Long-term follow up of serum HBeAg in the animals injected with the pHBV1.3-B6 replicon DNA.

<p>Serum HBeAg was monitored regularly following hydrodynamic injection of pHBV1.3-B6 replicon DNA (10 µg/mouse) in BALB/c (A), C57BL/6 (B), and FVB/N (C) mice. Each line represents one animal. The levels of HBeAg in 10-fold diluted sera are shown as S/CO, signal-to-control ratio. The dotted gray lines represent the cut-off value of HBeAg. (D) The kinetics of HBeAg clearance in the three mouse strains over a period of 50 weeks are shown and were compared using the logrank test. The HBeAg-positive rates are as follows: BALB/cP<0.01), BALB/cP<0.001), and C57BL/6P<0.001).</p

Histological analysis of the livers from different mouse strains injected with HBV DNA.

<p>BALB/c, C57BL/6, and FVB/N mice were injected with the pHBV1.3-B6 replicon DNA. The mice were sacrificed at the time points indicated, and their livers were fixed with formalin, embedded in paraffin, and stained with hematoxylin and eosin. The arrows indicate the infiltrating inflammatory cells. The scale bar represents 100 µm. The images are displayed at 200× magnification.</p

Mutation sites of the amino acid residues in the six sub-clones compared to the B6 consensus sequence.

<p>Mutation sites of the amino acid residues in the six sub-clones compared to the B6 consensus sequence.</p

The kinetics of ALT elevation, infiltration of activated CTLs, and the expression of cytokines and chemokines in different mouse strains after DNA injection.

<p>Mice were injected with pHBV1.3-B6 DNA (10 µg/mouse), and three animals were sacrificed at the indicated time points after DNA injection. (A) The serum ALT levels of the mice were measured. The gray dashed lines represent the normal serum ALT levels, 40 U/L. (B) Flow cytometric analysis of the levels of activated CTLs in the livers. Intrahepatic lymphocytes were isolated and stained for CD3 (PerCP), CD8 (FITC), and CD69 (PE). The percentages of CD69-positive cells in the CD3- and CD8-double positive lymphocytes were determined by flow cytometry. In addition, the liver expression of (C) IFN-γ, (D) TNF-α, (E) CXCL9, and (F) CXCL10 was analyzed by qRT-PCR. Values are shown as the mean ± SD.</p

Different persistence rates conferred by a single amino acid change in the HBV surface protein.

<p>The mutant replicon DNAs, including (A) pHBV1.3-B6.2S, containing an N214S point mutation in surface protein; (B) pHBV1.3-B6.2P, containing an H337Y point mutation in polymerase; and (C) pHBV1.3-B6.2SP, containing both N214S and H337Y mutations, were injected into FVB/N mice. The levels of HBeAg in 10-fold diluted sera are shown as S/CO, signal-to-control ratio. Each line represents one animal. The dotted gray lines represent the cut-off value of HBeAg. (D) The kinetics of HBeAg clearance in the mice injected with the wild-type sub-clone B6.2 or the three mutants (B6.2S, B6.2P, and B6.2SP) are shown and were compared using the logrank test. The HBeAg-positive rates are B6.2SP<0.01), B6.2SPP<0.05), and B6.2P <i>vs.</i> B6.2, no significance.</p

The duration of HBV replicative intermediates present in the livers of the replicon DNA-injected animals.

<p>BALB/c (A), C57BL/6 (B), and FVB/N (C) mice were hydrodynamically injected with 10 µg of the pHBV1.3-B6 replicon DNA. At the indicated time points, two animals were sacrificed. The liver genomic DNA was extracted and digested with <i>Hind</i>III (a single cut in the HBV replicon DNA), and then analyzed using Southern blot analysis with an HBV-specific probe. Each lane represents one animal. The sizes corresponding to the injected DNA, and the replicative intermediates are indicated on the right. (D) The IHC staining of HBcAg and HBsAg in the livers of the BALB/c, C57BL/6, and FVB/N mice injected with pHBV1.3-B6 DNA. Livers were harvested at the time points indicated. IHC staining was performed using anti-HBc or anti-HBs antibody on frozen sections. The scale bar represents 100 µm. The images are displayed at 200× magnification.</p

Different persistence rates of different HBV isolates.

<p>(A) The replicon DNA used for injection included pHBV1.3-B6, -B21, -B22, and -B29 isolated from different patients. The kinetics of HBeAg clearance over a period of 20 weeks are shown. (B) The replicon DNA of the six sub-clones (B6.1, B6.2, B6.3, B6.7, B6.8, and B6.9) contained in the B6 clone was injected separately into the FVB/N mice. Serum HBeAg was monitored for 20 weeks. The dotted gray lines represent the cut-off value of HBeAg. (C) The HBeAg-positive rate of each sub-clone is shown at 20 weeks post injection.</p

The levels of ALT, activated CTLs, and the liver expression of cytokines and chemokines in FVB/N mice following injection with pHBV1.3-B6.2 or -B6.2S DNA.

<p>Mice were injected with pHBV1.3-B6.2 or -B6.2S DNA (10 µg/mouse). Three animals were sacrificed at the time points indicated. (A) Serum ALT levels of the mice were measured. The gray dashed lines represent the normal serum ALT levels, 40 U/L. (B) Flow cytometric analysis of activated CTLs in the livers. Staining of activated CTLs was performed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036984#pone-0036984-g003" target="_blank">Fig. 3B</a>. The liver expression of (C) IFN-γ, (D) TNF-α, (E) CXCL9, and (F) CXCL10 was analyzed by qRT-PCR. Values are shown as the mean ± SD of three mice. *<i>P</i><0.05; **<i>P</i><0.01.</p

Histological analysis of the livers of FVB/N mice injected with pHBV1.3-B6.2 or -B6.2S replicon DNA.

<p>FVB/N mice were injected with pHBV1.3-B6.2 or -B6.2S replicon DNA. The mice were sacrificed at the indicated time points, and their livers were fixed with formalin, embedded in paraffin, and stained with hematoxylin and eosin. The arrows indicate the infiltrating inflammatory cells. The scale bar represents 50 µm. The images are displayed at 400× magnification.</p

Analysis of precore and basal core promoter wild types and mutants in chronic carriers and hepatocellular carcinoma (HCC) patients

<p><b>Copyright information:</b></p><p>Taken from "Basal core promoter T1762/A1764 and precore A1896 gene mutations in hepatitis B surface antigen-positive hepatocellular carcinoma: a comparison with chronic carriers"</p><p></p><p>Liver International 2007;27(10):1356-1363.</p><p>Published online Jan 2007</p><p>PMCID:PMC2229667.</p><p>© 2007 The Authors. Journal compilation © 2007 Blackwell Munksgaard</p