin resistance the link?

Metabolic syndrome (MetS) is a disease composed of different risk factors such as obesity, type 2 diabetes or dyslipidemia. The prevalence of this syndrome is increasing worldwide in parallel with the rise in obesity. Nonalcoholic fatty liver disease (NAFLD) is now the most frequent chronic liver disease in western countries, affecting more than 30% of the general population. NAFLD encompasses a spectrum of liver manifestations ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis, which may ultimately progress to hepatocellular carcinoma. There is accumulating evidence supporting an association between NAFLD and MetS. Indeed, NAFLD is recognized as the liver manifestation of MetS. Insulin resistance is increasingly recognized as a key factor linking MetS and NAFLD. Insulin resistance is associated with excessive fat accumulation in ectopic tissues, such as the liver, and increased circulating free fatty acids, which can further promote inflammation and endoplasmic reticulum stress. This in turn aggravates and maintains the insulin resistant state, constituting a vicious cycle. Importantly, evidence shows that most of the patients developing NAFLD present at least one of the MetS traits. This review will define MetS and NAFLD, provide an overview of the common pathophysiological mechanisms linking MetS and NAFLD, and give a perspective regarding treatment of these ever growing metabolic diseases.

When the non-contrast-enhanced phase is unnecessary in abdominal computed tomography scans? A retrospective analysis of 244 cases

Objective: To evaluate the necessity of the non contrast-enhanced phase in abdominal computed tomography scans. Materials and Methods: A retrospective, cross-sectional, observational study was developed, evaluating 244 consecutive abdominal computed tomography scans both with and without contrast injection. Initially, the contrast-enhanced images were analyzed (first analysis). Subsequently, the observers had access to the non-contrast-enhanced images for a second analysis. The primary and secondary diagnoses were established as a function of the clinical indications for each study (such as tumor staging, acute abdomen, investigation for abdominal collection and hepatocellular carcinoma, among others). Finally, the changes in the diagnoses resulting from the addition of the non-contrast-enhanced phase were evaluated. Results: Only one (0.4%; p > 0.999; non-statistically significant) out of the 244 reviewed cases had the diagnosis changed after the reading of non-contrast-enhanced images. As the secondary diagnoses are considered, 35 (14%) cases presented changes after the second analysis, as follows: nephrolithiasis (10%), steatosis (3%), adrenal nodule (0.7%) and cholelithiasis (0.3%). Conclusion: For the clinical indications of tumor staging, acute abdomen, investigation of abdominal collections and hepatocellular carcinoma, the non-contrast-enhanced phase can be excluded from abdominal computed tomography studies with no significant impact on the diagnosis.