Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Expression Plasmids Used in This Study

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Expressing, Plasmid Preparation, CRISPR, Sequencing, Construct, Luciferase

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Species specificity of mouse and human type III IFNs. (A) Table showing the relative antiviral activity of mouse and human IFN-λ on mouse LKR10 and human A549 cells. Species specificity index was calculated as the ratio between the relative antiviral activity on cells of homologous to nonhomologous species (eg, for mouse IFN: relative activity in mouse cells/relative activity in human cells). Relative activities in a given cell line were calculated as the IFN dilutions (starting from 1 ng/mL) that yielded similar antiviral activities. *Due to the low amount of IFN-λ4 available, the initial concentration of this IFN was estimated by comparison with human IFN-λ3 antiviral activity that was reported to have a similar specific activity. (B) Western blot showing STAT1 phosphorylation in mouse LKR10 cells 30 min after treatment with control medium (mock) or IFN-λ. Concentrations of human (Hu) IFN-λ3 (615 pg/mL) and IFN-λ4 that yielded the same antiviral activity on human A549 cells were used. As a control, mouse IFN-λ3 was used at a concentration (2.5 ng/mL) showing equivalent antiviral activity as human IFN-λ4 on mouse cells. Results are representative of 3 experiments.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Activity Assay, Concentration Assay, Comparison, Western Blot, Control

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: IFN-stimulated gene expression, STAT1 phosphorylation, and antiviral activity in response to IFN-αA and IFN-λ3 treatment in LKR10 derived cells. (A, B) Amounts of Oasl2 (A) and Usp18 (B) transcripts per 10 6 β-actin copies detected in WT and IFNAR2-KO LKR10 cells 24 h after treatment with mouse IFN-αA, IFN-λ3 (mIFN-λ3), or control medium (mock). (C) Percentage of mCherry-positive cells in WT and IFNAR2-KO LKR10 cells measured by flow cytometry 24 h postinfection with 0.5 PFU/cell TM967. Cells were pretreated with mouse IFN-αA, mIFN-λ3, or mock for 7 h before infection. (D) Western blot showing STAT1 phosphorylation and expression in LKR10 cells and derivatives. Cells were treated for 30 min before protein extraction. Reproducible results were obtained in 3 independent experiments. (A–D) 100 U/mL IFN-αA and 700 pg/mL mIFN-λ3 were used for treatment. Student's t -test: ***indicates a significant difference of the IFN-treated groups compared to the mock-treated group (A–C) . ns, nonsignificant. WT, wild-type.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Expressing, Activity Assay, Derivative Assay, Control, Flow Cytometry, Infection, Western Blot, Protein Extraction

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Mouse IFN-λ titration using Fawa-λ-luc reporter cells and ELISA. (A ) Luciferase activity detected in Fawa-λ-luc cells treated for the indicated time with 700 pg/mL mouse IFN-λ3 (mIFN-λ3). (B) Quantification by ELISA of mouse IFN-λ2 (mIFN-λ2) and mIFN-λ3 in cell supernatants. Two fold serial dilutions (800–12,800-fold) were quantified in quintuplicate. (C, D) Dose–response of mIFN-λ2 and mIFN-λ3 supernatants was measured in triplicate Fawa-λ-luc cells and is representative of at least 3 independent experiments. Cells were treated for 6 h with 2-fold serial dilutions. Data points in the linear range of the assays were plotted , and linear regression analysis was performed. LOD is based on the mean of mock treated cell signal, plus 3 standard deviations. ELISA, enzyme-linked immunosorbent assay; LOD, limit of detection; RLU, relative light units; R 2 , coefficient of regression.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Titration, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Human IFN-λ response in the mouse Fawa-λ-luc reporter cells. (A–D) Dose–response of human (hu) recombinant (A) IFN-λ1, (B) IFN-λ2, (C) IFN-λ3, and (D) IFN-λ4 as measured in triplicate. (A–D) Data points in the linear range of the assays were plotted , and linear regression analysis was performed. LOD is based on the mean of mock treated cell signal, plus 3 standard deviations.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Recombinant

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Sensitivity of IFN-λ to UV exposure and influence of serum on reporter cell activity and ELISA detection. (A, B) UV sensitivity of mouse and human IFN-λ. (A) Relative luciferase activities of Fawa-λ-luc cells treated with 250 pg/mL of mouse IFN-λ2 (mIFN-λ2) or IFN-λ3 (mIFN-λ3) supernatants irradiated ( n = 3) under 0.25, 0.5, 1 and 2 J/cm 2 . (B) Percentage of IFN-λ activity (mean and SD) remaining after UV treatment ( n = 3) at 2 J/cm 2 . IFN activity was measured in Fawa-λ-luc cells for IFN concentrations that yielded equivalent luciferase activities (20,000 RLU) before UV treatment (250 pg/mL mIFN-λ2 and mIFN-λ3, 125 pg/mL human IFN-λ1, 10 ng/mL huIFN-λ2, 500 pg/mL huIFN-λ3, and 62.5 pg/mL huIFN-λ4). (C, D) Influence of serum dilution on IFN-λ detection by Fawa-λ-luc cells (C) and ELISA (D) . (C) Fawa-λ-luc cells were treated in triplicate with fixed doses of 15 and 250 pg/mL mIFN-λ2 supernatant and with 2-fold serial dilutions (2–128-fold) of control mouse serum. (D) 125 pg/mL recombinant mIFN-λ3 was mixed with 2-fold serial dilutions of control mouse serum (2.5–10-fold) before detection by ELISA. (A–C) Reporter cells were exposed to IFN for 6 h before luciferase assay. Student's t -test: */**/***denotes a significant difference in signal compared to no UV exposure (A) or the absence of serum (C) . UV, ultraviolet light.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Irradiation, Control, Recombinant

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Detection of type III IFN in biological samples by ELISA and bioassay. (A, B) IFN-λ2/3 detection by ELISA (A) and bioassay (B) in the serum of AG129 mice 2 or 4 days after electroinjection of mouse IFN-λ3 (mIFN-λ3) expressing (pCS59) or empty (pcDNA3) plasmids, 2 days after injection of pCS59, or 3 days after infection with mouse norovirus. IFN-λ detection in the serum by ELISA was performed without UV exposure to keep maximal sensitivity. (C, D) IFN-λ2/3 detection by ELISA (C) and bioassay (D) in the bronchoalveolar lavage of BALB/C mice, 5 days postinfection with RSV, compared to control mice (mock). BALF were UV-exposed before testing. (A–D) The horizontal dotted line represents the LOD. Mann–Whitney: */**indicates a significant difference compared to pcDNA3 group at days 2 or 4 (A, B) or compared to mock (D) . BALF, bronchoalveolar lavage fluid; RSV, respiratory syncytial virus.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Enzyme-linked Immunosorbent Assay, Bioassay, Expressing, Injection, Infection, Control, MANN-WHITNEY, Virus

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: IFN-λ sequence alignments. Sequences of human and mouse IFN-λ regions implicated in receptor binding were aligned. Key amino acid residues involved in receptor activation that differs between mouse and human IFN-λ2 and IFN-λ3 sequences are indicated in bold in mouse sequences. Residues unique to mouse IFN-λ3 in helices A and F are indicated in bold red . *Indicates identical amino acids between human IFN-λ3 and IFN-λ4. Color images available online at www.liebertpub.com/jir

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Sequencing, Binding Assay, Activation Assay