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2.
Methods Mol Biol ; 2769: 15-25, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38315386

RESUMO

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.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Humanos , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Dietilnitrosamina/toxicidade , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Carcinogênese/induzido quimicamente , Carcinogênese/genética , Hepatócitos/patologia , Camundongos Endogâmicos C57BL
3.
Methods Mol Biol ; 2769: 129-141, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38315394

RESUMO

Tissue-resident and recruited immune cells are essential mediators of natural and therapy-induced immunosurveillance of liver neoplasia. This idea has been recently reinforced by the clinical approval of immune checkpoint inhibitors for the immunotherapy of hepatocellular carcinoma and cholangiocarcinoma. Such research progress relies on the in-depth characterization of the immune populations that are present in pre-neoplastic and neoplastic hepatic lesions. A convenient technology for advancing along this path is high-dimensional cytometry.In this chapter, we present a protocol to assess the subtype and differentiation state of hepatic lymphocyte populations by multicolor immunofluorescence staining and flow cytometry. We detail the steps required for viability assessment and immune cell phenotyping of single-cell suspensions of liver cells by means of surface and intracellular staining of more than a dozen markers of interest. This protocol does not require prior removal of debris and dead cells and allows to process multiple samples in parallel. The procedure includes the use of a fixative-resistant viability dye that allows cell fixation and permeabilization after cell surface staining and before intracellular staining and data acquisition on a flow cytometer. Moreover, we provide a panel of fluorochrome-labeled antibodies designed for the characterization of lymphocytic subsets that can be adapted to distinct experimental settings. Finally, we present an overview of the post-staining pipeline, including data acquisition on a flow cytometer and tools for post-acquisition analyses.


Assuntos
Neoplasias dos Ductos Biliares , Neoplasias Hepáticas , Humanos , Citometria de Fluxo/métodos , Subpopulações de Linfócitos , Ductos Biliares Intra-Hepáticos
4.
Hepatol Int ; 16(6): 1259-1272, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35927368

RESUMO

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).


Assuntos
Hepatite B Crônica , Hepatite B , Ácidos Nucleicos , Humanos , Vírus da Hepatite B/genética , Ácidos Nucleicos/uso terapêutico , Estudos Prospectivos , DNA Viral/genética , Hepatite B Crônica/tratamento farmacológico , Replicação Viral , RNA , RNA Viral/análise
5.
Gut ; 70(12): 2373-2382, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34535538

RESUMO

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.


Assuntos
Processamento Alternativo , Vírus da Hepatite B/genética , Splicing de RNA , Genoma Viral , Humanos , Proteômica
6.
Artigo em Inglês | MEDLINE | ID: mdl-30987320

RESUMO

Exposure to particulate matter (PM) is leading to various respiratory health outcomes. Compared to coarse and fine particles, less is known about the effects of chronic exposure to ultrafine particles, despite their higher number and reactivity. In the present study, we performed a time-course experiment in mice to better analyze the lung impact of atmospheric ultrafine particles, with regard to the effects induced by fine particles collected on the same site. Trace element and PAH analysis demonstrated the almost similar chemical composition of both particle fractions. Mice were exposed intranasally to FF or UFP according to acute (10, 50 or 100 µg of PM) and repeated (10 µg of PM 3 times a week during 1 or 3 months) exposure protocols. More particle-laden macrophages and even greater chronic inflammation were observed in the UFP-exposed mice lungs. Histological analyses revealed that about 50% of lung tissues were damaged in mice exposed to UFP for three months versus only 35% in FF-exposed mice. These injuries were characterized by alveolar wall thickening, macrophage infiltrations, and cystic lesions. Taken together, these results strongly motivate the update of current regulations regarding ambient PM concentrations to include UFP and limit their emission.


Assuntos
Poluentes Atmosféricos/efeitos adversos , Exposição Ambiental/efeitos adversos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Material Particulado/efeitos adversos , Síndrome de Resposta Inflamatória Sistêmica/induzido quimicamente , Síndrome de Resposta Inflamatória Sistêmica/patologia , Poluentes Atmosféricos/análise , Animais , Modelos Animais de Doenças , Exposição Ambiental/análise , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Material Particulado/administração & dosagem , Fatores de Tempo
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