Journal: Journal of Innate Immunity

Article Title: L-Ficolin Binds to the Glycoproteins Hemagglutinin and Neuraminidase and Inhibits Influenza A Virus Infection Both in vitro and in vivo

doi: 10.1159/000335670

Figure Lengend Snippet: L-ficolin protects BABL/c mice against H1N1 (A/Yamagata/120/86) virus infection. Western blot analysis of L-ficolin in mouse muscle (a) and lung tissue (b) using the anti-L-ficolin mAb GN5. a BALB/c mice were injected with pcDNA3.1A-L-ficolin (lanes 1, 3 and 5) or control vector pcDNA3.1A (lanes 2, 4 and 6; 10 µg DNA per mouse) through intramuscular electroporation. Muscle was harvested from each mouse 0, 4 or 7 days after injection. b BALB/c mice were injected with pcDNA3.1A-L-ficolin and mouse lung was harvested from each mouse 0, 4, 7 or 10 days after injection. c L-ficolin concentrations in sera taken from mice injected with pcDNA3.1A-L-ficolin (* p < 0.05, day 0 group vs. other groups). d Viral hemagglutination assay of mouse lung tissue homogenates. Hemagglutination activity was expressed as a titer, i.e. the reciprocal of the highest dilution showing complete agglutination (* p < 0.05, L-ficolin group vs. pcDNA3.1A group). Data are shown as the mean ± SEM of at least 3 independent experiments. e A graph of the body weights of infected mice after the administration of L-ficolin plasmid treatment. f–i Histopathologic analyses of lung tissue. Lung tissue sections were analyzed using HE and were evaluated by light microscopy (×400).

Article Snippet: Mouse monoclonal anti-human L-ficolin GN5 (1:1,000 dilution; HyCult Biotechnology) was added to each well and incubated at 37°C for 1 h. After washing three times, 100 µl of labeled HRP-conjugated goat anti-mouse IgG (1:1,000 dilution) were added.

Techniques: Infection, Western Blot, Injection, Plasmid Preparation, Electroporation, Hemagglutination Assay, Activity Assay, Agglutination, Light Microscopy

Journal: Journal of Innate Immunity

Article Title: L-Ficolin Binds to the Glycoproteins Hemagglutinin and Neuraminidase and Inhibits Influenza A Virus Infection Both in vitro and in vivo

doi: 10.1159/000335670

Figure Lengend Snippet: Neutralization of H1N1 A/Yamagata/120/86 infectivity. a Virus was incubated with L-ficolin for 30 min. The mixture was added to MDCK cells. The infected MDCK cells were collected for FQ-RT-PCR to detect the expression of the M gene of the H1N1 virus. The neutralization activity of L-ficolin was determined by percent inhibition: (M gene copies in the absence of L-ficolin – M gene copies with L-ficolin)/M gene copies in the absence of L-ficolin (* p < 0.05, L-ficolin + H1N1 virus vs. control group). This experiment was repeated 3 times. b FQ-RT-PCR analysis showed that neutralization effects of L-ficolin were reversed in cell culture by adding different doses of anti-L-ficolin mAb. FQ-RT-PCR (c) and flow-cytometric (d) analysis showed that L-ficolin did not bind to other elements or molecules on the cell surface and inhibition effects of L-ficolin on viral infection were not due to the binding of L-ficolin to MDCK cells. FITC anti-GST mAb was used in the flow-cytometric analysis.

Article Snippet: Mouse monoclonal anti-human L-ficolin GN5 (1:1,000 dilution; HyCult Biotechnology) was added to each well and incubated at 37°C for 1 h. After washing three times, 100 µl of labeled HRP-conjugated goat anti-mouse IgG (1:1,000 dilution) were added.

Techniques: Neutralization, Infection, Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing, Activity Assay, Inhibition, Cell Culture, Binding Assay

Journal: Journal of Innate Immunity

Article Title: L-Ficolin Binds to the Glycoproteins Hemagglutinin and Neuraminidase and Inhibits Influenza A Virus Infection Both in vitro and in vivo

doi: 10.1159/000335670

Figure Lengend Snippet: L-ficolin protects FCNA-KO mice against H1N1 (A/PR/8/34) virus infection. a Western blot analysis of L-ficolin or FCNA expression in the muscle of FCNA-KO mice using monoclonal anti-L-ficolin antibody GN5 or polyclonal anti-FCNA antibody. Male FCNA-KO mice in a C57BL/6 background (8 weeks old) were injected with pcDNA3.1A-L-ficolin (lanes 1, 3 and 5), pcDNA3.1A (lanes 2, 4 and 6), pVAX-1-FCNA (lanes 7, 9 and 11) or pVAX-1 (lanes 8, 10 and 12; 10 µg DNA/mouse) through intramuscular electroporation. Muscle tissue was harvested from each mouse 0, 4 or 7 days after injection. b Survival rate of mice infected with H1N1 A/PR/8/34 (* p < 0.05, vs. FCNA-KO + PBS and FCNA-KO + pVAX-1). The Kaplan-Meier method was used to plot survival curves for each infected group of mice. A Breslow test was used for statistical analysis of the survival curves. Differences were considered to be statistically significant for p = 0.05. c Real-time RT-PCR analysis of viral RNA expression (* p < 0.05, FCNA-KO + L-ficolin or FCNA-KO + FCNA group vs. other FCNA-KO control groups). Data are shown as the mean ± SEM of at least 3 independent experiments. d Viral hemagglutination assays from mouse lung tissue homogenates 3 days after infection. Hemagglutination activity was expressed as HA titer, i.e. the reciprocal of the highest dilution showing complete agglutination (*p < 0.05, FCNA-KO + L-ficolin or FCNA-KO + FCNA group vs. other FCNA-KO control groups). Data are shown as the mean ± SEM of at least 3 independent experiments. e TCID50 of mouse lung tissue homogenates 3 and 5 days after infection (* p < 0.05, FCNA-KO + L-ficolin or FCNA-KO + FCNA group vs. other FCNA-KO control groups). The plaque assay was used to determine the lung viral titer and TCID50. f Histopathological analysis of lung tissue. The lung tissue sections were stained with HE and light-microscopically evaluated (×400).

Article Snippet: Mouse monoclonal anti-human L-ficolin GN5 (1:1,000 dilution; HyCult Biotechnology) was added to each well and incubated at 37°C for 1 h. After washing three times, 100 µl of labeled HRP-conjugated goat anti-mouse IgG (1:1,000 dilution) were added.

Techniques: Infection, Western Blot, Expressing, Injection, Electroporation, Quantitative RT-PCR, RNA Expression, Activity Assay, Agglutination, Plaque Assay, Staining

Journal: Journal of Innate Immunity

Article Title: L-Ficolin Binds to the Glycoproteins Hemagglutinin and Neuraminidase and Inhibits Influenza A Virus Infection Both in vitro and in vivo

doi: 10.1159/000335670

Figure Lengend Snippet: Recombinant L-ficolin bound to influenza virus particles. Influenza viruses (A/chicken/Hubei/489/2004, A/Yamagata/ 120/86 and A/PR/8/34) were coated onto 96-well plates, and recombinant GST-L-ficolin or GST was added. The binding ability of L-ficolin to the viruses was determined as a ratio: OD value (GST-L-ficolin + influenza virus)/OD value (GST + influenza virus). a Recombinant L-ficolin bound to H1N1 A/Yamagata/120/86 virus (** p < 0.01, * p < 0.05, 0 µM GST-L-ficolin group vs. various GST-L-ficolin concentration groups). b Recombinant L-ficolin bound to H5N1 A/chicken/Hubei/489/2004 virus (** p < 0.01, * p < 0.05, 0 µM GST-L-ficolin group vs. various L-ficolin-GST concentration groups). c Recombinant L-ficolin bound to H1N1 A/PR/8/34 virus (** p < 0.01, * p < 0.05, 0 µM GST-L-ficolin group vs. various GST-L-ficolin concentration groups). d Effects of GlcNAc and mannan on the binding between recombinant L-ficolin-GST and H1N1 A/PR/8/34 virus. 0.28 mM recombinant GST-L-ficolin or GST were added, along with GlcNAc or mannan, to a plate coated with H1N1 A/PR/8/34 virus. All data are shown as the mean ± SEM of at least 3 independent experiments.

Article Snippet: Mouse monoclonal anti-human L-ficolin GN5 (1:1,000 dilution; HyCult Biotechnology) was added to each well and incubated at 37°C for 1 h. After washing three times, 100 µl of labeled HRP-conjugated goat anti-mouse IgG (1:1,000 dilution) were added.

Techniques: Recombinant, Binding Assay, Concentration Assay

Journal: Journal of Innate Immunity

Article Title: L-Ficolin Binds to the Glycoproteins Hemagglutinin and Neuraminidase and Inhibits Influenza A Virus Infection Both in vitro and in vivo

doi: 10.1159/000335670

Figure Lengend Snippet: Binding assay between L-ficolin and HA or NA. Flow-cytometric analyses of HA expression in HA-CT26 (a) and NA expression in NA-CT26 (b) using anti-flag antibody. c Western blot analysis of recombinant GST-L-ficolin fusion proteins using anti-GST antibody. Lane 1 = GST-L-ficolin; lane 2 = GST. d Binding assay between L-ficolin and HA or NA by GST pull-down and Western blot analysis using anti-HA, anti-NA and anti-L-ficolin mAbs. β-Actin, a housekeeping gene with a constant expression, was used as an internal control.

Article Snippet: Mouse monoclonal anti-human L-ficolin GN5 (1:1,000 dilution; HyCult Biotechnology) was added to each well and incubated at 37°C for 1 h. After washing three times, 100 µl of labeled HRP-conjugated goat anti-mouse IgG (1:1,000 dilution) were added.

Techniques: Binding Assay, Expressing, Western Blot, Recombinant

Journal: Journal of Innate Immunity

Article Title: L-Ficolin Binds to the Glycoproteins Hemagglutinin and Neuraminidase and Inhibits Influenza A Virus Infection Both in vitro and in vivo

doi: 10.1159/000335670

Figure Lengend Snippet: Binding of L-ficolin to influenza A virus leads to the activation of the complement lectin pathway.

Article Snippet: Mouse monoclonal anti-human L-ficolin GN5 (1:1,000 dilution; HyCult Biotechnology) was added to each well and incubated at 37°C for 1 h. After washing three times, 100 µl of labeled HRP-conjugated goat anti-mouse IgG (1:1,000 dilution) were added.

Techniques: Binding Assay, Activation Assay