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    Thermo Fisher h5n1 viruses
    Influenza A virus (IAV) infection inhibits expression of DNA methyltransferase 1 (DNMT1), resulting in up-regulation of miR-203. ( A ) A549 cells were infected with <t>H5N1</t> (multiplicity of infection (MOI) = 2) viruses, and abundance of mRNAs encoding three DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) was measured at different times (0, 2, 4, 6, 12, and 24 h). ( B ) Expression of DNMT1, DNMT3a, and DNMT3b protein in A549 cells during H5N1 (MOI = 2) infection was assayed by western blotting. Full-length blots are presented in Supplementary Figures S2 – S4 . ( C ) A549 cells were transfected for 24 h with pCMV3-DNMT1 or DNMT1-specific siRNA (si-DNMT1). Expression of DNMT1 (pCMV3-DNMT1) and efficiency of si-DNMT1 was measured by qPCR and western blotting. Full-length blots are presented in Supplementary Figure S5 . ( D and E ) A549 cells were transfected for 24 h with the pCMV3-DNMT1 plasmid ( D ) or si-DNMT1 ( E ). Next, cells were mock-infected or infected with H5N1 (MOI = 2) viruses for another 24 h to detect abundance of miR-203. The empty vector (pCMV3-empty) and si-control served as negative controls. Data are expressed as the mean + SD of three independent experiments. * p
    H5n1 Viruses, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    h5n1 viruses - by Bioz Stars, 2021-07
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    92
    Sino Biological h5n1
    Influenza A virus (IAV) infection inhibits expression of DNA methyltransferase 1 (DNMT1), resulting in up-regulation of miR-203. ( A ) A549 cells were infected with <t>H5N1</t> (multiplicity of infection (MOI) = 2) viruses, and abundance of mRNAs encoding three DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) was measured at different times (0, 2, 4, 6, 12, and 24 h). ( B ) Expression of DNMT1, DNMT3a, and DNMT3b protein in A549 cells during H5N1 (MOI = 2) infection was assayed by western blotting. Full-length blots are presented in Supplementary Figures S2 – S4 . ( C ) A549 cells were transfected for 24 h with pCMV3-DNMT1 or DNMT1-specific siRNA (si-DNMT1). Expression of DNMT1 (pCMV3-DNMT1) and efficiency of si-DNMT1 was measured by qPCR and western blotting. Full-length blots are presented in Supplementary Figure S5 . ( D and E ) A549 cells were transfected for 24 h with the pCMV3-DNMT1 plasmid ( D ) or si-DNMT1 ( E ). Next, cells were mock-infected or infected with H5N1 (MOI = 2) viruses for another 24 h to detect abundance of miR-203. The empty vector (pCMV3-empty) and si-control served as negative controls. Data are expressed as the mean + SD of three independent experiments. * p
    H5n1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h5n1/product/Sino Biological
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    h5n1 - by Bioz Stars, 2021-07
    92/100 stars
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    94
    Sino Biological influenza a h5n1 hemagglutinin ha protein
    Influenza A virus (IAV) infection inhibits expression of DNA methyltransferase 1 (DNMT1), resulting in up-regulation of miR-203. ( A ) A549 cells were infected with <t>H5N1</t> (multiplicity of infection (MOI) = 2) viruses, and abundance of mRNAs encoding three DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) was measured at different times (0, 2, 4, 6, 12, and 24 h). ( B ) Expression of DNMT1, DNMT3a, and DNMT3b protein in A549 cells during H5N1 (MOI = 2) infection was assayed by western blotting. Full-length blots are presented in Supplementary Figures S2 – S4 . ( C ) A549 cells were transfected for 24 h with pCMV3-DNMT1 or DNMT1-specific siRNA (si-DNMT1). Expression of DNMT1 (pCMV3-DNMT1) and efficiency of si-DNMT1 was measured by qPCR and western blotting. Full-length blots are presented in Supplementary Figure S5 . ( D and E ) A549 cells were transfected for 24 h with the pCMV3-DNMT1 plasmid ( D ) or si-DNMT1 ( E ). Next, cells were mock-infected or infected with H5N1 (MOI = 2) viruses for another 24 h to detect abundance of miR-203. The empty vector (pCMV3-empty) and si-control served as negative controls. Data are expressed as the mean + SD of three independent experiments. * p
    Influenza A H5n1 Hemagglutinin Ha Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    influenza a h5n1 hemagglutinin ha protein - by Bioz Stars, 2021-07
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    Recombinant Influenza A A Hong Kong 483 97 H5N1 Hemagglutinin HA protein produced in HEK293 cells Protein contains a C terminal His tag
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    Recombinant Influenza A A Thailand 1 KAN 1 2004 H5N1 Neuraminidase NA protein produced in HEK293 cells Protein contains a C terminal His tag
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    H5N1 Influenza A Virus Indonesia 05 05 Recombinant 10 ug
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    Influenza A virus (IAV) infection inhibits expression of DNA methyltransferase 1 (DNMT1), resulting in up-regulation of miR-203. ( A ) A549 cells were infected with H5N1 (multiplicity of infection (MOI) = 2) viruses, and abundance of mRNAs encoding three DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) was measured at different times (0, 2, 4, 6, 12, and 24 h). ( B ) Expression of DNMT1, DNMT3a, and DNMT3b protein in A549 cells during H5N1 (MOI = 2) infection was assayed by western blotting. Full-length blots are presented in Supplementary Figures S2 – S4 . ( C ) A549 cells were transfected for 24 h with pCMV3-DNMT1 or DNMT1-specific siRNA (si-DNMT1). Expression of DNMT1 (pCMV3-DNMT1) and efficiency of si-DNMT1 was measured by qPCR and western blotting. Full-length blots are presented in Supplementary Figure S5 . ( D and E ) A549 cells were transfected for 24 h with the pCMV3-DNMT1 plasmid ( D ) or si-DNMT1 ( E ). Next, cells were mock-infected or infected with H5N1 (MOI = 2) viruses for another 24 h to detect abundance of miR-203. The empty vector (pCMV3-empty) and si-control served as negative controls. Data are expressed as the mean + SD of three independent experiments. * p

    Journal: Scientific Reports

    Article Title: Up-regulation of microRNA-203 in influenza A virus infection inhibits viral replication by targeting DR1

    doi: 10.1038/s41598-018-25073-9

    Figure Lengend Snippet: Influenza A virus (IAV) infection inhibits expression of DNA methyltransferase 1 (DNMT1), resulting in up-regulation of miR-203. ( A ) A549 cells were infected with H5N1 (multiplicity of infection (MOI) = 2) viruses, and abundance of mRNAs encoding three DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) was measured at different times (0, 2, 4, 6, 12, and 24 h). ( B ) Expression of DNMT1, DNMT3a, and DNMT3b protein in A549 cells during H5N1 (MOI = 2) infection was assayed by western blotting. Full-length blots are presented in Supplementary Figures S2 – S4 . ( C ) A549 cells were transfected for 24 h with pCMV3-DNMT1 or DNMT1-specific siRNA (si-DNMT1). Expression of DNMT1 (pCMV3-DNMT1) and efficiency of si-DNMT1 was measured by qPCR and western blotting. Full-length blots are presented in Supplementary Figure S5 . ( D and E ) A549 cells were transfected for 24 h with the pCMV3-DNMT1 plasmid ( D ) or si-DNMT1 ( E ). Next, cells were mock-infected or infected with H5N1 (MOI = 2) viruses for another 24 h to detect abundance of miR-203. The empty vector (pCMV3-empty) and si-control served as negative controls. Data are expressed as the mean + SD of three independent experiments. * p

    Article Snippet: Then the data were normalized (average normalization) using IlluminaGUI in R. For mRNA expression profiling analysis, A549 cells and miR-203 KO A549 cells were infected with H5N1 viruses (MOI = 2) for 48 h and total RNA was extracted with TRIzol® Reagent (Thermo Fisher Scientific).

    Techniques: Infection, Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation

    Type I interferon (IFN) participates in transcriptional regulation of miR-203 to promote its expression directly. ( A ) A549 cells were infected with H5N1 (MOI = 2) viruses and harvested at different times (2, 6, 12, and 24 h). The abundance of IFN-α and IFN-β mRNA was measured by quantitative real-time PCR (qPCR). ( B and C ) A549 cells ( B ) and Vero cells ( C ) were treated with IFN-α (2000 units/ml) for 12 h, and expression of miR-203 was measured by qPCR. (D) A549 cells were co-transfected for 24 h with luciferase reporter vectors (pGL3-promoter) and a pRL-TK plasmid (pGL3-basic vectors were used as a negative control). Then, cells were treated with IFN-α for another 12 h followed by measurement of luciferase activity in a dual-luciferase assay. The results are presented as the normalized ratio of Firefly to Renilla luciferase activity. ( E ) Vero cells were mock-infected or infected with H5N1 (MOI = 2) viruses. Cells were harvested at different times (12, 24, 36, and 48 h), and abundance of miR-203 was measured by qPCR. Data are expressed as the mean + SD of three independent experiments. * p

    Journal: Scientific Reports

    Article Title: Up-regulation of microRNA-203 in influenza A virus infection inhibits viral replication by targeting DR1

    doi: 10.1038/s41598-018-25073-9

    Figure Lengend Snippet: Type I interferon (IFN) participates in transcriptional regulation of miR-203 to promote its expression directly. ( A ) A549 cells were infected with H5N1 (MOI = 2) viruses and harvested at different times (2, 6, 12, and 24 h). The abundance of IFN-α and IFN-β mRNA was measured by quantitative real-time PCR (qPCR). ( B and C ) A549 cells ( B ) and Vero cells ( C ) were treated with IFN-α (2000 units/ml) for 12 h, and expression of miR-203 was measured by qPCR. (D) A549 cells were co-transfected for 24 h with luciferase reporter vectors (pGL3-promoter) and a pRL-TK plasmid (pGL3-basic vectors were used as a negative control). Then, cells were treated with IFN-α for another 12 h followed by measurement of luciferase activity in a dual-luciferase assay. The results are presented as the normalized ratio of Firefly to Renilla luciferase activity. ( E ) Vero cells were mock-infected or infected with H5N1 (MOI = 2) viruses. Cells were harvested at different times (12, 24, 36, and 48 h), and abundance of miR-203 was measured by qPCR. Data are expressed as the mean + SD of three independent experiments. * p

    Article Snippet: Then the data were normalized (average normalization) using IlluminaGUI in R. For mRNA expression profiling analysis, A549 cells and miR-203 KO A549 cells were infected with H5N1 viruses (MOI = 2) for 48 h and total RNA was extracted with TRIzol® Reagent (Thermo Fisher Scientific).

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Plasmid Preparation, Negative Control, Activity Assay

    IAV-mediated demethylation of the miR-203 promoter region up-regulates expression of miR-203. ( A ) Bisulfite sequencing PCR of the miR-203 promoter region was performed in A549 cells mock-infected or infected with H5N1 (MOI = 2) viruses for 24 h. The sequenced region covered −600 to −10 bp within the miR-203 promoter region. Three lines denote three replicates in per group. Open circles, unmethylated; solid circles, methylated. ( B ) A549 cells were treated for 48 h with 5-aza-2′-deoxycytidine, a methylation inhibitor, at a final concentration of 0.5, 1, or 2 μM, and total RNA was purified to analyze abundance of miR-203 by quantitative real-time PCR (qPCR). ( C ) Vero cells were treated for 48 h with 5-aza-2′-deoxycytidine (final concentration, 1 μM) and total RNA purified. The abundance of miR-203 was measured by qPCR. Data are expressed as the mean + SD of three independent experiments. * p

    Journal: Scientific Reports

    Article Title: Up-regulation of microRNA-203 in influenza A virus infection inhibits viral replication by targeting DR1

    doi: 10.1038/s41598-018-25073-9

    Figure Lengend Snippet: IAV-mediated demethylation of the miR-203 promoter region up-regulates expression of miR-203. ( A ) Bisulfite sequencing PCR of the miR-203 promoter region was performed in A549 cells mock-infected or infected with H5N1 (MOI = 2) viruses for 24 h. The sequenced region covered −600 to −10 bp within the miR-203 promoter region. Three lines denote three replicates in per group. Open circles, unmethylated; solid circles, methylated. ( B ) A549 cells were treated for 48 h with 5-aza-2′-deoxycytidine, a methylation inhibitor, at a final concentration of 0.5, 1, or 2 μM, and total RNA was purified to analyze abundance of miR-203 by quantitative real-time PCR (qPCR). ( C ) Vero cells were treated for 48 h with 5-aza-2′-deoxycytidine (final concentration, 1 μM) and total RNA purified. The abundance of miR-203 was measured by qPCR. Data are expressed as the mean + SD of three independent experiments. * p

    Article Snippet: Then the data were normalized (average normalization) using IlluminaGUI in R. For mRNA expression profiling analysis, A549 cells and miR-203 KO A549 cells were infected with H5N1 viruses (MOI = 2) for 48 h and total RNA was extracted with TRIzol® Reagent (Thermo Fisher Scientific).

    Techniques: Expressing, Methylation Sequencing, Polymerase Chain Reaction, Infection, Methylation, Concentration Assay, Purification, Real-time Polymerase Chain Reaction

    Influenza A virus (IAV) infection of A549 cells up-regulates expression of miR-203. ( A ) Venn diagram illustrates the numbers of miRNAs identified in each group and the number of common miRNAs within the four groups. ( B ) Heatmap of miRNA expression in A549 cells infected with H1N1 (501) and H5N1 viruses for 24 or 48 h. Each value for the virus/mock average signal ratio was expressed as log10 when drawing the Heatmap. Red denotes up-regulation, while green denotes down-regulation. ( C ) A549 cells were mock-infected or infected with H1N1 (501), H1N1 (PR8), H3N2, H5N1, or H7N9 for 24 or 48 h, and abundance of miR-203 was assessed by quantitative real-time PCR (qPCR). ( D and E ) A549 cells were mock-infected or infected with H1N1 (501) ( D ) or H5N1 viruses ( E ). Cells were harvested at different times (12, 24, 36, and 48 h), and dynamic changes in miR-203 expression were assessed by qPCR. Data are expressed as the mean ± SD of three independent experiments. * p

    Journal: Scientific Reports

    Article Title: Up-regulation of microRNA-203 in influenza A virus infection inhibits viral replication by targeting DR1

    doi: 10.1038/s41598-018-25073-9

    Figure Lengend Snippet: Influenza A virus (IAV) infection of A549 cells up-regulates expression of miR-203. ( A ) Venn diagram illustrates the numbers of miRNAs identified in each group and the number of common miRNAs within the four groups. ( B ) Heatmap of miRNA expression in A549 cells infected with H1N1 (501) and H5N1 viruses for 24 or 48 h. Each value for the virus/mock average signal ratio was expressed as log10 when drawing the Heatmap. Red denotes up-regulation, while green denotes down-regulation. ( C ) A549 cells were mock-infected or infected with H1N1 (501), H1N1 (PR8), H3N2, H5N1, or H7N9 for 24 or 48 h, and abundance of miR-203 was assessed by quantitative real-time PCR (qPCR). ( D and E ) A549 cells were mock-infected or infected with H1N1 (501) ( D ) or H5N1 viruses ( E ). Cells were harvested at different times (12, 24, 36, and 48 h), and dynamic changes in miR-203 expression were assessed by qPCR. Data are expressed as the mean ± SD of three independent experiments. * p

    Article Snippet: Then the data were normalized (average normalization) using IlluminaGUI in R. For mRNA expression profiling analysis, A549 cells and miR-203 KO A549 cells were infected with H5N1 viruses (MOI = 2) for 48 h and total RNA was extracted with TRIzol® Reagent (Thermo Fisher Scientific).

    Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction

    Mice infected with H5N1 (2:6) mount robust T cell responses but show delayed viral clearance. (A-E) C57BL/6 mice (n=3-4/group) were infected with 100 PFU of H1N1 or H5N1 (2:6) and on day 8 pi, T cells from the lungs were isolated and evaluated in various assays. (A-C) Comparative analysis of lung CD8+ T cells from H5N1 (2:6) or H1N1 infected mice by NP or PA tetramer staining. (A) Representative FACS plots for NP or PA tetramer staining. (B) Relative frequency of tetramer positive CD8+ T cells. (C) Absolute numbers of virus specific CD8+ T cells. (D-E) Comparative analysis of cytokine production in T cells isolated from the lungs of H5N1 (2:6) or H1N1 infected mice. T cells were co-cultured with BMDC either infected with X31 (H3N2) or pulsed with NP peptide, and the frequencies of IFNγ and GrB producing T cells were analyzed by flow cytometry. (D) Relative frequency of cytokine producing CD8+ T cells. (E) Relative frequency of cytokine producing CD4+ T cells stimulated with NP peptide. (F) Evaluation of viral loads in the lungs of infected mice. C57BL/6 mice were infected with 100 PFU of H1N1 or H5N1 (2:6) and at various times pi, viral loads in the lungs were measured by standard plaque assay. (G-H) Ex vivo analysis of cytotoxic T cell functions. CFSE labeled splenocytes pulsed with NP peptide were co-cultured with lung CD8+ T cells for 8hrs, followed by staining with 7-AAD. Ex vivo cytotoxic effects of CD8+ T cells were evaluated by analyzing 7-AAD positive splenocytes. (G) Representative FACS plots for 7-AAD positive cells and (H) relative level of killing by T cells shown as percentage of 7-AAD positive cells. (I-J) In vivo analysis of cytotoxic T cell functions. (I) Representative FACS plots for in vivo killing of adoptively transferred NP pulsed splenocytes in H1N1 or H5N1 (2:6) virus infected mice and (J) relative level of kiiling by T cells. The values are expressed as mean ± SD. *, **, *** denotes significance of

    Journal: bioRxiv

    Article Title: Suppression of Cytotoxic T Cell Functions and Decreased Levels of Tissue Resident Memory T cell During H5N1 infection

    doi: 10.1101/2020.01.09.901132

    Figure Lengend Snippet: Mice infected with H5N1 (2:6) mount robust T cell responses but show delayed viral clearance. (A-E) C57BL/6 mice (n=3-4/group) were infected with 100 PFU of H1N1 or H5N1 (2:6) and on day 8 pi, T cells from the lungs were isolated and evaluated in various assays. (A-C) Comparative analysis of lung CD8+ T cells from H5N1 (2:6) or H1N1 infected mice by NP or PA tetramer staining. (A) Representative FACS plots for NP or PA tetramer staining. (B) Relative frequency of tetramer positive CD8+ T cells. (C) Absolute numbers of virus specific CD8+ T cells. (D-E) Comparative analysis of cytokine production in T cells isolated from the lungs of H5N1 (2:6) or H1N1 infected mice. T cells were co-cultured with BMDC either infected with X31 (H3N2) or pulsed with NP peptide, and the frequencies of IFNγ and GrB producing T cells were analyzed by flow cytometry. (D) Relative frequency of cytokine producing CD8+ T cells. (E) Relative frequency of cytokine producing CD4+ T cells stimulated with NP peptide. (F) Evaluation of viral loads in the lungs of infected mice. C57BL/6 mice were infected with 100 PFU of H1N1 or H5N1 (2:6) and at various times pi, viral loads in the lungs were measured by standard plaque assay. (G-H) Ex vivo analysis of cytotoxic T cell functions. CFSE labeled splenocytes pulsed with NP peptide were co-cultured with lung CD8+ T cells for 8hrs, followed by staining with 7-AAD. Ex vivo cytotoxic effects of CD8+ T cells were evaluated by analyzing 7-AAD positive splenocytes. (G) Representative FACS plots for 7-AAD positive cells and (H) relative level of killing by T cells shown as percentage of 7-AAD positive cells. (I-J) In vivo analysis of cytotoxic T cell functions. (I) Representative FACS plots for in vivo killing of adoptively transferred NP pulsed splenocytes in H1N1 or H5N1 (2:6) virus infected mice and (J) relative level of kiiling by T cells. The values are expressed as mean ± SD. *, **, *** denotes significance of

    Article Snippet: Briefly, 0.5 μg of each of the six pDZ plasmids representing PB2, PB1, PA, NP, NS and M from A/Puerto Rico/8/1934 (PR8) and two pPol-I plasmids representing the HA (low pathogenic) and NA segments of H5N1 were transfected into a cell mixture containing 293T-MDCK using Lipofectamine 2000 (Invitrogen).

    Techniques: Mouse Assay, Infection, Isolation, Staining, FACS, Cell Culture, Flow Cytometry, Plaque Assay, Ex Vivo, Labeling, In Vivo

    H5N1 (2:6) infection induces higher expression of PD-1 and IL-10 in cytotoxic T cells. C57BL/6 mice were infected with H5N1 (2:6) or H1N1 virus and on day 8pi, expression of PD-1 and production of IL-10 in CD8+ T cells were measured ex vivo upon co-culture with infected DC or peptide pulsed DC by flow cytometry. PD-L1 expression on inflammatory monocytes was also measured by flow cytometry. (A) Representative histograms showing expression of PD-1 on CD8+ T cells and PD-L1 on Ly6C + inflammatory monocytes. (B) quantification for PD-1 expression in CD8 T cells as MFI. (C) Quantification of PD-L1 expression in inflammatory monocytes and inflammatory DCs. (D) Absolute numbers of inflammatory monocytes and inflammatory DCs. (E) Quantification of IL-10 producing CD8+ T cell frequencies in X-31 infected DC-T cell co-culture (upper panel) and NP peptide pulsed DC-T cell co-culture (lower panel). (E) Cytokine production in CD4+ T cells. Frequencies of IFNγ and IL-10 or IL-10 alone producing CD4+ T cells in X-31 infected DC-T cell co-culture (left panels) and NP peptide pulsed DC-T cell co-culture (right panels). The values are expressed as mean ± SD. *, **, *** denotes significance of

    Journal: bioRxiv

    Article Title: Suppression of Cytotoxic T Cell Functions and Decreased Levels of Tissue Resident Memory T cell During H5N1 infection

    doi: 10.1101/2020.01.09.901132

    Figure Lengend Snippet: H5N1 (2:6) infection induces higher expression of PD-1 and IL-10 in cytotoxic T cells. C57BL/6 mice were infected with H5N1 (2:6) or H1N1 virus and on day 8pi, expression of PD-1 and production of IL-10 in CD8+ T cells were measured ex vivo upon co-culture with infected DC or peptide pulsed DC by flow cytometry. PD-L1 expression on inflammatory monocytes was also measured by flow cytometry. (A) Representative histograms showing expression of PD-1 on CD8+ T cells and PD-L1 on Ly6C + inflammatory monocytes. (B) quantification for PD-1 expression in CD8 T cells as MFI. (C) Quantification of PD-L1 expression in inflammatory monocytes and inflammatory DCs. (D) Absolute numbers of inflammatory monocytes and inflammatory DCs. (E) Quantification of IL-10 producing CD8+ T cell frequencies in X-31 infected DC-T cell co-culture (upper panel) and NP peptide pulsed DC-T cell co-culture (lower panel). (E) Cytokine production in CD4+ T cells. Frequencies of IFNγ and IL-10 or IL-10 alone producing CD4+ T cells in X-31 infected DC-T cell co-culture (left panels) and NP peptide pulsed DC-T cell co-culture (right panels). The values are expressed as mean ± SD. *, **, *** denotes significance of

    Article Snippet: Briefly, 0.5 μg of each of the six pDZ plasmids representing PB2, PB1, PA, NP, NS and M from A/Puerto Rico/8/1934 (PR8) and two pPol-I plasmids representing the HA (low pathogenic) and NA segments of H5N1 were transfected into a cell mixture containing 293T-MDCK using Lipofectamine 2000 (Invitrogen).

    Techniques: Infection, Expressing, Mouse Assay, Ex Vivo, Co-Culture Assay, Flow Cytometry

    H5N1 virus stimulates higher activation of dendritic cells in the lungs. (A-D) C57BL/6 mice (n=3-4 per group) were infected with 5×10 4 PFU of H1N1-GFP or H5N1-GFP and cell surface expression of co-stimulatory molecule CD86 was measured flow cytometry. (A) Representative histograms comparing CD86 expression on lung DC subsets. (B) Quantification of CD86 expression on lung DC subsets. CD86 expression levels are shown as mean fluorescent intensity (MFI). (C-D) Comparison of CD86 expression on inflammatory DC and monocytes. (C) Histogram plot of CD86 expression. (D) Quantification of CD86 expression. (E-F) Comparison of CD86 expression on DC subsets in mice infected with H5N1 (2:6) and H1N1. C57BL/6 mice were infected with 100 PFU of H1N1 or H5N1 (2:6) and CD86 expression was measured flow cytometry. (E) Representative histograms of CD86 expression on lung DC subsets. (F) Quantification for panel E shown as MFI. (G) Comparison of Mx1, ISG15, and IFNβ expressions between H5N1 (2:6) and H1N1 infected lungs. Total RNA was extracted from lung homogenates of infected mice isolated on day 4 pi and subjected to qRT-PCR analysis. The values are expressed as mean ± SD. *, **, *** denotes significance of

    Journal: bioRxiv

    Article Title: Suppression of Cytotoxic T Cell Functions and Decreased Levels of Tissue Resident Memory T cell During H5N1 infection

    doi: 10.1101/2020.01.09.901132

    Figure Lengend Snippet: H5N1 virus stimulates higher activation of dendritic cells in the lungs. (A-D) C57BL/6 mice (n=3-4 per group) were infected with 5×10 4 PFU of H1N1-GFP or H5N1-GFP and cell surface expression of co-stimulatory molecule CD86 was measured flow cytometry. (A) Representative histograms comparing CD86 expression on lung DC subsets. (B) Quantification of CD86 expression on lung DC subsets. CD86 expression levels are shown as mean fluorescent intensity (MFI). (C-D) Comparison of CD86 expression on inflammatory DC and monocytes. (C) Histogram plot of CD86 expression. (D) Quantification of CD86 expression. (E-F) Comparison of CD86 expression on DC subsets in mice infected with H5N1 (2:6) and H1N1. C57BL/6 mice were infected with 100 PFU of H1N1 or H5N1 (2:6) and CD86 expression was measured flow cytometry. (E) Representative histograms of CD86 expression on lung DC subsets. (F) Quantification for panel E shown as MFI. (G) Comparison of Mx1, ISG15, and IFNβ expressions between H5N1 (2:6) and H1N1 infected lungs. Total RNA was extracted from lung homogenates of infected mice isolated on day 4 pi and subjected to qRT-PCR analysis. The values are expressed as mean ± SD. *, **, *** denotes significance of

    Article Snippet: Briefly, 0.5 μg of each of the six pDZ plasmids representing PB2, PB1, PA, NP, NS and M from A/Puerto Rico/8/1934 (PR8) and two pPol-I plasmids representing the HA (low pathogenic) and NA segments of H5N1 were transfected into a cell mixture containing 293T-MDCK using Lipofectamine 2000 (Invitrogen).

    Techniques: Activation Assay, Mouse Assay, Infection, Expressing, Flow Cytometry, Isolation, Quantitative RT-PCR

    H5N1 (2:6) infection results in decreased numbers of tissue resident memory T cells in the lung parenchyma. C57BL/6 mice (n=3) were infected with H5N1 (2:6) or H1N1 virus and on day 30 pi, the frequency and absolute number of lung resident memory cells was analyzed by flow cytometry. (A) Lung resident memory CD8+ T cell responses. Representative FACS plots for lung resident memory CD8+ T cells (gated on NP+ 366-374 CD44+ CD8α+ CD8β-T cells) that display CD44+ CD69+ CD103 hi phenotype (left) and the absolute numbers of tissue resident memory CD8+ T cells (right). (B) Lung resident memory CD4+ T cell responses. Representative FACS plots (left) and absolute numbers of CD4+ T cells (right) are shown. (C) Heterologous challenges with H3N2 (X-31) virus. Mice previously infected with 50 PFU of H1N1 or H5N2(2:6) virus were challenged with H3N2 (X-31) strain at a dose of 5×10 6 PFU. The values are expressed as mean ± SD. * denotes statistical significance of

    Journal: bioRxiv

    Article Title: Suppression of Cytotoxic T Cell Functions and Decreased Levels of Tissue Resident Memory T cell During H5N1 infection

    doi: 10.1101/2020.01.09.901132

    Figure Lengend Snippet: H5N1 (2:6) infection results in decreased numbers of tissue resident memory T cells in the lung parenchyma. C57BL/6 mice (n=3) were infected with H5N1 (2:6) or H1N1 virus and on day 30 pi, the frequency and absolute number of lung resident memory cells was analyzed by flow cytometry. (A) Lung resident memory CD8+ T cell responses. Representative FACS plots for lung resident memory CD8+ T cells (gated on NP+ 366-374 CD44+ CD8α+ CD8β-T cells) that display CD44+ CD69+ CD103 hi phenotype (left) and the absolute numbers of tissue resident memory CD8+ T cells (right). (B) Lung resident memory CD4+ T cell responses. Representative FACS plots (left) and absolute numbers of CD4+ T cells (right) are shown. (C) Heterologous challenges with H3N2 (X-31) virus. Mice previously infected with 50 PFU of H1N1 or H5N2(2:6) virus were challenged with H3N2 (X-31) strain at a dose of 5×10 6 PFU. The values are expressed as mean ± SD. * denotes statistical significance of

    Article Snippet: Briefly, 0.5 μg of each of the six pDZ plasmids representing PB2, PB1, PA, NP, NS and M from A/Puerto Rico/8/1934 (PR8) and two pPol-I plasmids representing the HA (low pathogenic) and NA segments of H5N1 were transfected into a cell mixture containing 293T-MDCK using Lipofectamine 2000 (Invitrogen).

    Techniques: Infection, Mouse Assay, Flow Cytometry, FACS

    H5N1 (2:6) infection induces higher upregulation of CCR7 and migration of lung DC. C57BL/6 mice (n=3-4 per group) were intranasally infected with 100 PFU of H1N1 or H5N1 (2:6) and lung DC activation and migration was analyzed by flow cytometry. (A) Representative histogram comparing the expression of CCR7 on CD103+ DC or CD11b+ DC subsets on day 2 pi. (B) Quantification for panel A. CCR7 expression levels are shown as MFI. (C-D) C57BL/6 mice were infected with 100 PFU of H5N1 (2:6) or H1N1 and instilled with 50μl of 8mM CFSE at day 2pi. After 16h, the number of CFSE+ migratory DC present in the MLN was analyzed by flow cytometry. (C) Representative FACS plots showing CFSE+ population in the MLN. (D) Relative levels of CFSE positive CD103+ and CD11b+ DC in the MLN. (E) Bar charts showing the number of CFSE+ DC subsets in the MLN. (F) Bar chart showing total numbers of DC subsets in the MLN. The values are expressed as mean ± SD. * denotes statistical significance of

    Journal: bioRxiv

    Article Title: Suppression of Cytotoxic T Cell Functions and Decreased Levels of Tissue Resident Memory T cell During H5N1 infection

    doi: 10.1101/2020.01.09.901132

    Figure Lengend Snippet: H5N1 (2:6) infection induces higher upregulation of CCR7 and migration of lung DC. C57BL/6 mice (n=3-4 per group) were intranasally infected with 100 PFU of H1N1 or H5N1 (2:6) and lung DC activation and migration was analyzed by flow cytometry. (A) Representative histogram comparing the expression of CCR7 on CD103+ DC or CD11b+ DC subsets on day 2 pi. (B) Quantification for panel A. CCR7 expression levels are shown as MFI. (C-D) C57BL/6 mice were infected with 100 PFU of H5N1 (2:6) or H1N1 and instilled with 50μl of 8mM CFSE at day 2pi. After 16h, the number of CFSE+ migratory DC present in the MLN was analyzed by flow cytometry. (C) Representative FACS plots showing CFSE+ population in the MLN. (D) Relative levels of CFSE positive CD103+ and CD11b+ DC in the MLN. (E) Bar charts showing the number of CFSE+ DC subsets in the MLN. (F) Bar chart showing total numbers of DC subsets in the MLN. The values are expressed as mean ± SD. * denotes statistical significance of

    Article Snippet: Briefly, 0.5 μg of each of the six pDZ plasmids representing PB2, PB1, PA, NP, NS and M from A/Puerto Rico/8/1934 (PR8) and two pPol-I plasmids representing the HA (low pathogenic) and NA segments of H5N1 were transfected into a cell mixture containing 293T-MDCK using Lipofectamine 2000 (Invitrogen).

    Techniques: Infection, Migration, Mouse Assay, Activation Assay, Flow Cytometry, Expressing, FACS