Hepatitis B virus （HBV） infection is a global public health problem. According to the data of WorldHealth Organization （WHO）, 2 billion people worldwide have been infected with HBV, and among them 350400 million a...Hepatitis B virus （HBV） infection is a global public health problem. According to the data of WorldHealth Organization （WHO）, 2 billion people worldwide have been infected with HBV, and among them 350400 million are chronic HBV carriers. Hepatitis B causes about 1 million deaths of HBV related liver failure, cirrhosis, and hepatocellular carcinoma annually.展开更多
AIM:To evaluate the covalently closed circle DNA (cccDNA) level of hepatitis B virus (HBV) in patients' liver and sera. METHODS:HBV DNA was isolated from patients' liver biopsies and sera.A sensitive real-time...AIM:To evaluate the covalently closed circle DNA (cccDNA) level of hepatitis B virus (HBV) in patients' liver and sera. METHODS:HBV DNA was isolated from patients' liver biopsies and sera.A sensitive real-time PCR method,which is capable of differentiation of HBV viral genomic DNA and cccDNA,was used to quantify the total HBV cccDNA.The total HBV viral DNA was quantitated by real-time PCR using a HBV diagnostic kit (PG Biotech,LTD,Shenzhen,China) described previously. RESULTS:For the first time,we measured the level of HBV DNA and cccDNA isolated from ten HBV patients' liver biopsies and sera.In the liver biopsies,cccDNA was detected from all the biopsy samples.The copy number of cccDNA ranged from from 0.03 to 173.1 per cell,the copy number of total HBV DNA ranged from 0.08 to 3 717 per cell.The ratio of total HBV DNA to cccDNA ranged from 1 to 3 406.In the sera, cccDNA was only detected from six samples whereas HBV viral DNA was detected from all ten samples.The ratio of cccDNA to total HBV DNA ranged from 0 to 1.77%.To further investigate the reason why cccDNA could only be detected in some patients' sera,we performed longitudinal studies.The cccDNA was detected from the patients' sera with HBV reactivation but not from the patients' sera without HBV reactivation.The level of cccDNA in the sera was correlated with ALT and viral load in the HBV reactivation patients. CONCLUSION:HBV cccDNA is actively transcribed and replicated in some patients' hepatoo/tes,which is reflected by a high ratio of HBV total DNA vs cccDNA.Detection of cccDNA in the liver biopsy will provide an end-point for the anti-HBV therapy.The occurrence of cccDNA in the sera is an early signal of liver damage,which may be another important clinical parameter.展开更多
乙型肝炎病毒(hepatitis B virus,HBV)感染是一个严重的公共卫生问题。据世界卫生组织(World Health Organization,WHO)报道,全球60亿人口中,约20亿人曾感染过HBV,其中3.5亿人为慢性HBV感染,每年约有1 00万人死于HBV感染所致的肝衰竭、...乙型肝炎病毒(hepatitis B virus,HBV)感染是一个严重的公共卫生问题。据世界卫生组织(World Health Organization,WHO)报道,全球60亿人口中,约20亿人曾感染过HBV,其中3.5亿人为慢性HBV感染,每年约有1 00万人死于HBV感染所致的肝衰竭、肝硬化和原发性肝细胞癌(肝癌)[1]。全球肝癌患者中,75%以上是HBV所致[2]。展开更多
AIM In this study we investigated the relationship of the X protein of HBV and nuclear factor-κB(NF-κB)and the expression of NF-κB in human hepatocellular carcinoma tissues. METHODS Immunohistochemistry SP method w...AIM In this study we investigated the relationship of the X protein of HBV and nuclear factor-κB(NF-κB)and the expression of NF-κB in human hepatocellular carcinoma tissues. METHODS Immunohistochemistry SP method was used to detect the expression of NF-κB and the X protein of HBV in human hepatocellular carcinoma tissues of 52 cases.Gene transfection mediated by lipofectamine was used to transfect the eukaryotic expression vector pCDNA3.1-HBX of HBV x gene into human hepatocellular carcinoma cell line HCC-9204 and NF-κB was detected. RESULTS NF-κB was widely expressed in human hepatocellular carcinoma tissues in a total of 52 cases and its expression was related to the X protein of HBV.NF-κB was localized both in the cytoplasm and the nuclei of hepatocellular carcinoma cells in 11 cases which were positive for the X protein of HBV while in 41 cases negative for the X protein of HBV,NF-κB was only localized in the cytoplasm of hepatocellular carcinoma cells but translocated to the nuclei of hepatocellular carcinoma cells after the eukaryotic expression vector pCDNA3.1-HBX was transfected into HCC-9204 cells. CONCLUSION This study strongly suggests that the nuclear factor NF-κB is widely expressed in hepatocellular carcinoma tissues in different styles according to the expression of the X protein of HBV.NF-κB is abnormally activated in hepatocellular carcinoma,which is probably rélated to the X protein of HBV.The X protein of HBV can activate NF-κB to translocate into nuclei of hepatocellular carcinoma cells.展开更多
Evidence suggests that exosomes can transfer genetic material between cells. However, their roles in hepatitis B virus （HBV） infection remain unclear. Here, we report that exosomes present in the sera of chronic hep...Evidence suggests that exosomes can transfer genetic material between cells. However, their roles in hepatitis B virus （HBV） infection remain unclear. Here, we report that exosomes present in the sera of chronic hepatitis B （CHB） patients contained both HBV nucleic acids and HBV proteins, and transferred HBV to hepatocytes in an active manner. Notably, HBV nucleic acids were detected in natural killer （NK） cells from both CHB patients and healthy donors after exposure to HBV-positive exosomes. Through real-time fluorescence microscopy and flow cytometry, 1, r-dioctadecyl-3,3,3＇,3＇,-tetramethylindodicarbocyanine, 4-chlorobenzenesulfnate salt （DiD）-Iabeled exosomes were observed to interact with NK cells and to be taken up by NK cells, which was enhanced by transforming growth factor-β treatment. Furthermore, HBV-positive exosomes impaired NK-cell functions, including interferon （IFN）-y production, cytolytic activity, NK-cell proliferation and survival, as well as the responsiveness of the cells to poly （I.C） stimulation. HBV infection suppressed the expression of pattern-recognition receptors, especially retinoic acid inducible gene I （RIG-I）, on NK cells, resulting in the dampening of the nuclear factor KB （NF-KB） and p38 mitogen-activated protein kinase pathways. Our results highlight a previously unappreciated role of exosomes in HBV transmission and NK-cell dysfunction during CHB infection.展开更多
Hepadnaviruses, including human hepatitis B virus （HBV）, replicate through reverse transcription of an RNA intermediate, the pregenomic RNA （pgRNA）. Despite this kinship to retroviruses, there are fundamental diff...Hepadnaviruses, including human hepatitis B virus （HBV）, replicate through reverse transcription of an RNA intermediate, the pregenomic RNA （pgRNA）. Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription： it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, ε, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV （DHBV）, now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cellfree systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the ~ RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.展开更多
文摘Hepatitis B virus （HBV） infection is a global public health problem. According to the data of WorldHealth Organization （WHO）, 2 billion people worldwide have been infected with HBV, and among them 350400 million are chronic HBV carriers. Hepatitis B causes about 1 million deaths of HBV related liver failure, cirrhosis, and hepatocellular carcinoma annually.
基金SuppoSed by CRCG grant from the University of Hong KongCERG grant from University Grant Council of Hong Kong Research Fund from Science and Technology Commission of Shanghai,China
文摘AIM:To evaluate the covalently closed circle DNA (cccDNA) level of hepatitis B virus (HBV) in patients' liver and sera. METHODS:HBV DNA was isolated from patients' liver biopsies and sera.A sensitive real-time PCR method,which is capable of differentiation of HBV viral genomic DNA and cccDNA,was used to quantify the total HBV cccDNA.The total HBV viral DNA was quantitated by real-time PCR using a HBV diagnostic kit (PG Biotech,LTD,Shenzhen,China) described previously. RESULTS:For the first time,we measured the level of HBV DNA and cccDNA isolated from ten HBV patients' liver biopsies and sera.In the liver biopsies,cccDNA was detected from all the biopsy samples.The copy number of cccDNA ranged from from 0.03 to 173.1 per cell,the copy number of total HBV DNA ranged from 0.08 to 3 717 per cell.The ratio of total HBV DNA to cccDNA ranged from 1 to 3 406.In the sera, cccDNA was only detected from six samples whereas HBV viral DNA was detected from all ten samples.The ratio of cccDNA to total HBV DNA ranged from 0 to 1.77%.To further investigate the reason why cccDNA could only be detected in some patients' sera,we performed longitudinal studies.The cccDNA was detected from the patients' sera with HBV reactivation but not from the patients' sera without HBV reactivation.The level of cccDNA in the sera was correlated with ALT and viral load in the HBV reactivation patients. CONCLUSION:HBV cccDNA is actively transcribed and replicated in some patients' hepatoo/tes,which is reflected by a high ratio of HBV total DNA vs cccDNA.Detection of cccDNA in the liver biopsy will provide an end-point for the anti-HBV therapy.The occurrence of cccDNA in the sera is an early signal of liver damage,which may be another important clinical parameter.
文摘乙型肝炎病毒(hepatitis B virus,HBV)感染是一个严重的公共卫生问题。据世界卫生组织(World Health Organization,WHO)报道,全球60亿人口中,约20亿人曾感染过HBV,其中3.5亿人为慢性HBV感染,每年约有1 00万人死于HBV感染所致的肝衰竭、肝硬化和原发性肝细胞癌(肝癌)[1]。全球肝癌患者中,75%以上是HBV所致[2]。
文摘AIM In this study we investigated the relationship of the X protein of HBV and nuclear factor-κB(NF-κB)and the expression of NF-κB in human hepatocellular carcinoma tissues. METHODS Immunohistochemistry SP method was used to detect the expression of NF-κB and the X protein of HBV in human hepatocellular carcinoma tissues of 52 cases.Gene transfection mediated by lipofectamine was used to transfect the eukaryotic expression vector pCDNA3.1-HBX of HBV x gene into human hepatocellular carcinoma cell line HCC-9204 and NF-κB was detected. RESULTS NF-κB was widely expressed in human hepatocellular carcinoma tissues in a total of 52 cases and its expression was related to the X protein of HBV.NF-κB was localized both in the cytoplasm and the nuclei of hepatocellular carcinoma cells in 11 cases which were positive for the X protein of HBV while in 41 cases negative for the X protein of HBV,NF-κB was only localized in the cytoplasm of hepatocellular carcinoma cells but translocated to the nuclei of hepatocellular carcinoma cells after the eukaryotic expression vector pCDNA3.1-HBX was transfected into HCC-9204 cells. CONCLUSION This study strongly suggests that the nuclear factor NF-κB is widely expressed in hepatocellular carcinoma tissues in different styles according to the expression of the X protein of HBV.NF-κB is abnormally activated in hepatocellular carcinoma,which is probably rélated to the X protein of HBV.The X protein of HBV can activate NF-κB to translocate into nuclei of hepatocellular carcinoma cells.
基金This work was supported by grants from the National Basic Research Program of China （No. 2013CB531503）, the Natural Science Foundation of China （Nos. 81172789 and 30972692）.
文摘Evidence suggests that exosomes can transfer genetic material between cells. However, their roles in hepatitis B virus （HBV） infection remain unclear. Here, we report that exosomes present in the sera of chronic hepatitis B （CHB） patients contained both HBV nucleic acids and HBV proteins, and transferred HBV to hepatocytes in an active manner. Notably, HBV nucleic acids were detected in natural killer （NK） cells from both CHB patients and healthy donors after exposure to HBV-positive exosomes. Through real-time fluorescence microscopy and flow cytometry, 1, r-dioctadecyl-3,3,3＇,3＇,-tetramethylindodicarbocyanine, 4-chlorobenzenesulfnate salt （DiD）-Iabeled exosomes were observed to interact with NK cells and to be taken up by NK cells, which was enhanced by transforming growth factor-β treatment. Furthermore, HBV-positive exosomes impaired NK-cell functions, including interferon （IFN）-y production, cytolytic activity, NK-cell proliferation and survival, as well as the responsiveness of the cells to poly （I.C） stimulation. HBV infection suppressed the expression of pattern-recognition receptors, especially retinoic acid inducible gene I （RIG-I）, on NK cells, resulting in the dampening of the nuclear factor KB （NF-KB） and p38 mitogen-activated protein kinase pathways. Our results highlight a previously unappreciated role of exosomes in HBV transmission and NK-cell dysfunction during CHB infection.
文摘Hepadnaviruses, including human hepatitis B virus （HBV）, replicate through reverse transcription of an RNA intermediate, the pregenomic RNA （pgRNA）. Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription： it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, ε, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV （DHBV）, now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cellfree systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the ~ RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.
