Animals ; DNA, Circular ; analysis ; Hepatitis B ; virology ; Hepatitis B virus ; genetics ; Humans

No abstract available.
DNA, Circular/*analysis ; Female ; Hepatitis B/*pathology ; Hepatitis B Surface Antigens/*genetics ; Hepatitis B virus/*metabolism ; Humans ; Male

OBJECTIVETo quantitatively detect intrahepatic HBV covalently closed circular DNA (cccDNA) and serum HBsAg; and to analyze the relationship between the two parameters and with serum HBV DNA level.

METHODSIntrahepatic cccDNA (copies/cell) was quantitated by plasmid-safe ATP-dependent Danes (PSAD) digestion in combination with rolling circle amplification and gap-spanning selective real-time PCR assay using formalin fixed paraffin-embedded liver biopsy samples. HBsAg was measured by chemiluminescence's reagent manufactured by Abbott Company using sera sampled at time-point of liver biopsy.

RESULTSIntrahepatic cccDNA level was positively correlated with serum HBsAg level (r = 0.459, P < 0.001), but not correlated with serum HBV DNA level. Serum HBsAg level was positively correlated with serum HBV DNA level (r = 0.328, P = 0.015), and reversely correlated with HBV replicative efficiency defined as the ratio of serum HBV DNA to cccDNA (r = -0.373, P = 0.005).

CONCLUSIONIn patients with chronic hepatitis B, intrahepatic cccDNA level is correlated with serum HBsAg level. The two parameters combined with serum HBV DNA may comprehensively reflect HBV replication activity and help evaluation of antiviral therapeutic efficacy.

DNA, Circular ; analysis ; DNA, Viral ; analysis ; Female ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; virology ; Humans ; Liver ; virology ; Male ; Viral Load

OBJECTIVETo determine the circular DNA level of patients with hand foot and mouth disease (HFMD) and evaluate its potential clinical value.

METHODSVenous blood in 30 healthy children and 78 patients with HFMD within 3 days of onset of illness and convalescent period was collected. The level of plasma circular DNA was detected by duplex real-time polymerase chain reaction assay. Blood sugar, high-sensitive CRP(hs-CRP) and leucocyte were also detected.

RESULTSThe level of circular DNA in control group was (6.57 +/- 4.67) ng/ml. The level of circular DNA in ordinary and severe HFMD patients was (11.51 +/- 7.75) ng/ml and (20.59 +/- 10.67) ng/ml before treatment, respectively. The levels of circular DNA in ordinary and severe HFMD patients were significantly higher than that in control group (P = 0.021; 0.000); the level of circular DNA in severe HFMD patients was significantly higher than that in ordinary HFMD patients (P = 0.011). The level of circular DNA in severe HFMD patients after treatment were significantly lower than that before treatment (P = 0.033). The level of circular DNA before treatment and after treatment in ordinary HFMD patients had no significant difference. The levels of blood sugar and hs-CRP in severe HFMD patients were higher than those in ordinary before treatment (P = 0.045; 0.011). The levels of blood sugar and hs-CRP before treatment and after treatment in ordinary HFMD patients had no significant change. There was significantly positive correlation between the level of circular DNA and that of hs-CRP in HFMD patient (P = 0.021), but there was no correlation between the level of circular DNA and that of blood sugar and leucocyte.

CONCLUSIONSThe level of circular DNA not only become an early identification marker of severe HFMD patients, but also become monitoring marker of effect of treatment.

Biomarkers ; Blood Glucose ; analysis ; C-Reactive Protein ; analysis ; Child, Preschool ; DNA, Circular ; blood ; Female ; Hand, Foot and Mouth Disease ; blood ; genetics ; Humans ; Infant ; Male

OBJECTIVETo establish an in vitro model of hepatitis B virus (HBV) replication and expression in primary rat hepatocytes (PRH) transfected with circular viral DNA for further study on the interaction of HBV with hepatocytes.

METHODSCircular viral DNA containing complete HBV genome were transfected into PRH by electroporation (transfected group, about 4mug of circular viral DNA/1 10(7)cells). From day 1 to day 10 after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH were measured with IMX system. HBcAg was assayed with western blotting, immunol dot blotting and immunocytochemistry. Meanwhile, HBV S-mRNA and X-mRNA were tested with RT-PCR, and replicative intermediates of HBV DNA were analyzed by southern blotting and dot blotting. Moreover, Transmission electron microscopy was used if viral particles were produced in transfected rat hepatocytes. PRH electroporated only was used as control group.

RESULTS(1) Viral antigen production in transfected rat hepatocytes: HBsAg in cell lysates was positive. P/N values ranged from 4.83 to 85.69, and could be maintained for 10 days after transfection. The average P/N values was 18.239 27.459. Whereas, HBsAg was negative in the supernatants of transfected group (P/N values, negative<2.1). HBeAg in the supernatants and lysates of transfected hepatocytes all was negative (P/N values<2.1) during 10 days following transfection. HBcAg was only found positive in transfected hepatocytes by immunol dot blotting. (2) Detection of viral transcripts: transcription of HBV DNA was investigated by preparing total RNA from rat hepatocytes 2 days after transfection and looking for S-mRNA and X-mRNA by RT-PCR. Results showed S-mRNA positive, X-mRNA negative. (3) HBV DNA replication analysis: intracellular total DNA was extracted 2 days after transfection and analysed by southern blotting. All replicative DNA intermediates, including relaxed circular (rcDNA), covalently closed circular (cccDNA), and single-stranded (ssDNA) linear HBV DNA forms, were indicated. Dot blotting showed intracellular HBV DNA positive in transfected group during 10 days after transfection. However, viral particles were not found in transfected hepatocytes during 3 days after transfection.

CONCLUSIONSCircular HBV DNA transfected into primary adult rat hepatocytes could obtain continuous replication and stable expression of HBV surface antigen. This in vitro model has high reproducibility and stability, and is useful for directly studying the interaction of HBV with hepatocytes.

Animals ; DNA Replication ; DNA, Circular ; genetics ; DNA, Viral ; genetics ; Gene Expression ; Hepatitis B Core Antigens ; analysis ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B e Antigens ; analysis ; Hepatitis B virus ; genetics ; Hepatocytes ; virology ; Male ; Rats ; Rats, Wistar ; Transfection ; Virus Replication

BACKGROUNDTo determine the presence of covalently closed circular DNA (cccDNA), and to investigate the expression kinetics of HBV DNA, HBsAg and HBeAg in 2.2.15 cell.

METHODSHBV cccDNA was assessed by polymerase chain reaction, HBV DNA was measured by Taqman quantitative PCR and HBsAg and HBeAg was measured by EIA.

RESULTSHBV cccDNA was found in both intracellular and extracellular space. There was a good correlation between HBsAg, HBeAg and HBV DNA in the supernatant of 2.2.15 cell (r= 0.833, P < 0.05 and r= 0.939, P < 0.01 for HBsAg and HBeAg, respectively), whereas there was no significant correlation between intracellular HBV DNA levels and virus antigen levels (r= 0.024, P= 0.955 and r= 0.177; P= 0.625 for HBsAg and HBeAg, respectively).

CONCLUSIONHBV cccDNA was detectable in the culture medium and intracellularly in 2.2.15 cells, and these data provided an indication of HBV replication in 2.2.15 cell.

Cell Line, Tumor ; DNA, Circular ; genetics ; DNA, Viral ; chemistry ; genetics ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B e Antigens ; analysis ; Hepatitis B virus ; genetics ; immunology ; Humans ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

OBJECTIVETo investigate the correlation of sera HBV DNA and serological makers with hepatic tissue HBVcccDNA in chronic HBV carriers.

METHODSReal time fluorescence quantitative polymerase chain reaction (RT-PCR) were used to detect HBV covalently closed circular DNA (cccDNA) and total intrahepatic HBV DNA from 30 needle-biopsy specimens as well as HBV DNA in sera in chronic HBV carriers. Quantification of the HBsAg, HBeAg in sera were quantified using Chemiluminescence immunoassay.

RESULTSHBVcccDNA can be detected in chronic HBV carriers, which rang from 3.15 x 10(3) copies/mg to 1.06 x 10(7) copies/mg. There was a positive correlation between the cccDNA and HBVtDNA (r = 0.375, P < 0.05), but there was no correlation between the cccDNA and sera HBV DNA (P = 0.174). There was a positive correlation between cccDNA and sera HBsAg quantification (r = 0.562, P < 0.001) but no correlation with sera HBeAg qantification (r = 0.152, P > 0.05).

CONCLUSIONHBV cccDNA can be replicated stably in hepatic tissue in all chronic HBV carriers. HBV DNA in sera can not be indicated hepatic tissue cccDNA level. While HBsAg quantification in sera can be used as a marker of cccDNA quantification in hepatic tissue to some extent.

Adolescent ; Adult ; Biopsy ; Carrier State ; blood ; pathology ; virology ; DNA, Circular ; analysis ; genetics ; DNA, Viral ; blood ; genetics ; Female ; Hepatitis B virus ; genetics ; isolation & purification ; Hepatitis B, Chronic ; blood ; pathology ; virology ; Humans ; Liver ; pathology ; virology ; Male ; Young Adult

OBJECTIVETo test if myricanone (C21H24O5), a cyclic diarylheptanoid, has anticancer effects on two different cancer cell lines HeLa and PC3. The present study was conducted with a note on the drug-DNA interaction and apoptotic signalling pathway.

METHODSSeveral studies like cytotoxicity, nuclear damage, annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)-labelled apoptotic assay and cell cycle arrest, immunoblot and reverse transcriptase-polymerase chain reaction (RT-PCR) were used following standard protocols. Circular dichroism (CD) spectroscopy was also done to evaluate whether myricanone effectively interacted with DNA to bring about conformational changes that could strongly inhibit the cancer cell proliferation.

RESULTSMyricanone showed a greater cytotoxic effect on PC3 cells than on HeLa cells. Myricanone promoted G0/G1 arrest in HeLa cells and S phase arrest in PC3 cells. Nuclear condensation and annexin V-FITC/PI studies revealed that myricanone promoted apoptotic cell death. CD spectroscopic data indicated that myricanone had an interaction with calf thymus DNA that changed DNA structural conformation. RT-PCR and immunoblot studies revealed that myricanone activated the apoptotic signalling cascades through down-regulation of transcription factors like nuclear factor-κB (NF-κB) (p65), and signal transducers and activators of transcription 3 (STAT3); cell cycle regulators like cyclin D1, and survivin and other signal proteins like Bcl-2 and up-regulation of Bax, caspase-9 and caspase-3.

CONCLUSIONMyricanone induced apoptosis in both types of cancer cells by triggering caspase activation, and suppression of cell proliferation by down-regulation of NF-κB and STAT3 signalling cascades, which makes it a suitable candidate for possible use in the formulation of therapeutic agent for combating cancer.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Circular Dichroism ; DNA ; metabolism ; Diarylheptanoids ; metabolism ; pharmacology ; Female ; Humans ; Male ; Myrica ; chemistry ; Plant Extracts ; analysis ; Signal Transduction ; Spectroscopy, Fourier Transform Infrared

OBJECTIVETo establish a method for detection of reverse transcriptase region of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to compare the pattern and frequency of drug-resistant mutations in the region between intrahepatic HBV cccDNA and serum HBV relax circle DNA (rcDNA).

METHODSHBV DNA were extracted from liver biopsy tissues of 20 patients with chronic hepatitis B. The RT region of HBV cccDNA was amplified by rolling circle amplification (RCA) followed by polymerase chain reaction (PCR) mediated by a pair of primers spanning across the gap region of HBV genome. The RT region of serum HBV rcDNA from the same patient was amplified by nested-PCR. The PCR products were directly sequenced and analyzed by Vector NTI Suite 8.0 and chromaslite 201 software. x2 test was used for statistical significance analysis of drug-resistant mutation occurrences between the HBV cccDNA and rcDNA.

RESULTSThe RT regions of HBV cccDNA were successfully amplified from liver tissues of all enrolled patients using the RCA plus PCR assay. Simultaneously, HBV the RT regions of rcDNA were amplified from these patients serum samples. Sequence analysis showed that the drug-resistant mutations were significantly more frequently detected in HBV rcDNA (40%) than in HBV cccDNA (10%) (P<0.05). Different mutational patterns were observed between the HBV cccDNA and rcDNA in a few cases.

CONCLUSIONThe RCA in combination with PCR is a practical method for the detection of drug-resistant mutation in the RT region of HBV cccDNA. Drug-resistant mutational patterns could be discrepant between HBV cccDNA and rcDNA.

DNA Primers ; genetics ; DNA, Circular ; genetics ; DNA, Viral ; genetics ; Drug Resistance, Viral ; genetics ; Genes, Viral ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Mutation ; Nucleic Acid Amplification Techniques ; methods ; Polymerase Chain Reaction ; methods ; RNA-Directed DNA Polymerase ; genetics ; Sequence Analysis, DNA

OBJECTIVETo explore the dynamics of hepatitis B virus covalently closed circular DNA (cccDNA) and optimal duration of treatment after serum virology response.

METHODSHBV cccDNA in liver biopsies and the serum HBV DNA were quantified by real time PCR, the serum makers were detected by enzyme-linked immunosorbent assay.

RESULTS(1) The cccDNA in biopsy samples continued to decrease after serum virology responded. (2) The longer the treatment after serum virology response, the lower the cccDNA level in liver tissue. (3) Anti-HBe positive patients had lower cccDNA in liver tissue than anti-HBe negative patients. (4) cccDNA in liver tissue was undetectable in 12 out of the 18 case anti-HBe(+) patients. Serum virology response lasted 35 months and anti-HBe(+) lasted 30 months.

CONCLUSIONAfter serum virology responded, the longer the treatment, the lower the liver cccDNA. The cccDNA is undetectable in about 2/3 of the patients if the serum virological clearance lasts more than 35 months and anti-HBe lasts more than 30 months.

Adult ; Aged ; Antiviral Agents ; therapeutic use ; Biopsy ; DNA, Circular ; analysis ; DNA, Viral ; analysis ; blood ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; drug therapy ; virology ; Humans ; Liver ; pathology ; virology ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; Time Factors ; Viral Load ; Young Adult