Browsing by Subject "Chronic hepatitis"

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    Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure
    (BMJ, 2023) Allweiss, Lena; Testoni, Barbara; Yu, Mei; Lucifora, Julie; Ko, Chunkyu; Qu, Bingqian; Lütgehetmann, Marc; Guo, Haitao; Urban, Stephan; Fletcher, Simon P.; Protzer, Ulrike; Levrero, Massimo; Zoulim, Fabien; Dandri, Maura; Microbiology and Immunology, School of Medicine
    Objectives: A major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA quantification is essential in preclinical and clinical studies. Southern blot (SB) permits cccDNA visualisation but lacks sensitivity and is very laborious. Quantitative PCR (qPCR) has no such limitations but inaccurate quantification due to codetection of viral replicative intermediates (RI) can occur. The use of different samples, preservation conditions, DNA extraction, nuclease digestion methods and qPCR strategies has hindered standardisation. Within the ICE-HBV consortium, available and novel protocols for cccDNA isolation and qPCR quantification in liver tissues and cell cultures were compared in six laboratories to develop evidence-based guidance for best practices. Design: Reference material (HBV-infected humanised mouse livers and HepG2-NTCP cells) was exchanged for cross-validation. Each group compared different DNA extraction methods (Hirt extraction, total DNA extraction with or without proteinase K treatment (+PK/-PK)) and nuclease digestion protocols (plasmid-safe ATP-dependent DNase (PSD), T5 exonuclease, exonucleases I/III). Samples were analysed by qPCR and SB. Results: Hirt and -PK extraction reduced coexisting RI forms. However, both cccDNA and the protein-free relaxed circular HBV DNA (pf-rcDNA) form were detected by qPCR. T5 and Exo I/III nucleases efficiently removed all RI forms. In contrast, PSD did not digest pf-rcDNA, but was less prone to induce cccDNA overdigestion. In stabilised tissues (eg, Allprotect), nucleases had detrimental effects on cccDNA. Conclusions: We present here a comprehensive evidence-based guidance for optimising, controlling and validating cccDNA measurements using available qPCR assays.
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    Regression of Hepatic Fibrosis and Evolution of Cirrhosis: A Concise Review
    (Wolters Kluwer, 2021-11) Khan, Shahbaz; Saxena, Romil; Pathology and Laboratory Medicine, School of Medicine
    Fibrosis is not a unidirectional, linear process, but a dynamic one resulting from an interplay of fibrogenesis and fibrolysis depending on the extent and severity of a biologic insult, or lack thereof. Regression of fibrosis has been documented best in patients treated with phlebotomies for hemochromatosis, and after successful suppression and eradication of chronic hepatitis B and C infections. This evidence mandates a reconsideration of the term “cirrhosis,” which implies an inevitable progression towards liver failure. Furthermore, it also necessitates a staging system that acknowledges the bidirectional nature of evolution of fibrosis, and has the ability to predict if the disease process is progressing or regressing. The Beijing classification attempts to fill this gap in contemporary practice. It is based on microscopic features termed “the hepatic repair complex,” defined originally by Wanless and colleagues. The elements of the hepatic repair complex represent the 3 processes of fragmentation and regression of scar, vascular remodeling (resolution), and parenchymal regeneration. However, regression of fibrosis does not imply resolution of cirrhosis, which is more than just a stage of fibrosis. So far, there is little to no evidence to suggest that large regions of parenchymal extinction can be repopulated by regenerating hepatocytes. Similarly, the vascular lesions of cirrhosis persist, and there is no evidence of complete return to normal microcirculation in cirrhotic livers. In addition, the risk of hepatocellular carcinoma is higher compared with the general population and these patients need continued screening and surveillance.