Diethylnitrosamine Induction of Hepatocarcinogenesis in Mice.

Diethylnitrosamine (DEN) is a chemical hepatocarcinogenic agent that triggers a large array of oncogenic mutations after a single injection. Initiated hepatocytes subsequently undergo clonal expansion within a proliferative environment, rendering the DEN model a comprehensive carcinogen. In rodent studies, DEN finds extensive utility in experimental liver cancer research, mimicking several aspects of human hepatocellular carcinoma (HCC), including angiogenesis, metabolic reprogramming, immune exhaustion, and the ability to metastasize. Beyond the wealth of scientific insights gleaned from this model, the objective of this chapter is to review morphological, genomic, and immunological characteristics associated to DEN-induced HCC. Furthermore, this chapter provides a detailed procedural guide to effectively induce hepatocarcinogenesis in mice through a single intraperitoneal injection of DEN.

Diversity of the nucleic acid forms of circulating HBV in chronically infected patients and its impact on viral cycle.

BACKGROUND: Besides the prototypical hepatitis B virus (HBV) infectious particle, which contains a full-length double-stranded DNA (flDNA), additional circulating virus-like particles, which carry pregenomic RNA (pgRNA), spliced1RNA (sp1RNA) or spliced-derived DNA (defDNA) forms have been described. We aimed to determine the level of these four circulating forms in patients and to evaluate their impact on viral lifecycle. METHODS: Chronic HBV untreated patients (n = 162), included in the HEPATHER cohort, were investigated. Pangenomic qPCRs were set up to quantify the four circulating forms of HBV nucleic acids (HBVnaf). In vitro infection assays were performed to address the impact of HBVnaf. RESULTS: Hierarchical clustering individualized two clusters of HBVnaf diversity among patients: (1) cluster 1 (C1) showing a predominance of flDNA; (2) cluster 2 (C2) showing various proportions of the different forms. HBeAg-positive chronic hepatitis phase and higher viral load (7.0 ± 6.4 vs 6.6 ± 6.2 Log10 copies/ml; p < 0.001) characterized C2 compared to C1 patients. Among the different HBVnaf, pgRNA was more prevalent in C1 patients with high vs low HBV viral load (22.1% ± 2.5% vs 4.1% ± 1.8% of HBVnaf, p < 0.0001) but remained highly prevalent in C2 patients, whatever the level of replication. C2 patients samples used in infection assays showed that: (1) HBVnaf secretion was independent of the viral strain; (2) the viral cycle efficiency differed according to the proportion of HBVnaf in the inoculum, independently of cccDNA formation. Inoculum enrichment before infection suggests that pgRNA-containing particles drive this impact on viral replication. CONCLUSION: Besides the critical role of HBV replication in circulating HBVnaf diversity, our data highlight an impact of this diversity on the dynamics of viral cycle. CLINICAL TRIAL REGISTRATION: Patients were included from a prospective multicenter French national cohort (ANRS CO22 HEPATHER, NCT01953458).

Alternative splicing of viral transcripts: the dark side of HBV.

Regulation of alternative splicing is one of the most efficient mechanisms to enlarge the proteomic diversity in eukaryotic organisms. Many viruses hijack the splicing machinery following infection to accomplish their replication cycle. Regarding the HBV, numerous reports have described alternative splicing events of the long viral transcript (pregenomic RNA), which also acts as a template for viral genome replication. Alternative splicing of HBV pregenomic RNAs allows the synthesis of at least 20 spliced variants. In addition, almost all these spliced forms give rise to defective particles, detected in the blood of infected patients. HBV-spliced RNAs have long been unconsidered, probably due to their uneasy detection in comparison to unspliced forms as well as for their dispensable role during viral replication. However, recent data highlighted the relevance of these HBV-spliced variants through (1) the trans-regulation of the alternative splicing of viral transcripts along the course of liver disease; (2) the ability to generate defective particle formation, putative biomarker of the liver disease progression; (3) modulation of viral replication; and (4) their intrinsic propensity to encode for novel viral proteins involved in liver pathogenesis and immune response. Altogether, tricky regulation of HBV alternative splicing may contribute to modulate multiple viral and cellular processes all along the course of HBV-related liver disease.