Journal: Journal of Veterinary Research

Article Title: IFNγ Production Profile in Turkeys of Different Immunological Status after TRT Vaccination

doi: 10.2478/jvetres-2020-0040

Figure Lengend Snippet: Summary of mean relative IFNγ gene expression in splenic mononuclear cells on different days post aMPV/A vaccination in experiments I (MDA+ vaccinated) and II (MDA− vaccinated) (A) and III (MDA+ vaccinated) and IV (MDA− vaccinated) (B). The relative expression of the IFNγ gene was calculated using the 2 −ΔΔCt method normalised to efficiency corrections, expression levels of the reference gene coding GAPDH and adequate control groups. Bars represent mean IFN gamma expression level against its expression in the adequate control group * Significant differences at different DPV ( t -test, P < 0.05)

Article Snippet: After incubation, the plates were washed four times with PBS-Tween 20 and once with PBS and incubated overnight at 4°C with Anti - Chicken IFNγ Biotinylated Polyclonal Antibody (PBB0448C-050; Kingfisher Biotech, St Paul.

Techniques: Gene Expression, Expressing, Control

Journal: Journal of Veterinary Research

Article Title: IFNγ Production Profile in Turkeys of Different Immunological Status after TRT Vaccination

doi: 10.2478/jvetres-2020-0040

Figure Lengend Snippet: Mean percentage of CD4 + IFNγ + cells ± SD within splenic mononuclear cells of turkeys of vaccinated (V) and not vaccinated (NV) groups of experiments I–IV at different DPV

Article Snippet: After incubation, the plates were washed four times with PBS-Tween 20 and once with PBS and incubated overnight at 4°C with Anti - Chicken IFNγ Biotinylated Polyclonal Antibody (PBB0448C-050; Kingfisher Biotech, St Paul.

Techniques:

Journal: Journal of Veterinary Research

Article Title: IFNγ Production Profile in Turkeys of Different Immunological Status after TRT Vaccination

doi: 10.2478/jvetres-2020-0040

Figure Lengend Snippet: Mean percentage of CD8 + IFNγ + cells ± SD within splenic mononuclear cells of turkeys of vaccinated (V) and not vaccinated (NV) groups of experiments I–IV at different DPV

Article Snippet: After incubation, the plates were washed four times with PBS-Tween 20 and once with PBS and incubated overnight at 4°C with Anti - Chicken IFNγ Biotinylated Polyclonal Antibody (PBB0448C-050; Kingfisher Biotech, St Paul.

Techniques:

Journal: Journal of Veterinary Research

Article Title: IFNγ Production Profile in Turkeys of Different Immunological Status after TRT Vaccination

doi: 10.2478/jvetres-2020-0040

Figure Lengend Snippet: Summary of mean contribution of anti-aMPV IFNγ-secreting cells within splenic mononuclear cells at different days post aMPV/A vaccination in experiments I (MDA+ vaccinated) and II (MDA− vaccinated) (A) and III (MDA+ vaccinated) and IV (MDA− vaccinated) (B) in relation to the mean contribution of anti-aMPV IFNγ-secreting cells in the spleen of adequate control birds (not vaccinated) * Significant differences in vaccinated groups of birds in comparison to adequate control groups, at different DPV ( t -test, P < 0.05)

Article Snippet: After incubation, the plates were washed four times with PBS-Tween 20 and once with PBS and incubated overnight at 4°C with Anti - Chicken IFNγ Biotinylated Polyclonal Antibody (PBB0448C-050; Kingfisher Biotech, St Paul.

Techniques: Control, Comparison

Journal: FASEB bioAdvances

Article Title: Manipulation of the tumor microenvironment by cytokine gene transfection enhances dendritic cell‐based immunotherapy

doi: 10.1096/fba.2019-00052

Figure Lengend Snippet: Lung metastasis in the LM8‐inoculated mice

Article Snippet: Cells infiltrating tumor tissues were evaluated by immunohistochemistry using following primary Abs: for IFNγ, Bioss Cat# bs‐0480R‐PE‐Cy7, RRID:AB_11092092; for CD40L, Biorbyt Cat# orb10326, RRID:AB_10746923; for CD8, clone 53‐6.7, Thermo Fisher Scientific Cat# 14‐0081‐81, RRID:AB_467086; for Foxp3, clone FJK‐16s, Thermo Fisher Scientific Cat# 14‐5773‐80, RRID:AB_467575; for Iba1, Wako Pure Chemical Industries Inc, Cat# 019‐19741, RRID:AB_839504; for granzyme B, Spring Bioscience Cat# E2580, RRID:AB_1661202.

Techniques:

Journal: FASEB bioAdvances

Article Title: Manipulation of the tumor microenvironment by cytokine gene transfection enhances dendritic cell‐based immunotherapy

doi: 10.1096/fba.2019-00052

Figure Lengend Snippet: Effect of IFNγ and CD40L (Comb) gene‐transfection on DC‐based therapy. (A) Survival of mice with the indicated treatments. (B) The relative tumor volume (with the volume on day 0 set at 1) of each mouse in the control groups. Black solid lines indicate the relative tumor volumes of the [Untreated] group; black dotted lines indicate those of the [Cont (IT)] group; gray solid lines indicate those of the [Cont (IV)] group. (C) The relative tumor volume of each mouse in the intratumoral (IT) gene‐administrated group. Black solid lines indicate relative tumor volumes of the [Comb (IT) +DC] group. Black dotted lines indicate those of the [Comb (IT)] group. Gray solid lines indicate those of the [Cont (IT) + DC] group. (D) The relative tumor volume of each mouse in the intravenous (IV) gene‐administrated group. Black solid lines indicate relative tumor volumes of the [Comb (IV) +DC] group. Black dotted lines indicate those of the [Comb (IV)] group. Gray solid lines indicate those of the [Cont (IV) + DC] group. Experiments were performed independently three times using a total of six mice in each group. All mice were humanely euthanized according to the criteria in Section . * P < .05, vs the [Cont (IT)] group. ** P < .05 vs the [Untreated] group, and P < .01, vs the [Cont (IV)] group. *** P < .05 vs the [Untreated] group, and P < .005, vs the [Cont (IT) +DC] group or the [Cont (IV) +DC] group, respectively

Article Snippet: Cells infiltrating tumor tissues were evaluated by immunohistochemistry using following primary Abs: for IFNγ, Bioss Cat# bs‐0480R‐PE‐Cy7, RRID:AB_11092092; for CD40L, Biorbyt Cat# orb10326, RRID:AB_10746923; for CD8, clone 53‐6.7, Thermo Fisher Scientific Cat# 14‐0081‐81, RRID:AB_467086; for Foxp3, clone FJK‐16s, Thermo Fisher Scientific Cat# 14‐5773‐80, RRID:AB_467575; for Iba1, Wako Pure Chemical Industries Inc, Cat# 019‐19741, RRID:AB_839504; for granzyme B, Spring Bioscience Cat# E2580, RRID:AB_1661202.

Techniques: Transfection

Journal: FASEB bioAdvances

Article Title: Manipulation of the tumor microenvironment by cytokine gene transfection enhances dendritic cell‐based immunotherapy

doi: 10.1096/fba.2019-00052

Figure Lengend Snippet: Effect of IFNγ and CD40L (Comb) gene‐transfection therapy on the infiltration of CD8 + cells into LM8 tumors. Tumor tissue was collected from mice with the indicated treatments at 7 d after the second treatment. Immunohistochemistry was performed to detect CD8 + cells present in the tumor tissue. (A) Typical photos of tumors collected from the indicated treatment groups are shown. Expression of CD8 is shown as brown color products. (B) Counts of CD8 + cells in 1000 cells in tumors are shown. Results are expressed as mean ± SE. The experiment was performed using three mice in each treatment group. All mice were humanely euthanized according to the criteria in Section . * P < .05, ** P < .01 by the Tukey‐Kramer test

Article Snippet: Cells infiltrating tumor tissues were evaluated by immunohistochemistry using following primary Abs: for IFNγ, Bioss Cat# bs‐0480R‐PE‐Cy7, RRID:AB_11092092; for CD40L, Biorbyt Cat# orb10326, RRID:AB_10746923; for CD8, clone 53‐6.7, Thermo Fisher Scientific Cat# 14‐0081‐81, RRID:AB_467086; for Foxp3, clone FJK‐16s, Thermo Fisher Scientific Cat# 14‐5773‐80, RRID:AB_467575; for Iba1, Wako Pure Chemical Industries Inc, Cat# 019‐19741, RRID:AB_839504; for granzyme B, Spring Bioscience Cat# E2580, RRID:AB_1661202.

Techniques: Transfection, Immunohistochemistry, Expressing

Journal: FASEB bioAdvances

Article Title: Manipulation of the tumor microenvironment by cytokine gene transfection enhances dendritic cell‐based immunotherapy

doi: 10.1096/fba.2019-00052

Figure Lengend Snippet: Effect of the IFNγ and CD40L (Comb) gene‐transfection therapy on tumor‐infiltrating cells. Tumor tissue was collected from mice with the indicated treatments at 7 d after the second treatment. The Iba 1 + activated macrophage and granzyme B + killer cells in the tumor tissue were detected by immunohistochemistry. Results are expressed as mean ± SE. The experiment was performed using three mice in each treatment group. All mice were humanely euthanized according to the criteria in Section . * P < .05, ** P < .01 by the Tukey‐Kramer test

Article Snippet: Cells infiltrating tumor tissues were evaluated by immunohistochemistry using following primary Abs: for IFNγ, Bioss Cat# bs‐0480R‐PE‐Cy7, RRID:AB_11092092; for CD40L, Biorbyt Cat# orb10326, RRID:AB_10746923; for CD8, clone 53‐6.7, Thermo Fisher Scientific Cat# 14‐0081‐81, RRID:AB_467086; for Foxp3, clone FJK‐16s, Thermo Fisher Scientific Cat# 14‐5773‐80, RRID:AB_467575; for Iba1, Wako Pure Chemical Industries Inc, Cat# 019‐19741, RRID:AB_839504; for granzyme B, Spring Bioscience Cat# E2580, RRID:AB_1661202.

Techniques: Transfection, Immunohistochemistry

Journal: FASEB bioAdvances

Article Title: Manipulation of the tumor microenvironment by cytokine gene transfection enhances dendritic cell‐based immunotherapy

doi: 10.1096/fba.2019-00052

Figure Lengend Snippet: Effect of IFNγ and CD40L (Comb) gene‐transfection therapy on tumor‐infiltrating cells. Tumor tissue was collected from mice with the indicated treatments at 7 d after the second treatment. Mature DCs (MHC II high CD83 + cells), MDSCs (CD11b + , Gr1 + cells), NK (NK1.1 + ) cells and PD‐L1 + cells in the tumor tissues were detected in FCM. Results are expressed as mean ± SE. The experiment was performed using three mice in each treatment group. All mice were humanely euthanized according to the criteria in Section . * P < .05, ** P < .01 by the Tukey‐Kramer test

Article Snippet: Cells infiltrating tumor tissues were evaluated by immunohistochemistry using following primary Abs: for IFNγ, Bioss Cat# bs‐0480R‐PE‐Cy7, RRID:AB_11092092; for CD40L, Biorbyt Cat# orb10326, RRID:AB_10746923; for CD8, clone 53‐6.7, Thermo Fisher Scientific Cat# 14‐0081‐81, RRID:AB_467086; for Foxp3, clone FJK‐16s, Thermo Fisher Scientific Cat# 14‐5773‐80, RRID:AB_467575; for Iba1, Wako Pure Chemical Industries Inc, Cat# 019‐19741, RRID:AB_839504; for granzyme B, Spring Bioscience Cat# E2580, RRID:AB_1661202.

Techniques: Transfection

Journal: FASEB bioAdvances

Article Title: Manipulation of the tumor microenvironment by cytokine gene transfection enhances dendritic cell‐based immunotherapy

doi: 10.1096/fba.2019-00052

Figure Lengend Snippet: Effect of IFNγ and CD40L (Comb) gene‐transfection therapy on systemic immune responses. Spleen cells were collected from the LM8 tumor‐bearing mice with the indicated treatments at 7 d after the second treatment. Cytotoxic activity of the cells against LM8 (A), BW5147 (B), and YAC‐1 (C) was evaluated in vitro. The experiments were repeated twice. Each experiment was performed using three mice in each treatment group. A typical result is shown. All mice were humanely euthanized according to the criteria in Section . Results are expressed as mean ± SE. * P < .05, ** P < .01 vs the [Untreated] group, † P < .05 vs the [Comb (IT) + DC] and [Comb (IV)] group at the E/T ratio

Article Snippet: Cells infiltrating tumor tissues were evaluated by immunohistochemistry using following primary Abs: for IFNγ, Bioss Cat# bs‐0480R‐PE‐Cy7, RRID:AB_11092092; for CD40L, Biorbyt Cat# orb10326, RRID:AB_10746923; for CD8, clone 53‐6.7, Thermo Fisher Scientific Cat# 14‐0081‐81, RRID:AB_467086; for Foxp3, clone FJK‐16s, Thermo Fisher Scientific Cat# 14‐5773‐80, RRID:AB_467575; for Iba1, Wako Pure Chemical Industries Inc, Cat# 019‐19741, RRID:AB_839504; for granzyme B, Spring Bioscience Cat# E2580, RRID:AB_1661202.

Techniques: Transfection, Activity Assay, In Vitro

Journal: FASEB bioAdvances

Article Title: Manipulation of the tumor microenvironment by cytokine gene transfection enhances dendritic cell‐based immunotherapy

doi: 10.1096/fba.2019-00052

Figure Lengend Snippet: Effect of IFNγ and CD40L (Comb) gene‐transfection therapy on spleen cells. Spleen was collected from mice with the indicated treatments at 7 days after the second treatment. Mature DCs (MHC II high CD83 + cells), MDSCs (CD11b + , Gr1 + cells), NK (NK1.1 + ) cells and PDL‐1 + cells in the tumor tissues were detected in FCM. Results are expressed as mean ± SE. The experiment was performed using three mice in each treatment group. All mice were humanely euthanized according to the criteria in Section . * P < .05, ** P < .01 by the Tukey‐Kramer test

Article Snippet: Cells infiltrating tumor tissues were evaluated by immunohistochemistry using following primary Abs: for IFNγ, Bioss Cat# bs‐0480R‐PE‐Cy7, RRID:AB_11092092; for CD40L, Biorbyt Cat# orb10326, RRID:AB_10746923; for CD8, clone 53‐6.7, Thermo Fisher Scientific Cat# 14‐0081‐81, RRID:AB_467086; for Foxp3, clone FJK‐16s, Thermo Fisher Scientific Cat# 14‐5773‐80, RRID:AB_467575; for Iba1, Wako Pure Chemical Industries Inc, Cat# 019‐19741, RRID:AB_839504; for granzyme B, Spring Bioscience Cat# E2580, RRID:AB_1661202.

Techniques: Transfection

Journal: bioRxiv

Article Title: Porcine Nasal Organoids as a model to study the interactions between the swine nasal microbiota and the host

doi: 10.1101/2024.08.13.606910

Figure Lengend Snippet: Differentiated PNOs were incubated with 10 CFU/ml of R. nasimurium (Rn), 10 CFU/ml of Moraxella pluranimalium (Mp), 10 CFU/ml of non-virulent Glaesserella parasuis (nvG) or with 10 CFU/ml of virulent G. parasuis (vG) individually or in combinations. After overnight incubation supernatants were recovered for cytokine quantification. Results are shown as the delta of each interleukin and cytokine, subtracting the negative control (PNOs without inoculation, basal levels of the secretion) in pg/ml. A) Secretion of interleukin 8 (IL-8), B) Secretion of interleukin 12 (IL-12), C) Secretion of Tumor Necrosis Factor alpha (TNFα) and D) Secretion of Interferon gamma (IFNγ). Each dot represents a replicate from three different experiments of PNOs coming from two animals. Two replicates were included in each experiment. Kruskal-Wallis multiple comparison with Benjamini, Krieger and Yekutieli post-hoc test was used to analyze the data.

Article Snippet: Interferon gamma (IFNγ) secretion was also assessed with an ELISA following manufacturer’s protocol (Swine IFN gamma Polyclonal Antibody, PB0157S-100; Swine IFN gamma Polyclonal Antibody Biotinylated, PBB0269S-050; Swine IFN gamma [Yeast-derived Recombinant Protein], RP0126S-005; Kingfisher Biotech, Inc).

Techniques: Incubation, Negative Control, Comparison

Journal: Virology Journal

Article Title: Pre-immune state induced by chicken interferon gamma inhibits the replication of H1N1 human and H9N2 avian influenza viruses in chicken embryo fibroblasts

doi: 10.1186/s12985-016-0527-1

Figure Lengend Snippet: Expression of chIFN-γ in CEFs and the cell culture medium. a CEFs were transfected with pcDNA-IFN-γ. Then, the cell culture medium was harvested at 6, 12, 24, and 48 h post transfection. The medium was centrifuged and concentrated using spin columns. Western blotting was performed as described in Methods. b At 24 h post transfection, the localization of chIFN-γ in CEFs was demonstrated using anti-chIFN-γ polyclonal rabbit antibody as the primary antibody and anti-rabbit IgG Alexa Fluor 488 goat antibody as the secondary antibody (green). Cell nuclei were stained with 4,6-diamidino-2-phenylindole (blue)

Article Snippet: The concentrated culture medium samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by western blotting using an anti-chIFN-γ rabbit polyclonal antibody (Kingfisher Biotech, St Paul, MN, USA) and peroxidase-conjugated anti-rabbit goat monoclonal secondary antibody (Sigma).

Techniques: Expressing, Cell Culture, Transfection, Western Blot, Staining

Journal: Virology Journal

Article Title: Pre-immune state induced by chicken interferon gamma inhibits the replication of H1N1 human and H9N2 avian influenza viruses in chicken embryo fibroblasts

doi: 10.1186/s12985-016-0527-1

Figure Lengend Snippet: Reduced viral titer of H1N1 and H9N2 influenza viruses in CEF expressing chIFN-γ. Twenty-four hours after transfection with pcDNA-IFN-γ, CEFs were infected with H1N1 and H9N2 virus. Forty-eight hours post infection, MDCK cells were treated with a H1N1- and b H9N2-infected CEF culture supernatants. TCID 50 titer was measured by end-point dilution assay. Statistical significance was determined by one-way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.001)

Article Snippet: The concentrated culture medium samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by western blotting using an anti-chIFN-γ rabbit polyclonal antibody (Kingfisher Biotech, St Paul, MN, USA) and peroxidase-conjugated anti-rabbit goat monoclonal secondary antibody (Sigma).

Techniques: Expressing, Transfection, Infection, Virus, End-point Dilution Assay

Journal: Virology Journal

Article Title: Pre-immune state induced by chicken interferon gamma inhibits the replication of H1N1 human and H9N2 avian influenza viruses in chicken embryo fibroblasts

doi: 10.1186/s12985-016-0527-1

Figure Lengend Snippet: Viral gene replication in CEF expressing chIFN-γ and suppressed by siRNA targeting chIFN-γ. CEFs were harvested and analyzed by real-time RT-PCR after H1N1 and H9N2 virus infection. a The mRNAs of viral glycoprotein (HA) and viral polymerase (PB2) were significantly reduced in pcDNA-chIFN-γ-transfected CEFs compared to the control. b HA and PB2 mRNA was significantly increased in siRNA targeting chIFN-γ transfected CEFs compared to the control. Statistical significance was determined by unpaired student t -test (* P < 0.05, ** P < 0.01, and *** P < 0.001)

Article Snippet: The concentrated culture medium samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by western blotting using an anti-chIFN-γ rabbit polyclonal antibody (Kingfisher Biotech, St Paul, MN, USA) and peroxidase-conjugated anti-rabbit goat monoclonal secondary antibody (Sigma).

Techniques: Expressing, Quantitative RT-PCR, Virus, Infection, Transfection, Control

Journal: Virology Journal

Article Title: Pre-immune state induced by chicken interferon gamma inhibits the replication of H1N1 human and H9N2 avian influenza viruses in chicken embryo fibroblasts

doi: 10.1186/s12985-016-0527-1

Figure Lengend Snippet: Relative expression of type I interferons and interferon-stimulated genes following H1N1 infection in CEFs. Twenty-four hours after transfection with pcDNA-IFN-γ, CEFs were infected with 0.01 MOI of H1N1 virus. At 0, 6, 12, 24, and 48 hpi, the total RNA content of CEFs was extracted and analyzed by real-time RT-PCR, as described in Methods. The expression levels of chIFN-α ( a ), chIFN-β ( b ), chMx ( c ), chPKR ( d ), and chOAS ( e ), and chRNaseL ( f ) were assessed. Statistical significance was determined by two-way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.001)

Article Snippet: The concentrated culture medium samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by western blotting using an anti-chIFN-γ rabbit polyclonal antibody (Kingfisher Biotech, St Paul, MN, USA) and peroxidase-conjugated anti-rabbit goat monoclonal secondary antibody (Sigma).

Techniques: Expressing, Infection, Transfection, Virus, Quantitative RT-PCR

Journal: Virology Journal

Article Title: Pre-immune state induced by chicken interferon gamma inhibits the replication of H1N1 human and H9N2 avian influenza viruses in chicken embryo fibroblasts

doi: 10.1186/s12985-016-0527-1

Figure Lengend Snippet: Relative expression of type I interferons and interferon-stimulated genes following H9N2 infection in CEFs. Twenty-four hours after transfection with pcDNA-IFN-γ, CEFs were infected with 0.01 MOI of H9N2 virus. At 0, 6, 12, 24, and 48 hpi, the total RNA content of CEFs was extracted and analyzed by real-time RT-PCR, as described in Methods. The expression levels of chIFN-α ( a ), chIFN-β ( b ), chMx ( c ), chPKR ( d ), and chOAS ( e ), and chRNaseL ( f ) were assessed. Statistical significance was determined by two-way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.001)

Article Snippet: The concentrated culture medium samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by western blotting using an anti-chIFN-γ rabbit polyclonal antibody (Kingfisher Biotech, St Paul, MN, USA) and peroxidase-conjugated anti-rabbit goat monoclonal secondary antibody (Sigma).

Techniques: Expressing, Infection, Transfection, Virus, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Effects of Chicken Interferon Gamma on Newcastle Disease Virus Vaccine Immunogenicity

doi: 10.1371/journal.pone.0159153

Figure Lengend Snippet: Plasmids expressing NDV F and chIFNγ genes were developed and characterized for their use as DNA vaccines and adjuvant, respectively. DF-1 cells were transfected with pTriEX, pTriEX-ZJ1-F and pTriEX- IFNγ. Cell culture supernatants were tested by western blotting for the presence of F protein (A) and chIFNγ (B) , respectively. Eighteen-day-old SPF ECEs were inoculated with TE buffer, pTriEX, pTriEX-ZJ1-F, or pTriEX-ZJ1-F plus pTriEX- IFNγ and boosted 2 weeks after hatched. Two weeks after booster vaccination, birds were challenged with vZJ1. Oropharyngeal (C) and cloacal (D) swab samples were collected 3 days after challenge to measure the amount of challenge virus shed into the environment. Viral titers were determined by quantitative real time reverse transcription polymerase chain reaction (qRRT-PCR). A standard was prepared with a vZJ1 virus stock of know concentration, this was included in every plate and was used to obtain viral titers expressed as EID 50 /mL. Morbidity (E) and mortality (F) were also evaluated. Viral shedding results were analyzed with One-way ANOVA followed by a multiple comparisons Tukey's test. Differences in morbidity among groups were evaluated using a two-tailed Z test for comparison of sample proportions. Survival curves were analyzed using the Long-Rank test. Statistical difference was considered with a P<0 . 05 . Significant differences are denoted by different letters.

Article Snippet: Cell lysates and supernatants were analyzed through western blotting using 8–16% polyacrylamide gels and an anti-chIFNγ polyclonal antibody (Cat.# PB0442C-100, KingFisher Biotech, Inc., Saint Paul, MN).

Techniques: Expressing, Vaccines, Adjuvant, Transfection, Cell Culture, Western Blot, Virus, Reverse Transcription, Polymerase Chain Reaction, Concentration Assay, Two Tailed Test, Comparison

Journal: PLoS ONE

Article Title: Effects of Chicken Interferon Gamma on Newcastle Disease Virus Vaccine Immunogenicity

doi: 10.1371/journal.pone.0159153

Figure Lengend Snippet: DF-1 cells and 10-day-old ECEs (B) were infected with live rZJ1*L and rZJ1*L/IFNγ to confirm in vitro and in vivo expression of chIFNγ. Cell culture supernatants were collected 24 hrs after infection and tested for chIFNγ by western blotting (A) . Infected allantoic fluids (AFs) were collected 24, 48, 72, and 96 hrs after infection; chIFNγ concentrations from infected AFs were determined by ELISA (B). Nineteen-day-old SPF ECEs were inoculated with BHI, LS, rZJ1*L or rZJ1*L/IFNγ and challenged two weeks after hatch with vZJ1. Sera were collected before and after the challenge for HI antibody titer determination (C) . Survival after challenge was recorded (D) . Four-week-old SPF chickens were also vaccinated with BHI, LS, rZJ1*L or rZJ1*L/IFNγ and challenged two weeks later with vZJ1 to record mortality (E) . HI antibody titers were analyzed with One-way ANOVA followed by a multiple comparisons Tukey's test. Survival curves were analyzed using the Long-Rank test. Statistical difference was considered with a P<0 . 05 . Significant differences are denoted by different letters.

Article Snippet: Cell lysates and supernatants were analyzed through western blotting using 8–16% polyacrylamide gels and an anti-chIFNγ polyclonal antibody (Cat.# PB0442C-100, KingFisher Biotech, Inc., Saint Paul, MN).

Techniques: Infection, In Vitro, In Vivo, Expressing, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Effects of Chicken Interferon Gamma on Newcastle Disease Virus Vaccine Immunogenicity

doi: 10.1371/journal.pone.0159153

Figure Lengend Snippet: Effect of chIFNγ on hatchability and survival after in ovo vaccination with rZJ1*L/IFNγ and challenge with vZJ1.

Article Snippet: Cell lysates and supernatants were analyzed through western blotting using 8–16% polyacrylamide gels and an anti-chIFNγ polyclonal antibody (Cat.# PB0442C-100, KingFisher Biotech, Inc., Saint Paul, MN).

Techniques: In Ovo

Journal: PLoS ONE

Article Title: Effects of Chicken Interferon Gamma on Newcastle Disease Virus Vaccine Immunogenicity

doi: 10.1371/journal.pone.0159153

Figure Lengend Snippet: Uninfected allantoic fluid (AF), rZJ1*L- and rZJ1*L/IFNγ-infected AFs were inactivated with BPL for inactivated vaccine preparation and tested for chIFNγ concentration by ELISA (A) . BPL-treated AFs were used to stimulate HD11 cells and confirm chIFNγ bio-activity through determination of nitrites as a sub-product of nitric oxide induced upon macrophage activation. Stimulated cells were incubated for 48 hrs at 37°C under a 5% CO 2 atmosphere. Cell culture was used to determine nitrite concentration using the Griess method (B) . Two-week-old SPF birds were vaccinated and boosted with inactivated Sham-vaccine, rZJ1*L or rZJ1*L/IFNγ. Two different vaccine doses were tested and serum samples were collected before and after challenge for antibody titer determination by HI test. Pre-challenge HI titers after vaccination with an EID 50 /mL of 10 8.1 (C) and 10 9.1 (D) , and post-challenge titers (E) are shown. Mortality was recorded daily for 2 weeks (F) . Oropharyngeal (G) and cloacal (H) swab samples were collected 2 and 4 dpc, viral titers were determined in 10-day-old ECEs and are expressed as EID 50 /mL. One week after booster vaccination, 6 birds from each vaccinated group were euthanized and the spleens were collected for the isolation of lymphocytes to be used in a proliferation assay to measure antigen-specific memory T cell response (I) and to determine T and B cell subpopulation by flow cytometric analysis (J) . Hemagglutination inhibition (HI) antibody titers, viral shedding and cell proliferation assay results were analyzed with One-way ANOVA followed by a multiple comparisons Tukey's test. Survival curves were analyzed using the Long-Rank test. Statistical difference was considered with a P<0 . 05 . Significant differences are denoted by different letters.

Article Snippet: Cell lysates and supernatants were analyzed through western blotting using 8–16% polyacrylamide gels and an anti-chIFNγ polyclonal antibody (Cat.# PB0442C-100, KingFisher Biotech, Inc., Saint Paul, MN).

Techniques: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Activation Assay, Incubation, Cell Culture, Isolation, Proliferation Assay, HI Assay

Journal: Scientific Reports

Article Title: CD11b deficiency suppresses intestinal tumor growth by reducing myeloid cell recruitment

doi: 10.1038/srep15948

Figure Lengend Snippet: The accumulation of Gr-1 + CD11b + MDSC cells in the tumor microenvironment of the Apc Min/ + mice compared with the normal intestine tissues from the C57 mice; the Gr-1 + cells were decreased in the tumor microenvironment of the Apc Min/ + ;CD11b −/− mice ( a ). The number of the Ly6G + Ly6C low subset of MDSCs (gated on CD45 + cells) in the bone marrow of C57, CD11b −/− , Apc Min/ + and Apc Min/ + ;CD11b −/− mice ( b ) and in the peripheral blood, spleen and tumor tissues of the Apc Min/ + and Apc Min/ + ;CD11b −/− mice ( c ) was measured by FACS. The results of the IF staining and FACS represent of 11 independent mice (all mice were 16–weeks-old). The proteins expression of IFN-γ and CXCL9 in the tumor tissues from the Apc Min/ + and Apc Min/ + ;CD11b −/− mice was examined by immunoblotting ( d ). The protein bands intensities were quantitated and normalized to those of GAPDH. The statistical data are expressed as the means ± S.D. * P <0.05, ** P < 0.01 and *** P < 0.001. Scale bars: 50 μm ( a ).

Article Snippet: The primary antibodies were used for immunohistochemical (IHC), immunofluorescence (IF) analysis and Western blotting (WB) assays: and mouse anti-BrdU antibody (RPN202, diluted at 1:150 for IHC) was obtained from GE Healthcare (Little Chalfond, UK); rabbit anti-CyclinD1 (BM0771, diluted at 1:100 for IHC and 1:400 for WB), rabbit anti-CD34 (BA0532, diluted at 1:100 for IHC), rabbit anti-CD45 (BA3371, diluted at 1:100 for IHC) and rabbit anti-p65 (BA0610, diluted at 1:100 for IHC) were obtained from Boster (Wuhan, China); mouse anti-TNF-α (ab10863, diluted at 1:5000 for WB) was obtained from Abcam (Cambridge, UK); rabbit anti-IFN-γ (bs-0480R), rabbit anti-CXCL-9 (bs-2551R) (both diluted at 1:500 for WB), rabbit anti-CD11b (bs-1014R) and rabbit anti-Gr-1 (bs-2576R, diluted at 1:100 for IF) were obtained from Bioss (Beijing, China); mouse anti-Cytokeratin 8 (CK8, ZM-0310, diluted at 1:100 for IF) was purchased from ZSGB-BIO (Beijing, China); mouse anti-CD11b (Santa Cruz, USA, diluted at 1:100 for IF); mouse anti-E-cadherin (#610181, diluted at 1:100 for IHC and 1:5000 for WB) and mouse anti-β-catenin (#610154, diluted at 1:100 for IHC, and 1:2000 for WB) were purchased from BD Transduction Laboratories (Franklin Lakes, NJ); rabbit anti-GAPDH (#2118s, diluted at 1:2000 for WB) was purchased from CST (USA); rabbit anti-pp65 (Ser276, sc-101749, diluted at 1:500 for IHC) was obtained from Santa Cruz Biotechnology Inc. (USA).

Techniques: Staining, Expressing, Western Blot