Journal: Antimicrobial Agents and Chemotherapy

Article Title: A Substituted Tetrahydro-Tetrazolo-Pyrimidine Is a Specific and Novel Inhibitor of Hepatitis B Virus Surface Antigen Secretion

doi: 10.1128/aac.00541-07

Figure Lengend Snippet: FIG. 2. Basic characteristics of HBF-0259. (A) Structural represen- tation. Most hydrogen atoms are left inferred. The tetrahydro-tetra- zolo-pyrimidine ring is conventionally numbered according to double ring positions, with bridge atoms ignored. R1, substituent at carbon 7; R2, substituent at carbon 5. (B) EC50 and CC50 determinations with HepDE19 cells by the HBsAg ELISA and the MTT assay, respectively. All data points are the means for duplicate wells, and error bars represent the standard deviations from the mean. The results shown are those of one typical experiment of five trials. The best-fit curve and calculation were carried out with XLfit software (version 4.0), with outliers removed if necessary to generate R2 values above 0.5. Com- pound concentrations are expressed on a log10 scale and were 0.0158, 0.05, 0.158, 0.5, 1.58, 5.0, 15.8, and 50.0 M (from left to right). The percent inhibition of secretion and the percent loss of viability were calculated against the addition of DMSO alone, for which the results were scored as 0% inhibition and 0% loss of viability, respectively.

Article Snippet: The antibodies used in the HBsAg enzyme-linked immunosorbent assay (ELISA) for screening were as follows: the primary capture antibody was an anti-HBsAg mouse monoclonal antibody (Fitzgerald Industries, Concord MA), and the detection antibody was a horseradish peroxidase-conjugated anti-surface antigen mouse monoclonal antibody (Abbott Diagnostics, Abbott Park, IL).

Techniques: Enzyme-linked Immunosorbent Assay, MTT Assay, Software, Inhibition

Journal: Antimicrobial Agents and Chemotherapy

Article Title: A Substituted Tetrahydro-Tetrazolo-Pyrimidine Is a Specific and Novel Inhibitor of Hepatitis B Virus Surface Antigen Secretion

doi: 10.1128/aac.00541-07

Figure Lengend Snippet: FIG. 3. Characterization of HBF-0259 activity by secondary assays. (A) HepG2.2.15 cells were treated with the indicated concentrations, as described in the text; and cell culture supernatants were analyzed by Western blotting for L, M, AGP, and -1-antitrypsin (Anti-Tryp.). Whole-cell lysates were analyzed by Western blotting for L, M and actin. p30, gp33, and gp36 represent nonglycosylated, singly glycosylated, and double glycosylated M, respectively; p39 and gp42 represent nonglycosylated and glycosylated L, respectively. The results represent multiple blotting of the same samples, and each assay was repeated (with some variations in the experimental setup) at least three times. (B) Culture supernatants from HepG2.2.15 cells treated with HBF-0259 or DMSO only, as described for panel A, were analyzed for total HBsAg by dot blot enzyme immunoassay under nondenaturing conditions. The results represent those of an experiment repeated twice. (C) Culture supernatants from HepG2.2.15 cells treated with the indicated concentrations of HBF-0259, 1 mM DTT, or DMSO alone were assayed for secreted HBeAg by capture ELISA. The percent inhibition of secretion was calculated by normalizing the reading for the DMSO-only-treated sample to 100% secretion or 0% inhibition. The results represent those of two trials. (D) Effects of extended incubation with HBF-0259 or DMSO alone on L, M, and AGP secretion from HepG2.2.15 cells. Culture supernatants were collected every 3 days from day 3 to day 24. The samples were analyzed by Western blotting, as described for panel B. The results represent the observations from experiments conducted with triplicate samples.

Article Snippet: The antibodies used in the HBsAg enzyme-linked immunosorbent assay (ELISA) for screening were as follows: the primary capture antibody was an anti-HBsAg mouse monoclonal antibody (Fitzgerald Industries, Concord MA), and the detection antibody was a horseradish peroxidase-conjugated anti-surface antigen mouse monoclonal antibody (Abbott Diagnostics, Abbott Park, IL).

Techniques: Activity Assay, Cell Culture, Western Blot, Dot Blot, Enzyme-linked Immunosorbent Assay, Inhibition, Incubation

Journal: AMB Express

Article Title: Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression

doi: 10.1186/s13568-017-0493-z

Figure Lengend Snippet: Effect of IFN-CSP or mu-IFN-CSP on HBV antigens secretion and HBV-DNA replication in HepG2.2.15 cells. HepG2.2.15 cells were cultured in the presence of IFN-CSP or mu-IFN-CSP for 6 days. Hepatitis B surface antigen (HBsAg; a ) and hepatitis B e antigen (HBeAg; b ) in the culture supernatants were analyzed by enzyme-linked immunosorbant assay (ELISA). Supernatant HBV-DNA ( c ) and intracellular HBV-DNA ( d ) were measured by real-time quantitative PCR. Data represent the mean ± SEM of three experiments. ***P < 0.001 drug group vs control group; # P < 0.05, ## P < 0.01 IFN-CSP group vs mu-IFN-CSP group

Article Snippet: The treated HepG2.2.15 cells were seeded on coverslip and the primary antibody and the second antibody were goat polyclonal anti-HBsAg antibody (1:200; Novus Biologicals, CO, USA) and Alexa Fluor Cy3-conjugated donkey anti-goat IgG (1:200; Beyotime, Guangzhou, China), respectively.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Control

Journal: AMB Express

Article Title: Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression

doi: 10.1186/s13568-017-0493-z

Figure Lengend Snippet: Effect of IFN-CSP or mu-IFN-CSP on HBsAg expression in HepG2.2.15 cells. HepG2.2.15 cells stained by immunofluorescent staining with anti-HBsAg antibody and representative photographs are captured by microscope. A Controls; B IFN-CSP; C mu-IFN-CSP; 1 red stained with HBsAg; 2 blue nuclear stained with DAPI; 3 merged images of 1 and 2. Bar, 100 μm and is the same for all photomicrographs

Article Snippet: The treated HepG2.2.15 cells were seeded on coverslip and the primary antibody and the second antibody were goat polyclonal anti-HBsAg antibody (1:200; Novus Biologicals, CO, USA) and Alexa Fluor Cy3-conjugated donkey anti-goat IgG (1:200; Beyotime, Guangzhou, China), respectively.

Techniques: Expressing, Staining, Microscopy

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Tapasin modification on the intracellular epitope HBcAg18-27 enhances HBV-specific CTL immune response and inhibits hepatitis B virus replication in vivo.

doi: 10.1038/labinvest.2014.6

Figure Lengend Snippet: Figure 3 Histopathologic changes and immunohistological analysis of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in hepatitis B virus (HBV) transgenic mice livers. (a) Liver sections were stained with hematoxylin and eosin, and examined by light microscopy. Some lymphocytes appeared in the liver of mice. Representative photographs are presented (original magnifications: 400). (b and c) Liver sections were subjected to immunohistological analysis of HBsAg (b) and HBcAg (c). Representative photographs are presented (original magnifications: 400).

Article Snippet: For histological analysis, deparaffinized 3–5-mm-thick sections of the liver tissue were treated with 0.3% H2O2 in methanol for 10 min to eliminate the endogenous peroxidase activity, rinsed with distilled water, and immersed in PBS for 5 min. After the sections were blocked with normal goat serum for 30 min at room temperature, a goat anti-HBsAg polyclonal antibody and a goat anti-HBcAg polyclonal antibody (Novus Biologicals, USA) was applied overnight at 4 1C, and following three times of washing in PBS, sections were incubated for 30 min with biotinyated secondary antibody (Boster, China) at 37 1C and then for 30 min with streptavidin– biotin–peroxidase complex before being revealed by diaminobenzidine (DAB) and counterstained with hematoxylin.21 The Levels of Cytokine IFN-g and IL-2 Splenocytes (2 106 cells/ml) harvested from immunized HLA-A2 transgenic mice were cultured in 24-well plates at 37 1C in the presence of 10mg/ml HBcAg (CalBioreagents, USA).

Techniques: Virus, Transgenic Assay, Staining, Light Microscopy

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Tapasin modification on the intracellular epitope HBcAg18-27 enhances HBV-specific CTL immune response and inhibits hepatitis B virus replication in vivo.

doi: 10.1038/labinvest.2014.6

Figure Lengend Snippet: Figure 4 The levels of hepatitis B surface antigen (HBsAg), hepatitis B virus (HBV) DNA, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in HBV transgenic mice. The data are the means±s.d. of six mice per group. The HBsAg (a) and HBV DNA (b) inhibitory rate in sera of mice treated with 100 mg CTP-HBcAg18–27-Tapasin was significantly higher than those in 50 mg CTP-HBcAg18–27-Tapasin group. Data represent the means±s.d. (– ¼ 6) (*Po0.05). Meanwhile, the AST (c) and ALT (d) in sera of mice treated with 100 mg CTP-HBcAg18–27-Tapasin was significantly higher than those in 50 mg CTP-HBcAg18–27-Tapasin group. Data represent the means±s.d. (n ¼ 6) (*Po0.05). CTP, cytoplasmic transduction peptide.

Article Snippet: For histological analysis, deparaffinized 3–5-mm-thick sections of the liver tissue were treated with 0.3% H2O2 in methanol for 10 min to eliminate the endogenous peroxidase activity, rinsed with distilled water, and immersed in PBS for 5 min. After the sections were blocked with normal goat serum for 30 min at room temperature, a goat anti-HBsAg polyclonal antibody and a goat anti-HBcAg polyclonal antibody (Novus Biologicals, USA) was applied overnight at 4 1C, and following three times of washing in PBS, sections were incubated for 30 min with biotinyated secondary antibody (Boster, China) at 37 1C and then for 30 min with streptavidin– biotin–peroxidase complex before being revealed by diaminobenzidine (DAB) and counterstained with hematoxylin.21 The Levels of Cytokine IFN-g and IL-2 Splenocytes (2 106 cells/ml) harvested from immunized HLA-A2 transgenic mice were cultured in 24-well plates at 37 1C in the presence of 10mg/ml HBcAg (CalBioreagents, USA).

Techniques: Virus, Transgenic Assay, Transduction