Journal: Journal of Leukocyte Biology

Article Title: The macrocyclic peptide rhesus theta defensin 1 activates interferon and antiviral pathways in human monocytes

doi: 10.1093/jleuko/qiaf150

Figure Lengend Snippet: Stimulation of ISRE reporter by RTD-1. The activity of Lucia luciferase secreted in the medium was assayed and the luminescence units are plotted. (A) THP-1 Dual cells were stimulated with HOAc (vehicle) or increasing concentrations of RTD-1. Results are from 3 experiments. * P < 0.05, 2-tailed t test, comparison with HOAc-treated sample. Error bars indicate standard deviation. (B) THP-1 Dual cells were pretreated with DMSO or Ruxo as indicated. Assay done in triplicate. * P < 0.05 when compared with HOAc-treated control, 2-tailed t test. # P < 0.05 when compared with RTD-1 treatment. Error bars indicate standard deviation. (C) THP-1 Dual cells were transferred to RPMI + 1% HI FBS + P/S + normocin medium and either 2.5 μg human IgG1 or anti-IFNAR1 antibodies were added to the culture. Cells were incubated for 2 h, and RTD-1 or IFN-β was added as indicated and the incubation continued for 18 h. Assay was done in triplicate. * P < 0.05 when compared with HOAc-treated control, 2-tailed t test. Error bars indicate standard deviation. (D) THP-1 Dual cells were treated with RTD-1, IFN-β, or both as shown. Assay was done in triplicate. * P < 0.05, 2-tailed t test. Error bars indicate standard deviation.

Article Snippet: IFN-β was purchased from R&D Systems and human IgG1 and human anti IFNAR1 antibodies were purchased from Selleck USA.

Techniques: Activity Assay, Luciferase, Comparison, Standard Deviation, Control, Incubation

Journal: Journal of Leukocyte Biology

Article Title: The macrocyclic peptide rhesus theta defensin 1 activates interferon and antiviral pathways in human monocytes

doi: 10.1093/jleuko/qiaf150

Figure Lengend Snippet: ISRE stimulation activity is distinct from RTD-1–mediated inhibition of NF-κB activation. THP-1 Dual cells were treated with LPS, RTD-1, or IFN-β or vehicle as shown and the medium was assayed for ISRE reporter activity (top) and NF-κB reporter activity (bottom) using Quanti-Luc and Quanti-Blue assays, respectively. Assay done in triplicate. # P > 0.05 (nonsignificant) when compared with respect to LPS treated samples, 2-tailed t test. Error bars indicate standard deviation.

Article Snippet: IFN-β was purchased from R&D Systems and human IgG1 and human anti IFNAR1 antibodies were purchased from Selleck USA.

Techniques: Activity Assay, Inhibition, Activation Assay, Standard Deviation

Journal: Scientific Reports

Article Title: An orally available, small-molecule interferon inhibits viral replication

doi: 10.1038/srep00259

Figure Lengend Snippet: (a, b) The anti-HCV replicon activity of RO8191 was attenuated by knockdown of IFNAR2 (b), but not IFNAR1 (a). Inhibition of HCV replicon replication by each agent is shown (the mean and SD from 3 experiments). The HCV replicon cells were transfected with 50 nM of the indicated siRNAs (blue, red, and yellow bars). Forty-eight hours after transfection, the HCV replicon cells were treated with 1.5 μM RO8191 or 3 IU/mL IFN-α for 24 h. Twenty-four hours after treatment with each agent, the replication levels of HCV RNA were analyzed using a luciferase assay. (c, d) U5A cells that lack IFNAR2 were transfected with either an empty vector or a vector expressing the IFNAR2 gene. (c) Forty-eight hours after transfection, the cells were treated with 50 μM RO8191 (red bars) or 100 IU/mL IFN-α (yellow bars). After an additional 8 h of incubation, total RNA was extracted from the U5A cells, and the OAS1 mRNA level was measured using real-time RT-PCR. The values shown are relative to the mRNA level of human β-actin . (d) Forty-eight hours after transfection, the cells were lysed, and the whole cell lysates were immunoblotted with the indicated antibodies. (e) Real-time kinetic SPR analysis of the binding of RO8191 to the IFNAR2 ECD (red and blue lines). The results are consistent with 1:1 binding. PEG-IFN-α2a was also injected as a positive interacting control for IFNAR2 (black line, K D : 30 nM). (f, g) The phosphorylation of STAT1 was attenuated by a knockdown of IFNAR2 (g) but not IFNAR1 (f). The HCV replicon cells were transfected with the indicated siRNAs (10 nM). Forty-eight hours after transfection, the cells were treated for 15 min with 10 μM RO8191 or 200 IU/mL IFN-α. The total lysates were subjected to western blot analysis to analyze the phosphorylated and total protein levels of STAT1. The data were statistically analyzed using Student's t -test.

Article Snippet: Anti-IFNAR1 mouse monoclonal (MAB245) and anti-IFNAR2 sheep polyclonal antibodies were purchased from R&D Systems.

Techniques: Activity Assay, Knockdown, Inhibition, Transfection, Luciferase, Plasmid Preparation, Expressing, Incubation, Quantitative RT-PCR, Binding Assay, Injection, Control, Phospho-proteomics, Western Blot

Journal: PLoS Pathogens

Article Title: Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

doi: 10.1371/journal.ppat.1002016

Figure Lengend Snippet: TetR and TetR-IE1 cells were treated with doxycycline for 72 h and with solvent (w/o), IFN-β or IFN-γ for 24 h. Doxycycline and IFN treatment was performed in the continuous presence of normal goat immunoglobulin G (IgG), goat anti-IFN-β or goat anti-IFN-γ antibodies. Relative mRNA expression levels were determined by qRT-PCR with primers specific for the CXCL10, CXCL11, GBP4, and IE1 genes. Results were normalized to TUBB and mean values with standard deviations from two biological and two technical replicates are shown. Expression is shown in comparison to normal IgG-treated cells (set to 1).

Article Snippet: Neutralizing goat antibodies to human IFN-β (no. AF814) or IFN-γ (no. AF-285-NA) and normal goat IgG (no. AB-108-C) were purchased from R&D Systems and used at concentrations of 1 μg/ml (anti-IFN-β) or 2 μg/ml (anti-IFN-γ, normal IgG).

Techniques: Solvent, Expressing, Quantitative RT-PCR, Comparison

Journal: Journal of clinical immunology

Article Title: Three copies of four interferon receptor genes underlie a mild type I interferonopathy in Down syndrome

doi: 10.1007/s10875-020-00803-9

Figure Lengend Snippet: (A). Schematic diagram of the structure of HSA21, with the IFN-R locus including IFNAR2, IL-10RB, IFNAR1 and IFNGR2 as well as genes for the corresponding agonists; (B-F). IFN-R expression was analyzed in EBV-B cells from healthy controls (WT, n=14) and DS patients (DS, n=16) with the corresponding specific antibodies, against IFN-γR1 (B), IFN-γR2 (C), IFN-αR1 (D), IFN-αR2 (E) and IL-10RB (F). ΔMFI was calculated by subtracting the MFI for the isotype control from that for the specific anti-IFN-R antibody. Significance was assessed by calculating p values in unpaired t tests. ■p>0.05; * p≤ 0.05; * p≤0.005; *** p≤ 0.0005; **** p≤ 0.0001.

Article Snippet: We then added 125 ng/ml anti-IFN-γR2-APC (FAB773A, R&D Systems), anti-IFN-γR1-PE (558934, BD), anti-IFN-αR1 (AA-3, from Dr. Sandra Pellegreni), anti-IFN-αR2-PE (21385-3, PBL) or anti-IL-10RB-FITC (FAB245F, R&D systems) antibody and incubated the cells for 30 minutes on ice before washing them twice with 1% FBS in PBS.

Techniques: Expressing, Control

Journal: Journal of clinical immunology

Article Title: Three copies of four interferon receptor genes underlie a mild type I interferonopathy in Down syndrome

doi: 10.1007/s10875-020-00803-9

Figure Lengend Snippet: (A). Representative histogram of the levels of IFN-γR1, IFN-γR2, IFN-αR2 and IL-10RB expressed on monocytes from three healthy controls (WT) and three DS patients (DS) in one representative experiment; (B). The relative levels of IFN-Rs on monocytes were compared between healthy controls (n=17) and DS patients (n=15). The p values were obtained in unpaired t tests. ■ p>0.05; **** p≤ 0.0001.

Article Snippet: We then added 125 ng/ml anti-IFN-γR2-APC (FAB773A, R&D Systems), anti-IFN-γR1-PE (558934, BD), anti-IFN-αR1 (AA-3, from Dr. Sandra Pellegreni), anti-IFN-αR2-PE (21385-3, PBL) or anti-IL-10RB-FITC (FAB245F, R&D systems) antibody and incubated the cells for 30 minutes on ice before washing them twice with 1% FBS in PBS.

Techniques: