María Mora González López Ledesma

Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis

Objectives We used a screening strategy of global serum microRNA (miRNA) profiling, followed by a second stage of independent replication and exploration of liver expression of selected miRNAs to study: (1) the circulating miRNA signature associated with non-alcoholic fatty liver disease (NAFLD) progression and predictive power, (2) the role of miRNAs in disease biology and (3) the association between circulating miRNAs and features of the metabolic syndrome. Methods The study used a case-control design and included patients with NAFLD proven through biopsy and healthy controls. Results Among 84 circulating miRNAs analysed, miR-122, miR-192, miR-19a and miR-19b, miR-125b, and miR-375 were upregulated >2-fold (p<0.05) either in simple steatosis (SS) or non-alcoholic steatohepatitis (NASH). The most dramatic and significant fold changes were observed in the serum levels of miR-122 (7.2-fold change in NASH vs controls and 3.1-fold change in NASH vs SS) and miR-192 (4.4-fold change in NASH vs controls); these results were replicated in the validation set. The majority of serum miR-122 circulate in argonaute2-free forms. Circulating miR-19a/b and miR-125b were correlated with biomarkers of atherosclerosis. Liver miR-122 expression was 10-fold (p<0.03) downregulated in NASH compared with SS and was preferentially expressed at the edge of lipid-laden hepatocytes. In vitro exploration showed that overexpression of miR-122 enhances alanine aminotransferase activity. Conclusions miR-122 plays a role of physiological significance in the biology of NAFLD; circulating miRNAs mirror the histological and molecular events occurring in the liver. NAFLD has a distinguishing circulating miRNA profile associated with a global dysmetabolic disease state and cardiovascular risk.

A targeted functional RNA interference screen uncovers glypican 5 as an entry factor for hepatitis B and D viruses

Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus and hepatitis delta virus use the sodium/taurocholate cotransporting polypeptide (a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high‐throughput infectious cell culture model enabling functional genomics of hepatitis delta virus entry and infection. Using a targeted RNA interference entry screen, we identified glypican 5 as a common host cell entry factor for hepatitis B and delta viruses. Conclusion: These findings advance our understanding of virus cell entry and open new avenues for curative therapies. As glypicans have been shown to play a role in the control of cell division and growth regulation, virus–glypican 5 interactions may also play a role in the pathogenesis of virus‐induced liver disease and cancer. (Hepatology 2016;63:35–48)

Characterization of the basal core promoter and precore regions in anti-HBe-positive inactive carriers of hepatitis B virus

BACKGROUND The study of hepatitis B virus (HBV) genomic heterogeneity has become a major issue in investigations aimed at understanding the relationship between HBV mutants and the wide spectrum of clinical and pathological conditions associated with HBV infection. Although most chronically infected HBV patients are inactive carriers, several virological aspects of this state remain unclear. METHODS In order to determine the prevalence and clinical significance of mutations in the basal core promoter (BCP) and precore (pC) regions among inactive carriers, the nucleotide sequences from 41 inactive carriers were analyzed and compared with those from 29 individuals with chronic active hepatitis. RESULTS Genotypes A (24.3%), D (37.1%), F1b (12.9%), and F4 (18.6%) were the most prevalent. Mutations in the BCP/pC regions were observed in most of the inactive carriers (92.7%) and in most of the patients with chronic active hepatitis (93.1%). The prevalence of mutation 1764(A) was significantly higher in patients with chronic active hepatitis (65.5%) than in inactive carriers (36.6%) (p=0.038), whereas the prevalences of mutations at the other positions analyzed were not significantly different. Older patients (>50 years) showed BCP/pC patterns with a higher number of substitutions. Mutations were found to be biased by genotype: the 1896(A) mutation was highly prevalent in genotypes D and F4, while alternative substitutions in the pC region were more prevalent in genotypes A and F1b. CONCLUSIONS Mutations in the BCP/pC regions are the hallmark of chronic anti-HBe-positive individuals; nevertheless, the even distribution of mutations in active and inactive carriers suggests that BCP/pC mutations may occur during HBV infection not strictly related to the HBV infection activity.

Hepatitis B virus genotype distribution and genotype-specific BCP/preCore substitutions in acute and chronic infections in Argentina.

Aim In order to assess Hepatitis B Virus genotype (g) and subgenotype (sg) implications in the course of infection, 234 HBsAg positive patients in different infection stages were characterized (66 acute infections, 63 HBeAg positive chronic infections and 105 anti-HBe positive chronic infections). Results Overall, sgA2 (17.9%), gD (20.9%), sgF1b (34.2%) and sgF4 (19.7%) were the most prevalent. Subgenotype F1b was overrepresented in acute and chronic HBeAg infections (56.1%), whereas gD was the most frequent (40.0%) in anti-HBe positive chronic infections. Among chronic infections, HBeAg positivity rates were 50.0, 12.5, 62.8 and 35.3% for sgA2, gD, sgF1b and sgF4, respectively (p <0.05). A bias toward BCP/preCore mutations was observed among genotypes. In anti-HBe positive chronic infections, sgF1b was more prone to have A1762T/G1764A mutation than sgA2, sgF4 and gD (75.0, 40.0, 33.3 and 31.8%, p<0.005), whereas in the pC region, gD and sgF4 were more likely to have G1896A than sgA2 and sgF1b (81.0, 72.7, 0.0 and 31.3%, p <0.001). The unexpected low frequency of the G1896A mutation in the sgF1b (despite carrying 1858T) prompted us to perform a further analysis in order to identify genotype-specific features that could justify the pattern mutations observed. A region encompassing nucleotides 1720 to 1920 showed the higher dissimilarity between sgF1b and sgF4. Genotypes and subgenotypes carrying the 1727G, 1740C and 1773T polymorphisms were prevented to mutate position 1896. Discussion HBeAg seroconversion is a critical event in the natural history of HBV infection. Differences in the HBeAg positivity rate might be relevant since different studies have observed that delayed HBeAg seroconversion is associated with a more severe clinical course of infection, highlighting the critical role that genotypes/subgenotypes might play in the progression of HBV infection. Polymorphisms in the regions 1720 to 1920 could be involved in the molecular mechanisms underlying seroconversion of each genotype/subgenotype.

Promoter DNA Methylation of Farnesoid X Receptor and Pregnane X Receptor Modulates the Intrahepatic Cholestasis of Pregnancy Phenotype

The intrahepatic cholestasis of pregnancy (ICP) is a multifactorial liver disorder which pathogenesis involves the interplay among abnormal bile acid (BA) levels, sex hormones, environmental factors, and genetic susceptibility. The dynamic nature of ICP that usually resolves soon after delivery suggests the possibility that its pathobiology is under epigenetic modulation. We explored the status of white blood peripheral cells-DNA methylation of CpG-enriched sites at the promoter of targeted genes (FXR/NR1H4, PXR/NR1I2, NR1I3, ESR1, and ABCC2) in a sample of 88 ICP patients and 173 healthy pregnant women in the third trimester of their pregnancies. CpG dinucleotides at the gene promoter of nuclear receptors subfamily 1 members and ABCC2 transporter were highly methylated during healthy pregnancy. We observed significant differences at the distal (−1890) and proximal promoter (−358) CpG sites of the FXR/NR1H4 and at the distal PXR/NR1I2 (−1224) promoter, which were consistently less methylated in ICP cases when compared with controls. In addition, we observed that methylation at FXR/NR1H4-1890 and PXR/NR1I2-1224 promoter sites was highly and positively correlated with BA profiling, particularly, conjugated BAs. Conversely, methylation level at the proximal FXR/NR1H4-358 CpG site was significantly and negatively correlated with the primary cholic and secondary deoxycholic acid. In vitro exploration showed that epiallopregnanolone sulfate, a reported FXR inhibitor, regulates the transcriptional activity of FXR/NR1H4 but seems to be not involved in the methylation changes. In conclusion, the identification of epigenetic marks in target genes provides a basis for the understanding of adverse liver-related pregnancy outcomes, including ICP.

A targeted functional RNAi screen uncovers Glypican 5 as an entry factor for hepatitis B and D viruses

Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus and hepatitis delta virus use the sodium/taurocholate cotransporting polypeptide (a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high‐throughput infectious cell culture model enabling functional genomics of hepatitis delta virus entry and infection. Using a targeted RNA interference entry screen, we identified glypican 5 as a common host cell entry factor for hepatitis B and delta viruses. Conclusion: These findings advance our understanding of virus cell entry and open new avenues for curative therapies. As glypicans have been shown to play a role in the control of cell division and growth regulation, virus–glypican 5 interactions may also play a role in the pathogenesis of virus‐induced liver disease and cancer. (Hepatology 2016;63:35–48)

Analysis of the Molecular Evolution of Hepatitis B Virus Genotypes in Symptomatic Acute Infections in Argentina.

Hepatitis B virus (HBV) is a globally distributed human pathogen that leads to both self-limited and chronic infections. At least eight genotypes (A-H) with distinct geographical allocations and phylodynamic behaviors have been described. They differ substantially in many virological and probably some clinical parameters. The aim of this study was to analyze full-length HBV genome sequences from individuals with symptomatic acute HBV infections using phylogenetic and coalescent methods. The phylogenetic analysis resulted in the following subgenotype distribution: F1b (52.7%), A2 (18.2%), F4 (18.2%) and A1, B2, D3 and F2a 1.8% each. These results contrast with those previously reported from chronic infections, where subgenotypes F1b, F4, A2 and genotype D were evenly distributed. This differential distribution might be related to recent internal migrations and/or intrinsic biological features of each viral genotype that could impact on the probability of transmission. The coalescence analysis showed that after a diversification process started in the 80s, the current sequences of subgenotype F1b were grouped in at least four highly supported lineages, whereas subgenotype F4 revealed a more limited diversification pattern with most lineages without offspring in the present. In addition, the genetic characterization of the studied sequences showed that only two of them presented mutations of clinical relevance at S codifyng region and none at the polymerase catalytic domains. Finally, since the acute infections could be an expression of the genotypes currently being transmitted to new hosts, the predominance of subgenotype F1b might have epidemiological, as well as, clinical relevance due to its potential adverse disease outcome among the chronic cases.

HBV DNA genome co-transfection procedure for the evaluation of relative fitness

Hepatitis B virus (HBV) has a high mutation rate and exists as a mixture of genetically different but closely related variants. We present a HBV DNA co-transfection fitness assay and use it to evaluate the relative fitness of different HBV variants in two scenarios: seroconversion process and occupation of an ecological niche. In the seroconversion experiment, subgenotype D1 (sgtD1) deletion (1763–1770) had significantly lower fitness comparing with both sgtD1 wild type and sgtD1mut G1896A, while, in the case of occupation of ecological niche experiment, the results showed the same relative fitness between all of the genotype combinations, except F1b-F4. In this case sgtF1b clearly overgrow sgtF4, which is in accordance with the observation that F1b is the most prevalent in the new infections in Argentina. In summary, we present a method aimed to evaluate HBV viral fitness which improve the analysis of the relative frequency of viral variants during the HBV infection process.

Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: Role of basal core promoter and preCore mutations

In the context of pathogenesis of HBV infection, HBV genotypes and mutants have been shown to affect the natural course of chronic infection and treatment outcomes. In this work, we studied the induction of apoptosis by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. Both subgenotypes F1b and F4 HBV genome transfections induced cell death by apoptosis in human hepatocytes. The BCPdm (A1762T/G1764A) and preCore (G1896A) mutants induced higher levels of apoptosis than the wt virus. This increase in apoptosis was not associated with the enhanced viral replication of the variants. HBV-mediated apoptosis was independent of viral subgenotypes, and associated with the modulation of members of the regulatory Bcl-2 family proteins expression in the mitochondrial apoptotic pathway. Finally, the apoptosis induction increase observed for the preCore mutants suggests that HBeAg might have an anti-apoptotic effect in human hepatocytes.

A targeted functional RNA interference screen uncovers glypican 5 as an entry factor for hepatitis B and D viruses: VIRAL HEPATITIS

Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus and hepatitis delta virus use the sodium/taurocholate cotransporting polypeptide (a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high‐throughput infectious cell culture model enabling functional genomics of hepatitis delta virus entry and infection. Using a targeted RNA interference entry screen, we identified glypican 5 as a common host cell entry factor for hepatitis B and delta viruses. Conclusion: These findings advance our understanding of virus cell entry and open new avenues for curative therapies. As glypicans have been shown to play a role in the control of cell division and growth regulation, virus–glypican 5 interactions may also play a role in the pathogenesis of virus‐induced liver disease and cancer. (Hepatology 2016;63:35–48)