Xinxin Zhang

Ruijin Hospital North, Shanghai, Shanghai Shi, China

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Publications (9)22.86 Total impact

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    ABSTRACT: Hepatitis B, which caused by Hepatitis B virus (HBV) infection, remains a major health threat worldwide. Hepatic injury and regeneration from chronic inflammation is the main driving factor of liver fibrosis and cirrhosis in chronic hepatitis B. Pro-inflammatory tumor necrosis factor (TNF)-α has been implicated as a major inducer of liver cell death during viral hepatitis. Here, we report that in hepatoma cell lines and in primary mouse and human hepatocytes, expression of hepatitis B core (HBc) protein made cells susceptible to TNF-α-induced apoptosis. We found that receptor of activated protein kinase C (RACK) 1 interacted with HBc by tandem affinity purification and mass spectrometry. RACK1 was recently reported as a scaffold protein that facilitates the phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7) by its upstream activators. Our study showed that HBc abrogated the interaction between MKK7 and RACK1 by competitively binding to RACK1, thereby downregulating TNF-α-induced phosphorylation of MKK7 and the activation of c-Jun N-terminal kinase. In line with this finding, specific knockdown of MKK7 increased the sensitivity of hepatocytes to TNF-α-induced apoptosis, while overexpression of RACK1 counteracted the pro-apoptotic activity of HBc. Capsid particle formation was not obligatory for HBc pro-apoptotic activity, as analyzed using an assembly-defective HBc mutant. In conclusion, the expression of HBc sensitized hepatocytes to TNF-α-induced apoptosis by disrupting the interaction between MKK7 and RACK1. Our study is thus a first indication of the pathogenic effects of HBc in liver injury during hepatitis B. Our study revealed a previously unappreciated role of HBc in TNF-α-mediated apoptosis. The pro-apoptotic activity of HBc is important for understanding hepatitis B pathogenesis. In particular, severe hepatitis-associated HBV variants may upregulate apoptosis of hepatocytes through enhanced HBc expression. Our study also found that MKK7 is centrally involved in TNF-α-induced hepatocyte apoptosis and revealed a multifaceted role for JNK signaling in this process. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Journal of virology. 11/2014;
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    ABSTRACT: It remains crucial to develop a laboratory model for studying hepatitis B virus (HBV) chronic infection. We hereby produced a recombinant (r) covalently closed circular (ccc) DNA in view of the key role of cccDNA in HBV persistence. A loxP-chimeric intron was engineered into a monomeric HBV genome in a precursor plasmid (prcccDNA), which was excised using Cre/loxP-mediated DNA recombination into a 3.3 kb rcccDNA in the nucleus of hepatocytes. The chimeric intron was spliced from RNA transcripts without interrupting the HBV life cycle. In cultured hepatoma cells, co-transfection of prcccDNA and pCMV-Cre (encoding Cre-recombinase) resulted in accumulation of nuclear rcccDNA that was heat-stable and epigenetically organized as a minichromosome. A mouse model of HBV infection was developed by hydrodynamic injection of prcccDNA. In the presence of Cre-recombinase, rcccDNA was induced in the mouse liver with effective viral replication and expression, triggering a compromised T-cell response against HBV. Significant T-cell hyporesponsiveness occurred in mice receiving 4 μg prcccDNA, resulting in prolonged HBV antigenemia for up to 9 weeks. Persistent liver injury was observed as elevated alanine transaminase activity in serum and sustained inflammatory infiltration in the liver. Although a T-cell dysfunction was induced similarly, mice injected with a plasmid containing linear HBV replicon showed rapid viral clearance within 2 weeks. Collectively, our study provides an innovative approach for producing a cccDNA surrogate that established HBV persistence in immunocompetent mice. It also represents a useful model system in vitro and in vivo for evaluating antiviral treatments against HBV cccDNA. (i) Unlike plasmids that contain a linear HBV replicon, rcccDNA established HBV persistence with sustained liver injury in immunocompetent mice. This method could be a prototype for developing a mouse model of chronic HBV infection. (ii) An exogenous intron was engineered into the HBV genome for functionally seamless DNA recombination. This original approach could be also extended to other viral studies. (iii) rcccDNA was substantially induced in the nucleus of hepatocytes, and could be easily distinguished by its exogenous intron using PCR. This convenient model system affords the opportunity to test antivirals directly targeting HBV cccDNA.
    Journal of Virology 05/2014; · 5.08 Impact Factor
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    ABSTRACT: We previously reported a proof-of-concept study for curing chronic hepatitis B virus (HBV) infection using a foreign antigen recombinant HBV (rHBV), as a gene therapy vector. Targeted elimination of wild-type (wt) HBV-infected cells could be achieved by functionally activating an in situ T-cell response against the foreign antigen. However, as chronic HBV infection spreads to all hepatocytes, specific targeting of virus-infected cells is thought to be less critical. It is also concerned that rHBV may not induce active immunization in a setting resembling natural infection. For this immunotherapeutic approach to be practically viable, in the present study, we used a recombinant adenovirus (rAd) vector for rHBV delivery. The rAd vector allowed efficient transduction of wtHBV-producing HepG2 cells, with transferred rHBV undergoing dominant viral replication. Progeny rHBV virions proved to be infectious as demonstrated in primary tupaia hepatocytes. These results largely expanded the antiviral capacity of the replication-defective rAd/rHBV in wtHBV-infected liver tissue. With prior priming in the periphery, transduction with rAd/rHBV attracted a substantial influx of the foreign antigen-specific T-effector cells into the liver. Despite the fully activated T-cell response, active expression of rHBV was observed for a prolonged time, which is essential for rHBV to achieve sustained expansion. In a mouse model of HBV persistence established by infection with a recombinant adeno-associated virus carrying wtHBV genome, rAd/rHBV-based immunotherapy elicited a foreign antigen-specific T-cell response that triggered effective viral clearance and subsequent seroconversion to HBV. It therefore represents an efficient strategy to overcome immune tolerance, thereby eliminating chronic HBV infection.
    Journal of Virology 12/2013; · 5.08 Impact Factor
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    ABSTRACT: BACKGROUND: The response rate to antiviral therapy varies greatly among individuals, and its prediction is still very challenging. OBJECTIVES: To evaluate the usefulness of serum hepatitis B virus large surface protein (LHBs) levels compared with HBsAg in prediction of the antiviral treatment effect. STUDY DESIGN: Quantification of LHBs, HBsAg and HBV DNA was carried out at baseline and during antiviral therapy (weeks 4, 12, 24, 36 and 48) in HBeAg-positive patients treated with peginterferon alfa-2a (n=21) or entecavir (n=41). RESULTS: The serum LHBs concentration was correlated positively with HBV DNA and HBsAg (r=0.635 and 0.588, respectively). LHBs and HBV DNA levels decreased significantly in a biphasic manner and HBsAg level tended to decrease slowly in both treatment groups. In peginterferon alfa-2a group, the cutoff of 88.46ng/ml in serum LHBs at week 4 gave the best AUC (=0.96) with positive and negative predictive values of 88.9% and 100%, in association with virological response (VR). Serum LHBs level at week 4 also showed an association with VR in entecavir group (AUC 0.78). The predictive model incorporating LHBs, HBsAg and HBV DNA could discriminate VR at baseline (AUC 0.79) and showed an association with serological response (SR) at week 12 (AUC 0.80) in peginterferon alfa-2a group. CONCLUSIONS: On-treatment quantification of serum LHBs may be a more useful parameter for predicting VR in patients on peginterferon alfa-2a than those on entecavir. Combining LHBs, HBsAg and HBV DNA can predict VR and SR more effectively and earlier.
    Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 04/2013; · 3.12 Impact Factor
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    ABSTRACT: FibroScan is one of the noninvasive techniques based on the transient elastography that can assess the progression of liver fibrosis in chronic hepatitis patients in daily clinical practice. Recently, LecT-Hepa was validated as a serological glycomarker correlating well with the fibrosis stage determined by liver biopsy, and was superior to many other noninvasive biochemical markers and tests. We compared the reliability of LecT-Hepa with that of FibroScan for evaluation of liver fibrosis. The effects of increased alanine aminotransferase (ALT) activities on LecT-Hepa and FibroScan were investigated. The areas under the receiver-operating characteristic curves, sensitivity and specificity for detecting cirrhosis, which is one of the outcomes of fibrosis estimation, were 0.82, 72.5% and 78.2% of LecT-Hepa, 0.85, 87.0% and 74.1% of FibroScan; these did not differ significantly. The count distribution of LecT-Hepa in non-cirrhosis group or cirrhosis group did not differ between the patients grouped according to their ALT levels, whereas that of FibroScan was substantially affected. LecT-Hepa was confirmed as a reliable noninvasive test for the evaluation of liver fibrosis in hepatitis B virus-infected patients with comparable performance to that of FibroScan and proved to be unaffected by inflammation.
    Clinica chimica acta; international journal of clinical chemistry 07/2012; 413(21-22):1796-9. · 2.54 Impact Factor
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    ABSTRACT: Long-term lamivudine treatment for chronic hepatitis B virus (HBV) infection induces the emergence of lamivudine resistant HBV YMDD mutant strains. The aim of the present study was to observe the clone evolution of YMDD wild type and mutant strains in pretreatment and post-treatment samples during lamivudine therapy and analyze their clinical significance. Ten serum samples (five before and five after 48 weeks of therapy) obtained from five patients chronically infected with HBV and treated with lamivudine were studied. Part of the HBV polymerase gene flanking the YMDD motif was sequenced after polymerase chain reaction (PCR) amplification. Meanwhile, 20-24 clones were selected at random from each sample and YMDD wild type and mutant strains were detected by real-time fluorimetry PCR established in our laboratory. The YMDD mutants were not detectable in all five patients before treatment and were found in four patients after 48 weeks of therapy by sequencing directly on PCR products. Analysis of individual clones showed that the ratios of mutant strains in each of the five patients were 0, 9.5, 0, 4.5 and 5.6%, respectively, before therapy and 100, 100, 65, 100 and 0%, respectively, after 48 weeks of therapy. M552I was detected before therapy in one patient but M552V became the domain strain after therapy. Until 52 weeks of therapy, serum HBV DNA and alanine aminotransferase (ALT) breakthrough were found in two of the four patients with YMDD mutations. The fifth patient experienced breakthrough of ALT but HBV DNA remained undetectable. The mutant strains of YMDD motif of HBV polymerase could be found in patients before lamivudine treatment, indicating that antiviral therapy allows the rapid selection of resistant strains. The replication ability of the M552V mutant strain might be stronger than that of the M552I mutant strain.
    Journal of Gastroenterology and Hepatology 01/2004; 18(12):1353-7. · 3.33 Impact Factor
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    ABSTRACT: The putative envelope protein E2 of hepatitis C virus (HCV) is the most variable antigenic fragment in the whole viral genome and is responsible mainly for the large inter- and intra-individual heterogeneity of the infecting virus. To analyze variation and immunogenicity of HCV envelope genes of HCV strains from China, the serum samples from 12 Chinese patients with chronic hepatitis C from 10 cities of China were amplified, sequenced directly, and compared with international strains reported previously. Variant analysis and immunogenicity prediction were then carried out with computer programs. The results suggested that HCV envelope genes show diversity in the north and the south of China and that they vary among different genotypes. Therefore, the diversity among different genotypes and derived areas should be calculated for HCV vaccine design; simultaneously, domains characterized by the hydrophilicity and the conserved immunogenicity could be targeted, for example, the domain of aa434-444, aa408-414, aa449-462 in the envelope region and aa399-406 in HVR1. These data may hold the key for future development of HCV vaccine in China.
    Journal of Medical Virology 09/2002; 67(4):490-500. · 2.37 Impact Factor
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    ABSTRACT: The envelope glycoprotein E2 of hepatitis C virus (HCV) has been shown to bind human target cells. Anti-E2 antibodies have been associated with both recovery from natural infection in humans and protection from challenge with homologous HCV in chimpanzees. Therefore E2 has become a major target for the development of anti-HCV vaccines. Two E2 fragments [amino acids (aa) 450–565 and aa 385–565] derived from a subtype 1b HCV genome were expressed as N-terminally hexahistidine-tagged proteins in Escherichia coli and purified to over 85% purity. Both proteins were specifically recognized by homologous hepatitis-C-patient's serum on Western blotting, suggesting that these E. coli-derived E2 proteins displayed E2-specific antigenicity. E2-116 (aa 450–565) elicited strong antibody responses in BALB/c mice and rabbits. Rabbit antiserum raised against renatured E2-116 (RE2–116R) was able to recognize subtype 1b and 1a E2 glycoproteins expressed in mammalian cells on Western blotting. E2-181 (aa 385–565) reacted with 40% of anti-HCV+ patients’ sera in ELISA. RE2-116R and E2-181 were successfully used in preliminary modified vaccinia virus Ankara- and DNA-based E2 vaccine studies for detecting antigen expression in vitro and assessing induced humoral immune responses in mice. The E2 proteins and rabbit antiserum reported here could find wider applications in the development of effective diagnostic, prophylactic and therapeutic measures against HCV.
    Biotechnology and Applied Biochemistry 09/2001; 34(2):109 - 119. · 1.35 Impact Factor