anti pig ifn α R&D Systems Search Results


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  • 95
    Millipore human ifn α
    Blockage of KLHL20-mediated DAPK degradation contributes to MM cell responsiveness to <t>IFN.</t> ( A , B ) <t>IFN-α</t> induces PML-NBs and KLHL20 relocation in H929 cells but not in XG1 cells. Cells treated with IFN-α (1000 U/ml) for 16 h were fixed,
    Human Ifn α, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ifn α
    Expression of <t>IFN-α4,</t> IFN-β, CXCL10, and IRF-7 in MEF cells. Expression of mouse <t>IFN-α</t> and IFN-β (A), the early IFN response gene CXCL10 (B), and the transcription factor IRF7 that functions as amplifier of the type I IFN
    Ifn α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1084 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Thermo Fisher ifn α levels
    Secretion of cytokines by PBMC following culture in the presence of TLR7L, TLR8L, or TLR7/8L. PBMC or sorted cell populations were incubated with either TLR7L, TLR8L, TLR7/8L, or a control compound for 18 h, and supernatants were collected for cytokine detection by ELISA as described in Materials and Methods. (A) <t>IFN-α;</t> (B) IL-12; (C) IL-18; (D) IL-15. (E) Neutralizing antibodies against IL-12, IL-15, or IFN-α do not completely inhibit the activation of porcine NK cells in PBMC stimulated with TLR7/8L. PBMC were cultured in the presence of TLR7/8L with the addition of antibodies against IL-12, IL-15, IFN-α, or a mixture of the three antibodies. An NK cytotoxicity assay against K562-GFP cells was performed 18 h later as described in Materials and Methods. Data are means ± standard deviations for three experiments ( n = 3).
    Ifn α Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 76/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ifn α
    <t>IFN-α</t> and -ß induces transcription of ISG in PHFG cells. Naïve PHFG cells or cells infected with JCV, were exposed to 100 U/mL of IFN-α or -ß for 15 consecutive days and at days 3, 5, 8, and 15 p.i., MxA, cig5 and
    Ifn α, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 691 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mab Technologies ifn alpha
    BST2 mRNA induction by type I interferon. a Fold induction of relative BST2 mRNA in PBMC from three uninfected rhesus macaques after stimulation with human Interferon <t>Alpha</t> A (Alpha 2a) for 16 h. Data are expressed as fold increase over baseline after normalization to pre-treatment values. Error bars represent standard deviation, b relative mRNA copies of BST2 in PBMC (shown in copy numbers per 100 copies of GAPDH) are illustrated in relation to plasma <t>IFN-alpha</t> levels from blood samples of 18 uninfected rhesus macaques 24 h after inoculation of replication incompetent adenovirus or fowl pox vectors. The black dashed line indicates the detection limit of the ELISA and c whole blood MX1 mRNA levels correlate with BST2 mRNA determined in 38 SIVmac251 infected rhesus macaques at 24 wpi. Relative mRNA levels are depicted as log-transformed copy numbers per 100 copies of GAPDH. Each data point represents one animal. Regression line is shown; r , Spearman’s correlation coefficient; p , p value
    Ifn Alpha, supplied by Mab Technologies, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    R&D Systems anti porcine ifn α
    BST2 mRNA induction by type I interferon. a Fold induction of relative BST2 mRNA in PBMC from three uninfected rhesus macaques after stimulation with human Interferon <t>Alpha</t> A (Alpha 2a) for 16 h. Data are expressed as fold increase over baseline after normalization to pre-treatment values. Error bars represent standard deviation, b relative mRNA copies of BST2 in PBMC (shown in copy numbers per 100 copies of GAPDH) are illustrated in relation to plasma <t>IFN-alpha</t> levels from blood samples of 18 uninfected rhesus macaques 24 h after inoculation of replication incompetent adenovirus or fowl pox vectors. The black dashed line indicates the detection limit of the ELISA and c whole blood MX1 mRNA levels correlate with BST2 mRNA determined in 38 SIVmac251 infected rhesus macaques at 24 wpi. Relative mRNA levels are depicted as log-transformed copy numbers per 100 copies of GAPDH. Each data point represents one animal. Regression line is shown; r , Spearman’s correlation coefficient; p , p value
    Anti Porcine Ifn α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 84/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Reprokine interferon alpha ifn α
    <t>IFN-α</t> is a potent inducer of HBD1 in monocytes in vitro and correlates with HBD1 transcription in vivo . (A, B) CD14+ monocytes (A n = 11 and B n = 5) were isolated from PBMCs and incubated with different concentrations of recombinant IFN-α2 or left untreated. Where indicated cells were treated with either an anti-IFN-α or an isotype control antibody 30 min before stimulation with 50 pg/ml recombinant IFN-α2. Relative expression of HBD1 and ISG15 was assessed using quantitative PCR. IFN-α2 was found to significantly upregulate HBD1 and ISG15. Each dot represents one independent experiment from a different healthy control subject. (C, D) Human PBMCs were isolated from whole blood from HIV-1 uninfected (HIV-) (C n = 5, D n = 9), HIV-1 untreated chronic progressors (PG) (C n = 9, D n = 14) and acutely HIV-1 infected (Acute) individuals (C n = 12, D n = 15). IFNα (IFNA) transcription of PBMCs was assessed by qPCR. IFNA and ISG15 were both significantly upregulated in acutely but not chronically infected subjects compared to HIV-uninfected control subjects (A-D) Kruskal-Wallis and Dunn’s multiple comparison test with graphs showing median and interquartile range. (E, F) HBD1 transcription in PBMCs of subjects with acute HIV-1 infection ( Fig 1A ) was plotted against the corresponding transcription of IFNA (E) or ISG15 (F) with each dot representing one subject. ISG15 and IFNA were found to significantly correlate with HBD1 transcription in acutely infected individuals (Spearman r correlation).
    Interferon Alpha Ifn α, supplied by Reprokine, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam anti ifn α
    <t>IFN-α</t> is a potent inducer of HBD1 in monocytes in vitro and correlates with HBD1 transcription in vivo . (A, B) CD14+ monocytes (A n = 11 and B n = 5) were isolated from PBMCs and incubated with different concentrations of recombinant IFN-α2 or left untreated. Where indicated cells were treated with either an anti-IFN-α or an isotype control antibody 30 min before stimulation with 50 pg/ml recombinant IFN-α2. Relative expression of HBD1 and ISG15 was assessed using quantitative PCR. IFN-α2 was found to significantly upregulate HBD1 and ISG15. Each dot represents one independent experiment from a different healthy control subject. (C, D) Human PBMCs were isolated from whole blood from HIV-1 uninfected (HIV-) (C n = 5, D n = 9), HIV-1 untreated chronic progressors (PG) (C n = 9, D n = 14) and acutely HIV-1 infected (Acute) individuals (C n = 12, D n = 15). IFNα (IFNA) transcription of PBMCs was assessed by qPCR. IFNA and ISG15 were both significantly upregulated in acutely but not chronically infected subjects compared to HIV-uninfected control subjects (A-D) Kruskal-Wallis and Dunn’s multiple comparison test with graphs showing median and interquartile range. (E, F) HBD1 transcription in PBMCs of subjects with acute HIV-1 infection ( Fig 1A ) was plotted against the corresponding transcription of IFNA (E) or ISG15 (F) with each dot representing one subject. ISG15 and IFNA were found to significantly correlate with HBD1 transcription in acutely infected individuals (Spearman r correlation).
    Anti Ifn α, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    R&D Systems anti ifn α
    Diarrhoea scores, percentage of piglets with jejunal pathology, serum interferon-α <t>(IFN-α)</t> and infectious virus shed after 10 focus-forming units (ffu) or 1000 ffu of human rotavirus (HRV). Diarrhoea scores (a), percentage of piglets with jejunal pathology (b), serum IFN-α (pg/ml, c) and infectious virus shedding (ffu/ml, d) are shown after 10 ffu (○) and 1000 ffu (▪) from post-infection day (PID) 0–6 ( x -axis). The number of piglets studied and the percentage of piglets that developed diarrhoea, shedding and jejunal pathology at each time-point is shown in each graph. Data-points with an asterisk differ significantly between doses at each time-point. The error bars denote the standard error of the mean (SEM) and values with an asterisk denote significant differences between groups at each PID. The HRV faecal shedding and diarrhoea scores were analysed using analysis of variance followed by Duncan's rank sum test, the percentage of pigs with diarrhoea and RV shedding was analysed using Fisher's exact test. Serum IFN-α was analysed using Kruskall–Wallis rank sum test. A P
    Anti Ifn α, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche ifn α
    Knockdown of ISG54 by shRNA decreases <t>IFN-α-induced</t> apoptosis. A , stable cell lines were established expressing shRNA plasmids directed at four different fragments of ISG54 cDNA or nonspecific ( ns ) shRNA plasmid. IFN-stimulated cells were analyzed
    Ifn α, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ifn α
    TRAIL induction is driven primarily by cytokines. (A) PBMC were infected with influenza. After infection, decreasing numbers of cells (10 6 – 1.25 × 10 5 cells) were aliquoted into 2 ml of media. After 24 h culture, <t>IFN-α</t> levels
    Ifn α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PBL Assay ifn α
    Influence of stimulatory conditions on the activation phenotype of specific CD8 + T cells. (A) Representative dot plots of HLA-DR and CD38 expression in unstimulated conditions (upper graph), after <t>IFN-α</t> stimulation (middle graph) or peptide stimulation (2 µM, lower graph) among specific (dark dots) and non-specific (gray dots) CD8 + T cells from healthy donors after a four-day culture period. CD38 and HLA-DR expression on bulk (C left panel) and specific CD8 + T cells (B and C right panel). (B) Flow cytometry histograms showing representative results for cells from one individual in unstimulated conditions (dark lines), after IFN-α stimulation (light gray histograms) or peptide stimulation (dark gray histograms). (C) Surface expression of CD38 (white bars) and HLA-DR (gray bars) on bulk and specific CD8 + T cells (n = 4).
    Ifn α, supplied by PBL Assay, used in various techniques. Bioz Stars score: 99/100, based on 1094 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti ifn α
    Expression of <t>IFN-α</t> and TNF-α in A498 xenografts after rIL-22 treatment. Western blot assay was performed to detect the expression of IFN-α and TNF-α in the A498 cell xenografts. Neither the expression of IFN-α nor TNF-α were increased significantly in A498 cell xenografts treated with rIL-22 (P > 0.05 compared with control). N = 2.
    Anti Ifn α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec anti ifn α
    Both <t>IFN-α</t> and IFN-γ have direct priming effect on purified pDCs. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. (B) TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. Data are mean ± S.E.M. of 3 independent experiments.
    Anti Ifn α, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PBL Biomedical Laboratories ifn α
    pDC retain largely normal function in response to TLR7 stimulation during acute SIV infection. (A) Left: Contour plots demonstrating the gating strategy used to identify TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys following stimulation with 3M-007. Numbers represent the percentage of TNF-α + pDC within the indicated gates. Right: Percent of TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys in response to media or 3M-007. Bars represent the median and error bars the 95% confidence interval. (B) Left: Representative contour plots demonstrating the gating strategy used to identify TNF-α + , <t>IFN-α</t> + , or TNF-α + / IFN-α + pDC in lymph nodes following 3M-007 stimulation in the absence or presence of Brefeldin A (BFA). Numbers in parentheses represent the percentage of cells expressing the respective cytokines. Right: Percent of lymph node pDC from SIV-naïve and SIV-infected monkeys expressing TNF-α, IFN-α, or both in response to TLR7/8 agonist. Bars represent the median and error bars the 95% confidence interval. NS = not significant.
    Ifn α, supplied by PBL Biomedical Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 769 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co ifn α
    Comparison of protein microarray performance with an established bead-based assay. A , Corresponding MFI values from protein microarrays and a bead-based assay were plotted for the targets GM-CSF and <t>IFN-α,</t> and Pearson correlation coefficients
    Ifn α, supplied by Merck & Co, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hycult Biotech anti ifn α
    Recruitment and activation of pDCs in inflamed LNs. (A) The frequency (top) and the absolute numbers (bottom) of B220 + CD11c + pDCs in PLNs (rt. PLN) from uninfected (d0) or HSV-infected mice on day 2 (d2). (B) Expression of CD86, CD40, and CD40L on blood pDC precursors from uninfected (d0) or HSV-infected mice on day 2 (d2). (C) Expression of CD86, CD40, and CD40L on LN CD11c + DCs from uninfected (d0) or HSV-infected mice on day 2 (d2). The percentages of cells are indicated. (D) <t>IFN-α</t> production by sorted pDC precursors from HSV-infected mice on day 2 (pDCpre) and PLN pDCs from uninfected (LN pDC d0) or HSV-infected mice on day 2 (LN pDC d2) after 16 h of incubation with irradiated HSV. (A and D) Representative values from three independent experiments are presented as the mean ± SD ( n = 6).
    Anti Ifn α, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad ifn α
    NV RNA replication is sensitive to type I and III <t>IFN</t> treatment. (A) Effect of type I and III IFN pretreatment on NV RNA replication. 293FT cells were pretreated with increasing doses of type I (100 and 1,000 U/ml <t>IFN-α</t> or IFN-β) or III (10 and 100 ng/ml IL-29) IFN for 24 h and then transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. NV VP1 in cell lysate was detected by Western blotting. Actin served as an equal loading control. (B) Effect of posttransfection IFN treatment on NV RNA replication. 293FT cells were transfected with NV RNA and treated with 1,000 U/ml IFN-β at 12 h post-RNA transfection. Cells were lysed at 48 h post-RNA transfection, and NV VP1 in cell lysate was detected by IP and Western blotting. (C) Immunofluorescence staining of NV VP1 in NV RNA-transfected 293FT cells either untreated (no IFN) or pretreated with 1,000 U/ml IFN-β. Cells were fixed at 48 h post-RNA transfection and stained with guinea pig antibody to NV VP1. Scale bars, 200 μm.
    Ifn α, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    HumanZyme ifn α
    NV RNA replication is sensitive to type I and III <t>IFN</t> treatment. (A) Effect of type I and III IFN pretreatment on NV RNA replication. 293FT cells were pretreated with increasing doses of type I (100 and 1,000 U/ml <t>IFN-α</t> or IFN-β) or III (10 and 100 ng/ml IL-29) IFN for 24 h and then transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. NV VP1 in cell lysate was detected by Western blotting. Actin served as an equal loading control. (B) Effect of posttransfection IFN treatment on NV RNA replication. 293FT cells were transfected with NV RNA and treated with 1,000 U/ml IFN-β at 12 h post-RNA transfection. Cells were lysed at 48 h post-RNA transfection, and NV VP1 in cell lysate was detected by IP and Western blotting. (C) Immunofluorescence staining of NV VP1 in NV RNA-transfected 293FT cells either untreated (no IFN) or pretreated with 1,000 U/ml IFN-β. Cells were fixed at 48 h post-RNA transfection and stained with guinea pig antibody to NV VP1. Scale bars, 200 μm.
    Ifn α, supplied by HumanZyme, used in various techniques. Bioz Stars score: 96/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Accurate Chemical & Scientific Corporation ifn α
    NV RNA replication is sensitive to type I and III <t>IFN</t> treatment. (A) Effect of type I and III IFN pretreatment on NV RNA replication. 293FT cells were pretreated with increasing doses of type I (100 and 1,000 U/ml <t>IFN-α</t> or IFN-β) or III (10 and 100 ng/ml IL-29) IFN for 24 h and then transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. NV VP1 in cell lysate was detected by Western blotting. Actin served as an equal loading control. (B) Effect of posttransfection IFN treatment on NV RNA replication. 293FT cells were transfected with NV RNA and treated with 1,000 U/ml IFN-β at 12 h post-RNA transfection. Cells were lysed at 48 h post-RNA transfection, and NV VP1 in cell lysate was detected by IP and Western blotting. (C) Immunofluorescence staining of NV VP1 in NV RNA-transfected 293FT cells either untreated (no IFN) or pretreated with 1,000 U/ml IFN-β. Cells were fixed at 48 h post-RNA transfection and stained with guinea pig antibody to NV VP1. Scale bars, 200 μm.
    Ifn α, supplied by Accurate Chemical & Scientific Corporation, used in various techniques. Bioz Stars score: 96/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend ifn α
    IAV downregulates PPAR-γ expression in AM. (A) Comparison of the expression levels of 84 transcription factors in AM (isolated and pooled from at least 3 mice) with or without IAV infection overnight in vitro by using an RT 2 profiler PCR array. Dotted line, fold cutoff of gene expression (1.5-fold); red dots, genes upregulated following IAV infection; green dots, genes downregulated following IAV infection. (B) List of up- or downregulated transcription factors in AM (isolated and pooled from at least 3 mice) following IAV infection in vitro overnight determined by using an RT 2 profiler PCR array. (C) Relative expression levels of Pparg in AM (isolated and pooled from at least 3 mice) with or without IAV infection overnight in vitro determined by qRT-PCR. (D) Western blot analysis of PPAR-γ levels in AM (isolated and pooled from at least 3 mice) with or without IAV infection overnight. The bar graph represents the relative density of the PPAR-γ band pooled from three independent experiments. (E) Relative expression levels of Pparg in sorted AM isolated from noninfected (day 0) or IAV-infected mice at 4, 6, 10, or 15 dpi. (F) Western blot analysis of PPAR-γ expression ex vivo in AM (isolated and pooled from at least 3 mice) from noninfected (day 0) or IAV-infected (6 dpi) lungs. The bar graph represents the relative density of the PPAR-γ band pooled from three independent experiments. (G) Western blot analysis of PPAR-γ expression in AM (isolated and pooled from at least 3 mice) with or without <t>IFN-α</t> treatment overnight. The bar graph represents the relative density of the PPAR-γ band pooled from three independent experiments. (H) Relative expression of Pparg in AM (isolated and pooled from at least 3 mice) with or without IAV infection in the presence or absence of anti-IFNAR1 overnight in vitro determined by qRT-PCR. (I) STAT1 binding to the Pparg locus in AM following overnight IFN-α treatment in vitro determined by ChIP analysis (data pooled from > 20 mice). Numbers in red are distances of the binding sites to the start codon. Data are representative of results from two to three independent experiments. *, P
    Ifn α, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Livzon ifn α
    Morphology of BMMNCs cultured at different time durations of the entire culture period. a BMMNCs before the culture. b CML-DCs cultured in the presence of GM-CSF/IL-4 for 8 days. c CML-DCs cultured in the presence of <t>GM-CSF/IFN-α</t> for 8 days.
    Ifn α, supplied by Livzon, used in various techniques. Bioz Stars score: 96/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    R&D Systems antibodies anti ifnα
    mRNA-promoted cytokine response and adjuvant effect. (a) PBMC were co-cultured with autologous MoDC loaded with either non-coding control mRNA (NC), spa mRNA, SpA protein or the lipofection control (LF). IFNγ was detected after overnight incubation by ELISpot. The individual results of the single donors (n = 7) were connected with lines and displayed as IFNγ spots. Analysis was carried out in technical duplicates. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. (b) Cytokine production by MoDC stimulated with mRNA-encoded staphylococcal antigens or the respective proteins and controls (lipofectamine (LF), non-coding control mRNA (NC), Influenza MP1 peptides (MP1) and tetanus toxoid (TT)) was measured in supernatants after 24 hours. The results of duplicates for TNF and <t>IFNα</t> are shown as mean values ± SEM of n = 4 donors. p≥ 0.05 n.s. (One-way ANOVA) (c) Stimulation of MoDC/CD8 + T cell co-cultures with PBP2a or SpA in the presence and absence of NC mRNA after overnight IFNγ ELISPOT analysis. The ELISPOT enzymatic activity relative to the unstimulated control is shown as mean values ± SEM of n = 8 independent donors. Statistical analysis was done using the Wilcoxon matched-pairs signed rank test. Experiments were carried out in duplicates. (d) Upon transfection of MoDC with spa mRNA and co-culture with CD8 + T cells, blocking antibodies against IFNα and TNFα alone or in combination were added and IFNγ secretion of duplicates was quantified by ELISPOT. IFNγ enzymatic activity of n = 5 independent donors is normalized to the unstimulated isotype control and displayed as mean ± SEM. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. p**
    Antibodies Anti Ifnα, supplied by R&D Systems, used in various techniques. Bioz Stars score: 80/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gag-VLPs inhibit HIV-1 replication in DC/T cell cocultures in an <t>IFN-α-dependent</t> manner. a Control DCs, LPS-DCs and VLP-DCs were pulsed with HIV-1 NL4–3 for 2 h. After washing, activated CD4 + T cells were added at a DC-to-T cell ratio of 1:5. Viral production was determined 5 days postinfection by measuring p24 Gag in culture supernatants. b <t>Anti-IFN-α</t> antibody was added to DC cultures before treatment with Gag-VLPs or LPS. DCs were then infected with HIV-1 NL4–3 for 2 h and CD4 + T cells were added at a DC-to-T cell ratio of 1:5. P24 Gag in culture supernatants was measured 5 days postinfection. c For the analysis of the kinetics of HIV infection, DCs and T cells were cocultured for 9 days and viral replication was measured by p24 Gag content at the indicated time points. Mean ± SD values of p24 Gag levels in 3 independent experiments using cells from different donors. Statistical analysis was performed using the Student t test (* p
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    Image Search Results


    Blockage of KLHL20-mediated DAPK degradation contributes to MM cell responsiveness to IFN. ( A , B ) IFN-α induces PML-NBs and KLHL20 relocation in H929 cells but not in XG1 cells. Cells treated with IFN-α (1000 U/ml) for 16 h were fixed,

    Journal: The EMBO Journal

    Article Title: The Cullin 3 substrate adaptor KLHL20 mediates DAPK ubiquitination to control interferon responses

    doi: 10.1038/emboj.2010.62

    Figure Lengend Snippet: Blockage of KLHL20-mediated DAPK degradation contributes to MM cell responsiveness to IFN. ( A , B ) IFN-α induces PML-NBs and KLHL20 relocation in H929 cells but not in XG1 cells. Cells treated with IFN-α (1000 U/ml) for 16 h were fixed,

    Article Snippet: Anti-Flag (M2) antibody, pS308-DAPK antibody, human IFN-α, and cycloheximide were purchased from Sigma.

    Techniques:

    Expression of IFN-α4, IFN-β, CXCL10, and IRF-7 in MEF cells. Expression of mouse IFN-α and IFN-β (A), the early IFN response gene CXCL10 (B), and the transcription factor IRF7 that functions as amplifier of the type I IFN

    Journal:

    Article Title: Deletion of Nonstructural Proteins NS1 and NS2 from Pneumonia Virus of Mice Attenuates Viral Replication and Reduces Pulmonary Cytokine Expression and Disease ▿

    doi: 10.1128/JVI.02041-08

    Figure Lengend Snippet: Expression of IFN-α4, IFN-β, CXCL10, and IRF-7 in MEF cells. Expression of mouse IFN-α and IFN-β (A), the early IFN response gene CXCL10 (B), and the transcription factor IRF7 that functions as amplifier of the type I IFN

    Article Snippet: Commercial ELISA kits were used to determine the concentrations of IFN-α and IFN-β (Invitrogen) and IP-10/CXCL10 (R & D Systems, Minneapolis, MN).

    Techniques: Expressing

    Blockade of CD4, but not of type I or type II IFN, inhibits AT-2 HIV-induced IDO . (A) IDO mRNA expression was measured in PBMCs from HIV-uninfected donors after exposure to AT-2 HIV in presence or absence of Ab against IFN-α, IFN-β, and/or

    Journal:

    Article Title: HIV inhibits CD4+ T-cell proliferation by inducing indoleamine 2,3-dioxygenase in plasmacytoid dendritic cells

    doi: 10.1182/blood-2006-07-034785

    Figure Lengend Snippet: Blockade of CD4, but not of type I or type II IFN, inhibits AT-2 HIV-induced IDO . (A) IDO mRNA expression was measured in PBMCs from HIV-uninfected donors after exposure to AT-2 HIV in presence or absence of Ab against IFN-α, IFN-β, and/or

    Article Snippet: Blocking anti–IFN-α (Invitrogen) and anti–IFN-β polyclonal Ab (Invitrogen) were used at a final concentration of 6 × 105 blocking units/mL and 2 × 105 blocking units/mL, respectively.

    Techniques: Expressing

    Response induced by viral entry is protective. (A) Graphical outline of the experimental setup. MDM treated with recombinant IFN-α or VLP (psPAX2 VSVG ) for 24 h were washed and challenged with increasing doses of HIV-1 NL-AD8. (B) Viperin mRNA expression at 24 h posttreatment. (C) Intracellular Nef staining at 48 h postchallenge. One representative FACS plot is shown. Pooled data are expressed as normalized infection rate, calculated as the percentage of Nef + cells, compared to the condition of medium with 1 μg/ml p24 of HIV-1 NL-AD8 for each donor (2 independent experiments, n = 5).

    Journal: Journal of Virology

    Article Title: Sensing of HIV-1 Entry Triggers a Type I Interferon Response in Human Primary Macrophages

    doi: 10.1128/JVI.00147-17

    Figure Lengend Snippet: Response induced by viral entry is protective. (A) Graphical outline of the experimental setup. MDM treated with recombinant IFN-α or VLP (psPAX2 VSVG ) for 24 h were washed and challenged with increasing doses of HIV-1 NL-AD8. (B) Viperin mRNA expression at 24 h posttreatment. (C) Intracellular Nef staining at 48 h postchallenge. One representative FACS plot is shown. Pooled data are expressed as normalized infection rate, calculated as the percentage of Nef + cells, compared to the condition of medium with 1 μg/ml p24 of HIV-1 NL-AD8 for each donor (2 independent experiments, n = 5).

    Article Snippet: Type I IFN neutralizing experiments were performed using a cocktail of four antibodies targeting IFN-α (1 μg/ml; EBI-1; eBioscience), IFN-β (1 μg/ml of A1IFNb [eBioscience] and A1 at 1 μg/ml [Millipore]), and the interferon-α/β receptor chain-2 (MMHAR-2 at 0.5 μg/ml; Millipore).

    Techniques: Recombinant, Expressing, Staining, FACS, Infection

    “Interferon-like” response to neutralizing antibodies by different EC variants or other cell types. (A, n=2) HDMECs stimulated with pro-inflammatory mediators such as LPS at 1 μg/ml or TNFα at 100ng/ml were concomitantly treated with 12 μg/ml of anti-IFN-α mAb clone #2 for 4 h. (B, n=2) EC populations isolated from blood (BECs) or lymphatic (LECs) skin vessels or from human umbilical veins (HUVECs) were exposed to 12 μg/ml of anti-IFN-α mAb clone #2 or mIgG 1 isotype control for 4 hours. (C, n=2) Comparably, freshly isolated PBMCs (peripheral blood mononuclear cells) or in vitro cultures of 293T and HT-29 cells were exposed to 12 μg/ml of anti-IFN-α mAb clone #2 for 4 hours. Total RNA and corresponding cDNA were analyzed for IFIT-1 mRNA expression by real-time PCR.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Neutralizing Type I Interferon Antibodies trigger an Interferon-Like Response in Endothelial Cells

    doi:

    Figure Lengend Snippet: “Interferon-like” response to neutralizing antibodies by different EC variants or other cell types. (A, n=2) HDMECs stimulated with pro-inflammatory mediators such as LPS at 1 μg/ml or TNFα at 100ng/ml were concomitantly treated with 12 μg/ml of anti-IFN-α mAb clone #2 for 4 h. (B, n=2) EC populations isolated from blood (BECs) or lymphatic (LECs) skin vessels or from human umbilical veins (HUVECs) were exposed to 12 μg/ml of anti-IFN-α mAb clone #2 or mIgG 1 isotype control for 4 hours. (C, n=2) Comparably, freshly isolated PBMCs (peripheral blood mononuclear cells) or in vitro cultures of 293T and HT-29 cells were exposed to 12 μg/ml of anti-IFN-α mAb clone #2 for 4 hours. Total RNA and corresponding cDNA were analyzed for IFIT-1 mRNA expression by real-time PCR.

    Article Snippet: The anti-IFN-α mAb #2 or mIgG1 isotype control antibody were subjected to ficin digest at 37 °C in the presence of 10 mM cysteine for Fab versus 1 mM cysteine for F(ab’)2 fragment generation according to the instructions of the “ImmunoPure IgG1 Fab and F(ab’)2 Preparation Kit” (Pierce Biotechnology, Rockford, IL).

    Techniques: Isolation, In Vitro, Expressing, Real-time Polymerase Chain Reaction

    Characterization of the mechanisms involved in the induction of interferon-responsive genes by neutralizing antibodies to type I IFN. (A, n=3) HDMECs were challenged for 4 h by single or combined treatment with 3 μg/ml of anti-IFN-α mAb clone #2 and anti-IFN-β mAb clone #3 (or mIgG 1 isotype control). Endothelial RNA and corresponding cDNA were then analyzed for IFIT-1 mRNA expression by real-time PCR which demonstrated an additive effect of anti-IFN-α and -β antibodies. (B, n=3) IFIT-1 induction by 3 μg/ml of anti-IFN-α mAb clone #2 was blocked by the addition of anti-IFNAR antibody at 1 μg/ml versus mIgG 2a isotype control. (C, n=2) Activation of the interferon-responsive transcription factor ISGF3 was evaluated by reporter gene assay measuring luciferase activity in relative light units (RLU) after EC stimulation for 4 h or 24 h with 6 μg/ml of anti-IFN-α mAb clone #2 or rIFN-α (100 pg/ml) for comparison. (D) Endothelial cultures after 0, 4 or 24 h of stimulation with 6 μg/ml of anti-IFN-α mAb clone #2 were investigated by phase contrast microscopy to document that no apparent changes in EC morphology were triggered by antibody treatment.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Neutralizing Type I Interferon Antibodies trigger an Interferon-Like Response in Endothelial Cells

    doi:

    Figure Lengend Snippet: Characterization of the mechanisms involved in the induction of interferon-responsive genes by neutralizing antibodies to type I IFN. (A, n=3) HDMECs were challenged for 4 h by single or combined treatment with 3 μg/ml of anti-IFN-α mAb clone #2 and anti-IFN-β mAb clone #3 (or mIgG 1 isotype control). Endothelial RNA and corresponding cDNA were then analyzed for IFIT-1 mRNA expression by real-time PCR which demonstrated an additive effect of anti-IFN-α and -β antibodies. (B, n=3) IFIT-1 induction by 3 μg/ml of anti-IFN-α mAb clone #2 was blocked by the addition of anti-IFNAR antibody at 1 μg/ml versus mIgG 2a isotype control. (C, n=2) Activation of the interferon-responsive transcription factor ISGF3 was evaluated by reporter gene assay measuring luciferase activity in relative light units (RLU) after EC stimulation for 4 h or 24 h with 6 μg/ml of anti-IFN-α mAb clone #2 or rIFN-α (100 pg/ml) for comparison. (D) Endothelial cultures after 0, 4 or 24 h of stimulation with 6 μg/ml of anti-IFN-α mAb clone #2 were investigated by phase contrast microscopy to document that no apparent changes in EC morphology were triggered by antibody treatment.

    Article Snippet: The anti-IFN-α mAb #2 or mIgG1 isotype control antibody were subjected to ficin digest at 37 °C in the presence of 10 mM cysteine for Fab versus 1 mM cysteine for F(ab’)2 fragment generation according to the instructions of the “ImmunoPure IgG1 Fab and F(ab’)2 Preparation Kit” (Pierce Biotechnology, Rockford, IL).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Activation Assay, Reporter Gene Assay, Luciferase, Activity Assay, Microscopy

    IFN-β gene silencing inhibits the “IFN-like” response of ECs to IFN-neutralizing mAbs. Following EC transfection with IFN-β siRNA or non-specific control siRNA, cells were challenged with 12 μg/ml of anti-IFN-α mAb clone #2 or anti-IFN-β mAb clone #3 for 2 h (A, n=3). To reverse the effect of IFN-β gene silencing, EC cultures were pretreated with “sub-threshold” concentrations of rIFN-β (1 pg/ml) for 2 h with or without subsequent addition of 12 μg/ml of IFN-β mAb clone #3 for another 2 h (B, n=2). Treatment of siRNA transfected ECs with high-dose (100 pg/ml) rIFN-α or -β for 4 h (C, n=4) was followed by RNA isolation and real-time RT-PCR detection of IFIT-1 expression. (D, n=3) Silencing efficiencies for IFN-β as well as IFN-α1 and IFN-α2 were determined by real-time RT-PCR and are shown for EC samples stimulated for 2 h with 12 μg/ml anti-IFN-β mAb clone #3. Comparably, the effect of IFN-β siRNA versus non-specific control siRNA was tested on transcript levels of non-related EC genes such as BCoR and β-actin for the same samples (E, n=3).

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Neutralizing Type I Interferon Antibodies trigger an Interferon-Like Response in Endothelial Cells

    doi:

    Figure Lengend Snippet: IFN-β gene silencing inhibits the “IFN-like” response of ECs to IFN-neutralizing mAbs. Following EC transfection with IFN-β siRNA or non-specific control siRNA, cells were challenged with 12 μg/ml of anti-IFN-α mAb clone #2 or anti-IFN-β mAb clone #3 for 2 h (A, n=3). To reverse the effect of IFN-β gene silencing, EC cultures were pretreated with “sub-threshold” concentrations of rIFN-β (1 pg/ml) for 2 h with or without subsequent addition of 12 μg/ml of IFN-β mAb clone #3 for another 2 h (B, n=2). Treatment of siRNA transfected ECs with high-dose (100 pg/ml) rIFN-α or -β for 4 h (C, n=4) was followed by RNA isolation and real-time RT-PCR detection of IFIT-1 expression. (D, n=3) Silencing efficiencies for IFN-β as well as IFN-α1 and IFN-α2 were determined by real-time RT-PCR and are shown for EC samples stimulated for 2 h with 12 μg/ml anti-IFN-β mAb clone #3. Comparably, the effect of IFN-β siRNA versus non-specific control siRNA was tested on transcript levels of non-related EC genes such as BCoR and β-actin for the same samples (E, n=3).

    Article Snippet: The anti-IFN-α mAb #2 or mIgG1 isotype control antibody were subjected to ficin digest at 37 °C in the presence of 10 mM cysteine for Fab versus 1 mM cysteine for F(ab’)2 fragment generation according to the instructions of the “ImmunoPure IgG1 Fab and F(ab’)2 Preparation Kit” (Pierce Biotechnology, Rockford, IL).

    Techniques: Transfection, Isolation, Quantitative RT-PCR, Expressing

    Enhanced response to rIFN-α or -β in the presence of neutralizing antibodies to type I IFN. (A, n=3) HDMECs were stimulated with rIFN-β (10 pg/ml) for 4 h in the absence or presence of anti-IFN-β mAb clone #3 or #12, or mIgG 1 isotype control at 12 μg/ml each. (B, n=2) EC treatment for 6 h with rIFN-α (10 pg/ml) was combined with 12 μg/ml of mIgG 1 isotype control, anti-IFN-α mAb clone #2 or anti-IFNAR antibody. (C, n=3) Comparably, ECs were exposed for 6 h to 10000 U/ml of rIFN-γ and 12 μg/ml of mIgG 1 isotype control, anti-IFN-γ or anti-IFNGR antibody. Endothelial RNA was analyzed for IFIT-1 mRNA expression by real-time RT-PCR.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Neutralizing Type I Interferon Antibodies trigger an Interferon-Like Response in Endothelial Cells

    doi:

    Figure Lengend Snippet: Enhanced response to rIFN-α or -β in the presence of neutralizing antibodies to type I IFN. (A, n=3) HDMECs were stimulated with rIFN-β (10 pg/ml) for 4 h in the absence or presence of anti-IFN-β mAb clone #3 or #12, or mIgG 1 isotype control at 12 μg/ml each. (B, n=2) EC treatment for 6 h with rIFN-α (10 pg/ml) was combined with 12 μg/ml of mIgG 1 isotype control, anti-IFN-α mAb clone #2 or anti-IFNAR antibody. (C, n=3) Comparably, ECs were exposed for 6 h to 10000 U/ml of rIFN-γ and 12 μg/ml of mIgG 1 isotype control, anti-IFN-γ or anti-IFNGR antibody. Endothelial RNA was analyzed for IFIT-1 mRNA expression by real-time RT-PCR.

    Article Snippet: The anti-IFN-α mAb #2 or mIgG1 isotype control antibody were subjected to ficin digest at 37 °C in the presence of 10 mM cysteine for Fab versus 1 mM cysteine for F(ab’)2 fragment generation according to the instructions of the “ImmunoPure IgG1 Fab and F(ab’)2 Preparation Kit” (Pierce Biotechnology, Rockford, IL).

    Techniques: Expressing, Quantitative RT-PCR

    Antibody domains involved in IFIT-1 induction by IFN-neutralizing antibodies. (A, n=4) HDMECs were exposed for 4 h to 1, 3 or 6 μg/ml of intact (control-treated) anti-IFN-α mAb clone #2 or a generated Fab or F(ab’) 2 fragment of clone #2. Incubation for 4 h was followed by real-time RT-PCR analysis of endothelial RNA for IFIT-1 mRNA expression. In comparison to the full-length antibody, the fragments were incapable of eliciting IFIT-1 mRNA expression (p≤0.029 for all concentrations of intact mAb versus fragments applied). (B, n=4) The full-length anti-IFN-α mAb clone #2 (1 μg/ml) was mixed at a ratio of 1:1 or 1:2 with the corresponding generated Fab fragment, a non-specific pool of unrelated Fab fragments or with an intact mIgG 1 isotype control. (C, n=3) HDMEC stimulation with recombinant IFN-α at 10 pg/ml for 4 h was challenged with concomitant administration of 1 μg/ml Fab or F(ab’) 2 fragments generated from anti-IFN-α mAb clone #2 or from a mIgG 1 isotype antibody.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Neutralizing Type I Interferon Antibodies trigger an Interferon-Like Response in Endothelial Cells

    doi:

    Figure Lengend Snippet: Antibody domains involved in IFIT-1 induction by IFN-neutralizing antibodies. (A, n=4) HDMECs were exposed for 4 h to 1, 3 or 6 μg/ml of intact (control-treated) anti-IFN-α mAb clone #2 or a generated Fab or F(ab’) 2 fragment of clone #2. Incubation for 4 h was followed by real-time RT-PCR analysis of endothelial RNA for IFIT-1 mRNA expression. In comparison to the full-length antibody, the fragments were incapable of eliciting IFIT-1 mRNA expression (p≤0.029 for all concentrations of intact mAb versus fragments applied). (B, n=4) The full-length anti-IFN-α mAb clone #2 (1 μg/ml) was mixed at a ratio of 1:1 or 1:2 with the corresponding generated Fab fragment, a non-specific pool of unrelated Fab fragments or with an intact mIgG 1 isotype control. (C, n=3) HDMEC stimulation with recombinant IFN-α at 10 pg/ml for 4 h was challenged with concomitant administration of 1 μg/ml Fab or F(ab’) 2 fragments generated from anti-IFN-α mAb clone #2 or from a mIgG 1 isotype antibody.

    Article Snippet: The anti-IFN-α mAb #2 or mIgG1 isotype control antibody were subjected to ficin digest at 37 °C in the presence of 10 mM cysteine for Fab versus 1 mM cysteine for F(ab’)2 fragment generation according to the instructions of the “ImmunoPure IgG1 Fab and F(ab’)2 Preparation Kit” (Pierce Biotechnology, Rockford, IL).

    Techniques: Generated, Incubation, Quantitative RT-PCR, Expressing, Recombinant

    Dose-dependent induction of interferon-responsive genes by neutralizing antibodies to type I IFN. Endothelial cell cultures were treated for 4 h with 0.8, 3 or 12 μg/ml of anti-IFN-α mAb clone #2 (A, n=7), clone #13 (B, n=2) or anti-IFN-β mAb clone #3 (C, n=3). The non-specific mIgG 1 isotype control was applied at 12 μg/ml. Real-time RT-PCR analysis of endothelial RNA was performed to evaluate mRNA levels of IFIT-1 and ISG15. Each sample was assayed in triplicate and the number of comparable experiments performed is given for each figure (n). IFIT-1 induction by antibody treatment was statistically significant (p≤0.03) for all mAbs and concentrations. Comparably, p-values ≤0.01 were recorded for ISG15 induction with the exception of anti-IFN-α mAb clone #13 at the lowest concentration (p=n.s.). Protein expression (D, n=2) of IFIT-1 and ISG15 was investigated by immunoblotting of whole cell extracts following 5 h of stimulation with anti-IFN-α mAb clone #2 at 12 μg/ml (*non-specific, cross-reactive protein).

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Neutralizing Type I Interferon Antibodies trigger an Interferon-Like Response in Endothelial Cells

    doi:

    Figure Lengend Snippet: Dose-dependent induction of interferon-responsive genes by neutralizing antibodies to type I IFN. Endothelial cell cultures were treated for 4 h with 0.8, 3 or 12 μg/ml of anti-IFN-α mAb clone #2 (A, n=7), clone #13 (B, n=2) or anti-IFN-β mAb clone #3 (C, n=3). The non-specific mIgG 1 isotype control was applied at 12 μg/ml. Real-time RT-PCR analysis of endothelial RNA was performed to evaluate mRNA levels of IFIT-1 and ISG15. Each sample was assayed in triplicate and the number of comparable experiments performed is given for each figure (n). IFIT-1 induction by antibody treatment was statistically significant (p≤0.03) for all mAbs and concentrations. Comparably, p-values ≤0.01 were recorded for ISG15 induction with the exception of anti-IFN-α mAb clone #13 at the lowest concentration (p=n.s.). Protein expression (D, n=2) of IFIT-1 and ISG15 was investigated by immunoblotting of whole cell extracts following 5 h of stimulation with anti-IFN-α mAb clone #2 at 12 μg/ml (*non-specific, cross-reactive protein).

    Article Snippet: The anti-IFN-α mAb #2 or mIgG1 isotype control antibody were subjected to ficin digest at 37 °C in the presence of 10 mM cysteine for Fab versus 1 mM cysteine for F(ab’)2 fragment generation according to the instructions of the “ImmunoPure IgG1 Fab and F(ab’)2 Preparation Kit” (Pierce Biotechnology, Rockford, IL).

    Techniques: Quantitative RT-PCR, Concentration Assay, Expressing

    Association between treatment response and anti-IFN-α antibodies, including binding antibodies (BAbs) ( A ) or neutralizing antibody (NAbs) ( B ). BAbs – binding antibodies; NAbs – neutralizing antibodies; CR – complete remission; PR – partial remission; NR – no response.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Anti-Interferon Alpha Antibodies in Patients with High-Risk BCR/ABL-Negative Myeloproliferative Neoplasms Treated with Recombinant Human Interferon-α

    doi: 10.12659/MSM.907876

    Figure Lengend Snippet: Association between treatment response and anti-IFN-α antibodies, including binding antibodies (BAbs) ( A ) or neutralizing antibody (NAbs) ( B ). BAbs – binding antibodies; NAbs – neutralizing antibodies; CR – complete remission; PR – partial remission; NR – no response.

    Article Snippet: Detection of binding antibodies against IFN-α Anti-IFN BAbs were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) Kit (Invitrogen, ThermoFisher Scientific, CA), following the manufacturer’s instructions.

    Techniques: Binding Assay

    Association between molecular response rates and anti-IFN-α antibodies, including binding antibodies (BAbs) ( A ) or neutralizing antibody (NAbs) ( B ). CMR – complete molecular response (undetected JAK2V617F); PMR – partial molecular response (≥50% JAK2V617F decrease); MMR – minor molecular response (20–49% JAK2V617F decrease); NR – no response (0–19% JAK2V617F decrease).

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Anti-Interferon Alpha Antibodies in Patients with High-Risk BCR/ABL-Negative Myeloproliferative Neoplasms Treated with Recombinant Human Interferon-α

    doi: 10.12659/MSM.907876

    Figure Lengend Snippet: Association between molecular response rates and anti-IFN-α antibodies, including binding antibodies (BAbs) ( A ) or neutralizing antibody (NAbs) ( B ). CMR – complete molecular response (undetected JAK2V617F); PMR – partial molecular response (≥50% JAK2V617F decrease); MMR – minor molecular response (20–49% JAK2V617F decrease); NR – no response (0–19% JAK2V617F decrease).

    Article Snippet: Detection of binding antibodies against IFN-α Anti-IFN BAbs were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) Kit (Invitrogen, ThermoFisher Scientific, CA), following the manufacturer’s instructions.

    Techniques: Binding Assay

    Secretion of cytokines by PBMC following culture in the presence of TLR7L, TLR8L, or TLR7/8L. PBMC or sorted cell populations were incubated with either TLR7L, TLR8L, TLR7/8L, or a control compound for 18 h, and supernatants were collected for cytokine detection by ELISA as described in Materials and Methods. (A) IFN-α; (B) IL-12; (C) IL-18; (D) IL-15. (E) Neutralizing antibodies against IL-12, IL-15, or IFN-α do not completely inhibit the activation of porcine NK cells in PBMC stimulated with TLR7/8L. PBMC were cultured in the presence of TLR7/8L with the addition of antibodies against IL-12, IL-15, IFN-α, or a mixture of the three antibodies. An NK cytotoxicity assay against K562-GFP cells was performed 18 h later as described in Materials and Methods. Data are means ± standard deviations for three experiments ( n = 3).

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Accessory-Cell-Mediated Activation of Porcine NK Cells by Toll-Like Receptor 7 (TLR7) and TLR8 Agonists ▿Accessory-Cell-Mediated Activation of Porcine NK Cells by Toll-Like Receptor 7 (TLR7) and TLR8 Agonists ▿ †

    doi: 10.1128/CVI.00035-09

    Figure Lengend Snippet: Secretion of cytokines by PBMC following culture in the presence of TLR7L, TLR8L, or TLR7/8L. PBMC or sorted cell populations were incubated with either TLR7L, TLR8L, TLR7/8L, or a control compound for 18 h, and supernatants were collected for cytokine detection by ELISA as described in Materials and Methods. (A) IFN-α; (B) IL-12; (C) IL-18; (D) IL-15. (E) Neutralizing antibodies against IL-12, IL-15, or IFN-α do not completely inhibit the activation of porcine NK cells in PBMC stimulated with TLR7/8L. PBMC were cultured in the presence of TLR7/8L with the addition of antibodies against IL-12, IL-15, IFN-α, or a mixture of the three antibodies. An NK cytotoxicity assay against K562-GFP cells was performed 18 h later as described in Materials and Methods. Data are means ± standard deviations for three experiments ( n = 3).

    Article Snippet: Coating and detecting antibodies against porcine IFN-α were purchased from Endogen/Pierce (Rockford, IL); a pIL-12 detection kit was purchased from R & D Systems Inc. (Minneapolis, MN); a pIL-15 detection kit was purchased from Biosource/Invitrogen (Camarillo, CA); and a pIL-18 detection kit was purchased from Bender MedSystems (Vienna, Austria).

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay, Cell Culture, Cytotoxicity Assay

    Possible pathways of porcine NK cell activation by TLR7/8L. (Arrow 1) Addition of TLR7/8L to a culture of porcine PBMC may induce cytokine production in DCs, Mφ/monocytes, or B cells. (Arrow 2) Cytokines such as IL-12, IL-15, IL-18, or IFN-α may then act on NK cells to enhance their cytolytic activity as well as their IFN-γ production. (Arrow 3) Once DCs are activated by TLR7/8L, they may express ligands such as MICA or MICB and thereby interact directly with NK cells through NKG2D to promote their cytotoxic and secretory functions. (Arrow 4) TLR7/8L themselves may directly activate the NK cells through direct engagement of their appropriate receptors expressed on the NK cells, thus driving them to increased NK cell activity.

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Accessory-Cell-Mediated Activation of Porcine NK Cells by Toll-Like Receptor 7 (TLR7) and TLR8 Agonists ▿Accessory-Cell-Mediated Activation of Porcine NK Cells by Toll-Like Receptor 7 (TLR7) and TLR8 Agonists ▿ †

    doi: 10.1128/CVI.00035-09

    Figure Lengend Snippet: Possible pathways of porcine NK cell activation by TLR7/8L. (Arrow 1) Addition of TLR7/8L to a culture of porcine PBMC may induce cytokine production in DCs, Mφ/monocytes, or B cells. (Arrow 2) Cytokines such as IL-12, IL-15, IL-18, or IFN-α may then act on NK cells to enhance their cytolytic activity as well as their IFN-γ production. (Arrow 3) Once DCs are activated by TLR7/8L, they may express ligands such as MICA or MICB and thereby interact directly with NK cells through NKG2D to promote their cytotoxic and secretory functions. (Arrow 4) TLR7/8L themselves may directly activate the NK cells through direct engagement of their appropriate receptors expressed on the NK cells, thus driving them to increased NK cell activity.

    Article Snippet: Coating and detecting antibodies against porcine IFN-α were purchased from Endogen/Pierce (Rockford, IL); a pIL-12 detection kit was purchased from R & D Systems Inc. (Minneapolis, MN); a pIL-15 detection kit was purchased from Biosource/Invitrogen (Camarillo, CA); and a pIL-18 detection kit was purchased from Bender MedSystems (Vienna, Austria).

    Techniques: Activation Assay, Activated Clotting Time Assay, Activity Assay

    IFN-α and -ß induces transcription of ISG in PHFG cells. Naïve PHFG cells or cells infected with JCV, were exposed to 100 U/mL of IFN-α or -ß for 15 consecutive days and at days 3, 5, 8, and 15 p.i., MxA, cig5 and

    Journal:

    Article Title: Interferon-? and -ss Restrict Polyomavirus JC Replication in Primary Human Fetal Glial Cells: Implications for Progressive Multifocal Leukoencephalopathy Therapy

    doi: 10.1086/520518

    Figure Lengend Snippet: IFN-α and -ß induces transcription of ISG in PHFG cells. Naïve PHFG cells or cells infected with JCV, were exposed to 100 U/mL of IFN-α or -ß for 15 consecutive days and at days 3, 5, 8, and 15 p.i., MxA, cig5 and

    Article Snippet: PHFG cells grown on 35-mm plates were treated with 100 U/mL of IFN-α (Sigma) or IFN-ß (PBL Laboratories) 24 hr prior to JCV inoculation.

    Techniques: Infection

    Anti-IFN-α reverses the inhibitory effect of IFN-α on JCV replication in PHFG cells. PHFG cells were exposed to anti-IFN-α prior to IFN treatment and (A) One milligram of total protein from day 15 JCV-infected PHFG cells with and

    Journal:

    Article Title: Interferon-? and -ss Restrict Polyomavirus JC Replication in Primary Human Fetal Glial Cells: Implications for Progressive Multifocal Leukoencephalopathy Therapy

    doi: 10.1086/520518

    Figure Lengend Snippet: Anti-IFN-α reverses the inhibitory effect of IFN-α on JCV replication in PHFG cells. PHFG cells were exposed to anti-IFN-α prior to IFN treatment and (A) One milligram of total protein from day 15 JCV-infected PHFG cells with and

    Article Snippet: PHFG cells grown on 35-mm plates were treated with 100 U/mL of IFN-α (Sigma) or IFN-ß (PBL Laboratories) 24 hr prior to JCV inoculation.

    Techniques: Infection

    Treatment with IFN-α, -ß and neutralizing anti-IFN-α is not toxic to PHFG cells. PHFG cells grown in 96-well plates were exposed to IFN-α, -ß and IFN-α with anti-IFN-α for 24 hr before infection.

    Journal:

    Article Title: Interferon-? and -ss Restrict Polyomavirus JC Replication in Primary Human Fetal Glial Cells: Implications for Progressive Multifocal Leukoencephalopathy Therapy

    doi: 10.1086/520518

    Figure Lengend Snippet: Treatment with IFN-α, -ß and neutralizing anti-IFN-α is not toxic to PHFG cells. PHFG cells grown in 96-well plates were exposed to IFN-α, -ß and IFN-α with anti-IFN-α for 24 hr before infection.

    Article Snippet: PHFG cells grown on 35-mm plates were treated with 100 U/mL of IFN-α (Sigma) or IFN-ß (PBL Laboratories) 24 hr prior to JCV inoculation.

    Techniques: Infection

    IFN-α and -ß inhibit JCV replication and transcription. PHFG cells were pretreated with IFN-α or -ß as described previously, and JCV T antigen and VP1 DNA copies ( A and B ) and mRNA transcripts ( C and D ) were measured in

    Journal:

    Article Title: Interferon-? and -ss Restrict Polyomavirus JC Replication in Primary Human Fetal Glial Cells: Implications for Progressive Multifocal Leukoencephalopathy Therapy

    doi: 10.1086/520518

    Figure Lengend Snippet: IFN-α and -ß inhibit JCV replication and transcription. PHFG cells were pretreated with IFN-α or -ß as described previously, and JCV T antigen and VP1 DNA copies ( A and B ) and mRNA transcripts ( C and D ) were measured in

    Article Snippet: PHFG cells grown on 35-mm plates were treated with 100 U/mL of IFN-α (Sigma) or IFN-ß (PBL Laboratories) 24 hr prior to JCV inoculation.

    Techniques:

    Production of interferon-α/β (IFN-α/β), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in response to Toll-like receptor (TLR) 1–9 agonists in non-parenchymal liver cells (NPC) and myeloid dendritic cells (mDC). The three types of cells were stimulated with TLR1–9 agonists for 20 hr, (a) IFN-α, (b) IFN-β, (c) TNF-α and (d) IL-6 secretion in the culture supernatants were measured by enzyme-linked immunosorbent assay. Data are shown as mean values of three independent experiments ± SEM.

    Journal: Immunology

    Article Title: Toll-like receptor-induced innate immune responses in non-parenchymal liver cells are cell type-specific

    doi: 10.1111/j.1365-2567.2009.03179.x

    Figure Lengend Snippet: Production of interferon-α/β (IFN-α/β), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in response to Toll-like receptor (TLR) 1–9 agonists in non-parenchymal liver cells (NPC) and myeloid dendritic cells (mDC). The three types of cells were stimulated with TLR1–9 agonists for 20 hr, (a) IFN-α, (b) IFN-β, (c) TNF-α and (d) IL-6 secretion in the culture supernatants were measured by enzyme-linked immunosorbent assay. Data are shown as mean values of three independent experiments ± SEM.

    Article Snippet: Neutralizing anti-IFN-α and anti-IFN-β rabbit antibodies were purchased from Calbiochem (Darmstadt, Germany).

    Techniques: Enzyme-linked Immunosorbent Assay

    Growth at low MOIs and spread of P/V-CPI − and P/V-CPI − -G3A are restricted by IFN in the PC3 prostate cancer cell line. (A) Effect of IFN on viral spread. PC3 cells were mock infected or infected at an MOI of 0.05 with P/V-CPI − or P/V-CPI − -G3A. Cells were incubated in medium with (+) or without (−) neutralizing IFN-α and IFN-β antibodies as described in Materials and Methods. At 24, 48, and 72 h p.i., microscopy pictures were taken. (B) Effect of IFN on viral growth. The medium from PC3 cells treated as described in panel A was harvested to assay infectious virus by plaque assay. Results are representative of two experiments. Ab, antibody.

    Journal: Journal of Virology

    Article Title: A Hyperfusogenic F Protein Enhances the Oncolytic Potency of a Paramyxovirus Simian Virus 5 P/V Mutant without Compromising Sensitivity to Type I Interferon ▿

    doi: 10.1128/JVI.01054-08

    Figure Lengend Snippet: Growth at low MOIs and spread of P/V-CPI − and P/V-CPI − -G3A are restricted by IFN in the PC3 prostate cancer cell line. (A) Effect of IFN on viral spread. PC3 cells were mock infected or infected at an MOI of 0.05 with P/V-CPI − or P/V-CPI − -G3A. Cells were incubated in medium with (+) or without (−) neutralizing IFN-α and IFN-β antibodies as described in Materials and Methods. At 24, 48, and 72 h p.i., microscopy pictures were taken. (B) Effect of IFN on viral growth. The medium from PC3 cells treated as described in panel A was harvested to assay infectious virus by plaque assay. Results are representative of two experiments. Ab, antibody.

    Article Snippet: In experiments involving neutralization of IFN, neutralizing antibodies against IFN-α (Millipore MAB411) and IFN-β (Chemicon International AB1431) were included in medium during infections at final concentrations of 5,000 neutralizing units/ml for each antibody.

    Techniques: Infection, Incubation, Microscopy, Plaque Assay

    BST2 mRNA induction by type I interferon. a Fold induction of relative BST2 mRNA in PBMC from three uninfected rhesus macaques after stimulation with human Interferon Alpha A (Alpha 2a) for 16 h. Data are expressed as fold increase over baseline after normalization to pre-treatment values. Error bars represent standard deviation, b relative mRNA copies of BST2 in PBMC (shown in copy numbers per 100 copies of GAPDH) are illustrated in relation to plasma IFN-alpha levels from blood samples of 18 uninfected rhesus macaques 24 h after inoculation of replication incompetent adenovirus or fowl pox vectors. The black dashed line indicates the detection limit of the ELISA and c whole blood MX1 mRNA levels correlate with BST2 mRNA determined in 38 SIVmac251 infected rhesus macaques at 24 wpi. Relative mRNA levels are depicted as log-transformed copy numbers per 100 copies of GAPDH. Each data point represents one animal. Regression line is shown; r , Spearman’s correlation coefficient; p , p value

    Journal: Retrovirology

    Article Title: Increased BST2 expression during simian immunodeficiency virus infection is not a determinant of disease progression in rhesus monkeys

    doi: 10.1186/s12977-015-0219-8

    Figure Lengend Snippet: BST2 mRNA induction by type I interferon. a Fold induction of relative BST2 mRNA in PBMC from three uninfected rhesus macaques after stimulation with human Interferon Alpha A (Alpha 2a) for 16 h. Data are expressed as fold increase over baseline after normalization to pre-treatment values. Error bars represent standard deviation, b relative mRNA copies of BST2 in PBMC (shown in copy numbers per 100 copies of GAPDH) are illustrated in relation to plasma IFN-alpha levels from blood samples of 18 uninfected rhesus macaques 24 h after inoculation of replication incompetent adenovirus or fowl pox vectors. The black dashed line indicates the detection limit of the ELISA and c whole blood MX1 mRNA levels correlate with BST2 mRNA determined in 38 SIVmac251 infected rhesus macaques at 24 wpi. Relative mRNA levels are depicted as log-transformed copy numbers per 100 copies of GAPDH. Each data point represents one animal. Regression line is shown; r , Spearman’s correlation coefficient; p , p value

    Article Snippet: In vitro, BST2 transcription seems to be specifically controlled by IFN-alpha as other cytokines such as IL-6, TNF-alpha did not influence BST2 expression [ ].

    Techniques: Standard Deviation, Enzyme-linked Immunosorbent Assay, Infection, Transformation Assay

    Resting levels of cellular proliferation and cell culture supernatant cytokines IFN-γ, IL-17, IL-10, IFN-α, and sCTLA-4 isolated from PBMC of SLE patients or age- and sex-matched healthy donors following incubation for 5 days ( n = 45 per group; * p

    Journal: Arthritis Research & Therapy

    Article Title: Immunoregulatory soluble CTLA-4 modifies effector T-cell responses in systemic lupus erythematosus

    doi: 10.1186/s13075-016-1075-1

    Figure Lengend Snippet: Resting levels of cellular proliferation and cell culture supernatant cytokines IFN-γ, IL-17, IL-10, IFN-α, and sCTLA-4 isolated from PBMC of SLE patients or age- and sex-matched healthy donors following incubation for 5 days ( n = 45 per group; * p

    Article Snippet: Cytokine standards (IL-10, IL-17, IFN-γ) were from Peprotech EC Ltd. (London, UK), and IFN-α was from Mabtech.

    Techniques: Cell Culture, Isolation, Incubation

    IFN-α is a potent inducer of HBD1 in monocytes in vitro and correlates with HBD1 transcription in vivo . (A, B) CD14+ monocytes (A n = 11 and B n = 5) were isolated from PBMCs and incubated with different concentrations of recombinant IFN-α2 or left untreated. Where indicated cells were treated with either an anti-IFN-α or an isotype control antibody 30 min before stimulation with 50 pg/ml recombinant IFN-α2. Relative expression of HBD1 and ISG15 was assessed using quantitative PCR. IFN-α2 was found to significantly upregulate HBD1 and ISG15. Each dot represents one independent experiment from a different healthy control subject. (C, D) Human PBMCs were isolated from whole blood from HIV-1 uninfected (HIV-) (C n = 5, D n = 9), HIV-1 untreated chronic progressors (PG) (C n = 9, D n = 14) and acutely HIV-1 infected (Acute) individuals (C n = 12, D n = 15). IFNα (IFNA) transcription of PBMCs was assessed by qPCR. IFNA and ISG15 were both significantly upregulated in acutely but not chronically infected subjects compared to HIV-uninfected control subjects (A-D) Kruskal-Wallis and Dunn’s multiple comparison test with graphs showing median and interquartile range. (E, F) HBD1 transcription in PBMCs of subjects with acute HIV-1 infection ( Fig 1A ) was plotted against the corresponding transcription of IFNA (E) or ISG15 (F) with each dot representing one subject. ISG15 and IFNA were found to significantly correlate with HBD1 transcription in acutely infected individuals (Spearman r correlation).

    Journal: PLoS ONE

    Article Title: Early type I Interferon response induces upregulation of human β-defensin 1 during acute HIV-1 infection

    doi: 10.1371/journal.pone.0173161

    Figure Lengend Snippet: IFN-α is a potent inducer of HBD1 in monocytes in vitro and correlates with HBD1 transcription in vivo . (A, B) CD14+ monocytes (A n = 11 and B n = 5) were isolated from PBMCs and incubated with different concentrations of recombinant IFN-α2 or left untreated. Where indicated cells were treated with either an anti-IFN-α or an isotype control antibody 30 min before stimulation with 50 pg/ml recombinant IFN-α2. Relative expression of HBD1 and ISG15 was assessed using quantitative PCR. IFN-α2 was found to significantly upregulate HBD1 and ISG15. Each dot represents one independent experiment from a different healthy control subject. (C, D) Human PBMCs were isolated from whole blood from HIV-1 uninfected (HIV-) (C n = 5, D n = 9), HIV-1 untreated chronic progressors (PG) (C n = 9, D n = 14) and acutely HIV-1 infected (Acute) individuals (C n = 12, D n = 15). IFNα (IFNA) transcription of PBMCs was assessed by qPCR. IFNA and ISG15 were both significantly upregulated in acutely but not chronically infected subjects compared to HIV-uninfected control subjects (A-D) Kruskal-Wallis and Dunn’s multiple comparison test with graphs showing median and interquartile range. (E, F) HBD1 transcription in PBMCs of subjects with acute HIV-1 infection ( Fig 1A ) was plotted against the corresponding transcription of IFNA (E) or ISG15 (F) with each dot representing one subject. ISG15 and IFNA were found to significantly correlate with HBD1 transcription in acutely infected individuals (Spearman r correlation).

    Article Snippet: CD14+ cells were stimulated with 5 ug/ml polyinosinic-polycytidylic acid (poly I:C; Invivogen), 200 ng/ml E . coli lipopolysaccharide (LPS) (Sigma), 2.5 μg/ml Imiquimod (Invivogen), 5 μM ODN2006 (Invivogen), 1 μg/ml 5’ppp-dsRNA (Invivogen) 10–100 pg/ml Interferon alpha (IFN-α) (Reprokine), 5 μg/ml Imiquimod / R837 (Invivogen), 10 pg/ml tumor necrosis factor alpha (TNFα) (Reprokine) or 10 pg/ml interleukin 1 2 beta (IL1-ß) (Reprokine) or 5 μg/ml flagellin from S . typhimurium (FLA-ST) (Invivogen).

    Techniques: In Vitro, In Vivo, Isolation, Incubation, Recombinant, Expressing, Real-time Polymerase Chain Reaction, Infection

    Diarrhoea scores, percentage of piglets with jejunal pathology, serum interferon-α (IFN-α) and infectious virus shed after 10 focus-forming units (ffu) or 1000 ffu of human rotavirus (HRV). Diarrhoea scores (a), percentage of piglets with jejunal pathology (b), serum IFN-α (pg/ml, c) and infectious virus shedding (ffu/ml, d) are shown after 10 ffu (○) and 1000 ffu (▪) from post-infection day (PID) 0–6 ( x -axis). The number of piglets studied and the percentage of piglets that developed diarrhoea, shedding and jejunal pathology at each time-point is shown in each graph. Data-points with an asterisk differ significantly between doses at each time-point. The error bars denote the standard error of the mean (SEM) and values with an asterisk denote significant differences between groups at each PID. The HRV faecal shedding and diarrhoea scores were analysed using analysis of variance followed by Duncan's rank sum test, the percentage of pigs with diarrhoea and RV shedding was analysed using Fisher's exact test. Serum IFN-α was analysed using Kruskall–Wallis rank sum test. A P

    Journal: Immunology

    Article Title: Innate immune responses to human rotavirus in the neonatal gnotobiotic piglet disease model

    doi: 10.1111/j.1365-2567.2010.03298.x

    Figure Lengend Snippet: Diarrhoea scores, percentage of piglets with jejunal pathology, serum interferon-α (IFN-α) and infectious virus shed after 10 focus-forming units (ffu) or 1000 ffu of human rotavirus (HRV). Diarrhoea scores (a), percentage of piglets with jejunal pathology (b), serum IFN-α (pg/ml, c) and infectious virus shedding (ffu/ml, d) are shown after 10 ffu (○) and 1000 ffu (▪) from post-infection day (PID) 0–6 ( x -axis). The number of piglets studied and the percentage of piglets that developed diarrhoea, shedding and jejunal pathology at each time-point is shown in each graph. Data-points with an asterisk differ significantly between doses at each time-point. The error bars denote the standard error of the mean (SEM) and values with an asterisk denote significant differences between groups at each PID. The HRV faecal shedding and diarrhoea scores were analysed using analysis of variance followed by Duncan's rank sum test, the percentage of pigs with diarrhoea and RV shedding was analysed using Fisher's exact test. Serum IFN-α was analysed using Kruskall–Wallis rank sum test. A P

    Article Snippet: Briefly, Nunc Maxisorp 96-well microtitre plates were coated with anti-IL-12, anti-TNF-α, anti-IL-6, anti-IL-10 or anti-IFN-α (R & D Systems) overnight at room temperature.

    Techniques: Infection

    Correlation coefficient between interferon-α (IFN-α) positive plasmacytoid dendritic cells (pDCs) and serum IFN-α. A Spearman's correlation coefficient was calculated between IFN-α + pDCs and serum IFN-α. The R, P and number of piglets included in the analysis are shown in the graph.

    Journal: Immunology

    Article Title: Innate immune responses to human rotavirus in the neonatal gnotobiotic piglet disease model

    doi: 10.1111/j.1365-2567.2010.03298.x

    Figure Lengend Snippet: Correlation coefficient between interferon-α (IFN-α) positive plasmacytoid dendritic cells (pDCs) and serum IFN-α. A Spearman's correlation coefficient was calculated between IFN-α + pDCs and serum IFN-α. The R, P and number of piglets included in the analysis are shown in the graph.

    Article Snippet: Briefly, Nunc Maxisorp 96-well microtitre plates were coated with anti-IL-12, anti-TNF-α, anti-IL-6, anti-IL-10 or anti-IFN-α (R & D Systems) overnight at room temperature.

    Techniques:

    Intestinal frequencies of interferon-α (IFN-α) positive plasmacytoid dendritic cells (pDCs), conventional dendritic cells (cDCs) and macrophages/monocytes after human rotavirus (HRV) infection. (a) HRV inoculated and (b) medium alone inoculated representative dot plots of SWC3 versus CD4 at post-infection days (PID) 2, 4 and 7 are shown. Gates on SWC3 low CD4 + DCs (pDCs), SWC3 high CD4 − DCs (macrophage/monocytes) and SWC3 low CD4 − DCs (cDCs) and plots on each gate show IFN-α + DCs. Mean frequencies of IFN-α + pDCs (c), IFN-α + cDCs (d) and IFN-α + macrophages/monocytes (e) in piglets that received HRV (○) or medium alone (▪) are summarized. For each experiment 100 000 events were acquired by the flow cytometer. The number of piglets studied at each PID is shown at the bottom of each graph. The error bars denote the standard error of the mean (SEM) and bars with an asterisk differ significantly from the controls at each PID (Kruskall–Wallis rank sum test P ≤ 0.05).

    Journal: Immunology

    Article Title: Innate immune responses to human rotavirus in the neonatal gnotobiotic piglet disease model

    doi: 10.1111/j.1365-2567.2010.03298.x

    Figure Lengend Snippet: Intestinal frequencies of interferon-α (IFN-α) positive plasmacytoid dendritic cells (pDCs), conventional dendritic cells (cDCs) and macrophages/monocytes after human rotavirus (HRV) infection. (a) HRV inoculated and (b) medium alone inoculated representative dot plots of SWC3 versus CD4 at post-infection days (PID) 2, 4 and 7 are shown. Gates on SWC3 low CD4 + DCs (pDCs), SWC3 high CD4 − DCs (macrophage/monocytes) and SWC3 low CD4 − DCs (cDCs) and plots on each gate show IFN-α + DCs. Mean frequencies of IFN-α + pDCs (c), IFN-α + cDCs (d) and IFN-α + macrophages/monocytes (e) in piglets that received HRV (○) or medium alone (▪) are summarized. For each experiment 100 000 events were acquired by the flow cytometer. The number of piglets studied at each PID is shown at the bottom of each graph. The error bars denote the standard error of the mean (SEM) and bars with an asterisk differ significantly from the controls at each PID (Kruskall–Wallis rank sum test P ≤ 0.05).

    Article Snippet: Briefly, Nunc Maxisorp 96-well microtitre plates were coated with anti-IL-12, anti-TNF-α, anti-IL-6, anti-IL-10 or anti-IFN-α (R & D Systems) overnight at room temperature.

    Techniques: Infection, Flow Cytometry, Cytometry

    Sn-targeted liposomes bind to IFN-α stimulated bone marrow derived macrophages (BMM)s. ( A ) Mature macrophages differentiated from wild type mouse bone marrow cells were stimulated with IFN-α or left untreated before staining with anti-Sn, anti-F4/80 antibodies ( filled gray ) and Sn-targeted liposomes ( filled gray ). Cells were thoroughly washed prior to FACS analysis. Isotype antibody or naked liposome stained cells were used as negative controls ( black lines ). ( B ) BMMs derived from wild type or Sn −/− mice were stained with fluorescent naked (blue lines) or Sn-targeted ( filled red ) liposomes prior to FACS analysis. Unstained cells ( black lines ) were used as a negative control. Data are representative of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Antigen Delivery to Macrophages Using Liposomal Nanoparticles Targeting Sialoadhesin/CD169

    doi: 10.1371/journal.pone.0039039

    Figure Lengend Snippet: Sn-targeted liposomes bind to IFN-α stimulated bone marrow derived macrophages (BMM)s. ( A ) Mature macrophages differentiated from wild type mouse bone marrow cells were stimulated with IFN-α or left untreated before staining with anti-Sn, anti-F4/80 antibodies ( filled gray ) and Sn-targeted liposomes ( filled gray ). Cells were thoroughly washed prior to FACS analysis. Isotype antibody or naked liposome stained cells were used as negative controls ( black lines ). ( B ) BMMs derived from wild type or Sn −/− mice were stained with fluorescent naked (blue lines) or Sn-targeted ( filled red ) liposomes prior to FACS analysis. Unstained cells ( black lines ) were used as a negative control. Data are representative of 3 independent experiments.

    Article Snippet: On day 7, IFN-α (500 IU/ml, R & D Systems) was added to the culture for 2 additional days to induce Sn/CD169 expression.

    Techniques: Derivative Assay, Staining, FACS, Mouse Assay, Negative Control

    The PI3K/AKT/mTOR signaling axis and HIF-1α play roles in cellular growth, invasion, vasculogenic mimicry, sphere formation activities in vitro and tumor growth in vivo induced by acute IFN-α exposure. a-c Pharmacological inhibitions of the JAK/PI3K/PTEN/mTOR/AKT, Ras/p38/MEK/ERK axes and HIF-1α significantly impacted on the IFN-α-stimulated anchorage-independent growth ( a ), scratch wound closure ( b ) and vasculogenic mimicry formation ( c ) in 769-P cells. d Inhibitors (as indicated) differentially affected expression of genes involved in EMT, cell survival and apoptotic cell death. e-f HIF-1α is needed for sphere colony formation ( e ), tumor formation and growth ( f ) of differentially educated 769-P cells; i.e. pSuper, pSuper + IFN, pshHIF-1α and pshHIF-1α + IFN cells. g Schematic illustration of the signaling pathways involved in the IFN-α-induced HIF-1α expression and stimulated tumorigenic propensities. The IFN-α-induced H IF-1α expression by first binding to the interferon alpha receptor 1 and 2 (IFNAR1/2), subsequent activation of JAK1 and TYK2, phosphorylation of PI3K, AKT and mTOR, those lead to promotion of HIF-1 α mRNA transcription and translation as well as corresponding tumorigenic activities including EMT, anchorage-independent growth, invasion and vasculogenic mimicry activities. **, P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Inflammatory interferon activates HIF-1α-mediated epithelial-to-mesenchymal transition via PI3K/AKT/mTOR pathway

    doi: 10.1186/s13046-018-0730-6

    Figure Lengend Snippet: The PI3K/AKT/mTOR signaling axis and HIF-1α play roles in cellular growth, invasion, vasculogenic mimicry, sphere formation activities in vitro and tumor growth in vivo induced by acute IFN-α exposure. a-c Pharmacological inhibitions of the JAK/PI3K/PTEN/mTOR/AKT, Ras/p38/MEK/ERK axes and HIF-1α significantly impacted on the IFN-α-stimulated anchorage-independent growth ( a ), scratch wound closure ( b ) and vasculogenic mimicry formation ( c ) in 769-P cells. d Inhibitors (as indicated) differentially affected expression of genes involved in EMT, cell survival and apoptotic cell death. e-f HIF-1α is needed for sphere colony formation ( e ), tumor formation and growth ( f ) of differentially educated 769-P cells; i.e. pSuper, pSuper + IFN, pshHIF-1α and pshHIF-1α + IFN cells. g Schematic illustration of the signaling pathways involved in the IFN-α-induced HIF-1α expression and stimulated tumorigenic propensities. The IFN-α-induced H IF-1α expression by first binding to the interferon alpha receptor 1 and 2 (IFNAR1/2), subsequent activation of JAK1 and TYK2, phosphorylation of PI3K, AKT and mTOR, those lead to promotion of HIF-1 α mRNA transcription and translation as well as corresponding tumorigenic activities including EMT, anchorage-independent growth, invasion and vasculogenic mimicry activities. **, P

    Article Snippet: Antibodies against fibronectin, vimentin, actin (Sigma), HIF-1α, HIF-1β, E-cadherin, N-cadherin, β-catenin, Bcl-2, MCL-1 (BD Bioscience), STAT1Y701 (Invitrogen), CA9, Glut1, PGK1, 4EBP1, MDR1, S6K, S6 and mTOR, Survivin (Genetex), IFN-alpha antibody (R & D systems, MN, USA), Bmi1 (Millipore Inc.) were obtained commercially.

    Techniques: In Vitro, In Vivo, Expressing, Binding Assay, Activation Assay

    Knockdown of ISG54 by shRNA decreases IFN-α-induced apoptosis. A , stable cell lines were established expressing shRNA plasmids directed at four different fragments of ISG54 cDNA or nonspecific ( ns ) shRNA plasmid. IFN-stimulated cells were analyzed

    Journal: The Journal of Biological Chemistry

    Article Title: The Interferon Stimulated Gene 54 Promotes Apoptosis *

    doi: 10.1074/jbc.M110.207068

    Figure Lengend Snippet: Knockdown of ISG54 by shRNA decreases IFN-α-induced apoptosis. A , stable cell lines were established expressing shRNA plasmids directed at four different fragments of ISG54 cDNA or nonspecific ( ns ) shRNA plasmid. IFN-stimulated cells were analyzed

    Article Snippet: HeLa cells were transfected with plasmids T7-ISG54 pcDNA3 or T7-pcDNA3 and were subsequently treated with 1000 units/ml IFN-α (gift from Roche Applied Science, Nutley, NJ) overnight.

    Techniques: shRNA, Stable Transfection, Expressing, Plasmid Preparation

    Glycerol gradient sedimentation of ISG protein complexes. HeLa cells were co-transfected with ISG54-V5, FLAG-ISG56, and FLAG-ISG60 plasmids and treated with IFN-α for 18 h. Lysates were sedimented through 25–40% glycerol gradients, and

    Journal: The Journal of Biological Chemistry

    Article Title: The Interferon Stimulated Gene 54 Promotes Apoptosis *

    doi: 10.1074/jbc.M110.207068

    Figure Lengend Snippet: Glycerol gradient sedimentation of ISG protein complexes. HeLa cells were co-transfected with ISG54-V5, FLAG-ISG56, and FLAG-ISG60 plasmids and treated with IFN-α for 18 h. Lysates were sedimented through 25–40% glycerol gradients, and

    Article Snippet: HeLa cells were transfected with plasmids T7-ISG54 pcDNA3 or T7-pcDNA3 and were subsequently treated with 1000 units/ml IFN-α (gift from Roche Applied Science, Nutley, NJ) overnight.

    Techniques: Sedimentation, Transfection

    TRAIL induction is driven primarily by cytokines. (A) PBMC were infected with influenza. After infection, decreasing numbers of cells (10 6 – 1.25 × 10 5 cells) were aliquoted into 2 ml of media. After 24 h culture, IFN-α levels

    Journal:

    Article Title: Influenza-induced expression of functional TNF-related apoptosis-inducing ligand (TRAIL) on human PBMC

    doi: 10.1016/j.humimm.2008.07.012

    Figure Lengend Snippet: TRAIL induction is driven primarily by cytokines. (A) PBMC were infected with influenza. After infection, decreasing numbers of cells (10 6 – 1.25 × 10 5 cells) were aliquoted into 2 ml of media. After 24 h culture, IFN-α levels

    Article Snippet: As a positive control for inducing TRAIL expression, uninfected PBMC were treated with 500ng/ml IFN-α (Cell Signaling Technologies, Danvers, MA) for 24 h.

    Techniques: Infection

    Influenza stimulates IFN-α and –γ production from PBMC. (A) PBMC were infected with viable or UV-inactivated influenza (UV-flu) or stimulated with the TLR agonists poly I:C, ssRNA, or CpG ODN. After 24 h, IFN-α and –γ

    Journal:

    Article Title: Influenza-induced expression of functional TNF-related apoptosis-inducing ligand (TRAIL) on human PBMC

    doi: 10.1016/j.humimm.2008.07.012

    Figure Lengend Snippet: Influenza stimulates IFN-α and –γ production from PBMC. (A) PBMC were infected with viable or UV-inactivated influenza (UV-flu) or stimulated with the TLR agonists poly I:C, ssRNA, or CpG ODN. After 24 h, IFN-α and –γ

    Article Snippet: As a positive control for inducing TRAIL expression, uninfected PBMC were treated with 500ng/ml IFN-α (Cell Signaling Technologies, Danvers, MA) for 24 h.

    Techniques: Infection

    Influence of stimulatory conditions on the activation phenotype of specific CD8 + T cells. (A) Representative dot plots of HLA-DR and CD38 expression in unstimulated conditions (upper graph), after IFN-α stimulation (middle graph) or peptide stimulation (2 µM, lower graph) among specific (dark dots) and non-specific (gray dots) CD8 + T cells from healthy donors after a four-day culture period. CD38 and HLA-DR expression on bulk (C left panel) and specific CD8 + T cells (B and C right panel). (B) Flow cytometry histograms showing representative results for cells from one individual in unstimulated conditions (dark lines), after IFN-α stimulation (light gray histograms) or peptide stimulation (dark gray histograms). (C) Surface expression of CD38 (white bars) and HLA-DR (gray bars) on bulk and specific CD8 + T cells (n = 4).

    Journal: PLoS ONE

    Article Title: Potential Role for HIV-Specific CD38−/HLA-DR+ CD8+ T Cells in Viral Suppression and Cytotoxicity in HIV Controllers

    doi: 10.1371/journal.pone.0101920

    Figure Lengend Snippet: Influence of stimulatory conditions on the activation phenotype of specific CD8 + T cells. (A) Representative dot plots of HLA-DR and CD38 expression in unstimulated conditions (upper graph), after IFN-α stimulation (middle graph) or peptide stimulation (2 µM, lower graph) among specific (dark dots) and non-specific (gray dots) CD8 + T cells from healthy donors after a four-day culture period. CD38 and HLA-DR expression on bulk (C left panel) and specific CD8 + T cells (B and C right panel). (B) Flow cytometry histograms showing representative results for cells from one individual in unstimulated conditions (dark lines), after IFN-α stimulation (light gray histograms) or peptide stimulation (dark gray histograms). (C) Surface expression of CD38 (white bars) and HLA-DR (gray bars) on bulk and specific CD8 + T cells (n = 4).

    Article Snippet: IFN-α (PBL InterferonSource) was used at 500 IU/mL.

    Techniques: Activation Assay, Expressing, Flow Cytometry, Cytometry

    Sortilin is involved in IFN-α secretion in pDCs. ( a ) Confirmation by qRT-PCR of siRNA knockdown efficiency of sortilin in pDCs. Data are mean ± SD (n = 3). * p

    Journal: Scientific Reports

    Article Title: TLR signals posttranscriptionally regulate the cytokine trafficking mediator sortilin

    doi: 10.1038/srep26566

    Figure Lengend Snippet: Sortilin is involved in IFN-α secretion in pDCs. ( a ) Confirmation by qRT-PCR of siRNA knockdown efficiency of sortilin in pDCs. Data are mean ± SD (n = 3). * p

    Article Snippet: The primary antibodies [anti-sortilin (R & D; 1:100); anti-IFN-α (Pestka Biomedical Laboratories, New Brunswick, NJ; 1:100); anti-syntaxin 6 (Cell Signaling Technology, Beverly, MA; 1:100)] and the secondary antibodies [DyLight 488-conjugated donkey-anti-goat IgG (Rockland, Gilbertsville, PA; 1:100); DyLight 649-conjugated donkey-anti-rabbit IgG (Rockland; 1:100)] were diluted in PBS and incubated for 18 h and 1 h, respectively.

    Techniques: Quantitative RT-PCR

    Sortilin interacts with various cytokines. The binding of cytokines to sortilin was measured by surface plasmon resonance analysis. Cytokine binding to immobilized recombinant sortilin was measured with a Biacore X instrument. Sensorgrams of surface plasmon resonance were obtained with various concentrations of recombinant IFN-α ( a) , IFN-γ ( b ), IL-6 ( c ), IL-10 ( d ), IL-12 ( e ), IL-17A ( f ) and IL-1β ( g ). BSA or GST was used as a negative control. All curves indicate the response after subtraction of non-specific binding, which was determined as the binding of analytes to a BSA-coated flow cell. The mean Kd values ± SEM (n = 3) are shown.

    Journal: Scientific Reports

    Article Title: TLR signals posttranscriptionally regulate the cytokine trafficking mediator sortilin

    doi: 10.1038/srep26566

    Figure Lengend Snippet: Sortilin interacts with various cytokines. The binding of cytokines to sortilin was measured by surface plasmon resonance analysis. Cytokine binding to immobilized recombinant sortilin was measured with a Biacore X instrument. Sensorgrams of surface plasmon resonance were obtained with various concentrations of recombinant IFN-α ( a) , IFN-γ ( b ), IL-6 ( c ), IL-10 ( d ), IL-12 ( e ), IL-17A ( f ) and IL-1β ( g ). BSA or GST was used as a negative control. All curves indicate the response after subtraction of non-specific binding, which was determined as the binding of analytes to a BSA-coated flow cell. The mean Kd values ± SEM (n = 3) are shown.

    Article Snippet: The primary antibodies [anti-sortilin (R & D; 1:100); anti-IFN-α (Pestka Biomedical Laboratories, New Brunswick, NJ; 1:100); anti-syntaxin 6 (Cell Signaling Technology, Beverly, MA; 1:100)] and the secondary antibodies [DyLight 488-conjugated donkey-anti-goat IgG (Rockland, Gilbertsville, PA; 1:100); DyLight 649-conjugated donkey-anti-rabbit IgG (Rockland; 1:100)] were diluted in PBS and incubated for 18 h and 1 h, respectively.

    Techniques: Binding Assay, SPR Assay, Recombinant, Negative Control, Flow Cytometry

    Infection with 1.5 and 150 HA U/ml of SeV results in distinct profiles of IFN-α subtype induction. U937 cells were infected with either 1.5 or 150 HA U/ml of SeV for 4, 8, 16, 24, and 48 p.i. RNA was harvested at each time point, and IFN-α and IFN-β subtype RNA was measured via qRT-PCR. (A) IFN-β and IFN-α subtypes in cells infected with 1.5 HA U/ml. (B) IFN-β and IFN-α subtypes in cells infected with 150 HA U/ml. The data are from 3 biological replicates performed in duplicate. Statistics were performed using one-way ANOVA with Bonferroni posttest analysis. *, P

    Journal: Journal of Virology

    Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

    doi: 10.1128/JVI.01727-15

    Figure Lengend Snippet: Infection with 1.5 and 150 HA U/ml of SeV results in distinct profiles of IFN-α subtype induction. U937 cells were infected with either 1.5 or 150 HA U/ml of SeV for 4, 8, 16, 24, and 48 p.i. RNA was harvested at each time point, and IFN-α and IFN-β subtype RNA was measured via qRT-PCR. (A) IFN-β and IFN-α subtypes in cells infected with 1.5 HA U/ml. (B) IFN-β and IFN-α subtypes in cells infected with 150 HA U/ml. The data are from 3 biological replicates performed in duplicate. Statistics were performed using one-way ANOVA with Bonferroni posttest analysis. *, P

    Article Snippet: Polyclonal anti-human leukocyte IFN-α antibody from calf antiserum (BEI Resources, VA) was used at a concentration of 1:100 (antibody/total cell culture volume), as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activities of 1,000 U/ml of all purified IFN-α subtypes (PBL Assay Science, Piscataway, NJ) to ensure an appropriate molar excess of antibody.

    Techniques: Infection, Quantitative RT-PCR

    IFN-β and IFN-α subtypes 1, 2, and 8 are induced in an NF-κB- and TBK1-dependent manner. (A) U937 cells were infected with 150 or 1.5 HA U/ml of SeV (S) for 3 h in the presence or absence of BX795 (BX), and pTBK1 and pIRF3 protein expression was measured via Western blotting. Actin was measured as a loading control. (B) Same as panel A, except cells were infected in the presence or absence of BAY11-7802 (BAY), and pIκB protein expression was measured. (C) U937 cells were infected with either 150 or 1.5 HA U/ml of SeV for 8 h in the presence or absence of the following inhibitors: BX795 (BX), BAY11-7802 (BAY), or chloroquine (Ch). (C to F) mRNA expression of IFN-α1 (C), IFN-α2 (D), IFN-α8 (E), and IFN-β (F) was measured via qRT-PCR. A one-way ANOVA with Bonferroni posttest analysis was performed. *, P

    Journal: Journal of Virology

    Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

    doi: 10.1128/JVI.01727-15

    Figure Lengend Snippet: IFN-β and IFN-α subtypes 1, 2, and 8 are induced in an NF-κB- and TBK1-dependent manner. (A) U937 cells were infected with 150 or 1.5 HA U/ml of SeV (S) for 3 h in the presence or absence of BX795 (BX), and pTBK1 and pIRF3 protein expression was measured via Western blotting. Actin was measured as a loading control. (B) Same as panel A, except cells were infected in the presence or absence of BAY11-7802 (BAY), and pIκB protein expression was measured. (C) U937 cells were infected with either 150 or 1.5 HA U/ml of SeV for 8 h in the presence or absence of the following inhibitors: BX795 (BX), BAY11-7802 (BAY), or chloroquine (Ch). (C to F) mRNA expression of IFN-α1 (C), IFN-α2 (D), IFN-α8 (E), and IFN-β (F) was measured via qRT-PCR. A one-way ANOVA with Bonferroni posttest analysis was performed. *, P

    Article Snippet: Polyclonal anti-human leukocyte IFN-α antibody from calf antiserum (BEI Resources, VA) was used at a concentration of 1:100 (antibody/total cell culture volume), as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activities of 1,000 U/ml of all purified IFN-α subtypes (PBL Assay Science, Piscataway, NJ) to ensure an appropriate molar excess of antibody.

    Techniques: Infection, Expressing, Western Blot, Quantitative RT-PCR

    IFN-β and IFN-α subtypes 1, 2 and 8 are initially induced early in an IFNAR-independent manner in both virus infections. U937 cells were infected with either 1.5 (A) or 150 (B) HA U/ml of SeV for 8 and 24 h in the presence or absence of IFNAR NA. mRNA expression of IFN-α1, -2, and -8 and IFN-β was measured via qRT-PCR. A one-way ANOVA with Bonferroni posttest analysis was performed. *, P

    Journal: Journal of Virology

    Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

    doi: 10.1128/JVI.01727-15

    Figure Lengend Snippet: IFN-β and IFN-α subtypes 1, 2 and 8 are initially induced early in an IFNAR-independent manner in both virus infections. U937 cells were infected with either 1.5 (A) or 150 (B) HA U/ml of SeV for 8 and 24 h in the presence or absence of IFNAR NA. mRNA expression of IFN-α1, -2, and -8 and IFN-β was measured via qRT-PCR. A one-way ANOVA with Bonferroni posttest analysis was performed. *, P

    Article Snippet: Polyclonal anti-human leukocyte IFN-α antibody from calf antiserum (BEI Resources, VA) was used at a concentration of 1:100 (antibody/total cell culture volume), as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activities of 1,000 U/ml of all purified IFN-α subtypes (PBL Assay Science, Piscataway, NJ) to ensure an appropriate molar excess of antibody.

    Techniques: Infection, Expressing, Quantitative RT-PCR

    IFN-α subtypes 4, 6, 7, 10, and 17 are induced in an IFNAR-dependent manner. (A) U937 cells were infected with 1.5 HA U/ml of SeV in the presence or absence of IFNAR2-neutralizing antibody (NA) for 8 and 24 h. qRT-PCR analysis was performed measuring IFN-α4, -6, -7, -10, and -17 mRNA. Statistics were performed using one-way ANOVA and Bonferroni posttest analysis. **, P

    Journal: Journal of Virology

    Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

    doi: 10.1128/JVI.01727-15

    Figure Lengend Snippet: IFN-α subtypes 4, 6, 7, 10, and 17 are induced in an IFNAR-dependent manner. (A) U937 cells were infected with 1.5 HA U/ml of SeV in the presence or absence of IFNAR2-neutralizing antibody (NA) for 8 and 24 h. qRT-PCR analysis was performed measuring IFN-α4, -6, -7, -10, and -17 mRNA. Statistics were performed using one-way ANOVA and Bonferroni posttest analysis. **, P

    Article Snippet: Polyclonal anti-human leukocyte IFN-α antibody from calf antiserum (BEI Resources, VA) was used at a concentration of 1:100 (antibody/total cell culture volume), as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activities of 1,000 U/ml of all purified IFN-α subtypes (PBL Assay Science, Piscataway, NJ) to ensure an appropriate molar excess of antibody.

    Techniques: Infection, Quantitative RT-PCR

    IFN-α subtypes 5, 14, 16, and 21 are induced in an IFNAR-dependent manner in cells infected with 1.5 HA U/ml and an IFNAR-independent manner in cells infected with 150 HA U/ml. (A and C) U937 cells were infected with either 1.5 (A) or 150 (C) HA U/ml of SeV for 8 and 24 h in the presence or absence of IFNAR NA. mRNA expression of IFN-α5, -14, -16, and -21 was measured via qRT-PCR. The data shown are from 3 biological replicates performed in duplicate technical replicates. (B) U937 cells were infected with 1.5 HA U/ml of SeV for 24 h in the presence or absence of neutralizing antibodies to total IFN-α, IFN-β, IFN-α plus IFN-β (IFNAB), or IFNAR2 (IFNAR NA). mRNA expression of IFN-α5, -14, -16, and -21 was measured via qRT-PCR. The data are from 2 biological replicates performed in duplicate. A one-way ANOVA with a Bonferroni posttest analysis was performed. ****, P

    Journal: Journal of Virology

    Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

    doi: 10.1128/JVI.01727-15

    Figure Lengend Snippet: IFN-α subtypes 5, 14, 16, and 21 are induced in an IFNAR-dependent manner in cells infected with 1.5 HA U/ml and an IFNAR-independent manner in cells infected with 150 HA U/ml. (A and C) U937 cells were infected with either 1.5 (A) or 150 (C) HA U/ml of SeV for 8 and 24 h in the presence or absence of IFNAR NA. mRNA expression of IFN-α5, -14, -16, and -21 was measured via qRT-PCR. The data shown are from 3 biological replicates performed in duplicate technical replicates. (B) U937 cells were infected with 1.5 HA U/ml of SeV for 24 h in the presence or absence of neutralizing antibodies to total IFN-α, IFN-β, IFN-α plus IFN-β (IFNAB), or IFNAR2 (IFNAR NA). mRNA expression of IFN-α5, -14, -16, and -21 was measured via qRT-PCR. The data are from 2 biological replicates performed in duplicate. A one-way ANOVA with a Bonferroni posttest analysis was performed. ****, P

    Article Snippet: Polyclonal anti-human leukocyte IFN-α antibody from calf antiserum (BEI Resources, VA) was used at a concentration of 1:100 (antibody/total cell culture volume), as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activities of 1,000 U/ml of all purified IFN-α subtypes (PBL Assay Science, Piscataway, NJ) to ensure an appropriate molar excess of antibody.

    Techniques: Infection, Expressing, Quantitative RT-PCR

    Expression of IFN-α and TNF-α in A498 xenografts after rIL-22 treatment. Western blot assay was performed to detect the expression of IFN-α and TNF-α in the A498 cell xenografts. Neither the expression of IFN-α nor TNF-α were increased significantly in A498 cell xenografts treated with rIL-22 (P > 0.05 compared with control). N = 2.

    Journal: PLoS ONE

    Article Title: Interleukin-22 Suppresses the Growth of A498 Renal Cell Carcinoma Cells via Regulation of STAT1 Pathway

    doi: 10.1371/journal.pone.0020382

    Figure Lengend Snippet: Expression of IFN-α and TNF-α in A498 xenografts after rIL-22 treatment. Western blot assay was performed to detect the expression of IFN-α and TNF-α in the A498 cell xenografts. Neither the expression of IFN-α nor TNF-α were increased significantly in A498 cell xenografts treated with rIL-22 (P > 0.05 compared with control). N = 2.

    Article Snippet: The membrane was immersed in blocking buffer (5% non-fat dry milk/1% Tween 20 in 20 mM TBS, pH 7.5) for 1.5 h at room temperature and incubated with the appropriate primary antibodies, mouse monoclonal anti-TNF-α (1∶ 50) and IFN-α (1∶100) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in blocking buffer overnight at 4°C, followed by incubation with complementary secondary antibody.

    Techniques: Expressing, Western Blot

    Both IFN-α and IFN-γ have direct priming effect on purified pDCs. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. (B) TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. Data are mean ± S.E.M. of 3 independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: Both IFN-α and IFN-γ have direct priming effect on purified pDCs. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. (B) TLR7/9-mediated IFN-α production by purified pDCs after pre-treatment with IFN-α and IFN-γ. Data are mean ± S.E.M. of 3 independent experiments.

    Article Snippet: All antibodies, except anti-IFN-α (clone LT27:295: Miltenyi Biotec), anti-IFN-β (PBL Assay Science) and anti-TLR7 (ThermoFischer), were purchased from BD biosciences and isotype-matched mouse IgG controls (BD biosciences) were used to evaluate the background.

    Techniques: Purification, Flow Cytometry, Cytometry

    TLR7-mediated IFN-α production correlates with disease activity in SLE patients. Correlation between TLR7/9-mediated IFN-α production and SLEDAI or anti-dsDNA Ab titer. Statistical analysis by the Spearman's correlation coefficient.

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: TLR7-mediated IFN-α production correlates with disease activity in SLE patients. Correlation between TLR7/9-mediated IFN-α production and SLEDAI or anti-dsDNA Ab titer. Statistical analysis by the Spearman's correlation coefficient.

    Article Snippet: All antibodies, except anti-IFN-α (clone LT27:295: Miltenyi Biotec), anti-IFN-β (PBL Assay Science) and anti-TLR7 (ThermoFischer), were purchased from BD biosciences and isotype-matched mouse IgG controls (BD biosciences) were used to evaluate the background.

    Techniques: Activity Assay

    TLR7/9 response of pDCs is regulated by the priming effects of types I and II IFNs in PBMCs of healthy subjects. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each cytokine. (B) TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each cytokine. (C) TLR7/9 expression in pDCs after pre-treatment with each cytokine. (D) TLR7/9-mediated IFN-α production in pDCs after pre-treatment with different doses of IFN-α and IFN-γ. Data are mean ± S.E.M. of 4 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: TLR7/9 response of pDCs is regulated by the priming effects of types I and II IFNs in PBMCs of healthy subjects. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each cytokine. (B) TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each cytokine. (C) TLR7/9 expression in pDCs after pre-treatment with each cytokine. (D) TLR7/9-mediated IFN-α production in pDCs after pre-treatment with different doses of IFN-α and IFN-γ. Data are mean ± S.E.M. of 4 independent experiments. * p

    Article Snippet: All antibodies, except anti-IFN-α (clone LT27:295: Miltenyi Biotec), anti-IFN-β (PBL Assay Science) and anti-TLR7 (ThermoFischer), were purchased from BD biosciences and isotype-matched mouse IgG controls (BD biosciences) were used to evaluate the background.

    Techniques: Flow Cytometry, Cytometry, Expressing

    TLR7 response was quickly up-regulated by IFN-α, but down-regulation of TLR9 response by IFN-γ was required long time. PBMCs were pre-treated with IFN-α and IFN-γ for 2, 12, and 24 h, followed by stimulation with TLR7/9 agonist for 5 h. TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each condition. Data are mean ± S.E.M. of 3 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: TLR7 response was quickly up-regulated by IFN-α, but down-regulation of TLR9 response by IFN-γ was required long time. PBMCs were pre-treated with IFN-α and IFN-γ for 2, 12, and 24 h, followed by stimulation with TLR7/9 agonist for 5 h. TLR7/9-mediated IFN-α production in pDCs after pre-treatment with each condition. Data are mean ± S.E.M. of 3 independent experiments. * p

    Article Snippet: All antibodies, except anti-IFN-α (clone LT27:295: Miltenyi Biotec), anti-IFN-β (PBL Assay Science) and anti-TLR7 (ThermoFischer), were purchased from BD biosciences and isotype-matched mouse IgG controls (BD biosciences) were used to evaluate the background.

    Techniques:

    Increased localization of TLR7 in late endosome and lysosome by IFN-α. (A) Representative images showing TLR7 (red) and indicated endosomal maturation markers (green) in pDCs pre-treated with or without IFN-α. White arrows indicate robust co-localization of TLR7 with Rab7 and LAMP1. (B) Quantification of co-localization between TLR7 and EEA1, Rab7, and LAMP-1. Data shows 10 cells per each condition in one of three independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: Increased localization of TLR7 in late endosome and lysosome by IFN-α. (A) Representative images showing TLR7 (red) and indicated endosomal maturation markers (green) in pDCs pre-treated with or without IFN-α. White arrows indicate robust co-localization of TLR7 with Rab7 and LAMP1. (B) Quantification of co-localization between TLR7 and EEA1, Rab7, and LAMP-1. Data shows 10 cells per each condition in one of three independent experiments.

    Article Snippet: All antibodies, except anti-IFN-α (clone LT27:295: Miltenyi Biotec), anti-IFN-β (PBL Assay Science) and anti-TLR7 (ThermoFischer), were purchased from BD biosciences and isotype-matched mouse IgG controls (BD biosciences) were used to evaluate the background.

    Techniques:

    Up-regulation of TLR7-mediated IFN-α production by pDCs in SLE patients. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production in Lin − HLA-DR + CD11c − CD123 + pDCs from healthy control subjects and SLE patients. (B) TLR7/9-mediated IFN-α production in pDCs of each group. (C) Representative flow cytometry plots showing TLR7/9 expression in pDCs of healthy control subjects and SLE patients. (D) TLR7/9 expression in pDCs of each group. Horizontal lines represent the mean value of each group. * p

    Journal: Frontiers in Immunology

    Article Title: Up-Regulation of TLR7-Mediated IFN-α Production by Plasmacytoid Dendritic Cells in Patients With Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01957

    Figure Lengend Snippet: Up-regulation of TLR7-mediated IFN-α production by pDCs in SLE patients. (A) Representative flow cytometry plots showing TLR7/9-mediated IFN-α production in Lin − HLA-DR + CD11c − CD123 + pDCs from healthy control subjects and SLE patients. (B) TLR7/9-mediated IFN-α production in pDCs of each group. (C) Representative flow cytometry plots showing TLR7/9 expression in pDCs of healthy control subjects and SLE patients. (D) TLR7/9 expression in pDCs of each group. Horizontal lines represent the mean value of each group. * p

    Article Snippet: All antibodies, except anti-IFN-α (clone LT27:295: Miltenyi Biotec), anti-IFN-β (PBL Assay Science) and anti-TLR7 (ThermoFischer), were purchased from BD biosciences and isotype-matched mouse IgG controls (BD biosciences) were used to evaluate the background.

    Techniques: Flow Cytometry, Cytometry, Expressing

    a Increased STAT1 phosphorylation in patients’ monocytes after IFN-α and IFN-γ treatment. PBMC from patients (solid line) and healthy controls (dashed lines) were stimulated for 15 min with IFN-α (500 U/ml) or IFN-γ (100 ng/ml). Cells were fixed and permeabilized prior to staining with anti-CD14 and anti-phospho-STAT1 (pY701) antibodies. The gate was set on CD14+ monocytes. b Increased STAT1 phosphorylation in patients’ CD4+ T cells following IFN-α and IL-27 stimulation. PBMC were stimulated with either IFN-α (10,000 U/ml) or IL-27 (200 ng/ml) for 7.5, 15 and 30 min. Cells were then treated as described in ( a ) and phospho-STAT1 levels were evaluated gating on CD4+ T cells (Red – unstimulated patient cells; blue – stimulated healthy control cells; orange – stimulated patient cells)

    Journal: Journal of Clinical Immunology

    Article Title: The Extended Clinical Phenotype of 26 Patients with Chronic Mucocutaneous Candidiasis due to Gain-of-Function Mutations in STAT1

    doi: 10.1007/s10875-015-0214-9

    Figure Lengend Snippet: a Increased STAT1 phosphorylation in patients’ monocytes after IFN-α and IFN-γ treatment. PBMC from patients (solid line) and healthy controls (dashed lines) were stimulated for 15 min with IFN-α (500 U/ml) or IFN-γ (100 ng/ml). Cells were fixed and permeabilized prior to staining with anti-CD14 and anti-phospho-STAT1 (pY701) antibodies. The gate was set on CD14+ monocytes. b Increased STAT1 phosphorylation in patients’ CD4+ T cells following IFN-α and IL-27 stimulation. PBMC were stimulated with either IFN-α (10,000 U/ml) or IL-27 (200 ng/ml) for 7.5, 15 and 30 min. Cells were then treated as described in ( a ) and phospho-STAT1 levels were evaluated gating on CD4+ T cells (Red – unstimulated patient cells; blue – stimulated healthy control cells; orange – stimulated patient cells)

    Article Snippet: Intracellular monocyte staining: 1 × 106 PBMC were stimulated for 15 min at 37 °C with IFN-α (500 U/ml; Miltenyi Biotec) or IFN-γ (100 ng/ml; Miltenyi Biotec).

    Techniques: Staining

    PBMC gene expression of TLR7 was higher in post-pubertal females. In healthy volunteers, PBMC gene expression was assessed by Nanostring and normalized gene transcript counts are shown ( n = 50). p -values represent significance of the coefficient for the variables shown after correcting for the other variable (sex or puberty). (A) There was an overall difference in TLR7 expression ( p = 0.016). After post hoc testing with Bonferroni correction, post-pubertal females had a higher TLR7 expression than post-pubertal males ( p = 0.024) and pre-pubertal females ( p = 0.03) (B) PBMC TLR9 gene expression was lower in post-pubertal volunteers. (C) There were no pubertal ( p = 0.274) differences in the PBMC IFN score. (D) After stimulation with IFNα, the IFN score (post stim IFN score) was significantly higher in post pubertal volunteers ( p = 0.001, n = 33). * p

    Journal: Frontiers in Immunology

    Article Title: Sex and Pubertal Differences in the Type 1 Interferon Pathway Associate With Both X Chromosome Number and Serum Sex Hormone Concentration

    doi: 10.3389/fimmu.2018.03167

    Figure Lengend Snippet: PBMC gene expression of TLR7 was higher in post-pubertal females. In healthy volunteers, PBMC gene expression was assessed by Nanostring and normalized gene transcript counts are shown ( n = 50). p -values represent significance of the coefficient for the variables shown after correcting for the other variable (sex or puberty). (A) There was an overall difference in TLR7 expression ( p = 0.016). After post hoc testing with Bonferroni correction, post-pubertal females had a higher TLR7 expression than post-pubertal males ( p = 0.024) and pre-pubertal females ( p = 0.03) (B) PBMC TLR9 gene expression was lower in post-pubertal volunteers. (C) There were no pubertal ( p = 0.274) differences in the PBMC IFN score. (D) After stimulation with IFNα, the IFN score (post stim IFN score) was significantly higher in post pubertal volunteers ( p = 0.001, n = 33). * p

    Article Snippet: Following incubation, cells were washed, fixed, permeabilized (Ebioscience IC fix 88-8824-00) and stained for detection of intracellular IFNα (IFNα APC, Miltenyi Biotec, Cat#130-092-602).

    Techniques: Expressing

    PBMC TLR7 gene expression was higher in males with jSLE. Healthy volunteers and young people with jSLE were included. p -values represent t -tests unless data was not normally distributed, when a Mann Whitney U test was done as indicated by “MW.” (A) PBMC IFN score was higher in jSLE ( p = 0.001, n = 79). (B) pDC tetherin expression was higher in jSLE ( p = 0.001, n = 136). (C) The sex difference in pDC tetherin expression seen in healthy volunteers ( p = 0.05) was lost in those with jSLE ( p = 0.623). (D) B cell tetherin expression was higher in jSLE ( p = 0.004, n = 136). (E) After R848 stimulation, there was no significant difference in the %pDC producing IFNα in jSLE ( p = 0.632, n = 138). Females with jSLE had a trend toward a higher % pDC producing IFNα than males with jSLE ( p = 0.057). (F–H) There were no differences in jSLE in the amount of IFNα ( p = 0.784) or IFNβ ( p = 0.699) produced by PBMC in supernatant after R848 stimulation, but there was more TNFα produced in jSLE ( p = 0.001, n = 108). (I–L) After stimulation with CpG, there was a reduction in the production of all cytokines in patients with jSLE ( n = 92). (M) PBMC TLR7 gene expression was not different overall in jSLE ( p = 0.636, n = 79) although the sex difference was lost in jSLE ( p = 0.440). Males with jSLE had a higher PBMC TLR7 gene expression ( p = 0.045) than healthy males. (N) There was a trend toward decreased PBMC gene expression of TLR9 in jSLE ( p = 0.053, n = 43). * p

    Journal: Frontiers in Immunology

    Article Title: Sex and Pubertal Differences in the Type 1 Interferon Pathway Associate With Both X Chromosome Number and Serum Sex Hormone Concentration

    doi: 10.3389/fimmu.2018.03167

    Figure Lengend Snippet: PBMC TLR7 gene expression was higher in males with jSLE. Healthy volunteers and young people with jSLE were included. p -values represent t -tests unless data was not normally distributed, when a Mann Whitney U test was done as indicated by “MW.” (A) PBMC IFN score was higher in jSLE ( p = 0.001, n = 79). (B) pDC tetherin expression was higher in jSLE ( p = 0.001, n = 136). (C) The sex difference in pDC tetherin expression seen in healthy volunteers ( p = 0.05) was lost in those with jSLE ( p = 0.623). (D) B cell tetherin expression was higher in jSLE ( p = 0.004, n = 136). (E) After R848 stimulation, there was no significant difference in the %pDC producing IFNα in jSLE ( p = 0.632, n = 138). Females with jSLE had a trend toward a higher % pDC producing IFNα than males with jSLE ( p = 0.057). (F–H) There were no differences in jSLE in the amount of IFNα ( p = 0.784) or IFNβ ( p = 0.699) produced by PBMC in supernatant after R848 stimulation, but there was more TNFα produced in jSLE ( p = 0.001, n = 108). (I–L) After stimulation with CpG, there was a reduction in the production of all cytokines in patients with jSLE ( n = 92). (M) PBMC TLR7 gene expression was not different overall in jSLE ( p = 0.636, n = 79) although the sex difference was lost in jSLE ( p = 0.440). Males with jSLE had a higher PBMC TLR7 gene expression ( p = 0.045) than healthy males. (N) There was a trend toward decreased PBMC gene expression of TLR9 in jSLE ( p = 0.053, n = 43). * p

    Article Snippet: Following incubation, cells were washed, fixed, permeabilized (Ebioscience IC fix 88-8824-00) and stained for detection of intracellular IFNα (IFNα APC, Miltenyi Biotec, Cat#130-092-602).

    Techniques: Expressing, MANN-WHITNEY, Produced

    In healthy volunteers, TLR7, and not TLR9 induced pDC IFNα production was higher in females and after puberty. (A) After stimulation with R848 or CpG, PBMC from healthy volunteers were assessed by flow cytometry as shown. p -values represent the significance of the coefficient the variable shown as estimated by linear regression after correcting for the other variable in the model (sex or puberty). (B,C) Upon R848 stimulation, females had a higher % pDC IFNα+ ( p = 0.008) than males, and post-pubertal volunteers had a higher % pDC IFNα+ ( p = 0.039), than pre-pubertal volunteers ( n = 109). (D) After CpG stimulation, there was no sex difference in the % pDC IFNα+ ( p = 0.535, n = 90). (E,F) After R848 stimulation, the same trend toward higher PBMC production of IFNα in females ( p = 0.074) and post-pubertal volunteers ( p = 0.092) was seen as in the pDC specific experiment, although this did not reach significance ( n = 86). (G) No sex differences were seen in PBMC IFNα production after CpG stimulation ( p = 0.107, n = 74). * p

    Journal: Frontiers in Immunology

    Article Title: Sex and Pubertal Differences in the Type 1 Interferon Pathway Associate With Both X Chromosome Number and Serum Sex Hormone Concentration

    doi: 10.3389/fimmu.2018.03167

    Figure Lengend Snippet: In healthy volunteers, TLR7, and not TLR9 induced pDC IFNα production was higher in females and after puberty. (A) After stimulation with R848 or CpG, PBMC from healthy volunteers were assessed by flow cytometry as shown. p -values represent the significance of the coefficient the variable shown as estimated by linear regression after correcting for the other variable in the model (sex or puberty). (B,C) Upon R848 stimulation, females had a higher % pDC IFNα+ ( p = 0.008) than males, and post-pubertal volunteers had a higher % pDC IFNα+ ( p = 0.039), than pre-pubertal volunteers ( n = 109). (D) After CpG stimulation, there was no sex difference in the % pDC IFNα+ ( p = 0.535, n = 90). (E,F) After R848 stimulation, the same trend toward higher PBMC production of IFNα in females ( p = 0.074) and post-pubertal volunteers ( p = 0.092) was seen as in the pDC specific experiment, although this did not reach significance ( n = 86). (G) No sex differences were seen in PBMC IFNα production after CpG stimulation ( p = 0.107, n = 74). * p

    Article Snippet: Following incubation, cells were washed, fixed, permeabilized (Ebioscience IC fix 88-8824-00) and stained for detection of intracellular IFNα (IFNα APC, Miltenyi Biotec, Cat#130-092-602).

    Techniques: Flow Cytometry, Cytometry

    X chromosome number and serum testosterone concentration was associated with TLR7 induced IFNα production. Pre- and post-pubertal healthy, transgender, and TUS volunteers were analyzed ( n = 128), p -values represent the significance of the coefficient the variable shown as estimated by linear regression after correcting for the other variables in the model (X chromosome number, serum testosterone, and oestradiol). (A) If two X chromosomes were present, a higher % pDC produced IFNα after R848 stimulation ( p = 0.003). (B) % pDC IFNα+ after R848 stimulation associated with serum testosterone ( p = 0.008) with a significant interaction term between X chromosome number and serum testosterone ( p = 0.002) as illustrated, holding oestradiol constant at 5pmol/L. (C) Transgender birth females ( p = 0.018) and males ( p = 0.047) both had a lower %pDC IFNα+ after R848 stimulation than their chromosomal counterparts when analyzed by ANOVA with post hoc analysis. (D–G) After R848 stimulation, if two X chromosomes were present, PBMC produced more IFNα ( p = 0.017) and IFNβ ( p = 0.03) with a significant interaction between testosterone and X chromosome number ( p = 0.028; p = 0.019 respectively). (H,I) After CpG stimulation, % pDC IFNα+ did not associate with X chromosome number ( p = 0.243) but did associate with serum testosterone concentration ( p = 0.007). pre F, Pre-pubertal female; post F, Post- pubertal female; Trans F, Transgender birth female; pre M, Pre-pubertal male; post M, Post-pubertal male; Trans M, transgender birth male; TUS, turners syndrome female. * p

    Journal: Frontiers in Immunology

    Article Title: Sex and Pubertal Differences in the Type 1 Interferon Pathway Associate With Both X Chromosome Number and Serum Sex Hormone Concentration

    doi: 10.3389/fimmu.2018.03167

    Figure Lengend Snippet: X chromosome number and serum testosterone concentration was associated with TLR7 induced IFNα production. Pre- and post-pubertal healthy, transgender, and TUS volunteers were analyzed ( n = 128), p -values represent the significance of the coefficient the variable shown as estimated by linear regression after correcting for the other variables in the model (X chromosome number, serum testosterone, and oestradiol). (A) If two X chromosomes were present, a higher % pDC produced IFNα after R848 stimulation ( p = 0.003). (B) % pDC IFNα+ after R848 stimulation associated with serum testosterone ( p = 0.008) with a significant interaction term between X chromosome number and serum testosterone ( p = 0.002) as illustrated, holding oestradiol constant at 5pmol/L. (C) Transgender birth females ( p = 0.018) and males ( p = 0.047) both had a lower %pDC IFNα+ after R848 stimulation than their chromosomal counterparts when analyzed by ANOVA with post hoc analysis. (D–G) After R848 stimulation, if two X chromosomes were present, PBMC produced more IFNα ( p = 0.017) and IFNβ ( p = 0.03) with a significant interaction between testosterone and X chromosome number ( p = 0.028; p = 0.019 respectively). (H,I) After CpG stimulation, % pDC IFNα+ did not associate with X chromosome number ( p = 0.243) but did associate with serum testosterone concentration ( p = 0.007). pre F, Pre-pubertal female; post F, Post- pubertal female; Trans F, Transgender birth female; pre M, Pre-pubertal male; post M, Post-pubertal male; Trans M, transgender birth male; TUS, turners syndrome female. * p

    Article Snippet: Following incubation, cells were washed, fixed, permeabilized (Ebioscience IC fix 88-8824-00) and stained for detection of intracellular IFNα (IFNα APC, Miltenyi Biotec, Cat#130-092-602).

    Techniques: Concentration Assay, Produced

    PBMC TLR7 gene expression did not associate with X chromosome number or serum sex hormone. Pre- and post-pubertal healthy, transgender and TUS volunteers were included ( n = 77), p -values represent the significance of the coefficient the variable shown as estimated by linear regression after correcting for the other variables in the model (X chromosome number, serum testosterone, and oestradiol). (A) PBMC TLR7 gene expression did not differ with X chromosome number ( p = 0.512). (B,C) PBMC TLR 9 gene expression did not differ with X chromosome number ( p = 0.318), but was negatively associated with serum testosterone concentration ( p = 0.014). (D) After cells were pre-stimulated with IFNα, IFN score was significantly associated with serum oestradiol ( p = 0.009, n = 58).

    Journal: Frontiers in Immunology

    Article Title: Sex and Pubertal Differences in the Type 1 Interferon Pathway Associate With Both X Chromosome Number and Serum Sex Hormone Concentration

    doi: 10.3389/fimmu.2018.03167

    Figure Lengend Snippet: PBMC TLR7 gene expression did not associate with X chromosome number or serum sex hormone. Pre- and post-pubertal healthy, transgender and TUS volunteers were included ( n = 77), p -values represent the significance of the coefficient the variable shown as estimated by linear regression after correcting for the other variables in the model (X chromosome number, serum testosterone, and oestradiol). (A) PBMC TLR7 gene expression did not differ with X chromosome number ( p = 0.512). (B,C) PBMC TLR 9 gene expression did not differ with X chromosome number ( p = 0.318), but was negatively associated with serum testosterone concentration ( p = 0.014). (D) After cells were pre-stimulated with IFNα, IFN score was significantly associated with serum oestradiol ( p = 0.009, n = 58).

    Article Snippet: Following incubation, cells were washed, fixed, permeabilized (Ebioscience IC fix 88-8824-00) and stained for detection of intracellular IFNα (IFNα APC, Miltenyi Biotec, Cat#130-092-602).

    Techniques: Expressing, Concentration Assay

    pDC retain largely normal function in response to TLR7 stimulation during acute SIV infection. (A) Left: Contour plots demonstrating the gating strategy used to identify TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys following stimulation with 3M-007. Numbers represent the percentage of TNF-α + pDC within the indicated gates. Right: Percent of TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys in response to media or 3M-007. Bars represent the median and error bars the 95% confidence interval. (B) Left: Representative contour plots demonstrating the gating strategy used to identify TNF-α + , IFN-α + , or TNF-α + / IFN-α + pDC in lymph nodes following 3M-007 stimulation in the absence or presence of Brefeldin A (BFA). Numbers in parentheses represent the percentage of cells expressing the respective cytokines. Right: Percent of lymph node pDC from SIV-naïve and SIV-infected monkeys expressing TNF-α, IFN-α, or both in response to TLR7/8 agonist. Bars represent the median and error bars the 95% confidence interval. NS = not significant.

    Journal: PLoS Pathogens

    Article Title: Rapid Influx and Death of Plasmacytoid Dendritic Cells in Lymph Nodes Mediate Depletion in Acute Simian Immunodeficiency Virus Infection

    doi: 10.1371/journal.ppat.1000413

    Figure Lengend Snippet: pDC retain largely normal function in response to TLR7 stimulation during acute SIV infection. (A) Left: Contour plots demonstrating the gating strategy used to identify TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys following stimulation with 3M-007. Numbers represent the percentage of TNF-α + pDC within the indicated gates. Right: Percent of TNF-α + pDC in PBMC from SIV-naïve and SIV-infected monkeys in response to media or 3M-007. Bars represent the median and error bars the 95% confidence interval. (B) Left: Representative contour plots demonstrating the gating strategy used to identify TNF-α + , IFN-α + , or TNF-α + / IFN-α + pDC in lymph nodes following 3M-007 stimulation in the absence or presence of Brefeldin A (BFA). Numbers in parentheses represent the percentage of cells expressing the respective cytokines. Right: Percent of lymph node pDC from SIV-naïve and SIV-infected monkeys expressing TNF-α, IFN-α, or both in response to TLR7/8 agonist. Bars represent the median and error bars the 95% confidence interval. NS = not significant.

    Article Snippet: Cells were stained with surface-labeling antibodies and fixed and permeabilized as above prior to incubation with antibodies to TNF-α (MAb11) and/or IFN- α (MMHA-2, PBL Biomedical Laboratories).

    Techniques: Infection, Expressing

    Comparison of protein microarray performance with an established bead-based assay. A , Corresponding MFI values from protein microarrays and a bead-based assay were plotted for the targets GM-CSF and IFN-α, and Pearson correlation coefficients

    Journal: The Journal of allergy and clinical immunology

    Article Title: Protein microarrays identify disease-specific anti-cytokine autoantibody profiles in the landscape of immunodeficiency

    doi: 10.1016/j.jaci.2015.07.032

    Figure Lengend Snippet: Comparison of protein microarray performance with an established bead-based assay. A , Corresponding MFI values from protein microarrays and a bead-based assay were plotted for the targets GM-CSF and IFN-α, and Pearson correlation coefficients

    Article Snippet: IFN-α (2000 IU per well, INTRONA; Merck, Kenilworth, NJ) or LPS (1 µg/mL; BioLabs, ■■■) alone or both stimuli were added.

    Techniques: Microarray, Bead-based Assay

    Recruitment and activation of pDCs in inflamed LNs. (A) The frequency (top) and the absolute numbers (bottom) of B220 + CD11c + pDCs in PLNs (rt. PLN) from uninfected (d0) or HSV-infected mice on day 2 (d2). (B) Expression of CD86, CD40, and CD40L on blood pDC precursors from uninfected (d0) or HSV-infected mice on day 2 (d2). (C) Expression of CD86, CD40, and CD40L on LN CD11c + DCs from uninfected (d0) or HSV-infected mice on day 2 (d2). The percentages of cells are indicated. (D) IFN-α production by sorted pDC precursors from HSV-infected mice on day 2 (pDCpre) and PLN pDCs from uninfected (LN pDC d0) or HSV-infected mice on day 2 (LN pDC d2) after 16 h of incubation with irradiated HSV. (A and D) Representative values from three independent experiments are presented as the mean ± SD ( n = 6).

    Journal: The Journal of Experimental Medicine

    Article Title: Plasmacytoid DCs help lymph node DCs to induce anti-HSV CTLs

    doi: 10.1084/jem.20041961

    Figure Lengend Snippet: Recruitment and activation of pDCs in inflamed LNs. (A) The frequency (top) and the absolute numbers (bottom) of B220 + CD11c + pDCs in PLNs (rt. PLN) from uninfected (d0) or HSV-infected mice on day 2 (d2). (B) Expression of CD86, CD40, and CD40L on blood pDC precursors from uninfected (d0) or HSV-infected mice on day 2 (d2). (C) Expression of CD86, CD40, and CD40L on LN CD11c + DCs from uninfected (d0) or HSV-infected mice on day 2 (d2). The percentages of cells are indicated. (D) IFN-α production by sorted pDC precursors from HSV-infected mice on day 2 (pDCpre) and PLN pDCs from uninfected (LN pDC d0) or HSV-infected mice on day 2 (LN pDC d2) after 16 h of incubation with irradiated HSV. (A and D) Representative values from three independent experiments are presented as the mean ± SD ( n = 6).

    Article Snippet: Equal numbers (105 ) of impaired LNDCs and pDCs were co-cultured for 16 h, supplemented with a neutralizing anti–IFN-α (Hycult Biotechnology), anti-CD2, anti-CD40L mAb, or 100 μg/ml of control Abs.

    Techniques: Activation Assay, Infection, Mouse Assay, Expressing, Incubation, Irradiation

    NV RNA replication is sensitive to type I and III IFN treatment. (A) Effect of type I and III IFN pretreatment on NV RNA replication. 293FT cells were pretreated with increasing doses of type I (100 and 1,000 U/ml IFN-α or IFN-β) or III (10 and 100 ng/ml IL-29) IFN for 24 h and then transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. NV VP1 in cell lysate was detected by Western blotting. Actin served as an equal loading control. (B) Effect of posttransfection IFN treatment on NV RNA replication. 293FT cells were transfected with NV RNA and treated with 1,000 U/ml IFN-β at 12 h post-RNA transfection. Cells were lysed at 48 h post-RNA transfection, and NV VP1 in cell lysate was detected by IP and Western blotting. (C) Immunofluorescence staining of NV VP1 in NV RNA-transfected 293FT cells either untreated (no IFN) or pretreated with 1,000 U/ml IFN-β. Cells were fixed at 48 h post-RNA transfection and stained with guinea pig antibody to NV VP1. Scale bars, 200 μm.

    Journal: Journal of Virology

    Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

    doi: 10.1128/JVI.01425-16

    Figure Lengend Snippet: NV RNA replication is sensitive to type I and III IFN treatment. (A) Effect of type I and III IFN pretreatment on NV RNA replication. 293FT cells were pretreated with increasing doses of type I (100 and 1,000 U/ml IFN-α or IFN-β) or III (10 and 100 ng/ml IL-29) IFN for 24 h and then transfected with carrier RNA (−) or NV RNA (+) and incubated for 48 h. NV VP1 in cell lysate was detected by Western blotting. Actin served as an equal loading control. (B) Effect of posttransfection IFN treatment on NV RNA replication. 293FT cells were transfected with NV RNA and treated with 1,000 U/ml IFN-β at 12 h post-RNA transfection. Cells were lysed at 48 h post-RNA transfection, and NV VP1 in cell lysate was detected by IP and Western blotting. (C) Immunofluorescence staining of NV VP1 in NV RNA-transfected 293FT cells either untreated (no IFN) or pretreated with 1,000 U/ml IFN-β. Cells were fixed at 48 h post-RNA transfection and stained with guinea pig antibody to NV VP1. Scale bars, 200 μm.

    Article Snippet: IFN-α (PHP108Z; AbD Serotec) and IFN-β (407318; CalBiochem) were used at 1,000 U/ml unless otherwise indicated.

    Techniques: Transfection, Incubation, Western Blot, Immunofluorescence, Staining

    NV RNA replication does not induce an IFN response. (A) 293FT-ISRE-Luc reporter cells have strong IFN responses to various stimuli and detect both IFN induction and signaling. Cells were pretreated with B18R protein (125 ng/ml) or carrier protein BSA (control) for 1 h and stimulated with the indicated reagents for 18 h. Doses of stimuli were as follows: SeV, 40 HA/ml; poly(I·C), 100 μg/ml; IFN-α and IFN-β, 1,000 U/ml; IL-29, 100 ng/ml. Luciferase activity was normalized to that of BSA-pretreated and unstimulated (mock) cells and is presented as fold induction. *, P

    Journal: Journal of Virology

    Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

    doi: 10.1128/JVI.01425-16

    Figure Lengend Snippet: NV RNA replication does not induce an IFN response. (A) 293FT-ISRE-Luc reporter cells have strong IFN responses to various stimuli and detect both IFN induction and signaling. Cells were pretreated with B18R protein (125 ng/ml) or carrier protein BSA (control) for 1 h and stimulated with the indicated reagents for 18 h. Doses of stimuli were as follows: SeV, 40 HA/ml; poly(I·C), 100 μg/ml; IFN-α and IFN-β, 1,000 U/ml; IL-29, 100 ng/ml. Luciferase activity was normalized to that of BSA-pretreated and unstimulated (mock) cells and is presented as fold induction. *, P

    Article Snippet: IFN-α (PHP108Z; AbD Serotec) and IFN-β (407318; CalBiochem) were used at 1,000 U/ml unless otherwise indicated.

    Techniques: Luciferase, Activity Assay

    NV RNA replication is not enhanced by neutralization of type I IFNs. (A) Western blotting of NV VP1 in 293FT cells pretreated with type I IFN neutralizing antibodies for 6 h and transfected with carrier RNA or NV RNA for 48 h. See Materials and Methods for the dilutions of the antibodies. Sh, sheep; Rb, rabbit; Ab, antibody. (B and C) Western blotting of NV VP1 and VPg in 293FT cells pretreated with BSA (−) or with 125 ng/ml B18R (+) for 1 h and transfected with carrier RNA or NV RNA for 48 h. Actin served as an equal loading control . (D) 293FT-ISRE-Luc reporter assay of type I IFN neutralizing antibodies. Spent medium (containing IFN neutralizing antibody) from the experiment shown in panel A was collected before lysis of cells and added to 293FT-ISRE-Luc reporter cells for 1 h, followed by stimulation with 500 U/ml IFN-α or IFN-β for 18 h. Luciferase activity (presented as fold induction) was normalized to that of the reporter cells that received medium from untreated (no antibody and no IFN) carrier RNA-transfected 293FT cells. * and ^, P

    Journal: Journal of Virology

    Article Title: Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response

    doi: 10.1128/JVI.01425-16

    Figure Lengend Snippet: NV RNA replication is not enhanced by neutralization of type I IFNs. (A) Western blotting of NV VP1 in 293FT cells pretreated with type I IFN neutralizing antibodies for 6 h and transfected with carrier RNA or NV RNA for 48 h. See Materials and Methods for the dilutions of the antibodies. Sh, sheep; Rb, rabbit; Ab, antibody. (B and C) Western blotting of NV VP1 and VPg in 293FT cells pretreated with BSA (−) or with 125 ng/ml B18R (+) for 1 h and transfected with carrier RNA or NV RNA for 48 h. Actin served as an equal loading control . (D) 293FT-ISRE-Luc reporter assay of type I IFN neutralizing antibodies. Spent medium (containing IFN neutralizing antibody) from the experiment shown in panel A was collected before lysis of cells and added to 293FT-ISRE-Luc reporter cells for 1 h, followed by stimulation with 500 U/ml IFN-α or IFN-β for 18 h. Luciferase activity (presented as fold induction) was normalized to that of the reporter cells that received medium from untreated (no antibody and no IFN) carrier RNA-transfected 293FT cells. * and ^, P

    Article Snippet: IFN-α (PHP108Z; AbD Serotec) and IFN-β (407318; CalBiochem) were used at 1,000 U/ml unless otherwise indicated.

    Techniques: Neutralization, Western Blot, Transfection, Reporter Assay, Lysis, Luciferase, Activity Assay

    IAV downregulates PPAR-γ expression in AM. (A) Comparison of the expression levels of 84 transcription factors in AM (isolated and pooled from at least 3 mice) with or without IAV infection overnight in vitro by using an RT 2 profiler PCR array. Dotted line, fold cutoff of gene expression (1.5-fold); red dots, genes upregulated following IAV infection; green dots, genes downregulated following IAV infection. (B) List of up- or downregulated transcription factors in AM (isolated and pooled from at least 3 mice) following IAV infection in vitro overnight determined by using an RT 2 profiler PCR array. (C) Relative expression levels of Pparg in AM (isolated and pooled from at least 3 mice) with or without IAV infection overnight in vitro determined by qRT-PCR. (D) Western blot analysis of PPAR-γ levels in AM (isolated and pooled from at least 3 mice) with or without IAV infection overnight. The bar graph represents the relative density of the PPAR-γ band pooled from three independent experiments. (E) Relative expression levels of Pparg in sorted AM isolated from noninfected (day 0) or IAV-infected mice at 4, 6, 10, or 15 dpi. (F) Western blot analysis of PPAR-γ expression ex vivo in AM (isolated and pooled from at least 3 mice) from noninfected (day 0) or IAV-infected (6 dpi) lungs. The bar graph represents the relative density of the PPAR-γ band pooled from three independent experiments. (G) Western blot analysis of PPAR-γ expression in AM (isolated and pooled from at least 3 mice) with or without IFN-α treatment overnight. The bar graph represents the relative density of the PPAR-γ band pooled from three independent experiments. (H) Relative expression of Pparg in AM (isolated and pooled from at least 3 mice) with or without IAV infection in the presence or absence of anti-IFNAR1 overnight in vitro determined by qRT-PCR. (I) STAT1 binding to the Pparg locus in AM following overnight IFN-α treatment in vitro determined by ChIP analysis (data pooled from > 20 mice). Numbers in red are distances of the binding sites to the start codon. Data are representative of results from two to three independent experiments. *, P

    Journal: Journal of Virology

    Article Title: PPAR-γ in Macrophages Limits Pulmonary Inflammation and Promotes Host Recovery following Respiratory Viral Infection

    doi: 10.1128/JVI.00030-19

    Figure Lengend Snippet: IAV downregulates PPAR-γ expression in AM. (A) Comparison of the expression levels of 84 transcription factors in AM (isolated and pooled from at least 3 mice) with or without IAV infection overnight in vitro by using an RT 2 profiler PCR array. Dotted line, fold cutoff of gene expression (1.5-fold); red dots, genes upregulated following IAV infection; green dots, genes downregulated following IAV infection. (B) List of up- or downregulated transcription factors in AM (isolated and pooled from at least 3 mice) following IAV infection in vitro overnight determined by using an RT 2 profiler PCR array. (C) Relative expression levels of Pparg in AM (isolated and pooled from at least 3 mice) with or without IAV infection overnight in vitro determined by qRT-PCR. (D) Western blot analysis of PPAR-γ levels in AM (isolated and pooled from at least 3 mice) with or without IAV infection overnight. The bar graph represents the relative density of the PPAR-γ band pooled from three independent experiments. (E) Relative expression levels of Pparg in sorted AM isolated from noninfected (day 0) or IAV-infected mice at 4, 6, 10, or 15 dpi. (F) Western blot analysis of PPAR-γ expression ex vivo in AM (isolated and pooled from at least 3 mice) from noninfected (day 0) or IAV-infected (6 dpi) lungs. The bar graph represents the relative density of the PPAR-γ band pooled from three independent experiments. (G) Western blot analysis of PPAR-γ expression in AM (isolated and pooled from at least 3 mice) with or without IFN-α treatment overnight. The bar graph represents the relative density of the PPAR-γ band pooled from three independent experiments. (H) Relative expression of Pparg in AM (isolated and pooled from at least 3 mice) with or without IAV infection in the presence or absence of anti-IFNAR1 overnight in vitro determined by qRT-PCR. (I) STAT1 binding to the Pparg locus in AM following overnight IFN-α treatment in vitro determined by ChIP analysis (data pooled from > 20 mice). Numbers in red are distances of the binding sites to the start codon. Data are representative of results from two to three independent experiments. *, P

    Article Snippet: AM were cultured in complete medium supplemented with 10 ng/ml GM-CSF in the presence of 50 ng/ml IFN-α (BioLegend) or the vehicle overnight.

    Techniques: Expressing, Isolation, Mouse Assay, Infection, In Vitro, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Ex Vivo, Binding Assay, Chromatin Immunoprecipitation

    Morphology of BMMNCs cultured at different time durations of the entire culture period. a BMMNCs before the culture. b CML-DCs cultured in the presence of GM-CSF/IL-4 for 8 days. c CML-DCs cultured in the presence of GM-CSF/IFN-α for 8 days.

    Journal: Cytotechnology

    Article Title: Clinical reagents of GM-CSF and IFN-? induce the generation of functional chronic myeloid leukemia dendritic cells in vitro

    doi: 10.1007/s10616-011-9393-2

    Figure Lengend Snippet: Morphology of BMMNCs cultured at different time durations of the entire culture period. a BMMNCs before the culture. b CML-DCs cultured in the presence of GM-CSF/IL-4 for 8 days. c CML-DCs cultured in the presence of GM-CSF/IFN-α for 8 days.

    Article Snippet: Briefly, BMMNCs were resuspended in RPMI1640 medium supplemented with 10% human AB serum and seeded in 24-well plates at 3–5 × 106 cells/well and cultured at 37 °C in 5% CO2 incubator for 2 h. Then the non-adherent cells were decanted and the adherent cells were cultured in RPMI-1640 medium supplemented with either GM-CSF (800 U/mL; PeproTech, London, UK) plus IL-4 (500 U/mL; PeproTech, London, UK) (Group A: represented by GM-CSF/IL-4) or GM-CSF (600 ng/mL; North China Pharmaceutical Group, Shijiazhuang, China) plus IFN-α (200 U/mL; Livzon Group, Suzhou Xinbao Pharmaceutical Factory, China) (Group B: represented by GM-CSF/IFN-α).

    Techniques: Cell Culture

    mRNA-promoted cytokine response and adjuvant effect. (a) PBMC were co-cultured with autologous MoDC loaded with either non-coding control mRNA (NC), spa mRNA, SpA protein or the lipofection control (LF). IFNγ was detected after overnight incubation by ELISpot. The individual results of the single donors (n = 7) were connected with lines and displayed as IFNγ spots. Analysis was carried out in technical duplicates. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. (b) Cytokine production by MoDC stimulated with mRNA-encoded staphylococcal antigens or the respective proteins and controls (lipofectamine (LF), non-coding control mRNA (NC), Influenza MP1 peptides (MP1) and tetanus toxoid (TT)) was measured in supernatants after 24 hours. The results of duplicates for TNF and IFNα are shown as mean values ± SEM of n = 4 donors. p≥ 0.05 n.s. (One-way ANOVA) (c) Stimulation of MoDC/CD8 + T cell co-cultures with PBP2a or SpA in the presence and absence of NC mRNA after overnight IFNγ ELISPOT analysis. The ELISPOT enzymatic activity relative to the unstimulated control is shown as mean values ± SEM of n = 8 independent donors. Statistical analysis was done using the Wilcoxon matched-pairs signed rank test. Experiments were carried out in duplicates. (d) Upon transfection of MoDC with spa mRNA and co-culture with CD8 + T cells, blocking antibodies against IFNα and TNFα alone or in combination were added and IFNγ secretion of duplicates was quantified by ELISPOT. IFNγ enzymatic activity of n = 5 independent donors is normalized to the unstimulated isotype control and displayed as mean ± SEM. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. p**

    Journal: PLoS Pathogens

    Article Title: Antigen delivery to dendritic cells shapes human CD4+ and CD8+ T cell memory responses to Staphylococcus aureus

    doi: 10.1371/journal.ppat.1006387

    Figure Lengend Snippet: mRNA-promoted cytokine response and adjuvant effect. (a) PBMC were co-cultured with autologous MoDC loaded with either non-coding control mRNA (NC), spa mRNA, SpA protein or the lipofection control (LF). IFNγ was detected after overnight incubation by ELISpot. The individual results of the single donors (n = 7) were connected with lines and displayed as IFNγ spots. Analysis was carried out in technical duplicates. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. (b) Cytokine production by MoDC stimulated with mRNA-encoded staphylococcal antigens or the respective proteins and controls (lipofectamine (LF), non-coding control mRNA (NC), Influenza MP1 peptides (MP1) and tetanus toxoid (TT)) was measured in supernatants after 24 hours. The results of duplicates for TNF and IFNα are shown as mean values ± SEM of n = 4 donors. p≥ 0.05 n.s. (One-way ANOVA) (c) Stimulation of MoDC/CD8 + T cell co-cultures with PBP2a or SpA in the presence and absence of NC mRNA after overnight IFNγ ELISPOT analysis. The ELISPOT enzymatic activity relative to the unstimulated control is shown as mean values ± SEM of n = 8 independent donors. Statistical analysis was done using the Wilcoxon matched-pairs signed rank test. Experiments were carried out in duplicates. (d) Upon transfection of MoDC with spa mRNA and co-culture with CD8 + T cells, blocking antibodies against IFNα and TNFα alone or in combination were added and IFNγ secretion of duplicates was quantified by ELISPOT. IFNγ enzymatic activity of n = 5 independent donors is normalized to the unstimulated isotype control and displayed as mean ± SEM. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. p**

    Article Snippet: Blocking antibodies anti-IFNα (#21100–1), anti-TNFα (#MAB210), isotype control anti-mouse IgG1 (#MAB002) were purchased from R & D Systems, Inc. (Minneapolis, U.S.).

    Techniques: Cell Culture, Incubation, Enzyme-linked Immunospot, Activity Assay, Transfection, Co-Culture Assay, Blocking Assay

    NOD. Ncf1 m1J BM-MΦ display hindered inflammatory and anti-viral responses upon CB3 virus infection NOD and NOD. Ncf1 m1J BM-MΦ were infected with 10–25 MOI CB3. Quantitative RT-PCR analysis was performed for Tnf , Cxcl10 Ccl5 , Ifnb1 , Isg15 , Tlr3 , Ifih1 and Ddx58 after 6 hours of infection (A) . Production of TNF-α (B) , CXCL10 (C) , CCL5 (D) , and IFN-β (E) were measured 24 hours post-infection via ELISA. Data shown represent average of 3 independent experiments performed in triplicate. ND, not detected; ***p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Superoxide Production by NADPH Oxidase Intensifies Macrophage Anti-Viral Responses during Diabetogenic Coxsackievirus Infection

    doi: 10.4049/jimmunol.1700478

    Figure Lengend Snippet: NOD. Ncf1 m1J BM-MΦ display hindered inflammatory and anti-viral responses upon CB3 virus infection NOD and NOD. Ncf1 m1J BM-MΦ were infected with 10–25 MOI CB3. Quantitative RT-PCR analysis was performed for Tnf , Cxcl10 Ccl5 , Ifnb1 , Isg15 , Tlr3 , Ifih1 and Ddx58 after 6 hours of infection (A) . Production of TNF-α (B) , CXCL10 (C) , CCL5 (D) , and IFN-β (E) were measured 24 hours post-infection via ELISA. Data shown represent average of 3 independent experiments performed in triplicate. ND, not detected; ***p

    Article Snippet: TNF-α, CXCL10, CCL5 (R & D Systems), and IFN-α/IFN-β (PBL Assay Science) was quantified by ELISAs.

    Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Gag-VLPs inhibit HIV-1 replication in DC/T cell cocultures in an IFN-α-dependent manner. a Control DCs, LPS-DCs and VLP-DCs were pulsed with HIV-1 NL4–3 for 2 h. After washing, activated CD4 + T cells were added at a DC-to-T cell ratio of 1:5. Viral production was determined 5 days postinfection by measuring p24 Gag in culture supernatants. b Anti-IFN-α antibody was added to DC cultures before treatment with Gag-VLPs or LPS. DCs were then infected with HIV-1 NL4–3 for 2 h and CD4 + T cells were added at a DC-to-T cell ratio of 1:5. P24 Gag in culture supernatants was measured 5 days postinfection. c For the analysis of the kinetics of HIV infection, DCs and T cells were cocultured for 9 days and viral replication was measured by p24 Gag content at the indicated time points. Mean ± SD values of p24 Gag levels in 3 independent experiments using cells from different donors. Statistical analysis was performed using the Student t test (* p

    Journal: Journal of Innate Immunity

    Article Title: HIV-1 Gag-Virus-Like Particles Inhibit HIV-1 Replication in Dendritic Cells and T Cells through IFN-α-Dependent Upregulation of APOBEC3G and 3F

    doi: 10.1159/000339402

    Figure Lengend Snippet: Gag-VLPs inhibit HIV-1 replication in DC/T cell cocultures in an IFN-α-dependent manner. a Control DCs, LPS-DCs and VLP-DCs were pulsed with HIV-1 NL4–3 for 2 h. After washing, activated CD4 + T cells were added at a DC-to-T cell ratio of 1:5. Viral production was determined 5 days postinfection by measuring p24 Gag in culture supernatants. b Anti-IFN-α antibody was added to DC cultures before treatment with Gag-VLPs or LPS. DCs were then infected with HIV-1 NL4–3 for 2 h and CD4 + T cells were added at a DC-to-T cell ratio of 1:5. P24 Gag in culture supernatants was measured 5 days postinfection. c For the analysis of the kinetics of HIV infection, DCs and T cells were cocultured for 9 days and viral replication was measured by p24 Gag content at the indicated time points. Mean ± SD values of p24 Gag levels in 3 independent experiments using cells from different donors. Statistical analysis was performed using the Student t test (* p

    Article Snippet: IFN-α in the DC culture medium was neutralized by adding anti-IFN-α antibody (5 µg/ml; PBL Biomedical Laboratories).

    Techniques: Infection

    Evolution of IFN-α levels (Limit of detection: 3.9 pg/mL)

    Journal: Porcine Health Management

    Article Title: Immune response development after vaccination of 1-day-old naïve pigs with a Porcine Reproductive and Respiratory Syndrome 1-based modified live virus vaccine

    doi: 10.1186/s40813-018-0112-7

    Figure Lengend Snippet: Evolution of IFN-α levels (Limit of detection: 3.9 pg/mL)

    Article Snippet: Briefly, plates were coated with anti-pig IFN-α (mAb K9, R & D systems) at 1.1 Ug/mL; 50 μl of each sample and biotinylated pig IFN-α (mAb F17, R & D systems) were added.

    Techniques: