[Show abstract][Hide abstract] ABSTRACT: Chronic hepatitis B virus (HBV) infection is a major global health burden. Functional exhaustion and numerical reduction of HBV-specific cytotoxic T lymphocytes (CTLs) in the liver and peripheral blood limit anti-HBV CTL activity in patients with chronic HBV infection (CHB). However, the ongoing anti-HBV CD8(+) T cell responses in the lymphoid organs are largely unknown due to the infeasibility of obtaining lymphoid organs from CHB patients. Here we demonstrate that the percentage of HBV-specific CD8(+) T cells is higher in the spleen of CHB patients than that from peripheral blood and liver. Although they do respond to TCR stimulation and produce IFNγ, the cells proliferate poorly. Furthermore, miR-720 expression is upregulated in HBV-specific CD8(+) T cells. Overexpression of miR-720 in primary human CD8(+) T cells inhibits TCR stimulation-induced proliferation. We also demonstrate that TGFβ sustains miR-720 upregulation after TCR stimulation, and blood TGFβ levels are associated with the outcome of type I interferon treatment of CHB patients. Thus, therapies targeting miR-720 may help restore impaired immunity in CHB patients.
[Show abstract][Hide abstract] ABSTRACT: T cell functional exhaustion during chronic hepatitis B virus (HBV) infection may contribute to the failed viral clearance; however, the underlying molecular mechanisms remain largely unknown. Here we demonstrate that jumonji domain-containing protein 6 (JMJD6) is a potential regulator of T cell proliferation during chronic HBV infection. The expression of JMJD6 was reduced in T lymphocytes in chronic hepatitis B (CHB) patients, and this reduction in JMJD6 expression was associated with impaired T cell proliferation. Moreover, silencing JMJD6 expression in primary human T cells impaired T cell proliferation. We found that JMJD6 promotes T cell proliferation by suppressing the mRNA expression of CDKN3. Furthermore, we have identified platelet derived growth factor-BB (PDGF-BB) as a regulator of JMJD6 expression. PDGF-BB downregulates JMJD6 expression and inhibits the proliferation of human primary T cells. Importantly, the expression levels of JMJD6 and PDGF-BB in lymphocytes from CHB patients were correlated with the degree of liver damage and the outcome of chronic HBV infection treatment. Our results demonstrate that PDGF-BB and JMJD6 regulate T cell function during chronic HBV infection and may provide insights for the treatment strategies for CHB patients.
[Show abstract][Hide abstract] ABSTRACT: Objective:
To investigate reliability of FibroScan (FS) in diagnosing size of oesophageal varices (OV) in patients with liver cirrhosis.
A total of 260 patients with liver cirrhosis were enrolled in the study. All patients underwent endoscopy to assess OV stage and FS to measure liver stiffness. Receiver operating characteristic (ROC) curves were generated to evaluate the diagnostic reliability of FS.
The FS values according to OV stage were 20.9 ± 10.3 kPa for patients without OV, 32.2 ± 13.5 kPa for patients with mild OV, 45.6 ± 18.3 kPa for patients with moderate OV, and 55.1 ± 15.6 kPa for patients with severe OV. Significant differences were found among the groups (P < 0.001) as well as between any two groups (t=6.574, 10.533, 13.247, 4.719, 7.072 and 2.171, P less than 0.05 respectively). ROC curves of FS for the diagnoses of patients with various OV stages showed the following:with vs. without OV, 0.824 (95% CI:77.5% to 87.4%); less than moderate vs. more than moderate OV, 0.849 (95% CI:79.6% to 90.2%); and less than severe vs. severe OV, 0.871 (95% CI:81.1% to 93.0%); the corresponding FS cut-off values were 22.8 kPa, 30.6 kPa, and 34.6 kPa.
Liver stiffness measurement by FibroScan allows prediction of the oesophageal varices stage in patients with liver cirrhosis.
No preview · Article · Aug 2014 · Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology
[Show abstract][Hide abstract] ABSTRACT: To study ability of FibroScan (FS) in diagnosing the size of oesophageal varices (OV) in patients with HBV-related cirrhosis.
A total of 158 patients with HBV-related liver cirrhosis were enrolled in the study. The relation between the presence of OV assessed by endoscopy, and liver stiffness measurement by Fibroscan was studied, and ROC curves were drawn to assess the diagnostic ability of FS value.
For the patients without OV, mild OV, moderate OV, and severe OV, their corresponding FS values were (21.7 +/- 9.9) kPa, (32.1 +/- 13.6) kPa, (42.3 +/- 20.0) kPa and (54.5 +/- 16.2) kPa, respectively. Significant difference was found among the groups (P < 0.001) and also between any two groups (P < 0.05). ROC curve for the diagnosis of with vs. without OV, moderate OV, and < severe vs. severe OV were 0.798 (95% CI: 73.1%-86.5%), 0.823 (95% CI: 74.5%-90.0%) and 0.879 (95% CI: 80.8%-95.0%), respectively, with corresponding FS cut-off value of 23.3 kPa, 31.5 kPa and 34.6 kPa.
Liver stiffness measurement allows to predict the sizes of oesophageal varices in patients with HBV-related cirrhosis.
No preview · Article · Dec 2012 · Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology
[Show abstract][Hide abstract] ABSTRACT: To explore whether the cellular apoptosis susceptibility (CAS) protein could serve as a pathologic marker for HCC diagnosis and the roles of CAS expression in HBV infection associated HCC.
The expression of CAS protein in HCC and its paracarcinoma tissues, non-tumor liver cirrhosis and hepatitis tissues were detected by immunohistochemistry. Meanwhile, HBsAg, HBcAg and HBV DNA in HCC tissues with HBV infection were examined by immunohistochemistry and in situ hybridization respectively.
The expression of CAS protein was significantly higher in HCC than in its paracarcinomas tissues (P < 0.01), and higher in paracarcinomas tissues than in non-tumor liver cirrhosis and hepatitis tissues (P < 0.01). Poorly differentiated tumors immunochemically stained stronger than moderately or well differentiated (P < 0.01). CAS protein expression was significantly higher in HBV-infected HCC tissues than that of in non-HBV infection (P < 0.01). Meanwhile, in HBV-infected HCC tissues, the staining intensity score of CAS protein in HBV DNA positive HCC tissues was significantly higher than HBV DNA negative tissues (P < 0.05).
Higher expression of CAS protein is found in HCC tissues,and the intensity of CAS protein expression is related closely to tumor differentiation. We suggested that CAS protein might serve as a marker for HCC diagnosis and differentiation estimation, and deduced that CAS might play an important role in the initiation of HBV infection associated HCC through upregulating expression of CAS.
No preview · Article · Aug 2012 · Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology
[Show abstract][Hide abstract] ABSTRACT: Inflammation caused by chronic hepatitis B virus (HBV) infection is associated with the development of cirrhosis and hepatocellular carcinoma; however, the mechanisms by which HBV infection induces inflammation and inflammatory cytokine production remain largely unknown. We analyzed the gene expression patterns of lymphocytes from chronic HBV-infected patients and found that the expression of ZFP36, an AU-rich element (ARE)-binding protein, was dramatically reduced in CD4(+) and CD8(+) T lymphocytes from chronic HBV patients. ZFP36 expression was also reduced in CD14(+) monocytes and in total PBMCs from chronic HBV patients. To investigate the functional consequences of reduced ZFP36 expression, we knocked down ZFP36 in PBMCs from healthy donors using siRNA. siRNA-mediated silencing of ZFP36 resulted in dramatically increased expression of multiple inflammatory cytokines, most of which were also increased in the plasma of chronic HBV patients. Furthermore, we found that IL-8 and RANTES induced ZFP36 downregulation, and this effect was mediated through protein kinase C. Importantly, we found that HBsAg stimulated PBMCs to express IL-8 and RANTES, resulting in decreased ZFP36 expression. Our results suggest that an inflammatory feedback loop involving HBsAg, ZFP36, and inflammatory cytokines may play a critical role in the pathogenesis of chronic HBV and further indicate that ZFP36 may be an important target for anti-inflammatory therapy during chronic HBV infection.
[Show abstract][Hide abstract] ABSTRACT: It remains unclear whether hepatitis B virus (HBV) reverse-transcriptase (RT) rtL229 substitutions influence HBV drug resistance.
The study was to investigate the association of HBV rtL229 substitutions with viral resistance to lamivudine (LAM).
Entire HBV RT genes were amplified by nested PCR and sequenced from sera of 6000 nucleos(t)ide analog-experienced patients with chronic HBV infection. The incidence and clinic relevance of rtL229 substitutions were analyzed. Replication-competent viral amplicons which harbored HBV genomes of wild-type, rtM204I, or rtM204I in conjunction with various rtL229 substitutions (rtL229F/W/M/V) were constructed. The amplicons were transfected into HepG2 cells for phenotyping of replication capacity and susceptibility to nucleos(t)ide analogs.
The rtL229 substitutions were detected in 6.57% (394/6000) of patients. Individual substitution incidences were 2.77%, 0.97%, 0.83% and 0.55% for rtL229V, rtL229F, rtL229M and rtL229W, respectively. The incidence of rtL229 substitutions was significantly higher in LAM-experienced patients (341/4220, 8.1%) than in LAM-naïve patients (53/1780, 3.0%), and were independently associated with genotypic LAM resistance (77.9% vs. 21.2%, OR 8.806, 95%CI 6.345-12.223) and low viral replication (HBV DNA <1000IU/mL) (4.60% vs. 24.2%, OR 0.478, 95%CI 0.254-0.898). Representative cases follow-up showed that rtL229F developed subsequent to rtM204I emergence during LAM treatment and regressed with rtM204I after LAM withdrawal. Functionally, rtL229F did not confer reduced susceptibility to LAM, but could restore replication capacity of rtM204I strain.
The rtL229 substitutions were potentially associated with LAM resistance in Chinese patients and rtL229F had characteristics of a compensatory mutation of rtM204I mutant.
No preview · Article · Mar 2012 · Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology
[Show abstract][Hide abstract] ABSTRACT: Various mutations in reverse-transcriptase domain (RT) of hepatitis B virus (HBV) polymerase may develop during antiviral therapy. The influence of these mutational patterns on HBV replication capacity remains to be fully clarified.
Nine clones containing complete HBV genomes were isolated from 5 patients with chronic hepatitis B who had received antiviral treatment. Viral replication capacity was measured by quantitation of HBV replicative intermediates using vector-free transfer of paired mutant and wild-type HBV genomes into human hepatoma cell lines HepG2 and Huh7. HBV pgRNA was quantitated by real-time PCR and Southern blot analysis.
A real-time PCR assay with high sensitivity and small variation was developed for quantitation of HBV replicative intermediates. Compared to wild-type counterpart, mutant rtL217P produced 1.98-fold higher replicative intermediate level, and mutant rtM204I+rtL217P increased the replicative intermediate level to 1.20 fold. Other mutational patterns (rtV173M, rtA181S/V, rtM204I, rtQ215H, rtL229M, rtN238H, rtV84M+rtA181S+rtM204I, rtV84M+rtM204I, rtA181S+rtM204I, rtA181V+rtL229M, rtQ215H+rtN238H) reduced viral replication capacity to different extents.
The study offers a practical measurement assay and novel information for replication features of mutant strains; especially, rtL217P substitution likely represents an energetic replication-compensatory mutation.
No preview · Article · Nov 2010 · Clinica chimica acta; international journal of clinical chemistry
[Show abstract][Hide abstract] ABSTRACT: To investigate the down-regulating effect of HBV pre-S2 protein upon insulin receptor (INSR) gene promoter.
Eukaryotic expression plasmid pcDNA3.1(-)-preS2 and report plasmid pCAT3-INSRp containing INSR gene promoter upstream of CAT gene were constructed with routine molecular biological methods and were confirmed by endonuclease digestion analysis and sequencing. Then HepG2 cells were transfected with pcDNA3.1(-)-preS2 plasmid and the total RNAs were examined with relative quantitative real-time PCR to confirm the expression of HBV pre-S2 protein. Later HepG2 and Huh7 cells were transfected with pCAT3-INSRp at various doses (0 - 2.0 microg). The choloraphenical acetyltransferase (CAT) activity of transfected cells was detected with CAT-ELISA to generate the dose-effect curve of pCAT3-INSRp. Lastly pCAT3-INSRp (1.0 microg) and pcDNA3.1(-)-preS2 (1.0, 1.5, 2.0, 2.5, 3.0 microg) were co-transfected into HepG2 and Huh7 cells respectively to study the regulation effect of pre-S2 protein upon INSR promoter. Meanwhile pre-S2 monoclonal antibody was added into additional cells transfected with 2.0 microg of pcDNA3.1(-)-preS2 plasmid to evaluate the effect when pre-S2 protein was blocked.
The mRNA of pre-S2 protein could be detected with real-time PCR indicating that pre-S2 protein was properly expressed in pcDNA3.1(-)-preS2-transfected cells. The expression of CAT increased proportionally with the incremental doses of pCAT3-INSRp. It suggested that the INSR gene promoter had its transcription activity. After co-transfection of pCAT3-INSRp and pcDNA3.1(-)-preS2, the CAT expression in HepG2 cells, comparing with that of controls, were 69.8%, 60.1%, 19.7%, 10.3%, 5.6% (36 h) and 68.6%, 56.0%, 10.3%, 8.6%, 3.2% (72 h) respectively. When pre-S2 monoclonal antibody was added into the supernatant of HepG2 cells transfected with 2.0 microg of pcDNA3.1(-)-preS2, the CAT expression was partly restored to 55.4%(36 h)and 69.7%(72 h)of controls. The similar results were observed in Huh7 cells.
The pre-S2 protein down-regulates the activity of INSR gene promoter so as to reduce the expression of INSR. It may partly elucidate the pathogenesis of hepatogenous diabetes at the molecular level.
No preview · Article · Nov 2009 · Zhonghua yi xue za zhi
[Show abstract][Hide abstract] ABSTRACT: To investigate the transactivating effect of pre-S2 protein of hepatitis B virus (HBV) and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization (SSH) technique, and to pave the way for elucidating the pathogenesis of HBV infection.
pcDNA3.1(-)-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1(-)-pre-S2/pSV-lacZ and empty pcDNA3.1(-)/pSV-lacZ. After 48 h, cells were collected and detected for the expression of beta-galactosidase (beta-gal). SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1(-)-pre-S2 and pcDNA3.1(-) empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.
The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1(-)-pre-S2 plasmid. The activity of beta-gal in HepG2 cells transfected with pcDNA3.1(-)-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid (P<0.01). The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positive clones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein.
The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transduction and cell apoptosis. This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.
Full-text · Article · Oct 2005 · World Journal of Gastroenterology
[Show abstract][Hide abstract] ABSTRACT: To screen and clone the genes of protein interacting with the N-terminal protein (TP) of hepatitis B virus DNA polymerase.
TP was amplified by polymerase chain reaction (PCR) and TP bait plasmid was constructed by using yeast two-hybrid system 3, then transformed into yeast AH 109. The transformed yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout medium (SD/-Trp-Leu-His-Ade) and that containing X-alpha-GAL for selecting two times and screening. Plasmids were extracted from blue colonies, and sequence analysis was performed by bioinformatics.
Forty-seven clones were obtained, these clones included human P36956 sterol regulatory element binding protein-1, RNA polymerase II subunit hsRPB7 mRNA, asialoglycoprotein receptor 2, transcript variant 3, ceruloplasmin (ferroxidase), transmembrane 4 superfamily member 2 and 19 of the hypothetical proteins and so on.
Genes encoding TP interacting proteins in hepatocytes were successfully cloned and the results suggest that TP has a wide variety of biological functions.
No preview · Article · Apr 2005 · Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology
[Show abstract][Hide abstract] ABSTRACT: AIM: To elucidate the clinical and pathological characteris- tics of steatohepatitis in the patients with chronic hepatitis C virus (HCV) infection. METHODS: The clinical and pathological data of 159 pa- tients with chronic HCV infection were analyzed retrospec- tively to elucidate the epidemiological rate and the charac- teristics of different pathological types and grades. The pathological typing was clarified as microvesicular, macrovesicular and mixed. The pathological grading was clarified as mild (less than 10%),moderate (less than 30%, but more than 10%) and severe (more than 30%). The significant analysis of multiple factors was conducted by using SAS statistic analysis software. RESULTS: The incidence of steatohepatitis in patients with chronic HCV infection was 82. 39% (131/159). The per- centages of mild,moderate and severe types of steatohepatitis were 29. 46% (33/112),60. 71% (68/112) and 9. 82% (11/112) respectively. The percentages of microvesicular,macrovesicular and mixed types in steatohepatitis were 47. 33% (62/131),3. 82% (5/131) and 48. 85% (64/131),respectively. The types and grades of steatohepatitis in patients with chronic HCV infection were not significantly related with the types of hepatitis viruses, HCV RNA positivity,transfusion history and age,but the gender. Generally,the steatohepatitis in male patients is sig- nificantly severe than that of female patients. CONCLUSION: Eighty-two percent of patients with chronic HCV infection were accompanied with steatihepatitis. The steatohepatitis of male patients was significant severe than that of female patients.