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

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

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

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

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

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

Journal: Immunity

Article Title: Human CD14dim Monocytes Patrol and Sense Nucleic Acids and Viruses via TLR7 and TLR8 Receptors

doi: 10.1016/j.immuni.2010.08.012

Figure Lengend Snippet: Monocyte Subset-Specific Response to Viruses (A and B) Cytokine production from two different donors after overnight incubation with viruses (A and B) (herpes simplex virus-1 (dsDNA, HSV-1), vesicular stomatitis virus [ss(−)RNA, VSV], measles virus [ss(−)RNA, MV], and Encephalomyocarditis virus [ss(+)RNA, EMCV'. (C) Cytokine production after overnight incubation with TLR agonists. (D and E) Cytokine production by CD14 dim monocytes (D) and CD14 + monocytes (E) from controls, MYD88-deficient patients and IRAK-4-deficient patients after exposure to measles virus (MV) or herpes simplex virus-1 (HSV-1). Data are representative of at least three donors. Bar graphs in (A)–(C) show mean (±SD) of triplicates from experiments representative of five healthy donors. Bar graphs in (D) and (E) show results representative of two IRAK −/− patients, three MYD88 −/− patients, and ten healthy donors. See also Figure S3 .

Article Snippet: Cell supernatant was collected and analyzed for cytokine production with the Biorad multiplex assay or ELISA tests.

Techniques: Incubation

Journal: Immunity

Article Title: Human CD14dim Monocytes Patrol and Sense Nucleic Acids and Viruses via TLR7 and TLR8 Receptors

doi: 10.1016/j.immuni.2010.08.012

Figure Lengend Snippet: Monocyte Subset-Specific Activation Pathways (A) Phosphorylated Akt, p38-MAPK, MEK1, and JNK in CD14 + (black bars) and CD14 lo monocytes (red bars) following treatment with TLR8 agonist (3M2, open bars) or TLR7 agonist (3M13, closed bars) for 0, 30, and 120 min at 37°C. Data are presented as mean fold increase from vehicle control treated cells from three independent experiments. (B) Cytokine production by CD14 + (open black bars) and CD14 dim (closed red bars) monocytes treated with vehicle, 3M2, or 3M13 with or without MEK inhibitor PD98059. Data are presented as mean ± SD in expression (pg/ml) from three independent experiments. See also Figure S4 .

Article Snippet: Cell supernatant was collected and analyzed for cytokine production with the Biorad multiplex assay or ELISA tests.

Techniques: Activation Assay, Expressing

Journal: Immunity

Article Title: Human CD14dim Monocytes Patrol and Sense Nucleic Acids and Viruses via TLR7 and TLR8 Receptors

doi: 10.1016/j.immuni.2010.08.012

Figure Lengend Snippet: Functional Analysis of Monocyte Subsets Human monocytes were obtained from whole blood from healthy donors and purified by flow cytometry as indicated in the Experimental Procedures (see Figure S1 ). (A) Percentage of cells that have phagocytosed 1 μm beads after the indicated time. The mean ± SD of triplicates is shown. A representative of five independent experiments is shown. (B) ROS production. (C) Cytokine production after overnight incubation with (+) or without (−) LPS (100 ng/ml). (D) Production of IL1-RA after 18 hr incubation. Bar graphs in (B)–(D) show mean ± SD of triplicates, from experiments representative of three to five donors. See also Figure S2 .

Article Snippet: Cell supernatant was collected and analyzed for cytokine production with the Biorad multiplex assay or ELISA tests.

Techniques: Functional Assay, Purification, Flow Cytometry, Cytometry, Incubation

Journal: Immunity

Article Title: Human CD14dim Monocytes Patrol and Sense Nucleic Acids and Viruses via TLR7 and TLR8 Receptors

doi: 10.1016/j.immuni.2010.08.012

Figure Lengend Snippet: CD14 dim Monocytes Detect Nucleic Acids and Mediate Inflammation in Lupus Patients (A) Immunostaining for IgG, CD68, CD16, CD14, CD15, and CD3ɛ, on tissue sections from kidney biopsy with IV-G lupus glomerulonephritis (left panels) and postinfectious glomerulonephritis (right panels). (B) Cytokine production by CD14 dim or CD14 + monocytes from healthy controls (n = 3) incubated with serum from Lupus patients with autoantibodies to RNPs (n = 3) and controls (n = 2). (C and D) Monocyte cytokine production after incubation with serum from patients without treatment (open bar), RNase+DNase treatment (red bar), addition of RNP (light yellow bar), depletion of immunoglobulins (green bar), depletion of immunoglobulins+RNP (blue bar), or depletion of immunoglobulins+RNase+DNase (orange bar). Two representative figures for each cytokine from at least three experiments are shown. ∗ p

Article Snippet: Cell supernatant was collected and analyzed for cytokine production with the Biorad multiplex assay or ELISA tests.

Techniques: Immunostaining, Incubation

Journal: Frontiers in Immunology

Article Title: In Vivo Chronic Stimulation Unveils Autoreactive Potential of Wiskott–Aldrich Syndrome Protein-Deficient B Cells

doi: 10.3389/fimmu.2017.00490

Figure Lengend Snippet: Cytokine production in Was −/− mice after in vivo chronic stimulations . The fold increase of IL6 (A) and IL17a (B) concentrations was calculated as the ratio between the mean value of cytokine production after the indicated in vivo treatment over the mean value before the treatment for each group of mice [LPS/CpG, n = 4 mice; Apo, n = 7–8 mice; lymphocytic choriomeningitis virus (LCMV), n = 5 mice].

Article Snippet: Cytokine Production Cytokine production was evaluated on serum samples from wt and Was −/− mice before and after administration of LPS, CpG, apoptotic cells, or infection with LCMV, by using the Bio-Plex Pro Mouse Cytokine Th17 Panel A 6-plex (IL6, IL17a, IFNg, IL1b, IL10, TNFa) (Biorad) and following the manufacturer’s instruction.

Techniques: Mouse Assay, In Vivo

Journal: Infection and Immunity

Article Title: Transforming Growth Factor ?1 Production by CD4+ CD25+ Regulatory T Cells in Peripheral Blood Mononuclear Cells from Healthy Subjects Stimulated with Leishmania guyanensis

doi: 10.1128/IAI.73.9.5908-5914.2005

Figure Lengend Snippet: Suppression of cytokine production in CD4 + CD25 − T-cell responses by CD4 + CD25 + T cells. CD4 + T cells were purified from PBMC using anti-CD4 magnetic beads (Dynal). This yielded 95% pure CD4 + T cells. After release of CD4 MAbs with Detachabeads (Dynal), CD4 + T cells were incubated with an anti-CD25 MAb (Pharmingen) and purified on magnetic beads with anti-mouse IgG antibodies. Purified CD4 + CD25 − T cells were then activated with either live L. guyanensis (A) or an anti-CD3 MAb (B) in the presence or absence of CD4 + CD25 + T cells as described in Materials and Methods. After 3 and 7 days of culture, supernatants were collected for analysis of cytokine production. Some cultures were carried out in the presence of either anti-IL-10, anti-TGF-β1, or an isotype control MAb (5 μg/ml) to analyze the role of these cytokines in the suppressor activity of CD4 + CD25 + T cells. n.d., not detected. *, statistical significance for cocultures of CD4 + CD25 + and CD4 + CD25 − T cells versus cultures of CD4 + CD25 − T cells alone ( P

Article Snippet: We analyzed cytokine production by specific IL-4, IL-10, IL-13, IFN-γ, and TGF-β1 enzyme-linked immunosorbent assays (ELISA) (Pharmingen) with a sensitivity of 10 pg/ml (except for TGF-β1, for which the assay sensitivity was 62.5 pg/ml).

Techniques: Purification, Magnetic Beads, Incubation, Activity Assay

Journal: Infection and Immunity

Article Title: Transforming Growth Factor ?1 Production by CD4+ CD25+ Regulatory T Cells in Peripheral Blood Mononuclear Cells from Healthy Subjects Stimulated with Leishmania guyanensis

doi: 10.1128/IAI.73.9.5908-5914.2005

Figure Lengend Snippet: Production of cytokines by PBMC in response to live Leishmania promastigotes. (A) Cytokines were produced by culture of PBMC (10 6 /ml) from healthy controls and LCL patients in the presence of live Leishmania guyanensis promastigotes (10 6 /ml) as described in Materials and Methods. Supernatants were harvested after 2 days for IL-2 and after 7 days for IFN-γ, IL-10, and TGF-β1 production. Cytokine production was analyzed by using specific ELISA. These ELISA had sensitivities of 10 pg/ml except for TGF-β1 (62.5 pg/ml). n.d., not detected. Production of cytokines by unstimulated cells was under the limit of detection for healthy controls. Unstimulated cells from LCL patients produced low levels of IFN-γ (54 ± 12 pg/ml) and IL-10 (12 ± 8 pg/ml), but IL-2 and TGF-β1 production was undetectable. (B) TGF-β1 production was analyzed by culture of PBMC from healthy controls in the presence of either live L. guyanensis or live L. major promastigotes or amastigotes (10 6 /ml) as described above.

Article Snippet: We analyzed cytokine production by specific IL-4, IL-10, IL-13, IFN-γ, and TGF-β1 enzyme-linked immunosorbent assays (ELISA) (Pharmingen) with a sensitivity of 10 pg/ml (except for TGF-β1, for which the assay sensitivity was 62.5 pg/ml).

Techniques: Produced, Enzyme-linked Immunosorbent Assay

Journal: Infection and Immunity

Article Title: Transforming Growth Factor ?1 Production by CD4+ CD25+ Regulatory T Cells in Peripheral Blood Mononuclear Cells from Healthy Subjects Stimulated with Leishmania guyanensis

doi: 10.1128/IAI.73.9.5908-5914.2005

Figure Lengend Snippet: Cytokine secretion profile of CD25 + and CD25 − CD4 + T cells. CD4 + T cells were purified from PBMC using anti-CD4 magnetic beads (Dynal). This yielded 94% pure CD4 + T cells. After release of CD4 MAbs with Detachabeads (Dynal), CD4 + T cells were incubated with an anti-CD25 MAb (Pharmingen) and purified on magnetic beads with anti-mouse IgG antibodies. CD4 + CD25 + and CD4 + CD25 − T cells (5 × 10 5 ) were stimulated with either anti-CD3 MAbs or live L. guyanensis . Supernatants were collected after 3 and 7 days of culture with anti-CD3 and live L. guyanensis , respectively. Data are means ± standard errors of the means for six healthy controls. n.d., not detected.

Article Snippet: We analyzed cytokine production by specific IL-4, IL-10, IL-13, IFN-γ, and TGF-β1 enzyme-linked immunosorbent assays (ELISA) (Pharmingen) with a sensitivity of 10 pg/ml (except for TGF-β1, for which the assay sensitivity was 62.5 pg/ml).

Techniques: Purification, Magnetic Beads, Incubation

Journal: The international journal of biochemistry & cell biology

Article Title: Structural Requirements for the Inhibition of Calcium Mobilization and Mast Cell Activation by the Pyrazole Derivative BTP2

doi: 10.1016/j.biocel.2011.04.016

Figure Lengend Snippet: BTP2 inhibits cytokine secretion of IgE/anti-IgE-activated mast cells

Article Snippet: A 3' enhancer in the IL-4 gene regulates cytokine production by Th2 cells and mast cells.

Techniques:

Journal: The international journal of biochemistry & cell biology

Article Title: Structural Requirements for the Inhibition of Calcium Mobilization and Mast Cell Activation by the Pyrazole Derivative BTP2

doi: 10.1016/j.biocel.2011.04.016

Figure Lengend Snippet: BTP2 does not affect preformed cytokine mRNA expression

Article Snippet: A 3' enhancer in the IL-4 gene regulates cytokine production by Th2 cells and mast cells.

Techniques: Expressing

Journal: The international journal of biochemistry & cell biology

Article Title: Structural Requirements for the Inhibition of Calcium Mobilization and Mast Cell Activation by the Pyrazole Derivative BTP2

doi: 10.1016/j.biocel.2011.04.016

Figure Lengend Snippet: BTP2 inhibits cytokine secretion of PMA/ionomycin-activated BMMCs

Article Snippet: A 3' enhancer in the IL-4 gene regulates cytokine production by Th2 cells and mast cells.

Techniques:

Journal: The international journal of biochemistry & cell biology

Article Title: Structural Requirements for the Inhibition of Calcium Mobilization and Mast Cell Activation by the Pyrazole Derivative BTP2

doi: 10.1016/j.biocel.2011.04.016

Figure Lengend Snippet: BTP2 inhibits cytokine secretion of IgE/antigen-mediated activation of mast cells

Article Snippet: A 3' enhancer in the IL-4 gene regulates cytokine production by Th2 cells and mast cells.

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: HRV16 Impairs Macrophages Cytokine Response to a Secondary Bacterial Trigger

doi: 10.3389/fimmu.2018.02908

Figure Lengend Snippet: Cytokine response of human macrophages exposed to HRV16 or TLR agonists for 48 h and then challenged with NTHi or LPS. Human macrophages were exposed to HRV16 (red bars), LPS (blue bars), CpG (orange bars) or MI (black bars) for 1 h and rested for 48 h. Then they were exposed to NTHi or LPS for 2 h and supernatants were collected and analyzed by MSD. MSD results for (A) IFNγ, (B) IL10, (C) IL12p70, (D) IL1β, (E) IL4, (F) IL6, (G) IL8, (H) TNFα. (I) Relative fold changes for cytokine production in HRV16, LPS or CpG exposed human macrophages. * p

Article Snippet: Cytokine production by macrophages was analyzed using the Meso Scale Discovery® technology according to the manufacturer's instructions.

Techniques:

Journal: Frontiers in Immunology

Article Title: HRV16 Impairs Macrophages Cytokine Response to a Secondary Bacterial Trigger

doi: 10.3389/fimmu.2018.02908

Figure Lengend Snippet: Human macrophages exposed to HRV16 cannot secrete elevated levels of pro-inflammatory and regulatory cytokines in response to NTHi. Human macrophages were exposed to HRV16 (red bars), HRV16 UV (open bars) or MI (black bars) for 1 h and rested overnight before being exposed to NTHi for 2 h. Supernatants were collected and analyzed by MSD. MSD results for (A) IFNγ, (B) IL10, (C) IL12p70, (D) IL1β, (E) IL4, (F) IL6, (G) IL8, (H) TNFα. (I) Relative fold changes for cytokine production in HRV16 and HRV16 UV exposed human macrophages vs NTHi. ** p

Article Snippet: Cytokine production by macrophages was analyzed using the Meso Scale Discovery® technology according to the manufacturer's instructions.

Techniques:

Journal: Frontiers in Immunology

Article Title: HRV16 Impairs Macrophages Cytokine Response to a Secondary Bacterial Trigger

doi: 10.3389/fimmu.2018.02908

Figure Lengend Snippet: Cytokine response of human macrophages exposed to HRV16 or TLR agonists for 24 h and then challenged with NTHi or LPS. Human macrophages were exposed to HRV16 (red bars), LPS (blue bars), CpG (orange bars) or MI (black bars) for 1 h and rested overnight. Then they were exposed to NTHi or LPS for 2 h and supernatants were collected and analyzed by MSD. MSD results for (A) IFNγ, (B) IL10, (C) IL12p70, (D) IL1β, (E) IL4, (F) IL6, (G) IL8, (H) TNFα. * p

Article Snippet: Cytokine production by macrophages was analyzed using the Meso Scale Discovery® technology according to the manufacturer's instructions.

Techniques:

Journal: Frontiers in Immunology

Article Title: HRV16 Impairs Macrophages Cytokine Response to a Secondary Bacterial Trigger

doi: 10.3389/fimmu.2018.02908

Figure Lengend Snippet: Cytokine response of human macrophages exposed to HRV16 or TLR agonists for 72 h and then challenged with NTHi or LPS. Human macrophages were exposed to HRV16 (red bars), LPS (blue bars), CpG (orange bars) or MI (black bars) for 1 h and rested for 72 h. Then they were exposed to NTHi or LPS for 2 h and supernatants were collected and analyzed by MSD. MSD results for (A) IFNγ, (B) IL10, (C) IL12p70, (D) IL1β, (E) IL4, (F) IL6, (G) IL8, (H) TNFα. (I) Relative fold changes for cytokine production in HRV16, LPS or CpG exposed human macrophages. * p

Article Snippet: Cytokine production by macrophages was analyzed using the Meso Scale Discovery® technology according to the manufacturer's instructions.

Techniques:

Journal: Frontiers in Immunology

Article Title: HRV16 Impairs Macrophages Cytokine Response to a Secondary Bacterial Trigger

doi: 10.3389/fimmu.2018.02908

Figure Lengend Snippet: Human macrophages release pro-inflammatory and regulatory cytokines in response to HRV16. (A) Experimental protocol: human macrophages differentiated from blood monocytes were exposed to HRV16 (red bars), HRV16 UV (open bars) or MI (black bars) for 1 h and rested overnight. Supernatants were collected and analyzed by MSD. MSD results for (B) IFNγ, (C) IL10, (D) IL12p70, (E) IL1β, (F) IL4, (G) IL6, (H) IL8, (I) TNFα, (J) Relative fold changes for cytokine production in HRV16 and HRV16 UV exposed human macrophages vs. MI. n = 5 independent experiments on different donors. Error bars represent standard error of the mean (SEM). * p

Article Snippet: Cytokine production by macrophages was analyzed using the Meso Scale Discovery® technology according to the manufacturer's instructions.

Techniques:

Journal: Influenza and Other Respiratory Viruses

Article Title: The mucosal and systemic immune responses elicited by a chitosan‐adjuvanted intranasal influenza H5N1 vaccine

doi: 10.1111/j.1750-2659.2011.00271.x

Figure Lengend Snippet: The Th1, Th2 and Th17 cytokine profiles after in vitro activation of spleen cells collected 3 weeks after second vaccine dose. Groups of five mice were intranasally immunised twice (21 days apart) with a subunit (SU) influenza A H5N1 vaccine. The control group consisted of unimmunised mice. Three groups were vaccinated with different antigen doses (7.5, 15 or 30 μg HA) of the chitosan‐adjuvanted SU vaccine. One group was vaccinated with a non‐adjuvanted SU vaccine with 30 μg HA, and a further group was immunised with a non‐adjuvanted 15 μg HA whole virus vaccine. Splenocytes were collected 3 weeks after the second immunisation, and the Bio‐Plex Pro cytokine assay was used to quantify the different cytokines secreted from the in vitro stimulated spleen cells. The data are presented as the mean cytokine concentration (pg/ml) in the supernatant from the stimulated splenocytes ± standard error of the mean for cytokines typically produced by Th1 cells (A), Th2 cells (B) and Th17 cells (C). HA, haemagglutinin.

Article Snippet: Cells were then stained for CD3, CD4, CD8 and intracellular cytokine production (IFN‐γ, IL‐2, TNF‐α), and live lymphocytes were acquired using a BD FACSCanto flow cytometer.

Techniques: In Vitro, Activation Assay, Mouse Assay, Cytokine Assay, Concentration Assay, Produced

Journal: Influenza and Other Respiratory Viruses

Article Title: The mucosal and systemic immune responses elicited by a chitosan‐adjuvanted intranasal influenza H5N1 vaccine

doi: 10.1111/j.1750-2659.2011.00271.x

Figure Lengend Snippet: The multifunctional influenza‐specific Th1 CD4+ cytokine‐secreting response. Groups of five mice were intranasally immunised twice (21 days apart) with a subunit (SU) influenza A H5N1 vaccine. The control group consisted of unimmunised mice. Three groups were vaccinated with different antigen doses (7.5, 15 or 30 μg HA) of the chitosan‐adjuvanted (+) SU vaccine. One group was vaccinated with a non‐adjuvanted (−) SU vaccine with 30 μg HA, and a further group was immunised with a non‐adjuvanted 15 μg HA whole virus (WV) vaccine. Splenocytes were activated in vitro with homologous H5N1 antigen and were intracellularly stained for cytokine products and analysed by flow cytometry. The bars show the mean frequencies of multifunctional cells expressing combinations of IFN‐γ, IL‐2 and TNF‐α. HA, haemagglutinin.

Article Snippet: Cells were then stained for CD3, CD4, CD8 and intracellular cytokine production (IFN‐γ, IL‐2, TNF‐α), and live lymphocytes were acquired using a BD FACSCanto flow cytometer.

Techniques: Mouse Assay, In Vitro, Staining, Flow Cytometry, Cytometry, Expressing

Journal: Cytometry

Article Title: Identification and visualization of multidimensional antigen‐specific T‐cell populations in polychromatic cytometry data

doi: 10.1002/cyto.a.22623

Figure Lengend Snippet: Application of t‐SNE to the TB data set. A : t‐SNE plots for the Ag‐specific T cells on the two selected peptide stimulations (Ag85B: MTB nonspecific, and ESAT‐6: MTB specific) with subjects stratified according to their MTB infection status. Colors of the points indicate the number of cytokines expressed by each single cell (degree of functionality). B : Similar to (A), but the colors indicate the fluorescent intensities of cytokine IFNγ at the single‐cell level. C : Similar to (A), but t‐SNE was applied to total CD4+ T‐cells. D : Similar to (B), but t‐SNE was applied to total CD4+ T‐cells.

Article Snippet: OpenCyto was used for automated gating of T‐cell subsets based on cytokine production, while gates for major phenotypic subsets were imported from FlowJo.

Techniques: Infection

Journal: Cytometry

Article Title: Identification and visualization of multidimensional antigen‐specific T‐cell populations in polychromatic cytometry data

doi: 10.1002/cyto.a.22623

Figure Lengend Snippet: Application of t‐SNE to the Ag‐specific T cells for HVTN078 data set. Left panel: colors indicate cell subsets of differing cytokine polyfunctionality (degree > 1). Condition‐specific differences are visible. Right panel: colors indicate degree 1 (single‐marker) expression of different cytokines in single cells. No condition‐specific differences are visible.

Article Snippet: OpenCyto was used for automated gating of T‐cell subsets based on cytokine production, while gates for major phenotypic subsets were imported from FlowJo.

Techniques: Marker, Expressing

Journal: Cytometry

Article Title: Identification and visualization of multidimensional antigen‐specific T‐cell populations in polychromatic cytometry data

doi: 10.1002/cyto.a.22623

Figure Lengend Snippet: Pipeline for visualizing t‐SNE projected T‐cell subsets. Using OpenCyto FCS files are first gated to extract major T‐cell populations (e.g., CD4 +) and their different cytokine producing subsets. The samples within each group are concatenated and subsampled such that each concatenated sample contains the same number of T‐cell events. t‐SNE is then used to project and visualize individual cell events from these concatenated samples into a two dimensional space.

Article Snippet: OpenCyto was used for automated gating of T‐cell subsets based on cytokine production, while gates for major phenotypic subsets were imported from FlowJo.

Techniques:

Journal: Cytometry

Article Title: Identification and visualization of multidimensional antigen‐specific T‐cell populations in polychromatic cytometry data

doi: 10.1002/cyto.a.22623

Figure Lengend Snippet: Application of t‐SNE to the HVTN078 data set. A : t‐SNE plots for Ag‐specific T cells (upper half of panel) and total CD4+ T cells (lower half of panel) for ENV stimulated samples from two different vaccine treatment groups (T1 and T2). Colors of the points indicate the number of cytokines expressed by each single cell (degree of functionality). B : Similar to (a), but the colors indicate the fluorescent intensities of cytokine IL2 at the single‐cell level.

Article Snippet: OpenCyto was used for automated gating of T‐cell subsets based on cytokine production, while gates for major phenotypic subsets were imported from FlowJo.

Techniques:

Journal: Scientific Reports

Article Title: Tolerogenic dendritic cells are efficiently generated using minocycline and dexamethasone

doi: 10.1038/s41598-017-15569-1

Figure Lengend Snippet: Suppressed cytokine production of minocycline/dexamethasone-conditioned tDCs. DCs (2 × 10 5 cells/well) were stimulated with LPS for TNF-α production, or IFN-γ plus TNF-α for the other cytokine production, for 24 h, and cytokine secretion in the supernatant was determined by ELISA. The data are presented as the mean ± SD of at least three independent experiments. One way ANOVA tests were performed in order to evaluate significance. # P

Article Snippet: To induce maturation or cytokine production, DCs were exposed to 50 ng/mL IFN-γ and 50 ng/mL TNF-α (both from PeproTech) for 24 h, except in the experiments that induce TNF-α production from DCs.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Antigen Is Required for the Activation of Effector Activities, whereas Interleukin 2 Is Required for the Maintenance of Memory in Ovalbumin-specific, CD8+ Cytotoxic T Lymphocytes

doi:

Figure Lengend Snippet: Metabolic inhibitors prevent cytokine production, but not cytolytic activity of CTLs. OVA-CTLs (10 6 /ml) were pretreated with 5 μg/ml actinomycin D, 15 μg/ml cycloheximide, or medium at 37°C for 1 h. The cells were then washed and incubated with 10 5 of E.G7-OVA. After incubation at 37°C for various periods of time, supernatants were tested for production of ( A ) IFN-γ and ( B ) TNF-α by ELISA. In C , pretreated OVA-CTLs were incubated with 51 Cr-labeled targets. After 4 h, supernatant fluids were collected and counted. The results of one of two similar experiments are shown as the mean of triplicates ± SD.

Article Snippet: Cytokine production was tested by two-site sandwich ELISA using a mouse IFN-γ kit (Genzyme, Cambridge, MA) or TNF-α kit ( Endogen, Inc. , Boston, MA) ( , ).

Techniques: Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Antigen Is Required for the Activation of Effector Activities, whereas Interleukin 2 Is Required for the Maintenance of Memory in Ovalbumin-specific, CD8+ Cytotoxic T Lymphocytes

doi:

Figure Lengend Snippet: Comparison of cytokine production between stimulated and unstimulated CTLs. Stimulated OVA-CTLs were stimulated weekly with irradiated E.G7-OVA, syngeneic filler cells, and Con A supernatant. Unstimulated OVA-CTLs were maintained by weekly replenishment with irradiated filler cells and Con A supernatant, but without E.G7-OVA. After 100 d, 10 6 of stimulated or unstimulated OVA-CTLs were incubated with 10 5 of EL4 or E.G7-OVA, or without targets. After incubation at 37°C, supernatants were tested for production of ( A ) IFN-γ and ( B ) TNF-α by ELISA. The results shown are the mean of triplicates ± SD of a representative experiment that has been repeated twice.

Article Snippet: Cytokine production was tested by two-site sandwich ELISA using a mouse IFN-γ kit (Genzyme, Cambridge, MA) or TNF-α kit ( Endogen, Inc. , Boston, MA) ( , ).

Techniques: Irradiation, Incubation, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: The immunoproteasome‐specific inhibitor ONX 0914 reverses susceptibility to acute viral myocarditis

doi: 10.15252/emmm.201708089

Figure Lengend Snippet: Impact of ONX 0914 on CVB 3 infection in C57 BL /6 mice Starting one day prior to CVB3 (Nancy) inoculation, C57BL/6 mice were treated daily until day 10 p.i. with either ONX 0914 or vehicle and further three times a week until day 28 p.i. Survival (A) as well as body weight (B) was monitored for 28 days. Average percentages of body weight loss relative to initial weight ± SEM are shown (vehicle n = 8, ONX 0914 n = 8). On day 8 p.i., where inflammation of the myocardium is expected to be fully developed, animals were sacrificed (C), and heart tissue was analyzed. Paraffin‐embedded heart sections were stained using hematoxylin and eosin. A representative image for each group is shown. Extent of myocardial infiltration was scored microscopically, and percentage of inflamed area was assessed and calculated using ImageJ (vehicle n = 13, ONX 0914 n = 11). Total heart tissue mRNA was isolated (D), reverse transcribed and cytokine/chemokine‐specific gene expression was determined by TaqMan qPCR (vehicle n = 10, ONX 0914 n = 10). Virus load (E) was determined by standard plaque assay assessing the amount of infectious virus particles (vehicle n = 13, ONX 0914 n = 10). IFN‐β serum levels (F) during early infection (day 2 p.i.) were determined by ELISA (control=uninfected animals, vehicle n = 7, ONX 0914 n = 7). CVB3‐neutralizing antibody titers in the serum (G) were determined after 8 and 28 days post CVB3 infection (day 8 vehicle n = 13, ONX 0914 n = 10, day 28 vehicle n = 8, ONX 0914 n = 7). To investigate the impact of ONX 0914 treatment on developing an immunological memory status, all mice were challenged with a second CVB3 infection on day 28 after the initial one. Animals received no further ONX 0914 treatment. Proportional body weight loss on day 8 after second challenge relative to the weight on day 28 after the initial challenge (H). Viral load of heart tissue (I) was determined by plaque assay after 8 days. Additionally, paraffin‐embedded heart tissue sections were prepared and Masson's trichrome staining was performed (J). Representative images for each group are shown (one experiment was carried out; vehicle n = 8, ONX 0914 n = 6). Data information: Data are mean ± SEM. Scale bars, 60 μm. P ‐values are indicated in each graph. Survival curves were estimated from the Kaplan–Meier procedure with the log‐rank (Mantel–Cox). For body weight course, two‐way ANOVA was used. Otherwise, unpaired t ‐test was conducted.

Article Snippet: These studies are based on the highly regulated immunoproteasome function of controlling cytokine production and antigen presentation (Muchamuel et al , ; Basler et al , , ; Groettrup et al , ; Kalim et al , ; Mundt et al , ), which is in turn intensively influenced through its inhibition.

Techniques: Infection, Mouse Assay, Staining, Isolation, Expressing, Real-time Polymerase Chain Reaction, Plaque Assay, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: The immunoproteasome‐specific inhibitor ONX 0914 reverses susceptibility to acute viral myocarditis

doi: 10.15252/emmm.201708089

Figure Lengend Snippet: ONX 0914‐mediated suppression of CVB 3‐induced pro‐inflammatory cytokines/chemokines in A/J mice A/J mice were infected with CVB3. ONX 0914 or vehicle treatment was carried out daily, starting one day prior to virus inoculation. Animals were sacrificed after 3‐day (A, B) and 8‐day p.i. (C). Total spleen mRNA (A) was used to determine cytokine/chemokine‐specific expression of the indicated genes by TaqMan qPCR. Means of 2 −Δ C t + SEM are shown (vehicle n = 7, ONX n = 9, t ‐tests). Serum concentration of the indicated cytokines/chemokines (B) was assessed using bead‐based multiplex immunoassay or ELISA, respectively. Depicted are means + SEM (four separate experiments were carried out; vehicle n = 3–7, ONX n = 3–7, t ‐tests). During the peak of heart muscle inflammation, total heart mRNA (C) was used to determine cytokine/chemokine‐specific expression of the indicated genes by TaqMan qPCR. Means of 2 −Δ C t + SEM are shown (E1 + E2; vehicle n = 4, ONX n = 12, t ‐tests). P ‐values are indicated in each graph.

Article Snippet: These studies are based on the highly regulated immunoproteasome function of controlling cytokine production and antigen presentation (Muchamuel et al , ; Basler et al , , ; Groettrup et al , ; Kalim et al , ; Mundt et al , ), which is in turn intensively influenced through its inhibition.

Techniques: Mouse Assay, Infection, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: The immunoproteasome‐specific inhibitor ONX 0914 reverses susceptibility to acute viral myocarditis

doi: 10.15252/emmm.201708089

Figure Lengend Snippet: ONX 0914 manipulates TLR 7 signaling leading to reduced MAPk inase phosphorylation Bone marrow‐derived macrophages (BMM) from A/J mice were cultivated. Mitogen‐activated protein kinase (MAPkinase) inhibitors were used to investigate the contribution of p38‐, ERK1/2‐, and JNK‐mediated signaling to cytokine/chemokine expression upon TLR7 engagement. Therefore, BMM were treated with the MAPkinase inhibitors SB203580 (p38), PD98059 (ERK1/2), SP600125 (JNK), or DMSO (control), respectively, for one hour prior to R848 stimulation. After 8 h, mRNA levels of the indicated genes were determined by TaqMan qPCR. Data represent fold change increased mRNA expression (ΔΔ C t normalized to stimulated DMSO control) and are mean of three independent experiments. Paired t ‐tests (inhibitor versus DMSO) were performed. BMM from A/J mice were treated with ONX 0914 or DMSO prior to stimulation with the TLR7 agonist R848. After 8 h, cytokine and chemokine expression was determined for the indicated genes by TaqMan qPCR. Data represent fold change increased mRNA expression (normalized to un‐stimulated DMSO control) and are mean of four independent experiments. Paired t ‐tests were performed. BMM from A/J mice were treated with ONX 0914 or DMSO prior to stimulation with R848 for the indicated points in time. Cytosolic protein extracts were subjected to Western blot analysis for detection of the phosphorylation status of p38 (Thr180/Tyr182), ERK1/2 (Thr202/Tyr204) and JNK (Thr183/Tyr185) (C). Antibodies directed against the respective total MAPkinases were used as respective loading control. The most intense phosphorylation status for all MAPkinases was observed after 15‐min TLR7 engagement. Signal intensity at this point in time was determined for DMSO‐ and ONX 0914‐treated cells (D), and signals of phosphorylated kinases were normalized to the respective non‐phosphorylated protein. For three independent experiments, thereby obtained data of ONX 0914‐treated cells were normalized to respective DMSO controls and plotted bar graphs demonstrate mean of the respective phosphorylated MAPkinases (unpaired t ‐test). Data information: Data presented as mean ± SEM. P ‐values are indicated in each graph. Source data are available online for this figure.

Article Snippet: These studies are based on the highly regulated immunoproteasome function of controlling cytokine production and antigen presentation (Muchamuel et al , ; Basler et al , , ; Groettrup et al , ; Kalim et al , ; Mundt et al , ), which is in turn intensively influenced through its inhibition.

Techniques: Derivative Assay, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Immunology Letters

Article Title: The role of individual protein kinase C isoforms in mouse mast cell function and their targeting by the immunomodulatory parasitic worm product, ES-62

doi: 10.1016/j.imlet.2015.09.001

Figure Lengend Snippet: FcϵRI-mediated cytokine release by PKC isoform −/− and matched WT BMMCs. BMMCs were sensitized with murine anti-DNP IgE antibodies (IgE; 0.5 μg/mL) for 24 h then stimulated with DNP-BSA to induce cross-linking (XL) for 24 h. Cell culture supernatants were harvested and cytokine levels of IL-6 and TNF-α release were measured by ELISA from PKC isoform-deficient and corresponding WT cells: PKC-α (a and b), PKC-β (c), PKC-ϵ (d) and PKC-θ (e and f) (Means ± SD, n = 3). The data presented are single experiments representative of at least 3 independent experiments apart from those involving PKC-α −/− and PKC–β −/− IL-6 release, which are representative of 2 individual statistically significant experiments. Statistical analysis was by ANOVA with Bonferoni post test where * P

Article Snippet: The statistical analysis for cytokine production was performed in Graphpad PRISM using either a one way ANOVA or a two way ANOVA with Bonferoni post test where *P < 0.05 **P < 0.005 *** P < 0.001 **** P < 0.0001.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Immunology Letters

Article Title: The role of individual protein kinase C isoforms in mouse mast cell function and their targeting by the immunomodulatory parasitic worm product, ES-62

doi: 10.1016/j.imlet.2015.09.001

Figure Lengend Snippet: TLR-4-mediated cytokine release by PKC isoform −/− and matched WT BMMCs (a: PKC-α; b and c: PKC-θ; d and e: PKC-β). BMMCs were incubated with LPS (1 μg/ml) for 24 h. IL-6 and TNF-α release were measured by ELISA. The data presented are single experiments representative of at least 3 independent experiments with the exception of those involving PKC-θ −/− TNF-α release, which are representative of 2 experiments. Statistical analysis was by ANOVA with Bonferoni post test where * P

Article Snippet: The statistical analysis for cytokine production was performed in Graphpad PRISM using either a one way ANOVA or a two way ANOVA with Bonferoni post test where *P < 0.05 **P < 0.005 *** P < 0.001 **** P < 0.0001.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Immunology Letters

Article Title: The role of individual protein kinase C isoforms in mouse mast cell function and their targeting by the immunomodulatory parasitic worm product, ES-62

doi: 10.1016/j.imlet.2015.09.001

Figure Lengend Snippet: Model of differential and interactive roles of PKC isoforms in mast cells. (a) The conventional PKC isoforms α and β along with the novel isoforms ϵ and θ exhibit differential functional roles in mast cells with respect to both antigen-induced FcϵRI cross-linking and LPS stimulation. PKC-α and PKC-θ serve similar functions through promoting IL-6 cytokine secretion following antigen stimulation and this is somewhat counter-regulated by PKC-β and ϵ. The specific role of a PKC isoform also appears to be context-dependent as, for example, PKC-α acts as a negative regulator of IL-6 production in response to LPS/TLR-4 signalling yet PKC-θ positively regulates this cytokine in response to LPS. Unlike the other PKC isoforms, the role that PKC-ϵ plays in mast cell functional responses seems to be restricted to the FcϵRI pathway. (b) The signalling mechanisms whereby ES-62 inhibits mast cell function have not been fully delineated, however, we indicate that ES-62 targets key signals involved in mast cell activation such as PKC-α. Through targeting this isoform, ES-62 may also have an indirect effect on other PKC isoforms such as PKC-ϵ (a negative regulator of BMMC responses) which in turn leads to a further reduction in the secretion of pro-inflammatory cytokines. We hypothesise that ES-62 may also target PKC-θ as this isoform has not only been shown to be crucial for full mast cell activation, but it may share redundant or similar roles to PKC-α. Additionally, the expression levels of PKC-θ appear to regulate those of PKC-α and therefore through targeting this isoform, ES-62 may again be indirectly targeting a network of PKC isoforms that includes, but is not restricted to, PKC-α.

Article Snippet: The statistical analysis for cytokine production was performed in Graphpad PRISM using either a one way ANOVA or a two way ANOVA with Bonferoni post test where *P < 0.05 **P < 0.005 *** P < 0.001 **** P < 0.0001.

Techniques: Functional Assay, Cell Function Assay, Activation Assay, Expressing

Journal: Journal of blood disorders & transfusion

Article Title: Characterization of Bone Marrow-Derived Dendritic Cells Developed in Serum-Free Media and their Ability to Prevent Type 1 Diabetes in Nonobese Diabetic Mice

doi: 10.4172/2155-9864.1000206

Figure Lengend Snippet: Splenic cytokine producing cells and pancreatic-draining lymph node specific Foxp3 expression. A–C. Two weeks following BM-DC treatment, 3×10 5 spleen cells were re-stimulated with GAD217-236 peptide for 4 days (n=3). Total spot forming units (SFU) were enumerated for the indicated cytokines with an automated spot counter. D,E. Two weeks following BM-DC treatment, Inguinal (IngLN) and Pancreatic-draining Lymph Node (PLN) CD4 + T cells were examined for Foxp3 expression (n=3). D. Representative dot plots of showing Foxp3 production in CD4 + T cells.

Article Snippet: In vitro antigen specific T cell proliferation and cytokine production CD4+ T cells were isolated from spleens of BDC2.5 NOD females using CD4 Microbeads (Miltenyi Biotec).

Techniques: Expressing