ABSTRACT
This study investigated the expression profiles of IL-10 gene in three human hepatoma cell lines including Huh7,HepG2,and HepG2 transfected with a plasmid containing hepatitis B virus (HBV) named HepG2.2.15.RT-PCR analysis demonstrated that IL-10 message RNA was absent in HepG2 and Huh7 cells,whereas it was present in HepG2.2.15 cells,which was consistent with ELISA result.Furthermore,except for lamivudine other antiviral treatments did not significantly decrease the HBV DNA level in HepG2.2.15 cells,while they had different effects on the expression of IL-10 protein,although stimulation by LPS had no significant effect.In addition,except for poly(I:C),the other treatments decreased the expression of IL-10 protein to different degrees,but had no significant effects on the expression of NF-κB and MyD88.Meanwhile,all treatments we used had effect on the expression of STAT1.In conclusion,IL-10 was expressed in HepG2.2.15 cells and STAT1 pathway might be involved in the regulation of IL-10 expression in HepG2.2.15 cells,but it was not the sole pathway,the exact mechanism warrants further study.
ABSTRACT
To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR Hlb) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH 1 a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector plRES2EGFP,pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRHla and H2c in 4-1-6 were confirmed by RT-PCR,Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H lb/pCDNA3.1 (neo)was transfected into cell line 4-1-6, Hlb did not down-regulate the ligand binding ability of ASGPR.The eukaryotic expression plasmid Hlb/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single Hlb nor Hlb and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both Hla and H2c stably was established. The new split variant Hlb has no effect on ASGPR binding to ASOR. ASGPRHlb alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.
ABSTRACT
Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are believed to play a major role in viral clearance and disease pathogenesis during HBV infection. To clarify the differ-ences in host immune responses between self-limited and chronic HBV infections, we constructed three HLA-A*0201/HBV tetramers with immunodominant epitopes of core18-27, polymerase 575-583 and envelope 335-343, and analyzed the HBV-specific CTLs in peripheral blood mononu-clear cells (PBMCs) from patients infected with HBV. The frequencies and expansion ability of HBV-specific CD8+ T cells in most self-limited HBV infected individuals were higher than those in chronic HBV-infected patients. HBV-specific CD8+ T cells could be induced by in vitro peptide stimulation from chronic patients with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. In chronic infection, no significant correlation was found either between the frequencies of HBV-specific CD8+ T cells and the viral load, or between the frequencies and the levels of alanine transaminase. Our results suggested that the frequencies of HBV-specific CTLs are not the main determinant of immune-mediated protection in chronic HBV infection and immuno-therapeutic approaches should be aimed at not only boosting a HBV-specific CD8+T response but also improving its function.
ABSTRACT
The pathogenesis of HBsAg (+)/HBsAb(+) double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Eukaryotic expression plasmids PCI-SY, PCI-adw, PCI-adr, PCI-ayw, which ex-pressed S gene product of different serotypes, were constructed and transfected into HepG2 cells. Re-combinant proteins were purified from the transfeeted cells. At the same time, HBsAg mouse antiserum was obtained by immunizing mice with PCI-SY plasmid. HBsAg/HBsAb coexistence was simulated using these antigens and antiserum. Furthermore, the expression plasmids expressing different serotypes of S gene product including PCI-adw, PCI-adr, and PCI-ayw were injected into mice via tail vein.HBsAg and HBsAb in mice sera were tested at the first and 7th day respectively after antigen plasmids injection. Both in vitro simulation and in vivo animal models demonstrated that HBsAg antigen and HBsAb of the same serotypes could not coexist, but HBsAg antigen and HBsAb of different serotype could coexist. HBsAg/HBsAb double positive hepatitis B virus infection could be due to infection of viruses of different serorypes.
ABSTRACT
The objective of this study was to characterize the genome structure of duck hepatitis B virus (DHBV) isolated from Hubei brown ducks. The natural carrier rate of DHBV in adult ducks from Hubei area was investigated and the DHBV DNA-positive serum screened out. The complete genome of a DHBV strain was amplified by polymerase chain reaction (PCR) and cloned into T vector and sequenced. The results showed that the carrier rate of DHBV in Hubei brown ducks was 10 %.This strain (GenBank accession number DQ276978) had a genome of 3024 nucleotides with three overlapping open reading frames encoding the surface, core and polymerase proteins respectively. Comparison of the strain with 17 DHBV strains registered in GenBank revealed a homology from 89.3 % to 93.5 % at the nucleotide level. The sequences of the structural and functional domains of these proteins were highly conserved. The strain was found to share more signature amino acids in the polymerase genes with the "Chinese" DHBV strains than those of the "Western" country strains.This finding was also corroborated by a phylogenetic tree analysis. Therefore, the DQ276978 might belong to a subtype of the Chinese DHBV strains.