cytokines Search Results


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  • 99
    Thermo Fisher cytokines
    NC410 therapy induces immune activity in the local tumor microenvironment 1 × 10 7 total human PBMCs were adoptively transferred intravenously to NSG mice (N = 6/group) on Day 0. 1 × 10 6 HT-29 tumor cells were injected subcutaneously with Matrigel on Day 1. Mice were treated with NC410 or control by intraperitoneal injection, Q4D x 4 doses followed by Q7D until endpoint. On day 27, tumor and spleen tissues were isolated for tumor infiltrating T cells and <t>cytokine</t> analysis. A) CD4 + and B) CD8 + TIL cell numbers in the tumor. The cell number was counted by flow cytometry and normalized to weight (gram) of tumor tissue. Asterisks indicate statistical significance (*P
    Cytokines, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11775 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cytokines
    NC410 therapy induces immune activity in the local tumor microenvironment 1 × 10 7 total human PBMCs were adoptively transferred intravenously to NSG mice (N = 6/group) on Day 0. 1 × 10 6 HT-29 tumor cells were injected subcutaneously with Matrigel on Day 1. Mice were treated with NC410 or control by intraperitoneal injection, Q4D x 4 doses followed by Q7D until endpoint. On day 27, tumor and spleen tissues were isolated for tumor infiltrating T cells and <t>cytokine</t> analysis. A) CD4 + and B) CD8 + TIL cell numbers in the tumor. The cell number was counted by flow cytometry and normalized to weight (gram) of tumor tissue. Asterisks indicate statistical significance (*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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    92
    Bio-Rad cytokines
    Relationship between the severity of diabetic retinopathy (DR) and the aqueous interleukin-8 (IL-8) level. The results by measuring <t>cytokines</t> in the aqueous humor samples shows that the IL-8 level in aqueous humor increased with increasing severity of DR, and this correlation was significant (r=0.309, p
    Cytokines, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 5709 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson cytokines
    New insights into the heterogeneity of human Th17 cells at homeostasis and during ART-controlled HIV-1 infection. In this work we identified two new subsets of CCR6 + T-cells, CCR6 + DN/CCR4 − CXCR3 − and CCR6 + DP/CCR4 + CXCR3 + , that share Th17 features with the previously described Th17/CCR4 + CXCR3 − and Th1Th17/CCR4 − CXCR3 + [ 5 ]. Despite these similarities, CCR6 + DN distinguished from the other three subsets by superior their ability to produce Th17 effector <t>cytokines</t> ( e.g., IL-17F, IL-8, and IL-21) and their predominant frequency/counts in the blood and lymph nodes HIV-infected individuals receiving ART. Finally, we demonstrate that CCR6 + DN harbor replication-competent HIV-DNA. Thus, we reveal the existence in humans of four Th17-polarized CCR6 + subsets that represent distinct stages of Th17 differentiation, with CCR6 + DN being the most predominant and contributing to HIV reservoir persistence under ART
    Cytokines, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 14656 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Meso Scale Diagnostics LLC cytokines
    <t>Cytokines</t> significantly induced during VZV infection in HFLs Cells and culture supernatants were harvested at 72 hpi from mock- or VZV-infected HFLs. (A) Flow cytometry analyses of VZV gE expression in mock- (blue) and VZV-infected (red) HFLs at 72 hpi showed that > 80% of cells were VZV gE+. Cells were gated using isotype controls. Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HFLs had significantly higher levels of IL-8 (B; p = 0.02), IL-6 (C; p = 0.02), VEGF-A (D; p = 0.01), TGF-β (E; p = 0.01), IL-15 (F; p = 0.04), and IL-4 (G; p = 0.05). All cytokine values are given in pg/ml. Bar graphs represent mean ± SD cytokine levels from 3 independent experiments. HFL = human fetal lung fibroblast; IL = interleukin; TGF-β = transforming growth factor β; VEGF-A = vascular endothelial growth factor A; VZV = varicella zoster virus.
    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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    92
    Becton Dickinson intracellular cytokine staining
    <t>Cytokine</t> production kinetics in T cells. ( A ) T cells that were rested for 3 d in the presence of IL-7 ( Top ), IL-2 ( Center ), or IL-15 ( Bottom ) were activated with 100 nM SIINFEKL (N4) or SIITFEKL (T4) OVA peptide for indicated time points ( n = 3 mice). Brefeldin A (BrfA) was added during the last 2 h of culture. ( B ) Representative dot plots for TNF-α and IFN-γ ( Upper ) and IFN-γ and IL-2 ( Lower ) production of T cells that were activated for indicated time with 100 nM OVA 257–264 peptide (N4) in the presence of BrfA during the last 2 h of stimulation. Numbers indicate percentage of cytokine-producing T cells. ( C ) mRNA levels of Tnfa , Ifng , and Il2 of T cells that were rested for 3 d in the presence of IL-7, IL-2, or IL-15.
    Intracellular Cytokine Staining, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 4683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen cytokines
    Influence of Pro-Th1 (IL-2 and IL-12) and Pro-Th2 (IL-4) <t>cytokines</t> on the proliferation and IFN-γ secretion by Th1 cells stimulated with anti-CD3 Ab and B7-1. pGL-10 Th1 cells were cultured with anti-CD3 Ab (0.01 μg/ml) in the presence of CHO-B7-1 (5 × 10 4 /well). Cytokines IL-2, IL-4 or IL-12 alone or in combinations were also added into the cultures. For proliferation (Fig. 5a), cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and were harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells. IFN-γ (Fig. 5b) was estimated by ELISA in the pooled culture supernatants collected from the triplicate wells after 48 h of the initiation of cultures. The control cultures comprising of Th1 cells, CHO-B7-1+Th1 cells, CHO+Th1 cells, CHO-B7-1, CHO showed no noticeable change. The data presented are from three independent experiments. '*' Represents p
    Cytokines, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 899 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson cytokine staining
    Flt3L potentiates both soluble and targeted vaccine priming and improves polyclonal CD8 + T cells responses and humoral immunity to OVA cross-presentation . Vaccine priming with αCD205-gag-p24 and soluble p24 at high (5 µg) and low (0.5 µg) doses in Flt3L-treated (red) versus PBS-treated (blue) mice. (A) Intracellular <t>cytokine</t> stain. (B) Proliferation of CD4 + T cells measured by CFSE-diluted IFN-γ + cells. One representative experiment ( n = 3 mice per group). Error bars show mean ± SEM. ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (C) CD8 + IFN-γ + intracellular cytokine staining from individual mice vaccinated with polyIC(LC) + 5 µg αCD205 OVA intraperitoneal route in spleen, lung, and lamina propria ( n = 4–6 mice pooled from 2 independent experiments). CD8 + IFN-γ + CFSE low/divided T cells at 96 h. One of two representative experiments ( n = 5). Error bars show standard error of the mean. CD8 + IFN-γ + intracellular cytokine staining. OVA serum total IgG, mean ELISA OD 450 . Error bars show mean ± SEM across 5 individual mice. ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
    Cytokine Staining, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 6235 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
    Reduction in <t>IL-4</t> availability by Il4 gene hemizygosity has a partial effect on the development of IL-4AC- and IL-4AC/IL-13DR-producing T cell subsets. 4C13R-IL-4 +/+ , 4C13R-IL-4 +/− , and 4C13R-IL-4 −/− mice were treated with 200 μg house dust mite i.d. in the ear pinnae. Ear draining lymph nodes and ear tissue were harvested from the mice 7 days posttreatment. (A) Lymph nodes were analyzed to determine the number of IL-4AC + T follicular helper (Tfh) and IL-4AC + <t>Th2</t> CD4 T cells. (B) Ear tissue was analyzed to determine the number of reporter + Th2 CD4 + T cells. (A) Data pooled from three experiments ( n = 18) for ear lymph node and (B) three experiments ( n = 15) for ear tissue. Data show mean + SEM (* p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 two-tailed t -test).
    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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    92
    Meso Scale Diagnostics LLC cytokine levels
    Tiotropium treatment of CS-exposed and H1N1-infected mice. (A) CS-exposed and H1N1-infected mice (black bars) were treated for a total of 10 days with 0.1 mg/ml or 0.3 mg/ml nebulized tiotropium (gray bars). White bars show the results from untreated negative control (NC) animals. (B) Body weight loss in NC mice (black dotted line), untreated CS-exposed, and H1N1-infected mice (red line); CS-exposed and H1N1-infected mice treated with 0.1 mg/ml (light green line); or 0.3 mg/ml (dark green line) nebulized tiotropium. (C) Lung function, resistance, compliance, and (D) <t>cytokine</t> levels in lung homogenate and (E) total cell, neutrophil, and macrophage numbers in BAL fluid. Mean values ± S.E.M. of n = 7 or 8 animals in the HIN1- and CS-exposed mice groups and n = 4 in the NC group. **** P
    Cytokine Levels, supplied by Meso Scale Diagnostics LLC, used in various techniques. Bioz Stars score: 92/100, based on 356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam cytokines
    Loss of tight-junctions by cytokine treatment and its rescue by trypsin inhibitor. A , Western blotting analysis of tight-junction proteins, zonula occludens (ZO) 1 and occludin after treatment of the cells with <t>cytokines</t> for 12 h in the absence and presence of 50 µM aprotinin. Actin as an internal control (Ctr). B , Representative example (from 3 separate experiments) of immunofluorescence showing decreased ZO-1 with cytokine treatment and its restoration by aprotinin. C , Increased permeability of the cells treated with cytokines and its rescue by aprotinin ( n =3). Data are mean value ± standard error of the mean. * P
    Cytokines, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 405 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Assay Biotechnology cytokines
    In vitro effects of ethanol with or without opioid receptor agonists on cytokine secretion from hypothalamic microglia in primary cultures. Showing the changes in the protein levels of pro-inflammatory <t>cytokines</t> TNF-α ( a ), IFN-γ ( b ), IL-1α ( c ), IL-1β ( d ), IL-6 ( e ), MIP-3α ( f ), MCP-1 ( g ), M-CSF ( h ), CXCL1 ( i ), and RANTES ( j ) and anti-inflammatory cytokines IL-4 ( k ) and IL-13 ( l ) in microglial conditioned medium following 24-h treatment with ethanol (50 mM) with or without MOR agonist (DAMGO, 50 μM) or DOR agonist (DPDPE, 10 nM). Data are represented as mean ± SEM ( n = 6). Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between control and other treatment groups or ethanol and ethanol and opioidergic drug groups are shown by lines with p values on the top of bar graphs
    Cytokines, supplied by Assay Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 388 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA cytokines
    <t>Cytokines</t> found to have significantly altered concentrations in the peripheral blood of cats with mycobacterial disease compared to healthy controls and/or cats hospitalised for other reasons. Statistically significant differences between groups were determined by Kruskal-Wallis test by ranks (P
    Cytokines, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 401 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cytokine staining
    <t>Cytokines</t> found to have significantly altered concentrations in the peripheral blood of cats with mycobacterial disease compared to healthy controls and/or cats hospitalised for other reasons. Statistically significant differences between groups were determined by Kruskal-Wallis test by ranks (P
    Cytokine Staining, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2931 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bio-Rad bio plex pro mouse cytokine 23 plex assay
    Higher production of tumor-promoting factors in miR-155−/− chimeric mice.  A–D , 23 cytokines in sera of WT and miR-155−/− chimeric mice were detected by Bio-Plex Pro™ Mouse Cytokine 23-plex Assay. Data are
    Bio Plex Pro Mouse Cytokine 23 Plex Assay, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen recombinant cytokines
    Influence of Pro-Th1 (IL-2 and IL-12) and Pro-Th2 (IL-4) <t>cytokines</t> on the proliferation and IFN-γ secretion by Th1 cells stimulated with anti-CD3 Ab and B7-1. pGL-10 Th1 cells were cultured with anti-CD3 Ab (0.01 μg/ml) in the presence of CHO-B7-1 (5 × 10 4 /well). Cytokines IL-2, IL-4 or IL-12 alone or in combinations were also added into the cultures. For proliferation (Fig. 5a), cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and were harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells. IFN-γ (Fig. 5b) was estimated by ELISA in the pooled culture supernatants collected from the triplicate wells after 48 h of the initiation of cultures. The control cultures comprising of Th1 cells, CHO-B7-1+Th1 cells, CHO+Th1 cells, CHO-B7-1, CHO showed no noticeable change. The data presented are from three independent experiments. '*' Represents p
    Recombinant Cytokines, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 279 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bender MedSystems cytokines
    Transcriptional changes in eYFP + CD4 + T cells (a) CCR6 + eYFP + CD4 + T cells from 2D2 IL-17-reporter mice were purified 4 days after MOG-CFA immunization and adoptively transferred into immunized Rag-deficient mice. FACS plots and bar graphs of IL-17A and IFN-γ expression in eYFP + CD4 + T cells from draining lymph nodes and spinal cord (day 16) restimulated with PdBU-ionomycin (upper panels) or MOG peptide (lower panels). Histograms show mean values for individual mice +/− SD. (b) CCR6 − and CCR6 + eYFP + CD4 + T cells from spinal cord were sorted for qPCR analysis. Representative FACS plots show expression of IL-17A and IFN-γ. Relative gene expression in sorted (not restimulated) cells normalized to the expression of Hprt is shown. (c) CD4 + eYFP − IFN-γ + , representing T H 1, (shaded gray), CD4 + eYFP + IFN-γ + (ex-T H 17 -dotted line), and CD4 + eYFP + IL-17A + (T H 17- solid line) from draining LN and spinal cord cells 15 days after MOG-CFA immunisation were gated and assessed for IL-1R1 expression. The data represent at least three independent experiments. (d) Cytokine levels measured in supernatant of purified eYFP + or eYFP − CD4 T cells from draining lymph nodes or spinal cord (day 15) restimulated with anti-CD3 +/− 20 ng/ml IL-1β for 24 hr. Data show mean values ± SD of <t>cytokines</t> from three individual mice. The data represent at least three independent experiments.
    Cytokines, supplied by Bender MedSystems, used in various techniques. Bioz Stars score: 92/100, based on 454 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    gp130 modulator activates JAK STAT signaling
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    N/A
    gp130 agonist brain penetrant and neuroprotectant
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    N/A
    MIF binder
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    IL 6  (Bachem)
    N/A
    IL 6 88 121 competitively inhibits the binding of human IL 6 to its receptor by interacting with the IL 6 receptor to a significant degree It is suggested that
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    Image Search Results


    NC410 therapy induces immune activity in the local tumor microenvironment 1 × 10 7 total human PBMCs were adoptively transferred intravenously to NSG mice (N = 6/group) on Day 0. 1 × 10 6 HT-29 tumor cells were injected subcutaneously with Matrigel on Day 1. Mice were treated with NC410 or control by intraperitoneal injection, Q4D x 4 doses followed by Q7D until endpoint. On day 27, tumor and spleen tissues were isolated for tumor infiltrating T cells and cytokine analysis. A) CD4 + and B) CD8 + TIL cell numbers in the tumor. The cell number was counted by flow cytometry and normalized to weight (gram) of tumor tissue. Asterisks indicate statistical significance (*P

    Journal: bioRxiv

    Article Title: Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking LAIR-collagen interaction

    doi: 10.1101/2020.10.21.349480

    Figure Lengend Snippet: NC410 therapy induces immune activity in the local tumor microenvironment 1 × 10 7 total human PBMCs were adoptively transferred intravenously to NSG mice (N = 6/group) on Day 0. 1 × 10 6 HT-29 tumor cells were injected subcutaneously with Matrigel on Day 1. Mice were treated with NC410 or control by intraperitoneal injection, Q4D x 4 doses followed by Q7D until endpoint. On day 27, tumor and spleen tissues were isolated for tumor infiltrating T cells and cytokine analysis. A) CD4 + and B) CD8 + TIL cell numbers in the tumor. The cell number was counted by flow cytometry and normalized to weight (gram) of tumor tissue. Asterisks indicate statistical significance (*P

    Article Snippet: The Singleplex Luminex™ Protein Assay Kit for each cytokine was from ThermoFisher.

    Techniques: Activity Assay, Mouse Assay, Injection, Isolation, Flow Cytometry

    Cellular immune response induced by vaccination. Splenocytes were harvested from three mice per group 2 weeks after the final immunization. After stimulation with 10 μg/ml of the recombinant proteins, i.e., HBc Δ , H82, HBc ΔH82 , H301, HBc ΔH301 , R82, HBc ΔR82 , R301, and HBc ΔR301 , cellular immune responses were analyzed. (A) Percentages of T lymphocyte subsets in the immunized mice. After 72 h of stimulation, the lymphocytes were stained with anti-mouse CD3-APC, anti-mouse CD4-FITC and anti-mouse CD8-PE and were analyzed by flow cytometry. (B) Cytokine production in the immunized mice. The interleukin (IL)-2 and IL-4 levels in the supernatants of splenocytes at 24 h, IL-10 levels at 72 h, and interferon (IFN)-γ levels at 96 h were assessed with ELISA. Splenocytes from three mice in each group were tested individually, and the data represent mean ± SD values. * p

    Journal: Frontiers in Immunology

    Article Title: Immunogenicity of a Virus-Like-Particle Vaccine Containing Multiple Antigenic Epitopes of Toxoplasma gondii Against Acute and Chronic Toxoplasmosis in Mice

    doi: 10.3389/fimmu.2019.00592

    Figure Lengend Snippet: Cellular immune response induced by vaccination. Splenocytes were harvested from three mice per group 2 weeks after the final immunization. After stimulation with 10 μg/ml of the recombinant proteins, i.e., HBc Δ , H82, HBc ΔH82 , H301, HBc ΔH301 , R82, HBc ΔR82 , R301, and HBc ΔR301 , cellular immune responses were analyzed. (A) Percentages of T lymphocyte subsets in the immunized mice. After 72 h of stimulation, the lymphocytes were stained with anti-mouse CD3-APC, anti-mouse CD4-FITC and anti-mouse CD8-PE and were analyzed by flow cytometry. (B) Cytokine production in the immunized mice. The interleukin (IL)-2 and IL-4 levels in the supernatants of splenocytes at 24 h, IL-10 levels at 72 h, and interferon (IFN)-γ levels at 96 h were assessed with ELISA. Splenocytes from three mice in each group were tested individually, and the data represent mean ± SD values. * p

    Article Snippet: The cell-free supernatants from cultured splenocytes were collected and assayed for interleukin (IL)-2 and IL-4 activities at 24 h, for IL-10 activity at 72 h, and for IFN-γ activity at 96 h. The cytokine levels were measured using a commercial ELISA kit (eBioscience) according to the manufacturer's instructions.

    Techniques: Mouse Assay, Recombinant, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Relationship between the severity of diabetic retinopathy (DR) and the aqueous interleukin-8 (IL-8) level. The results by measuring cytokines in the aqueous humor samples shows that the IL-8 level in aqueous humor increased with increasing severity of DR, and this correlation was significant (r=0.309, p

    Journal: Molecular Vision

    Article Title: Study of 27 aqueous humor cytokines in patients with type 2 diabetes with or without retinopathy

    doi:

    Figure Lengend Snippet: Relationship between the severity of diabetic retinopathy (DR) and the aqueous interleukin-8 (IL-8) level. The results by measuring cytokines in the aqueous humor samples shows that the IL-8 level in aqueous humor increased with increasing severity of DR, and this correlation was significant (r=0.309, p

    Article Snippet: Multiplex analysis of cytokines in aqueous humor samples A Bio-Plex multiplex assay (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA) was used to measure the concentrations of 27 human aqueous humor cytokines: interleukin-1β (IL-1β), IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, basic fibroblast growth factor (b-FGF), EOTAXIN, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GMCSF), interferon-gamma (IFN-γ), interferon-induced protein-10 (IP-10 or CXCL10), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3), macrophage inflammatory protein-1β (MIP-1β or CCL4), platelet-derived growth factor-BB (PDGF-BB), regulated upon activation normal T-cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

    Techniques:

    Relationship between the severity of diabetic retinopathy (DR) and the aqueous interleukin-12 (IL-12) level. The results by measuring cytokines in the aqueous humor samples shows that the IL-12 level in aqueous humor decreased with increasing severity of DR, and this correlation was significant (r=–0.482, p

    Journal: Molecular Vision

    Article Title: Study of 27 aqueous humor cytokines in patients with type 2 diabetes with or without retinopathy

    doi:

    Figure Lengend Snippet: Relationship between the severity of diabetic retinopathy (DR) and the aqueous interleukin-12 (IL-12) level. The results by measuring cytokines in the aqueous humor samples shows that the IL-12 level in aqueous humor decreased with increasing severity of DR, and this correlation was significant (r=–0.482, p

    Article Snippet: Multiplex analysis of cytokines in aqueous humor samples A Bio-Plex multiplex assay (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA) was used to measure the concentrations of 27 human aqueous humor cytokines: interleukin-1β (IL-1β), IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, basic fibroblast growth factor (b-FGF), EOTAXIN, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GMCSF), interferon-gamma (IFN-γ), interferon-induced protein-10 (IP-10 or CXCL10), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3), macrophage inflammatory protein-1β (MIP-1β or CCL4), platelet-derived growth factor-BB (PDGF-BB), regulated upon activation normal T-cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

    Techniques:

    Relationship between the severity of diabetic retinopathy (DR) and the aqueous interleukin-6 (IL-6) level. The results by measuring cytokines in the aqueous humor samples shows that the IL-6 level in aqueous humor increased with increasing severity of DR, and this correlation was significant (r=0.468, p

    Journal: Molecular Vision

    Article Title: Study of 27 aqueous humor cytokines in patients with type 2 diabetes with or without retinopathy

    doi:

    Figure Lengend Snippet: Relationship between the severity of diabetic retinopathy (DR) and the aqueous interleukin-6 (IL-6) level. The results by measuring cytokines in the aqueous humor samples shows that the IL-6 level in aqueous humor increased with increasing severity of DR, and this correlation was significant (r=0.468, p

    Article Snippet: Multiplex analysis of cytokines in aqueous humor samples A Bio-Plex multiplex assay (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA) was used to measure the concentrations of 27 human aqueous humor cytokines: interleukin-1β (IL-1β), IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, basic fibroblast growth factor (b-FGF), EOTAXIN, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GMCSF), interferon-gamma (IFN-γ), interferon-induced protein-10 (IP-10 or CXCL10), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3), macrophage inflammatory protein-1β (MIP-1β or CCL4), platelet-derived growth factor-BB (PDGF-BB), regulated upon activation normal T-cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

    Techniques:

    Relationship between the severity of diabetic retinopathy (DR) and the aqueous interferon gamma-induced protein-10 (IP-10) level. The results by measuring cytokines in the aqueous humor samples shows that the IP-10 level in aqueous humor increased with increasing severity of DR, and this correlation was significant (r=0.674, p

    Journal: Molecular Vision

    Article Title: Study of 27 aqueous humor cytokines in patients with type 2 diabetes with or without retinopathy

    doi:

    Figure Lengend Snippet: Relationship between the severity of diabetic retinopathy (DR) and the aqueous interferon gamma-induced protein-10 (IP-10) level. The results by measuring cytokines in the aqueous humor samples shows that the IP-10 level in aqueous humor increased with increasing severity of DR, and this correlation was significant (r=0.674, p

    Article Snippet: Multiplex analysis of cytokines in aqueous humor samples A Bio-Plex multiplex assay (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA) was used to measure the concentrations of 27 human aqueous humor cytokines: interleukin-1β (IL-1β), IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, basic fibroblast growth factor (b-FGF), EOTAXIN, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GMCSF), interferon-gamma (IFN-γ), interferon-induced protein-10 (IP-10 or CXCL10), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3), macrophage inflammatory protein-1β (MIP-1β or CCL4), platelet-derived growth factor-BB (PDGF-BB), regulated upon activation normal T-cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

    Techniques:

    Relationship between the severity of diabetic retinopathy (DR) and the aqueous monocyte chemoattractant protein-1 (MCP-1) level. The results by measuring cytokines in the aqueous humor samples shows that the MCP-1 level in aqueous humor increased with increasing severity of DR, and this correlation was significant (r=0.693, p

    Journal: Molecular Vision

    Article Title: Study of 27 aqueous humor cytokines in patients with type 2 diabetes with or without retinopathy

    doi:

    Figure Lengend Snippet: Relationship between the severity of diabetic retinopathy (DR) and the aqueous monocyte chemoattractant protein-1 (MCP-1) level. The results by measuring cytokines in the aqueous humor samples shows that the MCP-1 level in aqueous humor increased with increasing severity of DR, and this correlation was significant (r=0.693, p

    Article Snippet: Multiplex analysis of cytokines in aqueous humor samples A Bio-Plex multiplex assay (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA) was used to measure the concentrations of 27 human aqueous humor cytokines: interleukin-1β (IL-1β), IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, basic fibroblast growth factor (b-FGF), EOTAXIN, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GMCSF), interferon-gamma (IFN-γ), interferon-induced protein-10 (IP-10 or CXCL10), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3), macrophage inflammatory protein-1β (MIP-1β or CCL4), platelet-derived growth factor-BB (PDGF-BB), regulated upon activation normal T-cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

    Techniques:

    Relationship between the severity of diabetic retinopathy (DR) and the aqueous vascular endothelial growth factor (VEGF) level. The results by measuring cytokines in the aqueous humor samples shows that the VEGF level in aqueous humor did not increase with increasing severity of DR, and this correlation was not significant (r=0.161, p=0.062). Table 3 shows the sampling sizes for each group according to the ETDRS retinopathy severity scale.

    Journal: Molecular Vision

    Article Title: Study of 27 aqueous humor cytokines in patients with type 2 diabetes with or without retinopathy

    doi:

    Figure Lengend Snippet: Relationship between the severity of diabetic retinopathy (DR) and the aqueous vascular endothelial growth factor (VEGF) level. The results by measuring cytokines in the aqueous humor samples shows that the VEGF level in aqueous humor did not increase with increasing severity of DR, and this correlation was not significant (r=0.161, p=0.062). Table 3 shows the sampling sizes for each group according to the ETDRS retinopathy severity scale.

    Article Snippet: Multiplex analysis of cytokines in aqueous humor samples A Bio-Plex multiplex assay (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA) was used to measure the concentrations of 27 human aqueous humor cytokines: interleukin-1β (IL-1β), IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, basic fibroblast growth factor (b-FGF), EOTAXIN, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GMCSF), interferon-gamma (IFN-γ), interferon-induced protein-10 (IP-10 or CXCL10), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3), macrophage inflammatory protein-1β (MIP-1β or CCL4), platelet-derived growth factor-BB (PDGF-BB), regulated upon activation normal T-cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

    Techniques: Sampling

    Relationship between the severity of diabetic retinopathy (DR) and the aqueous interleukin-1β level. The results by measuring cytokines in the aqueous humor samples shows that the interleukin-1β (IL-1β) level in aqueous humor increased with increasing severity of DR, and this correlation was significant (r=0.298, p

    Journal: Molecular Vision

    Article Title: Study of 27 aqueous humor cytokines in patients with type 2 diabetes with or without retinopathy

    doi:

    Figure Lengend Snippet: Relationship between the severity of diabetic retinopathy (DR) and the aqueous interleukin-1β level. The results by measuring cytokines in the aqueous humor samples shows that the interleukin-1β (IL-1β) level in aqueous humor increased with increasing severity of DR, and this correlation was significant (r=0.298, p

    Article Snippet: Multiplex analysis of cytokines in aqueous humor samples A Bio-Plex multiplex assay (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA) was used to measure the concentrations of 27 human aqueous humor cytokines: interleukin-1β (IL-1β), IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, basic fibroblast growth factor (b-FGF), EOTAXIN, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GMCSF), interferon-gamma (IFN-γ), interferon-induced protein-10 (IP-10 or CXCL10), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3), macrophage inflammatory protein-1β (MIP-1β or CCL4), platelet-derived growth factor-BB (PDGF-BB), regulated upon activation normal T-cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

    Techniques:

    Relationship between the severity of diabetic retinopathy (DR) and the aqueous interleukin-10 (IL-10) level. The results by measuring cytokines in the aqueous humor samples shows that the IL-10 level in aqueous humor decreased with increasing severity of DR, and this correlation was significant (r=–0.206, p=0.016). Table 4 shows the sampling sizes for each group according to the ETDRS retinopathy severity scale.

    Journal: Molecular Vision

    Article Title: Study of 27 aqueous humor cytokines in patients with type 2 diabetes with or without retinopathy

    doi:

    Figure Lengend Snippet: Relationship between the severity of diabetic retinopathy (DR) and the aqueous interleukin-10 (IL-10) level. The results by measuring cytokines in the aqueous humor samples shows that the IL-10 level in aqueous humor decreased with increasing severity of DR, and this correlation was significant (r=–0.206, p=0.016). Table 4 shows the sampling sizes for each group according to the ETDRS retinopathy severity scale.

    Article Snippet: Multiplex analysis of cytokines in aqueous humor samples A Bio-Plex multiplex assay (Bio-Plex Human Cytokine 27-plex panel; Bio-Rad, Hercules, CA) was used to measure the concentrations of 27 human aqueous humor cytokines: interleukin-1β (IL-1β), IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, basic fibroblast growth factor (b-FGF), EOTAXIN, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GMCSF), interferon-gamma (IFN-γ), interferon-induced protein-10 (IP-10 or CXCL10), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3), macrophage inflammatory protein-1β (MIP-1β or CCL4), platelet-derived growth factor-BB (PDGF-BB), regulated upon activation normal T-cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

    Techniques: Sampling

    The tumor necrosis factor-like cytokine 1A (TL1a) receptor death receptor 3 (DR3) is expressed at high levels on interleukin-18 receptor alpha (IL-18Rα) + CD45RO + CD4 + T cells in cytokine cultures. ( a – c ) DR3, IL-18Rα, CD161, and interferon-γ (IFN-γ) expression was assessed on peripheral blood CD45RO + CD4 + T cells by flow cytometry at day 0 or day 4 after culture (1 × 10 6 cells ml −1 , 200 μl per well) with interleukin-12 (IL-12) (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ), and TL1a (100 ng ml −1 ) or medium alone (control). ( a ) Representative plots showing DR3 expression (black line, unfilled histogram) or fluorescence minus one (FMO) control (shaded histogram) on CD45RO + CD4 + T cells. ( b ) DR3 and IL-18Rα expression on CD45RO + CD4 + T cells. Boxes within plots are gates defining IL-18Rα + or IL-18Rα − (day 0) and IL-18Rα + DR3 hi or IL-18Rα − DR3 lo (day 4) CD4 + T cells. ( c ) IFN-γ and CD161 expression on IL-18Rα + DR3 hi or IL-18Rα − DR3 lo CD4 + T cells as defined in b . Quadrants are set based on isotype control (IFN-γ) and FMO control (CD161) staining of IL-18Rα + DR3 hi and IL-18Rα − DR3 lo CD4 + T cells under each of the cytokine culture conditions. Results are representative of 3 (day 0) or 7 (day 4) biological replicates.

    Journal: Mucosal Immunology

    Article Title: A major population of mucosal memory CD4+ T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality

    doi: 10.1038/mi.2014.87

    Figure Lengend Snippet: The tumor necrosis factor-like cytokine 1A (TL1a) receptor death receptor 3 (DR3) is expressed at high levels on interleukin-18 receptor alpha (IL-18Rα) + CD45RO + CD4 + T cells in cytokine cultures. ( a – c ) DR3, IL-18Rα, CD161, and interferon-γ (IFN-γ) expression was assessed on peripheral blood CD45RO + CD4 + T cells by flow cytometry at day 0 or day 4 after culture (1 × 10 6 cells ml −1 , 200 μl per well) with interleukin-12 (IL-12) (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ), and TL1a (100 ng ml −1 ) or medium alone (control). ( a ) Representative plots showing DR3 expression (black line, unfilled histogram) or fluorescence minus one (FMO) control (shaded histogram) on CD45RO + CD4 + T cells. ( b ) DR3 and IL-18Rα expression on CD45RO + CD4 + T cells. Boxes within plots are gates defining IL-18Rα + or IL-18Rα − (day 0) and IL-18Rα + DR3 hi or IL-18Rα − DR3 lo (day 4) CD4 + T cells. ( c ) IFN-γ and CD161 expression on IL-18Rα + DR3 hi or IL-18Rα − DR3 lo CD4 + T cells as defined in b . Quadrants are set based on isotype control (IFN-γ) and FMO control (CD161) staining of IL-18Rα + DR3 hi and IL-18Rα − DR3 lo CD4 + T cells under each of the cytokine culture conditions. Results are representative of 3 (day 0) or 7 (day 4) biological replicates.

    Article Snippet: Cytokine levels in culture supernatants were assessed by Bio-Plex Pro Human Cytokine 17-plex Assay or Bio-Plex Pro Human Th17 Cytokine IFN-γ Set/Bio-Plex Pro Human Th17 Cytokine IL-10 Set (Bio-Rad, Hercules, CA) according to the manufacturer's instructions using the Bio-Plex 200 System (Bio-Rad).

    Techniques: Expressing, Flow Cytometry, Cytometry, Fluorescence, Staining

    The ability of tumor necrosis factor-like cytokine 1A (TL1a) to inhibit interleukin-15 (IL-15)-mediated IL-10 production is IL-18 dependent. ( a – d ) Peripheral blood CD45RO + CD4 + T cells (1 × 10 6 cells ml −1 , 200 μl per well) were cultured in medium alone (control) or with IL-12 (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ), and TL1a (100 ng ml −1 ) as indicated, and IL-10 levels were determined in culture supernatants at day 4. ( a ) IL-15 induces IL-10 production in CD45RO + CD4 + T cells that is inhibited by TL1a. Results are mean (s.e.m.) of 13 biological replicates. ( b ) IL-15-mediated induction of IL-10 requires IL-12 but not IL-18. Results are mean (s.e.m.) of 5 biological replicates. ( c ) TL1a-mediated inhibition of IL-10 is IL-18 dependent. Results are mean (s.e.m.) of 9 biological replicates. ( d ) TL1a-mediated inhibition of IL-10 is DR3 dependent. CD45RO + CD4 + T cells were cultured with the indicated cytokines in the presence of DR3 Fab′ or isotype control Fab′ (5 μg ml −1 ). Results are the mean (s.e.m.) of 14 biological replicates. IL-15 induces IL-10 production in ( e ) IL-18Rα + and IL-18Rα − CD45RO + CD4 + T cells and ( f ) FoxP3 + and FoxP3 − CD45RO + CD4 + T cells. CD45RO + CD4 + T cells were cultured as indicated for 4 days, stimulated 4 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and stained for IL-10. Results are representative of 3 biological replicates. ( g ) IL-15Rα expression on IL-18Rα + and IL-18Rα − CD4 + T cells 4 days after culture with the indicated cytokine cocktails (black line, unfilled histogram). Results are representative plots from 3 biological replicates. Isotype, shaded histogram.

    Journal: Mucosal Immunology

    Article Title: A major population of mucosal memory CD4+ T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality

    doi: 10.1038/mi.2014.87

    Figure Lengend Snippet: The ability of tumor necrosis factor-like cytokine 1A (TL1a) to inhibit interleukin-15 (IL-15)-mediated IL-10 production is IL-18 dependent. ( a – d ) Peripheral blood CD45RO + CD4 + T cells (1 × 10 6 cells ml −1 , 200 μl per well) were cultured in medium alone (control) or with IL-12 (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ), and TL1a (100 ng ml −1 ) as indicated, and IL-10 levels were determined in culture supernatants at day 4. ( a ) IL-15 induces IL-10 production in CD45RO + CD4 + T cells that is inhibited by TL1a. Results are mean (s.e.m.) of 13 biological replicates. ( b ) IL-15-mediated induction of IL-10 requires IL-12 but not IL-18. Results are mean (s.e.m.) of 5 biological replicates. ( c ) TL1a-mediated inhibition of IL-10 is IL-18 dependent. Results are mean (s.e.m.) of 9 biological replicates. ( d ) TL1a-mediated inhibition of IL-10 is DR3 dependent. CD45RO + CD4 + T cells were cultured with the indicated cytokines in the presence of DR3 Fab′ or isotype control Fab′ (5 μg ml −1 ). Results are the mean (s.e.m.) of 14 biological replicates. IL-15 induces IL-10 production in ( e ) IL-18Rα + and IL-18Rα − CD45RO + CD4 + T cells and ( f ) FoxP3 + and FoxP3 − CD45RO + CD4 + T cells. CD45RO + CD4 + T cells were cultured as indicated for 4 days, stimulated 4 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and stained for IL-10. Results are representative of 3 biological replicates. ( g ) IL-15Rα expression on IL-18Rα + and IL-18Rα − CD4 + T cells 4 days after culture with the indicated cytokine cocktails (black line, unfilled histogram). Results are representative plots from 3 biological replicates. Isotype, shaded histogram.

    Article Snippet: Cytokine levels in culture supernatants were assessed by Bio-Plex Pro Human Cytokine 17-plex Assay or Bio-Plex Pro Human Th17 Cytokine IFN-γ Set/Bio-Plex Pro Human Th17 Cytokine IL-10 Set (Bio-Rad, Hercules, CA) according to the manufacturer's instructions using the Bio-Plex 200 System (Bio-Rad).

    Techniques: Cell Culture, Inhibition, Staining, Expressing

    Interleukin-15 (IL-15) and tumor necrosis factor-like cytokine 1A (TL1a) induction of proinflammatory cytokines in CD45RO + CD4 + T cells requires synergy with IL-18. Peripheral blood CD45RO + CD4 + T cells (1 × 10 6 cells per ml, 200 μl per well) were cultured with IL-12 (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ), and TL1a (100 ng ml −1 ) as indicated, and cytokine levels were determined in culture supernatants at day 1 (interferon-γ (IFN-γ)) or day 4 (other cytokines). ( a ) TL1a and IL-15 synergy in inducing IFN-γ, IL-6, and TNF-α requires both IL-12 and IL-18. Results are the mean (s.e.m.) of 4 (IFN-γ) and 9 (IL-6 and TNF-α) biological replicates. ( b ) TL1a and IL-15 synergy in inducing GM-CSF, IL-5, IL-13, and IL-22 is IL-12 independent and IL-18 dependent. Results are the mean (s.e.m.) of 6 (IL-22) or 9 (other cytokines) biological replicates.

    Journal: Mucosal Immunology

    Article Title: A major population of mucosal memory CD4+ T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality

    doi: 10.1038/mi.2014.87

    Figure Lengend Snippet: Interleukin-15 (IL-15) and tumor necrosis factor-like cytokine 1A (TL1a) induction of proinflammatory cytokines in CD45RO + CD4 + T cells requires synergy with IL-18. Peripheral blood CD45RO + CD4 + T cells (1 × 10 6 cells per ml, 200 μl per well) were cultured with IL-12 (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ), and TL1a (100 ng ml −1 ) as indicated, and cytokine levels were determined in culture supernatants at day 1 (interferon-γ (IFN-γ)) or day 4 (other cytokines). ( a ) TL1a and IL-15 synergy in inducing IFN-γ, IL-6, and TNF-α requires both IL-12 and IL-18. Results are the mean (s.e.m.) of 4 (IFN-γ) and 9 (IL-6 and TNF-α) biological replicates. ( b ) TL1a and IL-15 synergy in inducing GM-CSF, IL-5, IL-13, and IL-22 is IL-12 independent and IL-18 dependent. Results are the mean (s.e.m.) of 6 (IL-22) or 9 (other cytokines) biological replicates.

    Article Snippet: Cytokine levels in culture supernatants were assessed by Bio-Plex Pro Human Cytokine 17-plex Assay or Bio-Plex Pro Human Th17 Cytokine IFN-γ Set/Bio-Plex Pro Human Th17 Cytokine IL-10 Set (Bio-Rad, Hercules, CA) according to the manufacturer's instructions using the Bio-Plex 200 System (Bio-Rad).

    Techniques: Cell Culture

    Interleukin-15 (IL-15) and tumor necrosis factor-like cytokine 1A (TL1a) synergize to induce cytokine production in tissue-resident IL-18Rα + DR3 + CD4 + T cells. Small intestinal lamina propria mononuclear cells ( a – d , f – h ) or purified CD4 + and CD4 − lamina propria cell fractions ( e ) were incubated (1 × 10 6 cells per ml, 200 μl per well) with the indicated cytokines or medium alone (control). ( a ) Number of live CD3 + CD4 + cells after 2 days of culture. The cytokine levels in ( b , d , e, and f ) cell culture supernatants, ( c ) intracellular cytokine staining, or ( g and h ) IL-10 secretion assay staining assessed after 2 days. ( c , g, and h ) Gating is on live CD3 + CD4 + T cells. Results are mean (s.e.m.) of ( a ) 3, ( b , d, and f ) 9, ( c ) 5, ( e ) 3 and ( g and h ) 3 small intestinal preparations.

    Journal: Mucosal Immunology

    Article Title: A major population of mucosal memory CD4+ T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality

    doi: 10.1038/mi.2014.87

    Figure Lengend Snippet: Interleukin-15 (IL-15) and tumor necrosis factor-like cytokine 1A (TL1a) synergize to induce cytokine production in tissue-resident IL-18Rα + DR3 + CD4 + T cells. Small intestinal lamina propria mononuclear cells ( a – d , f – h ) or purified CD4 + and CD4 − lamina propria cell fractions ( e ) were incubated (1 × 10 6 cells per ml, 200 μl per well) with the indicated cytokines or medium alone (control). ( a ) Number of live CD3 + CD4 + cells after 2 days of culture. The cytokine levels in ( b , d , e, and f ) cell culture supernatants, ( c ) intracellular cytokine staining, or ( g and h ) IL-10 secretion assay staining assessed after 2 days. ( c , g, and h ) Gating is on live CD3 + CD4 + T cells. Results are mean (s.e.m.) of ( a ) 3, ( b , d, and f ) 9, ( c ) 5, ( e ) 3 and ( g and h ) 3 small intestinal preparations.

    Article Snippet: Cytokine levels in culture supernatants were assessed by Bio-Plex Pro Human Cytokine 17-plex Assay or Bio-Plex Pro Human Th17 Cytokine IFN-γ Set/Bio-Plex Pro Human Th17 Cytokine IL-10 Set (Bio-Rad, Hercules, CA) according to the manufacturer's instructions using the Bio-Plex 200 System (Bio-Rad).

    Techniques: Purification, Incubation, Cell Culture, Staining

    Tumor necrosis factor (TNF)-like cytokine 1A (TL1a) and interleukin-15 (IL-15) synergize to induce proliferation of IL-18Rα + CD4 + T cells in IL-12/IL-18 cultures. Peripheral blood CD45RO + CD4 + T cells (1 × 10 6 cells ml −1 , 200 μl per well) were cultured in medium alone (control) or with IL-12 (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ), and TL1a (100 ng ml −1 ) as indicated. Representative flow cytometry plots depicting interferon-γ (IFN-γ) expression by IL-18Rα + CD4 + T cells ( a ), total number of live CD4 + cells (left) and total number of live CD4 + IL-18Rα + cells (right) ( b ) and percent of IFN-γ + cells within the IL-18Rα + CD4 + T cell population ( c ) after one day of culture. Results are mean (s.e.m.) from 4 biological replicates. ( d ) Representative flow cytometry plots showing IFN-γ-, TNF-α-, IL-6-, and granulocyte–macrophage colony-stimulating factor (GM-CSF)-expressing CD4 + T cells (top row) and isotype controls (IFN-γ, IL-6, and GM-CSF) and fluorescence minus one (FMO) (TNF-α) (lower row) as assessed by intracellular cytokine staining after 4 days of culture with the indicated cytokine cocktail. Brefeldin A was added to the cultures 4 h before analysis of TNF-α, IL-6, and GM-CSF, whereas cultures for IFN-γ analysis were left untreated. Numbers in quadrants represent the mean (s.e.m.) from 3 biological replicates. ( e ) Number of IL-18Rα + CD4 + T cells at day 0 and after 4 days of culture with the indicated cytokines. Results are mean (s.e.m.) of 6 biological replicates. ( f and g ) IL-15 and TL1a induce selective proliferation of IL-18Rα + cells. 5-Bromodeoxyuridine (BrdU) was added at the start of the culture and incorporation assessed 4 days later by flow cytometry. ( f ) Representative flow cytometry plot and ( g ) percent BrdU + cells within the IL-18Rα + CD4 + population after incubation with the indicated cytokine cocktails or medium alone (control). Results are the mean (s.e.m.) of 6 biological replicates. ( h ) Total number of live memory CD4 + T cells after 4 days of culture. Results are mean (s.e.m.) of 5 biological replicates. ( i ) Representative flow cytometry plot assessing BrdU incorporation in IFN-γ + CD4 + T cells after 4 days of culture with the indicated cytokine cocktail. Results are representative of 3 biological replicates.

    Journal: Mucosal Immunology

    Article Title: A major population of mucosal memory CD4+ T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality

    doi: 10.1038/mi.2014.87

    Figure Lengend Snippet: Tumor necrosis factor (TNF)-like cytokine 1A (TL1a) and interleukin-15 (IL-15) synergize to induce proliferation of IL-18Rα + CD4 + T cells in IL-12/IL-18 cultures. Peripheral blood CD45RO + CD4 + T cells (1 × 10 6 cells ml −1 , 200 μl per well) were cultured in medium alone (control) or with IL-12 (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ), and TL1a (100 ng ml −1 ) as indicated. Representative flow cytometry plots depicting interferon-γ (IFN-γ) expression by IL-18Rα + CD4 + T cells ( a ), total number of live CD4 + cells (left) and total number of live CD4 + IL-18Rα + cells (right) ( b ) and percent of IFN-γ + cells within the IL-18Rα + CD4 + T cell population ( c ) after one day of culture. Results are mean (s.e.m.) from 4 biological replicates. ( d ) Representative flow cytometry plots showing IFN-γ-, TNF-α-, IL-6-, and granulocyte–macrophage colony-stimulating factor (GM-CSF)-expressing CD4 + T cells (top row) and isotype controls (IFN-γ, IL-6, and GM-CSF) and fluorescence minus one (FMO) (TNF-α) (lower row) as assessed by intracellular cytokine staining after 4 days of culture with the indicated cytokine cocktail. Brefeldin A was added to the cultures 4 h before analysis of TNF-α, IL-6, and GM-CSF, whereas cultures for IFN-γ analysis were left untreated. Numbers in quadrants represent the mean (s.e.m.) from 3 biological replicates. ( e ) Number of IL-18Rα + CD4 + T cells at day 0 and after 4 days of culture with the indicated cytokines. Results are mean (s.e.m.) of 6 biological replicates. ( f and g ) IL-15 and TL1a induce selective proliferation of IL-18Rα + cells. 5-Bromodeoxyuridine (BrdU) was added at the start of the culture and incorporation assessed 4 days later by flow cytometry. ( f ) Representative flow cytometry plot and ( g ) percent BrdU + cells within the IL-18Rα + CD4 + population after incubation with the indicated cytokine cocktails or medium alone (control). Results are the mean (s.e.m.) of 6 biological replicates. ( h ) Total number of live memory CD4 + T cells after 4 days of culture. Results are mean (s.e.m.) of 5 biological replicates. ( i ) Representative flow cytometry plot assessing BrdU incorporation in IFN-γ + CD4 + T cells after 4 days of culture with the indicated cytokine cocktail. Results are representative of 3 biological replicates.

    Article Snippet: Cytokine levels in culture supernatants were assessed by Bio-Plex Pro Human Cytokine 17-plex Assay or Bio-Plex Pro Human Th17 Cytokine IFN-γ Set/Bio-Plex Pro Human Th17 Cytokine IL-10 Set (Bio-Rad, Hercules, CA) according to the manufacturer's instructions using the Bio-Plex 200 System (Bio-Rad).

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Expressing, Fluorescence, Staining, Incubation, BrdU Incorporation Assay

    Tumor necrosis factor-like cytokine 1A (TL1a) and interleukin-15 (IL-15) synergize to induce proinflammatory cytokine production in peripheral blood CD45RO + CD4 + T cells. Peripheral blood CD45RO + CD4 + T cells (1 × 10 6 cells ml −1 , 200 μl per well) were cultured in medium alone (control) or with IL-12 (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ), and TL1a (100 ng ml −1 ) as indicated. ( a ) Representative intracellular staining (top) and percent (bottom row) of CD4 + T cells expressing interferon-γ (IFN-γ) 4 days after incubation with indicated cytokines or medium alone (control). Results are the mean (s.e.m.) of 10 biological replicates. Cells were pregated on live single cells. ( b ) Cytokine levels in culture supernatants were assessed after 1 day (IFN-γ) and 4 days (other cytokines). Results are the mean (s.e.m.) of 4 (IFN-γ), 6 (IL-22), or 13 (other cytokines) biological replicates.

    Journal: Mucosal Immunology

    Article Title: A major population of mucosal memory CD4+ T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality

    doi: 10.1038/mi.2014.87

    Figure Lengend Snippet: Tumor necrosis factor-like cytokine 1A (TL1a) and interleukin-15 (IL-15) synergize to induce proinflammatory cytokine production in peripheral blood CD45RO + CD4 + T cells. Peripheral blood CD45RO + CD4 + T cells (1 × 10 6 cells ml −1 , 200 μl per well) were cultured in medium alone (control) or with IL-12 (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ), and TL1a (100 ng ml −1 ) as indicated. ( a ) Representative intracellular staining (top) and percent (bottom row) of CD4 + T cells expressing interferon-γ (IFN-γ) 4 days after incubation with indicated cytokines or medium alone (control). Results are the mean (s.e.m.) of 10 biological replicates. Cells were pregated on live single cells. ( b ) Cytokine levels in culture supernatants were assessed after 1 day (IFN-γ) and 4 days (other cytokines). Results are the mean (s.e.m.) of 4 (IFN-γ), 6 (IL-22), or 13 (other cytokines) biological replicates.

    Article Snippet: Cytokine levels in culture supernatants were assessed by Bio-Plex Pro Human Cytokine 17-plex Assay or Bio-Plex Pro Human Th17 Cytokine IFN-γ Set/Bio-Plex Pro Human Th17 Cytokine IL-10 Set (Bio-Rad, Hercules, CA) according to the manufacturer's instructions using the Bio-Plex 200 System (Bio-Rad).

    Techniques: Cell Culture, Staining, Expressing, Incubation

    Interleukin-18 receptor alpha (IL-18Rα) + death receptor 3 (DR3) + CD4 + T cells are present at barrier surfaces. ( a ) Representative flow cytometry plot and ( b ) percentage of IL-18Rα + DR3 + cells in the indicated organs. IL-18Rα and DR3 expression on CD4 + T cells was assessed after incubation in culture medium for 2 days to allow re-expression of DR3. Gating is on live, CD3 + CD4 + cells. ( b ) Results are mean (s.e.m.) of 7 (small intestine, SI), 3 (nasal polyp, NP), 5 (colon), and 3 (skin) stainings performed. ( c and d ) DR3 + CD4 + cells are diffusely distributed throughout the healthy human small intestine. ( c ) Small intestinal sections were stained with DR3 antibody. ( d ) Sections were stained with DR3 and CD4 antibody together with 4',6-diamidino-2-phenylindole (DAPI) to identify cell nuclei and analyzed by confocal microscopy. Arrowheads depict DR3 + CD4 + cells. Bars=( c ) 0.1 mm and ( d ) 50 μm. Images are from one representative tissue of at least ( c ) 20 and ( d ) 3 analyzed. ( e ) Cytokines induce tissue-resident IL-18Rα + CD4 + T cells to produce interferon-γ (IFN-γ). SI and NP cell suspensions were cultured in medium alone (control) or with interleukin (IL)-12 (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ) and tumor necrosis factor-like cytokine 1A (TL1a) (100 ng ml −1 ) for 2 days before analysis. Cells were gated on live CD3 + CD4 + cells (left panels). Results are representative of 5 (small intestine) and 3 (NP) biologic replicates.

    Journal: Mucosal Immunology

    Article Title: A major population of mucosal memory CD4+ T cells, coexpressing IL-18Rα and DR3, display innate lymphocyte functionality

    doi: 10.1038/mi.2014.87

    Figure Lengend Snippet: Interleukin-18 receptor alpha (IL-18Rα) + death receptor 3 (DR3) + CD4 + T cells are present at barrier surfaces. ( a ) Representative flow cytometry plot and ( b ) percentage of IL-18Rα + DR3 + cells in the indicated organs. IL-18Rα and DR3 expression on CD4 + T cells was assessed after incubation in culture medium for 2 days to allow re-expression of DR3. Gating is on live, CD3 + CD4 + cells. ( b ) Results are mean (s.e.m.) of 7 (small intestine, SI), 3 (nasal polyp, NP), 5 (colon), and 3 (skin) stainings performed. ( c and d ) DR3 + CD4 + cells are diffusely distributed throughout the healthy human small intestine. ( c ) Small intestinal sections were stained with DR3 antibody. ( d ) Sections were stained with DR3 and CD4 antibody together with 4',6-diamidino-2-phenylindole (DAPI) to identify cell nuclei and analyzed by confocal microscopy. Arrowheads depict DR3 + CD4 + cells. Bars=( c ) 0.1 mm and ( d ) 50 μm. Images are from one representative tissue of at least ( c ) 20 and ( d ) 3 analyzed. ( e ) Cytokines induce tissue-resident IL-18Rα + CD4 + T cells to produce interferon-γ (IFN-γ). SI and NP cell suspensions were cultured in medium alone (control) or with interleukin (IL)-12 (2 ng ml −1 ), IL-18 (50 ng ml −1 ), IL-15 (25 ng ml −1 ) and tumor necrosis factor-like cytokine 1A (TL1a) (100 ng ml −1 ) for 2 days before analysis. Cells were gated on live CD3 + CD4 + cells (left panels). Results are representative of 5 (small intestine) and 3 (NP) biologic replicates.

    Article Snippet: Cytokine levels in culture supernatants were assessed by Bio-Plex Pro Human Cytokine 17-plex Assay or Bio-Plex Pro Human Th17 Cytokine IFN-γ Set/Bio-Plex Pro Human Th17 Cytokine IL-10 Set (Bio-Rad, Hercules, CA) according to the manufacturer's instructions using the Bio-Plex 200 System (Bio-Rad).

    Techniques: Flow Cytometry, Cytometry, Expressing, Incubation, Staining, Confocal Microscopy, Cell Culture

    NAG-1 does not affect inflammatory cytokine production from Kupffer cells. (a) Kuppfer cells isolated from the livers of WT mice were plated in triplicate onto 6-well plates and cultured for 24 hrs in RPMI. Media were collected at the beginning of an experiment and cells were then refed and incubated with media containing recombinant human GDF-15 (50 ng/mL) or vehicle for 2 hours and then LPS (10 ng/mL) or vehicle for 6 hours, prior to media collection. TNF- α and IL-6 levels in the media were measured in duplicate using cytokine-specific ELISAs. The mean concentrations for each treatment are plotted. Error bars represent standard error. (b) Kupffer cells isolated from the livers of WT or NAG -1 Tg / Lox mice were plated in triplicate onto 6-well plates and cultured for 24 hrs in RPMI. Media were collected at the beginning of an experiment and cells were re-fed and incubated with media containing LPS (10 ng/mL) or vehicle and incubated for 6 hours prior to collection of media. TNF- α and IL-6 levels in the media were measured in duplicate using cytokine-specific ELISAs. The mean concentrations for each treatment are plotted. Error bars represent the standard error of the mean.

    Journal: Mediators of Inflammation

    Article Title: NAG-1/GDF15 Transgenic Mouse Has Less White Adipose Tissue and a Reduced Inflammatory Response

    doi: 10.1155/2013/641851

    Figure Lengend Snippet: NAG-1 does not affect inflammatory cytokine production from Kupffer cells. (a) Kuppfer cells isolated from the livers of WT mice were plated in triplicate onto 6-well plates and cultured for 24 hrs in RPMI. Media were collected at the beginning of an experiment and cells were then refed and incubated with media containing recombinant human GDF-15 (50 ng/mL) or vehicle for 2 hours and then LPS (10 ng/mL) or vehicle for 6 hours, prior to media collection. TNF- α and IL-6 levels in the media were measured in duplicate using cytokine-specific ELISAs. The mean concentrations for each treatment are plotted. Error bars represent standard error. (b) Kupffer cells isolated from the livers of WT or NAG -1 Tg / Lox mice were plated in triplicate onto 6-well plates and cultured for 24 hrs in RPMI. Media were collected at the beginning of an experiment and cells were re-fed and incubated with media containing LPS (10 ng/mL) or vehicle and incubated for 6 hours prior to collection of media. TNF- α and IL-6 levels in the media were measured in duplicate using cytokine-specific ELISAs. The mean concentrations for each treatment are plotted. Error bars represent the standard error of the mean.

    Article Snippet: The serum was used for cytokine analysis using the Multiplex cytokine detection assay system according to the manufacturer's instructions (Bio-Plex mouse cytokine assay, Bio-Rad).

    Techniques: Isolation, Mouse Assay, Cell Culture, Incubation, Recombinant

    NAG-1 does not inhibit LPS-induced inflammatory cytokine formation in macrophages. (a) RAW 264.7 cells were plated in 60 mm dishes. After 24 hr, cells were treated with NAG-1 protein (R D) at the concentrations indicated for 5 hrs and then treated with LPS (5 ng/mL) for 20 hrs. RNA was isolated from the collected cells and used for real-time PCR analysis, using primers specific to the genes indicated. The data are expressed as fold increases compared to untreated cells. Error bars represent standard deviation. (b) RAW 264.7 cells were transfected with empty vector (−) or NAG-1 expression vector (+), and then the cells were treated with 1 μ g/mL LPS for 6 hours. RNA was extracted and used for real-time PCR analysis, using primers specific to the genes indicated, using m β -actin as a control gene. The data are expressed as fold increases compared to untreated cells. Error bars represent standard error.

    Journal: Mediators of Inflammation

    Article Title: NAG-1/GDF15 Transgenic Mouse Has Less White Adipose Tissue and a Reduced Inflammatory Response

    doi: 10.1155/2013/641851

    Figure Lengend Snippet: NAG-1 does not inhibit LPS-induced inflammatory cytokine formation in macrophages. (a) RAW 264.7 cells were plated in 60 mm dishes. After 24 hr, cells were treated with NAG-1 protein (R D) at the concentrations indicated for 5 hrs and then treated with LPS (5 ng/mL) for 20 hrs. RNA was isolated from the collected cells and used for real-time PCR analysis, using primers specific to the genes indicated. The data are expressed as fold increases compared to untreated cells. Error bars represent standard deviation. (b) RAW 264.7 cells were transfected with empty vector (−) or NAG-1 expression vector (+), and then the cells were treated with 1 μ g/mL LPS for 6 hours. RNA was extracted and used for real-time PCR analysis, using primers specific to the genes indicated, using m β -actin as a control gene. The data are expressed as fold increases compared to untreated cells. Error bars represent standard error.

    Article Snippet: The serum was used for cytokine analysis using the Multiplex cytokine detection assay system according to the manufacturer's instructions (Bio-Plex mouse cytokine assay, Bio-Rad).

    Techniques: Isolation, Real-time Polymerase Chain Reaction, Standard Deviation, Transfection, Plasmid Preparation, Expressing

    Peritoneal macrophages from WT and NAG -1 Tg / Lox mice show no difference in the production of inflammatory cytokines in response to LPS. Peritoneal macrophages were isolated from WT and NAG -1 Tg / Lox mice after 3 days of thioglycollate injection and plated on 60 mm dishes. After 24 hrs, cells were treated with LPS (1 ng/mL) and culture media were collected at the indicated time points. The levels of TNF- α and IL-6 from 3 dishes for each treatment were measured, in duplicate, using cytokine-specific ELISAs (R D). The mean ± standard deviation is reported.

    Journal: Mediators of Inflammation

    Article Title: NAG-1/GDF15 Transgenic Mouse Has Less White Adipose Tissue and a Reduced Inflammatory Response

    doi: 10.1155/2013/641851

    Figure Lengend Snippet: Peritoneal macrophages from WT and NAG -1 Tg / Lox mice show no difference in the production of inflammatory cytokines in response to LPS. Peritoneal macrophages were isolated from WT and NAG -1 Tg / Lox mice after 3 days of thioglycollate injection and plated on 60 mm dishes. After 24 hrs, cells were treated with LPS (1 ng/mL) and culture media were collected at the indicated time points. The levels of TNF- α and IL-6 from 3 dishes for each treatment were measured, in duplicate, using cytokine-specific ELISAs (R D). The mean ± standard deviation is reported.

    Article Snippet: The serum was used for cytokine analysis using the Multiplex cytokine detection assay system according to the manufacturer's instructions (Bio-Plex mouse cytokine assay, Bio-Rad).

    Techniques: Mouse Assay, Isolation, Injection, Standard Deviation

    NA G -1 Tg / Lox mice have lower basal and LPS-induced serum leptin concentrations and abdominal fat leptin expression than WT littermates. The serum collected from the terminal bleeds ( Figure 5 ) was analyzed for leptin using a cytokine-specific ELISA. RNA was isolated from the isolated abdominal fat tissue, which was analyzed for relative expression of mouse leptin mRNA using RT-PCR.

    Journal: Mediators of Inflammation

    Article Title: NAG-1/GDF15 Transgenic Mouse Has Less White Adipose Tissue and a Reduced Inflammatory Response

    doi: 10.1155/2013/641851

    Figure Lengend Snippet: NA G -1 Tg / Lox mice have lower basal and LPS-induced serum leptin concentrations and abdominal fat leptin expression than WT littermates. The serum collected from the terminal bleeds ( Figure 5 ) was analyzed for leptin using a cytokine-specific ELISA. RNA was isolated from the isolated abdominal fat tissue, which was analyzed for relative expression of mouse leptin mRNA using RT-PCR.

    Article Snippet: The serum was used for cytokine analysis using the Multiplex cytokine detection assay system according to the manufacturer's instructions (Bio-Plex mouse cytokine assay, Bio-Rad).

    Techniques: Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    PTX, DEX and AZI inhibit LPS-, R848-, and LPS/ATP-induced cytokine production in newborn cord blood. Human cord blood (n = 10) was pretreated with (a) PTX (50–200 μM), (b) DEX (10 −10 –10 −7 M), (c) AZI (2.5–20 μM) or vehicle control for 2 hours. Samples were stimulated with 10 ng/ml LPS, 1 μg/ml R848, or LPS followed by 5 mM ATP for inflammasome induction, and cultured for 6 hours at 37°C in 5% CO 2 . Supernatant cytokine concentrations were measured, and results expressed as a percentage (mean ± SEM) compared to TLRAs alone, which were defined as 100%. The corresponding mean cytokine concentrations in pg/ml induced by TLRAs alone were represented in brackets next to each cytokine as part of the graph legends.

    Journal: PLoS ONE

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro

    doi: 10.1371/journal.pone.0196352

    Figure Lengend Snippet: PTX, DEX and AZI inhibit LPS-, R848-, and LPS/ATP-induced cytokine production in newborn cord blood. Human cord blood (n = 10) was pretreated with (a) PTX (50–200 μM), (b) DEX (10 −10 –10 −7 M), (c) AZI (2.5–20 μM) or vehicle control for 2 hours. Samples were stimulated with 10 ng/ml LPS, 1 μg/ml R848, or LPS followed by 5 mM ATP for inflammasome induction, and cultured for 6 hours at 37°C in 5% CO 2 . Supernatant cytokine concentrations were measured, and results expressed as a percentage (mean ± SEM) compared to TLRAs alone, which were defined as 100%. The corresponding mean cytokine concentrations in pg/ml induced by TLRAs alone were represented in brackets next to each cytokine as part of the graph legends.

    Article Snippet: Measurement of cytokine concentrations in culture supernatants Supernatant cytokine concentrations were determined with Bio-Plex Pro magnetic multiplex assays (Bio-Rad; Hercules, CA) and analyzed on the Bio-Plex 200 system with Bio-Plex Manager 5.0 software (Bio-Rad), which uses the Brendan five-parameter logistic regression for standard curve fitting.

    Techniques: Cell Culture

    (PTX+DEX) and (PTX+AZI) synergistically inhibit TLR-mediated pro-inflammatory cytokine production in whole blood assays. Newborn cord and adult peripheral blood (n = 8 per cohort for (PTX+DEX) and n = 5 per cohort for (PTX+AZI), analyzed combined) was pretreated with vehicle control, PTX (50–100 μM), DEX (10 −8 –10 −7 M), AZI (5–20 μM) or combined (PTX+DEX) or (PTX+AZI) for 2 hours. Samples were stimulated with 10 ng/ml LPS or 1 μg/ml R848, and cultured for 6 hours at 37°C in 5% CO 2 . Supernatant cytokine concentrations were measured, and results expressed as a percentage (mean ± SEM) compared to TLRAs alone, which were defined as 100% (see S8 Data file for absolute cytokine concentrations). Synergistic inhibition of LPS- and R848-induced (a) TNF and (b) IL-1β production employing selected concentrations of (PTX+DEX) and (c) IL-1β production with (PTX+AZI). Significant differences based on paired t-tests between single anti-inflammatory agents and their corresponding combinations of agents at the same concentrations as the individual compounds were indicated: *p

    Journal: PLoS ONE

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro

    doi: 10.1371/journal.pone.0196352

    Figure Lengend Snippet: (PTX+DEX) and (PTX+AZI) synergistically inhibit TLR-mediated pro-inflammatory cytokine production in whole blood assays. Newborn cord and adult peripheral blood (n = 8 per cohort for (PTX+DEX) and n = 5 per cohort for (PTX+AZI), analyzed combined) was pretreated with vehicle control, PTX (50–100 μM), DEX (10 −8 –10 −7 M), AZI (5–20 μM) or combined (PTX+DEX) or (PTX+AZI) for 2 hours. Samples were stimulated with 10 ng/ml LPS or 1 μg/ml R848, and cultured for 6 hours at 37°C in 5% CO 2 . Supernatant cytokine concentrations were measured, and results expressed as a percentage (mean ± SEM) compared to TLRAs alone, which were defined as 100% (see S8 Data file for absolute cytokine concentrations). Synergistic inhibition of LPS- and R848-induced (a) TNF and (b) IL-1β production employing selected concentrations of (PTX+DEX) and (c) IL-1β production with (PTX+AZI). Significant differences based on paired t-tests between single anti-inflammatory agents and their corresponding combinations of agents at the same concentrations as the individual compounds were indicated: *p

    Article Snippet: Measurement of cytokine concentrations in culture supernatants Supernatant cytokine concentrations were determined with Bio-Plex Pro magnetic multiplex assays (Bio-Rad; Hercules, CA) and analyzed on the Bio-Plex 200 system with Bio-Plex Manager 5.0 software (Bio-Rad), which uses the Brendan five-parameter logistic regression for standard curve fitting.

    Techniques: Cell Culture, Inhibition

    Timing of anti-inflammatory treatment effects extent of inhibition of LPS-induced inflammatory cytokine production. Newborn cord and adult peripheral blood were stimulated with 10 ng/ml LPS. Samples were treated with PTX (50 or 200 μM), DEX (10 −8 or 10 −7 M), AZI (2.5 or 20 μM) or vehicle control, which were added either 2 hours before, simultaneously, or 2 hours after TLR stimulation. Following LPS stimulation, samples were cultured for further 6 hours. Supernatant cytokines were expressed as a percentage compared to TLRAs alone, defined as 100%. For delayed treatment samples, cytokine concentrations measured at the start of anti-inflammatory treatment were subtracted from the corresponding delayed treatment samples, in order not to underestimate the delayed treatment effects. Effects of timing and concentration of (a) PTX [Fig 2a adapted from 20, Speer EM , et al . Pediatr Res . 2017;81 : 806–816 ], (b) DEX, and (c) AZI on LPS-induced cytokine production (mean ± SEM) in newborn and adult blood combined. To determine significant differences between time points (*p

    Journal: PLoS ONE

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro

    doi: 10.1371/journal.pone.0196352

    Figure Lengend Snippet: Timing of anti-inflammatory treatment effects extent of inhibition of LPS-induced inflammatory cytokine production. Newborn cord and adult peripheral blood were stimulated with 10 ng/ml LPS. Samples were treated with PTX (50 or 200 μM), DEX (10 −8 or 10 −7 M), AZI (2.5 or 20 μM) or vehicle control, which were added either 2 hours before, simultaneously, or 2 hours after TLR stimulation. Following LPS stimulation, samples were cultured for further 6 hours. Supernatant cytokines were expressed as a percentage compared to TLRAs alone, defined as 100%. For delayed treatment samples, cytokine concentrations measured at the start of anti-inflammatory treatment were subtracted from the corresponding delayed treatment samples, in order not to underestimate the delayed treatment effects. Effects of timing and concentration of (a) PTX [Fig 2a adapted from 20, Speer EM , et al . Pediatr Res . 2017;81 : 806–816 ], (b) DEX, and (c) AZI on LPS-induced cytokine production (mean ± SEM) in newborn and adult blood combined. To determine significant differences between time points (*p

    Article Snippet: Measurement of cytokine concentrations in culture supernatants Supernatant cytokine concentrations were determined with Bio-Plex Pro magnetic multiplex assays (Bio-Rad; Hercules, CA) and analyzed on the Bio-Plex 200 system with Bio-Plex Manager 5.0 software (Bio-Rad), which uses the Brendan five-parameter logistic regression for standard curve fitting.

    Techniques: Inhibition, Cell Culture, Concentration Assay

    PTX, DEX and AZI demonstrate distinct inhibition of TLR-mediated cytokine mRNA and intracellular protein expression. Whole blood was pretreated for 2 hours with PTX (200 μM), DEX (10 −7 M), AZI (20 μM) or vehicle control (V), either alone or in combination. Samples were stimulated with 10 ng/ml LPS or 1 μM R848, and cultured for 2 hours (mRNA expression) or 6 hours (flow cytometry) at 37°C in 5% CO 2 , in the presence (TNF, IL-6 and IL-10) or absence (IL-1β) of Brefeldin A for flow cytometric assays. MFI of monocytes, gated with forward and side scatter as CD45 + CD14 + cells, was measured as described in Methods . (a) LPS-induced relative mRNA expression (mean ± SEM) in cord and adult blood (n = 5 each, analyzed combined) in response to anti-inflammatory agents compared to LPS-stimulation alone, defined as 100%. Effects of anti-inflammatory treatment on (b) LPS- and (c) R848-induced intracellular cytokines in newborn monocytes (n = 8), plotted as MFI fold changes (± SEM) compared to TLRA stimulation alone (see S6 Data file for raw MFI data). Significant differences based on linear mixed models were indicated: *p

    Journal: PLoS ONE

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro

    doi: 10.1371/journal.pone.0196352

    Figure Lengend Snippet: PTX, DEX and AZI demonstrate distinct inhibition of TLR-mediated cytokine mRNA and intracellular protein expression. Whole blood was pretreated for 2 hours with PTX (200 μM), DEX (10 −7 M), AZI (20 μM) or vehicle control (V), either alone or in combination. Samples were stimulated with 10 ng/ml LPS or 1 μM R848, and cultured for 2 hours (mRNA expression) or 6 hours (flow cytometry) at 37°C in 5% CO 2 , in the presence (TNF, IL-6 and IL-10) or absence (IL-1β) of Brefeldin A for flow cytometric assays. MFI of monocytes, gated with forward and side scatter as CD45 + CD14 + cells, was measured as described in Methods . (a) LPS-induced relative mRNA expression (mean ± SEM) in cord and adult blood (n = 5 each, analyzed combined) in response to anti-inflammatory agents compared to LPS-stimulation alone, defined as 100%. Effects of anti-inflammatory treatment on (b) LPS- and (c) R848-induced intracellular cytokines in newborn monocytes (n = 8), plotted as MFI fold changes (± SEM) compared to TLRA stimulation alone (see S6 Data file for raw MFI data). Significant differences based on linear mixed models were indicated: *p

    Article Snippet: Measurement of cytokine concentrations in culture supernatants Supernatant cytokine concentrations were determined with Bio-Plex Pro magnetic multiplex assays (Bio-Rad; Hercules, CA) and analyzed on the Bio-Plex 200 system with Bio-Plex Manager 5.0 software (Bio-Rad), which uses the Brendan five-parameter logistic regression for standard curve fitting.

    Techniques: Inhibition, Expressing, Cell Culture, Flow Cytometry, Cytometry

    Timing of anti-inflammatory treatment effects extent of inhibition of R848-induced inflammatory cytokine production. Newborn cord and adult peripheral blood were stimulated with 1 μg/ml R848. Samples were treated with PTX (50 or 200 μM), DEX (10 −8 or 10 −7 M), AZI (2.5 or 20 μM) or vehicle control, which were added either 2 hours before, simultaneously, or 2 hours after TLR stimulation. Following R848 stimulation, samples were cultured for further 6 hours. Supernatant cytokines were expressed in percent compared to TLRAs alone, defined as 100%. For delayed treatment samples, cytokine concentrations measured at the start of anti-inflammatory treatment were subtracted from the corresponding delayed treatment samples, in order not to underestimate the delayed treatment effects. Effects of timing and concentration of (a) PTX [Fig 3a adapted from 20, Speer EM , et al . Pediatr Res . 2017;81 : 806–816 ], (b) DEX, and (c) AZI on R848-induced cytokine production (mean ± SEM) in newborn and adult blood combined. To determine significant differences between time points (*p

    Journal: PLoS ONE

    Article Title: Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro

    doi: 10.1371/journal.pone.0196352

    Figure Lengend Snippet: Timing of anti-inflammatory treatment effects extent of inhibition of R848-induced inflammatory cytokine production. Newborn cord and adult peripheral blood were stimulated with 1 μg/ml R848. Samples were treated with PTX (50 or 200 μM), DEX (10 −8 or 10 −7 M), AZI (2.5 or 20 μM) or vehicle control, which were added either 2 hours before, simultaneously, or 2 hours after TLR stimulation. Following R848 stimulation, samples were cultured for further 6 hours. Supernatant cytokines were expressed in percent compared to TLRAs alone, defined as 100%. For delayed treatment samples, cytokine concentrations measured at the start of anti-inflammatory treatment were subtracted from the corresponding delayed treatment samples, in order not to underestimate the delayed treatment effects. Effects of timing and concentration of (a) PTX [Fig 3a adapted from 20, Speer EM , et al . Pediatr Res . 2017;81 : 806–816 ], (b) DEX, and (c) AZI on R848-induced cytokine production (mean ± SEM) in newborn and adult blood combined. To determine significant differences between time points (*p

    Article Snippet: Measurement of cytokine concentrations in culture supernatants Supernatant cytokine concentrations were determined with Bio-Plex Pro magnetic multiplex assays (Bio-Rad; Hercules, CA) and analyzed on the Bio-Plex 200 system with Bio-Plex Manager 5.0 software (Bio-Rad), which uses the Brendan five-parameter logistic regression for standard curve fitting.

    Techniques: Inhibition, Cell Culture, Concentration Assay

    New insights into the heterogeneity of human Th17 cells at homeostasis and during ART-controlled HIV-1 infection. In this work we identified two new subsets of CCR6 + T-cells, CCR6 + DN/CCR4 − CXCR3 − and CCR6 + DP/CCR4 + CXCR3 + , that share Th17 features with the previously described Th17/CCR4 + CXCR3 − and Th1Th17/CCR4 − CXCR3 + [ 5 ]. Despite these similarities, CCR6 + DN distinguished from the other three subsets by superior their ability to produce Th17 effector cytokines ( e.g., IL-17F, IL-8, and IL-21) and their predominant frequency/counts in the blood and lymph nodes HIV-infected individuals receiving ART. Finally, we demonstrate that CCR6 + DN harbor replication-competent HIV-DNA. Thus, we reveal the existence in humans of four Th17-polarized CCR6 + subsets that represent distinct stages of Th17 differentiation, with CCR6 + DN being the most predominant and contributing to HIV reservoir persistence under ART

    Journal: Retrovirology

    Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

    doi: 10.1186/s12977-016-0293-6

    Figure Lengend Snippet: New insights into the heterogeneity of human Th17 cells at homeostasis and during ART-controlled HIV-1 infection. In this work we identified two new subsets of CCR6 + T-cells, CCR6 + DN/CCR4 − CXCR3 − and CCR6 + DP/CCR4 + CXCR3 + , that share Th17 features with the previously described Th17/CCR4 + CXCR3 − and Th1Th17/CCR4 − CXCR3 + [ 5 ]. Despite these similarities, CCR6 + DN distinguished from the other three subsets by superior their ability to produce Th17 effector cytokines ( e.g., IL-17F, IL-8, and IL-21) and their predominant frequency/counts in the blood and lymph nodes HIV-infected individuals receiving ART. Finally, we demonstrate that CCR6 + DN harbor replication-competent HIV-DNA. Thus, we reveal the existence in humans of four Th17-polarized CCR6 + subsets that represent distinct stages of Th17 differentiation, with CCR6 + DN being the most predominant and contributing to HIV reservoir persistence under ART

    Article Snippet: Intracellular cytokine staining Intracellular expression of cytokines was measured by FACS [ ] using the BD Cytofix/Cytoperm kit (BD Biosciences) and specific Abs (See table above).

    Techniques: Infection

    CCR6 + DN carry replication competent HIV-DNA. a , b The four memory CCR6 + subsets as well as Th1 cells from PBMCs of chronically infected receiving viral suppressive ART (CI on ART) individuals were sorted by FACS. a Levels of integrated HIV-DNA were quantified by nested real-time PCR in sorted cells ex vivo (mean ± SD of triplicate wells; n = 4 CI on ART individuals). b , c FACS-sorted memory subsets were stimulated via CD3/CD28 and cultured as described in S6A Figure legend for up to 14 days. b HIV-RNA levels were quantified by real-time RT-PCR in culture supernatant of cells stimulated via CD3/CD28 for 4 days (n = 4 CI on ART individuals). c , d At day 13, cells were stimulated with PMA and Ionomycin in the presence of Brefeldin A for 6 h. Intracellular staining was performed with cytokine-specific Abs and HIV-p24. c Shown are flow cytometry dot plots illustrating the co-expression of IL-17A and HIV-p24 (n = 3). d Shown are pie charts representations generated with the SPICE software illustrating the poly-functional profile of HIV-p24 + CCR6 + DN cells; all possible combinations of one ( blue ), two ( green ), three ( orange ) and four ( yellow ) or no ( purple ) cytokines are depicted (n = 3 CI on ART individuals). e At day 13, CCR6 + DN subsets were stained with CFSE and cultured for 5 additional days in the presence of either IL-2 (5 ng/ml) or CD3/CD28 (1 µg/ml). Cells expressing or not intracellular HIV-p24 were then analyzed for their ability to proliferate (CFSE low )

    Journal: Retrovirology

    Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

    doi: 10.1186/s12977-016-0293-6

    Figure Lengend Snippet: CCR6 + DN carry replication competent HIV-DNA. a , b The four memory CCR6 + subsets as well as Th1 cells from PBMCs of chronically infected receiving viral suppressive ART (CI on ART) individuals were sorted by FACS. a Levels of integrated HIV-DNA were quantified by nested real-time PCR in sorted cells ex vivo (mean ± SD of triplicate wells; n = 4 CI on ART individuals). b , c FACS-sorted memory subsets were stimulated via CD3/CD28 and cultured as described in S6A Figure legend for up to 14 days. b HIV-RNA levels were quantified by real-time RT-PCR in culture supernatant of cells stimulated via CD3/CD28 for 4 days (n = 4 CI on ART individuals). c , d At day 13, cells were stimulated with PMA and Ionomycin in the presence of Brefeldin A for 6 h. Intracellular staining was performed with cytokine-specific Abs and HIV-p24. c Shown are flow cytometry dot plots illustrating the co-expression of IL-17A and HIV-p24 (n = 3). d Shown are pie charts representations generated with the SPICE software illustrating the poly-functional profile of HIV-p24 + CCR6 + DN cells; all possible combinations of one ( blue ), two ( green ), three ( orange ) and four ( yellow ) or no ( purple ) cytokines are depicted (n = 3 CI on ART individuals). e At day 13, CCR6 + DN subsets were stained with CFSE and cultured for 5 additional days in the presence of either IL-2 (5 ng/ml) or CD3/CD28 (1 µg/ml). Cells expressing or not intracellular HIV-p24 were then analyzed for their ability to proliferate (CFSE low )

    Article Snippet: Intracellular cytokine staining Intracellular expression of cytokines was measured by FACS [ ] using the BD Cytofix/Cytoperm kit (BD Biosciences) and specific Abs (See table above).

    Techniques: Infection, FACS, Real-time Polymerase Chain Reaction, Ex Vivo, Cell Culture, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry, Expressing, Generated, Software, Functional Assay

    Lineage commitment versus plasticity of the four CM CCR6 + subsets in vitro. FACS-sorted CM (CD45RA − CCR7 + ) subsets isolated from the peripheral blood of HIV-uninfected individuals were analyzed for the expression of lineage-specific cytokines upon Th17/Th1-polarization in vitro. a CM subsets were stimulated via CD3/CD28 and cultured under Th17- (IL-1β, IL-6, and IL-23, and anti-IL-4 and anti-IFN-γ Abs) and Th1-polarizing conditions (IL-12 and anti-IL-4 Abs) for 4 and 14 days. Cells were stimulated with PMA and Ionomycin in the presence of Brefeldin A for 16 h. Intracellular staining was performed with cytokine-specific Abs. b , c Shown are statistical analyses of cytokines expressed by the distinct CM subsets cultured for 4 ( b ) or 14 days ( c ) under Th17- ( black bars ) and Th1-polarizing conditions ( grey bars ). Results (mean ± SEM) were generated with matches samples from n = 3 different donors. Paired t-Test p -values are indicated on the figures (Th17- versus Th1-polarization)

    Journal: Retrovirology

    Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

    doi: 10.1186/s12977-016-0293-6

    Figure Lengend Snippet: Lineage commitment versus plasticity of the four CM CCR6 + subsets in vitro. FACS-sorted CM (CD45RA − CCR7 + ) subsets isolated from the peripheral blood of HIV-uninfected individuals were analyzed for the expression of lineage-specific cytokines upon Th17/Th1-polarization in vitro. a CM subsets were stimulated via CD3/CD28 and cultured under Th17- (IL-1β, IL-6, and IL-23, and anti-IL-4 and anti-IFN-γ Abs) and Th1-polarizing conditions (IL-12 and anti-IL-4 Abs) for 4 and 14 days. Cells were stimulated with PMA and Ionomycin in the presence of Brefeldin A for 16 h. Intracellular staining was performed with cytokine-specific Abs. b , c Shown are statistical analyses of cytokines expressed by the distinct CM subsets cultured for 4 ( b ) or 14 days ( c ) under Th17- ( black bars ) and Th1-polarizing conditions ( grey bars ). Results (mean ± SEM) were generated with matches samples from n = 3 different donors. Paired t-Test p -values are indicated on the figures (Th17- versus Th1-polarization)

    Article Snippet: Intracellular cytokine staining Intracellular expression of cytokines was measured by FACS [ ] using the BD Cytofix/Cytoperm kit (BD Biosciences) and specific Abs (See table above).

    Techniques: In Vitro, FACS, Isolation, Expressing, Cell Culture, Staining, Generated

    Two new subsets of memory CD4 + T-cells express Th17 lineage markers. a Memory CD4 + T-cells (CD3 + CD4 + CD45RA − ) isolated from the peripheral blood of HIV-uninfected individuals were analyzed for their differential expression of CCR6, CCR4, and CXCR3. CCR6 + subsets included: CCR4 + CXCR3 − (Th17), CCR4 − CXCR3 − (double positive, CCR6 + DP), CCR4 + CXCR3 + (double negative, CCR6 + DN), and CCR4 − CXCR3 + (Th1Th17). CCR6 − subsets included: CCR4 + CXCR3 − (Th2), CCR4 + CXCR3 + (CCR6 − DP), CCR4 − CXCR3 − (CCR6 − DN) and CCR4 − CXCR3 + (Th1). Shown is the frequency of CCR6 + and CCR6 − subsets ( b ; n = 30) and their expression of CD161 ( c ; n = 8). Each symbol represents a distinct subject. Paired t-Test p -values are indicated on the figures. Horizontal bars indicate median values. d Shown are median frequencies of central (CM, CCR7 + CD27 + ), transitional (TM, CCR7 − CD27 + ) and effector (EM, CCR7 − CD27 − ) memory cells per CCR6 + subset (n = 10). e – g FACS-sorted memory subsets (S1 Figure) were stimulated via CD3/CD28 for 4 days. e The production of the lineage-specific cytokines IL-17A, IFN-γ, and IL-5 was quantified by ELISA. Shown are results (mean ± SEM) on matched Th17, Th1Th17, CCR6 + DN, CCR6 + DP, and CCR6- (n = 3–7). Paired t Test p -values are indicated on the figures . f Transcriptional profiling were generated using the HumanHT-12 v4 Expression BeadChip; (Illumina). The heat map depicts differential expression of well-established Th17 and Th1 transcripts (identified as being up/down regulated in Th17 versus CCR6 − DN, p value

    Journal: Retrovirology

    Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

    doi: 10.1186/s12977-016-0293-6

    Figure Lengend Snippet: Two new subsets of memory CD4 + T-cells express Th17 lineage markers. a Memory CD4 + T-cells (CD3 + CD4 + CD45RA − ) isolated from the peripheral blood of HIV-uninfected individuals were analyzed for their differential expression of CCR6, CCR4, and CXCR3. CCR6 + subsets included: CCR4 + CXCR3 − (Th17), CCR4 − CXCR3 − (double positive, CCR6 + DP), CCR4 + CXCR3 + (double negative, CCR6 + DN), and CCR4 − CXCR3 + (Th1Th17). CCR6 − subsets included: CCR4 + CXCR3 − (Th2), CCR4 + CXCR3 + (CCR6 − DP), CCR4 − CXCR3 − (CCR6 − DN) and CCR4 − CXCR3 + (Th1). Shown is the frequency of CCR6 + and CCR6 − subsets ( b ; n = 30) and their expression of CD161 ( c ; n = 8). Each symbol represents a distinct subject. Paired t-Test p -values are indicated on the figures. Horizontal bars indicate median values. d Shown are median frequencies of central (CM, CCR7 + CD27 + ), transitional (TM, CCR7 − CD27 + ) and effector (EM, CCR7 − CD27 − ) memory cells per CCR6 + subset (n = 10). e – g FACS-sorted memory subsets (S1 Figure) were stimulated via CD3/CD28 for 4 days. e The production of the lineage-specific cytokines IL-17A, IFN-γ, and IL-5 was quantified by ELISA. Shown are results (mean ± SEM) on matched Th17, Th1Th17, CCR6 + DN, CCR6 + DP, and CCR6- (n = 3–7). Paired t Test p -values are indicated on the figures . f Transcriptional profiling were generated using the HumanHT-12 v4 Expression BeadChip; (Illumina). The heat map depicts differential expression of well-established Th17 and Th1 transcripts (identified as being up/down regulated in Th17 versus CCR6 − DN, p value

    Article Snippet: Intracellular cytokine staining Intracellular expression of cytokines was measured by FACS [ ] using the BD Cytofix/Cytoperm kit (BD Biosciences) and specific Abs (See table above).

    Techniques: Isolation, Expressing, FACS, Enzyme-linked Immunosorbent Assay, Generated

    CCR6 + DN proliferate in response to C.albicans but not CMV. a FACS-sorted T-cell subsets isolated from the peripheral blood of HIV-uninfected individuals were stained with CFSE, co-cultured with antigen-loaded autologous monocyte-derived dendritic cells (MDDC), and analyzed for their ability to proliferate (CFSE low ) and produce cytokines. b Shown is the frequency of T-cells proliferating in response to C. albicans hyphae, CMV, or SEB at day 5 post co-culture. c Shown is the frequency of cytokine-expressing T-cells proliferating in response to C. albicans within each subset. d – f C. albicans -specific T-cells were further analyzed for the co-expression of IL-17A with IFN-γ or TNF-α. d Results are from one donor representative of results obtained with matched subsets from four different donors. (e – f ) Shown is the frequency and MFI of C. albicans -specific T-cells expressing IL-17A either alone (IL-17A + IFN-γ − ) ( e ) or in combination with IFN-γ (IL-17A + IFN-γ + ) ( f ). b – f The positivity gates were defined based on FMO controls. b , c and e , f Shown are results (mean ± SEM) on matched samples from n = 4 different. Paired t -Test p -values are indicated in the figures . ND not determined

    Journal: Retrovirology

    Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

    doi: 10.1186/s12977-016-0293-6

    Figure Lengend Snippet: CCR6 + DN proliferate in response to C.albicans but not CMV. a FACS-sorted T-cell subsets isolated from the peripheral blood of HIV-uninfected individuals were stained with CFSE, co-cultured with antigen-loaded autologous monocyte-derived dendritic cells (MDDC), and analyzed for their ability to proliferate (CFSE low ) and produce cytokines. b Shown is the frequency of T-cells proliferating in response to C. albicans hyphae, CMV, or SEB at day 5 post co-culture. c Shown is the frequency of cytokine-expressing T-cells proliferating in response to C. albicans within each subset. d – f C. albicans -specific T-cells were further analyzed for the co-expression of IL-17A with IFN-γ or TNF-α. d Results are from one donor representative of results obtained with matched subsets from four different donors. (e – f ) Shown is the frequency and MFI of C. albicans -specific T-cells expressing IL-17A either alone (IL-17A + IFN-γ − ) ( e ) or in combination with IFN-γ (IL-17A + IFN-γ + ) ( f ). b – f The positivity gates were defined based on FMO controls. b , c and e , f Shown are results (mean ± SEM) on matched samples from n = 4 different. Paired t -Test p -values are indicated in the figures . ND not determined

    Article Snippet: Intracellular cytokine staining Intracellular expression of cytokines was measured by FACS [ ] using the BD Cytofix/Cytoperm kit (BD Biosciences) and specific Abs (See table above).

    Techniques: FACS, Isolation, Staining, Cell Culture, Derivative Assay, Co-Culture Assay, Expressing

    CCR6 + DN cells are a major source of IL-17F, IL-8, and IL-21. Culture supernatants harvested from Th17, Th1Th17, and CCR6 + DN (stimulated as in Fig. 1 e) isolated from the peripheral blood of HIV-uninfected individuals were screened for the expression of 34 T-helper lineage-specific cytokines using the Human Th1/Th2/Th17 Antibody Array C series (RayBiotec). a , b Shown are results from one experiment with matched Th17 versus CCR6 + DN and Th1Th17 versus CCR6 + DN subsets: membrane blot ( left panels ) and relative density quantification ( right panels ). Results are representative of experiments performed with cells from two different donors: c , d Levels of IL-17F, IL-22, CCL20, IL-10, IL-13, TNF-α, IL-8, and IL-21 were quantified by ELISA. Shown are results on matched Th17, Th1Th17, CCR6 + DN, CCR6 + DP, and CCR6 − samples from different individuals (n = 3–7, mean ± SEM). Paired t -Test p -values are indicated in the graphs

    Journal: Retrovirology

    Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

    doi: 10.1186/s12977-016-0293-6

    Figure Lengend Snippet: CCR6 + DN cells are a major source of IL-17F, IL-8, and IL-21. Culture supernatants harvested from Th17, Th1Th17, and CCR6 + DN (stimulated as in Fig. 1 e) isolated from the peripheral blood of HIV-uninfected individuals were screened for the expression of 34 T-helper lineage-specific cytokines using the Human Th1/Th2/Th17 Antibody Array C series (RayBiotec). a , b Shown are results from one experiment with matched Th17 versus CCR6 + DN and Th1Th17 versus CCR6 + DN subsets: membrane blot ( left panels ) and relative density quantification ( right panels ). Results are representative of experiments performed with cells from two different donors: c , d Levels of IL-17F, IL-22, CCL20, IL-10, IL-13, TNF-α, IL-8, and IL-21 were quantified by ELISA. Shown are results on matched Th17, Th1Th17, CCR6 + DN, CCR6 + DP, and CCR6 − samples from different individuals (n = 3–7, mean ± SEM). Paired t -Test p -values are indicated in the graphs

    Article Snippet: Intracellular cytokine staining Intracellular expression of cytokines was measured by FACS [ ] using the BD Cytofix/Cytoperm kit (BD Biosciences) and specific Abs (See table above).

    Techniques: Isolation, Expressing, Ab Array, Enzyme-linked Immunosorbent 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

    PLY enhances cytokine production by splenocytes independently of TLR4. (A) Spleen cells from C3H/HeN and C3H/HeJ mice were incubated for 72 hours with PLY (0.32–200 ng/ml) (58,500 HU/mg) or W433F in the presence or absence of HkSp (1 bacterium:1 spleen cell). Splenocytes were then stimulated with PMA (5 µg/ml) and ionomycin (300 ng/ml) for a further 24 hours to activate the cells. IFN-γ concentrations were determined in supernatants. ** P

    Journal: PLoS Pathogens

    Article Title: Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4

    doi: 10.1371/journal.ppat.1001191

    Figure Lengend Snippet: PLY enhances cytokine production by splenocytes independently of TLR4. (A) Spleen cells from C3H/HeN and C3H/HeJ mice were incubated for 72 hours with PLY (0.32–200 ng/ml) (58,500 HU/mg) or W433F in the presence or absence of HkSp (1 bacterium:1 spleen cell). Splenocytes were then stimulated with PMA (5 µg/ml) and ionomycin (300 ng/ml) for a further 24 hours to activate the cells. IFN-γ concentrations were determined in supernatants. ** P

    Article Snippet: Effect of PLY on cytokine production by spleen cells Splenocytes were isolated from C3H/HeN or C3H/HeJ mice by passing spleens through a 70 µm cell strainer (BD Falcon).

    Techniques: Mouse Assay, Incubation

    In contrast to PLY-induced IL-1β secretion, the enhancement of TNF-α, IL-6, IL-10 and IL-12 by PLY is independent of NLRP3. DC (6.25×10 5 /ml) from C57BL/6 or NLRP3 −/− mice were incubated with medium alone or with various concentrations of PLY (0.2–0.75 µg/ml) for 1 hour before the addition of CpG (4 µg/ml). After 24 hours supernatants were assayed for IL-1β, TNF-α, IL-6, IL-23, IL-10 and IL-12p70. Results are mean cytokine concentrations (+ SEM) for triplicate samples. ** P

    Journal: PLoS Pathogens

    Article Title: Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4

    doi: 10.1371/journal.ppat.1001191

    Figure Lengend Snippet: In contrast to PLY-induced IL-1β secretion, the enhancement of TNF-α, IL-6, IL-10 and IL-12 by PLY is independent of NLRP3. DC (6.25×10 5 /ml) from C57BL/6 or NLRP3 −/− mice were incubated with medium alone or with various concentrations of PLY (0.2–0.75 µg/ml) for 1 hour before the addition of CpG (4 µg/ml). After 24 hours supernatants were assayed for IL-1β, TNF-α, IL-6, IL-23, IL-10 and IL-12p70. Results are mean cytokine concentrations (+ SEM) for triplicate samples. ** P

    Article Snippet: Effect of PLY on cytokine production by spleen cells Splenocytes were isolated from C3H/HeN or C3H/HeJ mice by passing spleens through a 70 µm cell strainer (BD Falcon).

    Techniques: Mouse Assay, Incubation

    Live S. pneumoniae promotes IL-1β secretion by DC in a NLRP3-dependent manner and this requires PLY. DC (6.25×10 5 /ml) from C57BL/6 or NLRP3 −/− mice were incubated for 24 hours with medium alone, with wild-type S. pneumoniae (D39; 10 bacteria:1DC) or with PLY-deficient bacteria (PLN; 10 bacteria:1 DC). As a positive control for IL-1β secretion and NLRP3 inflammasome activation, DC were primed with LPS (100 ng/ml) for 23 hours prior to one hour stimulation with ATP (2.7 mg/ml). Following incubation, supernatants were removed and assayed for IL-1β, IL-1α, TNF-α and IL-6. Results are mean cytokine concentrations (+ SEM) for triplicate samples. *** P

    Journal: PLoS Pathogens

    Article Title: Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4

    doi: 10.1371/journal.ppat.1001191

    Figure Lengend Snippet: Live S. pneumoniae promotes IL-1β secretion by DC in a NLRP3-dependent manner and this requires PLY. DC (6.25×10 5 /ml) from C57BL/6 or NLRP3 −/− mice were incubated for 24 hours with medium alone, with wild-type S. pneumoniae (D39; 10 bacteria:1DC) or with PLY-deficient bacteria (PLN; 10 bacteria:1 DC). As a positive control for IL-1β secretion and NLRP3 inflammasome activation, DC were primed with LPS (100 ng/ml) for 23 hours prior to one hour stimulation with ATP (2.7 mg/ml). Following incubation, supernatants were removed and assayed for IL-1β, IL-1α, TNF-α and IL-6. Results are mean cytokine concentrations (+ SEM) for triplicate samples. *** P

    Article Snippet: Effect of PLY on cytokine production by spleen cells Splenocytes were isolated from C3H/HeN or C3H/HeJ mice by passing spleens through a 70 µm cell strainer (BD Falcon).

    Techniques: Mouse Assay, Incubation, Positive Control, Activation Assay

    PLY is required for IFN-γ and IL-17A induction following infection with S. pneumoniae . (A) IFN-γ concentrations in lungs of infected mice in a model of acute pneumonia. MF1 mice were infected intranasally with wild-type (WT) or PLY-deficient (PLN-A) pneumococci. IFN-γ concentrations were determined in lung homogenates at 0 hours, 24 hours or 48 hours after infection. Results are mean cytokine concentration (+ SEM) for 5 mice per group. *** P

    Journal: PLoS Pathogens

    Article Title: Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4

    doi: 10.1371/journal.ppat.1001191

    Figure Lengend Snippet: PLY is required for IFN-γ and IL-17A induction following infection with S. pneumoniae . (A) IFN-γ concentrations in lungs of infected mice in a model of acute pneumonia. MF1 mice were infected intranasally with wild-type (WT) or PLY-deficient (PLN-A) pneumococci. IFN-γ concentrations were determined in lung homogenates at 0 hours, 24 hours or 48 hours after infection. Results are mean cytokine concentration (+ SEM) for 5 mice per group. *** P

    Article Snippet: Effect of PLY on cytokine production by spleen cells Splenocytes were isolated from C3H/HeN or C3H/HeJ mice by passing spleens through a 70 µm cell strainer (BD Falcon).

    Techniques: Infection, Mouse Assay, Concentration Assay

    PLY synergizes with TLR agonists to enhance pro-inflammatory cytokine secretion by DC. (A) DC from C3H/HeN or C3H/HeJ mice were incubated with PLY (1 µg/ml) for 1 hour before the addition of HkSp (10 bacteria:1 DC). After 24 hours supernatants were assayed for IL-12p40, IL-6, IL-23 and TNF-α. Results are mean cytokine concentrations (+ SEM) for triplicate samples. *** P

    Journal: PLoS Pathogens

    Article Title: Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4

    doi: 10.1371/journal.ppat.1001191

    Figure Lengend Snippet: PLY synergizes with TLR agonists to enhance pro-inflammatory cytokine secretion by DC. (A) DC from C3H/HeN or C3H/HeJ mice were incubated with PLY (1 µg/ml) for 1 hour before the addition of HkSp (10 bacteria:1 DC). After 24 hours supernatants were assayed for IL-12p40, IL-6, IL-23 and TNF-α. Results are mean cytokine concentrations (+ SEM) for triplicate samples. *** P

    Article Snippet: Effect of PLY on cytokine production by spleen cells Splenocytes were isolated from C3H/HeN or C3H/HeJ mice by passing spleens through a 70 µm cell strainer (BD Falcon).

    Techniques: Mouse Assay, Incubation

    Endotoxin-free PLY does not induce cytokine production by DC or macrophages but does enhance DC maturation in a TLR4-independent manner. DC (A) or BMDM (B) from C57BL/6 mice were incubated with PLY (1 µg/ml or 0.5 µg/ml) or Pam3CSK (10 µg/ml) or LPS (500 pg/ml) for 24 hours. Supernatants were analyzed for IL-12p40, TNF-α and IL-6. Results are presented as mean cytokine concentrations (+ SEM) for triplicate samples. (C) DC from C3H/HeN or C3H/HeJ mice were incubated with medium, PLY (1 µg/ml) or LPS (500 pg/ml). After 24 hours, cells were washed and stained with antibodies specific for CD80, CD86, CD40 and MHC Class II. Immunofluorescence is shown for PLY- or LPS-treated DC (black line) compared to untreated cells incubated with medium (grey histograms). Plots are representative of three independent experiments. (D) Adoptive transfer of DC incubated with antigen and PLY promotes antigen-specific T cell responses. BALB/c mice were immunized subcutaneously in the footpad with DC (5×10 5 cells/mouse) that had been incubated overnight with medium only as a control or with KLH antigen (10 µg/ml) in the presence or absence of PLY (1 µg/ml). 7 days later splenocytes were isolated and stimulated ex vivo with KLH (2 or 50 µg/ml). Proliferation was measured by [ 3 H]-thymidine incorporation after 4 days of culture and is expressed as mean cpm (+ SEM; n = 5). *** P

    Journal: PLoS Pathogens

    Article Title: Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4

    doi: 10.1371/journal.ppat.1001191

    Figure Lengend Snippet: Endotoxin-free PLY does not induce cytokine production by DC or macrophages but does enhance DC maturation in a TLR4-independent manner. DC (A) or BMDM (B) from C57BL/6 mice were incubated with PLY (1 µg/ml or 0.5 µg/ml) or Pam3CSK (10 µg/ml) or LPS (500 pg/ml) for 24 hours. Supernatants were analyzed for IL-12p40, TNF-α and IL-6. Results are presented as mean cytokine concentrations (+ SEM) for triplicate samples. (C) DC from C3H/HeN or C3H/HeJ mice were incubated with medium, PLY (1 µg/ml) or LPS (500 pg/ml). After 24 hours, cells were washed and stained with antibodies specific for CD80, CD86, CD40 and MHC Class II. Immunofluorescence is shown for PLY- or LPS-treated DC (black line) compared to untreated cells incubated with medium (grey histograms). Plots are representative of three independent experiments. (D) Adoptive transfer of DC incubated with antigen and PLY promotes antigen-specific T cell responses. BALB/c mice were immunized subcutaneously in the footpad with DC (5×10 5 cells/mouse) that had been incubated overnight with medium only as a control or with KLH antigen (10 µg/ml) in the presence or absence of PLY (1 µg/ml). 7 days later splenocytes were isolated and stimulated ex vivo with KLH (2 or 50 µg/ml). Proliferation was measured by [ 3 H]-thymidine incorporation after 4 days of culture and is expressed as mean cpm (+ SEM; n = 5). *** P

    Article Snippet: Effect of PLY on cytokine production by spleen cells Splenocytes were isolated from C3H/HeN or C3H/HeJ mice by passing spleens through a 70 µm cell strainer (BD Falcon).

    Techniques: Mouse Assay, Incubation, Staining, Immunofluorescence, Adoptive Transfer Assay, Isolation, Ex Vivo

    Cytokines significantly induced during VZV infection in HFLs Cells and culture supernatants were harvested at 72 hpi from mock- or VZV-infected HFLs. (A) Flow cytometry analyses of VZV gE expression in mock- (blue) and VZV-infected (red) HFLs at 72 hpi showed that > 80% of cells were VZV gE+. Cells were gated using isotype controls. Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HFLs had significantly higher levels of IL-8 (B; p = 0.02), IL-6 (C; p = 0.02), VEGF-A (D; p = 0.01), TGF-β (E; p = 0.01), IL-15 (F; p = 0.04), and IL-4 (G; p = 0.05). All cytokine values are given in pg/ml. Bar graphs represent mean ± SD cytokine levels from 3 independent experiments. HFL = human fetal lung fibroblast; IL = interleukin; TGF-β = transforming growth factor β; VEGF-A = vascular endothelial growth factor A; VZV = varicella zoster virus.

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    Article Title: Varicella zoster virus–infected cerebrovascular cells produce a proinflammatory environment

    doi: 10.1212/NXI.0000000000000382

    Figure Lengend Snippet: Cytokines significantly induced during VZV infection in HFLs Cells and culture supernatants were harvested at 72 hpi from mock- or VZV-infected HFLs. (A) Flow cytometry analyses of VZV gE expression in mock- (blue) and VZV-infected (red) HFLs at 72 hpi showed that > 80% of cells were VZV gE+. Cells were gated using isotype controls. Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HFLs had significantly higher levels of IL-8 (B; p = 0.02), IL-6 (C; p = 0.02), VEGF-A (D; p = 0.01), TGF-β (E; p = 0.01), IL-15 (F; p = 0.04), and IL-4 (G; p = 0.05). All cytokine values are given in pg/ml. Bar graphs represent mean ± SD cytokine levels from 3 independent experiments. HFL = human fetal lung fibroblast; IL = interleukin; TGF-β = transforming growth factor β; VEGF-A = vascular endothelial growth factor A; VZV = varicella zoster virus.

    Article Snippet: Conditioned supernatant from mock- and VZV-infected human brain vascular adventitial fibroblasts (HBVAFs), human perineurial cells (HPNCs), human brain vascular smooth muscle cells (HBVSMCs), and human fetal lung fibroblasts (HFLs) were collected at 72 hours postinfection and analyzed for levels of 30 proinflammatory cytokines using the Meso Scale Discovery Multiplex ELISA platform.

    Techniques: Infection, Flow Cytometry, Cytometry, Expressing, Cell Culture

    Specific cytokines significantly altered during VZV infection in HBVAFs Cells and cell culture supernatants were harvested at 72 hpi from mock- or VZV-infected HBVAFs. (A) Flow cytometry analyses of VZV gE expression in mock- (blue) and VZV-infected (red) HBVAFs at 72 hpi showed that > 80% of cells were VZV gE+. Cells were gated using isotype controls. Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HBVAFs had significantly higher levels of IL-8 (B; p = 0.02), IL-6 (C; p = 0.03), VEGF-A (D; p = 0.001), TGF-β (E; p = 0.04), and IL-16 (F; p = 0.01). Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HBVAFs had significantly lower levels of Eotaxin-1 (G; p = 0.002), MCP-1 (H; p = 0.003), IP-10 (I; p = 0.02), and GM-CSF (J; p = 0.03). All cytokine values are given in picograms per milliliter. Bar graphs represent mean ± SD cytokine levels from 3 independent experiments. GM-CSF = granulocyte macrophage colony-stimulating factor; HBVAF = human brain vascular adventitial fibroblast; IL = interleukin; TGF-β = transforming growth factor β; VEGF-A = vascular endothelial growth factor A; VZV = varicella zoster virus.

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    Article Title: Varicella zoster virus–infected cerebrovascular cells produce a proinflammatory environment

    doi: 10.1212/NXI.0000000000000382

    Figure Lengend Snippet: Specific cytokines significantly altered during VZV infection in HBVAFs Cells and cell culture supernatants were harvested at 72 hpi from mock- or VZV-infected HBVAFs. (A) Flow cytometry analyses of VZV gE expression in mock- (blue) and VZV-infected (red) HBVAFs at 72 hpi showed that > 80% of cells were VZV gE+. Cells were gated using isotype controls. Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HBVAFs had significantly higher levels of IL-8 (B; p = 0.02), IL-6 (C; p = 0.03), VEGF-A (D; p = 0.001), TGF-β (E; p = 0.04), and IL-16 (F; p = 0.01). Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HBVAFs had significantly lower levels of Eotaxin-1 (G; p = 0.002), MCP-1 (H; p = 0.003), IP-10 (I; p = 0.02), and GM-CSF (J; p = 0.03). All cytokine values are given in picograms per milliliter. Bar graphs represent mean ± SD cytokine levels from 3 independent experiments. GM-CSF = granulocyte macrophage colony-stimulating factor; HBVAF = human brain vascular adventitial fibroblast; IL = interleukin; TGF-β = transforming growth factor β; VEGF-A = vascular endothelial growth factor A; VZV = varicella zoster virus.

    Article Snippet: Conditioned supernatant from mock- and VZV-infected human brain vascular adventitial fibroblasts (HBVAFs), human perineurial cells (HPNCs), human brain vascular smooth muscle cells (HBVSMCs), and human fetal lung fibroblasts (HFLs) were collected at 72 hours postinfection and analyzed for levels of 30 proinflammatory cytokines using the Meso Scale Discovery Multiplex ELISA platform.

    Techniques: Infection, Cell Culture, Flow Cytometry, Cytometry, Expressing

    Specific cytokines significantly altered during VZV infection in HPNCs Cells and cell culture supernatants were harvested at 72 hpi from mock- or VZV-infected HPNCs. (A) Flow cytometry analyses of VZV gE expression in mock- (blue) and VZV-infected (red) HPNCs at 72 hpi showed that > 85% of cells were VZV gE+. Cells were gated using isotype controls. Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HPNCs had significantly higher levels of IL-8 (B; p = 0.05), IL-6 (C; p = 0.006), IL-2 (D; p = 0.03), and GM-CSF (E; p = 0.02). Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HPNCs had significantly lower levels of VEGF-A (F; p = 0.003). All cytokine values are given in picograms per milliliter. Bar graphs represent mean ± SD cytokine levels from 3 independent experiments. GM-CSF = granulocyte macrophage colony-stimulating factor; HPNC = human perineurial cell; IL = interleukin; VEGF-A = vascular endothelial growth factor A; VZV = varicella zoster virus.

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    Article Title: Varicella zoster virus–infected cerebrovascular cells produce a proinflammatory environment

    doi: 10.1212/NXI.0000000000000382

    Figure Lengend Snippet: Specific cytokines significantly altered during VZV infection in HPNCs Cells and cell culture supernatants were harvested at 72 hpi from mock- or VZV-infected HPNCs. (A) Flow cytometry analyses of VZV gE expression in mock- (blue) and VZV-infected (red) HPNCs at 72 hpi showed that > 85% of cells were VZV gE+. Cells were gated using isotype controls. Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HPNCs had significantly higher levels of IL-8 (B; p = 0.05), IL-6 (C; p = 0.006), IL-2 (D; p = 0.03), and GM-CSF (E; p = 0.02). Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HPNCs had significantly lower levels of VEGF-A (F; p = 0.003). All cytokine values are given in picograms per milliliter. Bar graphs represent mean ± SD cytokine levels from 3 independent experiments. GM-CSF = granulocyte macrophage colony-stimulating factor; HPNC = human perineurial cell; IL = interleukin; VEGF-A = vascular endothelial growth factor A; VZV = varicella zoster virus.

    Article Snippet: Conditioned supernatant from mock- and VZV-infected human brain vascular adventitial fibroblasts (HBVAFs), human perineurial cells (HPNCs), human brain vascular smooth muscle cells (HBVSMCs), and human fetal lung fibroblasts (HFLs) were collected at 72 hours postinfection and analyzed for levels of 30 proinflammatory cytokines using the Meso Scale Discovery Multiplex ELISA platform.

    Techniques: Infection, Cell Culture, Flow Cytometry, Cytometry, Expressing

    HBVAFs secrete the highest level of cytokines among vascular and lung cells Cell culture supernatants from mock-infected HBVAFs, HPNCs, HBVSMCs, and HFLs were harvested at 72 hpi and analyzed for IL-1β, IL-1α, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-12/23p40, IL-13, IL-15, IL-16, IL-17a, IFNγ, TNF-α, TNF-β, Eotaxin-1, Eotaxin-3, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, TARC, GM-CSF, TGF-β, and VEGF-A levels. Only significant fold-change in HBVAFs cytokine levels compared with HPNCs (red), HBVSMCs (blue), or HFLs (green) were plotted. Bar graphs represent mean ± SEM fold-change in cytokine levels from triplicate experiments. (a) p

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    Article Title: Varicella zoster virus–infected cerebrovascular cells produce a proinflammatory environment

    doi: 10.1212/NXI.0000000000000382

    Figure Lengend Snippet: HBVAFs secrete the highest level of cytokines among vascular and lung cells Cell culture supernatants from mock-infected HBVAFs, HPNCs, HBVSMCs, and HFLs were harvested at 72 hpi and analyzed for IL-1β, IL-1α, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-12/23p40, IL-13, IL-15, IL-16, IL-17a, IFNγ, TNF-α, TNF-β, Eotaxin-1, Eotaxin-3, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, TARC, GM-CSF, TGF-β, and VEGF-A levels. Only significant fold-change in HBVAFs cytokine levels compared with HPNCs (red), HBVSMCs (blue), or HFLs (green) were plotted. Bar graphs represent mean ± SEM fold-change in cytokine levels from triplicate experiments. (a) p

    Article Snippet: Conditioned supernatant from mock- and VZV-infected human brain vascular adventitial fibroblasts (HBVAFs), human perineurial cells (HPNCs), human brain vascular smooth muscle cells (HBVSMCs), and human fetal lung fibroblasts (HFLs) were collected at 72 hours postinfection and analyzed for levels of 30 proinflammatory cytokines using the Meso Scale Discovery Multiplex ELISA platform.

    Techniques: Cell Culture, Infection

    Cytokines significantly altered during VZV infection in HBVSMCs Cells and cell culture supernatants were harvested at 72 hpi from mock- or VZV-infected HBVSMCs. (A) Flow cytometry analyses of VZV gE expression in mock- (blue) and VZV-infected (red) HBVSMCs at 72 hpi showed that > 50% of cells were VZV gE+. Cells were gated using isotype controls. Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HBVSMCs had significantly higher levels of IL-8 (B; p = 0.002) and VEGF-A (C; p = 0.0004). Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HBVSMCs had significantly lower levels of IL-15 (D; p = 0.006), Eotaxin-3 (E; p = 0.004), IP-10 (F; p = 0.02), and MCP-1 (G; p = 0.02). All cytokine values are given in picograms per milliliter. Bar graphs represent mean ± SD cytokine levels from 3 independent experiments. HBVSMC = human brain vascular smooth muscle cell; IL = interleukin; VEGF-A = vascular endothelial growth factor A; VZV = varicella zoster virus.

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    Article Title: Varicella zoster virus–infected cerebrovascular cells produce a proinflammatory environment

    doi: 10.1212/NXI.0000000000000382

    Figure Lengend Snippet: Cytokines significantly altered during VZV infection in HBVSMCs Cells and cell culture supernatants were harvested at 72 hpi from mock- or VZV-infected HBVSMCs. (A) Flow cytometry analyses of VZV gE expression in mock- (blue) and VZV-infected (red) HBVSMCs at 72 hpi showed that > 50% of cells were VZV gE+. Cells were gated using isotype controls. Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HBVSMCs had significantly higher levels of IL-8 (B; p = 0.002) and VEGF-A (C; p = 0.0004). Compared with cell culture supernatants from mock-infected cells, supernatants from VZV-infected HBVSMCs had significantly lower levels of IL-15 (D; p = 0.006), Eotaxin-3 (E; p = 0.004), IP-10 (F; p = 0.02), and MCP-1 (G; p = 0.02). All cytokine values are given in picograms per milliliter. Bar graphs represent mean ± SD cytokine levels from 3 independent experiments. HBVSMC = human brain vascular smooth muscle cell; IL = interleukin; VEGF-A = vascular endothelial growth factor A; VZV = varicella zoster virus.

    Article Snippet: Conditioned supernatant from mock- and VZV-infected human brain vascular adventitial fibroblasts (HBVAFs), human perineurial cells (HPNCs), human brain vascular smooth muscle cells (HBVSMCs), and human fetal lung fibroblasts (HFLs) were collected at 72 hours postinfection and analyzed for levels of 30 proinflammatory cytokines using the Meso Scale Discovery Multiplex ELISA platform.

    Techniques: Infection, Cell Culture, Flow Cytometry, Cytometry, Expressing

    Cytokine production kinetics in T cells. ( A ) T cells that were rested for 3 d in the presence of IL-7 ( Top ), IL-2 ( Center ), or IL-15 ( Bottom ) were activated with 100 nM SIINFEKL (N4) or SIITFEKL (T4) OVA peptide for indicated time points ( n = 3 mice). Brefeldin A (BrfA) was added during the last 2 h of culture. ( B ) Representative dot plots for TNF-α and IFN-γ ( Upper ) and IFN-γ and IL-2 ( Lower ) production of T cells that were activated for indicated time with 100 nM OVA 257–264 peptide (N4) in the presence of BrfA during the last 2 h of stimulation. Numbers indicate percentage of cytokine-producing T cells. ( C ) mRNA levels of Tnfa , Ifng , and Il2 of T cells that were rested for 3 d in the presence of IL-7, IL-2, or IL-15.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: Cytokine production kinetics in T cells. ( A ) T cells that were rested for 3 d in the presence of IL-7 ( Top ), IL-2 ( Center ), or IL-15 ( Bottom ) were activated with 100 nM SIINFEKL (N4) or SIITFEKL (T4) OVA peptide for indicated time points ( n = 3 mice). Brefeldin A (BrfA) was added during the last 2 h of culture. ( B ) Representative dot plots for TNF-α and IFN-γ ( Upper ) and IFN-γ and IL-2 ( Lower ) production of T cells that were activated for indicated time with 100 nM OVA 257–264 peptide (N4) in the presence of BrfA during the last 2 h of stimulation. Numbers indicate percentage of cytokine-producing T cells. ( C ) mRNA levels of Tnfa , Ifng , and Il2 of T cells that were rested for 3 d in the presence of IL-7, IL-2, or IL-15.

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Mouse Assay

    Preformed cytokine mRNA promotes early T cell responsiveness. ( A ) Tnfa , Ifng , and Il2 mRNA levels of resting T cells were measured by RT-PCR. ( B – D ) T cells were pretreated for 30 min with 1 μg/mL ActD or 10 μg/mL CHX and then stimulated for indicated time points with 100 nM OVA peptide in the presence of BrfA. Graphs depict the relative percentage of TNF-α ( B ), IFN-γ ( C ), and IL-2 ( D )-producing T cells compared with T cells activated in the absence of inhibitors. Data are presented as mean ± SD of three independently performed experiments. (One-way ANOVA corrected for multiple comparisons with a Tukey test; n = 7 mice; * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: Preformed cytokine mRNA promotes early T cell responsiveness. ( A ) Tnfa , Ifng , and Il2 mRNA levels of resting T cells were measured by RT-PCR. ( B – D ) T cells were pretreated for 30 min with 1 μg/mL ActD or 10 μg/mL CHX and then stimulated for indicated time points with 100 nM OVA peptide in the presence of BrfA. Graphs depict the relative percentage of TNF-α ( B ), IFN-γ ( C ), and IL-2 ( D )-producing T cells compared with T cells activated in the absence of inhibitors. Data are presented as mean ± SD of three independently performed experiments. (One-way ANOVA corrected for multiple comparisons with a Tukey test; n = 7 mice; * P

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mouse Assay

    Regulation of cytokine production through PKC and Ca 2+ -dependent signaling. ( A ) TNF-α and IFN-γ ( Upper ), and IL-2 and IFN-γ ( Lower ) production of CD44 hi memory-like T cells activated for 4 h with indicated stimuli. Unstimulated T cells were used as negative control. Representative of four mice from two independently performed experiments. ( B ) T cells were stimulated with PMA/ionomycin for indicated time points. The percentage of TNF-α, IFN-γ, and IL-2 protein producing T cells was determined by intracellular cytokine staining (mean ± SD; n = 7 mice). ( C and D ) IL-2 production of PMA/ionomycin-stimulated T cells ( C ), and IFN-γ and IL-2 production of OVA 257–264 -stimulated T cells ( D ) that were pretreated with the indicated inhibitor. Representative of six mice from three independently performed experiments. ( E ) Tnfa , Ifng , and Il2 mRNA levels (mean ± SD) of resting T cells that were stimulated for 2 h as indicated (one-way ANOVA with Tukey’s multiple comparison; n = 7 mice; ns, nonsignificant; * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: Regulation of cytokine production through PKC and Ca 2+ -dependent signaling. ( A ) TNF-α and IFN-γ ( Upper ), and IL-2 and IFN-γ ( Lower ) production of CD44 hi memory-like T cells activated for 4 h with indicated stimuli. Unstimulated T cells were used as negative control. Representative of four mice from two independently performed experiments. ( B ) T cells were stimulated with PMA/ionomycin for indicated time points. The percentage of TNF-α, IFN-γ, and IL-2 protein producing T cells was determined by intracellular cytokine staining (mean ± SD; n = 7 mice). ( C and D ) IL-2 production of PMA/ionomycin-stimulated T cells ( C ), and IFN-γ and IL-2 production of OVA 257–264 -stimulated T cells ( D ) that were pretreated with the indicated inhibitor. Representative of six mice from three independently performed experiments. ( E ) Tnfa , Ifng , and Il2 mRNA levels (mean ± SD) of resting T cells that were stimulated for 2 h as indicated (one-way ANOVA with Tukey’s multiple comparison; n = 7 mice; ns, nonsignificant; * P

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Negative Control, Mouse Assay, Staining

    TNF-α, IFN-γ, and IL-2 follow individual production kinetics. ( A and B ) Cytokine production of resting CD8 + OT-I T cells stimulated with 100 nM OVA peptide. Brefeldin A (BrfA) was present throughout the stimulation ( A ) or added for the last 0.5, 1, or 2 h of activation ( B ). ( C ) Cytokine profile analysis of T cells activated for 2, 4, or 6 h from B . ( Upper ) Percentage of T cells that produce at least one cytokine. ( Lower ) Cytokine production of responding T cells. Pooled data from three independently performed experiments (mean ± SD; n = 7 mice). ( D ) FACS-sorted CD44 hi memory-like OT-I T cells activated with peptide-loaded bone marrow-derived dendritic cells. N4, SIINFEKL; T4, SIITFEKL ( n = 5 mice). ( E ) Effector memory (EM, Left ) and central memory (CM; Right ) T cells from LCMV-infected mice (d60) were reactivated with GP33 and NP396 peptide ( n = 5 mice).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: TNF-α, IFN-γ, and IL-2 follow individual production kinetics. ( A and B ) Cytokine production of resting CD8 + OT-I T cells stimulated with 100 nM OVA peptide. Brefeldin A (BrfA) was present throughout the stimulation ( A ) or added for the last 0.5, 1, or 2 h of activation ( B ). ( C ) Cytokine profile analysis of T cells activated for 2, 4, or 6 h from B . ( Upper ) Percentage of T cells that produce at least one cytokine. ( Lower ) Cytokine production of responding T cells. Pooled data from three independently performed experiments (mean ± SD; n = 7 mice). ( D ) FACS-sorted CD44 hi memory-like OT-I T cells activated with peptide-loaded bone marrow-derived dendritic cells. N4, SIINFEKL; T4, SIITFEKL ( n = 5 mice). ( E ) Effector memory (EM, Left ) and central memory (CM; Right ) T cells from LCMV-infected mice (d60) were reactivated with GP33 and NP396 peptide ( n = 5 mice).

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Activation Assay, Mouse Assay, FACS, Derivative Assay, Infection

    Role of PKC and Ca 2+ -dependent signaling in cytokine production. ( A ) Resting T cells were stimulated or not for 4 h with PMA, ionomycin, or with both (PMA/iono). Numbers in dot plots indicate percentage of TNF-α + and/or IFN-γ + T cells. Histogram depicts IL-2 production of the same sample. ( B and C ) T cells were pretreated or not with indicated inhibitor before activation with PMA/ionomycin ( B ), or with OVA peptide ( C ). Representative of three independently performed experiments. ( D – F ) TNF-α ( D ), IFN-γ ( E ), and IL-2 ( F ) protein levels ( Left ) and mRNA levels ( Right ) of CD44 hi memory-like T cells were determined after 4 h and 2 h of indicated stimulation, respectively ( n = 4 mice) (one-way ANOVA with Tukey’s comparison; * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: Role of PKC and Ca 2+ -dependent signaling in cytokine production. ( A ) Resting T cells were stimulated or not for 4 h with PMA, ionomycin, or with both (PMA/iono). Numbers in dot plots indicate percentage of TNF-α + and/or IFN-γ + T cells. Histogram depicts IL-2 production of the same sample. ( B and C ) T cells were pretreated or not with indicated inhibitor before activation with PMA/ionomycin ( B ), or with OVA peptide ( C ). Representative of three independently performed experiments. ( D – F ) TNF-α ( D ), IFN-γ ( E ), and IL-2 ( F ) protein levels ( Left ) and mRNA levels ( Right ) of CD44 hi memory-like T cells were determined after 4 h and 2 h of indicated stimulation, respectively ( n = 4 mice) (one-way ANOVA with Tukey’s comparison; * P

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Activation Assay, Mouse Assay

    Signal strength determines cytokine specific pathway for production. ( A – C ) TNF-α ( A ), IFN-γ ( B ), and IL-2 ( C ) protein production upon 6 h of stimulation with 0.1 nM (low), 1 nM (intermediate), or 100 nM (high) OVA peptide. Representative of four independently performed experiments ( n = 6 mice). ( D – F , Left ) Tnfa ( D ), Ifng ( E ), and Il2 ( F ) mRNA levels after 2 h of stimulation. Mean ± SD of three ( D and F ) and four ( E ) independently performed experiments [ n = 5 ( D and F ); n = 6 ( E ) mice] (one-way ANOVA with Tukey’s comparison; * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells

    doi: 10.1073/pnas.1704227114

    Figure Lengend Snippet: Signal strength determines cytokine specific pathway for production. ( A – C ) TNF-α ( A ), IFN-γ ( B ), and IL-2 ( C ) protein production upon 6 h of stimulation with 0.1 nM (low), 1 nM (intermediate), or 100 nM (high) OVA peptide. Representative of four independently performed experiments ( n = 6 mice). ( D – F , Left ) Tnfa ( D ), Ifng ( E ), and Il2 ( F ) mRNA levels after 2 h of stimulation. Mean ± SD of three ( D and F ) and four ( E ) independently performed experiments [ n = 5 ( D and F ); n = 6 ( E ) mice] (one-way ANOVA with Tukey’s comparison; * P

    Article Snippet: Intracellular cytokine staining was performed with BD Cytofix/Cytoperm kit (BD Biosciences). pmTOR and pS6 was measured upon 4% PFA fixation and methanol permeabilization.

    Techniques: Mouse Assay

    Influence of Pro-Th1 (IL-2 and IL-12) and Pro-Th2 (IL-4) cytokines on the proliferation and IFN-γ secretion by Th1 cells stimulated with anti-CD3 Ab and B7-1. pGL-10 Th1 cells were cultured with anti-CD3 Ab (0.01 μg/ml) in the presence of CHO-B7-1 (5 × 10 4 /well). Cytokines IL-2, IL-4 or IL-12 alone or in combinations were also added into the cultures. For proliferation (Fig. 5a), cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and were harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells. IFN-γ (Fig. 5b) was estimated by ELISA in the pooled culture supernatants collected from the triplicate wells after 48 h of the initiation of cultures. The control cultures comprising of Th1 cells, CHO-B7-1+Th1 cells, CHO+Th1 cells, CHO-B7-1, CHO showed no noticeable change. The data presented are from three independent experiments. '*' Represents p

    Journal: BMC Immunology

    Article Title: Regulatory role of pro-Th1 and pro-Th2 cytokines in modulating the activity of Th1 and Th2 cells when B cell and macrophages are used as antigen presenting cells

    doi: 10.1186/1471-2172-7-17

    Figure Lengend Snippet: Influence of Pro-Th1 (IL-2 and IL-12) and Pro-Th2 (IL-4) cytokines on the proliferation and IFN-γ secretion by Th1 cells stimulated with anti-CD3 Ab and B7-1. pGL-10 Th1 cells were cultured with anti-CD3 Ab (0.01 μg/ml) in the presence of CHO-B7-1 (5 × 10 4 /well). Cytokines IL-2, IL-4 or IL-12 alone or in combinations were also added into the cultures. For proliferation (Fig. 5a), cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and were harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells. IFN-γ (Fig. 5b) was estimated by ELISA in the pooled culture supernatants collected from the triplicate wells after 48 h of the initiation of cultures. The control cultures comprising of Th1 cells, CHO-B7-1+Th1 cells, CHO+Th1 cells, CHO-B7-1, CHO showed no noticeable change. The data presented are from three independent experiments. '*' Represents p

    Article Snippet: Estimation of cytokines The cytokines were estimated as per manufacturer instructions (Pharmingen) and expressed as pg/ml using recombinant cytokines as a standard (Pharmingen).

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

    Influence of Pro-Th1 (IL-2, IFN-γ) and Pro-Th2 (IL-1, IL-4, IL-5) cytokines on the proliferation and IL-4 and IL-5 secretion by Th2 cells when B cells, splenic macrophages and peritoneal macrophages were used as APC. D10G4.1 Th2 cells were cultured with B cells, splenic and peritoneal macrophages and conalbumin (100 μg/ml). Cytokines IL-1, IL-2, IL-4 or IL-5 and IFN-γ were also added in the cultures. Cultures with peritoneal macrophages also received 1 mM aminoguanidine. For proliferation, the cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells (Fig. 2a). For IL-4 (Fig. 2b) and IL-5 (Fig. 2c), the supernatants were collected from the triplicate wells after 48 h of the initiation of cultures, pooled and estimated by ELISA. The data are expressed as pg/ml. The control cultures comprising Th2 cells, Th2 cell+Ag, APCs+Ag, APCs+Th2 cells showed no apparent change. The data presented are from three independent experiments. '*' Represents p

    Journal: BMC Immunology

    Article Title: Regulatory role of pro-Th1 and pro-Th2 cytokines in modulating the activity of Th1 and Th2 cells when B cell and macrophages are used as antigen presenting cells

    doi: 10.1186/1471-2172-7-17

    Figure Lengend Snippet: Influence of Pro-Th1 (IL-2, IFN-γ) and Pro-Th2 (IL-1, IL-4, IL-5) cytokines on the proliferation and IL-4 and IL-5 secretion by Th2 cells when B cells, splenic macrophages and peritoneal macrophages were used as APC. D10G4.1 Th2 cells were cultured with B cells, splenic and peritoneal macrophages and conalbumin (100 μg/ml). Cytokines IL-1, IL-2, IL-4 or IL-5 and IFN-γ were also added in the cultures. Cultures with peritoneal macrophages also received 1 mM aminoguanidine. For proliferation, the cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells (Fig. 2a). For IL-4 (Fig. 2b) and IL-5 (Fig. 2c), the supernatants were collected from the triplicate wells after 48 h of the initiation of cultures, pooled and estimated by ELISA. The data are expressed as pg/ml. The control cultures comprising Th2 cells, Th2 cell+Ag, APCs+Ag, APCs+Th2 cells showed no apparent change. The data presented are from three independent experiments. '*' Represents p

    Article Snippet: Estimation of cytokines The cytokines were estimated as per manufacturer instructions (Pharmingen) and expressed as pg/ml using recombinant cytokines as a standard (Pharmingen).

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

    Influence of Pro-Th1 (IL-2 and IFN-γ) and Pro-Th2 (IL-4) cytokines on the proliferation and IL-4 and IL-5 secretion by Th2 cells stimulated with anti-CD3 Ab and B7-1. D10G4.1 Th2 cells were cultured with anti-CD3 Ab (0.01 μg/ml) in the presence of CHO-B7-1 (5 × 10 4 /well). Cytokines IL-2, IL-4 or IFN-γ alone or in combinations were also added in to the cultures. For the proliferation (Fig. 6a), cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and were harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells. IL-4 (Fig. 6b) and IL-5 (Fig. 6c) were estimated by ELISA in the pooled supernatants collected from the triplicate wells after 48 h of the initiation of the cultures. The control cultures comprising Th2 cells, CHO-B7-1+Th2 cells, CHO+Th2 cells, CHO-B7-1, CHO showed no considerable change except that IL-5 secretion was 1995 pg/ml when D10G4.1 Th2 cells were incubated with CHO-B7.1 in the absence of anti-CD3 Ab. The data presented are from three independent experiments. '*' Represents p

    Journal: BMC Immunology

    Article Title: Regulatory role of pro-Th1 and pro-Th2 cytokines in modulating the activity of Th1 and Th2 cells when B cell and macrophages are used as antigen presenting cells

    doi: 10.1186/1471-2172-7-17

    Figure Lengend Snippet: Influence of Pro-Th1 (IL-2 and IFN-γ) and Pro-Th2 (IL-4) cytokines on the proliferation and IL-4 and IL-5 secretion by Th2 cells stimulated with anti-CD3 Ab and B7-1. D10G4.1 Th2 cells were cultured with anti-CD3 Ab (0.01 μg/ml) in the presence of CHO-B7-1 (5 × 10 4 /well). Cytokines IL-2, IL-4 or IFN-γ alone or in combinations were also added in to the cultures. For the proliferation (Fig. 6a), cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and were harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells. IL-4 (Fig. 6b) and IL-5 (Fig. 6c) were estimated by ELISA in the pooled supernatants collected from the triplicate wells after 48 h of the initiation of the cultures. The control cultures comprising Th2 cells, CHO-B7-1+Th2 cells, CHO+Th2 cells, CHO-B7-1, CHO showed no considerable change except that IL-5 secretion was 1995 pg/ml when D10G4.1 Th2 cells were incubated with CHO-B7.1 in the absence of anti-CD3 Ab. The data presented are from three independent experiments. '*' Represents p

    Article Snippet: Estimation of cytokines The cytokines were estimated as per manufacturer instructions (Pharmingen) and expressed as pg/ml using recombinant cytokines as a standard (Pharmingen).

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

    Influence of Pro-Th1 (IL-2, IL-12, IFN-γ) and Pro-Th2 (IL-4) cytokines on the proliferation and IFN-γ secretion when B cells, splenic macrophages and peritoneal macrophages were used as APCs. pGL-10 Th1 cells were cultured with B cells, splenic and peritoneal macrophages and ovalbumin (200 μg/ml). Cytokines IL-2, IL-4, IL-12 and IFN-γ were also added in the cultures. For proliferation, the cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells (Fig. 1a). For IFN-γ, the supernatants were collected from the triplicate wells after 48 h of the initiation of cultures, pooled and estimated by ELISA. (Fig. 1b). The control cultures comprising of Th1 cells, Th1 cell+Ag, APCs+Ag, APCs+Th1 cells showed no discernible change. The data presented are from three independent experiments. '*' Represents p

    Journal: BMC Immunology

    Article Title: Regulatory role of pro-Th1 and pro-Th2 cytokines in modulating the activity of Th1 and Th2 cells when B cell and macrophages are used as antigen presenting cells

    doi: 10.1186/1471-2172-7-17

    Figure Lengend Snippet: Influence of Pro-Th1 (IL-2, IL-12, IFN-γ) and Pro-Th2 (IL-4) cytokines on the proliferation and IFN-γ secretion when B cells, splenic macrophages and peritoneal macrophages were used as APCs. pGL-10 Th1 cells were cultured with B cells, splenic and peritoneal macrophages and ovalbumin (200 μg/ml). Cytokines IL-2, IL-4, IL-12 and IFN-γ were also added in the cultures. For proliferation, the cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells (Fig. 1a). For IFN-γ, the supernatants were collected from the triplicate wells after 48 h of the initiation of cultures, pooled and estimated by ELISA. (Fig. 1b). The control cultures comprising of Th1 cells, Th1 cell+Ag, APCs+Ag, APCs+Th1 cells showed no discernible change. The data presented are from three independent experiments. '*' Represents p

    Article Snippet: Estimation of cytokines The cytokines were estimated as per manufacturer instructions (Pharmingen) and expressed as pg/ml using recombinant cytokines as a standard (Pharmingen).

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

    Flt3L potentiates both soluble and targeted vaccine priming and improves polyclonal CD8 + T cells responses and humoral immunity to OVA cross-presentation . Vaccine priming with αCD205-gag-p24 and soluble p24 at high (5 µg) and low (0.5 µg) doses in Flt3L-treated (red) versus PBS-treated (blue) mice. (A) Intracellular cytokine stain. (B) Proliferation of CD4 + T cells measured by CFSE-diluted IFN-γ + cells. One representative experiment ( n = 3 mice per group). Error bars show mean ± SEM. ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (C) CD8 + IFN-γ + intracellular cytokine staining from individual mice vaccinated with polyIC(LC) + 5 µg αCD205 OVA intraperitoneal route in spleen, lung, and lamina propria ( n = 4–6 mice pooled from 2 independent experiments). CD8 + IFN-γ + CFSE low/divided T cells at 96 h. One of two representative experiments ( n = 5). Error bars show standard error of the mean. CD8 + IFN-γ + intracellular cytokine staining. OVA serum total IgG, mean ELISA OD 450 . Error bars show mean ± SEM across 5 individual mice. ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Journal: The Journal of Experimental Medicine

    Article Title: Classical Flt3L-dependent dendritic cells control immunity to protein vaccine

    doi: 10.1084/jem.20131397

    Figure Lengend Snippet: Flt3L potentiates both soluble and targeted vaccine priming and improves polyclonal CD8 + T cells responses and humoral immunity to OVA cross-presentation . Vaccine priming with αCD205-gag-p24 and soluble p24 at high (5 µg) and low (0.5 µg) doses in Flt3L-treated (red) versus PBS-treated (blue) mice. (A) Intracellular cytokine stain. (B) Proliferation of CD4 + T cells measured by CFSE-diluted IFN-γ + cells. One representative experiment ( n = 3 mice per group). Error bars show mean ± SEM. ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (C) CD8 + IFN-γ + intracellular cytokine staining from individual mice vaccinated with polyIC(LC) + 5 µg αCD205 OVA intraperitoneal route in spleen, lung, and lamina propria ( n = 4–6 mice pooled from 2 independent experiments). CD8 + IFN-γ + CFSE low/divided T cells at 96 h. One of two representative experiments ( n = 5). Error bars show standard error of the mean. CD8 + IFN-γ + intracellular cytokine staining. OVA serum total IgG, mean ELISA OD 450 . Error bars show mean ± SEM across 5 individual mice. ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Article Snippet: IFN-γ staining was performed after cell surface label of T cell markers by intracellular cytokine staining using Fix/Perm and Perm/Wash buffers (BD).

    Techniques: Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay

    Blockade of migratory DCs does not impair immunity after s.c. or i.d. protein immunization. (A) Representative gating of inguinal LN taken from CCR7 −/− and Flt3L-treated and untreated vaccine mice. (B) CCR7 −/− mice versus B6 controls were immunized with 5 or 0.5 µg of αCD205 gag-p24 in separate experiments ( n = 3 mice per group). Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Red, Flt3L treated; blue, PBS treated. Intracellular cytokine staining for spleen, LN, and lung parenchyma and CFSE dilution of splenocytes after 4 d in culture with p24. (C) i.d. immunization with 0.5 µg αCD205 gag-p24: proximal draining LN and spleen intracellular cytokine staining and CFSE dilution of splenocytes after 4 d in culture with p24. Pooled from 3 independent experiments of n = 5 mice per group. Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D) CD8 + CFSE divided IFN-γ T cells after DEC-OVA SQ immunization. Pooled from 2 experiments, n = 4–5 mice per group. Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (E) Mixed bone marrow chimera after SQ immunization with 0.5 µg αCD205 gag-p24 in the presence or absence of DT, CFSE dilution shown. Pooled from 2 independent experiments with n = 4–5 mice per group for CCR7 + ZDTR chimeras compared plus and minus DT; n = 1 experiment for 5 mice per group for CCR7 + CD45.1 plus and minus DT controls. Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Journal: The Journal of Experimental Medicine

    Article Title: Classical Flt3L-dependent dendritic cells control immunity to protein vaccine

    doi: 10.1084/jem.20131397

    Figure Lengend Snippet: Blockade of migratory DCs does not impair immunity after s.c. or i.d. protein immunization. (A) Representative gating of inguinal LN taken from CCR7 −/− and Flt3L-treated and untreated vaccine mice. (B) CCR7 −/− mice versus B6 controls were immunized with 5 or 0.5 µg of αCD205 gag-p24 in separate experiments ( n = 3 mice per group). Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Red, Flt3L treated; blue, PBS treated. Intracellular cytokine staining for spleen, LN, and lung parenchyma and CFSE dilution of splenocytes after 4 d in culture with p24. (C) i.d. immunization with 0.5 µg αCD205 gag-p24: proximal draining LN and spleen intracellular cytokine staining and CFSE dilution of splenocytes after 4 d in culture with p24. Pooled from 3 independent experiments of n = 5 mice per group. Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D) CD8 + CFSE divided IFN-γ T cells after DEC-OVA SQ immunization. Pooled from 2 experiments, n = 4–5 mice per group. Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (E) Mixed bone marrow chimera after SQ immunization with 0.5 µg αCD205 gag-p24 in the presence or absence of DT, CFSE dilution shown. Pooled from 2 independent experiments with n = 4–5 mice per group for CCR7 + ZDTR chimeras compared plus and minus DT; n = 1 experiment for 5 mice per group for CCR7 + CD45.1 plus and minus DT controls. Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Article Snippet: IFN-γ staining was performed after cell surface label of T cell markers by intracellular cytokine staining using Fix/Perm and Perm/Wash buffers (BD).

    Techniques: Mouse Assay, Staining

    Langerin + DC, including LCs and CD103 + DC, are not required for CD4 s.c. protein immunization. (A) Deletion of Langerin + migratory DC subsets after a single dose (1 µg) DT administered 24 h before harvest. (B) Deletion of Langerin + subsets in Flt3L-treated Langerin DTR mice administered Flt3L daily. DT was administered −3 and −1 d before harvest at day 8. (C) Schema: 1 µg DT was administered to Langerin DTR mice versus WT controls day −3 or −1 to s.c. vaccine prime or boost with GLA plus 5 or 0.5 µg αCD205 gag-p24. (D) Spleen, (E) LN, (F) Lung- intracellular cytokine staining. (G) CD4 + IFN-γ + CFSE divided cells in Langerin DTR versus C57BL/6 WT mice in Flt3L-treated (red Flt3L treated, 1 representative experiment at 5 µg, or pooled from 2–3 independent experiments at 0.5 µg) or PBS-treated controls (blue, pooled from 2–3 independent experiments at 5 or 0.5μγ; ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (H) Serum HIV gag-p24 IgG titers in Langerin DTR versus WT mice treated with Flt3L versus PBS (one representative experiment of three ( n = 5 mice), *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001). (I–K) 1 µg DT was administered to Langerin DTR mice versus WT controls day −3 or −1 to vaccine prime or boost with s.c. polyIC(LC) plus 0.5 µg αCD205 OVA. In vitro challenge of OVA versus control peptide. (I) Spleen, (J) LN intracellular cytokine staining, and (K) CD8 + IFN-γ + CFSE divided cells ( n = 4–7 mice total per group pooled from 2 independent experiments). (L) Capture of αCD205 by classical CD8α + LN cDCs 3 h after footpad injection (7.5 µg total) is improved by Flt3L but not altered after Langerin + DC ablation. 1 µg of DT was administered at −3 and −1 d, with injection and harvest on day 0, after 9–10 d of PBS or Flt3L treatment. (left) WT (green), Langerin-DTR mice (red). (right) % αCD205uptake by cDCs and migDCs in the popliteal LNs. Pooled from two independent experiments ( n = 5–6 mice total; ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

    Journal: The Journal of Experimental Medicine

    Article Title: Classical Flt3L-dependent dendritic cells control immunity to protein vaccine

    doi: 10.1084/jem.20131397

    Figure Lengend Snippet: Langerin + DC, including LCs and CD103 + DC, are not required for CD4 s.c. protein immunization. (A) Deletion of Langerin + migratory DC subsets after a single dose (1 µg) DT administered 24 h before harvest. (B) Deletion of Langerin + subsets in Flt3L-treated Langerin DTR mice administered Flt3L daily. DT was administered −3 and −1 d before harvest at day 8. (C) Schema: 1 µg DT was administered to Langerin DTR mice versus WT controls day −3 or −1 to s.c. vaccine prime or boost with GLA plus 5 or 0.5 µg αCD205 gag-p24. (D) Spleen, (E) LN, (F) Lung- intracellular cytokine staining. (G) CD4 + IFN-γ + CFSE divided cells in Langerin DTR versus C57BL/6 WT mice in Flt3L-treated (red Flt3L treated, 1 representative experiment at 5 µg, or pooled from 2–3 independent experiments at 0.5 µg) or PBS-treated controls (blue, pooled from 2–3 independent experiments at 5 or 0.5μγ; ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (H) Serum HIV gag-p24 IgG titers in Langerin DTR versus WT mice treated with Flt3L versus PBS (one representative experiment of three ( n = 5 mice), *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001). (I–K) 1 µg DT was administered to Langerin DTR mice versus WT controls day −3 or −1 to vaccine prime or boost with s.c. polyIC(LC) plus 0.5 µg αCD205 OVA. In vitro challenge of OVA versus control peptide. (I) Spleen, (J) LN intracellular cytokine staining, and (K) CD8 + IFN-γ + CFSE divided cells ( n = 4–7 mice total per group pooled from 2 independent experiments). (L) Capture of αCD205 by classical CD8α + LN cDCs 3 h after footpad injection (7.5 µg total) is improved by Flt3L but not altered after Langerin + DC ablation. 1 µg of DT was administered at −3 and −1 d, with injection and harvest on day 0, after 9–10 d of PBS or Flt3L treatment. (left) WT (green), Langerin-DTR mice (red). (right) % αCD205uptake by cDCs and migDCs in the popliteal LNs. Pooled from two independent experiments ( n = 5–6 mice total; ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

    Article Snippet: IFN-γ staining was performed after cell surface label of T cell markers by intracellular cytokine staining using Fix/Perm and Perm/Wash buffers (BD).

    Techniques: Mouse Assay, Staining, In Vitro, Injection

    s.c. immunity is Flt3L dependent. (A) Serum from n = 3–5 mice taken before (0 h) or at 1, 3, 6, 24, or 48 h after s.c. immunization with 0.5 µg of αCD205 gag-p24 and GLA (blue) or polyIC(LC) (red). Error bars show mean ± SD. (B) CD4 + IFN-γ + intracellular cytokine staining from 3 pooled experiments of 4–5 individual WT versus Flt3 −/− mice after s.c. vaccination with GLA + 0.5 µg αCD205 gag-p24. Open, filled, and patterned symbols depict individual experiments. Error bars show mean ± SEM (P ≤ 001). (C) Schematic of vaccine immunization schedule and Flt3L versus PBS treatment. (D) CD4 + T cell immunity at lymphoid and mucosal sites and humoral immunity after protein immunization to HIV-gag with multiple adjuvants: GLA (D) and polyIC(LC) (E). CD4 + IFN-γ + intracellular cytokine staining from splenocytes of individual mice s.c. vaccinated with adjuvant + 5 µg αCD205 gag-p24 in spleen, LN, lamina propria, and lung. CD4 + IFN-γ + + CFSE divided T cells after 96 h from individual immunized mice depicted in spleen. Circles represent individual mice, open versus filled (pooled from 2 independent experiments, 3–5 mice per group). Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Gag-p24 serum total IgG in Flt3L-treated versus untreated mice, mean ELISA OD 450 , unimmunized controls. Error bars show mean ± SEM across 5 individual mice from 1 representative experiment. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Journal: The Journal of Experimental Medicine

    Article Title: Classical Flt3L-dependent dendritic cells control immunity to protein vaccine

    doi: 10.1084/jem.20131397

    Figure Lengend Snippet: s.c. immunity is Flt3L dependent. (A) Serum from n = 3–5 mice taken before (0 h) or at 1, 3, 6, 24, or 48 h after s.c. immunization with 0.5 µg of αCD205 gag-p24 and GLA (blue) or polyIC(LC) (red). Error bars show mean ± SD. (B) CD4 + IFN-γ + intracellular cytokine staining from 3 pooled experiments of 4–5 individual WT versus Flt3 −/− mice after s.c. vaccination with GLA + 0.5 µg αCD205 gag-p24. Open, filled, and patterned symbols depict individual experiments. Error bars show mean ± SEM (P ≤ 001). (C) Schematic of vaccine immunization schedule and Flt3L versus PBS treatment. (D) CD4 + T cell immunity at lymphoid and mucosal sites and humoral immunity after protein immunization to HIV-gag with multiple adjuvants: GLA (D) and polyIC(LC) (E). CD4 + IFN-γ + intracellular cytokine staining from splenocytes of individual mice s.c. vaccinated with adjuvant + 5 µg αCD205 gag-p24 in spleen, LN, lamina propria, and lung. CD4 + IFN-γ + + CFSE divided T cells after 96 h from individual immunized mice depicted in spleen. Circles represent individual mice, open versus filled (pooled from 2 independent experiments, 3–5 mice per group). Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Gag-p24 serum total IgG in Flt3L-treated versus untreated mice, mean ELISA OD 450 , unimmunized controls. Error bars show mean ± SEM across 5 individual mice from 1 representative experiment. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Article Snippet: IFN-γ staining was performed after cell surface label of T cell markers by intracellular cytokine staining using Fix/Perm and Perm/Wash buffers (BD).

    Techniques: Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay

    ZBTB46-dependent classical DCs are required for s.c. protein immunization. (A–C) CD45.2 mice were irradiated and reconstituted with bone marrow as indicated. Chimeras received DT on day −3 and −1 to prime and boost immunization with 0.5 µg αCD205 gag-p24. Intracellular cytokine staining immediately ex vivo for (A) Spleen and (B) LN. Black shows staining for p17 peptide control (plus DT) versus red or blue (plus DT) p24 peptide challenge. (C) CFSE dilution of splenocytes after 4 d in culture. (A–C) Pooled from 4–5 independent experiments ( n = 3–5 mice per group, based on survival after DT administration). No DT controls (blue) performed once across all 4 groups ( n = 4–5 mice per group). Error bars show mean ± SEM. ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D–F) CD11b DTR versus B6 control mice were administered DT on day −3 and −1 to prime-boost immunization with 0.5 µg αCD205 gag-p24. CFSE dilution of splenocytes after 4 d in culture (D) and intracellular cytokine stainings (E–F). Black shows staining for p17 peptide control (plus DT), red or blue (plus DT) show p24 challenge in wild-type and controls. One representative experiment of n = 4–5 mice per group. Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Journal: The Journal of Experimental Medicine

    Article Title: Classical Flt3L-dependent dendritic cells control immunity to protein vaccine

    doi: 10.1084/jem.20131397

    Figure Lengend Snippet: ZBTB46-dependent classical DCs are required for s.c. protein immunization. (A–C) CD45.2 mice were irradiated and reconstituted with bone marrow as indicated. Chimeras received DT on day −3 and −1 to prime and boost immunization with 0.5 µg αCD205 gag-p24. Intracellular cytokine staining immediately ex vivo for (A) Spleen and (B) LN. Black shows staining for p17 peptide control (plus DT) versus red or blue (plus DT) p24 peptide challenge. (C) CFSE dilution of splenocytes after 4 d in culture. (A–C) Pooled from 4–5 independent experiments ( n = 3–5 mice per group, based on survival after DT administration). No DT controls (blue) performed once across all 4 groups ( n = 4–5 mice per group). Error bars show mean ± SEM. ^, P ≤ 0.1; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D–F) CD11b DTR versus B6 control mice were administered DT on day −3 and −1 to prime-boost immunization with 0.5 µg αCD205 gag-p24. CFSE dilution of splenocytes after 4 d in culture (D) and intracellular cytokine stainings (E–F). Black shows staining for p17 peptide control (plus DT), red or blue (plus DT) show p24 challenge in wild-type and controls. One representative experiment of n = 4–5 mice per group. Error bars show mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Article Snippet: IFN-γ staining was performed after cell surface label of T cell markers by intracellular cytokine staining using Fix/Perm and Perm/Wash buffers (BD).

    Techniques: Mouse Assay, Irradiation, Staining, Ex Vivo

    Reduction in IL-4 availability by Il4 gene hemizygosity has a partial effect on the development of IL-4AC- and IL-4AC/IL-13DR-producing T cell subsets. 4C13R-IL-4 +/+ , 4C13R-IL-4 +/− , and 4C13R-IL-4 −/− mice were treated with 200 μg house dust mite i.d. in the ear pinnae. Ear draining lymph nodes and ear tissue were harvested from the mice 7 days posttreatment. (A) Lymph nodes were analyzed to determine the number of IL-4AC + T follicular helper (Tfh) and IL-4AC + Th2 CD4 T cells. (B) Ear tissue was analyzed to determine the number of reporter + Th2 CD4 + T cells. (A) Data pooled from three experiments ( n = 18) for ear lymph node and (B) three experiments ( n = 15) for ear tissue. Data show mean + SEM (* p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 two-tailed t -test).

    Journal: Frontiers in Immunology

    Article Title: IL-4 Is a Key Requirement for IL-4- and IL-4/IL-13-Expressing CD4 Th2 Subsets in Lung and Skin

    doi: 10.3389/fimmu.2018.01211

    Figure Lengend Snippet: Reduction in IL-4 availability by Il4 gene hemizygosity has a partial effect on the development of IL-4AC- and IL-4AC/IL-13DR-producing T cell subsets. 4C13R-IL-4 +/+ , 4C13R-IL-4 +/− , and 4C13R-IL-4 −/− mice were treated with 200 μg house dust mite i.d. in the ear pinnae. Ear draining lymph nodes and ear tissue were harvested from the mice 7 days posttreatment. (A) Lymph nodes were analyzed to determine the number of IL-4AC + T follicular helper (Tfh) and IL-4AC + Th2 CD4 T cells. (B) Ear tissue was analyzed to determine the number of reporter + Th2 CD4 + T cells. (A) Data pooled from three experiments ( n = 18) for ear lymph node and (B) three experiments ( n = 15) for ear tissue. Data show mean + SEM (* p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 two-tailed t -test).

    Article Snippet: Kopf M, Le Gros G, Bachmann M, Lamers MC, Bluethmann H, Köhler G. Disruption of the murine IL-4 gene blocks Th2 cytokine responses .

    Techniques: Mouse Assay, Two Tailed Test

    IL-4 plays a role in the development of IL-4AC-expressing lymph node Th2 cells but not IL-4AC-expressing T follicular helper (Tfh) cells. 4C13R-IL-4 +/+ and 4C13R-IL-4 −/− mice were treated with 200 μg house dust mite (HDM) i.d. in the ear. The ear draining lymph nodes were harvested from mice 7 days posttreatment, and lymph nodes were analyzed by flow cytometry. (A) FACS plots showing the proportion of IL-4AC + CD4 T cells and then the proportion of CXCR5 + PD-1 + Tfh and CXCR5 lo PD-1 lo , CXCR5 + PD-1 lo , and CXCR5 lo PD-1 + Th2 cells within this population. (B) Proportion of IL-4AC + CD4 T cells that are Tfh or Th2 cells. (C) Numbers of IL-4AC + Tfh and IL-4AC + Th2 CD4 T cells and (D) median fluorescent intensity (MFI) of IL-4AC of IL-4AC + Tfh and IL-4AC + Th2 CD4 T cells. (A) FACS plots from a representative experiment ( n = 6). (B,C) Data pooled from three experiments ( n = 18). (D) Data from a representative experiment ( n = 6). Data show mean + SEM (* p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 two-tailed t -test).

    Journal: Frontiers in Immunology

    Article Title: IL-4 Is a Key Requirement for IL-4- and IL-4/IL-13-Expressing CD4 Th2 Subsets in Lung and Skin

    doi: 10.3389/fimmu.2018.01211

    Figure Lengend Snippet: IL-4 plays a role in the development of IL-4AC-expressing lymph node Th2 cells but not IL-4AC-expressing T follicular helper (Tfh) cells. 4C13R-IL-4 +/+ and 4C13R-IL-4 −/− mice were treated with 200 μg house dust mite (HDM) i.d. in the ear. The ear draining lymph nodes were harvested from mice 7 days posttreatment, and lymph nodes were analyzed by flow cytometry. (A) FACS plots showing the proportion of IL-4AC + CD4 T cells and then the proportion of CXCR5 + PD-1 + Tfh and CXCR5 lo PD-1 lo , CXCR5 + PD-1 lo , and CXCR5 lo PD-1 + Th2 cells within this population. (B) Proportion of IL-4AC + CD4 T cells that are Tfh or Th2 cells. (C) Numbers of IL-4AC + Tfh and IL-4AC + Th2 CD4 T cells and (D) median fluorescent intensity (MFI) of IL-4AC of IL-4AC + Tfh and IL-4AC + Th2 CD4 T cells. (A) FACS plots from a representative experiment ( n = 6). (B,C) Data pooled from three experiments ( n = 18). (D) Data from a representative experiment ( n = 6). Data show mean + SEM (* p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 two-tailed t -test).

    Article Snippet: Kopf M, Le Gros G, Bachmann M, Lamers MC, Bluethmann H, Köhler G. Disruption of the murine IL-4 gene blocks Th2 cytokine responses .

    Techniques: Expressing, Mouse Assay, Flow Cytometry, Cytometry, FACS, Two Tailed Test

    IL-4 is required for the development of IL-4AC- and IL-4AC/IL-13DR-expressing Th2 subsets but not the IL-13DR-expressing CD4 + T cell subset in ear tissue. 4C13R-IL-4 +/+ and 4C13R-IL-4 −/− mice were treated with either (A–E) 200 μg house dust mite (HDM) or (F–J) 600 dead L3 Nippostrongylus braziliensis ( Nb ) i.d. in the ear pinnae. Ear tissue was harvested 7 days posttreatment. Tissues were analyzed to determine (A,B,F,G) proportion of IL-4AC + and IL-13DR + CD4 T cells, (C,H) number of IL-4AC + and IL-13DR + CD4 T cells, and (D,E,I,J) median fluorescent intensity (MFI) of IL-4AC and IL-13DR for each of the Th2 cell subsets. Data representative of seven experiments ( n = 35–37) for HDM and two experiments ( n = 6) for dead Nb . (A) FACS plots concatenated from a single representative experiment ( n = 6). (B,C) Data pooled from seven experiments ( n = 37). (D,E) Data from a single representative experiment ( n = 6). (F) FACS plots concatenated from a single representative experiment ( n = 3). (G,H) Data pooled from two experiments ( n = 6). (I,J) Data from a single representative experiment ( n = 3). Data show mean + SEM (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001 two-tailed t -test).

    Journal: Frontiers in Immunology

    Article Title: IL-4 Is a Key Requirement for IL-4- and IL-4/IL-13-Expressing CD4 Th2 Subsets in Lung and Skin

    doi: 10.3389/fimmu.2018.01211

    Figure Lengend Snippet: IL-4 is required for the development of IL-4AC- and IL-4AC/IL-13DR-expressing Th2 subsets but not the IL-13DR-expressing CD4 + T cell subset in ear tissue. 4C13R-IL-4 +/+ and 4C13R-IL-4 −/− mice were treated with either (A–E) 200 μg house dust mite (HDM) or (F–J) 600 dead L3 Nippostrongylus braziliensis ( Nb ) i.d. in the ear pinnae. Ear tissue was harvested 7 days posttreatment. Tissues were analyzed to determine (A,B,F,G) proportion of IL-4AC + and IL-13DR + CD4 T cells, (C,H) number of IL-4AC + and IL-13DR + CD4 T cells, and (D,E,I,J) median fluorescent intensity (MFI) of IL-4AC and IL-13DR for each of the Th2 cell subsets. Data representative of seven experiments ( n = 35–37) for HDM and two experiments ( n = 6) for dead Nb . (A) FACS plots concatenated from a single representative experiment ( n = 6). (B,C) Data pooled from seven experiments ( n = 37). (D,E) Data from a single representative experiment ( n = 6). (F) FACS plots concatenated from a single representative experiment ( n = 3). (G,H) Data pooled from two experiments ( n = 6). (I,J) Data from a single representative experiment ( n = 3). Data show mean + SEM (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001 two-tailed t -test).

    Article Snippet: Kopf M, Le Gros G, Bachmann M, Lamers MC, Bluethmann H, Köhler G. Disruption of the murine IL-4 gene blocks Th2 cytokine responses .

    Techniques: Expressing, Mouse Assay, FACS, Two Tailed Test

    IL-4- and IL-13-expressing Th2 subsets in the lymph node and ear tissue after allergen priming. Following house dust mite (HDM) priming in the ear skin, dendritic cells take up allergen and transport it to the ear draining lymph node where they present it to naïve CD4 T cells. Naïve CD4 T cells differentiate into either (i) IL-4-expressing Th2 cells in a process that is partially dependent on IL-4 or (ii) IL-4 independent IL-4-expressing T follicular helper (Tfh) cells. IL-4 single-positive CD4 T cell and IL-4/IL-13 double-positive-expressing CD4 T cell subsets in the skin tissue are very dependent on IL-4 while IL-13 single-positive-expressing Th2 cells are independent of IL-4. The broken line represent the as of yet unproven but potential link between the lymph node Th2 cells and those in the tissue.

    Journal: Frontiers in Immunology

    Article Title: IL-4 Is a Key Requirement for IL-4- and IL-4/IL-13-Expressing CD4 Th2 Subsets in Lung and Skin

    doi: 10.3389/fimmu.2018.01211

    Figure Lengend Snippet: IL-4- and IL-13-expressing Th2 subsets in the lymph node and ear tissue after allergen priming. Following house dust mite (HDM) priming in the ear skin, dendritic cells take up allergen and transport it to the ear draining lymph node where they present it to naïve CD4 T cells. Naïve CD4 T cells differentiate into either (i) IL-4-expressing Th2 cells in a process that is partially dependent on IL-4 or (ii) IL-4 independent IL-4-expressing T follicular helper (Tfh) cells. IL-4 single-positive CD4 T cell and IL-4/IL-13 double-positive-expressing CD4 T cell subsets in the skin tissue are very dependent on IL-4 while IL-13 single-positive-expressing Th2 cells are independent of IL-4. The broken line represent the as of yet unproven but potential link between the lymph node Th2 cells and those in the tissue.

    Article Snippet: Kopf M, Le Gros G, Bachmann M, Lamers MC, Bluethmann H, Köhler G. Disruption of the murine IL-4 gene blocks Th2 cytokine responses .

    Techniques: Expressing

    Tiotropium treatment of CS-exposed and H1N1-infected mice. (A) CS-exposed and H1N1-infected mice (black bars) were treated for a total of 10 days with 0.1 mg/ml or 0.3 mg/ml nebulized tiotropium (gray bars). White bars show the results from untreated negative control (NC) animals. (B) Body weight loss in NC mice (black dotted line), untreated CS-exposed, and H1N1-infected mice (red line); CS-exposed and H1N1-infected mice treated with 0.1 mg/ml (light green line); or 0.3 mg/ml (dark green line) nebulized tiotropium. (C) Lung function, resistance, compliance, and (D) cytokine levels in lung homogenate and (E) total cell, neutrophil, and macrophage numbers in BAL fluid. Mean values ± S.E.M. of n = 7 or 8 animals in the HIN1- and CS-exposed mice groups and n = 4 in the NC group. **** P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Tiotropium Attenuates Virus-Induced Pulmonary Inflammation in Cigarette Smoke–Exposed Mice

    doi: 10.1124/jpet.116.232009

    Figure Lengend Snippet: Tiotropium treatment of CS-exposed and H1N1-infected mice. (A) CS-exposed and H1N1-infected mice (black bars) were treated for a total of 10 days with 0.1 mg/ml or 0.3 mg/ml nebulized tiotropium (gray bars). White bars show the results from untreated negative control (NC) animals. (B) Body weight loss in NC mice (black dotted line), untreated CS-exposed, and H1N1-infected mice (red line); CS-exposed and H1N1-infected mice treated with 0.1 mg/ml (light green line); or 0.3 mg/ml (dark green line) nebulized tiotropium. (C) Lung function, resistance, compliance, and (D) cytokine levels in lung homogenate and (E) total cell, neutrophil, and macrophage numbers in BAL fluid. Mean values ± S.E.M. of n = 7 or 8 animals in the HIN1- and CS-exposed mice groups and n = 4 in the NC group. **** P

    Article Snippet: Cytokine levels in lung homogenate were assessed using Meso Scale Discovery multiplex technology (Meso Scale Discovery, Gaithersburg, MD) according to the manufacturer’s instructions.

    Techniques: Infection, Mouse Assay, Negative Control

    Model for CS/H1N1 induced exacerbation of pulmonary inflammation in mice. (A) Mice were exposed to cigarette smoke for a total of 10 days (light gray bars). Additional mice were infected with only H1N1 on day 8 (dark gray bars) or CS-exposed and infected with H1N1 on day 8 (black bars). White bars show the results from untreated negative control (NC) animals. (B) Body weight loss in NC animals (black dotted line), CS-exposed (blue line), H1N1-infected (green line), or CS-exposed and H1N1-infected (red line) mice. (C) Lung function; (D) cytokine levels in lung homogenate; (E) total cell, neutrophils, and macrophage numbers in BAL fluid; and (F) viral load in lung homogenate. Mean values ± S.E.M. of n = 7 or 8 animals in the HIN1- and CS-exposed mice groups and n = 4 in the NC group are shown. **** P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Tiotropium Attenuates Virus-Induced Pulmonary Inflammation in Cigarette Smoke–Exposed Mice

    doi: 10.1124/jpet.116.232009

    Figure Lengend Snippet: Model for CS/H1N1 induced exacerbation of pulmonary inflammation in mice. (A) Mice were exposed to cigarette smoke for a total of 10 days (light gray bars). Additional mice were infected with only H1N1 on day 8 (dark gray bars) or CS-exposed and infected with H1N1 on day 8 (black bars). White bars show the results from untreated negative control (NC) animals. (B) Body weight loss in NC animals (black dotted line), CS-exposed (blue line), H1N1-infected (green line), or CS-exposed and H1N1-infected (red line) mice. (C) Lung function; (D) cytokine levels in lung homogenate; (E) total cell, neutrophils, and macrophage numbers in BAL fluid; and (F) viral load in lung homogenate. Mean values ± S.E.M. of n = 7 or 8 animals in the HIN1- and CS-exposed mice groups and n = 4 in the NC group are shown. **** P

    Article Snippet: Cytokine levels in lung homogenate were assessed using Meso Scale Discovery multiplex technology (Meso Scale Discovery, Gaithersburg, MD) according to the manufacturer’s instructions.

    Techniques: Mouse Assay, Infection, Negative Control

    Fluticasone treatment of CS-exposed and H1N1-infected mice. (A) CS-exposed and H1N1- infected mice (black bars) were treated with 0.3 mg/kg or 0.5 mg/kg fluticasone (gray bars). White bars show the results from untreated negative control (NC) animals. (B) Body weight loss in NC mice (black dotted line), untreated CS-exposed, and H1N1-infected mice (red line); CS-exposed and H1N1-infected mice treated with 0.3 mg/kg (light green line) or 0.5 mg/kg (dark green line) fluticasone. (C) Lung function, resistance, compliance; (D) cytokine levels in lung homogenate; (E) total cell, neutrophil, and macrophage numbers in BAL fluid. Mean values ± S.E.M. of n = 4–8 animals in the HIN1- and CS-exposed mice groups and n = 4 in the NC group. **** P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Tiotropium Attenuates Virus-Induced Pulmonary Inflammation in Cigarette Smoke–Exposed Mice

    doi: 10.1124/jpet.116.232009

    Figure Lengend Snippet: Fluticasone treatment of CS-exposed and H1N1-infected mice. (A) CS-exposed and H1N1- infected mice (black bars) were treated with 0.3 mg/kg or 0.5 mg/kg fluticasone (gray bars). White bars show the results from untreated negative control (NC) animals. (B) Body weight loss in NC mice (black dotted line), untreated CS-exposed, and H1N1-infected mice (red line); CS-exposed and H1N1-infected mice treated with 0.3 mg/kg (light green line) or 0.5 mg/kg (dark green line) fluticasone. (C) Lung function, resistance, compliance; (D) cytokine levels in lung homogenate; (E) total cell, neutrophil, and macrophage numbers in BAL fluid. Mean values ± S.E.M. of n = 4–8 animals in the HIN1- and CS-exposed mice groups and n = 4 in the NC group. **** P

    Article Snippet: Cytokine levels in lung homogenate were assessed using Meso Scale Discovery multiplex technology (Meso Scale Discovery, Gaithersburg, MD) according to the manufacturer’s instructions.

    Techniques: Infection, Mouse Assay, Negative Control

    Roflumilast treatment of CS-exposed and H1N1-infected mice. (A) CS-exposed and H1N1-infected mice (black bars) were treated with 0.2 mg/kg or 1.0 mg/kg roflumilast (gray bars). White bars show the results from untreated negative control (NC) animals. (B) Body weight loss in NC mice (black dotted line), untreated CS-exposed and H1N1-infected mice (red line), CS-exposed and H1N1-infected mice treated with 0.2 mg/kg (light green line), or 1.0 mg/kg (dark green line) roflumilast. (C) Lung function, resistance, compliance and (D) cytokine levels in lung homogenate (E) total cell, neutrophil, and macrophage numbers in BAL fluid. Mean values ± S.E.M. of n = 7 or 8 animals in the HIN1- and CS-exposed mice groups and n = 4 in the NC group. **** P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Tiotropium Attenuates Virus-Induced Pulmonary Inflammation in Cigarette Smoke–Exposed Mice

    doi: 10.1124/jpet.116.232009

    Figure Lengend Snippet: Roflumilast treatment of CS-exposed and H1N1-infected mice. (A) CS-exposed and H1N1-infected mice (black bars) were treated with 0.2 mg/kg or 1.0 mg/kg roflumilast (gray bars). White bars show the results from untreated negative control (NC) animals. (B) Body weight loss in NC mice (black dotted line), untreated CS-exposed and H1N1-infected mice (red line), CS-exposed and H1N1-infected mice treated with 0.2 mg/kg (light green line), or 1.0 mg/kg (dark green line) roflumilast. (C) Lung function, resistance, compliance and (D) cytokine levels in lung homogenate (E) total cell, neutrophil, and macrophage numbers in BAL fluid. Mean values ± S.E.M. of n = 7 or 8 animals in the HIN1- and CS-exposed mice groups and n = 4 in the NC group. **** P

    Article Snippet: Cytokine levels in lung homogenate were assessed using Meso Scale Discovery multiplex technology (Meso Scale Discovery, Gaithersburg, MD) according to the manufacturer’s instructions.

    Techniques: Infection, Mouse Assay, Negative Control

    Loss of tight-junctions by cytokine treatment and its rescue by trypsin inhibitor. A , Western blotting analysis of tight-junction proteins, zonula occludens (ZO) 1 and occludin after treatment of the cells with cytokines for 12 h in the absence and presence of 50 µM aprotinin. Actin as an internal control (Ctr). B , Representative example (from 3 separate experiments) of immunofluorescence showing decreased ZO-1 with cytokine treatment and its restoration by aprotinin. C , Increased permeability of the cells treated with cytokines and its rescue by aprotinin ( n =3). Data are mean value ± standard error of the mean. * P

    Journal: The Journal of Infectious Diseases

    Article Title: Influenza Virus—Cytokine-Protease Cycle in the Pathogenesis of Vascular Hyperpermeability in Severe Influenza

    doi: 10.1086/656044

    Figure Lengend Snippet: Loss of tight-junctions by cytokine treatment and its rescue by trypsin inhibitor. A , Western blotting analysis of tight-junction proteins, zonula occludens (ZO) 1 and occludin after treatment of the cells with cytokines for 12 h in the absence and presence of 50 µM aprotinin. Actin as an internal control (Ctr). B , Representative example (from 3 separate experiments) of immunofluorescence showing decreased ZO-1 with cytokine treatment and its restoration by aprotinin. C , Increased permeability of the cells treated with cytokines and its rescue by aprotinin ( n =3). Data are mean value ± standard error of the mean. * P

    Article Snippet: The cells were infected by influenza A virus WSN at amultiplicity of infection of 0.5 or treated with recombinant human IL-6, TNF-α, and IL-1β (10 ng/mL of each) (PeproTec) in the presence or absence of antibodies against these cytokines (Abcam).

    Techniques: Western Blot, Immunofluorescence, Permeability

    Effects of cytokines trypsin, and protease-activated receptor (PAR) 2 agonist on [Ca 2+ ] i and rescue of the increase in [Ca 2+ ] i by PAR-2 antagonist and aprotinin in endothelial cells. The cells were treated for 10 h with 1 µg/mL trypsin, 10 µM PAR-2 agonist, 10 mM calcium ionophore A23187, and 2 mM CaCl 2 . The cells were stimulated without (control) or with 10 ng/mL tumor necrosis factor (TNF) α, interleukin (IL) 1β, and IL-6. For inhibition studies on [Ca 2+ ] i , the cells were pretreated for 30 min with 20 µM PAR-2 antagonist FSY-NH 2 or 50 µM aprotinin and then treated with these cytokines. Fluorescence images of the cells were analyzed by a confocal microscope. [Ca 2+ ]i values (nM) are displayed using a color scale. Scale bars are 25 µm.

    Journal: The Journal of Infectious Diseases

    Article Title: Influenza Virus—Cytokine-Protease Cycle in the Pathogenesis of Vascular Hyperpermeability in Severe Influenza

    doi: 10.1086/656044

    Figure Lengend Snippet: Effects of cytokines trypsin, and protease-activated receptor (PAR) 2 agonist on [Ca 2+ ] i and rescue of the increase in [Ca 2+ ] i by PAR-2 antagonist and aprotinin in endothelial cells. The cells were treated for 10 h with 1 µg/mL trypsin, 10 µM PAR-2 agonist, 10 mM calcium ionophore A23187, and 2 mM CaCl 2 . The cells were stimulated without (control) or with 10 ng/mL tumor necrosis factor (TNF) α, interleukin (IL) 1β, and IL-6. For inhibition studies on [Ca 2+ ] i , the cells were pretreated for 30 min with 20 µM PAR-2 antagonist FSY-NH 2 or 50 µM aprotinin and then treated with these cytokines. Fluorescence images of the cells were analyzed by a confocal microscope. [Ca 2+ ]i values (nM) are displayed using a color scale. Scale bars are 25 µm.

    Article Snippet: The cells were infected by influenza A virus WSN at amultiplicity of infection of 0.5 or treated with recombinant human IL-6, TNF-α, and IL-1β (10 ng/mL of each) (PeproTec) in the presence or absence of antibodies against these cytokines (Abcam).

    Techniques: Inhibition, Fluorescence, Microscopy

    Upregulation of cytokines and trypsin, increase in viral RNA after influenza A virus infection in mice, and effects of nuclear factor-kappa B (NF −κ B) and activator protein 1 inhibitors on the upregulation and survival of infected mice. A , Mice were infected with 250 plaque-forming units (PFU) of influenza A WSN/33(H1N1) virus (WSN) with and without treatment with pyrrolidine dithiocarbamate (PDTC), N-acetyl-L-cysteine (NAC), and nordihydroguaiaretic acid (NDGA). Levels of tumor necrosis factor (TNF) α, interleukin (IL) 6, and IL-1β in lung homogenates ( n =3) were analyzed before (Control) and at day 2 (WSN-D2), day 4 (WSN-D4), and day 6 (WSN-D6) after infection. Cytokine levels in lungs of animals treated once daily for 4 days with PDTC (PDTC-D4), NAC (NAC-D4), and NDGA (NDGA-D4) were also measured. Data are mean value ± standard error of the mean (SEM). ** P

    Journal: The Journal of Infectious Diseases

    Article Title: Influenza Virus—Cytokine-Protease Cycle in the Pathogenesis of Vascular Hyperpermeability in Severe Influenza

    doi: 10.1086/656044

    Figure Lengend Snippet: Upregulation of cytokines and trypsin, increase in viral RNA after influenza A virus infection in mice, and effects of nuclear factor-kappa B (NF −κ B) and activator protein 1 inhibitors on the upregulation and survival of infected mice. A , Mice were infected with 250 plaque-forming units (PFU) of influenza A WSN/33(H1N1) virus (WSN) with and without treatment with pyrrolidine dithiocarbamate (PDTC), N-acetyl-L-cysteine (NAC), and nordihydroguaiaretic acid (NDGA). Levels of tumor necrosis factor (TNF) α, interleukin (IL) 6, and IL-1β in lung homogenates ( n =3) were analyzed before (Control) and at day 2 (WSN-D2), day 4 (WSN-D4), and day 6 (WSN-D6) after infection. Cytokine levels in lungs of animals treated once daily for 4 days with PDTC (PDTC-D4), NAC (NAC-D4), and NDGA (NDGA-D4) were also measured. Data are mean value ± standard error of the mean (SEM). ** P

    Article Snippet: The cells were infected by influenza A virus WSN at amultiplicity of infection of 0.5 or treated with recombinant human IL-6, TNF-α, and IL-1β (10 ng/mL of each) (PeproTec) in the presence or absence of antibodies against these cytokines (Abcam).

    Techniques: Infection, Mouse Assay

    In vitro effects of ethanol with or without opioid receptor agonists on cytokine secretion from hypothalamic microglia in primary cultures. Showing the changes in the protein levels of pro-inflammatory cytokines TNF-α ( a ), IFN-γ ( b ), IL-1α ( c ), IL-1β ( d ), IL-6 ( e ), MIP-3α ( f ), MCP-1 ( g ), M-CSF ( h ), CXCL1 ( i ), and RANTES ( j ) and anti-inflammatory cytokines IL-4 ( k ) and IL-13 ( l ) in microglial conditioned medium following 24-h treatment with ethanol (50 mM) with or without MOR agonist (DAMGO, 50 μM) or DOR agonist (DPDPE, 10 nM). Data are represented as mean ± SEM ( n = 6). Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between control and other treatment groups or ethanol and ethanol and opioidergic drug groups are shown by lines with p values on the top of bar graphs

    Journal: Journal of Neuroinflammation

    Article Title: Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol

    doi: 10.1186/s12974-017-0844-3

    Figure Lengend Snippet: In vitro effects of ethanol with or without opioid receptor agonists on cytokine secretion from hypothalamic microglia in primary cultures. Showing the changes in the protein levels of pro-inflammatory cytokines TNF-α ( a ), IFN-γ ( b ), IL-1α ( c ), IL-1β ( d ), IL-6 ( e ), MIP-3α ( f ), MCP-1 ( g ), M-CSF ( h ), CXCL1 ( i ), and RANTES ( j ) and anti-inflammatory cytokines IL-4 ( k ) and IL-13 ( l ) in microglial conditioned medium following 24-h treatment with ethanol (50 mM) with or without MOR agonist (DAMGO, 50 μM) or DOR agonist (DPDPE, 10 nM). Data are represented as mean ± SEM ( n = 6). Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between control and other treatment groups or ethanol and ethanol and opioidergic drug groups are shown by lines with p values on the top of bar graphs

    Article Snippet: Quantification of chemokines and cytokines in microglial supernatant Supernatant of microglial cells treated with agents was analyzed for multiple cytokines and chemokines using Bio-Plex Pro rat cytokine assay (Bio-Rad Laboratories, Hercules, CA).

    Techniques: In Vitro

    The effect of opioid agonists and antagonists and immunoneutralization of inflammatory cytokines TNF-α and IL-6 on the ability of ethanol-or opioid-activated microglial conditioned media to induce apoptosis of POMC neurons. POMC neurons were differentiated from neural stem cells in culture and maintained in T25 flasks (1 × 10 6 /well) for 2 days and then treated for 24 h with microglial conditioned media exposed to opioidergic agents for 24 h before determining neuronal apoptosis using a nucleosome assay. Bar graphs are showing the apoptotic effects of ethanol (50 mM) with or without DAMGO (50 μM), naltrexone (10 ng/ml), or DAMGO and naltrexone ( a ); ethanol (50 mM) with or without DPDPE (10 nM), naltrindole (50 μM), or DPDPE and naltrindole ( b ). Microglial conditioned media from ethanol with or without opioidergic agonist-treated cultures were mixed with antibody to TNF-α (1 ng/ml ( c )), antibody to IL-6 (0.5 ng/ml ( d )), antibody to IL-4 (1 ng/ml ( e )), or antibody to IL-13 (5 ng/ml ( f )) and added to POMC neuron cultures for 24 h to determine neuronal apoptosis. The effects of immunoneutralization of inflammatory and anti-inflammatory cytokines on ethanol with or without opioid-activated POMC neuronal apoptosis are shown in bar graphs. Each bar represents mean ± SEM of 5–8 samples. Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between groups are shown by lines with p values on the top of bar graphs

    Journal: Journal of Neuroinflammation

    Article Title: Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol

    doi: 10.1186/s12974-017-0844-3

    Figure Lengend Snippet: The effect of opioid agonists and antagonists and immunoneutralization of inflammatory cytokines TNF-α and IL-6 on the ability of ethanol-or opioid-activated microglial conditioned media to induce apoptosis of POMC neurons. POMC neurons were differentiated from neural stem cells in culture and maintained in T25 flasks (1 × 10 6 /well) for 2 days and then treated for 24 h with microglial conditioned media exposed to opioidergic agents for 24 h before determining neuronal apoptosis using a nucleosome assay. Bar graphs are showing the apoptotic effects of ethanol (50 mM) with or without DAMGO (50 μM), naltrexone (10 ng/ml), or DAMGO and naltrexone ( a ); ethanol (50 mM) with or without DPDPE (10 nM), naltrindole (50 μM), or DPDPE and naltrindole ( b ). Microglial conditioned media from ethanol with or without opioidergic agonist-treated cultures were mixed with antibody to TNF-α (1 ng/ml ( c )), antibody to IL-6 (0.5 ng/ml ( d )), antibody to IL-4 (1 ng/ml ( e )), or antibody to IL-13 (5 ng/ml ( f )) and added to POMC neuron cultures for 24 h to determine neuronal apoptosis. The effects of immunoneutralization of inflammatory and anti-inflammatory cytokines on ethanol with or without opioid-activated POMC neuronal apoptosis are shown in bar graphs. Each bar represents mean ± SEM of 5–8 samples. Data were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between groups are shown by lines with p values on the top of bar graphs

    Article Snippet: Quantification of chemokines and cytokines in microglial supernatant Supernatant of microglial cells treated with agents was analyzed for multiple cytokines and chemokines using Bio-Plex Pro rat cytokine assay (Bio-Rad Laboratories, Hercules, CA).

    Techniques:

    Schematic diagram illustrating the proposed mechanism by which ethanol interacts with opioid receptors to control POMC neuronal apoptosis in the hypothalamus. Ethanol activation of MOR induces microglial polarization towards the M1 phenotype to produce overabundance of inflammatory cytokines and inflammation. Chronic inflammation and overproduction of TNF-α and IL-6 cytokines are cytotoxic to POMC producing neurons. DOR activation increases production of anti-inflammatory cytokines and helps microglial polarization towards the M2 phenotypes. Elevated productions of IL-4 and IL-13 cytokines from M2 microglia reduce ethanol’s ability to increase POMC neuronal apoptosis. DOR agonist may have therapeutic potential to prevent ethanol neurotoxic action

    Journal: Journal of Neuroinflammation

    Article Title: Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol

    doi: 10.1186/s12974-017-0844-3

    Figure Lengend Snippet: Schematic diagram illustrating the proposed mechanism by which ethanol interacts with opioid receptors to control POMC neuronal apoptosis in the hypothalamus. Ethanol activation of MOR induces microglial polarization towards the M1 phenotype to produce overabundance of inflammatory cytokines and inflammation. Chronic inflammation and overproduction of TNF-α and IL-6 cytokines are cytotoxic to POMC producing neurons. DOR activation increases production of anti-inflammatory cytokines and helps microglial polarization towards the M2 phenotypes. Elevated productions of IL-4 and IL-13 cytokines from M2 microglia reduce ethanol’s ability to increase POMC neuronal apoptosis. DOR agonist may have therapeutic potential to prevent ethanol neurotoxic action

    Article Snippet: Quantification of chemokines and cytokines in microglial supernatant Supernatant of microglial cells treated with agents was analyzed for multiple cytokines and chemokines using Bio-Plex Pro rat cytokine assay (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Activation Assay

    Effect of fetal alcohol exposure on microglia and proopiomelanocortin neuron interaction in the hypothalamus. Showing the changes in the protein level of inflammatory cytokines TNF-α ( a ), mRNA level of cytokine MCP1 ( b ), cytokine receptors CSFR1 ( c ), and TLR4 ( d ) in the mediobasal hypothalamus (MBH) of alcohol-fed (AF), pair-fed (PF) and ad lib-fed rats on postnatal day (PND) 6 as determined through Western blot and q-RTPCR, respectively. Representative photographs of IBA-1-positive cells ( e ) and histograms representing the mean ± SEM number of IBA-1-positive cells ( f ) in the MBH of AF, PF, and AD rats on PND 6. Scale bars shown in three photographs of panel e are 200 μm/each. Characterization of IBA-1-stained microglial cells in the mediobasal hypothalamus based on circularity (partially ramified ( g ); partially amoeboid ( h ); fully amoeboid ( i )) in AD, PF, and AF of rat pups on PND 6. Scale bars in these figures are 20 μm/each. Representative 3D rendering of IBA-1 microglia and GFP-POMC neurons interacting ( j ). Scale bars are 20 μm/each. Quantification of soma/process interaction of microglia with POMC neurons ( k ). Representative images of POMC-stained neurons in the arcuate nucleus ( l ) and histograms representing the mean ± SEM number of POMC-positive cells in the arcuate nucleus of AF, PF, and AD rats on PND 6 ( m ). Scale bars are 200 μm/each. Representative images of ß-endorphin-stained neurons in the arcuate nucleus ( n ) and histograms representing the mean ± SEM number of ß-endorphin-positive cells in the arcuate nucleus of AD, PF, and AF and AF + M (minocycline-treated and alcohol-fed) rats on PND 6 ( o ). Scale bars are 200 μm/each. Data are represented as mean ± SEM ( n = 5–7). The differences between AD, PF, and AF were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. * p

    Journal: Journal of Neuroinflammation

    Article Title: Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol

    doi: 10.1186/s12974-017-0844-3

    Figure Lengend Snippet: Effect of fetal alcohol exposure on microglia and proopiomelanocortin neuron interaction in the hypothalamus. Showing the changes in the protein level of inflammatory cytokines TNF-α ( a ), mRNA level of cytokine MCP1 ( b ), cytokine receptors CSFR1 ( c ), and TLR4 ( d ) in the mediobasal hypothalamus (MBH) of alcohol-fed (AF), pair-fed (PF) and ad lib-fed rats on postnatal day (PND) 6 as determined through Western blot and q-RTPCR, respectively. Representative photographs of IBA-1-positive cells ( e ) and histograms representing the mean ± SEM number of IBA-1-positive cells ( f ) in the MBH of AF, PF, and AD rats on PND 6. Scale bars shown in three photographs of panel e are 200 μm/each. Characterization of IBA-1-stained microglial cells in the mediobasal hypothalamus based on circularity (partially ramified ( g ); partially amoeboid ( h ); fully amoeboid ( i )) in AD, PF, and AF of rat pups on PND 6. Scale bars in these figures are 20 μm/each. Representative 3D rendering of IBA-1 microglia and GFP-POMC neurons interacting ( j ). Scale bars are 20 μm/each. Quantification of soma/process interaction of microglia with POMC neurons ( k ). Representative images of POMC-stained neurons in the arcuate nucleus ( l ) and histograms representing the mean ± SEM number of POMC-positive cells in the arcuate nucleus of AF, PF, and AD rats on PND 6 ( m ). Scale bars are 200 μm/each. Representative images of ß-endorphin-stained neurons in the arcuate nucleus ( n ) and histograms representing the mean ± SEM number of ß-endorphin-positive cells in the arcuate nucleus of AD, PF, and AF and AF + M (minocycline-treated and alcohol-fed) rats on PND 6 ( o ). Scale bars are 200 μm/each. Data are represented as mean ± SEM ( n = 5–7). The differences between AD, PF, and AF were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. * p

    Article Snippet: Quantification of chemokines and cytokines in microglial supernatant Supernatant of microglial cells treated with agents was analyzed for multiple cytokines and chemokines using Bio-Plex Pro rat cytokine assay (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining

    In vitro dose-response effects of ethanol on cytokine secretion from microglia in primary cultures. Showing the changes in the protein levels of pro-inflammatory cytokines TNF-α ( a ), IFN-γ ( b ), IL-1α ( c ), L-1β ( d ), IL-6 ( e ), MIP-3α ( f ), MCP-1 ( g ), M-CSF ( h ), CXCL1 ( i ), and RANTES ( j ) and anti-inflammatory cytokines IL-4 ( k ) and IL-13 ( l ) in microglial conditioned medium following 24-h treatment with various doses of ethanol (25, 50, 100 mM). Data are represented as mean ± SEM ( n = 6). Data of all groups were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between groups are shown by lines with p values on the top of bar graphs

    Journal: Journal of Neuroinflammation

    Article Title: Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol

    doi: 10.1186/s12974-017-0844-3

    Figure Lengend Snippet: In vitro dose-response effects of ethanol on cytokine secretion from microglia in primary cultures. Showing the changes in the protein levels of pro-inflammatory cytokines TNF-α ( a ), IFN-γ ( b ), IL-1α ( c ), L-1β ( d ), IL-6 ( e ), MIP-3α ( f ), MCP-1 ( g ), M-CSF ( h ), CXCL1 ( i ), and RANTES ( j ) and anti-inflammatory cytokines IL-4 ( k ) and IL-13 ( l ) in microglial conditioned medium following 24-h treatment with various doses of ethanol (25, 50, 100 mM). Data are represented as mean ± SEM ( n = 6). Data of all groups were compared by one-way analysis of variance (ANOVA) and the Newman-Keuls posttest. Differences between groups are shown by lines with p values on the top of bar graphs

    Article Snippet: Quantification of chemokines and cytokines in microglial supernatant Supernatant of microglial cells treated with agents was analyzed for multiple cytokines and chemokines using Bio-Plex Pro rat cytokine assay (Bio-Rad Laboratories, Hercules, CA).

    Techniques: In Vitro

    Cytokines found to have significantly altered concentrations in the peripheral blood of cats with mycobacterial disease compared to healthy controls and/or cats hospitalised for other reasons. Statistically significant differences between groups were determined by Kruskal-Wallis test by ranks (P

    Journal: Scientific Reports

    Article Title: Cytokine and Chemokine Concentrations as Biomarkers of Feline Mycobacteriosis

    doi: 10.1038/s41598-018-35571-5

    Figure Lengend Snippet: Cytokines found to have significantly altered concentrations in the peripheral blood of cats with mycobacterial disease compared to healthy controls and/or cats hospitalised for other reasons. Statistically significant differences between groups were determined by Kruskal-Wallis test by ranks (P

    Article Snippet: Cytokine and chemokine concentrations were measured in all of the samples using a commercial, feline specific, antibody-coated microsphere-based multiplex cytokine immunoassay shown to be able to quantify 19 cytokines contemporaneously using 25 ul of each patient’s serum (FCTYOMAG-20K MILLIPLEX MAP Feline Cytokine/Chemokine Magnetic Bead Panel, Premix 19 Plex kit, MERCK Millipore Corporation, Billerica, MA, USA).

    Techniques:

    Cytokines found to have significantly increased concentrations in the peripheral blood of cats with mycobacterial disease compared to healthy controls. Data are shown as the median for the group with error bars indicting the 95% confidence interval.

    Journal: Scientific Reports

    Article Title: Cytokine and Chemokine Concentrations as Biomarkers of Feline Mycobacteriosis

    doi: 10.1038/s41598-018-35571-5

    Figure Lengend Snippet: Cytokines found to have significantly increased concentrations in the peripheral blood of cats with mycobacterial disease compared to healthy controls. Data are shown as the median for the group with error bars indicting the 95% confidence interval.

    Article Snippet: Cytokine and chemokine concentrations were measured in all of the samples using a commercial, feline specific, antibody-coated microsphere-based multiplex cytokine immunoassay shown to be able to quantify 19 cytokines contemporaneously using 25 ul of each patient’s serum (FCTYOMAG-20K MILLIPLEX MAP Feline Cytokine/Chemokine Magnetic Bead Panel, Premix 19 Plex kit, MERCK Millipore Corporation, Billerica, MA, USA).

    Techniques:

    Cytokines found to have significantly altered concentrations in the peripheral blood of cats with mycobacterial disease compared to healthy controls and/or cats hospitalised for other reasons. Data are shown as the median for the group with error bars indicting the 95% confidence interval.

    Journal: Scientific Reports

    Article Title: Cytokine and Chemokine Concentrations as Biomarkers of Feline Mycobacteriosis

    doi: 10.1038/s41598-018-35571-5

    Figure Lengend Snippet: Cytokines found to have significantly altered concentrations in the peripheral blood of cats with mycobacterial disease compared to healthy controls and/or cats hospitalised for other reasons. Data are shown as the median for the group with error bars indicting the 95% confidence interval.

    Article Snippet: Cytokine and chemokine concentrations were measured in all of the samples using a commercial, feline specific, antibody-coated microsphere-based multiplex cytokine immunoassay shown to be able to quantify 19 cytokines contemporaneously using 25 ul of each patient’s serum (FCTYOMAG-20K MILLIPLEX MAP Feline Cytokine/Chemokine Magnetic Bead Panel, Premix 19 Plex kit, MERCK Millipore Corporation, Billerica, MA, USA).

    Techniques:

    Cytokines found to have significantly altered concentrations in the peripheral blood of cats with mycobacterial disease due to M . bovis or M . microti , respectively, compared to healthy controls and cats infected with non-tuberculous mycobacterial (NTM) species. Data are shown as the median for the group with error bars indicting the 95% confidence interval.

    Journal: Scientific Reports

    Article Title: Cytokine and Chemokine Concentrations as Biomarkers of Feline Mycobacteriosis

    doi: 10.1038/s41598-018-35571-5

    Figure Lengend Snippet: Cytokines found to have significantly altered concentrations in the peripheral blood of cats with mycobacterial disease due to M . bovis or M . microti , respectively, compared to healthy controls and cats infected with non-tuberculous mycobacterial (NTM) species. Data are shown as the median for the group with error bars indicting the 95% confidence interval.

    Article Snippet: Cytokine and chemokine concentrations were measured in all of the samples using a commercial, feline specific, antibody-coated microsphere-based multiplex cytokine immunoassay shown to be able to quantify 19 cytokines contemporaneously using 25 ul of each patient’s serum (FCTYOMAG-20K MILLIPLEX MAP Feline Cytokine/Chemokine Magnetic Bead Panel, Premix 19 Plex kit, MERCK Millipore Corporation, Billerica, MA, USA).

    Techniques: Infection

    Cytokines found to have significantly reduced concentrations in the peripheral blood of cats with mycobacterial disease compared to healthy controls. Data are shown as the median for the group with error bars indicting the 95% confidence interval.

    Journal: Scientific Reports

    Article Title: Cytokine and Chemokine Concentrations as Biomarkers of Feline Mycobacteriosis

    doi: 10.1038/s41598-018-35571-5

    Figure Lengend Snippet: Cytokines found to have significantly reduced concentrations in the peripheral blood of cats with mycobacterial disease compared to healthy controls. Data are shown as the median for the group with error bars indicting the 95% confidence interval.

    Article Snippet: Cytokine and chemokine concentrations were measured in all of the samples using a commercial, feline specific, antibody-coated microsphere-based multiplex cytokine immunoassay shown to be able to quantify 19 cytokines contemporaneously using 25 ul of each patient’s serum (FCTYOMAG-20K MILLIPLEX MAP Feline Cytokine/Chemokine Magnetic Bead Panel, Premix 19 Plex kit, MERCK Millipore Corporation, Billerica, MA, USA).

    Techniques:

    Cytokines found to have significantly altered concentrations in the peripheral blood of cats with mycobacterial disease due to TB-complex infections ( M . bovis or M . microti respectively), compared to healthy controls and cats infected with non-tuberculous mycobacterial (NTM) species. Data are shown as the median for the group with error bars indicting the 95% confidence interval.

    Journal: Scientific Reports

    Article Title: Cytokine and Chemokine Concentrations as Biomarkers of Feline Mycobacteriosis

    doi: 10.1038/s41598-018-35571-5

    Figure Lengend Snippet: Cytokines found to have significantly altered concentrations in the peripheral blood of cats with mycobacterial disease due to TB-complex infections ( M . bovis or M . microti respectively), compared to healthy controls and cats infected with non-tuberculous mycobacterial (NTM) species. Data are shown as the median for the group with error bars indicting the 95% confidence interval.

    Article Snippet: Cytokine and chemokine concentrations were measured in all of the samples using a commercial, feline specific, antibody-coated microsphere-based multiplex cytokine immunoassay shown to be able to quantify 19 cytokines contemporaneously using 25 ul of each patient’s serum (FCTYOMAG-20K MILLIPLEX MAP Feline Cytokine/Chemokine Magnetic Bead Panel, Premix 19 Plex kit, MERCK Millipore Corporation, Billerica, MA, USA).

    Techniques: Infection

    Higher production of tumor-promoting factors in miR-155−/− chimeric mice.  A–D , 23 cytokines in sera of WT and miR-155−/− chimeric mice were detected by Bio-Plex Pro™ Mouse Cytokine 23-plex Assay. Data are

    Journal: Molecular cancer research : MCR

    Article Title: microRNA-155 deficiency in bone marrow results in enhanced tumor metastasis

    doi: 10.1158/1541-7786.MCR-12-0686

    Figure Lengend Snippet: Higher production of tumor-promoting factors in miR-155−/− chimeric mice. A–D , 23 cytokines in sera of WT and miR-155−/− chimeric mice were detected by Bio-Plex Pro™ Mouse Cytokine 23-plex Assay. Data are

    Article Snippet: Concentrations of 23 mouse cytokines/chemokines were measured by Bio-Plex Pro™ Mouse Cytokine 23-plex Assay (Bio-rad, M60-009RDPD) following the manufacturer's instructions.

    Techniques: Mouse Assay, Plex Assay

    Influence of Pro-Th1 (IL-2 and IL-12) and Pro-Th2 (IL-4) cytokines on the proliferation and IFN-γ secretion by Th1 cells stimulated with anti-CD3 Ab and B7-1. pGL-10 Th1 cells were cultured with anti-CD3 Ab (0.01 μg/ml) in the presence of CHO-B7-1 (5 × 10 4 /well). Cytokines IL-2, IL-4 or IL-12 alone or in combinations were also added into the cultures. For proliferation (Fig. 5a), cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and were harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells. IFN-γ (Fig. 5b) was estimated by ELISA in the pooled culture supernatants collected from the triplicate wells after 48 h of the initiation of cultures. The control cultures comprising of Th1 cells, CHO-B7-1+Th1 cells, CHO+Th1 cells, CHO-B7-1, CHO showed no noticeable change. The data presented are from three independent experiments. '*' Represents p

    Journal: BMC Immunology

    Article Title: Regulatory role of pro-Th1 and pro-Th2 cytokines in modulating the activity of Th1 and Th2 cells when B cell and macrophages are used as antigen presenting cells

    doi: 10.1186/1471-2172-7-17

    Figure Lengend Snippet: Influence of Pro-Th1 (IL-2 and IL-12) and Pro-Th2 (IL-4) cytokines on the proliferation and IFN-γ secretion by Th1 cells stimulated with anti-CD3 Ab and B7-1. pGL-10 Th1 cells were cultured with anti-CD3 Ab (0.01 μg/ml) in the presence of CHO-B7-1 (5 × 10 4 /well). Cytokines IL-2, IL-4 or IL-12 alone or in combinations were also added into the cultures. For proliferation (Fig. 5a), cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and were harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells. IFN-γ (Fig. 5b) was estimated by ELISA in the pooled culture supernatants collected from the triplicate wells after 48 h of the initiation of cultures. The control cultures comprising of Th1 cells, CHO-B7-1+Th1 cells, CHO+Th1 cells, CHO-B7-1, CHO showed no noticeable change. The data presented are from three independent experiments. '*' Represents p

    Article Snippet: Estimation of cytokines The cytokines were estimated as per manufacturer instructions (Pharmingen) and expressed as pg/ml using recombinant cytokines as a standard (Pharmingen).

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

    Influence of Pro-Th1 (IL-2, IFN-γ) and Pro-Th2 (IL-1, IL-4, IL-5) cytokines on the proliferation and IL-4 and IL-5 secretion by Th2 cells when B cells, splenic macrophages and peritoneal macrophages were used as APC. D10G4.1 Th2 cells were cultured with B cells, splenic and peritoneal macrophages and conalbumin (100 μg/ml). Cytokines IL-1, IL-2, IL-4 or IL-5 and IFN-γ were also added in the cultures. Cultures with peritoneal macrophages also received 1 mM aminoguanidine. For proliferation, the cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells (Fig. 2a). For IL-4 (Fig. 2b) and IL-5 (Fig. 2c), the supernatants were collected from the triplicate wells after 48 h of the initiation of cultures, pooled and estimated by ELISA. The data are expressed as pg/ml. The control cultures comprising Th2 cells, Th2 cell+Ag, APCs+Ag, APCs+Th2 cells showed no apparent change. The data presented are from three independent experiments. '*' Represents p

    Journal: BMC Immunology

    Article Title: Regulatory role of pro-Th1 and pro-Th2 cytokines in modulating the activity of Th1 and Th2 cells when B cell and macrophages are used as antigen presenting cells

    doi: 10.1186/1471-2172-7-17

    Figure Lengend Snippet: Influence of Pro-Th1 (IL-2, IFN-γ) and Pro-Th2 (IL-1, IL-4, IL-5) cytokines on the proliferation and IL-4 and IL-5 secretion by Th2 cells when B cells, splenic macrophages and peritoneal macrophages were used as APC. D10G4.1 Th2 cells were cultured with B cells, splenic and peritoneal macrophages and conalbumin (100 μg/ml). Cytokines IL-1, IL-2, IL-4 or IL-5 and IFN-γ were also added in the cultures. Cultures with peritoneal macrophages also received 1 mM aminoguanidine. For proliferation, the cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells (Fig. 2a). For IL-4 (Fig. 2b) and IL-5 (Fig. 2c), the supernatants were collected from the triplicate wells after 48 h of the initiation of cultures, pooled and estimated by ELISA. The data are expressed as pg/ml. The control cultures comprising Th2 cells, Th2 cell+Ag, APCs+Ag, APCs+Th2 cells showed no apparent change. The data presented are from three independent experiments. '*' Represents p

    Article Snippet: Estimation of cytokines The cytokines were estimated as per manufacturer instructions (Pharmingen) and expressed as pg/ml using recombinant cytokines as a standard (Pharmingen).

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

    Influence of Pro-Th1 (IL-2 and IFN-γ) and Pro-Th2 (IL-4) cytokines on the proliferation and IL-4 and IL-5 secretion by Th2 cells stimulated with anti-CD3 Ab and B7-1. D10G4.1 Th2 cells were cultured with anti-CD3 Ab (0.01 μg/ml) in the presence of CHO-B7-1 (5 × 10 4 /well). Cytokines IL-2, IL-4 or IFN-γ alone or in combinations were also added in to the cultures. For the proliferation (Fig. 6a), cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and were harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells. IL-4 (Fig. 6b) and IL-5 (Fig. 6c) were estimated by ELISA in the pooled supernatants collected from the triplicate wells after 48 h of the initiation of the cultures. The control cultures comprising Th2 cells, CHO-B7-1+Th2 cells, CHO+Th2 cells, CHO-B7-1, CHO showed no considerable change except that IL-5 secretion was 1995 pg/ml when D10G4.1 Th2 cells were incubated with CHO-B7.1 in the absence of anti-CD3 Ab. The data presented are from three independent experiments. '*' Represents p

    Journal: BMC Immunology

    Article Title: Regulatory role of pro-Th1 and pro-Th2 cytokines in modulating the activity of Th1 and Th2 cells when B cell and macrophages are used as antigen presenting cells

    doi: 10.1186/1471-2172-7-17

    Figure Lengend Snippet: Influence of Pro-Th1 (IL-2 and IFN-γ) and Pro-Th2 (IL-4) cytokines on the proliferation and IL-4 and IL-5 secretion by Th2 cells stimulated with anti-CD3 Ab and B7-1. D10G4.1 Th2 cells were cultured with anti-CD3 Ab (0.01 μg/ml) in the presence of CHO-B7-1 (5 × 10 4 /well). Cytokines IL-2, IL-4 or IFN-γ alone or in combinations were also added in to the cultures. For the proliferation (Fig. 6a), cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and were harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells. IL-4 (Fig. 6b) and IL-5 (Fig. 6c) were estimated by ELISA in the pooled supernatants collected from the triplicate wells after 48 h of the initiation of the cultures. The control cultures comprising Th2 cells, CHO-B7-1+Th2 cells, CHO+Th2 cells, CHO-B7-1, CHO showed no considerable change except that IL-5 secretion was 1995 pg/ml when D10G4.1 Th2 cells were incubated with CHO-B7.1 in the absence of anti-CD3 Ab. The data presented are from three independent experiments. '*' Represents p

    Article Snippet: Estimation of cytokines The cytokines were estimated as per manufacturer instructions (Pharmingen) and expressed as pg/ml using recombinant cytokines as a standard (Pharmingen).

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

    Influence of Pro-Th1 (IL-2, IL-12, IFN-γ) and Pro-Th2 (IL-4) cytokines on the proliferation and IFN-γ secretion when B cells, splenic macrophages and peritoneal macrophages were used as APCs. pGL-10 Th1 cells were cultured with B cells, splenic and peritoneal macrophages and ovalbumin (200 μg/ml). Cytokines IL-2, IL-4, IL-12 and IFN-γ were also added in the cultures. For proliferation, the cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells (Fig. 1a). For IFN-γ, the supernatants were collected from the triplicate wells after 48 h of the initiation of cultures, pooled and estimated by ELISA. (Fig. 1b). The control cultures comprising of Th1 cells, Th1 cell+Ag, APCs+Ag, APCs+Th1 cells showed no discernible change. The data presented are from three independent experiments. '*' Represents p

    Journal: BMC Immunology

    Article Title: Regulatory role of pro-Th1 and pro-Th2 cytokines in modulating the activity of Th1 and Th2 cells when B cell and macrophages are used as antigen presenting cells

    doi: 10.1186/1471-2172-7-17

    Figure Lengend Snippet: Influence of Pro-Th1 (IL-2, IL-12, IFN-γ) and Pro-Th2 (IL-4) cytokines on the proliferation and IFN-γ secretion when B cells, splenic macrophages and peritoneal macrophages were used as APCs. pGL-10 Th1 cells were cultured with B cells, splenic and peritoneal macrophages and ovalbumin (200 μg/ml). Cytokines IL-2, IL-4, IL-12 and IFN-γ were also added in the cultures. For proliferation, the cultures were pulsed after 48 h with 3 H-thymidine (0.5 μCi/well) and harvested after last 16 h of incubation period. Data are expressed as mean ± SEM of triplicate wells (Fig. 1a). For IFN-γ, the supernatants were collected from the triplicate wells after 48 h of the initiation of cultures, pooled and estimated by ELISA. (Fig. 1b). The control cultures comprising of Th1 cells, Th1 cell+Ag, APCs+Ag, APCs+Th1 cells showed no discernible change. The data presented are from three independent experiments. '*' Represents p

    Article Snippet: Estimation of cytokines The cytokines were estimated as per manufacturer instructions (Pharmingen) and expressed as pg/ml using recombinant cytokines as a standard (Pharmingen).

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

    Transcriptional changes in eYFP + CD4 + T cells (a) CCR6 + eYFP + CD4 + T cells from 2D2 IL-17-reporter mice were purified 4 days after MOG-CFA immunization and adoptively transferred into immunized Rag-deficient mice. FACS plots and bar graphs of IL-17A and IFN-γ expression in eYFP + CD4 + T cells from draining lymph nodes and spinal cord (day 16) restimulated with PdBU-ionomycin (upper panels) or MOG peptide (lower panels). Histograms show mean values for individual mice +/− SD. (b) CCR6 − and CCR6 + eYFP + CD4 + T cells from spinal cord were sorted for qPCR analysis. Representative FACS plots show expression of IL-17A and IFN-γ. Relative gene expression in sorted (not restimulated) cells normalized to the expression of Hprt is shown. (c) CD4 + eYFP − IFN-γ + , representing T H 1, (shaded gray), CD4 + eYFP + IFN-γ + (ex-T H 17 -dotted line), and CD4 + eYFP + IL-17A + (T H 17- solid line) from draining LN and spinal cord cells 15 days after MOG-CFA immunisation were gated and assessed for IL-1R1 expression. The data represent at least three independent experiments. (d) Cytokine levels measured in supernatant of purified eYFP + or eYFP − CD4 T cells from draining lymph nodes or spinal cord (day 15) restimulated with anti-CD3 +/− 20 ng/ml IL-1β for 24 hr. Data show mean values ± SD of cytokines from three individual mice. The data represent at least three independent experiments.

    Journal: Nature immunology

    Article Title: Fate mapping of interleukin 17-producing T cells in inflammatory responses

    doi: 10.1038/ni.1993

    Figure Lengend Snippet: Transcriptional changes in eYFP + CD4 + T cells (a) CCR6 + eYFP + CD4 + T cells from 2D2 IL-17-reporter mice were purified 4 days after MOG-CFA immunization and adoptively transferred into immunized Rag-deficient mice. FACS plots and bar graphs of IL-17A and IFN-γ expression in eYFP + CD4 + T cells from draining lymph nodes and spinal cord (day 16) restimulated with PdBU-ionomycin (upper panels) or MOG peptide (lower panels). Histograms show mean values for individual mice +/− SD. (b) CCR6 − and CCR6 + eYFP + CD4 + T cells from spinal cord were sorted for qPCR analysis. Representative FACS plots show expression of IL-17A and IFN-γ. Relative gene expression in sorted (not restimulated) cells normalized to the expression of Hprt is shown. (c) CD4 + eYFP − IFN-γ + , representing T H 1, (shaded gray), CD4 + eYFP + IFN-γ + (ex-T H 17 -dotted line), and CD4 + eYFP + IL-17A + (T H 17- solid line) from draining LN and spinal cord cells 15 days after MOG-CFA immunisation were gated and assessed for IL-1R1 expression. The data represent at least three independent experiments. (d) Cytokine levels measured in supernatant of purified eYFP + or eYFP − CD4 T cells from draining lymph nodes or spinal cord (day 15) restimulated with anti-CD3 +/− 20 ng/ml IL-1β for 24 hr. Data show mean values ± SD of cytokines from three individual mice. The data represent at least three independent experiments.

    Article Snippet: Determination of cytokines in supernatants of FACS purified cells from lymph node or spinal cord at the adjusted concentration of 5×104 /ml was done after overnight culture with plate-bound anti-CD3 and anti-CD28 in the absence or presence of IL-1β (20ng/ml) using FlowCytomix (Bender Medsystems).

    Techniques: Mouse Assay, Purification, FACS, Expressing, Real-time Polymerase Chain Reaction

    IFN-γ expression and antigen specificity in eYFP + and eYFP − CD4 T cells (a) Draining LN cells from MOG/CFA immunized mice were stained for CD4 and γδ TCR and assessed for eYFP and intracellular IFNγ at the indicated days after immunization. (b) bar graphs showing % of eYFP + or eYFP − CD4 T cells from draining lymph nodes at day 12 expressing cytokines following restimulation with PdBU-ionomycin (left panel) or MOG peptide (right panel). Mean values +/− SD of three individual mice are shown. (c) Representative FACS plots showing expression of eYFP and IFN-γ of infiltrating CD4 + and γδ T cells in the spinal cord. (d) bar graphs showing % of eYFP + or eYFP − CD4 T cells from spinal cord at day 15 expressing cytokines following restimulation with PdBU-ionomycin (left panel) or MOG peptide (right panel).

    Journal: Nature immunology

    Article Title: Fate mapping of interleukin 17-producing T cells in inflammatory responses

    doi: 10.1038/ni.1993

    Figure Lengend Snippet: IFN-γ expression and antigen specificity in eYFP + and eYFP − CD4 T cells (a) Draining LN cells from MOG/CFA immunized mice were stained for CD4 and γδ TCR and assessed for eYFP and intracellular IFNγ at the indicated days after immunization. (b) bar graphs showing % of eYFP + or eYFP − CD4 T cells from draining lymph nodes at day 12 expressing cytokines following restimulation with PdBU-ionomycin (left panel) or MOG peptide (right panel). Mean values +/− SD of three individual mice are shown. (c) Representative FACS plots showing expression of eYFP and IFN-γ of infiltrating CD4 + and γδ T cells in the spinal cord. (d) bar graphs showing % of eYFP + or eYFP − CD4 T cells from spinal cord at day 15 expressing cytokines following restimulation with PdBU-ionomycin (left panel) or MOG peptide (right panel).

    Article Snippet: Determination of cytokines in supernatants of FACS purified cells from lymph node or spinal cord at the adjusted concentration of 5×104 /ml was done after overnight culture with plate-bound anti-CD3 and anti-CD28 in the absence or presence of IL-1β (20ng/ml) using FlowCytomix (Bender Medsystems).

    Techniques: Expressing, Mouse Assay, Staining, FACS

    Cytokine expression in eYFP + and eYFP − CD4 + T cells in the draining LN and spinal cord (a) Lymph nodes from non-immune mice (day 0) as well as draining LN and spinal cord cells 6 and 15 days after EAE induction, respectively, were stained for CD4 and intracellular cytokines as indicated. The dot plots show cytokine expression profiles in gated eYFP + CD4 + T cells (a) and gated eYFP − CD4 + T cells in (b ). The data are representative for at least three independent experiments. (c) Cytokine concentrations measured in supernatant of 2×10 4 sorted eYFP + or eYFP − CD4 T cells isolated from day 6 lymph nodes or day 15 spinal cord and restimulated in vitro with anti-CD3/anti-CD28 for 24h. Data show mean values +/− SD of cytokines measured from three individual mice.

    Journal: Nature immunology

    Article Title: Fate mapping of interleukin 17-producing T cells in inflammatory responses

    doi: 10.1038/ni.1993

    Figure Lengend Snippet: Cytokine expression in eYFP + and eYFP − CD4 + T cells in the draining LN and spinal cord (a) Lymph nodes from non-immune mice (day 0) as well as draining LN and spinal cord cells 6 and 15 days after EAE induction, respectively, were stained for CD4 and intracellular cytokines as indicated. The dot plots show cytokine expression profiles in gated eYFP + CD4 + T cells (a) and gated eYFP − CD4 + T cells in (b ). The data are representative for at least three independent experiments. (c) Cytokine concentrations measured in supernatant of 2×10 4 sorted eYFP + or eYFP − CD4 T cells isolated from day 6 lymph nodes or day 15 spinal cord and restimulated in vitro with anti-CD3/anti-CD28 for 24h. Data show mean values +/− SD of cytokines measured from three individual mice.

    Article Snippet: Determination of cytokines in supernatants of FACS purified cells from lymph node or spinal cord at the adjusted concentration of 5×104 /ml was done after overnight culture with plate-bound anti-CD3 and anti-CD28 in the absence or presence of IL-1β (20ng/ml) using FlowCytomix (Bender Medsystems).

    Techniques: Expressing, Mouse Assay, Staining, Isolation, In Vitro

    Induction of fate reporter eYFP + cells in IL-17-producing cells ( a ) Naïve CD4 + CD44 lo CD25 − T cells were cultured under T H 1, T H 2, T H 9, T H 17 or iTreg conditions for 4 days and stained for indicated intracellular cytokines. Dot plots show intracellular cytokine expression vs eYFP (b) Schematic representation of PCR primer location used for assessment of Cre-mediated recombination at the ROSA26 eYFP locus. Cells cultured under T H 17 conditions in vitro were sorted according to the gates indicated and DNA from sorted populations were tested for recombination at the ROSA26 eYFP locus. (c) mean fluorescence intensity for IL-17A(top) and Cre (bottom) in IL-17A + eYFP − and IL-17A + eYFP + populations. Mean values and SD are shown. *denotes P value 0.03, ** denotes P value 0.01 (d) LN cells from non-immune mice were stained for CD4 and γδ TCR, followed by intracellular IL-17A and IFN-γ staining. Dot plots show intracellular staining vs eYFP expression. The FACS plots are representative for three independent experiments and bar graphs showing experimental variability are shown in (e) .

    Journal: Nature immunology

    Article Title: Fate mapping of interleukin 17-producing T cells in inflammatory responses

    doi: 10.1038/ni.1993

    Figure Lengend Snippet: Induction of fate reporter eYFP + cells in IL-17-producing cells ( a ) Naïve CD4 + CD44 lo CD25 − T cells were cultured under T H 1, T H 2, T H 9, T H 17 or iTreg conditions for 4 days and stained for indicated intracellular cytokines. Dot plots show intracellular cytokine expression vs eYFP (b) Schematic representation of PCR primer location used for assessment of Cre-mediated recombination at the ROSA26 eYFP locus. Cells cultured under T H 17 conditions in vitro were sorted according to the gates indicated and DNA from sorted populations were tested for recombination at the ROSA26 eYFP locus. (c) mean fluorescence intensity for IL-17A(top) and Cre (bottom) in IL-17A + eYFP − and IL-17A + eYFP + populations. Mean values and SD are shown. *denotes P value 0.03, ** denotes P value 0.01 (d) LN cells from non-immune mice were stained for CD4 and γδ TCR, followed by intracellular IL-17A and IFN-γ staining. Dot plots show intracellular staining vs eYFP expression. The FACS plots are representative for three independent experiments and bar graphs showing experimental variability are shown in (e) .

    Article Snippet: Determination of cytokines in supernatants of FACS purified cells from lymph node or spinal cord at the adjusted concentration of 5×104 /ml was done after overnight culture with plate-bound anti-CD3 and anti-CD28 in the absence or presence of IL-1β (20ng/ml) using FlowCytomix (Bender Medsystems).

    Techniques: Cell Culture, Staining, Expressing, Polymerase Chain Reaction, In Vitro, Fluorescence, Mouse Assay, FACS