cytokines Search Results


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  • 99
    Millipore cytokines
    Pleural injection of carrageenan caused by 4 h an increase in the release of the <t>cytokines,</t> tumour necrosis factor alpha (TNFα, A), interleukin-1β (IL-1β, B), interleukin-6 (IL-6, C) and interleukin-10 (IL-10, D). When tested at the highest dose, M40403 (20 mg kg −1 , intraperitoneally) inhibited TNFα, IL-1β and IL-6 (A – C) but had no effect on IL-10 (D). Each value is the mean±s.e.mean for n =10 experiments. * P
    Cytokines, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7384 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson cytokines
    The effect of P2X 7 receptor inhibition in E. coli -induced sepsis . ARD6 was injected iv into anesthetized balb/cj mice (low dose—41 million). Two hours prior to ARD6 injection the animals were administered the P2X 7 antagonist BBG or vehicle (50 mg kg −1 , subcutaneously). (A) Kaplan-Meier plot shows survival, n = 6 for controls and 10–12 for ARD6 with or without BBG. (B) Absorbance of plasma as an indication of plasma haemolysis 2.5 h after ARD6 injection, n = 13 for all four groups. (C) Colony forming units from blood samples 2.5 h after a low number of bacteria (D) Levels of the <t>cytokines</t> TNFα, KC, IL-1β, and IL-6 2.5 h after ARD6 injection, n = 14 for ARD6 and 13 for ARD6 + BBG. * p
    Cytokines, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 15278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad cytokines
    Several <t>cytokines</t> are increased significantly in the sera of infected STAT1 KO mice. In WT mice, a multiplex assay showed small to moderate but significant changes for CCL4, CCL2, and IL-2 levels at day 3 postinfection (A) and for G-CSF, CCL2, IL-5, IL-12,
    Cytokines, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 5575 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson intracellular cytokine staining
    Extracellular potassium mediated T cell suppression requires intact PP2A activity and is associated with elevations in intracellular potassium ( a ) Selected phosphatase inhibitors from a pharmacologic screen for IFN-γ production of CD8 + T cells in the presence of elevated [K + ]e, depicted as the ratio of IFN-γ expression in Ctrl / elevated [K + ]e conditions in the presence of indicated phosphatase inhibitors ( b ) CD8 + T cell phosphorylation of pAkt S473 and pS6 S235/6 10 minutes following TCR cross-linking in the indicated conditions. ( c ) Compiled analysis of IFN-γ production by retrovirally transduced CD8 + Thy1.1 + T cells following TCR stimulation in the indicated conditions. ( d ) Pictorial representation of the resultant intracellular changes in plasma membrane potential ( V m ) and intracellular potassium concentration ([K + ]i) in the presence of elevated extracellular potassium ([K + ]e). ( e ) Relative cytoplasmic V m of CD8 + T cells in the indicated conditions represented as relative fluorescence of the voltage-sensitive fluorescent indicator DiSBAC 4 (3). ( f ) Pictorial representation of the resultant intracellular changes in plasma membrane potential ( V m ) and intracellular potassium concentration ([K + ]i) in the presence of the ionophore gramicidin. ( g ) Relative cytoplasmic V m of CD8 + T cells in the indicated conditions assayed as in ( e ) with representative flow cytometry in ( h ). ( i ) IFN-γ production by CD8 + T cells following TCR induced stimulation in the indicated conditions. ( j ) Representative flow cytometry representing [K + ]i of CD8 + T cells as relative fluorescence of the potassium sensitive dye Asante-Green 4. ( k ) Relative [K + ]i of CD8 + T cells in the indicated conditions assayed as relative fluorescence of the potassium sensitive dye Asante-Green 4. ( l ) Representative flow cytometry of <t>cytokine</t> expression by CD8 + T cells following TCR stimulation in the indicated conditions with compiled quantification in ( m ). Error bars represent mean ± SEM. *P
    Intracellular Cytokine Staining, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 4683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson cytokine staining
    <t>Cytokine</t> production and transcription factor expression in antigen reactive CD4 + T cells. At day 10 days post first type II collagen (CII) immunization, draining lymph node (LN) cells from C57BL/6 mice (n = 3 or 4) and RORγt Tg mice (n = 3 or 4) were cultured with or without 100 μg/mL of denatured CII for 72 h. (A) IL-17 and IFNγ levels in supernatants measured by ELISA. (B) Cells were analyzed by staining for intracellular IL-17 and IFNγ. Data represent gated CD3 + and CD4 + . Bar graphs show percentages of cytokine-positive cells. (C) The expression levels of RORγt and T-bet were evaluated by intracellular staining. Data represent gated CD3 + and CD4 + . In the histograms, the gray shaded area represents data for the isotype control. Bar graphs show mean fluorescence intensity (MFI) of T-bet and RORγt Tg. (D) IL-10 levels in supernatants measured by ELISA. (E , F) Cells were analyzed by staining for intracellular IL-17 and IL-10 (E) , and propium iodide (PI) and Annexin V (F) . Data represent gated CD3 + and CD4 + . Data are representative of two independent experiments with similar results, and are mean ± standard error of the mean. * P
    Cytokine Staining, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 6235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Meso Scale Diagnostics LLC cytokines
    In vitro Stimulation of cytokine release in human PBMCs by REP 9 and REP 9C . Human PBMCs were exposed to no compound, CPG 7909, REP 9, and REP 9C, and levels of secreted <t>cytokines</t> after 48 h of induction were determined as described in the material and methods. Data is depicted on different scales (7, 70, 300, and 20,000 pg/ml) to demonstrate differences in cytokine levels measured. Values plotted are mean +/- standard deviation (n = 3). * = statistically insignificant difference in cytokine concentrations compared to untreated controls, + = statistically significant reduction in cytokine concentrations (REP 9C versus REP 9) (p
    Cytokines, supplied by Meso Scale Diagnostics LLC, used in various techniques. Bioz Stars score: 92/100, based on 1481 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Pharmingen cytokines
    Quantification of the <t>cytokines</t> secreted by DCs in presence of NPs DCs were incubated at 37°C in the presence of LPS (2μg/ml) and/or gold NPs (0.5mM) for 24 hours, then the supernatants were collected and used to determine the concentration of IL-6 and IL-12p70 as indicated in the material and methods section. A control was performed without additive: LPS and NPs. The results correspond to the mean value of three independents experiments. ** denotes a significant difference between the indicated experimental groups (p
    Cytokines, supplied by Pharmingen, used in various techniques. Bioz Stars score: 93/100, based on 899 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher cytokines
    The antimicrobial peptide cathelicidin induces IL-17 production by CD4 + T cells. Splenocytes isolated from C57BL/6J mice were cultured in Th1-driving conditions for 4 days, Th2-driving for 4 days or Th17-driving for 2 days in the presence or absence of 2.5μM murine cathelicidin. (A,B) Expression of <t>cytokines</t> was determined by intracellular flow cytometry. (C) sorted CD4 + T cells were cultured alone in Th17-driving conditions with 2.5μM cathelicidin. (D) Production of IL-17A following increasing doses of cathelicidin (at 48 hours) and (E) production over increasing days in culture was assessed. (F) Geometric mean of IL-17A expression was also assessed on day 2 of culture. (G) After 3 days in culture the culture medium was collected and ELISA used to quantify IL-17A protein. (H) Alternative cathelicidins were used – human LL-37 and the D-enantiomer D-LL37. (I) Representative flow cytometry plots showing RORγt expression - (J) RORγt expression increased over time following cathelicidin exposure and (K) in the presence of TGF-β. Data shown are individual mice used in separate experiments, with line at median. Statistical significance was determined using a two-way repeated measures ANOVA with a Bonferroni multiple comparison post-test (E, J), a two-way ANOVA with Bonferroni correction (K), a paired t-test (B, C, F, G) or a one-way ANOVA with a Dunnett’s multiple comparison post-test (D. H).
    Cytokines, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 11463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson cytokine bead array
    Blocking the interaction between CD160 and HVEM enhances CMV and HIV-specific CD8 T cell proliferation and <t>cytokine</t> production. HIV-infected individuals(n = 11) were stimulated with HLA-restricted CMV and HIV peptides in the presence of blocking antibodies to HVEM and/or PD-L1. Dying cells were eliminated with an amine-reactive viability dye and PBMCs were stained at day 6 with HLA class I matched-tetramers and mAbs to CD3 and CD8. (A) Representative flow cytometry plots of an HIV-infected patient stimulated for 6 days with a CMV and HIV peptide in the presence of isotype, αPD-L1 and/or αHVEM blocking antibodies. Scatter plots represent the median fold increase in (B) CMV and (C) HIV-specific proliferation (Tetramer + /CFSE low ) compared to the isotype control. Each dot represents a CMV or HIV tetramer-specific response. P -values were determined by the Wilcoxon matched pairs test.
    Cytokine Bead Array, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 679 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    4Gene murine il 4 gene blocks th2 cytokine responses
    Histologic staining for mucin after <t>cytokine</t> blockade. Light micrographs of lung sections stained with AB/PAS from IL-9 transgene-negative ( a ) or –positive ( b – f ) mice that were housed for 14 days with doxycycline-supplemented food. During the entire time of IL-9 transgene induction selected groups of animals received Ab’s: ( c ) control Ab’s, hIgG; ( d ) <t>anti–IL-4;</t> ( e ) sIL-13Rα-Fc; ( f ) anti–IL-4 and sIL-13Rα-Fc. Lung sections from transgene-positive mice that received anti–IL-4 Ab’s, control Ab’s, or no Ab’s showed strong positive (magenta) staining for mucin in airway epithelial cells (arrows) and accumulation of inflammatory cells near blood vessels and airways ( b – d ). Infiltration of lung tissue with inflammatory cells or positive staining for mucin was not observed in sections from transgene-positive mice that received sIL-13Rα-Fc alone ( e ), or in combination with anti–IL-4 ( f ), or in transgene-negative mice ( a ). Original magnification ×300.
    Murine Il 4 Gene Blocks Th2 Cytokine Responses, supplied by 4Gene, used in various techniques. Bioz Stars score: 90/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    RayBiotech human cytokine antibody array
    <t>Cytokine</t> chip analysis delineates a different cytokine production signature of CTCs. a The cytokine profile array from the cell culture supernatants of the CTC-TJH-01, 95-D and A549 cells were detected by capture antibodies spotted in duplicate on nitrocellulose membranes. b The quantitative analysis of cytokine profile in A. c The fold changes of CX3CL1 and CXCL5 in the CTC-TJH-01, 95-D and A549 cells. d Verification of CX3CL1 and CXCL5 expression in the CTC-TJH-01, 95-D and A549 cells by western blot. e Real-time PCR and f western blotting were conducted to examine the mRNA and protein expression of CXCL5 in the CTC-TJH-01 cells transfected with the CXCL5-specific siRNA or a non-specific siRNA as the negative control (NC), respectively. g CCK-8 assay and h cell apoptosis assay were used to determine cell proliferation and apoptosis. i , j A transwell assay was used to determine cell migration and invasion. k ELISA assay was used to determine the CX3CL1 secretion level of cell supernatant. l , m After co-culture of CTC-TJH-01, 95-D and A549 cells with lymphocytes; flow cytometry was used to detect lymphocyte recruitment. Each bar represents the mean ± SD of three separate experiments. * P
    Human Cytokine Antibody Array, supplied by RayBiotech, used in various techniques. Bioz Stars score: 97/100, based on 621 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson cytokine secretion
    Analysis of EV function after floatation on iodixanol gradient A–C Pellets obtained after 2K, 10K and 100K centrifugations from 80–120 × 10 6 cells were floated into an overlayed iodixanol gradient (A). Five fractions were collected and analysed by WB (representative of five experiments) (B), showing the floatation of MHC II‐ and CD9‐enriched EVs in fraction F3–4 and fraction F5–6. Densities of recovered fractions, as measured by refractometry, are shown below the WB figure (mean density of five independent gradients). Quantification of the distribution of MHC II relative abundance in all fractions of each centrifugation pellet analysed by WB of five independent experiments (C). For each pellet, arbitrary units (AU) = (SI fraction )/sum(SI F1–2 + SI F3–4 + SI F5–6 + SI F7–8 + SI F9–10 ) where SI = signal intensity (i.e. ratio of signal intensity in the given pellet to the total secreted protein) ( n = 5, one symbol per donor). Red line indicates median. D IL‐13 and IFN‐γ secretion was measured in supernatants after 6 days of total CD4 + T‐cell culture with the different fractions of the iodixanol gradients of the 2K, 10K and 100K pellets. The graph indicates the relative contribution of each fraction to the total <t>cytokine</t> secretion induced by each pellet. The relative contribution for each donor was calculated as CC fraction /sum(CC F1–2 + CC F3–4 + CC F5–6 + CC F7–8 + CC F9–10 ) for each pellet, where CC is cytokine concentration. Mean + SEM is shown. Below each graph, the sum of the cytokine concentration in all the fractions for each pellet is shown (median of 14 individual DC‐EV:T‐cell combinations). Source data are available online for this figure.
    Cytokine Secretion, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson recombinant cytokines
    IL-24 increases the expression of anti-inflammatory <t>cytokines</t> and neuroprotective factors by primary murine astrocytes. a Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/ml) for 4 h prior to N. meningitidis infection (Nm; MOI of 10:1 bacteria to each astrocyte) or vehicle control. At 24 or 48 h postinfection, IL-10 protein release was assessed by specific capture ELISA. Asterisks and dagger indicate a statistically significant difference from uninfected cells and similarly challenged cells in the absence of IL-24, respectively ( n = 3; p
    Recombinant Cytokines, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Assay Biotechnology cytokines
    TCR-CAR can redirect NK-92 cells. ( a ) NK-92 cells were non transfected (grey) or transfected with DMF5 TCR-CAR TCR-CAR (red), Radium-1 (blue) and stained with specific antibody or multimer, respectively. Shown is a single staining representative of two separate stainings. ( b ) Stimulation of plain NK-92 cells (white) or transfected with TCR-CAR constructs (DMF5, black and Radium-1, grey) with Granta-519 loaded (+) or not (−) with the cognate peptide was performed for 6 hours at a E:T ratio of 1:2. Presence of CD107a was detected by flow cytometry and the MFI of the signal was plotted. Mean ± SEM, N = 3. Unpaired t-test was used as statistical analysis between indicated groups and similar trends were depicted as a group. For <t>cytokines</t> analysis, NK-92 cells transduced (black and grey) or not (white) were co-cultured with target cells loaded (+) or not (−) with the cognate peptide at 1:2 E:T ratio for 24 hours. Non transfected NK-92 cells alone (NK-92 only) were also tested. Supernatants from each condition were collected and presence of TNF-α and IFN-γ was performed with the Bio-Rad Bio-Plex 100 system. Measurements were made in triplicate from three separate supernatants per condition. Cytokine concentrations are shown in picograms per milliliter (pg/mL). Mean ± SEM, N = 3. Unpaired t-test was used as statistical analysis between indicated groups and similar trends were depicted as a group. ( c ) Specific lysis of target cells loaded with the indicated peptide (MART-1 peptide, black, TGFbR2 peptide, white, no peptide, grey) by plain NK-92 (circles) or NK-92 expressing TCR-CAR (squares) at different E:T ratios in a BLI cytotoxic assay. The specific lysis luminescence readings were collected after 10 hours of co-culture. Mean ± SEM, N = 3. Unpaired t-test performed between indicated group and corresponding non-transduced NK-92 group. Ranges for unpaired t-test were as follows *P
    Cytokines, supplied by Assay Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 388 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam cytokines
    Changes in <t>cytokines</t> and adiponectin. There was a significant difference in the change in TNF-α and adiponectin levels between the KRG and placebo groups. The mean TNF-α and adiponectin levels in patients with a BMI of 25 kg/m 2 or more showed particular improvement after KRG therapy. BMI, body mass index; IL-6, interleukin-6; KR G, Korean Red Ginseng; ns, not significant; TNF-α, tumor necrosis factor-α.
    Cytokines, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 405 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Merck KGaA cytokines
    Evaluation of circulating <t>cytokines/chemokines</t> in CVI patients 6 months after surgical hemodynamic correction. The circulating levels of cytokines/chemokines were assessed in plasma samples harvested from forearm vein of healthy subjects (controls) and of CVI patients before (baseline) and after (6 months) CHIVA. In (a), the 11 cytokines/chemokines displaying lower levels after CHIVA, compared to baseline levels, are shown; * P
    Cytokines, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 94/100, based on 401 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Pleural injection of carrageenan caused by 4 h an increase in the release of the cytokines, tumour necrosis factor alpha (TNFα, A), interleukin-1β (IL-1β, B), interleukin-6 (IL-6, C) and interleukin-10 (IL-10, D). When tested at the highest dose, M40403 (20 mg kg −1 , intraperitoneally) inhibited TNFα, IL-1β and IL-6 (A – C) but had no effect on IL-10 (D). Each value is the mean±s.e.mean for n =10 experiments. * P

    Journal: British Journal of Pharmacology

    Article Title: Pharmacological manipulation of the inflammatory cascade by the superoxide dismutase mimetic, M40403

    doi: 10.1038/sj.bjp.0703841

    Figure Lengend Snippet: Pleural injection of carrageenan caused by 4 h an increase in the release of the cytokines, tumour necrosis factor alpha (TNFα, A), interleukin-1β (IL-1β, B), interleukin-6 (IL-6, C) and interleukin-10 (IL-10, D). When tested at the highest dose, M40403 (20 mg kg −1 , intraperitoneally) inhibited TNFα, IL-1β and IL-6 (A – C) but had no effect on IL-10 (D). Each value is the mean±s.e.mean for n =10 experiments. * P

    Article Snippet: Cytokines (TNFα, IL-1β, IL-6 and I1-10) and PGE2 were measured in the exudates by ELISA's using commercially available kits (Calbiochem-Novabiochem Corporation, U.S.A.).

    Techniques: Injection

    The effect of P2X 7 receptor inhibition in E. coli -induced sepsis . ARD6 was injected iv into anesthetized balb/cj mice (low dose—41 million). Two hours prior to ARD6 injection the animals were administered the P2X 7 antagonist BBG or vehicle (50 mg kg −1 , subcutaneously). (A) Kaplan-Meier plot shows survival, n = 6 for controls and 10–12 for ARD6 with or without BBG. (B) Absorbance of plasma as an indication of plasma haemolysis 2.5 h after ARD6 injection, n = 13 for all four groups. (C) Colony forming units from blood samples 2.5 h after a low number of bacteria (D) Levels of the cytokines TNFα, KC, IL-1β, and IL-6 2.5 h after ARD6 injection, n = 14 for ARD6 and 13 for ARD6 + BBG. * p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: P2X1, P2X4, and P2X7 Receptor Knock Out Mice Expose Differential Outcome of Sepsis Induced by α-Haemolysin Producing Escherichia coli

    doi: 10.3389/fcimb.2017.00113

    Figure Lengend Snippet: The effect of P2X 7 receptor inhibition in E. coli -induced sepsis . ARD6 was injected iv into anesthetized balb/cj mice (low dose—41 million). Two hours prior to ARD6 injection the animals were administered the P2X 7 antagonist BBG or vehicle (50 mg kg −1 , subcutaneously). (A) Kaplan-Meier plot shows survival, n = 6 for controls and 10–12 for ARD6 with or without BBG. (B) Absorbance of plasma as an indication of plasma haemolysis 2.5 h after ARD6 injection, n = 13 for all four groups. (C) Colony forming units from blood samples 2.5 h after a low number of bacteria (D) Levels of the cytokines TNFα, KC, IL-1β, and IL-6 2.5 h after ARD6 injection, n = 14 for ARD6 and 13 for ARD6 + BBG. * p

    Article Snippet: CBA flex sets for measuring cytokines were from BD Biosciences.

    Techniques: Inhibition, Injection, Mouse Assay

    CHIKV patient convalescence was associated with increasing levels of TNF-α, IL-5, IL-1β, IL-12, IFN-γ and IL-10. Cytokine Bead Array analysis of CHIKV patient serum samples showed that following the acute phase of CHIKV disease patients had increasing levels of TNF-α, IL-5, IL-1β, IL-12, IFN-γ and IL-10. Six-month and one-year cytokine levels were analysed for statistical significance using the Wilcoxon test for Significance by comparing with acute phase values. As well, samples were analyzed for significance against healthy controls by the Mann-Whitney U test. The cross symbol indicates a p-value (Wilcoxon test) less than 0.05 for 6- and 12-month groups compared to acute values and star symbol indicates a p-value (Mann-Whitney U test) less than 0.05 acute, 6- and 12-month groups compared to control values. The dotted line indicates the median of healthy control cytokine levels. Acute (A), 6-month follow-up (6), and 12-month follow-up (12).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity

    doi: 10.1371/journal.pntd.0001279

    Figure Lengend Snippet: CHIKV patient convalescence was associated with increasing levels of TNF-α, IL-5, IL-1β, IL-12, IFN-γ and IL-10. Cytokine Bead Array analysis of CHIKV patient serum samples showed that following the acute phase of CHIKV disease patients had increasing levels of TNF-α, IL-5, IL-1β, IL-12, IFN-γ and IL-10. Six-month and one-year cytokine levels were analysed for statistical significance using the Wilcoxon test for Significance by comparing with acute phase values. As well, samples were analyzed for significance against healthy controls by the Mann-Whitney U test. The cross symbol indicates a p-value (Wilcoxon test) less than 0.05 for 6- and 12-month groups compared to acute values and star symbol indicates a p-value (Mann-Whitney U test) less than 0.05 acute, 6- and 12-month groups compared to control values. The dotted line indicates the median of healthy control cytokine levels. Acute (A), 6-month follow-up (6), and 12-month follow-up (12).

    Article Snippet: Next we sought to determine if the high levels of IgG in the follow-up phases were also significantly associated with cytokine levels compared to the cytokine levels of patients with low levels of IgG.

    Techniques: MANN-WHITNEY

    The stages of CHIKV Acute phase were marked by changes in CXCL10 and IL-10. Acute CHIKV patients were categorized into Viral stage (V), Antibody Initiation stage (AI) or Seroconversion stage (SC) according to the presence of CHIKV, IgM and IgG antibodies. Cytokine Bead Array analysis of the serum samples showed a significant decrease in CXCL10 and IL-10 from the Viral stage to the Seroconversion stage of the Acute phase. A Mann-Whitney U test was used to determine significance among the phases. The star symbol indicates a p-value less than 0.05 compared to the Viral phase.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity

    doi: 10.1371/journal.pntd.0001279

    Figure Lengend Snippet: The stages of CHIKV Acute phase were marked by changes in CXCL10 and IL-10. Acute CHIKV patients were categorized into Viral stage (V), Antibody Initiation stage (AI) or Seroconversion stage (SC) according to the presence of CHIKV, IgM and IgG antibodies. Cytokine Bead Array analysis of the serum samples showed a significant decrease in CXCL10 and IL-10 from the Viral stage to the Seroconversion stage of the Acute phase. A Mann-Whitney U test was used to determine significance among the phases. The star symbol indicates a p-value less than 0.05 compared to the Viral phase.

    Article Snippet: Next we sought to determine if the high levels of IgG in the follow-up phases were also significantly associated with cytokine levels compared to the cytokine levels of patients with low levels of IgG.

    Techniques: MANN-WHITNEY

    IL-2, IL-8 and IL-4 were not associated with acute or convalescence phase of CHIKV disease. Cytokine Bead Array analysis of CHIKV patient serum samples showed that IL-2, IL-8 and IL-4 were not associated with acute phase or convalescence of CHIKV disease in patients. Six-month and one-year cytokine levels were analysed for statistical significance using the Wilcoxon test for Significance by comparing with acute phase values. Samples were analyzed for significance against healthy controls by the Mann-Whitney U test. The star symbol indicates a p-value (Mann-Whitney U test) less than 0.05 acute, 6- and 12-month groups compared to control values. The dotted line indicates the median of healthy control cytokine levels. Acute (A), 6-month follow-up (6), and 12-month follow-up (12).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity

    doi: 10.1371/journal.pntd.0001279

    Figure Lengend Snippet: IL-2, IL-8 and IL-4 were not associated with acute or convalescence phase of CHIKV disease. Cytokine Bead Array analysis of CHIKV patient serum samples showed that IL-2, IL-8 and IL-4 were not associated with acute phase or convalescence of CHIKV disease in patients. Six-month and one-year cytokine levels were analysed for statistical significance using the Wilcoxon test for Significance by comparing with acute phase values. Samples were analyzed for significance against healthy controls by the Mann-Whitney U test. The star symbol indicates a p-value (Mann-Whitney U test) less than 0.05 acute, 6- and 12-month groups compared to control values. The dotted line indicates the median of healthy control cytokine levels. Acute (A), 6-month follow-up (6), and 12-month follow-up (12).

    Article Snippet: Next we sought to determine if the high levels of IgG in the follow-up phases were also significantly associated with cytokine levels compared to the cytokine levels of patients with low levels of IgG.

    Techniques: MANN-WHITNEY

    Acute phase CHIKV disease was associated with high levels of IL-6, CXCL9, CCL2, CXCL10. Cytokine Bead Array analysis of CHIKV patient serum samples showed high levels of IL-6, CXCL9, CCL2 and CXCL-10 are associated with acute disease phase and decreased with patient convalescence. Six-month and one-year cytokine levels were analysed for statistical significance using the Wilcoxon test for Significance by comparing with acute levels. All samples were also analyzed for significance against healthy controls by the Mann-Whitney U test. The cross symbol indicates a p-value less than 0.05 for 6- and 12-month groups compared to acute values and star symbol indicates a p-value less than 0.05 for acute, 6- and 12-month groups compared to control values. The dotted line indicates the median of healthy control cytokine levels. Acute (A), 6-month follow-up (6), and 12-month follow-up (12).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity

    doi: 10.1371/journal.pntd.0001279

    Figure Lengend Snippet: Acute phase CHIKV disease was associated with high levels of IL-6, CXCL9, CCL2, CXCL10. Cytokine Bead Array analysis of CHIKV patient serum samples showed high levels of IL-6, CXCL9, CCL2 and CXCL-10 are associated with acute disease phase and decreased with patient convalescence. Six-month and one-year cytokine levels were analysed for statistical significance using the Wilcoxon test for Significance by comparing with acute levels. All samples were also analyzed for significance against healthy controls by the Mann-Whitney U test. The cross symbol indicates a p-value less than 0.05 for 6- and 12-month groups compared to acute values and star symbol indicates a p-value less than 0.05 for acute, 6- and 12-month groups compared to control values. The dotted line indicates the median of healthy control cytokine levels. Acute (A), 6-month follow-up (6), and 12-month follow-up (12).

    Article Snippet: Next we sought to determine if the high levels of IgG in the follow-up phases were also significantly associated with cytokine levels compared to the cytokine levels of patients with low levels of IgG.

    Techniques: MANN-WHITNEY

    CHIKV disease severity is associated with high CXCL10, CXCL9 and IgG levels at the 6-month time point. CHIKV patients were determined to be nonsymptomatic (N), to have mild symptoms (M) or to have severe symptoms (S). The cytokine and IgG levels were then grouped by symptom level and a Mann-Whitney U test was used to determine significance among the severity groups. CXCL10, CXCL9 and IgG were found to be significantly increased in the patients with mild and severe symptoms at 6 months following initial infection. The cross symbol indicates a p-value less than 0.05.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity

    doi: 10.1371/journal.pntd.0001279

    Figure Lengend Snippet: CHIKV disease severity is associated with high CXCL10, CXCL9 and IgG levels at the 6-month time point. CHIKV patients were determined to be nonsymptomatic (N), to have mild symptoms (M) or to have severe symptoms (S). The cytokine and IgG levels were then grouped by symptom level and a Mann-Whitney U test was used to determine significance among the severity groups. CXCL10, CXCL9 and IgG were found to be significantly increased in the patients with mild and severe symptoms at 6 months following initial infection. The cross symbol indicates a p-value less than 0.05.

    Article Snippet: Next we sought to determine if the high levels of IgG in the follow-up phases were also significantly associated with cytokine levels compared to the cytokine levels of patients with low levels of IgG.

    Techniques: MANN-WHITNEY, Infection

    The stages of CHIKV 6- and 12-month follow-up phases are marked by differentials in CXCL10, CXCL9, IL-6 and CXCL9, IL-10 respectively. IgG levels in 6- and 12-month patient serum samples were determined by ELISA. The samples were then grouped by IgG level; a low IgG level group (L) and a high IgG level group (H). Cytokine Bead Array analysis of the serum samples showed a significant difference in CXCL9, CXCL10 and IL-6 between patients with high and low IgG levels at the 6-month time point. Twelve-month follow-up CHIKV patient samples showed a significant difference in CXCL9 and IL-10 between low (1) and high (2) IgG groups. A Mann-Whitney U test was used to determine significance among the IgG groups. The cross symbol indicates a p-value less than 0.05.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity

    doi: 10.1371/journal.pntd.0001279

    Figure Lengend Snippet: The stages of CHIKV 6- and 12-month follow-up phases are marked by differentials in CXCL10, CXCL9, IL-6 and CXCL9, IL-10 respectively. IgG levels in 6- and 12-month patient serum samples were determined by ELISA. The samples were then grouped by IgG level; a low IgG level group (L) and a high IgG level group (H). Cytokine Bead Array analysis of the serum samples showed a significant difference in CXCL9, CXCL10 and IL-6 between patients with high and low IgG levels at the 6-month time point. Twelve-month follow-up CHIKV patient samples showed a significant difference in CXCL9 and IL-10 between low (1) and high (2) IgG groups. A Mann-Whitney U test was used to determine significance among the IgG groups. The cross symbol indicates a p-value less than 0.05.

    Article Snippet: Next we sought to determine if the high levels of IgG in the follow-up phases were also significantly associated with cytokine levels compared to the cytokine levels of patients with low levels of IgG.

    Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Effect of IL-22 (CC10-IL-22) on cytokine and chemokine production in OVA-induced allergic asthma. Th1 cytokine, IFN-γ, and Th2 cytokines, IL-4 and IL-13, Th17 cytokine IL-17A, and chemokine eotaxin in the BAL were measured by ELISA. The number of animals in each group was indicated and data were shown as Mean±SEM. ** P

    Journal: PLoS ONE

    Article Title: Immune Modulatory Effects of IL-22 on Allergen-Induced Pulmonary Inflammation

    doi: 10.1371/journal.pone.0107454

    Figure Lengend Snippet: Effect of IL-22 (CC10-IL-22) on cytokine and chemokine production in OVA-induced allergic asthma. Th1 cytokine, IFN-γ, and Th2 cytokines, IL-4 and IL-13, Th17 cytokine IL-17A, and chemokine eotaxin in the BAL were measured by ELISA. The number of animals in each group was indicated and data were shown as Mean±SEM. ** P

    Article Snippet: Cytokine production by lymphocytes from draining lymph nodes (DLN) and spleen Lymphocytes from DLN (mediastinal and hilar) and purified splenic CD4+ cells were cultured in RPMI1640 medium (5% FCS) and were stimulated with medium as control, OVA (50 µg/ml) for 5 days or anti-CD3/CD28 (BD Bioscience) (5 µg/ml) for 3 days.

    Techniques: Enzyme-linked Immunosorbent Assay

    Effect of IL-22 (SPC-IL-22) on OVA-induced systemic and local immune responses. Splenocytes and lymphocytes from peribronchial draining lymph nodes (DLN) from IL-22 Tg(+) and Tg(−) mice after OVA challenge were cultured and stimulated with medium control, OVA or CD3/CD28. Th1 cytokine, IFN-γ, and Th2 cytokine, IL-13 in the supernatant were measured by ELISA. The number of animals used in the experiments was indicated and data were shown as Mean±SEM. * P

    Journal: PLoS ONE

    Article Title: Immune Modulatory Effects of IL-22 on Allergen-Induced Pulmonary Inflammation

    doi: 10.1371/journal.pone.0107454

    Figure Lengend Snippet: Effect of IL-22 (SPC-IL-22) on OVA-induced systemic and local immune responses. Splenocytes and lymphocytes from peribronchial draining lymph nodes (DLN) from IL-22 Tg(+) and Tg(−) mice after OVA challenge were cultured and stimulated with medium control, OVA or CD3/CD28. Th1 cytokine, IFN-γ, and Th2 cytokine, IL-13 in the supernatant were measured by ELISA. The number of animals used in the experiments was indicated and data were shown as Mean±SEM. * P

    Article Snippet: Cytokine production by lymphocytes from draining lymph nodes (DLN) and spleen Lymphocytes from DLN (mediastinal and hilar) and purified splenic CD4+ cells were cultured in RPMI1640 medium (5% FCS) and were stimulated with medium as control, OVA (50 µg/ml) for 5 days or anti-CD3/CD28 (BD Bioscience) (5 µg/ml) for 3 days.

    Techniques: Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    IL-22 alleviated OVA-induced eosinophilic inflammation in the lung. (A) High levels of IL-22 cytokine were seen in the BAL of PBS and OVA-stimulated IL-22 Tg(+) mice without difference between the two groups ( P > 0.05). When compared to Tg(−) mice, IL-22 concentrations in the BAL of Tg(+) mice were much higher than that in PBS and OVA-stimulated control groups ( P

    Journal: PLoS ONE

    Article Title: Immune Modulatory Effects of IL-22 on Allergen-Induced Pulmonary Inflammation

    doi: 10.1371/journal.pone.0107454

    Figure Lengend Snippet: IL-22 alleviated OVA-induced eosinophilic inflammation in the lung. (A) High levels of IL-22 cytokine were seen in the BAL of PBS and OVA-stimulated IL-22 Tg(+) mice without difference between the two groups ( P > 0.05). When compared to Tg(−) mice, IL-22 concentrations in the BAL of Tg(+) mice were much higher than that in PBS and OVA-stimulated control groups ( P

    Article Snippet: Cytokine production by lymphocytes from draining lymph nodes (DLN) and spleen Lymphocytes from DLN (mediastinal and hilar) and purified splenic CD4+ cells were cultured in RPMI1640 medium (5% FCS) and were stimulated with medium as control, OVA (50 µg/ml) for 5 days or anti-CD3/CD28 (BD Bioscience) (5 µg/ml) for 3 days.

    Techniques: Mouse Assay

    The expression of cytokines in the re-challenged control group and the re-challenged anti-Vγ 4 antibody group. (A) IMQ application increased the IL-17A-producing Vγ 4 + γδ T cells in the dermis and (B) the percent of IL-17A + Vγ 4 - cells and IL-17A + cells. (C) The IFN-γ-producing Vγ 4 + γδ T cells were detected by FACS. (D) The percent of γ-IFN-producing Vγ 4 - γδ T cells and γ-IFN + cells on different days. (E) The IL-22-producing Vγ 4 + γδ T cells on day 6 and (F) the IL-22-producing Vγ 4 - γδ T cells were detected by FACS. The values represent the mean ± SEM. *p

    Journal: American Journal of Translational Research

    Article Title: Dermal Vγ4+T cells enhance the IMQ-induced psoriasis-like skin inflammatidon in re-challenged mice

    doi:

    Figure Lengend Snippet: The expression of cytokines in the re-challenged control group and the re-challenged anti-Vγ 4 antibody group. (A) IMQ application increased the IL-17A-producing Vγ 4 + γδ T cells in the dermis and (B) the percent of IL-17A + Vγ 4 - cells and IL-17A + cells. (C) The IFN-γ-producing Vγ 4 + γδ T cells were detected by FACS. (D) The percent of γ-IFN-producing Vγ 4 - γδ T cells and γ-IFN + cells on different days. (E) The IL-22-producing Vγ 4 + γδ T cells on day 6 and (F) the IL-22-producing Vγ 4 - γδ T cells were detected by FACS. The values represent the mean ± SEM. *p

    Article Snippet: To stain intracellular cytokines, the cells were stimulated with a cell stimulation cocktail (eBiosicence) for 4 h. The cells were then stained for surface antigens and then fixed with BD Cytofix Buffer, permeabilized with Perm/Wash reagent (BD Biosciences), and stained with anti-IL-22, anti-IL-17A, and anti-IFN-γ.

    Techniques: Expressing, FACS

    IL-17C-IL-17RE Regulates IκBζ Expression (A) ELISA of cytokine production in CD4 + T cells retrovirally transduced with IL-17RE or control viral vector and treated with IL-17C for 2 days in Th17 cell condition. Sorted GFP + cells were activated with anti-CD3 for 24 hr. (B) Real-time RT-PCR analysis of cytokine and transcription factor expression in CD4 + T cells retrovirally transduced and cultured either in neutral or Th17 cell conditions. The cells were activated with anti-CD3 for 4 hr for cytokine mRNA detection. (C) Real-time RT-PCR analysis of Nfkbiz prepared as above. *p

    Journal: Immunity

    Article Title: Interleukin-17C Promotes Th17 Cell Responses and Autoimmune Disease via Interleukin-17 Receptor E

    doi: 10.1016/j.immuni.2011.09.010

    Figure Lengend Snippet: IL-17C-IL-17RE Regulates IκBζ Expression (A) ELISA of cytokine production in CD4 + T cells retrovirally transduced with IL-17RE or control viral vector and treated with IL-17C for 2 days in Th17 cell condition. Sorted GFP + cells were activated with anti-CD3 for 24 hr. (B) Real-time RT-PCR analysis of cytokine and transcription factor expression in CD4 + T cells retrovirally transduced and cultured either in neutral or Th17 cell conditions. The cells were activated with anti-CD3 for 4 hr for cytokine mRNA detection. (C) Real-time RT-PCR analysis of Nfkbiz prepared as above. *p

    Article Snippet: For intracellular cytokine analysis, cells were restimulated with 500 ng/ml of ionomycin and 50 ng/ml of PMA in the presence of Golgi Stop (BD Phar-Mingen) for 5 hr.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Transduction, Plasmid Preparation, Quantitative RT-PCR, Cell Culture

    IL-17C-IL-17RE Enhanced Polarized Th17 Cell Response (A) Representative flow cytometry plot of Th17 cells from IL-17F RFP mice polarized with TGF-β and IL-6 at day 5. (B) ELISA of IL-17A production in polarized Th17 cells with or without IL-17C in the presence of increasing concentration of αCD3. (C) ELISA of cytokine production in polarized Th17 cells cultured with irradiated splenocytes in the presence of increasing concentration of IL-17C. (D) Real-time RT-PCR analysis of Il17c in CNS and spleen from normal and EAE mice. All comparisons presented were statistically significant (***p

    Journal: Immunity

    Article Title: Interleukin-17C Promotes Th17 Cell Responses and Autoimmune Disease via Interleukin-17 Receptor E

    doi: 10.1016/j.immuni.2011.09.010

    Figure Lengend Snippet: IL-17C-IL-17RE Enhanced Polarized Th17 Cell Response (A) Representative flow cytometry plot of Th17 cells from IL-17F RFP mice polarized with TGF-β and IL-6 at day 5. (B) ELISA of IL-17A production in polarized Th17 cells with or without IL-17C in the presence of increasing concentration of αCD3. (C) ELISA of cytokine production in polarized Th17 cells cultured with irradiated splenocytes in the presence of increasing concentration of IL-17C. (D) Real-time RT-PCR analysis of Il17c in CNS and spleen from normal and EAE mice. All comparisons presented were statistically significant (***p

    Article Snippet: For intracellular cytokine analysis, cells were restimulated with 500 ng/ml of ionomycin and 50 ng/ml of PMA in the presence of Golgi Stop (BD Phar-Mingen) for 5 hr.

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Irradiation, Quantitative RT-PCR

    IL-17C-IL-17RE Regulates Th17 Cell Differentiation (A) Intracellular cytokine staining of IL-17A and IFN-γ. After naive CD4 + T cells were retrovirally transduced with control viral vector or IL-17RE, different IL-17 family cytokines were added during Th17 cell differentiation. The cells were restimulated with PMA and ionomycin for cytokine staining. (B) Statistical analysis of IL-17A-positive cells. (C) IL-17C induced IL-17A expression in Th17 cells over-expressing IL-17RE but not other IL-17R family members. CD4 + T cells were retrovirally transduced with each IL-17R-IRES-GFP and were cultured with IL-17C in Th17 cell polarizing condition. Cells were restimulated with PMA and ionomycin and GFP-positive cells were gated to show expression of IL-17A and IFN-γ. (D) Intracellular cytokine staining of WT and Il17ra −/− T cells retrovirally transduced with IL-17RE and cultured with IL-17C for 2 days, followed by 5 hr of PMA and ionomycin. Data shown are gated GFP + cells. Data shown are representative of at least three experiments. (E) Coimmunoprecipitation of IL-17RA with HA-tagged IL-17RE. 293T cells were transfected with IL-17RA or HA-tagged IL-17RE or both and immunoprecipitated with anti-HA. (F) Intracellular cytokine staining of WT and Act1 −/− T cells retrovirally transduced with IL-17RE or control viral vector (RV-GFP) and followed by 5 hr of PMA and ionomycin. Data shown are gated GFP + cells. Data shown are representative of at least three experiments. .

    Journal: Immunity

    Article Title: Interleukin-17C Promotes Th17 Cell Responses and Autoimmune Disease via Interleukin-17 Receptor E

    doi: 10.1016/j.immuni.2011.09.010

    Figure Lengend Snippet: IL-17C-IL-17RE Regulates Th17 Cell Differentiation (A) Intracellular cytokine staining of IL-17A and IFN-γ. After naive CD4 + T cells were retrovirally transduced with control viral vector or IL-17RE, different IL-17 family cytokines were added during Th17 cell differentiation. The cells were restimulated with PMA and ionomycin for cytokine staining. (B) Statistical analysis of IL-17A-positive cells. (C) IL-17C induced IL-17A expression in Th17 cells over-expressing IL-17RE but not other IL-17R family members. CD4 + T cells were retrovirally transduced with each IL-17R-IRES-GFP and were cultured with IL-17C in Th17 cell polarizing condition. Cells were restimulated with PMA and ionomycin and GFP-positive cells were gated to show expression of IL-17A and IFN-γ. (D) Intracellular cytokine staining of WT and Il17ra −/− T cells retrovirally transduced with IL-17RE and cultured with IL-17C for 2 days, followed by 5 hr of PMA and ionomycin. Data shown are gated GFP + cells. Data shown are representative of at least three experiments. (E) Coimmunoprecipitation of IL-17RA with HA-tagged IL-17RE. 293T cells were transfected with IL-17RA or HA-tagged IL-17RE or both and immunoprecipitated with anti-HA. (F) Intracellular cytokine staining of WT and Act1 −/− T cells retrovirally transduced with IL-17RE or control viral vector (RV-GFP) and followed by 5 hr of PMA and ionomycin. Data shown are gated GFP + cells. Data shown are representative of at least three experiments. .

    Article Snippet: For intracellular cytokine analysis, cells were restimulated with 500 ng/ml of ionomycin and 50 ng/ml of PMA in the presence of Golgi Stop (BD Phar-Mingen) for 5 hr.

    Techniques: Cell Differentiation, Staining, Transduction, Plasmid Preparation, Expressing, Cell Culture, Transfection, Immunoprecipitation

    Vasculotide (VT) suppresses cecal ligation and puncture (CLP)-induced upregulation of proinflammatory cytokines in vivo but does not affect cytokine release from macrophages in vitro . (A) Mice were pretreated with VT (200 ng intraperitoneally) or phosphate-buffered saline (PBS) at 16 hours and 1 hour prior to CLP or sham surgery, respectively. Levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), keratinocyte-derived chemokine/interleukin-8 homologue (KC), macrophage inflammatory protein-2 (MIP-2), and monocyte chemotactic protein-1 (MCP-1) in serum and peritoneal lavage (PL) fluid samples at 18 hours after CLP or sham surgery were quantified by bead-based flow cytometry or enzyme-linked immunosorbent assay (ELISA), respectively. Data are expressed as mean ± standard error of the mean (SEM) (n = 7 to 10 mice per group). * P

    Journal: Critical Care

    Article Title: The synthetic Tie2 agonist peptide vasculotide protects against vascular leakage and reduces mortality in murine abdominal sepsis

    doi: 10.1186/cc10523

    Figure Lengend Snippet: Vasculotide (VT) suppresses cecal ligation and puncture (CLP)-induced upregulation of proinflammatory cytokines in vivo but does not affect cytokine release from macrophages in vitro . (A) Mice were pretreated with VT (200 ng intraperitoneally) or phosphate-buffered saline (PBS) at 16 hours and 1 hour prior to CLP or sham surgery, respectively. Levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), keratinocyte-derived chemokine/interleukin-8 homologue (KC), macrophage inflammatory protein-2 (MIP-2), and monocyte chemotactic protein-1 (MCP-1) in serum and peritoneal lavage (PL) fluid samples at 18 hours after CLP or sham surgery were quantified by bead-based flow cytometry or enzyme-linked immunosorbent assay (ELISA), respectively. Data are expressed as mean ± standard error of the mean (SEM) (n = 7 to 10 mice per group). * P

    Article Snippet: Cytokine detection in serum and peritoneal lavage samples Serum levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and macrophage chemoattractant protein-1 (MCP-1) were quantified by bead-based flow cytometry assay (CBA Kit; BD Biosciences, Heidelberg, Germany) in accordance with the instructions of the manufacturer.

    Techniques: Ligation, In Vivo, In Vitro, Mouse Assay, Derivative Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Bar graphs showing the intravitreal concentration of pro-inflammatory cytokines in RCS control (n=24) and RCS dystrophic (n=48) rat eyes over time. The data are plotted as mean ± standard error of the mean; * indicates significant difference between control and dystrophic groups (p≤0.05). A : IL-1α, B: IL-4, C : IL-6, D: IL-8, E : TNF, F : IFN-γ.

    Journal: Molecular Vision

    Article Title: Basal membrane complex architecture is disrupted during posterior subcapsular cataract formation in Royal College of Surgeons rats

    doi:

    Figure Lengend Snippet: Bar graphs showing the intravitreal concentration of pro-inflammatory cytokines in RCS control (n=24) and RCS dystrophic (n=48) rat eyes over time. The data are plotted as mean ± standard error of the mean; * indicates significant difference between control and dystrophic groups (p≤0.05). A : IL-1α, B: IL-4, C : IL-6, D: IL-8, E : TNF, F : IFN-γ.

    Article Snippet: Intravitreal cytokine analysis using multiplexed bead-based immunoassay and flow cytometry The vitreous samples that were collected from RCS rat lenses were processed as per the manufacturer’s instructions and analyzed for the presence of inflammatory cytokines (specifically, interleukin [IL]-1α, IL-4, IL-6, IL-8, tumor necrosis factor [TNF], and interferon [IFN]-γ) using the BD-Cytometric Bead Array (BD-CBA; Becton Dickinson, San Jose, CA).

    Techniques: Concentration Assay

    Principle of the microdialysis experiments: 100 kDa cut-off membranes perfused with crystalloid or colloid solutions (perfusates) were placed in the dermal skin using a microdialysis pump at flow rates of 0.2, 0.5, or 1 µL/min. The dialysates were collected in fractions after an exchange process within dermal skin between the interstitial fluid and the perfusate via a microdialysis membrane (scheme, A ). Clinical pictures of linear microdialysis cut-off membranes (CMA) 66 catheters placed in human skin ( B ). Retrodialysis of porcine skin indicated cytokine release from the catheter to the dermis. Signs of inflammation in the catheter areas included rubor and tumefaction ( C , D ) as well as dermal oedema surrounding the microdialysis catheter ( E ), as demonstrated by high-resolution 22 MHz ultrasound (Taberna Pro Medicum, Luneburg, Germany). After placement of the microdialysis catheters into the dermal skin of a human forearm, UV irradiation and skin erythema were used to generate cytokines and chemokines ( F – H ). Dialysates were collected for 48 h after UVB irradiation in this clinical experiment ( I) . Dotted lines indicate virtual intradermal placement of microdialysis membranes.

    Journal: Materials

    Article Title: Cytokine and Chemokine Recovery Is Increased by Colloid Perfusates during Dermal Microdialysis

    doi: 10.3390/ma11050682

    Figure Lengend Snippet: Principle of the microdialysis experiments: 100 kDa cut-off membranes perfused with crystalloid or colloid solutions (perfusates) were placed in the dermal skin using a microdialysis pump at flow rates of 0.2, 0.5, or 1 µL/min. The dialysates were collected in fractions after an exchange process within dermal skin between the interstitial fluid and the perfusate via a microdialysis membrane (scheme, A ). Clinical pictures of linear microdialysis cut-off membranes (CMA) 66 catheters placed in human skin ( B ). Retrodialysis of porcine skin indicated cytokine release from the catheter to the dermis. Signs of inflammation in the catheter areas included rubor and tumefaction ( C , D ) as well as dermal oedema surrounding the microdialysis catheter ( E ), as demonstrated by high-resolution 22 MHz ultrasound (Taberna Pro Medicum, Luneburg, Germany). After placement of the microdialysis catheters into the dermal skin of a human forearm, UV irradiation and skin erythema were used to generate cytokines and chemokines ( F – H ). Dialysates were collected for 48 h after UVB irradiation in this clinical experiment ( I) . Dotted lines indicate virtual intradermal placement of microdialysis membranes.

    Article Snippet: Sample Concentrations Calculations To determine the cytokine concentrations in the samples, cytokine-specific bead populations were further analysed with FCAP Array software version 1.0.1 (BD Bioscience, Heidelberg, Germany).

    Techniques: Flow Cytometry, Irradiation

    Several cytokines are increased significantly in the sera of infected STAT1 KO mice. In WT mice, a multiplex assay showed small to moderate but significant changes for CCL4, CCL2, and IL-2 levels at day 3 postinfection (A) and for G-CSF, CCL2, IL-5, IL-12,

    Journal: Journal of Virology

    Article Title: Mice Deficient in STAT1 but Not STAT2 or IRF9 Develop a Lethal CD4+ T-Cell-Mediated Disease following Infection with Lymphocytic Choriomeningitis Virus

    doi: 10.1128/JVI.07147-11

    Figure Lengend Snippet: Several cytokines are increased significantly in the sera of infected STAT1 KO mice. In WT mice, a multiplex assay showed small to moderate but significant changes for CCL4, CCL2, and IL-2 levels at day 3 postinfection (A) and for G-CSF, CCL2, IL-5, IL-12,

    Article Snippet: To determine cytokine levels in serum, a Luminex assay for 23 cytokines (Bioplex Mouse Cytokine 23-Plex Panel; Bio-Rad, Gladesville, Australia) was performed according to the manufacturer's instructions.

    Techniques: Infection, Mouse Assay, Multiplex Assay

    Keratinocyte cell death and dermal macrophage infiltration are early events in cpdm dermatitis. ( A ) BioPlex cytokine analysis of skin lysates from mice of indicated genotypes. Data are represented as mean +S.E.M. *p ≤ 0.05. ( B ) F4/80 staining (brown) of skin sections counterstained with hematoxylin (blue). Control: Shpn +/+ or +/m . ( C ) Cleaved caspase-3 staining (brown) of skin, liver and spleen sections counterstained with hematoxylin (blue). Black arrows show examples of cleaved-caspase-3 positive cells. Control: Shpn +/+ or +/m . ( A – C ) Three mice were analyzed for each genotype or group. Scale bars: 50 µm. DOI: http://dx.doi.org/10.7554/eLife.03464.004

    Journal: eLife

    Article Title: TNFR1-dependent cell death drives inflammation in Sharpin-deficient mice

    doi: 10.7554/eLife.03464

    Figure Lengend Snippet: Keratinocyte cell death and dermal macrophage infiltration are early events in cpdm dermatitis. ( A ) BioPlex cytokine analysis of skin lysates from mice of indicated genotypes. Data are represented as mean +S.E.M. *p ≤ 0.05. ( B ) F4/80 staining (brown) of skin sections counterstained with hematoxylin (blue). Control: Shpn +/+ or +/m . ( C ) Cleaved caspase-3 staining (brown) of skin, liver and spleen sections counterstained with hematoxylin (blue). Black arrows show examples of cleaved-caspase-3 positive cells. Control: Shpn +/+ or +/m . ( A – C ) Three mice were analyzed for each genotype or group. Scale bars: 50 µm. DOI: http://dx.doi.org/10.7554/eLife.03464.004

    Article Snippet: Keratinocyte cell death and dermal macrophage infiltration are early events in Shpn m/m dermatitis To gain insight into the pathogenesis of cpdm dermatitis we assessed cytokine levels in skin lysates from 6-week-old cpdm and control mice using a BioPlex kit (BioRad) ( ) to determine which cytokines were elevated early in the disease process.

    Techniques: Mouse Assay, Staining

    Extracellular potassium mediated T cell suppression requires intact PP2A activity and is associated with elevations in intracellular potassium ( a ) Selected phosphatase inhibitors from a pharmacologic screen for IFN-γ production of CD8 + T cells in the presence of elevated [K + ]e, depicted as the ratio of IFN-γ expression in Ctrl / elevated [K + ]e conditions in the presence of indicated phosphatase inhibitors ( b ) CD8 + T cell phosphorylation of pAkt S473 and pS6 S235/6 10 minutes following TCR cross-linking in the indicated conditions. ( c ) Compiled analysis of IFN-γ production by retrovirally transduced CD8 + Thy1.1 + T cells following TCR stimulation in the indicated conditions. ( d ) Pictorial representation of the resultant intracellular changes in plasma membrane potential ( V m ) and intracellular potassium concentration ([K + ]i) in the presence of elevated extracellular potassium ([K + ]e). ( e ) Relative cytoplasmic V m of CD8 + T cells in the indicated conditions represented as relative fluorescence of the voltage-sensitive fluorescent indicator DiSBAC 4 (3). ( f ) Pictorial representation of the resultant intracellular changes in plasma membrane potential ( V m ) and intracellular potassium concentration ([K + ]i) in the presence of the ionophore gramicidin. ( g ) Relative cytoplasmic V m of CD8 + T cells in the indicated conditions assayed as in ( e ) with representative flow cytometry in ( h ). ( i ) IFN-γ production by CD8 + T cells following TCR induced stimulation in the indicated conditions. ( j ) Representative flow cytometry representing [K + ]i of CD8 + T cells as relative fluorescence of the potassium sensitive dye Asante-Green 4. ( k ) Relative [K + ]i of CD8 + T cells in the indicated conditions assayed as relative fluorescence of the potassium sensitive dye Asante-Green 4. ( l ) Representative flow cytometry of cytokine expression by CD8 + T cells following TCR stimulation in the indicated conditions with compiled quantification in ( m ). Error bars represent mean ± SEM. *P

    Journal: Nature

    Article Title: Ionic immune suppression within the tumour microenvironment limits T cell effector function

    doi: 10.1038/nature19364

    Figure Lengend Snippet: Extracellular potassium mediated T cell suppression requires intact PP2A activity and is associated with elevations in intracellular potassium ( a ) Selected phosphatase inhibitors from a pharmacologic screen for IFN-γ production of CD8 + T cells in the presence of elevated [K + ]e, depicted as the ratio of IFN-γ expression in Ctrl / elevated [K + ]e conditions in the presence of indicated phosphatase inhibitors ( b ) CD8 + T cell phosphorylation of pAkt S473 and pS6 S235/6 10 minutes following TCR cross-linking in the indicated conditions. ( c ) Compiled analysis of IFN-γ production by retrovirally transduced CD8 + Thy1.1 + T cells following TCR stimulation in the indicated conditions. ( d ) Pictorial representation of the resultant intracellular changes in plasma membrane potential ( V m ) and intracellular potassium concentration ([K + ]i) in the presence of elevated extracellular potassium ([K + ]e). ( e ) Relative cytoplasmic V m of CD8 + T cells in the indicated conditions represented as relative fluorescence of the voltage-sensitive fluorescent indicator DiSBAC 4 (3). ( f ) Pictorial representation of the resultant intracellular changes in plasma membrane potential ( V m ) and intracellular potassium concentration ([K + ]i) in the presence of the ionophore gramicidin. ( g ) Relative cytoplasmic V m of CD8 + T cells in the indicated conditions assayed as in ( e ) with representative flow cytometry in ( h ). ( i ) IFN-γ production by CD8 + T cells following TCR induced stimulation in the indicated conditions. ( j ) Representative flow cytometry representing [K + ]i of CD8 + T cells as relative fluorescence of the potassium sensitive dye Asante-Green 4. ( k ) Relative [K + ]i of CD8 + T cells in the indicated conditions assayed as relative fluorescence of the potassium sensitive dye Asante-Green 4. ( l ) Representative flow cytometry of cytokine expression by CD8 + T cells following TCR stimulation in the indicated conditions with compiled quantification in ( m ). Error bars represent mean ± SEM. *P

    Article Snippet: Antibodies for surface staining and intracellular cytokine staining were purchased from BD Biosciences and eBiosciences.

    Techniques: Activity Assay, Expressing, Concentration Assay, Fluorescence, Flow Cytometry, Cytometry

    Depletion of intracellular potassium restores T cell cytokine production in the presence of elevated extracellular [K + ]. ( a,b ) Intracellular [K + ] of CD8 + T cells in the indicated conditions assayed via relative fluorescence of the potassium sensitive dye Asante-Green 4. ( c ) Flow cytometry analysis of IFN-γ production by primed CD8 + T cells following immobilized anti-CD3/28 based activation in the indicated conditions. ( d ) Plasma membrane potential ( V m ) of CD8 + T cells in the indicated conditions assayed with the voltage-sensitive fluorescent indicator DiSBAC 4 ( e ) Relative intracellular [K + ] of CD8 + T cells in the indicated conditions assayed with the potassium sensitive dye Asante-Green 4. ( f ) Pictorial representation of the resultant intracellular changes in plasma membrane potential ( V m ) and intracellular potassium concentration ([K + ]i) in the presence of the ionophore Valinomycin. ( g ) Flow cytometry analysis of CD8 + T cells following immobilized anti-CD3/28 based re-activated in the indicated conditions. ( h ) Plasma membrane potential ( V m ) of CD8 + T cells in the indicated conditions assayed with the voltage-sensitive fluorescent indicator DiSBAC 4 (3) ( i ) Relative intracellular [K + ] of CD8 + T cells in the indicated conditions assayed with the potassium sensitive dye Asante-Green 4. ( j ) Pictorial representation of the resultant intracellular changes in plasma membrane potential ( V m ) and intracellular potassium concentration ([K + ]i) in the presence of the Na + , K + ATPase inhibitor Ouabain. ( k ) Flow cytometry analysis of CD8 + T cells following immobilized anti-CD3/28 based re-activated in the indicated conditions. Error bars represent mean ± SEM. *P

    Journal: Nature

    Article Title: Ionic immune suppression within the tumour microenvironment limits T cell effector function

    doi: 10.1038/nature19364

    Figure Lengend Snippet: Depletion of intracellular potassium restores T cell cytokine production in the presence of elevated extracellular [K + ]. ( a,b ) Intracellular [K + ] of CD8 + T cells in the indicated conditions assayed via relative fluorescence of the potassium sensitive dye Asante-Green 4. ( c ) Flow cytometry analysis of IFN-γ production by primed CD8 + T cells following immobilized anti-CD3/28 based activation in the indicated conditions. ( d ) Plasma membrane potential ( V m ) of CD8 + T cells in the indicated conditions assayed with the voltage-sensitive fluorescent indicator DiSBAC 4 ( e ) Relative intracellular [K + ] of CD8 + T cells in the indicated conditions assayed with the potassium sensitive dye Asante-Green 4. ( f ) Pictorial representation of the resultant intracellular changes in plasma membrane potential ( V m ) and intracellular potassium concentration ([K + ]i) in the presence of the ionophore Valinomycin. ( g ) Flow cytometry analysis of CD8 + T cells following immobilized anti-CD3/28 based re-activated in the indicated conditions. ( h ) Plasma membrane potential ( V m ) of CD8 + T cells in the indicated conditions assayed with the voltage-sensitive fluorescent indicator DiSBAC 4 (3) ( i ) Relative intracellular [K + ] of CD8 + T cells in the indicated conditions assayed with the potassium sensitive dye Asante-Green 4. ( j ) Pictorial representation of the resultant intracellular changes in plasma membrane potential ( V m ) and intracellular potassium concentration ([K + ]i) in the presence of the Na + , K + ATPase inhibitor Ouabain. ( k ) Flow cytometry analysis of CD8 + T cells following immobilized anti-CD3/28 based re-activated in the indicated conditions. Error bars represent mean ± SEM. *P

    Article Snippet: Antibodies for surface staining and intracellular cytokine staining were purchased from BD Biosciences and eBiosciences.

    Techniques: Fluorescence, Flow Cytometry, Cytometry, Activation Assay, Concentration Assay

    Potassium induced T cell suppression is functionally non-redundant to CTLA-4 and PD-L1 co-inhibitory signals and is present in TIL neoantigen responses. ( a-b ) IFN-γ + of CD8 + in the indicated conditions. ( c ) Flow cytometry analysis of cytokine production by CD4 + T cells polarized to under ( c ) T H 1 or ( d ) T H 17 conditions and subsequently re-activated via immobilized anti-CD3/28 in the indicated experimental conditions. ( e ) Annexin V and Propidium Iodide (PI) staining following activation of primed CD8 + T cells in the indicated conditions. ( f ) Representative flow cytometry plots and quantification of human neo-antigen selected TIL from 3 patients stimulated in the indicated conditions with cognate mutated (mut) neo-antigen peptide pulsed target cells (autologous B-cells). ( g ) Relevant somatic mutation induced neoepitope for Pts. A-C in ( f ). ( h ) Representative flow cytometry and quantification of peripheral blood leukocytes from 3 patients transduced with an HLA*A201 restricted NY-ESO-1 TCR were assayed in the indicated conditions for IFN-γ production. Additional [K + ]e equal to 40 mM for ( a - e ) 50mM for ( f,h ), three culture replicates per patient per data point, representative of two independent experiments. Error bars represent mean ± SEM. *P

    Journal: Nature

    Article Title: Ionic immune suppression within the tumour microenvironment limits T cell effector function

    doi: 10.1038/nature19364

    Figure Lengend Snippet: Potassium induced T cell suppression is functionally non-redundant to CTLA-4 and PD-L1 co-inhibitory signals and is present in TIL neoantigen responses. ( a-b ) IFN-γ + of CD8 + in the indicated conditions. ( c ) Flow cytometry analysis of cytokine production by CD4 + T cells polarized to under ( c ) T H 1 or ( d ) T H 17 conditions and subsequently re-activated via immobilized anti-CD3/28 in the indicated experimental conditions. ( e ) Annexin V and Propidium Iodide (PI) staining following activation of primed CD8 + T cells in the indicated conditions. ( f ) Representative flow cytometry plots and quantification of human neo-antigen selected TIL from 3 patients stimulated in the indicated conditions with cognate mutated (mut) neo-antigen peptide pulsed target cells (autologous B-cells). ( g ) Relevant somatic mutation induced neoepitope for Pts. A-C in ( f ). ( h ) Representative flow cytometry and quantification of peripheral blood leukocytes from 3 patients transduced with an HLA*A201 restricted NY-ESO-1 TCR were assayed in the indicated conditions for IFN-γ production. Additional [K + ]e equal to 40 mM for ( a - e ) 50mM for ( f,h ), three culture replicates per patient per data point, representative of two independent experiments. Error bars represent mean ± SEM. *P

    Article Snippet: Antibodies for surface staining and intracellular cytokine staining were purchased from BD Biosciences and eBiosciences.

    Techniques: Flow Cytometry, Cytometry, Staining, Activation Assay, Mutagenesis, Transduction

    Extracellular K + release from apoptotic and necrotic cells inhibits T cell effector function ( a ) Potassium concentration in TIF, RPMI, and mouse serum ( b ) Ratio of indicated ions in normal human tissue in comparison to serum measured on the day of tissue collection in cancer patients undergoing resection of nearby cancers originating from the same tissue type. ( c ) Representative flow cytometry plots of B16 melanoma tumour cells following the indicated treatment. ( d ) Extracellular [K + ] quantification following the indicated treatment. ( e,f) Representative flow cytometry plots following anti-CD3/28 stimulated CD8 + T cells cultured in isotonic or hypertonic to RPMI in the indicated conditions ( g ) quantification of ( f ). ( h ) Quantification of cytokine production by CD8 + T cells following stimulation in the indicated conditions, elevated Ca 2+ and Mg 2+ conditions equal to 2mM, in comparison to 0.4 mM for control conditions. ( i ) Cytokine production by T cells across a titration of anti-CD3 in the indicated conditions. ( j ) Representative flow cytometry plots and quantification following anti-CD3/CD28 titration based activation of CD8 + T cells in the indicated conditions. Error bars represent mean ± SEM. *P

    Journal: Nature

    Article Title: Ionic immune suppression within the tumour microenvironment limits T cell effector function

    doi: 10.1038/nature19364

    Figure Lengend Snippet: Extracellular K + release from apoptotic and necrotic cells inhibits T cell effector function ( a ) Potassium concentration in TIF, RPMI, and mouse serum ( b ) Ratio of indicated ions in normal human tissue in comparison to serum measured on the day of tissue collection in cancer patients undergoing resection of nearby cancers originating from the same tissue type. ( c ) Representative flow cytometry plots of B16 melanoma tumour cells following the indicated treatment. ( d ) Extracellular [K + ] quantification following the indicated treatment. ( e,f) Representative flow cytometry plots following anti-CD3/28 stimulated CD8 + T cells cultured in isotonic or hypertonic to RPMI in the indicated conditions ( g ) quantification of ( f ). ( h ) Quantification of cytokine production by CD8 + T cells following stimulation in the indicated conditions, elevated Ca 2+ and Mg 2+ conditions equal to 2mM, in comparison to 0.4 mM for control conditions. ( i ) Cytokine production by T cells across a titration of anti-CD3 in the indicated conditions. ( j ) Representative flow cytometry plots and quantification following anti-CD3/CD28 titration based activation of CD8 + T cells in the indicated conditions. Error bars represent mean ± SEM. *P

    Article Snippet: Antibodies for surface staining and intracellular cytokine staining were purchased from BD Biosciences and eBiosciences.

    Techniques: Concentration Assay, Flow Cytometry, Cytometry, Cell Culture, Titration, Activation Assay

    Kcna3 mediated augmented K + efflux lowers intracellular [K + ], enhances Akt-mTOR signalling, and augments anti-tumour effector function to improve tumour clearance and host survival. ( a ) Pictorial representation of the potassium channel K v 1.3 ( Kcna3 ) ( b ) Representative immunoblot analysis of K v 1.3 protein abundance in CD8 + Pmel-1 cells following retroviral transduction with Ctrl (Empty-Thy1.1) or Kcna3 -Thy1.1 constructs. ( c , d ) Thy1.1 + Pmel-1 CD45.1 + CD8 + TIL 6-8 days following transfer into B16 melanoma-bearing mice were analysed for indicated phospho-residues ( c ) or in vivo IFN-γ production 6 hours after injection with Brefeldin A ( d ). ( e ) Relative cytokine expression of human TIL originating from the indicated histology following TCR stimulation in the indicated conditions. ( f,g ) Analysis of intracellular [K + ] ( f ) and representative flow cytometry for the expression of the indicated cytokines ( g ) of CD8 + Thy1.1 + T cells following transduction with retrovirus expressing Ctrl, Kcna3 , or Kcna3_PD Thy1.1 + constructs. ( h , i ) Rates of tumour growth ( h ) and host survival ( i ) represented over time following receipt of Pmel-1 CD8 + T cells transduced as in ( f,g ). 2-tailed Student’s t tests ( c - e ), 2-way ANOVA ( f ), Wilcox rank-sum analysis ( h ), and Log-rank of Kaplan-meier survival estimates ( i ). Error bars represent mean ± SEM. *P

    Journal: Nature

    Article Title: Ionic immune suppression within the tumour microenvironment limits T cell effector function

    doi: 10.1038/nature19364

    Figure Lengend Snippet: Kcna3 mediated augmented K + efflux lowers intracellular [K + ], enhances Akt-mTOR signalling, and augments anti-tumour effector function to improve tumour clearance and host survival. ( a ) Pictorial representation of the potassium channel K v 1.3 ( Kcna3 ) ( b ) Representative immunoblot analysis of K v 1.3 protein abundance in CD8 + Pmel-1 cells following retroviral transduction with Ctrl (Empty-Thy1.1) or Kcna3 -Thy1.1 constructs. ( c , d ) Thy1.1 + Pmel-1 CD45.1 + CD8 + TIL 6-8 days following transfer into B16 melanoma-bearing mice were analysed for indicated phospho-residues ( c ) or in vivo IFN-γ production 6 hours after injection with Brefeldin A ( d ). ( e ) Relative cytokine expression of human TIL originating from the indicated histology following TCR stimulation in the indicated conditions. ( f,g ) Analysis of intracellular [K + ] ( f ) and representative flow cytometry for the expression of the indicated cytokines ( g ) of CD8 + Thy1.1 + T cells following transduction with retrovirus expressing Ctrl, Kcna3 , or Kcna3_PD Thy1.1 + constructs. ( h , i ) Rates of tumour growth ( h ) and host survival ( i ) represented over time following receipt of Pmel-1 CD8 + T cells transduced as in ( f,g ). 2-tailed Student’s t tests ( c - e ), 2-way ANOVA ( f ), Wilcox rank-sum analysis ( h ), and Log-rank of Kaplan-meier survival estimates ( i ). Error bars represent mean ± SEM. *P

    Article Snippet: Antibodies for surface staining and intracellular cytokine staining were purchased from BD Biosciences and eBiosciences.

    Techniques: Transduction, Construct, Mouse Assay, In Vivo, Injection, Expressing, Flow Cytometry, Cytometry

    Cytokine production and transcription factor expression in antigen reactive CD4 + T cells. At day 10 days post first type II collagen (CII) immunization, draining lymph node (LN) cells from C57BL/6 mice (n = 3 or 4) and RORγt Tg mice (n = 3 or 4) were cultured with or without 100 μg/mL of denatured CII for 72 h. (A) IL-17 and IFNγ levels in supernatants measured by ELISA. (B) Cells were analyzed by staining for intracellular IL-17 and IFNγ. Data represent gated CD3 + and CD4 + . Bar graphs show percentages of cytokine-positive cells. (C) The expression levels of RORγt and T-bet were evaluated by intracellular staining. Data represent gated CD3 + and CD4 + . In the histograms, the gray shaded area represents data for the isotype control. Bar graphs show mean fluorescence intensity (MFI) of T-bet and RORγt Tg. (D) IL-10 levels in supernatants measured by ELISA. (E , F) Cells were analyzed by staining for intracellular IL-17 and IL-10 (E) , and propium iodide (PI) and Annexin V (F) . Data represent gated CD3 + and CD4 + . Data are representative of two independent experiments with similar results, and are mean ± standard error of the mean. * P

    Journal: Arthritis Research & Therapy

    Article Title: Involvement of RORγt-overexpressing T cells in the development of autoimmune arthritis in mice

    doi: 10.1186/s13075-015-0606-5

    Figure Lengend Snippet: Cytokine production and transcription factor expression in antigen reactive CD4 + T cells. At day 10 days post first type II collagen (CII) immunization, draining lymph node (LN) cells from C57BL/6 mice (n = 3 or 4) and RORγt Tg mice (n = 3 or 4) were cultured with or without 100 μg/mL of denatured CII for 72 h. (A) IL-17 and IFNγ levels in supernatants measured by ELISA. (B) Cells were analyzed by staining for intracellular IL-17 and IFNγ. Data represent gated CD3 + and CD4 + . Bar graphs show percentages of cytokine-positive cells. (C) The expression levels of RORγt and T-bet were evaluated by intracellular staining. Data represent gated CD3 + and CD4 + . In the histograms, the gray shaded area represents data for the isotype control. Bar graphs show mean fluorescence intensity (MFI) of T-bet and RORγt Tg. (D) IL-10 levels in supernatants measured by ELISA. (E , F) Cells were analyzed by staining for intracellular IL-17 and IL-10 (E) , and propium iodide (PI) and Annexin V (F) . Data represent gated CD3 + and CD4 + . Data are representative of two independent experiments with similar results, and are mean ± standard error of the mean. * P

    Article Snippet: For intracellular cytokine staining, cells were cultured with or without CII for 72 h, and GolgiStop (BD PharMingen) was added during the last 4 h of each culture.

    Techniques: Expressing, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence

    In vitro Stimulation of cytokine release in human PBMCs by REP 9 and REP 9C . Human PBMCs were exposed to no compound, CPG 7909, REP 9, and REP 9C, and levels of secreted cytokines after 48 h of induction were determined as described in the material and methods. Data is depicted on different scales (7, 70, 300, and 20,000 pg/ml) to demonstrate differences in cytokine levels measured. Values plotted are mean +/- standard deviation (n = 3). * = statistically insignificant difference in cytokine concentrations compared to untreated controls, + = statistically significant reduction in cytokine concentrations (REP 9C versus REP 9) (p

    Journal: Virology Journal

    Article Title: Amphipathic DNA polymers exhibit antiviral activity against systemic Murine Cytomegalovirus infection

    doi: 10.1186/1743-422X-6-214

    Figure Lengend Snippet: In vitro Stimulation of cytokine release in human PBMCs by REP 9 and REP 9C . Human PBMCs were exposed to no compound, CPG 7909, REP 9, and REP 9C, and levels of secreted cytokines after 48 h of induction were determined as described in the material and methods. Data is depicted on different scales (7, 70, 300, and 20,000 pg/ml) to demonstrate differences in cytokine levels measured. Values plotted are mean +/- standard deviation (n = 3). * = statistically insignificant difference in cytokine concentrations compared to untreated controls, + = statistically significant reduction in cytokine concentrations (REP 9C versus REP 9) (p

    Article Snippet: The following cytokines were measured using kits from Meso Scale Discovery (MSD, Inc., Gaithersburg, MD): Human IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8 IL-10, IL-12p70, IL-13, TNF-α and IFN Inducible Protein 10 (IP-10).

    Techniques: In Vitro, Standard Deviation

    Altered cytokine production by myeloid cells from R6/2 mice. (A) CD11b + myeloid cells were isolated from pooled blood samples obtained from 12-week old R6/2 or wild-type mice. Cell cultures primed with IFNγ and stimulated with LPS for 24 h showed significant differences in IL-6 and TNFα production. (B) R6/2 spleen and (C) R6/2 bone marrow myeloid cells were isolated using anti-CD11b magnetic beads, and individual cultures from each mouse were stimulated with IFNγ and LPS for 24 h. Measuring cytokine production in supernatants by multiplex ELISA, R6/2 spleen myeloid cells demonstrated changes in IL-1β levels, whilst bone marrow myeloid cells from R6/2 mice showed the same cytokine levels as wild-type cells. Graphs show mean concentrations corrected to protein content ± SEM. Unpaired two-tailed Student's t -tests. (A) n = technical replicates of pooled samples from different mice, (B + C) n = biological replicates representing individual mice.

    Journal: Neurobiology of Disease

    Article Title: Characterisation of immune cell function in fragment and full-length Huntington's disease mouse models

    doi: 10.1016/j.nbd.2014.10.012

    Figure Lengend Snippet: Altered cytokine production by myeloid cells from R6/2 mice. (A) CD11b + myeloid cells were isolated from pooled blood samples obtained from 12-week old R6/2 or wild-type mice. Cell cultures primed with IFNγ and stimulated with LPS for 24 h showed significant differences in IL-6 and TNFα production. (B) R6/2 spleen and (C) R6/2 bone marrow myeloid cells were isolated using anti-CD11b magnetic beads, and individual cultures from each mouse were stimulated with IFNγ and LPS for 24 h. Measuring cytokine production in supernatants by multiplex ELISA, R6/2 spleen myeloid cells demonstrated changes in IL-1β levels, whilst bone marrow myeloid cells from R6/2 mice showed the same cytokine levels as wild-type cells. Graphs show mean concentrations corrected to protein content ± SEM. Unpaired two-tailed Student's t -tests. (A) n = technical replicates of pooled samples from different mice, (B + C) n = biological replicates representing individual mice.

    Article Snippet: After 24 h, supernatants were collected for analysis of cytokine levels using multiplex ELISAs (mouse pro-inflammatory 7-Plex tissue culture kit measuring IFNγ, IL-1β, IL-10, IL-12p70, IL-6, mKC, TNFα) according to the manufacturer's instructions (Meso Scale Discovery).

    Techniques: Mouse Assay, Isolation, Magnetic Beads, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Altered cytokine production by macrophages from YAC128 mice. Peritoneal macrophages obtained via peritoneal lavage were stimulated with CSE for 24 h before IL-6 levels were measured using ELISA. A significant increase in cytokine production by YAC128 peritoneal macrophages was seen when compared with wild-type. Macrophages were also differentiated from bone marrow monocytes of three month old YAC128 and wild-type mice using M-CSF before stimulation with CSE for 24 h. Measuring IL-6 levels using ELISA showed no difference in cytokine production from YAC128 bone marrow-derived cells compared with wild-type controls. Graphs show mean concentrations corrected to protein content ± SEM. Unpaired two-tailed Student's t -tests.

    Journal: Neurobiology of Disease

    Article Title: Characterisation of immune cell function in fragment and full-length Huntington's disease mouse models

    doi: 10.1016/j.nbd.2014.10.012

    Figure Lengend Snippet: Altered cytokine production by macrophages from YAC128 mice. Peritoneal macrophages obtained via peritoneal lavage were stimulated with CSE for 24 h before IL-6 levels were measured using ELISA. A significant increase in cytokine production by YAC128 peritoneal macrophages was seen when compared with wild-type. Macrophages were also differentiated from bone marrow monocytes of three month old YAC128 and wild-type mice using M-CSF before stimulation with CSE for 24 h. Measuring IL-6 levels using ELISA showed no difference in cytokine production from YAC128 bone marrow-derived cells compared with wild-type controls. Graphs show mean concentrations corrected to protein content ± SEM. Unpaired two-tailed Student's t -tests.

    Article Snippet: After 24 h, supernatants were collected for analysis of cytokine levels using multiplex ELISAs (mouse pro-inflammatory 7-Plex tissue culture kit measuring IFNγ, IL-1β, IL-10, IL-12p70, IL-6, mKC, TNFα) according to the manufacturer's instructions (Meso Scale Discovery).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Two Tailed Test

    Altered cytokine production by myeloid cells from HdhQ150 mice. (A) Blood, (B) spleen and (C) bone marrow myeloid cells were isolated from 22-month old Hdh Q150 and wild type mice using anti-CD11b magnetic beads and seeded in culture for stimulation with IFNγ and LPS for 24 h. Cytokine production was analysed by multiplex ELISA. Whilst bone marrow cells produced similar levels of cytokine compared with wild-type, both blood and spleen myeloid cells produced significantly higher levels of pro-inflammatory cytokines. Graphs show mean concentrations corrected to protein content ± SEM. Unpaired two-tailed Student's t -tests. n = biological replicates representing individual mice.

    Journal: Neurobiology of Disease

    Article Title: Characterisation of immune cell function in fragment and full-length Huntington's disease mouse models

    doi: 10.1016/j.nbd.2014.10.012

    Figure Lengend Snippet: Altered cytokine production by myeloid cells from HdhQ150 mice. (A) Blood, (B) spleen and (C) bone marrow myeloid cells were isolated from 22-month old Hdh Q150 and wild type mice using anti-CD11b magnetic beads and seeded in culture for stimulation with IFNγ and LPS for 24 h. Cytokine production was analysed by multiplex ELISA. Whilst bone marrow cells produced similar levels of cytokine compared with wild-type, both blood and spleen myeloid cells produced significantly higher levels of pro-inflammatory cytokines. Graphs show mean concentrations corrected to protein content ± SEM. Unpaired two-tailed Student's t -tests. n = biological replicates representing individual mice.

    Article Snippet: After 24 h, supernatants were collected for analysis of cytokine levels using multiplex ELISAs (mouse pro-inflammatory 7-Plex tissue culture kit measuring IFNγ, IL-1β, IL-10, IL-12p70, IL-6, mKC, TNFα) according to the manufacturer's instructions (Meso Scale Discovery).

    Techniques: Mouse Assay, Isolation, Magnetic Beads, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Produced, Two Tailed Test

    Quantification of the cytokines secreted by DCs in presence of NPs DCs were incubated at 37°C in the presence of LPS (2μg/ml) and/or gold NPs (0.5mM) for 24 hours, then the supernatants were collected and used to determine the concentration of IL-6 and IL-12p70 as indicated in the material and methods section. A control was performed without additive: LPS and NPs. The results correspond to the mean value of three independents experiments. ** denotes a significant difference between the indicated experimental groups (p

    Journal: Journal of Nanoparticle Research

    Article Title: Analysis of the toxicity of gold nano particles on the immune system: effect on dendritic cell functions

    doi: 10.1007/s11051-009-9692-0

    Figure Lengend Snippet: Quantification of the cytokines secreted by DCs in presence of NPs DCs were incubated at 37°C in the presence of LPS (2μg/ml) and/or gold NPs (0.5mM) for 24 hours, then the supernatants were collected and used to determine the concentration of IL-6 and IL-12p70 as indicated in the material and methods section. A control was performed without additive: LPS and NPs. The results correspond to the mean value of three independents experiments. ** denotes a significant difference between the indicated experimental groups (p

    Article Snippet: Cytokines were measured in the supernatant of cell cultures by immuno assays using OptEIA for mouse cytokines (Pharmingen) according to the procedures recommended by the manufacturer.

    Techniques: Incubation, Concentration Assay

    The antimicrobial peptide cathelicidin induces IL-17 production by CD4 + T cells. Splenocytes isolated from C57BL/6J mice were cultured in Th1-driving conditions for 4 days, Th2-driving for 4 days or Th17-driving for 2 days in the presence or absence of 2.5μM murine cathelicidin. (A,B) Expression of cytokines was determined by intracellular flow cytometry. (C) sorted CD4 + T cells were cultured alone in Th17-driving conditions with 2.5μM cathelicidin. (D) Production of IL-17A following increasing doses of cathelicidin (at 48 hours) and (E) production over increasing days in culture was assessed. (F) Geometric mean of IL-17A expression was also assessed on day 2 of culture. (G) After 3 days in culture the culture medium was collected and ELISA used to quantify IL-17A protein. (H) Alternative cathelicidins were used – human LL-37 and the D-enantiomer D-LL37. (I) Representative flow cytometry plots showing RORγt expression - (J) RORγt expression increased over time following cathelicidin exposure and (K) in the presence of TGF-β. Data shown are individual mice used in separate experiments, with line at median. Statistical significance was determined using a two-way repeated measures ANOVA with a Bonferroni multiple comparison post-test (E, J), a two-way ANOVA with Bonferroni correction (K), a paired t-test (B, C, F, G) or a one-way ANOVA with a Dunnett’s multiple comparison post-test (D. H).

    Journal: bioRxiv

    Article Title: The neutrophil antimicrobial peptide cathelicidin promotes Th17 differentiation

    doi: 10.1101/2020.04.10.035543

    Figure Lengend Snippet: The antimicrobial peptide cathelicidin induces IL-17 production by CD4 + T cells. Splenocytes isolated from C57BL/6J mice were cultured in Th1-driving conditions for 4 days, Th2-driving for 4 days or Th17-driving for 2 days in the presence or absence of 2.5μM murine cathelicidin. (A,B) Expression of cytokines was determined by intracellular flow cytometry. (C) sorted CD4 + T cells were cultured alone in Th17-driving conditions with 2.5μM cathelicidin. (D) Production of IL-17A following increasing doses of cathelicidin (at 48 hours) and (E) production over increasing days in culture was assessed. (F) Geometric mean of IL-17A expression was also assessed on day 2 of culture. (G) After 3 days in culture the culture medium was collected and ELISA used to quantify IL-17A protein. (H) Alternative cathelicidins were used – human LL-37 and the D-enantiomer D-LL37. (I) Representative flow cytometry plots showing RORγt expression - (J) RORγt expression increased over time following cathelicidin exposure and (K) in the presence of TGF-β. Data shown are individual mice used in separate experiments, with line at median. Statistical significance was determined using a two-way repeated measures ANOVA with a Bonferroni multiple comparison post-test (E, J), a two-way ANOVA with Bonferroni correction (K), a paired t-test (B, C, F, G) or a one-way ANOVA with a Dunnett’s multiple comparison post-test (D. H).

    Article Snippet: 200,000 cells were plated per well of round-bottom 96-well plates in complete medium (RPMI, 10% fetal calf serum, 10 units/mL penicillin, 10 μg/mL streptomycin and 2 mM L-glutamine, all supplied by Gibco, ThermoFisher UK), with the correct combination of cytokines and neutralizing antibodies as follows.

    Techniques: Isolation, Mouse Assay, Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    Cathelicidin suppresses Th1 differentiation in the presence of TGF-β1. Splenocytes isolated from C57BL/6J mice were cultured in Th17-driving conditions for 48 hours in the presence or absence of 2.5μM murine cathelicidin. (A) production of IL-2 was assessed by ELISA on day 2 of culture. (B-D) Expression of Tbet and IFN-γ were assessed by flow cytometry. (E) Tbet expression was assessed following incubation with individual cytokines in the presence of cathelicidin. Data shown are individual mice used in separate experiments and analysed by paired two-tailed t tests.

    Journal: bioRxiv

    Article Title: The neutrophil antimicrobial peptide cathelicidin promotes Th17 differentiation

    doi: 10.1101/2020.04.10.035543

    Figure Lengend Snippet: Cathelicidin suppresses Th1 differentiation in the presence of TGF-β1. Splenocytes isolated from C57BL/6J mice were cultured in Th17-driving conditions for 48 hours in the presence or absence of 2.5μM murine cathelicidin. (A) production of IL-2 was assessed by ELISA on day 2 of culture. (B-D) Expression of Tbet and IFN-γ were assessed by flow cytometry. (E) Tbet expression was assessed following incubation with individual cytokines in the presence of cathelicidin. Data shown are individual mice used in separate experiments and analysed by paired two-tailed t tests.

    Article Snippet: 200,000 cells were plated per well of round-bottom 96-well plates in complete medium (RPMI, 10% fetal calf serum, 10 units/mL penicillin, 10 μg/mL streptomycin and 2 mM L-glutamine, all supplied by Gibco, ThermoFisher UK), with the correct combination of cytokines and neutralizing antibodies as follows.

    Techniques: Isolation, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Incubation, Two Tailed Test

    Blocking the interaction between CD160 and HVEM enhances CMV and HIV-specific CD8 T cell proliferation and cytokine production. HIV-infected individuals(n = 11) were stimulated with HLA-restricted CMV and HIV peptides in the presence of blocking antibodies to HVEM and/or PD-L1. Dying cells were eliminated with an amine-reactive viability dye and PBMCs were stained at day 6 with HLA class I matched-tetramers and mAbs to CD3 and CD8. (A) Representative flow cytometry plots of an HIV-infected patient stimulated for 6 days with a CMV and HIV peptide in the presence of isotype, αPD-L1 and/or αHVEM blocking antibodies. Scatter plots represent the median fold increase in (B) CMV and (C) HIV-specific proliferation (Tetramer + /CFSE low ) compared to the isotype control. Each dot represents a CMV or HIV tetramer-specific response. P -values were determined by the Wilcoxon matched pairs test.

    Journal: PLoS Pathogens

    Article Title: CD160 and PD-1 Co-Expression on HIV-Specific CD8 T Cells Defines a Subset with Advanced Dysfunction

    doi: 10.1371/journal.ppat.1002840

    Figure Lengend Snippet: Blocking the interaction between CD160 and HVEM enhances CMV and HIV-specific CD8 T cell proliferation and cytokine production. HIV-infected individuals(n = 11) were stimulated with HLA-restricted CMV and HIV peptides in the presence of blocking antibodies to HVEM and/or PD-L1. Dying cells were eliminated with an amine-reactive viability dye and PBMCs were stained at day 6 with HLA class I matched-tetramers and mAbs to CD3 and CD8. (A) Representative flow cytometry plots of an HIV-infected patient stimulated for 6 days with a CMV and HIV peptide in the presence of isotype, αPD-L1 and/or αHVEM blocking antibodies. Scatter plots represent the median fold increase in (B) CMV and (C) HIV-specific proliferation (Tetramer + /CFSE low ) compared to the isotype control. Each dot represents a CMV or HIV tetramer-specific response. P -values were determined by the Wilcoxon matched pairs test.

    Article Snippet: Supernatants harvested following a 6-day CFSE assay were used to assess cytokine production by cytokine bead array in the presence or absence of PD-1 and or CD160 engagement by their respective ligands.

    Techniques: Blocking Assay, Infection, Staining, Flow Cytometry, Cytometry

    Histologic staining for mucin after cytokine blockade. Light micrographs of lung sections stained with AB/PAS from IL-9 transgene-negative ( a ) or –positive ( b – f ) mice that were housed for 14 days with doxycycline-supplemented food. During the entire time of IL-9 transgene induction selected groups of animals received Ab’s: ( c ) control Ab’s, hIgG; ( d ) anti–IL-4; ( e ) sIL-13Rα-Fc; ( f ) anti–IL-4 and sIL-13Rα-Fc. Lung sections from transgene-positive mice that received anti–IL-4 Ab’s, control Ab’s, or no Ab’s showed strong positive (magenta) staining for mucin in airway epithelial cells (arrows) and accumulation of inflammatory cells near blood vessels and airways ( b – d ). Infiltration of lung tissue with inflammatory cells or positive staining for mucin was not observed in sections from transgene-positive mice that received sIL-13Rα-Fc alone ( e ), or in combination with anti–IL-4 ( f ), or in transgene-negative mice ( a ). Original magnification ×300.

    Journal: The Journal of Clinical Investigation

    Article Title: Pulmonary overexpression of IL-9 induces Th2 cytokine expression, leading to immune pathology

    doi: 10.1172/JCI13696

    Figure Lengend Snippet: Histologic staining for mucin after cytokine blockade. Light micrographs of lung sections stained with AB/PAS from IL-9 transgene-negative ( a ) or –positive ( b – f ) mice that were housed for 14 days with doxycycline-supplemented food. During the entire time of IL-9 transgene induction selected groups of animals received Ab’s: ( c ) control Ab’s, hIgG; ( d ) anti–IL-4; ( e ) sIL-13Rα-Fc; ( f ) anti–IL-4 and sIL-13Rα-Fc. Lung sections from transgene-positive mice that received anti–IL-4 Ab’s, control Ab’s, or no Ab’s showed strong positive (magenta) staining for mucin in airway epithelial cells (arrows) and accumulation of inflammatory cells near blood vessels and airways ( b – d ). Infiltration of lung tissue with inflammatory cells or positive staining for mucin was not observed in sections from transgene-positive mice that received sIL-13Rα-Fc alone ( e ), or in combination with anti–IL-4 ( f ), or in transgene-negative mice ( a ). Original magnification ×300.

    Article Snippet: Kopf M, et al. Disruption of the murine IL-4 gene blocks Th2 cytokine responses.

    Techniques: Staining, Mouse Assay

    Characterization of inflammatory cells in lung lavage fluid after cytokine blockade. IL-9 transgene-negative (TG – ) or -positive (TG + ) mice were housed with (+Dox) or without (–Dox) doxycycline-supplemented food for 14 days. During the entire time of IL-9 transgene induction, selected groups of animals received anti–IL-4 Ab’s, sIL-13Rα-Fc, or isotype control Ab’s (hIgG) as described in Methods. At day 14, mice were sacrificed and the lung lavaged. ( a ) Total lung lavage cell counts. ( b ) Differential cell counts on lung lavage cells. Results are expressed as total cell numbers and were obtained from at least 200 cell counts. Data are expressed as mean ± SD. Significant differences in total cell number ( a ) or number of eosinophils ( b ) compared with that of transgene-positive mice that received doxycycline but no Ab’s are indicated with an asterisk.

    Journal: The Journal of Clinical Investigation

    Article Title: Pulmonary overexpression of IL-9 induces Th2 cytokine expression, leading to immune pathology

    doi: 10.1172/JCI13696

    Figure Lengend Snippet: Characterization of inflammatory cells in lung lavage fluid after cytokine blockade. IL-9 transgene-negative (TG – ) or -positive (TG + ) mice were housed with (+Dox) or without (–Dox) doxycycline-supplemented food for 14 days. During the entire time of IL-9 transgene induction, selected groups of animals received anti–IL-4 Ab’s, sIL-13Rα-Fc, or isotype control Ab’s (hIgG) as described in Methods. At day 14, mice were sacrificed and the lung lavaged. ( a ) Total lung lavage cell counts. ( b ) Differential cell counts on lung lavage cells. Results are expressed as total cell numbers and were obtained from at least 200 cell counts. Data are expressed as mean ± SD. Significant differences in total cell number ( a ) or number of eosinophils ( b ) compared with that of transgene-positive mice that received doxycycline but no Ab’s are indicated with an asterisk.

    Article Snippet: Kopf M, et al. Disruption of the murine IL-4 gene blocks Th2 cytokine responses.

    Techniques: Mouse Assay

    Analysis of cytokine expression in total lung tissue. ( a ) Levels of various mRNAs encoding predominantly Th2 cytokines from individual IL-9 transgene-negative (lane 1, 2) and -positive (lane 3, 4) mice after transgene induction by doxycycline were compared by RPA. The P 32 -labeled multiprobe template set (T) was used as a size marker. Levels of L32 and GAPDH mRNAs encoding housekeeping genes were used to ensure equal loading of samples. ( b ) Levels of mRNA encoding IL-4 were evaluated by RT-PCR using total lung RNA isolated from transgene-negative (lane 1, 2) and -positive (lane 3, 4) mice 14 days after IL-9 transgene induction by doxycycline. Levels of mRNA encoding HPRT, a housekeeping gene, were used to demonstrate equal loading of samples. Fragment sizes of a molecular-weight marker are indicated in base pairs.

    Journal: The Journal of Clinical Investigation

    Article Title: Pulmonary overexpression of IL-9 induces Th2 cytokine expression, leading to immune pathology

    doi: 10.1172/JCI13696

    Figure Lengend Snippet: Analysis of cytokine expression in total lung tissue. ( a ) Levels of various mRNAs encoding predominantly Th2 cytokines from individual IL-9 transgene-negative (lane 1, 2) and -positive (lane 3, 4) mice after transgene induction by doxycycline were compared by RPA. The P 32 -labeled multiprobe template set (T) was used as a size marker. Levels of L32 and GAPDH mRNAs encoding housekeeping genes were used to ensure equal loading of samples. ( b ) Levels of mRNA encoding IL-4 were evaluated by RT-PCR using total lung RNA isolated from transgene-negative (lane 1, 2) and -positive (lane 3, 4) mice 14 days after IL-9 transgene induction by doxycycline. Levels of mRNA encoding HPRT, a housekeeping gene, were used to demonstrate equal loading of samples. Fragment sizes of a molecular-weight marker are indicated in base pairs.

    Article Snippet: Kopf M, et al. Disruption of the murine IL-4 gene blocks Th2 cytokine responses.

    Techniques: Expressing, Mouse Assay, Recombinase Polymerase Amplification, Labeling, Marker, Reverse Transcription Polymerase Chain Reaction, Isolation, Molecular Weight

    Cytokine chip analysis delineates a different cytokine production signature of CTCs. a The cytokine profile array from the cell culture supernatants of the CTC-TJH-01, 95-D and A549 cells were detected by capture antibodies spotted in duplicate on nitrocellulose membranes. b The quantitative analysis of cytokine profile in A. c The fold changes of CX3CL1 and CXCL5 in the CTC-TJH-01, 95-D and A549 cells. d Verification of CX3CL1 and CXCL5 expression in the CTC-TJH-01, 95-D and A549 cells by western blot. e Real-time PCR and f western blotting were conducted to examine the mRNA and protein expression of CXCL5 in the CTC-TJH-01 cells transfected with the CXCL5-specific siRNA or a non-specific siRNA as the negative control (NC), respectively. g CCK-8 assay and h cell apoptosis assay were used to determine cell proliferation and apoptosis. i , j A transwell assay was used to determine cell migration and invasion. k ELISA assay was used to determine the CX3CL1 secretion level of cell supernatant. l , m After co-culture of CTC-TJH-01, 95-D and A549 cells with lymphocytes; flow cytometry was used to detect lymphocyte recruitment. Each bar represents the mean ± SD of three separate experiments. * P

    Journal: Cancer Cell International

    Article Title: Establishment and characterization of a patient-derived circulating lung tumor cell line in vitro and in vivo

    doi: 10.1186/s12935-019-0735-z

    Figure Lengend Snippet: Cytokine chip analysis delineates a different cytokine production signature of CTCs. a The cytokine profile array from the cell culture supernatants of the CTC-TJH-01, 95-D and A549 cells were detected by capture antibodies spotted in duplicate on nitrocellulose membranes. b The quantitative analysis of cytokine profile in A. c The fold changes of CX3CL1 and CXCL5 in the CTC-TJH-01, 95-D and A549 cells. d Verification of CX3CL1 and CXCL5 expression in the CTC-TJH-01, 95-D and A549 cells by western blot. e Real-time PCR and f western blotting were conducted to examine the mRNA and protein expression of CXCL5 in the CTC-TJH-01 cells transfected with the CXCL5-specific siRNA or a non-specific siRNA as the negative control (NC), respectively. g CCK-8 assay and h cell apoptosis assay were used to determine cell proliferation and apoptosis. i , j A transwell assay was used to determine cell migration and invasion. k ELISA assay was used to determine the CX3CL1 secretion level of cell supernatant. l , m After co-culture of CTC-TJH-01, 95-D and A549 cells with lymphocytes; flow cytometry was used to detect lymphocyte recruitment. Each bar represents the mean ± SD of three separate experiments. * P

    Article Snippet: Cytokine chip assay To study the underlying mechanism of metastasis in CTC-TJH-01 cells, we used a human cytokine antibody array (AAH-CYT-1000, RayBiotech, Inc.) to detect cytokine secretion in CTC-TJH-01, A549, and 95-D cell culture supernatants.

    Techniques: Chromatin Immunoprecipitation, Cell Culture, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Negative Control, CCK-8 Assay, Apoptosis Assay, Transwell Assay, Migration, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Flow Cytometry, Cytometry

    Analysis of EV function after floatation on iodixanol gradient A–C Pellets obtained after 2K, 10K and 100K centrifugations from 80–120 × 10 6 cells were floated into an overlayed iodixanol gradient (A). Five fractions were collected and analysed by WB (representative of five experiments) (B), showing the floatation of MHC II‐ and CD9‐enriched EVs in fraction F3–4 and fraction F5–6. Densities of recovered fractions, as measured by refractometry, are shown below the WB figure (mean density of five independent gradients). Quantification of the distribution of MHC II relative abundance in all fractions of each centrifugation pellet analysed by WB of five independent experiments (C). For each pellet, arbitrary units (AU) = (SI fraction )/sum(SI F1–2 + SI F3–4 + SI F5–6 + SI F7–8 + SI F9–10 ) where SI = signal intensity (i.e. ratio of signal intensity in the given pellet to the total secreted protein) ( n = 5, one symbol per donor). Red line indicates median. D IL‐13 and IFN‐γ secretion was measured in supernatants after 6 days of total CD4 + T‐cell culture with the different fractions of the iodixanol gradients of the 2K, 10K and 100K pellets. The graph indicates the relative contribution of each fraction to the total cytokine secretion induced by each pellet. The relative contribution for each donor was calculated as CC fraction /sum(CC F1–2 + CC F3–4 + CC F5–6 + CC F7–8 + CC F9–10 ) for each pellet, where CC is cytokine concentration. Mean + SEM is shown. Below each graph, the sum of the cytokine concentration in all the fractions for each pellet is shown (median of 14 individual DC‐EV:T‐cell combinations). Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Qualitative differences in T‐cell activation by dendritic cell‐derived extracellular vesicle subtypes

    doi: 10.15252/embj.201696003

    Figure Lengend Snippet: Analysis of EV function after floatation on iodixanol gradient A–C Pellets obtained after 2K, 10K and 100K centrifugations from 80–120 × 10 6 cells were floated into an overlayed iodixanol gradient (A). Five fractions were collected and analysed by WB (representative of five experiments) (B), showing the floatation of MHC II‐ and CD9‐enriched EVs in fraction F3–4 and fraction F5–6. Densities of recovered fractions, as measured by refractometry, are shown below the WB figure (mean density of five independent gradients). Quantification of the distribution of MHC II relative abundance in all fractions of each centrifugation pellet analysed by WB of five independent experiments (C). For each pellet, arbitrary units (AU) = (SI fraction )/sum(SI F1–2 + SI F3–4 + SI F5–6 + SI F7–8 + SI F9–10 ) where SI = signal intensity (i.e. ratio of signal intensity in the given pellet to the total secreted protein) ( n = 5, one symbol per donor). Red line indicates median. D IL‐13 and IFN‐γ secretion was measured in supernatants after 6 days of total CD4 + T‐cell culture with the different fractions of the iodixanol gradients of the 2K, 10K and 100K pellets. The graph indicates the relative contribution of each fraction to the total cytokine secretion induced by each pellet. The relative contribution for each donor was calculated as CC fraction /sum(CC F1–2 + CC F3–4 + CC F5–6 + CC F7–8 + CC F9–10 ) for each pellet, where CC is cytokine concentration. Mean + SEM is shown. Below each graph, the sum of the cytokine concentration in all the fractions for each pellet is shown (median of 14 individual DC‐EV:T‐cell combinations). Source data are available online for this figure.

    Article Snippet: At the end of the T cell/EV or DC culture, cells were centrifuged and the supernatants were collected and frozen at −20°C before analysis of cytokine secretion by cytometric bead array (BD Biosciences) following manufacturer indications.

    Techniques: Western Blot, Centrifugation, Cell Culture, Concentration Assay

    IL-24 increases the expression of anti-inflammatory cytokines and neuroprotective factors by primary murine astrocytes. a Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/ml) for 4 h prior to N. meningitidis infection (Nm; MOI of 10:1 bacteria to each astrocyte) or vehicle control. At 24 or 48 h postinfection, IL-10 protein release was assessed by specific capture ELISA. Asterisks and dagger indicate a statistically significant difference from uninfected cells and similarly challenged cells in the absence of IL-24, respectively ( n = 3; p

    Journal: Journal of Neuroinflammation

    Article Title: Murine astrocytes produce IL-24 and are susceptible to the immunosuppressive effects of this cytokine

    doi: 10.1186/s12974-019-1444-1

    Figure Lengend Snippet: IL-24 increases the expression of anti-inflammatory cytokines and neuroprotective factors by primary murine astrocytes. a Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/ml) for 4 h prior to N. meningitidis infection (Nm; MOI of 10:1 bacteria to each astrocyte) or vehicle control. At 24 or 48 h postinfection, IL-10 protein release was assessed by specific capture ELISA. Asterisks and dagger indicate a statistically significant difference from uninfected cells and similarly challenged cells in the absence of IL-24, respectively ( n = 3; p

    Article Snippet: A standard curve was constructed using varying dilutions of recombinant cytokines (BD Biosciences), and the cytokine content of culture supernatants determined by extrapolation of absorbances to the standard curve.

    Techniques: Expressing, Recombinant, Infection, Enzyme-linked Immunosorbent Assay

    TCR-CAR can redirect NK-92 cells. ( a ) NK-92 cells were non transfected (grey) or transfected with DMF5 TCR-CAR TCR-CAR (red), Radium-1 (blue) and stained with specific antibody or multimer, respectively. Shown is a single staining representative of two separate stainings. ( b ) Stimulation of plain NK-92 cells (white) or transfected with TCR-CAR constructs (DMF5, black and Radium-1, grey) with Granta-519 loaded (+) or not (−) with the cognate peptide was performed for 6 hours at a E:T ratio of 1:2. Presence of CD107a was detected by flow cytometry and the MFI of the signal was plotted. Mean ± SEM, N = 3. Unpaired t-test was used as statistical analysis between indicated groups and similar trends were depicted as a group. For cytokines analysis, NK-92 cells transduced (black and grey) or not (white) were co-cultured with target cells loaded (+) or not (−) with the cognate peptide at 1:2 E:T ratio for 24 hours. Non transfected NK-92 cells alone (NK-92 only) were also tested. Supernatants from each condition were collected and presence of TNF-α and IFN-γ was performed with the Bio-Rad Bio-Plex 100 system. Measurements were made in triplicate from three separate supernatants per condition. Cytokine concentrations are shown in picograms per milliliter (pg/mL). Mean ± SEM, N = 3. Unpaired t-test was used as statistical analysis between indicated groups and similar trends were depicted as a group. ( c ) Specific lysis of target cells loaded with the indicated peptide (MART-1 peptide, black, TGFbR2 peptide, white, no peptide, grey) by plain NK-92 (circles) or NK-92 expressing TCR-CAR (squares) at different E:T ratios in a BLI cytotoxic assay. The specific lysis luminescence readings were collected after 10 hours of co-culture. Mean ± SEM, N = 3. Unpaired t-test performed between indicated group and corresponding non-transduced NK-92 group. Ranges for unpaired t-test were as follows *P

    Journal: Scientific Reports

    Article Title: A TCR-based Chimeric Antigen Receptor

    doi: 10.1038/s41598-017-11126-y

    Figure Lengend Snippet: TCR-CAR can redirect NK-92 cells. ( a ) NK-92 cells were non transfected (grey) or transfected with DMF5 TCR-CAR TCR-CAR (red), Radium-1 (blue) and stained with specific antibody or multimer, respectively. Shown is a single staining representative of two separate stainings. ( b ) Stimulation of plain NK-92 cells (white) or transfected with TCR-CAR constructs (DMF5, black and Radium-1, grey) with Granta-519 loaded (+) or not (−) with the cognate peptide was performed for 6 hours at a E:T ratio of 1:2. Presence of CD107a was detected by flow cytometry and the MFI of the signal was plotted. Mean ± SEM, N = 3. Unpaired t-test was used as statistical analysis between indicated groups and similar trends were depicted as a group. For cytokines analysis, NK-92 cells transduced (black and grey) or not (white) were co-cultured with target cells loaded (+) or not (−) with the cognate peptide at 1:2 E:T ratio for 24 hours. Non transfected NK-92 cells alone (NK-92 only) were also tested. Supernatants from each condition were collected and presence of TNF-α and IFN-γ was performed with the Bio-Rad Bio-Plex 100 system. Measurements were made in triplicate from three separate supernatants per condition. Cytokine concentrations are shown in picograms per milliliter (pg/mL). Mean ± SEM, N = 3. Unpaired t-test was used as statistical analysis between indicated groups and similar trends were depicted as a group. ( c ) Specific lysis of target cells loaded with the indicated peptide (MART-1 peptide, black, TGFbR2 peptide, white, no peptide, grey) by plain NK-92 (circles) or NK-92 expressing TCR-CAR (squares) at different E:T ratios in a BLI cytotoxic assay. The specific lysis luminescence readings were collected after 10 hours of co-culture. Mean ± SEM, N = 3. Unpaired t-test performed between indicated group and corresponding non-transduced NK-92 group. Ranges for unpaired t-test were as follows *P

    Article Snippet: Cytokines in supernatants were measured by using the Bio-Plex ProTM Human Cytokine 17-plex Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to manufacturer’s protocol on a Bio-Rad Bio-Plex 100 system.

    Techniques: Transfection, Staining, Construct, Flow Cytometry, Cytometry, Cell Culture, Lysis, Expressing, Co-Culture Assay

    Changes in cytokines and adiponectin. There was a significant difference in the change in TNF-α and adiponectin levels between the KRG and placebo groups. The mean TNF-α and adiponectin levels in patients with a BMI of 25 kg/m 2 or more showed particular improvement after KRG therapy. BMI, body mass index; IL-6, interleukin-6; KR G, Korean Red Ginseng; ns, not significant; TNF-α, tumor necrosis factor-α.

    Journal: Journal of Ginseng Research

    Article Title: Anti-inflammatory and antifatigue effect of Korean Red Ginseng in patients with nonalcoholic fatty liver disease

    doi: 10.1016/j.jgr.2015.07.006

    Figure Lengend Snippet: Changes in cytokines and adiponectin. There was a significant difference in the change in TNF-α and adiponectin levels between the KRG and placebo groups. The mean TNF-α and adiponectin levels in patients with a BMI of 25 kg/m 2 or more showed particular improvement after KRG therapy. BMI, body mass index; IL-6, interleukin-6; KR G, Korean Red Ginseng; ns, not significant; TNF-α, tumor necrosis factor-α.

    Article Snippet: 2.3 Cytokines and adiponectin For the measurements of cytokines and adiponectin, serum was processed using the TNF alpha human ELISA kit (ab100654; Abcam, Inc., Cambridge, United Kingdom), the IL-6 human ELISA kit (ab100572; Abcam, Inc.), and the adiponectin human ELISA kit (ab99968; Abcam, Inc.).

    Techniques:

    Evaluation of circulating cytokines/chemokines in CVI patients 6 months after surgical hemodynamic correction. The circulating levels of cytokines/chemokines were assessed in plasma samples harvested from forearm vein of healthy subjects (controls) and of CVI patients before (baseline) and after (6 months) CHIVA. In (a), the 11 cytokines/chemokines displaying lower levels after CHIVA, compared to baseline levels, are shown; * P

    Journal: Journal of Immunology Research

    Article Title: Modulation of Circulating Cytokine-Chemokine Profile in Patients Affected by Chronic Venous Insufficiency Undergoing Surgical Hemodynamic Correction

    doi: 10.1155/2014/473765

    Figure Lengend Snippet: Evaluation of circulating cytokines/chemokines in CVI patients 6 months after surgical hemodynamic correction. The circulating levels of cytokines/chemokines were assessed in plasma samples harvested from forearm vein of healthy subjects (controls) and of CVI patients before (baseline) and after (6 months) CHIVA. In (a), the 11 cytokines/chemokines displaying lower levels after CHIVA, compared to baseline levels, are shown; * P

    Article Snippet: Analysis of Cytokines and Chemokines in Plasma Samples Plasma samples were frozen and thawed only once before performing the MILLIPLEX MAP Human Cytokine/Chemokine Panel (Merck Millipore, Billerica, MA), a bead-based multiplex immunoassay, which allows the simultaneous quantification of the following 29 human cytokines: IL-1α , IL-1β , IL-1 receptor antagonist (ra), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17A, EGF, Eotaxin, G-CSF, GM-CSF, IFN-α 2, IFN-γ , CXCL10, MCP-1, MIP-1α , MIP-1β , TNF-α , TNF-β , and VEGF.

    Techniques:

    Network analysis of the cytokines/chemokines differentially expressed in the context of CVI. The identified panel of cytokines/chemokines characterizing CVI was assessed for network analysis. In (a), the top-score biological network generated by using the analysis network algorithm illustrating the connections among the differentially expressed cytokine/chemokine highlighting the role of RANTES as hub. In (b), proteins network between PDGF and EGF generated by the shortest path algorithm is shown.

    Journal: Journal of Immunology Research

    Article Title: Modulation of Circulating Cytokine-Chemokine Profile in Patients Affected by Chronic Venous Insufficiency Undergoing Surgical Hemodynamic Correction

    doi: 10.1155/2014/473765

    Figure Lengend Snippet: Network analysis of the cytokines/chemokines differentially expressed in the context of CVI. The identified panel of cytokines/chemokines characterizing CVI was assessed for network analysis. In (a), the top-score biological network generated by using the analysis network algorithm illustrating the connections among the differentially expressed cytokine/chemokine highlighting the role of RANTES as hub. In (b), proteins network between PDGF and EGF generated by the shortest path algorithm is shown.

    Article Snippet: Analysis of Cytokines and Chemokines in Plasma Samples Plasma samples were frozen and thawed only once before performing the MILLIPLEX MAP Human Cytokine/Chemokine Panel (Merck Millipore, Billerica, MA), a bead-based multiplex immunoassay, which allows the simultaneous quantification of the following 29 human cytokines: IL-1α , IL-1β , IL-1 receptor antagonist (ra), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17A, EGF, Eotaxin, G-CSF, GM-CSF, IFN-α 2, IFN-γ , CXCL10, MCP-1, MIP-1α , MIP-1β , TNF-α , TNF-β , and VEGF.

    Techniques: Generated

    Comparison between the levels of cytokines/chemokines measured in CVI patients in the varicose vein blood and in the systemic circulation . The circulating levels of 31 cytokines/chemokines were measured in paired blood samples collected from the forearm and the varicose vein of CVI patients ( n = 6). Data are expressed as ratio between local (varicose) and systemic (forearm) values. In (a), 7 representative cytokines/chemokines displaying the same levels at the varicose vein site and at systemic level are shown. In (b), the 3 cytokines displaying higher circulating levels (* P

    Journal: Journal of Immunology Research

    Article Title: Modulation of Circulating Cytokine-Chemokine Profile in Patients Affected by Chronic Venous Insufficiency Undergoing Surgical Hemodynamic Correction

    doi: 10.1155/2014/473765

    Figure Lengend Snippet: Comparison between the levels of cytokines/chemokines measured in CVI patients in the varicose vein blood and in the systemic circulation . The circulating levels of 31 cytokines/chemokines were measured in paired blood samples collected from the forearm and the varicose vein of CVI patients ( n = 6). Data are expressed as ratio between local (varicose) and systemic (forearm) values. In (a), 7 representative cytokines/chemokines displaying the same levels at the varicose vein site and at systemic level are shown. In (b), the 3 cytokines displaying higher circulating levels (* P

    Article Snippet: Analysis of Cytokines and Chemokines in Plasma Samples Plasma samples were frozen and thawed only once before performing the MILLIPLEX MAP Human Cytokine/Chemokine Panel (Merck Millipore, Billerica, MA), a bead-based multiplex immunoassay, which allows the simultaneous quantification of the following 29 human cytokines: IL-1α , IL-1β , IL-1 receptor antagonist (ra), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17A, EGF, Eotaxin, G-CSF, GM-CSF, IFN-α 2, IFN-γ , CXCL10, MCP-1, MIP-1α , MIP-1β , TNF-α , TNF-β , and VEGF.

    Techniques:

    Modulation of circulating cytokines/chemokines in CVI patients after

    Journal: Journal of Immunology Research

    Article Title: Modulation of Circulating Cytokine-Chemokine Profile in Patients Affected by Chronic Venous Insufficiency Undergoing Surgical Hemodynamic Correction

    doi: 10.1155/2014/473765

    Figure Lengend Snippet: Modulation of circulating cytokines/chemokines in CVI patients after

    Article Snippet: Analysis of Cytokines and Chemokines in Plasma Samples Plasma samples were frozen and thawed only once before performing the MILLIPLEX MAP Human Cytokine/Chemokine Panel (Merck Millipore, Billerica, MA), a bead-based multiplex immunoassay, which allows the simultaneous quantification of the following 29 human cytokines: IL-1α , IL-1β , IL-1 receptor antagonist (ra), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17A, EGF, Eotaxin, G-CSF, GM-CSF, IFN-α 2, IFN-γ , CXCL10, MCP-1, MIP-1α , MIP-1β , TNF-α , TNF-β , and VEGF.

    Techniques: