Volume 39, Issue 5, Pages 834-842 (November 2003) The role of inducible nitric oxide synthase in a murine acute hepatitis B virus (HBV) infection model induced by hydrodynamics-based in vivo transfection of HBV-DNA  Wen-Wei Chang, Ih-Jen Su, Ming-Derg Lai, Wen-Tsan Chang, Wenya Huang, Huan-Yao Lei  Journal of Hepatology  Volume 39, Issue 5, Pages 834-842 (November 2003) DOI: 10.1016/S0168-8278(03)00389-1

Fig. 1 HBV antigen expression in the murine liver after hydrodynamics-based transfection of pHBV3.6. Groups of four BALB/c mice were injected intravenously with 10 μg of plasmid by hydrodynamics-based transfection. The liver tissues were collected at 3 days post injection to detect the gene expression. Anti-HBcAb was used to stain HBcAg expression (A–C) while anti-HBsAb detected HBsAg expression (D–F). A and D, pSGCLacZ; B and E, pHBV3.6; C, pHBVρPSX; and F, p(3A)SAg. The insets in B, C, E and F represent 2-fold magnification of positive staining hepatocytes. Arrow indicates positive stain. (Original magnification ×200). Journal of Hepatology 2003 39, 834-842DOI: (10.1016/S0168-8278(03)00389-1)

Fig. 2 H&E stain of liver after hydrodynamics-based transfection of pHBV3.6. Groups of four BALB/c mice were injected intravenously with 10 μg of plasmid by hydrodynamics-based transfection. The liver tissues were collected at various days post injection, and H&E stain was analyzed for its morphological changes. A, normal saline, D3; B, pHBV3.6, D2; C, pHBV3.6, D3; D, pHBV3.6, D5; E, p(3A)SAg, D3; and F, pHBVρPSX, D3. The arrow in B–D indicates the lipomorphogenic changes while the arrowhead in C–D showed the damaged hepatocytes. The liver tissues after injection of p(3A)SAg (E) or pHBVρPSX (F) have minor lipomorphogenic change while the saline control (A) remains intact. (Original magnification ×200). Journal of Hepatology 2003 39, 834-842DOI: (10.1016/S0168-8278(03)00389-1)

Fig. 3 HBV replication in the liver after hydrodynamics-based transfection of pHBV3.6. Groups of four BALB/c mice were injected intravenously with 10 μg of pHBV3.6 by hydrodynamics-based transfection. (A) Northern and Southern blot analysis. Liver tissues were collected at 2, 3 or 5 days of primary injection or at 2 days of secondary injection. Thirty μg of total hepatic RNA or cytoplasmic DNA were isolated from 30 mg of liver tissue as described in Section 2. The membranes were hybridized with 32P-labeled HBV-specific DNA probe. The 3.5 or 2.1/2.4 kb RNA fragments as HBV transcripts were indicated. The sALT at each time point was represented as mean±(SD). The relaxed circular (RC), double stranded (DS) or single stranded (SS) HBV DNA replicative forms were indicated. 28S and 18S rRNA were used as RNA loading control. N, PBS-injected control; 2′, secondary injection. (B) PCR analysis of HBV-DNA in serum. DNA was purified from mouse serum after treatment with 20 U of DNase I to remove retaining plasmid and used as template to amplify the preS region of HBV genome. The specific PCR product of 654 bp was detected in mouse serum at 2, 3, or 5 days after single injection, but not at day 2 after secondary injection of 10 μg of pHBV3.6. M, 100 bp DNA ladder; N, no template control. Journal of Hepatology 2003 39, 834-842DOI: (10.1016/S0168-8278(03)00389-1)

Fig. 4 HBV antigenemia in BALB/c after hydrodynamics-based transfection of pHBV3.6. Groups of four BALB/c mice were injected intravenously with 10 μg of pHBV3.6 by hydrodynamics-based transfection. Serum samples were collected at indicated time after injection, and HBsAg or HBeAg level was determinated by ELISA. Relative concentration of HBeAg is shown by [(A490 of sample – absorbance of background)/absorbance of background]. A and C, pHBV3.6; B, p(3A)SAg; D, pHBVeAg. Journal of Hepatology 2003 39, 834-842DOI: (10.1016/S0168-8278(03)00389-1)

Fig. 5 HBV antigenemia in B6 or iNOS−/− mice after hydrodynamics-based transfection of pHBV3.6. Groups of four B6 (A and B) or iNOS−/− (C and D) mice were injected intravenously with 10 μg of pHBV3.6 by hydrodynamics-based transfection. Serial sections of frozen liver tissue were made and stained with anti-HBs antibody (A and C) or anti-iNOS antibody (B and D). Arrows indicate positive staining. The iNOS mRNA expression was also analyzed by RT-PCR method and GAPDH was used as internal control (E). M, 100 bp DNA ladder; N, PBS-injected control. The antigenemia of HBsAg (F) or HBeAg (G) in iNOS−/− mice (open circle) or B6 mice (closed circle) was determined as described in Section 2. Journal of Hepatology 2003 39, 834-842DOI: (10.1016/S0168-8278(03)00389-1)

Fig. 6 HBV replication in B6 or iNOS−/− mice after hydrodynamics-based transfection of pHBV3.6. Groups of four B6 or iNOS−/− mice were injected intravenously with 10 μg of pHBV3.6 by hydrodynamics-based transfection. (A) Northern and Southern blot analysis. Thirty μg of total hepatic RNA or cytoplasmic DNA from 30 mg liver tissue of B6 or iNOS−/− mice were isolated at 2 or 5 days. 28S and 18S rRNA were used as RNA loading control. The sALT at each time point was represented as mean±(SD). RC, relaxed circular; DS, double stranded; SS, single stranded. (B) PCR analysis of HBV-DNA in serum. DNA was purified from B6 or iNOS−/− mouse serum after treatment with 20 U of DNase I to remove retaining plasmid and used as template to amplify the preS region of HBV genome. The upper panel was used as HBV dose standard curve created by indicated amount of pHBV3.6. The intensity of PCR product of samples from iNOS−/− mice at days 2, 3 or 5 was higher than from B6 mice. Journal of Hepatology 2003 39, 834-842DOI: (10.1016/S0168-8278(03)00389-1)

Fig. 7 Immunophenotyping of IHLs in B6 or iNOS−/− mice after hydrodynamics-based transfection of pHBV3.6. Groups of four B6 or iNOS−/− mice were injected intravenously with 10 μg of pHBV3.6 by hydrodynamics-based transfection. Mice were sacrificed at day 7 post injection, and total IHLs isolated from metrizamide gradient were stained with various antibodies as described in Section 2. The number of each cell types was normalized with the liver weight. Statistical significance was evaluated by unpaired Student's t-test. *, P<0.05. Journal of Hepatology 2003 39, 834-842DOI: (10.1016/S0168-8278(03)00389-1)

Fig. 8 F4/80, CD80, and Gr-1 expression of IHLs in B6 or iNOS−/− mice after hydrodynamics-based transfection of pHBV3.6. Groups of four B6 or iNOS−/− mice were injected intravenously with 10 μg of pHBV3.6 by hydrodynamics-based transfection. Mice were sacrificed at day 7 post injection and total IHLs isolated from metrizamide gradient were stained with anti-F4/80 (A); anti-CD80 (B); and anti-Gr-1 (C) antibodies as described in Section 2. Percentage (parenthesis) and mean fluorescent intensity of positive cells were represented. Dot line indicates isotype control. Journal of Hepatology 2003 39, 834-842DOI: (10.1016/S0168-8278(03)00389-1)