1. Volume 126, Issue 3, Pages 859-872 (March 2004)
Impaired clearance of virus-infected hepatocytes in transgenic mice expressing the hepatitis C virus polyprotein 
Olivier Disson, Delphine Haouzi, Solange Desagher, Kim Loesch, Michael Hahne, Eric J. Kremer, Chantal Jacquet, Stanley M. Lemon, Urszula Hibner, Hervé Lerat 
Gastroenterology 
Volume 126, Issue 3, Pages (March 2004)
DOI: /j.gastro

2. Figure 1
HCV+ mice mount a normal cell-mediated immune response to adenoviral infection. Eleven HCV+ mice and 11 control littermates were injected with 2.5 × 109 pfu of β-galactosidase encoding adenovirus. Each animal was subjected to a liver biopsy 2 days postinfection and groups of animals (3 animals at day 3, 7, and 21; 2 animals at day 14) were sacrificed at times indicated. (A) Schematic representation of the transgene in the FL/N-35 transgenic mice.11 (B) Mononuclear cells were purified from livers of transgenic (solid lines and open squares) and control mice (dashed lines and open squares) as described in the Materials and Methods section and counted. (C) Subpopulations of intrahepatic mononuclear cells were analyzed by flow cytometry according to the surface expression of CD3 (dotted), CD3 and CD4 (hatched), or CD3 and CD8 (open bars) markers. (D) Splenocytes isolated from HCV+ (solid line) or control littermates (dotted) at day 7, 14, or 21 postinfection were stimulated by a β-galactosidase-derived peptide epitope. The secretion of γ-interferon was assayed by enzyme-linked immunosorbent assay. AU, arbitrary units.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
HCV+ mice are defective in the clearance of adenovirus-infected hepatocytes. (A) β-galactosidase staining was performed on liver sections to qualitatively assess the level of infection in situ. Adenovirus-infected hepatocytes are visible in blue. Forty to 50% hepatocytes stain positive for β-galactosidase at day 3 both in transgenic and control samples. Positive cells are readily detectable at day 21 postinfection in the HCV+ livers; they are absent from the control samples. The bar represents 100 μ. (B) Crude protein extracts from HCV+ (hatched) and control (empty) livers and spleens were assayed for β-galactosidase activity at day 3 and 21 postinfection. Results are presented as mean ± standard error of the mean. (C) β-galactosidase activity was measured in crude extracts from of HCV+ (solid) and control (dashed) mice sacrificed at day 3, 7, 14, or 21 postinfection. The results are normalized to β-galactosidase activity measured in biopsies taken at day 2 postinfection and presented as mean ± standard error of the mean. The data in B and C come from 2 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
HCV gene expression protects hepatocytes from Fas-induced apoptosis. Agonistic anti-Fas antibody (Jo2) was injected into the tail vein of 4 HCV transgenic and 3 control mice. Animals were sacrificed 2 hours postinjection, and the histology of fixed, paraffin-embedded liver sections was analyzed by H&E staining (A). Panels 1 and 2: control mice; panels 3 and 4: HCV transgenic mice. Liver destruction and hemorrhage are severe in the control samples. Darker staining cells with condensed nuclei, typical of apoptotic morphology, are visible at higher magnification (panel 2). Scale bars = 20 μ. (B) Sections of control (empty) and transgenic (hatched) livers were stained with the Hoechst dye. Highly condensed, apoptotic, and normal nuclei were counted in randomly chosen fields. Bars are mean ± standard error of the mean. (C) Primary cultures of 8 transgenic (hatched) and 4 nontransgenic (empty) hepatocytes were treated with Jo2. Twenty-two hours later, cells were stained for activated caspases with Z-VAD-FITC and for nuclear morphology with Hoechst Apoptotic and normal cells were scored in randomly chosen fields. Bars are mean ± standard error of the mean. (D) Caspase 3 activation was assayed by immunoblotting in cell lysates prepared from primary hepatocyte cultures stimulated by anti-Fas antibody for the times indicated. (E) Crude protein extracts from 2 HCV+ and 2 control primary hepatocyte cultures were analyzed by Western blotting using a polyclonal anticaspase 3 antibody recognizing the unprocessed procaspase. The amount of protein loaded on the gel was normalized using an anti-GAPDH antibody. A representative Western blot is shown.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Apoptosis resistance of primary HCV transgenic hepatocytes is specific to the Fas pathway. Primary hepatocyte cultures from 4 HCV transgenic (solid or hatched) and 4 control (dashed or empty) mice were treated with indicated concentrations of anti-Fas antibody Jo2 (A), recombinant Fas ligand (B), TNF-α and, as positive control, Jo2 (0.1 μg/mL) (C) and lonidamine (D). Cell viability was assayed by a colorimetric XTT test in A, B, and D. Apoptosis was quantified in C by fluorescence microscopy of cytospin slides labeled with the Hoechst All results are presented as mean ± standard error of the mean of at least 3 measurements.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
HCV gene expression inhibits the Fas pathway at the level of mitochondrial cytochrome c release. (A) Primary hepatocytes isolated from HCV+ and control mice were cultured in the presence or absence of Jo2. Twenty-two hours later, the cells were collected, lysed and the mitochondrial (M) and cytosoluble (C) fractions were separated. The release of cytochrome c from the mitochondria was assayed by immunoblotting. (B) Fractionated liver extracts from 4 HCV+ (hatched) and 3 control (empty) animals treated in vivo Jo2 were analyzed by immunoblotting. The cytoplasmic release of cytochrome c was quantified by densitometry and normalized to the level of expression of GAPDH in the cytosol. Results are presented as mean ± standard error of the mean.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
Down-regulation of Bid expression in the livers of the HCV transgenic mice. (A) Expression of Bid mRNA in 7 control (empty) and 6 HCV+ (hatched) livers was measured by quantitative RT-PCR and normalized to the GAPDH mRNA expression levels. Results are presented as mean ± standard error of the mean. (B, C) Liver, kidney, and spleen extracts from 10 HCV+ (hatched) and 10 control (empty) mice were analyzed by immunoblotting. A representative Western blot with anti-Bid and anti-GAPDH antibodies is shown in B. A densitometric analysis of Bid expression in livers, normalized to the GAPDH expression level, is shown in C. Results are presented as mean ± standard error of the mean. (D) Bid complementation assay: cytosolic fractions from HCV+ and control hepatocytes were incubated with intact mitochondria in the presence or absence of recombinant, active, caspase 8, and recombinant Bid (rBid). Release of cytochrome c from the mitochondria was measured by Western blot of the cytosolic supernatant. The total content of cytochrome c is shown in the first lane, which corresponds to the mitochondrial fraction.
Gastroenterology  , DOI: ( /j.gastro )