Pub Date : 2024-06-01DOI: 10.1016/j.jhepr.2024.101032
Xiangyu Zhai , Hao Zhang , Zhijia Xia , Mingkun Liu , Gang Du , Zhengchen Jiang , Huaxin Zhou , Dan Luo , Dandan Dou , Jingxin Li , Wei Wang , Xiaosong Li , Bin Jin
Background & Aims
Previous studies demonstrated oxytocin treatment effectiveness in reducing mortality and reversing liver fibrosis in mice. However, the underlying mechanism remains obscure, given the absence of oxytocin receptor expression in hepatic stellate cells, the primary liver fibrosis effector cells.
Methods
A comprehensive map of cell populations in fibrotic liver was generated using single-cell sequencing. The map enabled our study of the target cells of oxytocin action in the liver in more dimensions. Furthermore, we elucidated the mechanism of the oxytocin signaling system in hepatic macrophages using oxytocin receptor-specific knockout mice and liver fibrosis animal models.
Results
The carbon tetrachloride-induced hepatic fibrosis and bile duct ligation hepatic fibrosis mouse models demonstrated that oxytocin reversed hepatic fibrosis in mice. The mapped liver cell populations demonstrated that oxytocin promoted the phenotypic switch from Ly6high to Ly6Clow in myeloid-derived macrophages. The phenotypic control of oxytocin signaling system activation on this phenotypic switch was validated using myeloid-specific oxytocin receptor knockout mice. Subsequent studies demonstrated that the calcium inward flow induced by oxytocin receptor activation activated the key orphan nuclear receptor NR4A1, which controls macrophage phenotypic switching. Specifically, calcium ions activated CREB, a key target regulator of NR4A1 expression.
Conclusions
The findings established hepatic macrophages as a hub responsible for the oxytocin-mediated alleviation of liver fibrosis. This study revealed a novel pathway where oxytocin regulates macrophage phenotype.
Impact and implications
Previous studies revealed for the first time the expression of oxytocin receptors in the liver. The present study shows that oxytocin reverses hepatic fibrosis and that hepatic macrophages are the central hub of oxytocin-mediated alleviation of hepatic fibrosis by promoting a phenotypic switch in hepatic macrophages, transitioning from Ly6high to Ly6Clow expression. The present study reveals a novel pathway by which oxytocin regulates macrophage phenotype. In addition, the potential applications of oxytocin and its analogues, as traditional drugs for clinical application, in the treatment of liver fibrosis deserve to be further explored.
{"title":"Oxytocin alleviates liver fibrosis via hepatic macrophages","authors":"Xiangyu Zhai , Hao Zhang , Zhijia Xia , Mingkun Liu , Gang Du , Zhengchen Jiang , Huaxin Zhou , Dan Luo , Dandan Dou , Jingxin Li , Wei Wang , Xiaosong Li , Bin Jin","doi":"10.1016/j.jhepr.2024.101032","DOIUrl":"10.1016/j.jhepr.2024.101032","url":null,"abstract":"<div><h3>Background & Aims</h3><p>Previous studies demonstrated oxytocin treatment effectiveness in reducing mortality and reversing liver fibrosis in mice. However, the underlying mechanism remains obscure, given the absence of oxytocin receptor expression in hepatic stellate cells, the primary liver fibrosis effector cells.</p></div><div><h3>Methods</h3><p>A comprehensive map of cell populations in fibrotic liver was generated using single-cell sequencing. The map enabled our study of the target cells of oxytocin action in the liver in more dimensions. Furthermore, we elucidated the mechanism of the oxytocin signaling system in hepatic macrophages using oxytocin receptor-specific knockout mice and liver fibrosis animal models.</p></div><div><h3>Results</h3><p>The carbon tetrachloride-induced hepatic fibrosis and bile duct ligation hepatic fibrosis mouse models demonstrated that oxytocin reversed hepatic fibrosis in mice. The mapped liver cell populations demonstrated that oxytocin promoted the phenotypic switch from Ly6<sup>high</sup> to Ly6C<sup>low</sup> in myeloid-derived macrophages. The phenotypic control of oxytocin signaling system activation on this phenotypic switch was validated using myeloid-specific oxytocin receptor knockout mice. Subsequent studies demonstrated that the calcium inward flow induced by oxytocin receptor activation activated the key orphan nuclear receptor NR4A1, which controls macrophage phenotypic switching. Specifically, calcium ions activated CREB, a key target regulator of NR4A1 expression.</p></div><div><h3>Conclusions</h3><p>The findings established hepatic macrophages as a hub responsible for the oxytocin-mediated alleviation of liver fibrosis. This study revealed a novel pathway where oxytocin regulates macrophage phenotype.</p></div><div><h3>Impact and implications</h3><p>Previous studies revealed for the first time the expression of oxytocin receptors in the liver. The present study shows that oxytocin reverses hepatic fibrosis and that hepatic macrophages are the central hub of oxytocin-mediated alleviation of hepatic fibrosis by promoting a phenotypic switch in hepatic macrophages, transitioning from Ly6<sup>high</sup> to Ly6C<sup>low</sup> expression. The present study reveals a novel pathway by which oxytocin regulates macrophage phenotype. In addition, the potential applications of oxytocin and its analogues, as traditional drugs for clinical application, in the treatment of liver fibrosis deserve to be further explored.</p></div>","PeriodicalId":14764,"journal":{"name":"JHEP Reports","volume":"6 6","pages":"Article 101032"},"PeriodicalIF":8.3,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2589555924000338/pdfft?md5=07e61a89be3469a172be222d513d2635&pid=1-s2.0-S2589555924000338-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139820330","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01DOI: 10.1016/j.jhepr.2024.101073
Mireille Khoury , Qianqian Guo , Kunimaro Furuta , Cristina Correia , Chady Meroueh , Hyun Se Kim Lee , Khaled Warasnhe , Lucía Valenzuela-Pérez , Andrew P. Mazar , Iljung Kim , Yung-Kyun Noh , Heather Holmes , Michael F. Romero , Caroline R. Sussman , Kevin D. Pavelko , Shahidul Islam , Adebowale O. Bamidele , Petra Hirsova , Hu Li , Samar H. Ibrahim
<div><h3>Background & Aims</h3><p>Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by excessive circulating toxic lipids, hepatic steatosis, and liver inflammation. Monocyte adhesion to liver sinusoidal endothelial cells (LSECs) and transendothelial migration (TEM) are crucial in the inflammatory process. Under lipotoxic stress, LSECs develop a proinflammatory phenotype known as endotheliopathy. However, mediators of endotheliopathy remain unclear.</p></div><div><h3>Methods</h3><p>Primary mouse LSECs isolated from C57BL/6J mice fed chow or MASH-inducing diets rich in fat, fructose, and cholesterol (FFC) were subjected to multi-omics profiling. Mice with established MASH resulting from a choline-deficient high-fat diet (CDHFD) or FFC diet were also treated with two structurally distinct GSK3 inhibitors (LY2090314 and elraglusib [9-ING-41]).</p></div><div><h3>Results</h3><p>Integrated pathway analysis of the mouse LSEC proteome and transcriptome indicated that leukocyte TEM and focal adhesion were the major pathways altered in MASH. Kinome profiling of the LSEC phosphoproteome identified glycogen synthase kinase (GSK)-3β as the major kinase hub in MASH. GSK3β-activating phosphorylation was increased in primary human LSECs treated with the toxic lipid palmitate and in human MASH. Palmitate upregulated the expression of C-X-C motif chemokine ligand 2, intracellular adhesion molecule 1, and phosphorylated focal adhesion kinase, via a GSK3-dependent mechanism. Congruently, the adhesive and transendothelial migratory capacities of primary human neutrophils and THP-1 monocytes through the LSEC monolayer under lipotoxic stress were reduced by GSK3 inhibition. Treatment with the GSK3 inhibitors LY2090314 and elraglusib ameliorated liver inflammation, injury, and fibrosis in FFC- and CDHFD-fed mice, respectively. Immunophenotyping using cytometry by mass cytometry by time of flight of intrahepatic leukocytes from CDHFD-fed mice treated with elraglusib showed reduced infiltration of proinflammatory monocyte-derived macrophages and monocyte-derived dendritic cells.</p></div><div><h3>Conclusion</h3><p>GSK3 inhibition attenuates lipotoxicity-induced LSEC endotheliopathy and could serve as a potential therapeutic strategy for treating human MASH.</p></div><div><h3>Impact and Implications</h3><p>LSECs under lipotoxic stress in MASH develop a proinflammatory phenotype known as endotheliopathy, with obscure mediators and functional outcomes. The current study identified GSK3 as the major driver of LSEC endotheliopathy, examined its pathogenic role in myeloid cell-associated liver inflammation, and defined the therapeutic efficacy of pharmacological GSK3 inhibitors in murine MASH. This study provides preclinical data for the future investigation of GSK3 pharmacological inhibitors in human MASH. The results of this study are important to hepatologists, vascular biologists, and investigators studying the mechanisms of inflammatory liver dis
{"title":"Glycogen synthase kinase 3 activity enhances liver inflammation in MASH","authors":"Mireille Khoury , Qianqian Guo , Kunimaro Furuta , Cristina Correia , Chady Meroueh , Hyun Se Kim Lee , Khaled Warasnhe , Lucía Valenzuela-Pérez , Andrew P. Mazar , Iljung Kim , Yung-Kyun Noh , Heather Holmes , Michael F. Romero , Caroline R. Sussman , Kevin D. Pavelko , Shahidul Islam , Adebowale O. Bamidele , Petra Hirsova , Hu Li , Samar H. Ibrahim","doi":"10.1016/j.jhepr.2024.101073","DOIUrl":"10.1016/j.jhepr.2024.101073","url":null,"abstract":"<div><h3>Background & Aims</h3><p>Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by excessive circulating toxic lipids, hepatic steatosis, and liver inflammation. Monocyte adhesion to liver sinusoidal endothelial cells (LSECs) and transendothelial migration (TEM) are crucial in the inflammatory process. Under lipotoxic stress, LSECs develop a proinflammatory phenotype known as endotheliopathy. However, mediators of endotheliopathy remain unclear.</p></div><div><h3>Methods</h3><p>Primary mouse LSECs isolated from C57BL/6J mice fed chow or MASH-inducing diets rich in fat, fructose, and cholesterol (FFC) were subjected to multi-omics profiling. Mice with established MASH resulting from a choline-deficient high-fat diet (CDHFD) or FFC diet were also treated with two structurally distinct GSK3 inhibitors (LY2090314 and elraglusib [9-ING-41]).</p></div><div><h3>Results</h3><p>Integrated pathway analysis of the mouse LSEC proteome and transcriptome indicated that leukocyte TEM and focal adhesion were the major pathways altered in MASH. Kinome profiling of the LSEC phosphoproteome identified glycogen synthase kinase (GSK)-3β as the major kinase hub in MASH. GSK3β-activating phosphorylation was increased in primary human LSECs treated with the toxic lipid palmitate and in human MASH. Palmitate upregulated the expression of C-X-C motif chemokine ligand 2, intracellular adhesion molecule 1, and phosphorylated focal adhesion kinase, via a GSK3-dependent mechanism. Congruently, the adhesive and transendothelial migratory capacities of primary human neutrophils and THP-1 monocytes through the LSEC monolayer under lipotoxic stress were reduced by GSK3 inhibition. Treatment with the GSK3 inhibitors LY2090314 and elraglusib ameliorated liver inflammation, injury, and fibrosis in FFC- and CDHFD-fed mice, respectively. Immunophenotyping using cytometry by mass cytometry by time of flight of intrahepatic leukocytes from CDHFD-fed mice treated with elraglusib showed reduced infiltration of proinflammatory monocyte-derived macrophages and monocyte-derived dendritic cells.</p></div><div><h3>Conclusion</h3><p>GSK3 inhibition attenuates lipotoxicity-induced LSEC endotheliopathy and could serve as a potential therapeutic strategy for treating human MASH.</p></div><div><h3>Impact and Implications</h3><p>LSECs under lipotoxic stress in MASH develop a proinflammatory phenotype known as endotheliopathy, with obscure mediators and functional outcomes. The current study identified GSK3 as the major driver of LSEC endotheliopathy, examined its pathogenic role in myeloid cell-associated liver inflammation, and defined the therapeutic efficacy of pharmacological GSK3 inhibitors in murine MASH. This study provides preclinical data for the future investigation of GSK3 pharmacological inhibitors in human MASH. The results of this study are important to hepatologists, vascular biologists, and investigators studying the mechanisms of inflammatory liver dis","PeriodicalId":14764,"journal":{"name":"JHEP Reports","volume":"6 6","pages":"Article 101073"},"PeriodicalIF":8.3,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2589555924000776/pdfft?md5=1daf44c0cb1dd9408f6b0a249cd88d18&pid=1-s2.0-S2589555924000776-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140405130","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-01DOI: 10.1016/j.jhepr.2024.101068
Linsey E. Jackson , Jennifer L. Tomlinson , Roberto Alva-Ruiz , Lindsey A. Gregory , Seul Kee Byeon , Amro M. Abdelrahman , Dong-Gi Mun , Caroline W. Grant , Zachary C. Fogarty , Chen Wang , Lewis R. Roberts , Rondell P. Graham , Mitesh J. Borad , Sumera I. Ilyas , Gregory J. Gores , Akhilesh Pandey , Arjun P. Athreya , Rory L. Smoot
Background & Aims
Metabolomic and lipidomic analyses provide an opportunity for novel biological insights. Cholangiocarcinoma (CCA) remains a highly lethal cancer with limited response to systemic, targeted, and immunotherapeutic approaches. Using a global metabolomics and lipidomics platform, this study aimed to discover and characterize metabolomic variations and associated pathway derangements in patients with CCA.
Methods
Leveraging a biospecimen collection, including samples from patients with digestive diseases and normal controls, global serum metabolomic and lipidomic profiling was performed on 213 patients with CCA and 98 healthy controls. The CCA cohort of patients included representation of intrahepatic, perihilar, and distal CCA tumours. Metabolome-wide association studies utilizing multivariable linear regression were used to perform case–control comparisons, followed by pathway enrichment analysis, CCA subtype analysis, and disease stage analysis. The impact of biliary obstruction was evaluated by repeating analyses in subsets of patients only with normal bilirubin levels.
Results
Of the 420 metabolites that discriminated patients with CCA from controls, decreased abundance of cysteine-glutathione disulfide was most closely associated with CCA. Additional conjugated bile acid species were found in increased abundance even in the absence of clinically relevant biliary obstruction denoted by elevated serum bilirubin levels. Pathway enrichment analysis also revealed alterations in caffeine metabolism and mitochondrial redox-associated pathways in the serum of patients with CCA.
Conclusions
The presented metabolomic and lipidomic profiling demonstrated multiple alterations in the serum of patients with CCA. These exploratory data highlight novel metabolic pathways in CCA and support future work in therapeutic targeting of these pathways and the development of a precision biomarker panel for diagnosis.
Impact and implications
Cholangiocarcinoma (CCA) is a highly lethal hepatobiliary cancer with limited treatment response, highlighting the need for a better understanding of the disease biology. Using a global metabolomics and lipidomics platform, we characterized distinct changes in the serum of 213 patients with CCA compared with healthy controls. The results of this study elucidate novel metabolic pathways in CCA. These findings benefit stakeholders in both the clinical and research realms by providing a foundation for improved disease diagnostics and identifying novel targets for therapeutic design.
背景& 目的代谢组学和脂质组学分析为深入了解新生物学提供了机会。胆管癌（CCA）仍然是一种致死率很高的癌症，对全身、靶向和免疫治疗方法的反应有限。本研究利用全球代谢组学和脂质组学平台，旨在发现和描述CCA患者的代谢组变异和相关通路失调。方法利用生物样本收集（包括消化系统疾病患者和正常对照组的样本），对213名CCA患者和98名健康对照组进行了全球血清代谢组学和脂质组学分析。CCA患者群包括肝内、肝周和远端CCA肿瘤。利用多变量线性回归的全代谢组关联研究进行了病例对照比较，随后进行了通路富集分析、CCA 亚型分析和疾病分期分析。结果 在区分 CCA 患者和对照组的 420 种代谢物中，半胱氨酸-谷胱甘肽二硫化物丰度的降低与 CCA 的关系最为密切。即使在血清胆红素水平升高、没有临床相关胆道梗阻的情况下，也能发现其他共轭胆汁酸种类的丰度增加。通路富集分析还揭示了 CCA 患者血清中咖啡因代谢和线粒体氧化还原相关通路的改变。这些探索性数据突显了 CCA 中的新型代谢通路，为今后针对这些通路进行治疗和开发用于诊断的精准生物标记物面板提供了支持。 影响和意义胆管癌（CCA）是一种致死率很高的肝胆癌，但治疗效果有限，因此需要更好地了解这种疾病的生物学特性。利用全球代谢组学和脂质组学平台，我们对 213 名 CCA 患者与健康对照组相比血清中的明显变化进行了表征。这项研究的结果阐明了 CCA 的新代谢途径。这些发现为改进疾病诊断和确定治疗设计的新靶点奠定了基础，使临床和研究领域的相关人员受益匪浅。
{"title":"Metabolome-wide association identifies altered metabolites and metabolic pathways in the serum of patients with cholangiocarcinoma","authors":"Linsey E. Jackson , Jennifer L. Tomlinson , Roberto Alva-Ruiz , Lindsey A. Gregory , Seul Kee Byeon , Amro M. Abdelrahman , Dong-Gi Mun , Caroline W. Grant , Zachary C. Fogarty , Chen Wang , Lewis R. Roberts , Rondell P. Graham , Mitesh J. Borad , Sumera I. Ilyas , Gregory J. Gores , Akhilesh Pandey , Arjun P. Athreya , Rory L. Smoot","doi":"10.1016/j.jhepr.2024.101068","DOIUrl":"10.1016/j.jhepr.2024.101068","url":null,"abstract":"<div><h3>Background & Aims</h3><p>Metabolomic and lipidomic analyses provide an opportunity for novel biological insights. Cholangiocarcinoma (CCA) remains a highly lethal cancer with limited response to systemic, targeted, and immunotherapeutic approaches. Using a global metabolomics and lipidomics platform, this study aimed to discover and characterize metabolomic variations and associated pathway derangements in patients with CCA.</p></div><div><h3>Methods</h3><p>Leveraging a biospecimen collection, including samples from patients with digestive diseases and normal controls, global serum metabolomic and lipidomic profiling was performed on 213 patients with CCA and 98 healthy controls. The CCA cohort of patients included representation of intrahepatic, perihilar, and distal CCA tumours. Metabolome-wide association studies utilizing multivariable linear regression were used to perform case–control comparisons, followed by pathway enrichment analysis, CCA subtype analysis, and disease stage analysis. The impact of biliary obstruction was evaluated by repeating analyses in subsets of patients only with normal bilirubin levels.</p></div><div><h3>Results</h3><p>Of the 420 metabolites that discriminated patients with CCA from controls, decreased abundance of cysteine-glutathione disulfide was most closely associated with CCA. Additional conjugated bile acid species were found in increased abundance even in the absence of clinically relevant biliary obstruction denoted by elevated serum bilirubin levels. Pathway enrichment analysis also revealed alterations in caffeine metabolism and mitochondrial redox-associated pathways in the serum of patients with CCA.</p></div><div><h3>Conclusions</h3><p>The presented metabolomic and lipidomic profiling demonstrated multiple alterations in the serum of patients with CCA. These exploratory data highlight novel metabolic pathways in CCA and support future work in therapeutic targeting of these pathways and the development of a precision biomarker panel for diagnosis.</p></div><div><h3>Impact and implications</h3><p>Cholangiocarcinoma (CCA) is a highly lethal hepatobiliary cancer with limited treatment response, highlighting the need for a better understanding of the disease biology. Using a global metabolomics and lipidomics platform, we characterized distinct changes in the serum of 213 patients with CCA compared with healthy controls. The results of this study elucidate novel metabolic pathways in CCA. These findings benefit stakeholders in both the clinical and research realms by providing a foundation for improved disease diagnostics and identifying novel targets for therapeutic design.</p></div>","PeriodicalId":14764,"journal":{"name":"JHEP Reports","volume":"6 6","pages":"Article 101068"},"PeriodicalIF":8.3,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2589555924000697/pdfft?md5=463262013cf7f20ac8c676890d1b802a&pid=1-s2.0-S2589555924000697-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140283580","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-25DOI: 10.1016/j.jhepr.2024.101124
Alexia Paturel , Francesca Casuscelli di Tocco , Delphine Bousquet , Marie-Laure Plissonnier , Xavier Grand , Hyosun Tak , Françoise Berby , Caroline Scholtès , Barbara Testoni , Fabien Zoulim , Massimo Levrero
Background & Aims
Circulating HBV RNAs have been proposed as a biomarker that reflects the transcriptional activity of covalently closed circular DNA (cccDNA) and may help to evaluate HBV treatment activity. Different research assays have been proposed and, although two PCR-based research use only investigational assays have been developed, the lack of standardized protocols represents an important limitation. Here we have designed and generated a stable clonal cell line producing an RNA-based standard for the calibration of PCR-based circulating HBV RNA assays.
Methods
HBV RNA-producing Huh7-derived stable cell lines were generated by transfecting pTriEX plasmids containing 1.1 unit length HBV DNA genomes carrying mutations in the catalytic site (YMAA mutation) and the TP domain (Y63F) of the polymerase, and the ε-loop of the pregenomic (pg)RNA (mutation A1G).
Results
The clonal cell line (Huh7-3D29), carrying a double YMAA and Y63F mutation, displayed, and maintained over several passages in culture, a high RNA secretion phenotype with negligible residual secreted HBV DNA. Density gradient centrifugation showed that most of the secreted HBV RNA from Huh7-3D29 cells was detected in naked capsid and virion-like particles and only a minority in small extracellular vescicles. Nanopore sequencing of 5’RACE products shows that the majority of the Huh7-3D29-secreted HBV RNAs start at the 5' end of pgRNA and pgRNA-derived spliced RNAs. Finally, Huh7-3D29 cells showed a high and up-scalable secreted RNA yield allowing 1,300 standard curves in 9 days from one flask.
Conclusion
We generated a clonal cell line that produces high quantities of HBV RNAs with very low quantities of contaminating HBV DNAs, representing a stable source of RNA standard for HBV RNA assay calibration.
Impact and implications:
Several investigational assays and two research use only assays have been developed to detect and quantify circulating HBV RNAs, an emerging biomarker of covalently closed circular DNA transcriptional activity and target engagement by new HBV treatments. The lack of a unique molecular standard for circulating HBV RNA quantification represents an important limitation. Here we describe the generation of a stable clonal cell line producing and secreting an RNA-based standard containing all the HBV RNA species found in HBV patients’ sera (e.g. pgRNA, HBx transcripts). This new RNA standard can be used to calibrate all PCR-based assays for circulating HBV RNA quantification to evaluate, in a non-invasive manner, the size of the transcriptionally active cccDNA pool and the activity of novel strategies aimed at curing HBV infection.
{"title":"A molecular standard for circulating HBV RNA detection and quantification assays in patients with chronic hepatitis B","authors":"Alexia Paturel , Francesca Casuscelli di Tocco , Delphine Bousquet , Marie-Laure Plissonnier , Xavier Grand , Hyosun Tak , Françoise Berby , Caroline Scholtès , Barbara Testoni , Fabien Zoulim , Massimo Levrero","doi":"10.1016/j.jhepr.2024.101124","DOIUrl":"10.1016/j.jhepr.2024.101124","url":null,"abstract":"<div><h3>Background & Aims</h3><p>Circulating HBV RNAs have been proposed as a biomarker that reflects the transcriptional activity of covalently closed circular DNA (cccDNA) and may help to evaluate HBV treatment activity. Different research assays have been proposed and, although two PCR-based research use only investigational assays have been developed, the lack of standardized protocols represents an important limitation. Here we have designed and generated a stable clonal cell line producing an RNA-based standard for the calibration of PCR-based circulating HBV RNA assays.</p></div><div><h3>Methods</h3><p>HBV RNA-producing Huh7-derived stable cell lines were generated by transfecting pTriEX plasmids containing 1.1 unit length HBV DNA genomes carrying mutations in the catalytic site (YMAA mutation) and the TP domain (Y63F) of the polymerase, and the ε-loop of the pregenomic (pg)RNA (mutation A1G).</p></div><div><h3>Results</h3><p>The clonal cell line (Huh7-3D29), carrying a double YMAA and Y63F mutation, displayed, and maintained over several passages in culture, a high RNA secretion phenotype with negligible residual secreted HBV DNA. Density gradient centrifugation showed that most of the secreted HBV RNA from Huh7-3D29 cells was detected in naked capsid and virion-like particles and only a minority in small extracellular vescicles. Nanopore sequencing of 5’RACE products shows that the majority of the Huh7-3D29-secreted HBV RNAs start at the 5' end of pgRNA and pgRNA-derived spliced RNAs. Finally, Huh7-3D29 cells showed a high and up-scalable secreted RNA yield allowing 1,300 standard curves in 9 days from one flask.</p></div><div><h3>Conclusion</h3><p>We generated a clonal cell line that produces high quantities of HBV RNAs with very low quantities of contaminating HBV DNAs, representing a stable source of RNA standard for HBV RNA assay calibration.</p></div><div><h3>Impact and implications:</h3><p>Several investigational assays and two research use only assays have been developed to detect and quantify circulating HBV RNAs, an emerging biomarker of covalently closed circular DNA transcriptional activity and target engagement by new HBV treatments. The lack of a unique molecular standard for circulating HBV RNA quantification represents an important limitation. Here we describe the generation of a stable clonal cell line producing and secreting an RNA-based standard containing all the HBV RNA species found in HBV patients’ sera (<em>e.g.</em> pgRNA, HBx transcripts). This new RNA standard can be used to calibrate all PCR-based assays for circulating HBV RNA quantification to evaluate, in a non-invasive manner, the size of the transcriptionally active cccDNA pool and the activity of novel strategies aimed at curing HBV infection.</p></div>","PeriodicalId":14764,"journal":{"name":"JHEP Reports","volume":"6 10","pages":"Article 101124"},"PeriodicalIF":9.5,"publicationDate":"2024-05-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2589555924001289/pdfft?md5=37299e3ae214fe07cfada1d254ac62ec&pid=1-s2.0-S2589555924001289-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142243910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-24DOI: 10.1016/j.jhepr.2024.101122
Background & Aims
A goal of the World Health Organization’s global hepatitis strategy is the elimination of chronic hepatitis C virus (HCV) infection by 2030. As part of its strategy, the Federal Joint Committee (Germany) decided to include hepatitis B and C screening in a preventive medical examination, which is performed at the primary care level in Germany. We investigated the results 1 year after implementation of screening between October 2021 and September 2022.
Methods
HBsAg/HBV DNA and anti-HCV/HCV RNA screenings were identified by billing categories in 286,192 individuals of 11 ambulatory healthcare centers.
Results
Compared to 30,106 HBsAg and 31,266 anti-HCV laboratory requisitions in the year 2018, the number of tests increased to 286,192 during the screening period. Compared to routine care, additional anti-HCV positive tests age dependently increased the tally by 98% (177 plus 170 positive cases in males) and 123% (96 plus 118 positive cases in females) in those aged 35-44 years up to 518% (17 plus 88 positive cases in males) and 514% (29 plus 149 positive cases in females) in those aged 75-84 years. Similar results were observed for HBsAg. Prevalences of HBsAg, anti-HCV and HCV RNA were 0.54%, 0.79% and 0.13%, respectively.
Conclusions
A structured hepatitis screening program at the primary care level has been successfully established and leads to age- and-sex-dependent large additional effects compared to routine care.
Impact and implications
Strategies to eliminate chronic hepatitis B and C virus infection are country specific and vary between clinical scenarios. Our analysis proves the efficacy of a screening program by primary care physicians compared to routine care in a low-prevalence country. This program should be accompanied by additional efforts in risk populations like people who inject drugs who are under-represented in the current screening approach.
{"title":"Successful hepatitis B and C screening in the health check-up in the German primary care setting","authors":"","doi":"10.1016/j.jhepr.2024.101122","DOIUrl":"10.1016/j.jhepr.2024.101122","url":null,"abstract":"<div><h3>Background & Aims</h3><p>A goal of the World Health Organization’s global hepatitis strategy is the elimination of chronic hepatitis C virus (HCV) infection by 2030. As part of its strategy, the Federal Joint Committee (Germany) decided to include hepatitis B and C screening in a preventive medical examination, which is performed at the primary care level in Germany. We investigated the results 1 year after implementation of screening between October 2021 and September 2022.</p></div><div><h3>Methods</h3><p>HBsAg/HBV DNA and anti-HCV/HCV RNA screenings were identified by billing categories in 286,192 individuals of 11 ambulatory healthcare centers.</p></div><div><h3>Results</h3><p>Compared to 30,106 HBsAg and 31,266 anti-HCV laboratory requisitions in the year 2018, the number of tests increased to 286,192 during the screening period. Compared to routine care, additional anti-HCV positive tests age dependently increased the tally by 98% (177 plus 170 positive cases in males) and 123% (96 plus 118 positive cases in females) in those aged 35-44 years up to 518% (17 plus 88 positive cases in males) and 514% (29 plus 149 positive cases in females) in those aged 75-84 years. Similar results were observed for HBsAg. Prevalences of HBsAg, anti-HCV and HCV RNA were 0.54%, 0.79% and 0.13%, respectively.</p></div><div><h3>Conclusions</h3><p>A structured hepatitis screening program at the primary care level has been successfully established and leads to age- and-sex-dependent large additional effects compared to routine care.</p></div><div><h3>Impact and implications</h3><p>Strategies to eliminate chronic hepatitis B and C virus infection are country specific and vary between clinical scenarios. Our analysis proves the efficacy of a screening program by primary care physicians compared to routine care in a low-prevalence country. This program should be accompanied by additional efforts in risk populations like people who inject drugs who are under-represented in the current screening approach.</p></div>","PeriodicalId":14764,"journal":{"name":"JHEP Reports","volume":"6 9","pages":"Article 101122"},"PeriodicalIF":9.5,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2589555924001265/pdfft?md5=288f05ab877132b99fd9cc829bbaed81&pid=1-s2.0-S2589555924001265-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141143776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-23DOI: 10.1016/j.jhepr.2024.101114
Gerardo V. Lo Russo , Stefano Carugo , Lorenzo Ridola , Vincenzo Cardinale
{"title":"Cirrhotic Cardiomyopathy And Beyond: Underscoring The Interaction Between The Liver And The Heart","authors":"Gerardo V. Lo Russo , Stefano Carugo , Lorenzo Ridola , Vincenzo Cardinale","doi":"10.1016/j.jhepr.2024.101114","DOIUrl":"10.1016/j.jhepr.2024.101114","url":null,"abstract":"","PeriodicalId":14764,"journal":{"name":"JHEP Reports","volume":"6 8","pages":"Article 101114"},"PeriodicalIF":9.5,"publicationDate":"2024-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2589555924001186/pdfft?md5=b77c68b8fc809e83e23f538b3110a902&pid=1-s2.0-S2589555924001186-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141131724","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-21DOI: 10.1016/j.jhepr.2024.101121
Background & Aims
HBV treatment is challenging due to the persistence of the covalently closed circular DNA replication pool, which remains unaffected by antiviral intervention. In this study, we determined whether targeting antigen-presenting cells via CD40 stimulation represents an appropriate therapeutic approach for achieving sustained HBV control in a mouse model of HBV replication.
Methods
Mice were transduced with an adeno-associated virus encoding the HBV genome (AAV-HBV) to initiate HBV replication and were administered agonistic CD40 antibody. CD4-depleting antibody was administered in addition to the CD40 antibody. Viral antigens in the blood were measured over time to determine HBV control. HBV-specific CD8+ T cells were quantified in the spleen and liver at the experimental endpoint.
Results
CD40 stimulation in CD4-depleted AAV-HBV mice resulted in the clearance of HBsAg and HBeAg, along with a reduction in liver HBV mRNA, contrasting with CD4-competent counterparts. CD8+ T cells were indispensable for CD40-mediated HBV control, determined by HBV persistence following their depletion. In CD4-replete mice, CD40 stimulation initially facilitated the expansion of HBV-specific CD8+ T cells, which subsequently could not control HBV. Finally, α-CD4/CD40 treatment reduced antigenemia and liver HBV mRNA levels in chronic AAV-HBV mice, with further enhancement through synergy with immunization by VSV-MHBs (vesicular stomatitis virus expressing middle HBsAg).
Conclusions
Our findings underscore the potential of CD40 stimulation as a targeted therapeutic strategy for achieving sustained HBV control and reveal a CD4+ T cell-dependent limitation on CD40-mediated antiviral efficacy.
Impact and implications:
Immunotherapy has the potential to overcome immune dysfunction in chronic HBV infection. Using a mouse model of HBV replication, this study shows that CD40 stimulation can induce sustained HBV control, which is dependent on CD8+ T cells and further enhanced by co-immunization. Unexpectedly, CD40-mediated HBV reduction was improved by the depletion of CD4+ cells. These findings suggest potential strategies for reversing HBV persistence in infected individuals.
{"title":"CD40 stimulation activates CD8+ T cells and controls HBV in CD4-depleted mice","authors":"","doi":"10.1016/j.jhepr.2024.101121","DOIUrl":"10.1016/j.jhepr.2024.101121","url":null,"abstract":"<div><h3>Background & Aims</h3><p>HBV treatment is challenging due to the persistence of the covalently closed circular DNA replication pool, which remains unaffected by antiviral intervention. In this study, we determined whether targeting antigen-presenting cells via CD40 stimulation represents an appropriate therapeutic approach for achieving sustained HBV control in a mouse model of HBV replication.</p></div><div><h3>Methods</h3><p>Mice were transduced with an adeno-associated virus encoding the HBV genome (AAV-HBV) to initiate HBV replication and were administered agonistic CD40 antibody. CD4-depleting antibody was administered in addition to the CD40 antibody. Viral antigens in the blood were measured over time to determine HBV control. HBV-specific CD8<sup>+</sup> T cells were quantified in the spleen and liver at the experimental endpoint.</p></div><div><h3>Results</h3><p>CD40 stimulation in CD4-depleted AAV-HBV mice resulted in the clearance of HBsAg and HBeAg, along with a reduction in liver HBV mRNA, contrasting with CD4-competent counterparts. CD8<sup>+</sup> T cells were indispensable for CD40-mediated HBV control, determined by HBV persistence following their depletion. In CD4-replete mice, CD40 stimulation initially facilitated the expansion of HBV-specific CD8<sup>+</sup> T cells, which subsequently could not control HBV. Finally, α-CD4/CD40 treatment reduced antigenemia and liver HBV mRNA levels in chronic AAV-HBV mice, with further enhancement through synergy with immunization by VSV-MHBs (vesicular stomatitis virus expressing middle HBsAg).</p></div><div><h3>Conclusions</h3><p>Our findings underscore the potential of CD40 stimulation as a targeted therapeutic strategy for achieving sustained HBV control and reveal a CD4<sup>+</sup> T cell-dependent limitation on CD40-mediated antiviral efficacy.</p></div><div><h3>Impact and implications:</h3><p>Immunotherapy has the potential to overcome immune dysfunction in chronic HBV infection. Using a mouse model of HBV replication, this study shows that CD40 stimulation can induce sustained HBV control, which is dependent on CD8<sup>+</sup> T cells and further enhanced by co-immunization. Unexpectedly, CD40-mediated HBV reduction was improved by the depletion of CD4<sup>+</sup> cells. These findings suggest potential strategies for reversing HBV persistence in infected individuals.</p></div>","PeriodicalId":14764,"journal":{"name":"JHEP Reports","volume":"6 9","pages":"Article 101121"},"PeriodicalIF":9.5,"publicationDate":"2024-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2589555924001253/pdfft?md5=01d66927efccecddb4b1e98ea0e78954&pid=1-s2.0-S2589555924001253-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141132032","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-16DOI: 10.1016/j.jhepr.2024.101120
Manon Allaire , Philippe Sultanik , Dominique Thabut
{"title":"Anticoagulation is not associated with an increased risk of variceal bleeding under systemic therapy for advanced HCC","authors":"Manon Allaire , Philippe Sultanik , Dominique Thabut","doi":"10.1016/j.jhepr.2024.101120","DOIUrl":"10.1016/j.jhepr.2024.101120","url":null,"abstract":"","PeriodicalId":14764,"journal":{"name":"JHEP Reports","volume":"6 8","pages":"Article 101120"},"PeriodicalIF":9.5,"publicationDate":"2024-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2589555924001241/pdfft?md5=7b5b3742b6effbd134f3060b7525bcb8&pid=1-s2.0-S2589555924001241-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141053791","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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