Journal: African Journal of Traditional, Complementary, and Alternative Medicines

Article Title: Anti-Tumour Effects of Polysaccharides Isolated from Artemisia Annua L by Inducing Cell Apoptosis and Immunomodulatory Anti-Hepatoma Effects of Polysaccharides

doi:

Figure Lengend Snippet: HQG treatment facilitates immunomodulation in lymphocytes: Prepared mice spleen lymphocytes from the tumour-bearing mice with (B) or without (A) HQG administration were fixed, stained with fluorescent-labelled CD4+ or CD8+ antibodies for 30 min followed by flow cytometry analysis to measure the percentage of CD4+ or CD8+ T-lymphocytes subpopulation. See detailed experimental protocol in Materials and Methods. UL means the percent of CD8+ cells, equal to the Th1 cells; LR means the percent of CD4+ cells, equal to the Th2 cells; LL means the CD4− CD8− cells; UR means the CD4+CD8+ cells. Prepared mice spleen lymphocytes from normal mice (C, a), tumour-bearing mice without (C, b, c) HQG or tumour-bearing mice with HQG administration (C, d, f) were fixed, stained and labelled antibodies against intracellular cytokines IL-4 and IFN-γ for 30 min followed by flow cytometry analysis to measure the percentage of cells secreting IL-4 (C, upper panel) or IFN-γ (C, lower panel). See detailed experimental protocol in Materials and Methods. UL: the percentage of IL-4+ cells; LR: the percentage of IFN-γ+ cells.

Article Snippet: Labelled antibodies against intracellular cytokines IL-4 and IFN-γ (BD Pharmingen; San Diego, CA, USA) were added in tubes with incubation for 30 min in darkness according to the antibody usage in instruction manual.

Techniques: Staining, Flow Cytometry

Journal: African Journal of Traditional, Complementary, and Alternative Medicines

Article Title: Anti-Tumour Effects of Polysaccharides Isolated from Artemisia Annua L by Inducing Cell Apoptosis and Immunomodulatory Anti-Hepatoma Effects of Polysaccharides

doi:

Figure Lengend Snippet: The average percentage of cells with IL-4 or IFN-γsecretion

Article Snippet: Labelled antibodies against intracellular cytokines IL-4 and IFN-γ (BD Pharmingen; San Diego, CA, USA) were added in tubes with incubation for 30 min in darkness according to the antibody usage in instruction manual.

Techniques:

Journal: Science signaling

Article Title: Instructive roles for agonist binding parameters in determining the functional bandwidth of cytokine receptor signaling

doi: 10.1126/scisignal.aab2677

Figure Lengend Snippet: (A) Surface staining of IL-13-displaying yeast with biotinylated IL-13Rα1 and IL-4Rα ECDs. IL-13Rα1 binds IL-13 displayed on yeast independent of IL-4Rα (second panel from the left), whereas IL-4Rα on the other hand requires the presence of IL-13Rα1 to bind IL-13 recapitulating the cooperativity that has been observed previously 15 (right two panels). (B) Crystal structure of the IL-13 ternary complex (IL-13Rα1 in green, IL-4Rα in blue, and IL-13 in gold). Magnifications of the site II (A–D helixes) and site III (C–D loop) binding interfaces highlighting the amino acids on IL-13 involved in IL-13Rα1 binding are shown. (C) Sequential enrichment of IL-13 agonists. The site-specifically mutagenized library was selected against decreasing concentrations of IL-13Rα1 for five rounds. Clones were selected from each round to isolate variants with a range of IL-13Rα1 binding affinities.

Article Snippet: Human IL-13, the IL-13Rα1 ectodomain (amino acids 1-310) and the IL-4Rα ectodomain (amino acids 1-202) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and a C-terminal hexahistidine tag and produced using the baculovirus expression system, as described in ( 15 ).

Techniques: Staining, Binding Assay, Clone Assay

Journal: Science signaling

Article Title: Instructive roles for agonist binding parameters in determining the functional bandwidth of cytokine receptor signaling

doi: 10.1126/scisignal.aab2677

Figure Lengend Snippet: (A) Binding isotherms of the IL-13 agonists displayed on the surface of yeast. Yeast-displayed IL-13 agonists were incubated with the indicated concentrations of biotinylated IL-13Rα1 ECD, stained with Alexa-647-coupled Streptavidin, and assessed via flow cytometry. Sigmoidal curves were fitted using Prism software (GraphPad). (B) Normalized KD binding affinities of the recombinant IL-13 agonists determined via surface plasmon resonance (SPR). The IL-13 KD was fixed to one and other agonists were normalized accordingly. (C) Summary of the amino acids mutated in the IL-13 agonists as well as the kon, koff and KD binding parameters values obtained from SPR experiments.

Article Snippet: Human IL-13, the IL-13Rα1 ectodomain (amino acids 1-310) and the IL-4Rα ectodomain (amino acids 1-202) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and a C-terminal hexahistidine tag and produced using the baculovirus expression system, as described in ( 15 ).

Techniques: Binding Assay, Incubation, Staining, Flow Cytometry, Software, Recombinant, SPR Assay

Journal: Science signaling

Article Title: Instructive roles for agonist binding parameters in determining the functional bandwidth of cytokine receptor signaling

doi: 10.1126/scisignal.aab2677

Figure Lengend Snippet: (A) Crystal structure of the IL-13 agonist A11 (brown) in complex with IL-13Rα1 (green) and IL-4Rα (blue). (B) Overlay of the IL-13 wt and IL-13 A11 ternary complex crystal structures. Note that IL-13 A11 engages the two receptor chains with identical geometry to that of IL-13 wt (RMSD=0.738). (C–D) Schematic representation of ligand-receptor contact interaction of IL-13Rα1 with IL-13 (top half) versus IL-13 A11 (bottom half). Black lines and arrows represent van der Waals interactions, blue lines represent hydrogen bonds and red lines designate aromatic amino acid interactions.

Article Snippet: Human IL-13, the IL-13Rα1 ectodomain (amino acids 1-310) and the IL-4Rα ectodomain (amino acids 1-202) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and a C-terminal hexahistidine tag and produced using the baculovirus expression system, as described in ( 15 ).

Techniques:

Journal: Science signaling

Article Title: Instructive roles for agonist binding parameters in determining the functional bandwidth of cytokine receptor signaling

doi: 10.1126/scisignal.aab2677

Figure Lengend Snippet: (A–C) IL-13 receptor dimerization detected by dual color single molecule imaging and co-locomotion-analysis. Images identifying an individual IL-13Rα1/IL-4Rα dimer in the presence of 200 nM IL-13 at different time points of a 500 frame (16 s) movie (A), the distance between the two molecules in each frame (B) and an overlay of the individual trajectories of IL-13Rα1 (magenta) and IL-4Rα (green) (C) are presented. Scale bars: 1μm in panels A and C. (D) Diffusion properties represented as step length distribution obtained for IL-13Rα1 in the absence (red) and presence (orange) of IL-13. For comparison, the step length distribution obtained for receptor dimers identified by co-locomotion analysis is shown (blue). (E) Cumulative probability of detecting an IL-13Rα1 molecule in the vicinity of an IL-4Rα molecule as a function of the squared distance, corrected for the statistical probability, in the presence and absence of IL-13 wt. For comparison, the cumulative correlation probability obtained in the presence of IL-13 DN is shown. (F) Correlation fraction for the IL-13Rα1 and IL-4Rα receptor subunit as a function of IL-13 agonist affinity. (G) Assembly and dissociation of an individual IL-13Rα1/IL-4Rα dimer in presence of agonist D7. Overlaying trajectories are shown in white. Scale bar 0.5 μm. (H) Average lifetime of the IL-13Rα1/IL-4Rα dimer as a function of agonist affinity.

Article Snippet: Human IL-13, the IL-13Rα1 ectodomain (amino acids 1-310) and the IL-4Rα ectodomain (amino acids 1-202) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and a C-terminal hexahistidine tag and produced using the baculovirus expression system, as described in ( 15 ).

Techniques: Imaging, Diffusion-based Assay

Journal: Science signaling

Article Title: Instructive roles for agonist binding parameters in determining the functional bandwidth of cytokine receptor signaling

doi: 10.1126/scisignal.aab2677

Figure Lengend Snippet: (A) A549 cells were stimulated with the indicated doses of IL-13 wt or IL-13 agonists and the levels of pSTAT6 were analyzed by flow cytometry, using anti-pSTAT6-specific antibodies coupled to fluorescent dyes. Sigmoidal curves were fitted with Prism software (GraphPad) (B) Dot plot graph in which the normalized pSTAT6 EC50 values are plotted against the normalized KD binding values for each IL-13 agonist. IL-13 pSTAT6 EC50 and KD values were set to one and agonist parameter values were normalized accordingly. Agonists binding within 100-fold of IL-13 wt in either direction lead to similar profiles of STAT6 activation, generating the denoted buffering region. (C) A549 cells were stimulated with 200 nM of IL-13 wt or the indicated IL-13 agonists and the kinetics of STAT6 phosphorylation were analyzed by flow cytometry. (D) Total levels of pSTAT6, calculated by measuring Area Under the Curve (AUC) from the kinetics studies shown in Figure S6. Decrease in ligand concentration only marginal affect total amount of pSTAT6 activated. (E) HeLa cells were stimulated for the indicated time-lengths with 200 nM of IL-13 or the indicated agonists and the translocation of mEGFP-STAT6 to the nucleus was measured via fluorescence microscopy. (F) A549 cells were transfected with different concentrations of IL-13Rα1 siRNA to obtain a range of IL-13Rα1 silencing. Cells were then stimulated with IL-13 wt or engineered agonists and pSTAT6 levels were quantified by flow cytometry. (G) TF-1 EC50 proliferation values obtained from fitting sigmoidal profiles to the dose-response curves shown in Fig. S10b. (H) Upregulation of CD86 in monocytes stimulated with the indicated doses of IL-13 wt and IL-13 mutants.

Article Snippet: Human IL-13, the IL-13Rα1 ectodomain (amino acids 1-310) and the IL-4Rα ectodomain (amino acids 1-202) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and a C-terminal hexahistidine tag and produced using the baculovirus expression system, as described in ( 15 ).

Techniques: Flow Cytometry, Software, Binding Assay, Activation Assay, Concentration Assay, Translocation Assay, Fluorescence, Microscopy, Transfection

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: Transcriptional profiling of PHH infected with HCVcc at an MOI of 0.2. (A) Relative expression of genes involved in the PAMP recognition pathway showing data from three replicates at 24 h or 48 h post-infection normalized to naive PHH at the same time points. HUGO nomenclature for the genes is to the left. Statistical analysis was performed using one-way ANOVA and P values reported in (B) Expression levels of IFNAR1 and IL28RA transcripts in naive (closed circle) and HCVcc-infected (open circle) PHH. The data points represent the RMA values from each individual analyte and horizontal bars indicate the mean levels from three biological replicates. The data shown is representative of two independent experiments.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Infection, Expressing

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: iHLCs were infected with HCVcc (MOI of 0.2), GT-1b HCVser or mock infected, and subsequently maintained in culture with medium replacement every 2 days. At 24 h post-infection, the NS3 PI ASV (0.5 μM) or vehicle control dimethyl sulfoxide (DMSO) was added to cell cultures during media replenishment. (A) Persistent HCVcc replication as measured by the detection of the virally-encoded core antigen and expression of type I (IFNAR1, IFNAR2) and type III IFN (IL28RA, IL10RB) co-receptor subunits in cells were monitored by Western immunoblotting at the indicated time points, with β-actin used as loading control. Right panel; Relative intensity of IFNAR1 detected in HCVcc infected cells maintained in the presence (open triangle) or absence (open square) of ASV treatment was quantified by densitometry analysis, normalized to β-actin levels in each sample, and expressed as a percentage relative to mock-infected cells. Data are representative of three independent Western immunoblot analyses. (B) Co-localization of IFNAR1 and HCV-core positive iHLCs were assessed by fluorescence microscopy. On Day 6 post-infection, naive and HCVcc-infected iHLCs were fixed, permeabilized and stained with antibodies to IFNAR1 (red) and HCV core antigen (green) as indicated. Nuclei were counterstained with Hoechst dye (blue) as shown in the overlays. (C) iHLC cultures infected in parallel with GT-1b HCVser or HCVcc were harvested at the indicated time points and IFNAR1 copy numbers estimated by quantitative RT-PCR following normalization to cellular GAPDH levels in each sample. Results are expressed as mean ± standard deviations (n = 4). Statistical analysis was performed by Bonferroni’s multiple comparison tests: **, P < 0.05; ***, P < 0.001. (D) Cell lysates were harvested at the indicated time points and protein levels of IFNAR1 and IL28RA were examined by Western immunoblotting. Susceptibility of HCVser to the NS3 PI ASV was determined using an antibody directed against NS3. Arrows indicate the presence of the processed and unprocessed forms of the HCV-encoded NS3/4A protease in infected iHLCs. Detection of β-actin served as loading control. Right panel; Relative intensity of IFNAR1 detected in HCVser infected cells maintained in the presence (open triangle) or absence (open square) of ASV treatment was quantified by densitometry analysis, normalized to β-actin levels in each sample, and expressed as a percentage relative to mock-infected cells. Data are representative of two independent Western immunoblot analyses.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Infection, Control, Expressing, Western Blot, Fluorescence, Microscopy, Staining, Quantitative RT-PCR, Comparison

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: (A) Naive and HCVcc infected iHLC cultures maintained in presence or absence of the IL28RA nAb were treated for 15 minutes with 10 ng/mL or 100 ng/mL of alfa or Lambda. Cell lysates were then prepared, and equal amounts of proteins subjected to Western immunoblotting to examine the levels of STAT1 phosphorylation using an antibody directed against phospho STAT1 (pSTAT1; Tyr701). Detection of total STAT1 served as loading control to ensure that equivalent amounts of protein were analyzed among samples. (B) Phosphorylation of STAT1 in iHLCs was evaluated upon stimulation using the Luminex bead-based assay. MFI values were reported as mean values of three independent cultures. Error bars show the standard deviations. Two-way ANOVA statistical analysis was performed using Bonferroni post test: ***, P < 0.001.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Infection, Western Blot, Phospho-proteomics, Control, Luminex, Bead-based Assay

Journal: PLoS ONE

Article Title: Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

doi: 10.1371/journal.pone.0121734

Figure Lengend Snippet: (A) Expression of Mx1 protein (red) and HCV-core antigen (green) was monitored in HCVcc-infected iHLC cultures maintained in presence or absence of the IL28RA nAb (10 μg/mL). Dual immunostaining was performed as described in the legend for . Nuclei were counterstained with Hoechst dye (blue) as shown in the overlays. (B) Induction of Mx1 expression (red) was assessed in Naive iHLCs 24 h following stimulation with 10 ng/mL of alfa (a) or Lambda (b). iHLC cultures infected with HCVcc were maintained in the presence (left panels) or absence (right panels) of the IL28RA nAb (10 μg/mL). On Day 6 post-infection, cells were treated as indicated with 10 ng/mL of alfa or Lambda for 24 h, and then dual immunostaining was performed using antibodies directed against Mx1 (red) and the HCV-core antigen (green). Arrows indicate examples of different HCV-Mx1 co-localization patterns in overlaid optical field. Scale bar, 60 μm.

Article Snippet: Briefly, iHLCs were infected with HCVcc at multiplicity of infection (MOI) of 0.2 and incubated for 4–6 days in presence or absence of a neutralizing monoclonal antibody directed against the extracellular domain of the IL28RA receptor subunit (ZymoGenetics).

Techniques: Expressing, Infection, Immunostaining