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  • 95
    Thermo Fisher total ige
    Effect of FAO inhibition on AHR and <t>IgE</t> production in <t>OVA-exposed</t> mice (A) A model for the treatment of OVA-induced asthma-like traits in mice. (B) One day after OVA challenge and treatment with etomoxir, AHR to inhaled methacholine was measured in unrestrained conscious C57BL/6J mice. (C) The experiment was repeated as in (B) except that mice were treated with ranolazine. AHR was measured one day after challenge and treatment. (D) The OVA sensitization and challenge followed by treatment with etomoxir or ranolazine were conducted on Balb/c mice. AHR was measured one day after allergen exposure and treatment. (E) Two days post OVA challenge and treatment with etomoxir in C57BL/6J mice, BAL fluids and sera were collected to measure the levels of OVA-specific IgE. (F) The levels of total IgE were measured in sera of the indicated conditions. Data = mean ± SEM; n = 5 mice/group; *, P
    Total Ige, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore ige
    Effect of FAO inhibition on AHR and <t>IgE</t> production in <t>OVA-exposed</t> mice (A) A model for the treatment of OVA-induced asthma-like traits in mice. (B) One day after OVA challenge and treatment with etomoxir, AHR to inhaled methacholine was measured in unrestrained conscious C57BL/6J mice. (C) The experiment was repeated as in (B) except that mice were treated with ranolazine. AHR was measured one day after challenge and treatment. (D) The OVA sensitization and challenge followed by treatment with etomoxir or ranolazine were conducted on Balb/c mice. AHR was measured one day after allergen exposure and treatment. (E) Two days post OVA challenge and treatment with etomoxir in C57BL/6J mice, BAL fluids and sera were collected to measure the levels of OVA-specific IgE. (F) The levels of total IgE were measured in sera of the indicated conditions. Data = mean ± SEM; n = 5 mice/group; *, P
    Ige, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 656 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Mimotopes immunoglobulin e ige allergen binding
    Peptide ELISA demonstrating the binding of Met e 1-specific <t>IgE</t> to the mimotopes. Mimotope KGIPNTKAP of Der p 1 and an IgE-binding epitope of Met e 1 (a.a. 251-270) were included as negative and positive controls, respectively. Met e 1-specific IgE could exclusively recognize the selected mimotopes from clusters 1–6, but not the irrelevant mimotope of Der p 1 ( P
    Immunoglobulin E Ige Allergen Binding, supplied by Mimotopes, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    4Gene ige stimulated upregulation
    Characterization of synthesized (−)-maackiain. (A) Separation of (−)-maackiain from (+)-maackiain by Chiral column chromatography. (B) Effect of synthesized (−)-maackiain and (+)-maackiain on PMA-induced <t>upregulation</t> of H1R mRNA expression in HeLa cells (a) and on <t>IgE/antigen-stimulated</t> upregulation of IL-4 mRNA expression in RBL-2H3 cells (b) (a), HeLa cells were serum-starved for 24 h, and stimulated with 100 nmol/L PMA for 3 h. Synthesized (−)- or (+)-maackiain was treated 24 h before PMA stimulation. After stimulation, total RNA was isolated, and the H1R mRNA levels were determined by real-time quantitative RT-PCR. (b), RBL-2H3 cells were treated with 100 ng/mL anti-DNP IgE for 18 h and then stimulated with 100 ng/mL DNP-HSA for 2 h. Synthesized (−)- or (+)-maackiain was treated 24 h before DNP-HSA stimulation. After stimulation, total RNA was isolated, and the IL-4 mRNA levels were determined by real-time quantitative RT-PCR. Data are presented as the mean ± SEM ( n = 3). **, P
    Ige Stimulated Upregulation, supplied by 4Gene, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore spe 7 ige
    Monovalent DNP inhibits <t>SPE-7</t> <t>IgE</t> induced LAD-2 cell activation. (A) Percentage degranulation of LAD-2 cells, induced by 30 nM SPE-7 IgE, was dose-dependently inhibited by addition of indicated molar excess of monovalent DNP-L-serine. (B) Data are represented as an inhibition curve. IC 50 of monovalent DNP-L-serine inhibition of SPE-7 IgE induced degranulation = 1.43 μM. (C) LAD-2 cell TNF-α release, induced by 30 nM SPE-7 IgE, was dose-dependently inhibited by addition of indicated molar excess of monovalent DNP-L-serine. (D) IC 50 of monovalent DNP-L-serine inhibition of SPE-7 IgE induced TNF-α release = 1.57 μM. Lower and upper dashed lines indicate cell-only background control and activation by SPE-7 IgE only, respectively. Means of 3 to 7 independent experiments ± SEM are shown. Statistically significant difference to SPE-7 IgE only was determined by one-way ANOVA with Dunnett's post-test; *** p
    Spe 7 Ige, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher in vitro ige detection
    ELISA data of 4 representative patients. A, Direct ELISA showing <t>IgE</t> binding to <t>rBet</t> v 1.0101, rApi g 1.0101, the chimeras, and a mix of all chimeras (nonnormalized OD values). B and C, Inhibition of IgE binding to immobilized rBet v 1.0101 (Fig 3, B ) or the chimeras (Fig 3, C ) by means of preincubation with rBet v 1.0101 (positive control), rApi g 1.0101, and the chimeras. n.d ., Not done.
    In Vitro Ige Detection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Glycoform lectin inhibition
    ELISA data of 4 representative patients. A, Direct ELISA showing <t>IgE</t> binding to <t>rBet</t> v 1.0101, rApi g 1.0101, the chimeras, and a mix of all chimeras (nonnormalized OD values). B and C, Inhibition of IgE binding to immobilized rBet v 1.0101 (Fig 3, B ) or the chimeras (Fig 3, C ) by means of preincubation with rBet v 1.0101 (positive control), rApi g 1.0101, and the chimeras. n.d ., Not done.
    Lectin Inhibition, supplied by Glycoform, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Serotech ige
    Antigen-specific <t>IgE/FcεRI-crosslinking</t> on activated DCs inhibits the production of proinflammatory cytokines and chemokines resulting in impaired migration of inflammatory cells. (a) IgE-crosslinking inhibits the papain-induced production of CCL-2, IL-6, and TNF-α by murine DCs. (b) Inhibition of CCL-2 production in human DCs by IgE/FcεRI-crosslinking. (c) The chemokine CCL-2 shows strong chemotactic activity for c-Kit + mast cell progenitors and CD11c + DC. Indicated concentrations of recombinant CCL-2 were added to the medium in the lower wells of transwell plates and the migration of cells from mast cell progenitor cultures towards the chemokine was determined. (d) Antigen-specific IgE/FcεRI-mediated DC activation (OVA plus IgE) inhibits migration of bone-marrow derived mast cell progenitors and DC. DCs were activated as indicated and DC-conditioned supernatants were used for chemotaxis assays. Migratory response of cKit + cells and CD11c + cells towards DC-conditioned supernatants was calculated from 3 independent experiments. Migratory response of DC stimulated with LPS or papain in the absence of IgE/FcεRI-mediated DC activation (OVA alone) was set at 100 %.
    Ige, supplied by Serotech, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher ige
    Antigen-specific <t>IgE/FcεRI-crosslinking</t> on activated DCs inhibits the production of proinflammatory cytokines and chemokines resulting in impaired migration of inflammatory cells. (a) IgE-crosslinking inhibits the papain-induced production of CCL-2, IL-6, and TNF-α by murine DCs. (b) Inhibition of CCL-2 production in human DCs by IgE/FcεRI-crosslinking. (c) The chemokine CCL-2 shows strong chemotactic activity for c-Kit + mast cell progenitors and CD11c + DC. Indicated concentrations of recombinant CCL-2 were added to the medium in the lower wells of transwell plates and the migration of cells from mast cell progenitor cultures towards the chemokine was determined. (d) Antigen-specific IgE/FcεRI-mediated DC activation (OVA plus IgE) inhibits migration of bone-marrow derived mast cell progenitors and DC. DCs were activated as indicated and DC-conditioned supernatants were used for chemotaxis assays. Migratory response of cKit + cells and CD11c + cells towards DC-conditioned supernatants was calculated from 3 independent experiments. Migratory response of DC stimulated with LPS or papain in the absence of IgE/FcεRI-mediated DC activation (OVA alone) was set at 100 %.
    Ige, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1875 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biogen Inc ige
    Antigen-specific <t>IgE/FcεRI-crosslinking</t> on activated DCs inhibits the production of proinflammatory cytokines and chemokines resulting in impaired migration of inflammatory cells. (a) IgE-crosslinking inhibits the papain-induced production of CCL-2, IL-6, and TNF-α by murine DCs. (b) Inhibition of CCL-2 production in human DCs by IgE/FcεRI-crosslinking. (c) The chemokine CCL-2 shows strong chemotactic activity for c-Kit + mast cell progenitors and CD11c + DC. Indicated concentrations of recombinant CCL-2 were added to the medium in the lower wells of transwell plates and the migration of cells from mast cell progenitor cultures towards the chemokine was determined. (d) Antigen-specific IgE/FcεRI-mediated DC activation (OVA plus IgE) inhibits migration of bone-marrow derived mast cell progenitors and DC. DCs were activated as indicated and DC-conditioned supernatants were used for chemotaxis assays. Migratory response of cKit + cells and CD11c + cells towards DC-conditioned supernatants was calculated from 3 independent experiments. Migratory response of DC stimulated with LPS or papain in the absence of IgE/FcεRI-mediated DC activation (OVA alone) was set at 100 %.
    Ige, supplied by Biogen Inc, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Novoprotein syk inhibition
    Spleen tyrosine kinase <t>(SYK)</t> inhibition in tonsillar B cells decreases viability, activation, and effector functions. Tonsillar cells activated in vitro by incubation with CD40L fibroblasts (1:10 ratio, fibroblasts: tonsillar cells), anti-μ antibody (10 µg/ml) and LPS (1 µg/ml) Non-activated tonsillar cells were cultured with CD32L fibroblasts (1:10 ratio, fibroblasts: tonsillar cells). (A) Following incubation for 20 min in the presence or absence of BAY61-3606 (5 µM), B cells were identified on the basis of their expression of CD19, and SYK phosphorylation status was determined by flow cytometry. (B) Following 3 days of culture, cells were stained with a fixable viability stain and identified on the basis of their CD19 expression status, by flow cytometry. Dead B cells were identified with a fixable viability stain. The boxplot shows the distribution between donors, and the quadrant indicates the values between the fifth and ninety-fifth percentiles, n = 7. (C) Following 3 days of culture, cells were stained with fixable viability stain, and for CD19, sIgD, CD38, CD80, and myeloid cell leukemia-1 (Mcl-1), and analyzed by flow cytometry. Viable cells were identified as fixable viability stain-negative. Mcl-1 levels were determined in CD19-positive cells and in pre-GC (BM2′) and GC B cells (BM3 + BM4) identified on the basis of sIgD and CD38 expression profiles. Activation state was determined by assessing CD80 expression in CD19-positive cells and in pre-GC (BM2′) and GC B-cells (BM3 + BM4). The boxplot represents the distribution between donors and the quadrant indicates the values between the fifth and ninety-fifth percentiles; significant P values are shown on the graphs for comparisons between all stimulated cells and those treated with BAY61-3606, n = 7. (D) The total immunoglobulin G (IgG) secreted by the cells during 3 days of culture was analyzed by ELISA. The boxplot represents the distribution between donors and the quadrant indicates the values between the fifth and ninety-fifth percentiles, n = 9. stim, stimulated; Bay, BAY61-3606; N-A, non-activated; CD, cluster of differentiation.
    Syk Inhibition, supplied by Novoprotein, used in various techniques. Bioz Stars score: 96/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson ige
    No cross-reactivity between bGST and HDM-GST. (A) <t>IgG1-reactivity</t> of sera (n = 3) collected from bGST-immunized mice at day 0 and day 59 to BPE and HDME. (B) <t>IgE-reactivity</t> of seven Der p 8-sensitized HDM-allergic patients to bGST and HDME. NHS, non-allergic control sera; O.D. optical density; dotted and dashed lines indicate the cut-off for positive IgE-reactivity.
    Ige, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1480 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    BioLegend pe conjugated ige
    Immunosuppression by Pregnenolone and Pregnenolone-Producing Cells (A–C) Pregnenolone inhibits Th cell proliferation. Naive Th cells were stained with CellTrace Violet and activated for Th1 (A) or Th2 (B) differentiation for 72 hr in the presence (blue histogram) or absence (red histogram) of pregnenolone (5 μM). The cell proliferation profile was captured by FACS based on dye decay. The histograms depicted are representative of three independent experiments with three to five mice each. (C) Mean division indices were calculated for the experiments described in (A) and (B). Division index is the average number of divisions that a cell (present in the starting population) has undergone. p values were calculated using unpaired two-tailed t test. (D) Ly6C + Th2 cells inhibit Th cell proliferation. CellTrace-Violet-stained naive Th cells (“responder cells”) were activated by anti-CD3e and anti-CD28 in the presence of FACS-sorted Ly6C + or Ly6C − Th2 cells and anti-IL-10 antibody and/or AG as indicated. The proliferation profile of the responder Th cells grown in the absence of Th2 (blue) was captured by FACS on the third day of activation. This was compared to the proliferation profile of responder Th cells activated in the presence of Th2 only (Ly6C + or Ly6C − ) (red). The Ly6C + cells were pretreated with neutralizing anti-IL-10 antibody (20 μg/ml) or with aminoglutethimide (AG, 250 μM) or with both for 24 hr and were used in the same culture conditions. The responder Th cell to Th2 (Ly6C + or Ly6C − ) cell ratio was 1:1. The histograms depicted are representative of three independent experiments with three mice in each experiment. Mean division indices ±SD are shown in the inset (right, bottom). p values were calculated using unpaired two-tailed t test. (E) Inhibition of Cyp11a1 activity negatively regulates IL-10 and TGF-β1 expression in Th2. In vitro polarized Th2 and Th1 cells (3 days activation, 2 days resting) in the presence or absence of aminoglutethimide (AG, 250 mM) were FACS analyzed after reactivation with PDBU/Ionomycin for intracellular cytokine (IL-10 and TGF-β1 for Th2; IFN-γ for Th1) expression. Data shown are representative of three independent experiments. (F) Effect of pregnenolone on B cell class switching to <t>IgG1</t> and <t>IgE.</t> Naive resting B cells were stained with CellTrace Violet. Class switch recombination (CSR) was induced with LPS and IL-4 in the presence of different concentrations of pregnenolone. Cell-surface expression of IgG1 and IgE were analyzed by FACS on day 5 of stimulation. Data shown are representative of three independent experiments with three mice each. p values were calculated using unpaired two-tailed t test and were
    Pe Conjugated Ige, supplied by BioLegend, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Mimotopes ige mimotopes
    Immunosuppression by Pregnenolone and Pregnenolone-Producing Cells (A–C) Pregnenolone inhibits Th cell proliferation. Naive Th cells were stained with CellTrace Violet and activated for Th1 (A) or Th2 (B) differentiation for 72 hr in the presence (blue histogram) or absence (red histogram) of pregnenolone (5 μM). The cell proliferation profile was captured by FACS based on dye decay. The histograms depicted are representative of three independent experiments with three to five mice each. (C) Mean division indices were calculated for the experiments described in (A) and (B). Division index is the average number of divisions that a cell (present in the starting population) has undergone. p values were calculated using unpaired two-tailed t test. (D) Ly6C + Th2 cells inhibit Th cell proliferation. CellTrace-Violet-stained naive Th cells (“responder cells”) were activated by anti-CD3e and anti-CD28 in the presence of FACS-sorted Ly6C + or Ly6C − Th2 cells and anti-IL-10 antibody and/or AG as indicated. The proliferation profile of the responder Th cells grown in the absence of Th2 (blue) was captured by FACS on the third day of activation. This was compared to the proliferation profile of responder Th cells activated in the presence of Th2 only (Ly6C + or Ly6C − ) (red). The Ly6C + cells were pretreated with neutralizing anti-IL-10 antibody (20 μg/ml) or with aminoglutethimide (AG, 250 μM) or with both for 24 hr and were used in the same culture conditions. The responder Th cell to Th2 (Ly6C + or Ly6C − ) cell ratio was 1:1. The histograms depicted are representative of three independent experiments with three mice in each experiment. Mean division indices ±SD are shown in the inset (right, bottom). p values were calculated using unpaired two-tailed t test. (E) Inhibition of Cyp11a1 activity negatively regulates IL-10 and TGF-β1 expression in Th2. In vitro polarized Th2 and Th1 cells (3 days activation, 2 days resting) in the presence or absence of aminoglutethimide (AG, 250 mM) were FACS analyzed after reactivation with PDBU/Ionomycin for intracellular cytokine (IL-10 and TGF-β1 for Th2; IFN-γ for Th1) expression. Data shown are representative of three independent experiments. (F) Effect of pregnenolone on B cell class switching to <t>IgG1</t> and <t>IgE.</t> Naive resting B cells were stained with CellTrace Violet. Class switch recombination (CSR) was induced with LPS and IL-4 in the presence of different concentrations of pregnenolone. Cell-surface expression of IgG1 and IgE were analyzed by FACS on day 5 of stimulation. Data shown are representative of three independent experiments with three mice each. p values were calculated using unpaired two-tailed t test and were
    Ige Mimotopes, supplied by Mimotopes, used in various techniques. Bioz Stars score: 88/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Siemens AG ige determination
    Frequency of typical <t>IgE</t> patterns obtained by western blot inhibition in <t>CAP</t> double sensitized patients. CCD: cross-reactive carbohydrate determinants, True DS: true double sensitization, WB: western blot, WB-I (western blot inhibition): To discriminate between IgE specific for peptide or carbohydrate epitopes, antibody binding to CCDs was inhibited by preincubating sera with MUXF-BSA. Among these patients the majority of DS was CCD-dependent. DS due to protein components of hyaluronidases played a minor role. n = 61.
    Ige Determination, supplied by Siemens AG, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Germfree Laboratories Inc hyper ige
    An increase in microbial diversity does not inhibit <t>hyper-IgE</t> despite pTreg induction. (A) Relative species abundance in the small intestine (SI) and cecum of a representative C3- and C4-colonized mouse, n = 3 mice per group. (B) Total serum IgE levels in C3 ( n = 31) and C4 ( n = 56) mice. The blue and orange horizontal lines represent the geometric mean of the GF and SPF cohorts, respectively (from Figure 1B ). (C) <t>SCFA</t> levels in the cecal contents of C3 ( n = 10–13) and C4 ( n = 7–9) mice. The orange and brown horizontal lines represent the mean of the SPF and C2 cohorts, respectively (from Figure 1C ). (D) Representative flow cytometry plots of RORγt + Helios − Tregs in different tissues (Spl, spleen; PP, Peyer's patches; MLN, mesenteric lymph nodes; SI, small intestinal lamina propria; Col, colon lamina propria) of GF, C3, C4 and SPF mice. (E) Frequencies of RORγt + Helios − Tregs among CD45 + TCRβ + CD4 + Foxp3 + T cells in different tissues of GF ( n = 10–15), C3 ( n = 4–9), C4 ( n = 4–8), and SPF ( n = 7–16) mice. All mice were 10–13 weeks old. SCFA and RORγt + Helios − Treg data are pooled from at least two independent experiments. Each symbol represents an individual mouse. Black horizontal lines depict the geometric mean (B) or mean (C,E) . * p
    Hyper Ige, supplied by Germfree Laboratories Inc, used in various techniques. Bioz Stars score: 95/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend pe anti mouse ige
    Antigen-specific <t>IgE/FcεRI-crosslinking</t> on activated DCs inhibits the production of proinflammatory cytokines and chemokines resulting in impaired migration of inflammatory cells. (a) IgE-crosslinking inhibits the papain-induced production of CCL-2, IL-6, and TNF-α by murine DCs. (b) Inhibition of CCL-2 production in human DCs by IgE/FcεRI-crosslinking. (c) The chemokine CCL-2 shows strong chemotactic activity for c-Kit + mast cell progenitors and <t>CD11c</t> + DC. Indicated concentrations of recombinant CCL-2 were added to the medium in the lower wells of transwell plates and the migration of cells from mast cell progenitor cultures towards the chemokine was determined. (d) Antigen-specific IgE/FcεRI-mediated DC activation (OVA plus IgE) inhibits migration of bone-marrow derived mast cell progenitors and DC. DCs were activated as indicated and DC-conditioned supernatants were used for chemotaxis assays. Migratory response of cKit + cells and CD11c + cells towards DC-conditioned supernatants was calculated from 3 independent experiments. Migratory response of DC stimulated with LPS or papain in the absence of IgE/FcεRI-mediated DC activation (OVA alone) was set at 100 %.
    Pe Anti Mouse Ige, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    InvivoGen tlr9 inhibition assay
    IgE-containing ICs deliver DNA to <t>TLR9</t> at the phagosome (a) Flow cytometry analysis of cell surface expression of FcεRI and FcεRII (red line) in pDCs compared isotype control (grey line). Data are representative of 3 independent experiments. (b) Mouse macrophages expressing TLR9-GFP and the alpha chain of human FcεRI (hFcεRI + ) were fed with beads coated with DNA+IgE D . TLR9-GFP localization in hFcεRI + and control macrophages (hFcεRI − ) was visualized by confocal microscopy 30 min. after bead internalization. Yellow arrows point to ingested beads. TLR9 + phagosomes were quantified ( n = 75 phagosomes per/group from three independent experiments). Data are presented as mean ± s.d. (c) Human pDCs were incubated with DNA+IgE D for 30 min. at 4°C (0 min) and then transferred to 37°C for 120 min. (120 min.). DNA+IgE D (green) and LAMP1 (red) intracellular localization was visualized by confocal microscopy and frames from one representative experiment out of three are shown. BF = bright field. Scale bars, 5μm. (d) Human pDCs were incubated for 5 h at 37°C in the presence of DNA+IgE D or untreated (ut). IRF7 intracellular localization was visualized by immunostaining using confocal microscopy. Frames from one representative experiment out of three are shown. (e) pDCs were stimulated with DNA+IgE D in the presence of 0.05 or 0.5 μg/ml of TLR9 oligodeoxynucleotide (ODN) inhibitor (TLR9 inhib) or control ODN (Control). After 16 h, IFN-α was measured by ELISA in supernatants. Data are presented as mean ± s.e.m. from three independent. * P
    Tlr9 Inhibition Assay, supplied by InvivoGen, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher nut specific ige
    IgE-containing ICs deliver DNA to <t>TLR9</t> at the phagosome (a) Flow cytometry analysis of cell surface expression of FcεRI and FcεRII (red line) in pDCs compared isotype control (grey line). Data are representative of 3 independent experiments. (b) Mouse macrophages expressing TLR9-GFP and the alpha chain of human FcεRI (hFcεRI + ) were fed with beads coated with DNA+IgE D . TLR9-GFP localization in hFcεRI + and control macrophages (hFcεRI − ) was visualized by confocal microscopy 30 min. after bead internalization. Yellow arrows point to ingested beads. TLR9 + phagosomes were quantified ( n = 75 phagosomes per/group from three independent experiments). Data are presented as mean ± s.d. (c) Human pDCs were incubated with DNA+IgE D for 30 min. at 4°C (0 min) and then transferred to 37°C for 120 min. (120 min.). DNA+IgE D (green) and LAMP1 (red) intracellular localization was visualized by confocal microscopy and frames from one representative experiment out of three are shown. BF = bright field. Scale bars, 5μm. (d) Human pDCs were incubated for 5 h at 37°C in the presence of DNA+IgE D or untreated (ut). IRF7 intracellular localization was visualized by immunostaining using confocal microscopy. Frames from one representative experiment out of three are shown. (e) pDCs were stimulated with DNA+IgE D in the presence of 0.05 or 0.5 μg/ml of TLR9 oligodeoxynucleotide (ODN) inhibitor (TLR9 inhib) or control ODN (Control). After 16 h, IFN-α was measured by ELISA in supernatants. Data are presented as mean ± s.e.m. from three independent. * P
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    Thermo Fisher allergen specific ige
    IgE-containing ICs deliver DNA to <t>TLR9</t> at the phagosome (a) Flow cytometry analysis of cell surface expression of FcεRI and FcεRII (red line) in pDCs compared isotype control (grey line). Data are representative of 3 independent experiments. (b) Mouse macrophages expressing TLR9-GFP and the alpha chain of human FcεRI (hFcεRI + ) were fed with beads coated with DNA+IgE D . TLR9-GFP localization in hFcεRI + and control macrophages (hFcεRI − ) was visualized by confocal microscopy 30 min. after bead internalization. Yellow arrows point to ingested beads. TLR9 + phagosomes were quantified ( n = 75 phagosomes per/group from three independent experiments). Data are presented as mean ± s.d. (c) Human pDCs were incubated with DNA+IgE D for 30 min. at 4°C (0 min) and then transferred to 37°C for 120 min. (120 min.). DNA+IgE D (green) and LAMP1 (red) intracellular localization was visualized by confocal microscopy and frames from one representative experiment out of three are shown. BF = bright field. Scale bars, 5μm. (d) Human pDCs were incubated for 5 h at 37°C in the presence of DNA+IgE D or untreated (ut). IRF7 intracellular localization was visualized by immunostaining using confocal microscopy. Frames from one representative experiment out of three are shown. (e) pDCs were stimulated with DNA+IgE D in the presence of 0.05 or 0.5 μg/ml of TLR9 oligodeoxynucleotide (ODN) inhibitor (TLR9 inhib) or control ODN (Control). After 16 h, IFN-α was measured by ELISA in supernatants. Data are presented as mean ± s.e.m. from three independent. * P
    Allergen Specific Ige, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MedImmune biotinylated ige
    dsDNA-specific <t>IgE</t> enhance pDC interferon responses through increased DNA phagocytosis (a) Serum concentration of dsDNA-specific IgG and dsDNA-specific IgE in SLE patients that tested positive for dsDNA-specific IgE ( n = 98). Mean values were 88.0 μg/ml for DNA-IgG and 4.8 μg/ml for DNA-IgE. Mean ± s.e.m are represented in red. (b) IFN-α in supernatants of pDCs stimulated with DNA-ICs containing a fixed amount of IgG D (10 μg/ml) and DNA (0.5 μg/ml) and increasing concentrations (as indicated in x-axis) of IgE D for 16 h. The ratio IgE D /IgG D in the immune complexes for each of the condition is indicated in red above each bar. Data are presented as fold increase over pDCs treated with ICs containing IgG D and DNA (red dotted line) and are mean ± s.e.m. of three independent experiments. (c) IFN-α in supernatants of pDCs treated with by <t>biotinylated</t> RNP (RNP) and biotinylated IgG, IgE or IgG + IgE in the presence of streptavidin to form RNA-containing immune complexes. Cells were incubated with the ICs for 16 h. Data are presented as mean ± s.e.m. of four independent experiments. (d) IFN-α in supernatant of pDCs treated with similar RNA immune complexes containing a fixed amount of IgG (10 μg/ml) and DNA (0.5 μg/ml) and increasing concentrations of IgE as indicated. The ratio IgE D /IgG D in the immune complexes for each of the condition is indicated in red above each bar. Data are represented as fold increase over the IFN-α obtained from cells treated with RNA-ICs containing IgG only (red dotted line). Data are presented as mean ± s.e.m. of five independent experiments. (e) Internalized DNA in pDCs treated with DNA-ICs. Representative frames from three independent experiments are shown. Scale bar = 5.0 μm. (f) Engulfment of DNA was quantified by FACS and the percentage of cells that were positive for phagocyted DNA is represented. Data are presented as mean ± s.e.m. from seven independent experiments. (g) DNA-ICs containing the indicated IgE D /IgG D ratio were fed to pDCs. Percentage of pDCs positive for engulfed DNA was measured by FACS. Data represent fold increase over cells incubated with DNA+ IgG D (open circle) and are presented as mean ± s.e.m. of five independent experiments. * P
    Biotinylated Ige, supplied by MedImmune, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Bio-Rad chimeric ige jw8
    dsDNA-specific <t>IgE</t> enhance pDC interferon responses through increased DNA phagocytosis (a) Serum concentration of dsDNA-specific IgG and dsDNA-specific IgE in SLE patients that tested positive for dsDNA-specific IgE ( n = 98). Mean values were 88.0 μg/ml for DNA-IgG and 4.8 μg/ml for DNA-IgE. Mean ± s.e.m are represented in red. (b) IFN-α in supernatants of pDCs stimulated with DNA-ICs containing a fixed amount of IgG D (10 μg/ml) and DNA (0.5 μg/ml) and increasing concentrations (as indicated in x-axis) of IgE D for 16 h. The ratio IgE D /IgG D in the immune complexes for each of the condition is indicated in red above each bar. Data are presented as fold increase over pDCs treated with ICs containing IgG D and DNA (red dotted line) and are mean ± s.e.m. of three independent experiments. (c) IFN-α in supernatants of pDCs treated with by <t>biotinylated</t> RNP (RNP) and biotinylated IgG, IgE or IgG + IgE in the presence of streptavidin to form RNA-containing immune complexes. Cells were incubated with the ICs for 16 h. Data are presented as mean ± s.e.m. of four independent experiments. (d) IFN-α in supernatant of pDCs treated with similar RNA immune complexes containing a fixed amount of IgG (10 μg/ml) and DNA (0.5 μg/ml) and increasing concentrations of IgE as indicated. The ratio IgE D /IgG D in the immune complexes for each of the condition is indicated in red above each bar. Data are represented as fold increase over the IFN-α obtained from cells treated with RNA-ICs containing IgG only (red dotted line). Data are presented as mean ± s.e.m. of five independent experiments. (e) Internalized DNA in pDCs treated with DNA-ICs. Representative frames from three independent experiments are shown. Scale bar = 5.0 μm. (f) Engulfment of DNA was quantified by FACS and the percentage of cells that were positive for phagocyted DNA is represented. Data are presented as mean ± s.e.m. from seven independent experiments. (g) DNA-ICs containing the indicated IgE D /IgG D ratio were fed to pDCs. Percentage of pDCs positive for engulfed DNA was measured by FACS. Data represent fold increase over cells incubated with DNA+ IgG D (open circle) and are presented as mean ± s.e.m. of five independent experiments. * P
    Chimeric Ige Jw8, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam recombinant ige
    Representative standard concentration curve for the IgG <t>anti-IgE</t> ELISA using omalizumab (A) . Comparison of the capacity of IgE-specific IgG autoantibodies to bind to “free” IgE (dark bars) and <t>FcεRI-bound</t> IgE (white bars) # analyte below detection limit of assay. Bars show mean/SD of 3 experiments using duplicate samples (B) . SDS-PAGE showing the purified IgG anti-IgE from serum compared with recombinant IgE and IgG (C) . Representative standard concentration curve for the IgG anti-FcεRI-bound ELISA using anti-IgE purified from human sera (D) .
    Recombinant Ige, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Collaborative Drug Discovery Inc ige antibodies
    Effect of the synthetic <t>CCD</t> blocker. Results for patients with anti-CCD <t>IgE</t> antibodies and one without such cross-reactive IgEs, as obtained on the Mediwiss test strip without ( n ) and with ( i ) an inhibitor. The left strips contained inhalant allergens (P, positive control; CCD, mixture of bromelain, horseradish peroxidase and ascorbate oxidase). Vegetable and animal foods as well as bromelain, horseradish peroxidase and ascorbate oxidase were applied on the right strip as CCD indicators. The symptoms of the CCD-positive patient were confined to perennial nasal congestion.
    Ige Antibodies, supplied by Collaborative Drug Discovery Inc, used in various techniques. Bioz Stars score: 91/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioreclamationIVT cross inhibition elisa
    Effect of the synthetic <t>CCD</t> blocker. Results for patients with anti-CCD <t>IgE</t> antibodies and one without such cross-reactive IgEs, as obtained on the Mediwiss test strip without ( n ) and with ( i ) an inhibitor. The left strips contained inhalant allergens (P, positive control; CCD, mixture of bromelain, horseradish peroxidase and ascorbate oxidase). Vegetable and animal foods as well as bromelain, horseradish peroxidase and ascorbate oxidase were applied on the right strip as CCD indicators. The symptoms of the CCD-positive patient were confined to perennial nasal congestion.
    Cross Inhibition Elisa, supplied by BioreclamationIVT, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of FAO inhibition on AHR and IgE production in OVA-exposed mice (A) A model for the treatment of OVA-induced asthma-like traits in mice. (B) One day after OVA challenge and treatment with etomoxir, AHR to inhaled methacholine was measured in unrestrained conscious C57BL/6J mice. (C) The experiment was repeated as in (B) except that mice were treated with ranolazine. AHR was measured one day after challenge and treatment. (D) The OVA sensitization and challenge followed by treatment with etomoxir or ranolazine were conducted on Balb/c mice. AHR was measured one day after allergen exposure and treatment. (E) Two days post OVA challenge and treatment with etomoxir in C57BL/6J mice, BAL fluids and sera were collected to measure the levels of OVA-specific IgE. (F) The levels of total IgE were measured in sera of the indicated conditions. Data = mean ± SEM; n = 5 mice/group; *, P

    Journal: Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology

    Article Title: Fueling the Mechanisms of Asthma: Increased Fatty Acid Oxidation in Inflammatory Immune Cells May Represent a Novel Therapeutic Target

    doi: 10.1111/cea.12947

    Figure Lengend Snippet: Effect of FAO inhibition on AHR and IgE production in OVA-exposed mice (A) A model for the treatment of OVA-induced asthma-like traits in mice. (B) One day after OVA challenge and treatment with etomoxir, AHR to inhaled methacholine was measured in unrestrained conscious C57BL/6J mice. (C) The experiment was repeated as in (B) except that mice were treated with ranolazine. AHR was measured one day after challenge and treatment. (D) The OVA sensitization and challenge followed by treatment with etomoxir or ranolazine were conducted on Balb/c mice. AHR was measured one day after allergen exposure and treatment. (E) Two days post OVA challenge and treatment with etomoxir in C57BL/6J mice, BAL fluids and sera were collected to measure the levels of OVA-specific IgE. (F) The levels of total IgE were measured in sera of the indicated conditions. Data = mean ± SEM; n = 5 mice/group; *, P

    Article Snippet: A sandwich ELISA was used to quantify OVA-specific IgE (Serotec) and total IgE (ebioscience), as described by the manufacturer.

    Techniques: Inhibition, Mouse Assay

    Peptide ELISA demonstrating the binding of Met e 1-specific IgE to the mimotopes. Mimotope KGIPNTKAP of Der p 1 and an IgE-binding epitope of Met e 1 (a.a. 251-270) were included as negative and positive controls, respectively. Met e 1-specific IgE could exclusively recognize the selected mimotopes from clusters 1–6, but not the irrelevant mimotope of Der p 1 ( P

    Journal: Cellular and Molecular Immunology

    Article Title: Screening and identification of mimotopes of the major shrimp allergen tropomyosin using one-bead-one-compound peptide libraries

    doi: 10.1038/cmi.2015.83

    Figure Lengend Snippet: Peptide ELISA demonstrating the binding of Met e 1-specific IgE to the mimotopes. Mimotope KGIPNTKAP of Der p 1 and an IgE-binding epitope of Met e 1 (a.a. 251-270) were included as negative and positive controls, respectively. Met e 1-specific IgE could exclusively recognize the selected mimotopes from clusters 1–6, but not the irrelevant mimotope of Der p 1 ( P

    Article Snippet: Increasing number of studies have demonstrated the success of mimotope-based therapy in various diseases., , , , Inspired by the unique property of mimotopes to induce epitope-specific antibodies, we investigated the capacity of mimotopes to inhibit immunoglobulin E (IgE)-allergen binding., , Due to their monovalent properties, they are safer than natural extracts/recombinant allergens in allergen-specific immunotherapy (SIT), and can prevent anaphylaxis caused by cross-linking of IgE and degranulation of mast cells.

    Techniques: Peptide ELISA, Binding Assay

    Characterization of synthesized (−)-maackiain. (A) Separation of (−)-maackiain from (+)-maackiain by Chiral column chromatography. (B) Effect of synthesized (−)-maackiain and (+)-maackiain on PMA-induced upregulation of H1R mRNA expression in HeLa cells (a) and on IgE/antigen-stimulated upregulation of IL-4 mRNA expression in RBL-2H3 cells (b) (a), HeLa cells were serum-starved for 24 h, and stimulated with 100 nmol/L PMA for 3 h. Synthesized (−)- or (+)-maackiain was treated 24 h before PMA stimulation. After stimulation, total RNA was isolated, and the H1R mRNA levels were determined by real-time quantitative RT-PCR. (b), RBL-2H3 cells were treated with 100 ng/mL anti-DNP IgE for 18 h and then stimulated with 100 ng/mL DNP-HSA for 2 h. Synthesized (−)- or (+)-maackiain was treated 24 h before DNP-HSA stimulation. After stimulation, total RNA was isolated, and the IL-4 mRNA levels were determined by real-time quantitative RT-PCR. Data are presented as the mean ± SEM ( n = 3). **, P

    Journal: Pharmacology Research & Perspectives

    Article Title: Maackiain is a novel antiallergic compound that suppresses transcriptional upregulation of the histamine H1 receptor and interleukin-4 genes

    doi: 10.1002/prp2.166

    Figure Lengend Snippet: Characterization of synthesized (−)-maackiain. (A) Separation of (−)-maackiain from (+)-maackiain by Chiral column chromatography. (B) Effect of synthesized (−)-maackiain and (+)-maackiain on PMA-induced upregulation of H1R mRNA expression in HeLa cells (a) and on IgE/antigen-stimulated upregulation of IL-4 mRNA expression in RBL-2H3 cells (b) (a), HeLa cells were serum-starved for 24 h, and stimulated with 100 nmol/L PMA for 3 h. Synthesized (−)- or (+)-maackiain was treated 24 h before PMA stimulation. After stimulation, total RNA was isolated, and the H1R mRNA levels were determined by real-time quantitative RT-PCR. (b), RBL-2H3 cells were treated with 100 ng/mL anti-DNP IgE for 18 h and then stimulated with 100 ng/mL DNP-HSA for 2 h. Synthesized (−)- or (+)-maackiain was treated 24 h before DNP-HSA stimulation. After stimulation, total RNA was isolated, and the IL-4 mRNA levels were determined by real-time quantitative RT-PCR. Data are presented as the mean ± SEM ( n = 3). **, P

    Article Snippet: Therefore, we tried to purify the active compounds from Kujin extract by partitioning the fractions and assessing the suppressive activity of each fraction on the IgE-stimulated upregulation of IL-4 gene expression in RBL-2H3 cells.

    Techniques: Synthesized, Column Chromatography, Expressing, Isolation, Quantitative RT-PCR

    Monovalent DNP inhibits SPE-7 IgE induced LAD-2 cell activation. (A) Percentage degranulation of LAD-2 cells, induced by 30 nM SPE-7 IgE, was dose-dependently inhibited by addition of indicated molar excess of monovalent DNP-L-serine. (B) Data are represented as an inhibition curve. IC 50 of monovalent DNP-L-serine inhibition of SPE-7 IgE induced degranulation = 1.43 μM. (C) LAD-2 cell TNF-α release, induced by 30 nM SPE-7 IgE, was dose-dependently inhibited by addition of indicated molar excess of monovalent DNP-L-serine. (D) IC 50 of monovalent DNP-L-serine inhibition of SPE-7 IgE induced TNF-α release = 1.57 μM. Lower and upper dashed lines indicate cell-only background control and activation by SPE-7 IgE only, respectively. Means of 3 to 7 independent experiments ± SEM are shown. Statistically significant difference to SPE-7 IgE only was determined by one-way ANOVA with Dunnett's post-test; *** p

    Journal: Scientific Reports

    Article Title: Mechanism of the Antigen-Independent Cytokinergic SPE-7 IgE Activation of Human Mast Cells in Vitro

    doi: 10.1038/srep09538

    Figure Lengend Snippet: Monovalent DNP inhibits SPE-7 IgE induced LAD-2 cell activation. (A) Percentage degranulation of LAD-2 cells, induced by 30 nM SPE-7 IgE, was dose-dependently inhibited by addition of indicated molar excess of monovalent DNP-L-serine. (B) Data are represented as an inhibition curve. IC 50 of monovalent DNP-L-serine inhibition of SPE-7 IgE induced degranulation = 1.43 μM. (C) LAD-2 cell TNF-α release, induced by 30 nM SPE-7 IgE, was dose-dependently inhibited by addition of indicated molar excess of monovalent DNP-L-serine. (D) IC 50 of monovalent DNP-L-serine inhibition of SPE-7 IgE induced TNF-α release = 1.57 μM. Lower and upper dashed lines indicate cell-only background control and activation by SPE-7 IgE only, respectively. Means of 3 to 7 independent experiments ± SEM are shown. Statistically significant difference to SPE-7 IgE only was determined by one-way ANOVA with Dunnett's post-test; *** p

    Article Snippet: Following washing with HBSS, cells were stimulated with 0.5–5 μg/ml SPE-7 IgE (Sigma-Aldrich).

    Techniques: Activation Assay, Inhibition

    Rat and human mast cell systems and activation by highly cytokinergic SPE-7 IgE. (A) The number of rat FcεRI molecules expressed per RBL-2H3 and human FcεRI molecules expressed per LAD-2 mast cells were quantified by Qifikit® (Dako). RBL-2H3 cells express 0.8 ± 0.2 × 10 5 rat FcεRI molecules per cell and naïve LAD-2 cells and those primed with 6 ng/ml IL-4 for 5 days express 0.7 ± 0.3 × 10 5 and 1.7 ± 0.2 × 10 5 human FcεRI molecules per cell, respectively (n = 4, 6, and 12, respectively). (B) RBL-2H3 degranulation (over 14% baseline; n = 3–6), (C) LAD-2 mast cell degranulation (over 9% baseline; n = 7), and (D) LAD-2 TNF-α release (over 42 ng/ml baseline; n = 7), evoked by highly cytokinergic SPE-7 IgE. All data are shown as mean ± SEM. Statistically significant difference was determined by one-way ANOVA with Tukey's post-test; *** p

    Journal: Scientific Reports

    Article Title: Mechanism of the Antigen-Independent Cytokinergic SPE-7 IgE Activation of Human Mast Cells in Vitro

    doi: 10.1038/srep09538

    Figure Lengend Snippet: Rat and human mast cell systems and activation by highly cytokinergic SPE-7 IgE. (A) The number of rat FcεRI molecules expressed per RBL-2H3 and human FcεRI molecules expressed per LAD-2 mast cells were quantified by Qifikit® (Dako). RBL-2H3 cells express 0.8 ± 0.2 × 10 5 rat FcεRI molecules per cell and naïve LAD-2 cells and those primed with 6 ng/ml IL-4 for 5 days express 0.7 ± 0.3 × 10 5 and 1.7 ± 0.2 × 10 5 human FcεRI molecules per cell, respectively (n = 4, 6, and 12, respectively). (B) RBL-2H3 degranulation (over 14% baseline; n = 3–6), (C) LAD-2 mast cell degranulation (over 9% baseline; n = 7), and (D) LAD-2 TNF-α release (over 42 ng/ml baseline; n = 7), evoked by highly cytokinergic SPE-7 IgE. All data are shown as mean ± SEM. Statistically significant difference was determined by one-way ANOVA with Tukey's post-test; *** p

    Article Snippet: Following washing with HBSS, cells were stimulated with 0.5–5 μg/ml SPE-7 IgE (Sigma-Aldrich).

    Techniques: Activation Assay

    SPE-7 IgE Fab inhibits SPE-7 IgE induced LAD-2 cell activation. (A) Percentage degranulation of LAD-2 cells, induced by 30 nM SPE-7 IgE, was dose-dependently inhibited by addition of indicated molar excess of recombinant SPE-7 IgE Fab. (B) Data are shown in an inhibition curve. IC 50 of SPE-7 IgE Fab inhibition of SPE-7 IgE induced degranulation = 190 nM. Lower and upper dashed lines indicated cell-only background control and activation by SPE-7 IgE only, respectively. (C) LAD-2 cell TNF-α release, evoked by 30 nM SPE-7 IgE, was dose-dependently inhibited by addition of indicated molar excess of recombinant SPE-7 IgE Fab. (D) IC 50 of recombinant SPE-7 IgE Fab inhibition of SPE-7 IgE evoked TNF-α release ~170 nM. Dashed line indicates cell-only background control. Means of 4 to 10 independent experiments ± SEM are shown. Statistically significant difference to SPE-7 IgE only was determined by one-way ANOVA with Dunnett's post-test; *** p

    Journal: Scientific Reports

    Article Title: Mechanism of the Antigen-Independent Cytokinergic SPE-7 IgE Activation of Human Mast Cells in Vitro

    doi: 10.1038/srep09538

    Figure Lengend Snippet: SPE-7 IgE Fab inhibits SPE-7 IgE induced LAD-2 cell activation. (A) Percentage degranulation of LAD-2 cells, induced by 30 nM SPE-7 IgE, was dose-dependently inhibited by addition of indicated molar excess of recombinant SPE-7 IgE Fab. (B) Data are shown in an inhibition curve. IC 50 of SPE-7 IgE Fab inhibition of SPE-7 IgE induced degranulation = 190 nM. Lower and upper dashed lines indicated cell-only background control and activation by SPE-7 IgE only, respectively. (C) LAD-2 cell TNF-α release, evoked by 30 nM SPE-7 IgE, was dose-dependently inhibited by addition of indicated molar excess of recombinant SPE-7 IgE Fab. (D) IC 50 of recombinant SPE-7 IgE Fab inhibition of SPE-7 IgE evoked TNF-α release ~170 nM. Dashed line indicates cell-only background control. Means of 4 to 10 independent experiments ± SEM are shown. Statistically significant difference to SPE-7 IgE only was determined by one-way ANOVA with Dunnett's post-test; *** p

    Article Snippet: Following washing with HBSS, cells were stimulated with 0.5–5 μg/ml SPE-7 IgE (Sigma-Aldrich).

    Techniques: Activation Assay, Recombinant, Inhibition

    Human Mast Cells are Activated by a Combination of FcεRI-Bound and ‘Free’ SPE-7 IgE. Percentage degranulation of LAD-2 cells, induced by indicated nM concentration of SPE-7, 102.1F10 and 27–74 IgE antibodies, in non-sensitised cells (A, n = 2–6) and those sensitised with SPE-7 IgE (B, n = 2–5), 27–74 IgE (C, n = 2–3) or 102.1F10 IgE (D, n = 2–6). Statistically significant difference to PBS background control was determined by one-way ANOVA with Dunnett's post-test; *** p

    Journal: Scientific Reports

    Article Title: Mechanism of the Antigen-Independent Cytokinergic SPE-7 IgE Activation of Human Mast Cells in Vitro

    doi: 10.1038/srep09538

    Figure Lengend Snippet: Human Mast Cells are Activated by a Combination of FcεRI-Bound and ‘Free’ SPE-7 IgE. Percentage degranulation of LAD-2 cells, induced by indicated nM concentration of SPE-7, 102.1F10 and 27–74 IgE antibodies, in non-sensitised cells (A, n = 2–6) and those sensitised with SPE-7 IgE (B, n = 2–5), 27–74 IgE (C, n = 2–3) or 102.1F10 IgE (D, n = 2–6). Statistically significant difference to PBS background control was determined by one-way ANOVA with Dunnett's post-test; *** p

    Article Snippet: Following washing with HBSS, cells were stimulated with 0.5–5 μg/ml SPE-7 IgE (Sigma-Aldrich).

    Techniques: Concentration Assay

    Proposed Mechanism of the Cytokinergic Activity of SPE-7 IgE. Complexes may be formed by ‘free’ SPE-7 IgE bridging FcεRI-bound SPE-7 IgE in an Fv-Fv manner (A). Interactions between the Fv region of receptor-bound IgE and Fc of ‘free’ IgE (B) or between the Fc of receptor-bound IgE and Fv regions of ‘free’ IgE (C) have been ruled out. Variable and constant IgE domains are shown in blue and purple, respectively. Extracellular FcεRIα subunits are shown in green. The cytoplasmic region of the α-chain, and the β- and γ-chains of FcεRI have been omitted for clarity.

    Journal: Scientific Reports

    Article Title: Mechanism of the Antigen-Independent Cytokinergic SPE-7 IgE Activation of Human Mast Cells in Vitro

    doi: 10.1038/srep09538

    Figure Lengend Snippet: Proposed Mechanism of the Cytokinergic Activity of SPE-7 IgE. Complexes may be formed by ‘free’ SPE-7 IgE bridging FcεRI-bound SPE-7 IgE in an Fv-Fv manner (A). Interactions between the Fv region of receptor-bound IgE and Fc of ‘free’ IgE (B) or between the Fc of receptor-bound IgE and Fv regions of ‘free’ IgE (C) have been ruled out. Variable and constant IgE domains are shown in blue and purple, respectively. Extracellular FcεRIα subunits are shown in green. The cytoplasmic region of the α-chain, and the β- and γ-chains of FcεRI have been omitted for clarity.

    Article Snippet: Following washing with HBSS, cells were stimulated with 0.5–5 μg/ml SPE-7 IgE (Sigma-Aldrich).

    Techniques: Activity Assay

    ELISA data of 4 representative patients. A, Direct ELISA showing IgE binding to rBet v 1.0101, rApi g 1.0101, the chimeras, and a mix of all chimeras (nonnormalized OD values). B and C, Inhibition of IgE binding to immobilized rBet v 1.0101 (Fig 3, B ) or the chimeras (Fig 3, C ) by means of preincubation with rBet v 1.0101 (positive control), rApi g 1.0101, and the chimeras. n.d ., Not done.

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy

    doi: 10.1016/j.jaci.2013.12.1073

    Figure Lengend Snippet: ELISA data of 4 representative patients. A, Direct ELISA showing IgE binding to rBet v 1.0101, rApi g 1.0101, the chimeras, and a mix of all chimeras (nonnormalized OD values). B and C, Inhibition of IgE binding to immobilized rBet v 1.0101 (Fig 3, B ) or the chimeras (Fig 3, C ) by means of preincubation with rBet v 1.0101 (positive control), rApi g 1.0101, and the chimeras. n.d ., Not done.

    Article Snippet: Patients were selected on the basis of a typical case history of birch pollen allergy, positive skin prick test responses to birch pollen, and/or in vitro IgE detection to rBet v 1 or birch pollen extract (≥0.35 kUA /L; ImmunoCAP, Thermo-Fisher, Uppsala, Sweden).

    Techniques: Enzyme-linked Immunosorbent Assay, Direct ELISA, Binding Assay, Inhibition, Positive Control

    Antigen-specific IgE/FcεRI-crosslinking on activated DCs inhibits the production of proinflammatory cytokines and chemokines resulting in impaired migration of inflammatory cells. (a) IgE-crosslinking inhibits the papain-induced production of CCL-2, IL-6, and TNF-α by murine DCs. (b) Inhibition of CCL-2 production in human DCs by IgE/FcεRI-crosslinking. (c) The chemokine CCL-2 shows strong chemotactic activity for c-Kit + mast cell progenitors and CD11c + DC. Indicated concentrations of recombinant CCL-2 were added to the medium in the lower wells of transwell plates and the migration of cells from mast cell progenitor cultures towards the chemokine was determined. (d) Antigen-specific IgE/FcεRI-mediated DC activation (OVA plus IgE) inhibits migration of bone-marrow derived mast cell progenitors and DC. DCs were activated as indicated and DC-conditioned supernatants were used for chemotaxis assays. Migratory response of cKit + cells and CD11c + cells towards DC-conditioned supernatants was calculated from 3 independent experiments. Migratory response of DC stimulated with LPS or papain in the absence of IgE/FcεRI-mediated DC activation (OVA alone) was set at 100 %.

    Journal: Mucosal immunology

    Article Title: Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites

    doi: 10.1038/mi.2014.85

    Figure Lengend Snippet: Antigen-specific IgE/FcεRI-crosslinking on activated DCs inhibits the production of proinflammatory cytokines and chemokines resulting in impaired migration of inflammatory cells. (a) IgE-crosslinking inhibits the papain-induced production of CCL-2, IL-6, and TNF-α by murine DCs. (b) Inhibition of CCL-2 production in human DCs by IgE/FcεRI-crosslinking. (c) The chemokine CCL-2 shows strong chemotactic activity for c-Kit + mast cell progenitors and CD11c + DC. Indicated concentrations of recombinant CCL-2 were added to the medium in the lower wells of transwell plates and the migration of cells from mast cell progenitor cultures towards the chemokine was determined. (d) Antigen-specific IgE/FcεRI-mediated DC activation (OVA plus IgE) inhibits migration of bone-marrow derived mast cell progenitors and DC. DCs were activated as indicated and DC-conditioned supernatants were used for chemotaxis assays. Migratory response of cKit + cells and CD11c + cells towards DC-conditioned supernatants was calculated from 3 independent experiments. Migratory response of DC stimulated with LPS or papain in the absence of IgE/FcεRI-mediated DC activation (OVA alone) was set at 100 %.

    Article Snippet: For the standard curves mouse anti-OVA specific IgE (AbD Serotech) or mouse anti-OVA IgG1 (Sigma Aldrich) were used.

    Techniques: Migration, Inhibition, Activity Assay, Recombinant, Activation Assay, Derivative Assay, Chemotaxis Assay

    IgE/FcεRI-mediated antigen presentation by DCs does not result in efficient generation of Th2-type effector T cells. (a) Induction of in vivo T cell proliferation. Splenic DCs were pulsed with NP-OVA in vitro (0.5 μg/ml antigen) in the presence (plus) or absence (no) of NP-specific IgE. Representative experiment (n=3). (b) Induction of in vitro T cell proliferation by DCs via IgE-mediated antigen uptake. Soluble OVA was present continuously; OVA-peptide 323-339 was used as control. Mean of triplicates +/− SEM, representative experiment (n≥5). (c) In vitro DC/T cells proliferation assay in the presence of increasing antigen concentrations. Triplicates of representative experiment (n=2). (d) IL-4 and IL13 in DC/T cell co-culture supernatant at day 3. (e) IL-4 and IFN-γ increase in culture supernatants when DC-bound IgE is used for antigen uptake. Expression levels of IL-4 mRNA are compared in T cells that were induced IgE-independently (no IgE) or via the IgE/ FcεRI-mediated uptake (plus IgE). ( f) Intracellular cytokine staining for IL-4 and IFN-γ in T cells. Recombinant IL-4 or LPS was added to the antigen presentation assays as indicated. Bar diagram shows percentages of IL-4 + and IFN-γ CD4 + T cells in DC/T cell co-cultures. Data represent triplicates of biological replicates +/− SEM of a representative experiment (n=3). Representative FACS blots depict gated CD4 + T cells at day 7. (g) Recombinant IL-12 was added to the co-cultures and priming of Th1 cells was determined by intracellular staining for IFN-γ at day 7. Data represent triplicates of biological replicates +/− SEM of a representative experiment (n=2).

    Journal: Mucosal immunology

    Article Title: Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites

    doi: 10.1038/mi.2014.85

    Figure Lengend Snippet: IgE/FcεRI-mediated antigen presentation by DCs does not result in efficient generation of Th2-type effector T cells. (a) Induction of in vivo T cell proliferation. Splenic DCs were pulsed with NP-OVA in vitro (0.5 μg/ml antigen) in the presence (plus) or absence (no) of NP-specific IgE. Representative experiment (n=3). (b) Induction of in vitro T cell proliferation by DCs via IgE-mediated antigen uptake. Soluble OVA was present continuously; OVA-peptide 323-339 was used as control. Mean of triplicates +/− SEM, representative experiment (n≥5). (c) In vitro DC/T cells proliferation assay in the presence of increasing antigen concentrations. Triplicates of representative experiment (n=2). (d) IL-4 and IL13 in DC/T cell co-culture supernatant at day 3. (e) IL-4 and IFN-γ increase in culture supernatants when DC-bound IgE is used for antigen uptake. Expression levels of IL-4 mRNA are compared in T cells that were induced IgE-independently (no IgE) or via the IgE/ FcεRI-mediated uptake (plus IgE). ( f) Intracellular cytokine staining for IL-4 and IFN-γ in T cells. Recombinant IL-4 or LPS was added to the antigen presentation assays as indicated. Bar diagram shows percentages of IL-4 + and IFN-γ CD4 + T cells in DC/T cell co-cultures. Data represent triplicates of biological replicates +/− SEM of a representative experiment (n=3). Representative FACS blots depict gated CD4 + T cells at day 7. (g) Recombinant IL-12 was added to the co-cultures and priming of Th1 cells was determined by intracellular staining for IFN-γ at day 7. Data represent triplicates of biological replicates +/− SEM of a representative experiment (n=2).

    Article Snippet: For the standard curves mouse anti-OVA specific IgE (AbD Serotech) or mouse anti-OVA IgG1 (Sigma Aldrich) were used.

    Techniques: In Vivo, In Vitro, Proliferation Assay, Co-Culture Assay, Expressing, Staining, Recombinant, FACS

    IgE/FcεRI-crosslinking does not induce phenotypic maturation or production of inflammatory cytokines in DCs. (a) Antigen-mediated IgE/FcεRI activation induces phosphorylation of Syk and Erk1/2 in DCs. Splenic DCs were loaded with NP-specific IgE prior to incubation with NP-OVA (see schematic). IgE R -TG DCs were also stimulated with CpG DNA, or antigen-crosslinking was omitted (NT = not treated). As a control, WT DCs were treated identically. Immunoblots for phospho-Syk, total Syk, phospho-Erk1/2 or total Erk1/2 are shown. (b) IgE/FcεRI-crosslinking fails to upregulate expression of maturation marker molecules in DCs from IgE R -TG mice and (c) human monocyte-derived DCs. (d) Absence of cytokine secretion by splenic DCs upon antigen-specific IgE/FcεRI-crosslinking. Mean of triplicates +/− SEM, representative experiment (n=2); below detection level (bd) (e) TNF-α secretion from bone-marrow derived mast cells upon antigen-specific IgE/FcεRI-crosslinking . (f) Absence of transcriptional responses in murine DCs after antigen-specific IgE/FcεRI-crosslinking. mRNA expression was determined after 8 h. OVA uptake in the presence of CpG-DNA or papain was compared to IgE/FcεRI-mediated OVA uptake. Fold change compared to DCs that received OVA was calculated, and the mean of triplicates +/− SEM is shown, representative experiment (n=2).

    Journal: Mucosal immunology

    Article Title: Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites

    doi: 10.1038/mi.2014.85

    Figure Lengend Snippet: IgE/FcεRI-crosslinking does not induce phenotypic maturation or production of inflammatory cytokines in DCs. (a) Antigen-mediated IgE/FcεRI activation induces phosphorylation of Syk and Erk1/2 in DCs. Splenic DCs were loaded with NP-specific IgE prior to incubation with NP-OVA (see schematic). IgE R -TG DCs were also stimulated with CpG DNA, or antigen-crosslinking was omitted (NT = not treated). As a control, WT DCs were treated identically. Immunoblots for phospho-Syk, total Syk, phospho-Erk1/2 or total Erk1/2 are shown. (b) IgE/FcεRI-crosslinking fails to upregulate expression of maturation marker molecules in DCs from IgE R -TG mice and (c) human monocyte-derived DCs. (d) Absence of cytokine secretion by splenic DCs upon antigen-specific IgE/FcεRI-crosslinking. Mean of triplicates +/− SEM, representative experiment (n=2); below detection level (bd) (e) TNF-α secretion from bone-marrow derived mast cells upon antigen-specific IgE/FcεRI-crosslinking . (f) Absence of transcriptional responses in murine DCs after antigen-specific IgE/FcεRI-crosslinking. mRNA expression was determined after 8 h. OVA uptake in the presence of CpG-DNA or papain was compared to IgE/FcεRI-mediated OVA uptake. Fold change compared to DCs that received OVA was calculated, and the mean of triplicates +/− SEM is shown, representative experiment (n=2).

    Article Snippet: For the standard curves mouse anti-OVA specific IgE (AbD Serotech) or mouse anti-OVA IgG1 (Sigma Aldrich) were used.

    Techniques: Activation Assay, Incubation, Western Blot, Expressing, Marker, Mouse Assay, Derivative Assay

    Intestinal DCs express FcεRI and allergic sensitization increases the DC-bound IgE pool of IgE R -TG animals. ( a) FcεRI expression (in red) in the small intestine of IgE R -TG mice is found on CD11c + DCs (in blue). CD11c + DCs from IgE R -TG are GFP positive because the transgenic construct contains an IRES-GPF reporter element. (b) FcεRI expression on CD11c + DCs in the human small intestine. Human FcεRIα (green, first panel), CD11c (red, second panel). Mast cells as depicted by c-kit are not found in non-inflamed human tissue using immunofluorescence (row two, red). DAPI-stain for nuclei (blue). (c) and (d) Analysis of OVA-specific IgE and IgG1 levels in serum of sensitized mice before antigen challenge. (e) and (f) Sensitization and antigen challenge increases the amount of surface-bound IgE on DCs of IgE R -TG mice but not WT animals. DCs were isolated from the spleens and mesenteric lymph nodes (MLNs) of OVA-challenged WT and IgE R -TG mice, respectively. Sensitized mice are indicated by (+), non-sensitized mice by (−). Symbols are representative of individual mice (n=2).

    Journal: Mucosal immunology

    Article Title: Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites

    doi: 10.1038/mi.2014.85

    Figure Lengend Snippet: Intestinal DCs express FcεRI and allergic sensitization increases the DC-bound IgE pool of IgE R -TG animals. ( a) FcεRI expression (in red) in the small intestine of IgE R -TG mice is found on CD11c + DCs (in blue). CD11c + DCs from IgE R -TG are GFP positive because the transgenic construct contains an IRES-GPF reporter element. (b) FcεRI expression on CD11c + DCs in the human small intestine. Human FcεRIα (green, first panel), CD11c (red, second panel). Mast cells as depicted by c-kit are not found in non-inflamed human tissue using immunofluorescence (row two, red). DAPI-stain for nuclei (blue). (c) and (d) Analysis of OVA-specific IgE and IgG1 levels in serum of sensitized mice before antigen challenge. (e) and (f) Sensitization and antigen challenge increases the amount of surface-bound IgE on DCs of IgE R -TG mice but not WT animals. DCs were isolated from the spleens and mesenteric lymph nodes (MLNs) of OVA-challenged WT and IgE R -TG mice, respectively. Sensitized mice are indicated by (+), non-sensitized mice by (−). Symbols are representative of individual mice (n=2).

    Article Snippet: For the standard curves mouse anti-OVA specific IgE (AbD Serotech) or mouse anti-OVA IgG1 (Sigma Aldrich) were used.

    Techniques: Expressing, Mouse Assay, Transgenic Assay, Construct, Immunofluorescence, Staining, Isolation

    Reduction of allergic inflammation is regulated by the DC-bound IgE pool. (a) Expression patterns of murine and/or human FcεRIα-chains in WT mice, IgE R -TG mice, and IgE R -TG mice backcrossed onto the murine α-chain knock strain (muα KO). Backcrossed animals are referred to as IgE R -TG mice x muα KO (b) DC-bound IgE is found exclusively on strains that express FcεRI on this cell type after OVA/alum sensitization. (c) Analysis of OVA-specific serum IgE after sensitization. (d) Comparison of intestinal mast cell-specific transcripts of MCPT1 and MCPT2 as readout for the severity of food allergy. Representative experiment (n=3); All animals used for experiments (a-d) were on the C57BL/6 background.

    Journal: Mucosal immunology

    Article Title: Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites

    doi: 10.1038/mi.2014.85

    Figure Lengend Snippet: Reduction of allergic inflammation is regulated by the DC-bound IgE pool. (a) Expression patterns of murine and/or human FcεRIα-chains in WT mice, IgE R -TG mice, and IgE R -TG mice backcrossed onto the murine α-chain knock strain (muα KO). Backcrossed animals are referred to as IgE R -TG mice x muα KO (b) DC-bound IgE is found exclusively on strains that express FcεRI on this cell type after OVA/alum sensitization. (c) Analysis of OVA-specific serum IgE after sensitization. (d) Comparison of intestinal mast cell-specific transcripts of MCPT1 and MCPT2 as readout for the severity of food allergy. Representative experiment (n=3); All animals used for experiments (a-d) were on the C57BL/6 background.

    Article Snippet: For the standard curves mouse anti-OVA specific IgE (AbD Serotech) or mouse anti-OVA IgG1 (Sigma Aldrich) were used.

    Techniques: Expressing, Mouse Assay

    Spleen tyrosine kinase (SYK) inhibition in tonsillar B cells decreases viability, activation, and effector functions. Tonsillar cells activated in vitro by incubation with CD40L fibroblasts (1:10 ratio, fibroblasts: tonsillar cells), anti-μ antibody (10 µg/ml) and LPS (1 µg/ml) Non-activated tonsillar cells were cultured with CD32L fibroblasts (1:10 ratio, fibroblasts: tonsillar cells). (A) Following incubation for 20 min in the presence or absence of BAY61-3606 (5 µM), B cells were identified on the basis of their expression of CD19, and SYK phosphorylation status was determined by flow cytometry. (B) Following 3 days of culture, cells were stained with a fixable viability stain and identified on the basis of their CD19 expression status, by flow cytometry. Dead B cells were identified with a fixable viability stain. The boxplot shows the distribution between donors, and the quadrant indicates the values between the fifth and ninety-fifth percentiles, n = 7. (C) Following 3 days of culture, cells were stained with fixable viability stain, and for CD19, sIgD, CD38, CD80, and myeloid cell leukemia-1 (Mcl-1), and analyzed by flow cytometry. Viable cells were identified as fixable viability stain-negative. Mcl-1 levels were determined in CD19-positive cells and in pre-GC (BM2′) and GC B cells (BM3 + BM4) identified on the basis of sIgD and CD38 expression profiles. Activation state was determined by assessing CD80 expression in CD19-positive cells and in pre-GC (BM2′) and GC B-cells (BM3 + BM4). The boxplot represents the distribution between donors and the quadrant indicates the values between the fifth and ninety-fifth percentiles; significant P values are shown on the graphs for comparisons between all stimulated cells and those treated with BAY61-3606, n = 7. (D) The total immunoglobulin G (IgG) secreted by the cells during 3 days of culture was analyzed by ELISA. The boxplot represents the distribution between donors and the quadrant indicates the values between the fifth and ninety-fifth percentiles, n = 9. stim, stimulated; Bay, BAY61-3606; N-A, non-activated; CD, cluster of differentiation.

    Journal: Frontiers in Immunology

    Article Title: SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production

    doi: 10.3389/fimmu.2018.00787

    Figure Lengend Snippet: Spleen tyrosine kinase (SYK) inhibition in tonsillar B cells decreases viability, activation, and effector functions. Tonsillar cells activated in vitro by incubation with CD40L fibroblasts (1:10 ratio, fibroblasts: tonsillar cells), anti-μ antibody (10 µg/ml) and LPS (1 µg/ml) Non-activated tonsillar cells were cultured with CD32L fibroblasts (1:10 ratio, fibroblasts: tonsillar cells). (A) Following incubation for 20 min in the presence or absence of BAY61-3606 (5 µM), B cells were identified on the basis of their expression of CD19, and SYK phosphorylation status was determined by flow cytometry. (B) Following 3 days of culture, cells were stained with a fixable viability stain and identified on the basis of their CD19 expression status, by flow cytometry. Dead B cells were identified with a fixable viability stain. The boxplot shows the distribution between donors, and the quadrant indicates the values between the fifth and ninety-fifth percentiles, n = 7. (C) Following 3 days of culture, cells were stained with fixable viability stain, and for CD19, sIgD, CD38, CD80, and myeloid cell leukemia-1 (Mcl-1), and analyzed by flow cytometry. Viable cells were identified as fixable viability stain-negative. Mcl-1 levels were determined in CD19-positive cells and in pre-GC (BM2′) and GC B cells (BM3 + BM4) identified on the basis of sIgD and CD38 expression profiles. Activation state was determined by assessing CD80 expression in CD19-positive cells and in pre-GC (BM2′) and GC B-cells (BM3 + BM4). The boxplot represents the distribution between donors and the quadrant indicates the values between the fifth and ninety-fifth percentiles; significant P values are shown on the graphs for comparisons between all stimulated cells and those treated with BAY61-3606, n = 7. (D) The total immunoglobulin G (IgG) secreted by the cells during 3 days of culture was analyzed by ELISA. The boxplot represents the distribution between donors and the quadrant indicates the values between the fifth and ninety-fifth percentiles, n = 9. stim, stimulated; Bay, BAY61-3606; N-A, non-activated; CD, cluster of differentiation.

    Article Snippet: We then investigated the effects of SYK inhibition on Ig production, by assessing IgG secretion after 3 days in culture.

    Techniques: Inhibition, Activation Assay, In Vitro, Incubation, Cell Culture, Expressing, Flow Cytometry, Cytometry, Staining, Enzyme-linked Immunosorbent Assay

    No cross-reactivity between bGST and HDM-GST. (A) IgG1-reactivity of sera (n = 3) collected from bGST-immunized mice at day 0 and day 59 to BPE and HDME. (B) IgE-reactivity of seven Der p 8-sensitized HDM-allergic patients to bGST and HDME. NHS, non-allergic control sera; O.D. optical density; dotted and dashed lines indicate the cut-off for positive IgE-reactivity.

    Journal: PLoS ONE

    Article Title: Glutathione-S-Transferase: A Minor Allergen in Birch Pollen due to Limited Release from Hydrated Pollen

    doi: 10.1371/journal.pone.0109075

    Figure Lengend Snippet: No cross-reactivity between bGST and HDM-GST. (A) IgG1-reactivity of sera (n = 3) collected from bGST-immunized mice at day 0 and day 59 to BPE and HDME. (B) IgE-reactivity of seven Der p 8-sensitized HDM-allergic patients to bGST and HDME. NHS, non-allergic control sera; O.D. optical density; dotted and dashed lines indicate the cut-off for positive IgE-reactivity.

    Article Snippet: Rat anti-mouse IgG1, IgG2a, IgG3 and IgE (BD Pharmingen, San Jose, CA, USA) and a HRP-conjugated goat anti-rat IgG (GE Healthcare, Vienna, Austria) were used for detection.

    Techniques: Mouse Assay

    Immunosuppression by Pregnenolone and Pregnenolone-Producing Cells (A–C) Pregnenolone inhibits Th cell proliferation. Naive Th cells were stained with CellTrace Violet and activated for Th1 (A) or Th2 (B) differentiation for 72 hr in the presence (blue histogram) or absence (red histogram) of pregnenolone (5 μM). The cell proliferation profile was captured by FACS based on dye decay. The histograms depicted are representative of three independent experiments with three to five mice each. (C) Mean division indices were calculated for the experiments described in (A) and (B). Division index is the average number of divisions that a cell (present in the starting population) has undergone. p values were calculated using unpaired two-tailed t test. (D) Ly6C + Th2 cells inhibit Th cell proliferation. CellTrace-Violet-stained naive Th cells (“responder cells”) were activated by anti-CD3e and anti-CD28 in the presence of FACS-sorted Ly6C + or Ly6C − Th2 cells and anti-IL-10 antibody and/or AG as indicated. The proliferation profile of the responder Th cells grown in the absence of Th2 (blue) was captured by FACS on the third day of activation. This was compared to the proliferation profile of responder Th cells activated in the presence of Th2 only (Ly6C + or Ly6C − ) (red). The Ly6C + cells were pretreated with neutralizing anti-IL-10 antibody (20 μg/ml) or with aminoglutethimide (AG, 250 μM) or with both for 24 hr and were used in the same culture conditions. The responder Th cell to Th2 (Ly6C + or Ly6C − ) cell ratio was 1:1. The histograms depicted are representative of three independent experiments with three mice in each experiment. Mean division indices ±SD are shown in the inset (right, bottom). p values were calculated using unpaired two-tailed t test. (E) Inhibition of Cyp11a1 activity negatively regulates IL-10 and TGF-β1 expression in Th2. In vitro polarized Th2 and Th1 cells (3 days activation, 2 days resting) in the presence or absence of aminoglutethimide (AG, 250 mM) were FACS analyzed after reactivation with PDBU/Ionomycin for intracellular cytokine (IL-10 and TGF-β1 for Th2; IFN-γ for Th1) expression. Data shown are representative of three independent experiments. (F) Effect of pregnenolone on B cell class switching to IgG1 and IgE. Naive resting B cells were stained with CellTrace Violet. Class switch recombination (CSR) was induced with LPS and IL-4 in the presence of different concentrations of pregnenolone. Cell-surface expression of IgG1 and IgE were analyzed by FACS on day 5 of stimulation. Data shown are representative of three independent experiments with three mice each. p values were calculated using unpaired two-tailed t test and were

    Journal: Cell Reports

    Article Title: Single-Cell RNA Sequencing Reveals T Helper Cells Synthesizing Steroids De Novo to Contribute to Immune Homeostasis

    doi: 10.1016/j.celrep.2014.04.011

    Figure Lengend Snippet: Immunosuppression by Pregnenolone and Pregnenolone-Producing Cells (A–C) Pregnenolone inhibits Th cell proliferation. Naive Th cells were stained with CellTrace Violet and activated for Th1 (A) or Th2 (B) differentiation for 72 hr in the presence (blue histogram) or absence (red histogram) of pregnenolone (5 μM). The cell proliferation profile was captured by FACS based on dye decay. The histograms depicted are representative of three independent experiments with three to five mice each. (C) Mean division indices were calculated for the experiments described in (A) and (B). Division index is the average number of divisions that a cell (present in the starting population) has undergone. p values were calculated using unpaired two-tailed t test. (D) Ly6C + Th2 cells inhibit Th cell proliferation. CellTrace-Violet-stained naive Th cells (“responder cells”) were activated by anti-CD3e and anti-CD28 in the presence of FACS-sorted Ly6C + or Ly6C − Th2 cells and anti-IL-10 antibody and/or AG as indicated. The proliferation profile of the responder Th cells grown in the absence of Th2 (blue) was captured by FACS on the third day of activation. This was compared to the proliferation profile of responder Th cells activated in the presence of Th2 only (Ly6C + or Ly6C − ) (red). The Ly6C + cells were pretreated with neutralizing anti-IL-10 antibody (20 μg/ml) or with aminoglutethimide (AG, 250 μM) or with both for 24 hr and were used in the same culture conditions. The responder Th cell to Th2 (Ly6C + or Ly6C − ) cell ratio was 1:1. The histograms depicted are representative of three independent experiments with three mice in each experiment. Mean division indices ±SD are shown in the inset (right, bottom). p values were calculated using unpaired two-tailed t test. (E) Inhibition of Cyp11a1 activity negatively regulates IL-10 and TGF-β1 expression in Th2. In vitro polarized Th2 and Th1 cells (3 days activation, 2 days resting) in the presence or absence of aminoglutethimide (AG, 250 mM) were FACS analyzed after reactivation with PDBU/Ionomycin for intracellular cytokine (IL-10 and TGF-β1 for Th2; IFN-γ for Th1) expression. Data shown are representative of three independent experiments. (F) Effect of pregnenolone on B cell class switching to IgG1 and IgE. Naive resting B cells were stained with CellTrace Violet. Class switch recombination (CSR) was induced with LPS and IL-4 in the presence of different concentrations of pregnenolone. Cell-surface expression of IgG1 and IgE were analyzed by FACS on day 5 of stimulation. Data shown are representative of three independent experiments with three mice each. p values were calculated using unpaired two-tailed t test and were

    Article Snippet: On day 3 or day 5 of stimulation, B cell Fc receptors were blocked with PBS containing 2% rat serum and 10 mM EGTA and stained with FITC-conjugated anti-IgG1 (BD Biosciences) PE-conjugated IgE (BioLegend).

    Techniques: Staining, FACS, Mouse Assay, Two Tailed Test, Activation Assay, Inhibition, Activity Assay, Expressing, In Vitro

    Frequency of typical IgE patterns obtained by western blot inhibition in CAP double sensitized patients. CCD: cross-reactive carbohydrate determinants, True DS: true double sensitization, WB: western blot, WB-I (western blot inhibition): To discriminate between IgE specific for peptide or carbohydrate epitopes, antibody binding to CCDs was inhibited by preincubating sera with MUXF-BSA. Among these patients the majority of DS was CCD-dependent. DS due to protein components of hyaluronidases played a minor role. n = 61.

    Journal: PLoS ONE

    Article Title: Inconsistent Results of Diagnostic Tools Hamper the Differentiation between Bee and Vespid Venom Allergy

    doi: 10.1371/journal.pone.0020842

    Figure Lengend Snippet: Frequency of typical IgE patterns obtained by western blot inhibition in CAP double sensitized patients. CCD: cross-reactive carbohydrate determinants, True DS: true double sensitization, WB: western blot, WB-I (western blot inhibition): To discriminate between IgE specific for peptide or carbohydrate epitopes, antibody binding to CCDs was inhibited by preincubating sera with MUXF-BSA. Among these patients the majority of DS was CCD-dependent. DS due to protein components of hyaluronidases played a minor role. n = 61.

    Article Snippet: For this purpose, we aimed to compare the outcomes of the BAT with the IDT (intradermal test) as well as with three different methods of IgE determination (CAP, ADVIA (ADVIA Centaur®, Siemens, Tarrytown, NY, USA), Immulite (Immulite 2000®, Siemens, Tarrytown, NY, USA)) regarding the frequency of double positive results.

    Techniques: Western Blot, Inhibition, Binding Assay

    An increase in microbial diversity does not inhibit hyper-IgE despite pTreg induction. (A) Relative species abundance in the small intestine (SI) and cecum of a representative C3- and C4-colonized mouse, n = 3 mice per group. (B) Total serum IgE levels in C3 ( n = 31) and C4 ( n = 56) mice. The blue and orange horizontal lines represent the geometric mean of the GF and SPF cohorts, respectively (from Figure 1B ). (C) SCFA levels in the cecal contents of C3 ( n = 10–13) and C4 ( n = 7–9) mice. The orange and brown horizontal lines represent the mean of the SPF and C2 cohorts, respectively (from Figure 1C ). (D) Representative flow cytometry plots of RORγt + Helios − Tregs in different tissues (Spl, spleen; PP, Peyer's patches; MLN, mesenteric lymph nodes; SI, small intestinal lamina propria; Col, colon lamina propria) of GF, C3, C4 and SPF mice. (E) Frequencies of RORγt + Helios − Tregs among CD45 + TCRβ + CD4 + Foxp3 + T cells in different tissues of GF ( n = 10–15), C3 ( n = 4–9), C4 ( n = 4–8), and SPF ( n = 7–16) mice. All mice were 10–13 weeks old. SCFA and RORγt + Helios − Treg data are pooled from at least two independent experiments. Each symbol represents an individual mouse. Black horizontal lines depict the geometric mean (B) or mean (C,E) . * p

    Journal: Frontiers in Immunology

    Article Title: Using Precisely Defined in vivo Microbiotas to Understand Microbial Regulation of IgE

    doi: 10.3389/fimmu.2019.03107

    Figure Lengend Snippet: An increase in microbial diversity does not inhibit hyper-IgE despite pTreg induction. (A) Relative species abundance in the small intestine (SI) and cecum of a representative C3- and C4-colonized mouse, n = 3 mice per group. (B) Total serum IgE levels in C3 ( n = 31) and C4 ( n = 56) mice. The blue and orange horizontal lines represent the geometric mean of the GF and SPF cohorts, respectively (from Figure 1B ). (C) SCFA levels in the cecal contents of C3 ( n = 10–13) and C4 ( n = 7–9) mice. The orange and brown horizontal lines represent the mean of the SPF and C2 cohorts, respectively (from Figure 1C ). (D) Representative flow cytometry plots of RORγt + Helios − Tregs in different tissues (Spl, spleen; PP, Peyer's patches; MLN, mesenteric lymph nodes; SI, small intestinal lamina propria; Col, colon lamina propria) of GF, C3, C4 and SPF mice. (E) Frequencies of RORγt + Helios − Tregs among CD45 + TCRβ + CD4 + Foxp3 + T cells in different tissues of GF ( n = 10–15), C3 ( n = 4–9), C4 ( n = 4–8), and SPF ( n = 7–16) mice. All mice were 10–13 weeks old. SCFA and RORγt + Helios − Treg data are pooled from at least two independent experiments. Each symbol represents an individual mouse. Black horizontal lines depict the geometric mean (B) or mean (C,E) . * p

    Article Snippet: These results indicate that although the microbial communities of C3 and C4 produced SCFA and induced RORγt+ Helios− pTregs, this was not sufficient to inhibit hyper-IgE.

    Techniques: Mouse Assay, Flow Cytometry

    Cooperation between an immunogenic and an acetate-producing species can suppress hyper-IgE. (A) Total serum IgE levels in E. faecalis KB1 ( n = 40), A. muciniphila YL44 ( n = 40), B. caecimuris YL58 ( n = 46), KB1 + YL58 ( n = 23), YL44 + YL58 ( n = 28), and KB1 + YL44 + YL58 ( n = 61) colonized mice. The blue, pink, and orange horizontal lines represent the geometric mean of the serum IgE levels from the GF, Oligo-MM 12 and SPF mice, respectively (from Figures 1B , 3B ). (B) Relative species abundance in the small intestine (SI) and cecum of a representative KB1 + YL58, YL44 + YL58 and KB1 + YL44 + YL58 mouse, n = 3–4 mice per group. (C) SCFA levels in the cecal contents of KB1 ( n = 4), YL44 ( n = 3–4), YL58 ( n = 4), KB1 + YL58 ( n = 6), YL44 + YL58 ( n = 6), and KB1 + YL44 + YL58 ( n = 8–9) colonized mice. The blue, pink, and orange horizontal lines represent the mean SCFA levels from the GF, Oligo-MM 12 and SPF cohorts, respectively (from Figures 1C , 3C ). All mice were 10–13 weeks old. SCFA data are pooled from at least two independent experiments. Each symbol represents an individual mouse. Black horizontal lines depict the geometric mean (A) or mean (C) . *** p

    Journal: Frontiers in Immunology

    Article Title: Using Precisely Defined in vivo Microbiotas to Understand Microbial Regulation of IgE

    doi: 10.3389/fimmu.2019.03107

    Figure Lengend Snippet: Cooperation between an immunogenic and an acetate-producing species can suppress hyper-IgE. (A) Total serum IgE levels in E. faecalis KB1 ( n = 40), A. muciniphila YL44 ( n = 40), B. caecimuris YL58 ( n = 46), KB1 + YL58 ( n = 23), YL44 + YL58 ( n = 28), and KB1 + YL44 + YL58 ( n = 61) colonized mice. The blue, pink, and orange horizontal lines represent the geometric mean of the serum IgE levels from the GF, Oligo-MM 12 and SPF mice, respectively (from Figures 1B , 3B ). (B) Relative species abundance in the small intestine (SI) and cecum of a representative KB1 + YL58, YL44 + YL58 and KB1 + YL44 + YL58 mouse, n = 3–4 mice per group. (C) SCFA levels in the cecal contents of KB1 ( n = 4), YL44 ( n = 3–4), YL58 ( n = 4), KB1 + YL58 ( n = 6), YL44 + YL58 ( n = 6), and KB1 + YL44 + YL58 ( n = 8–9) colonized mice. The blue, pink, and orange horizontal lines represent the mean SCFA levels from the GF, Oligo-MM 12 and SPF cohorts, respectively (from Figures 1C , 3C ). All mice were 10–13 weeks old. SCFA data are pooled from at least two independent experiments. Each symbol represents an individual mouse. Black horizontal lines depict the geometric mean (A) or mean (C) . *** p

    Article Snippet: These results indicate that although the microbial communities of C3 and C4 produced SCFA and induced RORγt+ Helios− pTregs, this was not sufficient to inhibit hyper-IgE.

    Techniques: Mouse Assay

    A butyrate-producing bacterial species cannot regulate serum IgE levels (A) Relative species abundance in the small intestine (SI) and cecum of a representative C1- and C2-colonized mouse, n = 3–4 mice per group. (B) Total serum IgE levels in germ-free (GF) ( n = 113), C1 ( n = 15), C2 ( n = 28), and SPF ( n = 80) mice. (C) SCFA levels in the cecal contents of GF ( n = 15–16), C1 ( n = 4), C2 ( n = 6–10), and SPF ( n = 13–14) mice. Each symbol represents an individual mouse. Black horizontal lines depict the geometric mean (B) or mean (C) . All mice were 10–13 weeks old. * p

    Journal: Frontiers in Immunology

    Article Title: Using Precisely Defined in vivo Microbiotas to Understand Microbial Regulation of IgE

    doi: 10.3389/fimmu.2019.03107

    Figure Lengend Snippet: A butyrate-producing bacterial species cannot regulate serum IgE levels (A) Relative species abundance in the small intestine (SI) and cecum of a representative C1- and C2-colonized mouse, n = 3–4 mice per group. (B) Total serum IgE levels in germ-free (GF) ( n = 113), C1 ( n = 15), C2 ( n = 28), and SPF ( n = 80) mice. (C) SCFA levels in the cecal contents of GF ( n = 15–16), C1 ( n = 4), C2 ( n = 6–10), and SPF ( n = 13–14) mice. Each symbol represents an individual mouse. Black horizontal lines depict the geometric mean (B) or mean (C) . All mice were 10–13 weeks old. * p

    Article Snippet: These results indicate that although the microbial communities of C3 and C4 produced SCFA and induced RORγt+ Helios− pTregs, this was not sufficient to inhibit hyper-IgE.

    Techniques: Mouse Assay

    A further increase in microbial diversity suppresses hyper-IgE and demonstrates a dynamic bacterial colonization profile in the small intestine and colon in early life. (A) Relative species abundance in the small intestine (SI) and cecum of a representative adult Oligo-MM 12 mouse, n = 4 mice. (B) Total serum IgE levels in Oligo-MM 12 ( n = 100) mice. The blue and orange horizontal lines represent the geometric mean of the serum IgE levels from the GF and SPF cohorts, respectively (from Figure 1B ). (C) SCFA levels in the cecal contents of Oligo-MM 12 ( n = 19) mice. The orange and purple horizontal lines represent the mean SCFA levels in the SPF and C4 cohorts, respectively (from Figures 1C , 2C ). (D) Representative flow cytometry plots of RORγt + Helios − Tregs in different tissues (Spl, spleen; PP, Peyer's patches; MLN, mesenteric lymph nodes; SI, small intestinal lamina propria; Col, colon lamina propria) of Oligo-MM 12 mice. (E) Frequencies of RORγt + Helios − Tregs among CD3ε + CD4 + Foxp3 + T cells in different tissues of Oligo-MM 12 ( n = 12–13) mice. The blue and orange horizontal lines represent the mean of the GF and SPF cohorts, respectively (from Figure 2E ). (F) Relative species abundances of Oligo-MM 12 bacterial species in the small intestine and colon of 8 to 43-day old Oligo-MM 12 mice. n = 2–5 mice per time point. All mice were 10–13 weeks old (A–E) . SCFA and RORγt + Helios − Treg data are pooled from at least two independent experiments. Each symbol represents an individual mouse. Black horizontal lines depict the geometric mean (B) or mean (C,E) . * p

    Journal: Frontiers in Immunology

    Article Title: Using Precisely Defined in vivo Microbiotas to Understand Microbial Regulation of IgE

    doi: 10.3389/fimmu.2019.03107

    Figure Lengend Snippet: A further increase in microbial diversity suppresses hyper-IgE and demonstrates a dynamic bacterial colonization profile in the small intestine and colon in early life. (A) Relative species abundance in the small intestine (SI) and cecum of a representative adult Oligo-MM 12 mouse, n = 4 mice. (B) Total serum IgE levels in Oligo-MM 12 ( n = 100) mice. The blue and orange horizontal lines represent the geometric mean of the serum IgE levels from the GF and SPF cohorts, respectively (from Figure 1B ). (C) SCFA levels in the cecal contents of Oligo-MM 12 ( n = 19) mice. The orange and purple horizontal lines represent the mean SCFA levels in the SPF and C4 cohorts, respectively (from Figures 1C , 2C ). (D) Representative flow cytometry plots of RORγt + Helios − Tregs in different tissues (Spl, spleen; PP, Peyer's patches; MLN, mesenteric lymph nodes; SI, small intestinal lamina propria; Col, colon lamina propria) of Oligo-MM 12 mice. (E) Frequencies of RORγt + Helios − Tregs among CD3ε + CD4 + Foxp3 + T cells in different tissues of Oligo-MM 12 ( n = 12–13) mice. The blue and orange horizontal lines represent the mean of the GF and SPF cohorts, respectively (from Figure 2E ). (F) Relative species abundances of Oligo-MM 12 bacterial species in the small intestine and colon of 8 to 43-day old Oligo-MM 12 mice. n = 2–5 mice per time point. All mice were 10–13 weeks old (A–E) . SCFA and RORγt + Helios − Treg data are pooled from at least two independent experiments. Each symbol represents an individual mouse. Black horizontal lines depict the geometric mean (B) or mean (C,E) . * p

    Article Snippet: These results indicate that although the microbial communities of C3 and C4 produced SCFA and induced RORγt+ Helios− pTregs, this was not sufficient to inhibit hyper-IgE.

    Techniques: Mouse Assay, Flow Cytometry

    Two early-life Oligo-MM 12 Clostridiales bacteria are unable to regulate serum IgE levels. (A) Relative species abundance in the small intestine (SI) and cecum of a representative C5- and C6-colonized mouse, n = 2–4 mice per group. (B) Total serum IgE levels in C5 ( n = 30) and C6 ( n = 49) mice. The blue and orange horizontal lines represent the geometric mean of the serum IgE levels from the GF and SPF cohorts, respectively (from Figure 1B ). (C) SCFA levels in the cecal contents of C5 ( n = 10–11) and C6 ( n = 9) mice. The orange and purple horizontal lines represent the mean SCFA levels in the SPF and C4 cohorts, respectively (from Figure 1C , 2C ). (D) Representative flow cytometry plots of RORγt + Helios − Tregs in different tissues (Spl, spleen; PP, Peyer's patches; MLN, mesenteric lymph nodes; SI, small intestinal lamina propria; Col, colon lamina propria) of C5 and C6 mice. (E) Frequencies of RORγt + Helios − Tregs among CD45 + TCRβ + CD4 + Foxp3 + cells in different tissues of C5 ( n = 4–9) and C6 ( n = 3–8) mice. The orange and blue horizontal lines represent the mean frequencies of RORγt + Helios − Tregs from the SPF and GF cohorts, respectively (from Figure 2E ). All mice were 10–13 weeks old. SCFA and RORγt + Helios − Treg data are pooled from at least two independent experiments. Each symbol represents an individual mouse. Black horizontal lines depict the geometric mean (B) or mean (C,E) . *** p

    Journal: Frontiers in Immunology

    Article Title: Using Precisely Defined in vivo Microbiotas to Understand Microbial Regulation of IgE

    doi: 10.3389/fimmu.2019.03107

    Figure Lengend Snippet: Two early-life Oligo-MM 12 Clostridiales bacteria are unable to regulate serum IgE levels. (A) Relative species abundance in the small intestine (SI) and cecum of a representative C5- and C6-colonized mouse, n = 2–4 mice per group. (B) Total serum IgE levels in C5 ( n = 30) and C6 ( n = 49) mice. The blue and orange horizontal lines represent the geometric mean of the serum IgE levels from the GF and SPF cohorts, respectively (from Figure 1B ). (C) SCFA levels in the cecal contents of C5 ( n = 10–11) and C6 ( n = 9) mice. The orange and purple horizontal lines represent the mean SCFA levels in the SPF and C4 cohorts, respectively (from Figure 1C , 2C ). (D) Representative flow cytometry plots of RORγt + Helios − Tregs in different tissues (Spl, spleen; PP, Peyer's patches; MLN, mesenteric lymph nodes; SI, small intestinal lamina propria; Col, colon lamina propria) of C5 and C6 mice. (E) Frequencies of RORγt + Helios − Tregs among CD45 + TCRβ + CD4 + Foxp3 + cells in different tissues of C5 ( n = 4–9) and C6 ( n = 3–8) mice. The orange and blue horizontal lines represent the mean frequencies of RORγt + Helios − Tregs from the SPF and GF cohorts, respectively (from Figure 2E ). All mice were 10–13 weeks old. SCFA and RORγt + Helios − Treg data are pooled from at least two independent experiments. Each symbol represents an individual mouse. Black horizontal lines depict the geometric mean (B) or mean (C,E) . *** p

    Article Snippet: These results indicate that although the microbial communities of C3 and C4 produced SCFA and induced RORγt+ Helios− pTregs, this was not sufficient to inhibit hyper-IgE.

    Techniques: Mouse Assay, Flow Cytometry

    Antigen-specific IgE/FcεRI-crosslinking on activated DCs inhibits the production of proinflammatory cytokines and chemokines resulting in impaired migration of inflammatory cells. (a) IgE-crosslinking inhibits the papain-induced production of CCL-2, IL-6, and TNF-α by murine DCs. (b) Inhibition of CCL-2 production in human DCs by IgE/FcεRI-crosslinking. (c) The chemokine CCL-2 shows strong chemotactic activity for c-Kit + mast cell progenitors and CD11c + DC. Indicated concentrations of recombinant CCL-2 were added to the medium in the lower wells of transwell plates and the migration of cells from mast cell progenitor cultures towards the chemokine was determined. (d) Antigen-specific IgE/FcεRI-mediated DC activation (OVA plus IgE) inhibits migration of bone-marrow derived mast cell progenitors and DC. DCs were activated as indicated and DC-conditioned supernatants were used for chemotaxis assays. Migratory response of cKit + cells and CD11c + cells towards DC-conditioned supernatants was calculated from 3 independent experiments. Migratory response of DC stimulated with LPS or papain in the absence of IgE/FcεRI-mediated DC activation (OVA alone) was set at 100 %.

    Journal: Mucosal immunology

    Article Title: Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites

    doi: 10.1038/mi.2014.85

    Figure Lengend Snippet: Antigen-specific IgE/FcεRI-crosslinking on activated DCs inhibits the production of proinflammatory cytokines and chemokines resulting in impaired migration of inflammatory cells. (a) IgE-crosslinking inhibits the papain-induced production of CCL-2, IL-6, and TNF-α by murine DCs. (b) Inhibition of CCL-2 production in human DCs by IgE/FcεRI-crosslinking. (c) The chemokine CCL-2 shows strong chemotactic activity for c-Kit + mast cell progenitors and CD11c + DC. Indicated concentrations of recombinant CCL-2 were added to the medium in the lower wells of transwell plates and the migration of cells from mast cell progenitor cultures towards the chemokine was determined. (d) Antigen-specific IgE/FcεRI-mediated DC activation (OVA plus IgE) inhibits migration of bone-marrow derived mast cell progenitors and DC. DCs were activated as indicated and DC-conditioned supernatants were used for chemotaxis assays. Migratory response of cKit + cells and CD11c + cells towards DC-conditioned supernatants was calculated from 3 independent experiments. Migratory response of DC stimulated with LPS or papain in the absence of IgE/FcεRI-mediated DC activation (OVA alone) was set at 100 %.

    Article Snippet: Following antibodies were used: Phycoerythrin-(PE), allophycocyanin-(APC) and PE/Cy7 anti-human FcεRIα mAb CRA1 (clone AER-37, eBioscience or BioLegend), PE-anti-mouse IgE (RME-1, Biolegend), APC-anti-CD11c, Alexa Fluor® 647 anti-mouse CD8α and Alexa Fluor® 647 anti-mouse CD4 from Biolegend (San Diego, CA), PE-anti-mouse CD86 (BD Pharmingen), APC-anti- CD117 (Biolegend), and PE- anti-mouse FcεRIα mAb (Mar1, Biolegend).

    Techniques: Migration, Inhibition, Activity Assay, Recombinant, Activation Assay, Derivative Assay, Chemotaxis Assay

    Intestinal DCs express FcεRI and allergic sensitization increases the DC-bound IgE pool of IgE R -TG animals. ( a) FcεRI expression (in red) in the small intestine of IgE R -TG mice is found on CD11c + DCs (in blue). CD11c + DCs from IgE R -TG are GFP positive because the transgenic construct contains an IRES-GPF reporter element. (b) FcεRI expression on CD11c + DCs in the human small intestine. Human FcεRIα (green, first panel), CD11c (red, second panel). Mast cells as depicted by c-kit are not found in non-inflamed human tissue using immunofluorescence (row two, red). DAPI-stain for nuclei (blue). (c) and (d) Analysis of OVA-specific IgE and IgG1 levels in serum of sensitized mice before antigen challenge. (e) and (f) Sensitization and antigen challenge increases the amount of surface-bound IgE on DCs of IgE R -TG mice but not WT animals. DCs were isolated from the spleens and mesenteric lymph nodes (MLNs) of OVA-challenged WT and IgE R -TG mice, respectively. Sensitized mice are indicated by (+), non-sensitized mice by (−). Symbols are representative of individual mice (n=2).

    Journal: Mucosal immunology

    Article Title: Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites

    doi: 10.1038/mi.2014.85

    Figure Lengend Snippet: Intestinal DCs express FcεRI and allergic sensitization increases the DC-bound IgE pool of IgE R -TG animals. ( a) FcεRI expression (in red) in the small intestine of IgE R -TG mice is found on CD11c + DCs (in blue). CD11c + DCs from IgE R -TG are GFP positive because the transgenic construct contains an IRES-GPF reporter element. (b) FcεRI expression on CD11c + DCs in the human small intestine. Human FcεRIα (green, first panel), CD11c (red, second panel). Mast cells as depicted by c-kit are not found in non-inflamed human tissue using immunofluorescence (row two, red). DAPI-stain for nuclei (blue). (c) and (d) Analysis of OVA-specific IgE and IgG1 levels in serum of sensitized mice before antigen challenge. (e) and (f) Sensitization and antigen challenge increases the amount of surface-bound IgE on DCs of IgE R -TG mice but not WT animals. DCs were isolated from the spleens and mesenteric lymph nodes (MLNs) of OVA-challenged WT and IgE R -TG mice, respectively. Sensitized mice are indicated by (+), non-sensitized mice by (−). Symbols are representative of individual mice (n=2).

    Article Snippet: Following antibodies were used: Phycoerythrin-(PE), allophycocyanin-(APC) and PE/Cy7 anti-human FcεRIα mAb CRA1 (clone AER-37, eBioscience or BioLegend), PE-anti-mouse IgE (RME-1, Biolegend), APC-anti-CD11c, Alexa Fluor® 647 anti-mouse CD8α and Alexa Fluor® 647 anti-mouse CD4 from Biolegend (San Diego, CA), PE-anti-mouse CD86 (BD Pharmingen), APC-anti- CD117 (Biolegend), and PE- anti-mouse FcεRIα mAb (Mar1, Biolegend).

    Techniques: Expressing, Mouse Assay, Transgenic Assay, Construct, Immunofluorescence, Staining, Isolation

    Reduction of allergic inflammation is regulated by the DC-bound IgE pool. (a) Expression patterns of murine and/or human FcεRIα-chains in WT mice, IgE R -TG mice, and IgE R -TG mice backcrossed onto the murine α-chain knock strain (muα KO). Backcrossed animals are referred to as IgE R -TG mice x muα KO (b) DC-bound IgE is found exclusively on strains that express FcεRI on this cell type after OVA/alum sensitization. (c) Analysis of OVA-specific serum IgE after sensitization. (d) Comparison of intestinal mast cell-specific transcripts of MCPT1 and MCPT2 as readout for the severity of food allergy. Representative experiment (n=3); All animals used for experiments (a-d) were on the C57BL/6 background.

    Journal: Mucosal immunology

    Article Title: Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites

    doi: 10.1038/mi.2014.85

    Figure Lengend Snippet: Reduction of allergic inflammation is regulated by the DC-bound IgE pool. (a) Expression patterns of murine and/or human FcεRIα-chains in WT mice, IgE R -TG mice, and IgE R -TG mice backcrossed onto the murine α-chain knock strain (muα KO). Backcrossed animals are referred to as IgE R -TG mice x muα KO (b) DC-bound IgE is found exclusively on strains that express FcεRI on this cell type after OVA/alum sensitization. (c) Analysis of OVA-specific serum IgE after sensitization. (d) Comparison of intestinal mast cell-specific transcripts of MCPT1 and MCPT2 as readout for the severity of food allergy. Representative experiment (n=3); All animals used for experiments (a-d) were on the C57BL/6 background.

    Article Snippet: Following antibodies were used: Phycoerythrin-(PE), allophycocyanin-(APC) and PE/Cy7 anti-human FcεRIα mAb CRA1 (clone AER-37, eBioscience or BioLegend), PE-anti-mouse IgE (RME-1, Biolegend), APC-anti-CD11c, Alexa Fluor® 647 anti-mouse CD8α and Alexa Fluor® 647 anti-mouse CD4 from Biolegend (San Diego, CA), PE-anti-mouse CD86 (BD Pharmingen), APC-anti- CD117 (Biolegend), and PE- anti-mouse FcεRIα mAb (Mar1, Biolegend).

    Techniques: Expressing, Mouse Assay

    Expression of FcεRI on DCs results in a DC-specific IgE pool as seen in non-allergic humans. (a) Human peripheral blood DCs of a non-allergic individual carry surface IgE. DCs (in blue) were identified by gating on CD1c + CD19 − cells to distinguish them from CD1c + CD19 + B cells (in green) and analyzed for cell surface-bound IgE. DCs carry significant amounts of IgE but less than CD203 + basophils and mast cells (in purple). (b) CD11c + DCs of IgE R -TG mice carry IgE at steady state. Representative FACS plots of WT - and IgE R -TG DCs isolated from spleens and quantification of the DC - bound IgE pool using mean fluorescence intensities (MFI) determined by flow cytometric analysis. (c) Baseline serum IgE levels are lower in non-sensitized IgE R - TG animals. Symbols in are representative of individual mice of ≥ 2 independent experiments (* p

    Journal: Mucosal immunology

    Article Title: Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites

    doi: 10.1038/mi.2014.85

    Figure Lengend Snippet: Expression of FcεRI on DCs results in a DC-specific IgE pool as seen in non-allergic humans. (a) Human peripheral blood DCs of a non-allergic individual carry surface IgE. DCs (in blue) were identified by gating on CD1c + CD19 − cells to distinguish them from CD1c + CD19 + B cells (in green) and analyzed for cell surface-bound IgE. DCs carry significant amounts of IgE but less than CD203 + basophils and mast cells (in purple). (b) CD11c + DCs of IgE R -TG mice carry IgE at steady state. Representative FACS plots of WT - and IgE R -TG DCs isolated from spleens and quantification of the DC - bound IgE pool using mean fluorescence intensities (MFI) determined by flow cytometric analysis. (c) Baseline serum IgE levels are lower in non-sensitized IgE R - TG animals. Symbols in are representative of individual mice of ≥ 2 independent experiments (* p

    Article Snippet: Following antibodies were used: Phycoerythrin-(PE), allophycocyanin-(APC) and PE/Cy7 anti-human FcεRIα mAb CRA1 (clone AER-37, eBioscience or BioLegend), PE-anti-mouse IgE (RME-1, Biolegend), APC-anti-CD11c, Alexa Fluor® 647 anti-mouse CD8α and Alexa Fluor® 647 anti-mouse CD4 from Biolegend (San Diego, CA), PE-anti-mouse CD86 (BD Pharmingen), APC-anti- CD117 (Biolegend), and PE- anti-mouse FcεRIα mAb (Mar1, Biolegend).

    Techniques: Expressing, Mouse Assay, FACS, Isolation, Fluorescence, Flow Cytometry

    IgE-containing ICs deliver DNA to TLR9 at the phagosome (a) Flow cytometry analysis of cell surface expression of FcεRI and FcεRII (red line) in pDCs compared isotype control (grey line). Data are representative of 3 independent experiments. (b) Mouse macrophages expressing TLR9-GFP and the alpha chain of human FcεRI (hFcεRI + ) were fed with beads coated with DNA+IgE D . TLR9-GFP localization in hFcεRI + and control macrophages (hFcεRI − ) was visualized by confocal microscopy 30 min. after bead internalization. Yellow arrows point to ingested beads. TLR9 + phagosomes were quantified ( n = 75 phagosomes per/group from three independent experiments). Data are presented as mean ± s.d. (c) Human pDCs were incubated with DNA+IgE D for 30 min. at 4°C (0 min) and then transferred to 37°C for 120 min. (120 min.). DNA+IgE D (green) and LAMP1 (red) intracellular localization was visualized by confocal microscopy and frames from one representative experiment out of three are shown. BF = bright field. Scale bars, 5μm. (d) Human pDCs were incubated for 5 h at 37°C in the presence of DNA+IgE D or untreated (ut). IRF7 intracellular localization was visualized by immunostaining using confocal microscopy. Frames from one representative experiment out of three are shown. (e) pDCs were stimulated with DNA+IgE D in the presence of 0.05 or 0.5 μg/ml of TLR9 oligodeoxynucleotide (ODN) inhibitor (TLR9 inhib) or control ODN (Control). After 16 h, IFN-α was measured by ELISA in supernatants. Data are presented as mean ± s.e.m. from three independent. * P

    Journal: Nature immunology

    Article Title: Self-reactive IgE exacerbates interferon responses associated with autoimmunity

    doi: 10.1038/ni.3326

    Figure Lengend Snippet: IgE-containing ICs deliver DNA to TLR9 at the phagosome (a) Flow cytometry analysis of cell surface expression of FcεRI and FcεRII (red line) in pDCs compared isotype control (grey line). Data are representative of 3 independent experiments. (b) Mouse macrophages expressing TLR9-GFP and the alpha chain of human FcεRI (hFcεRI + ) were fed with beads coated with DNA+IgE D . TLR9-GFP localization in hFcεRI + and control macrophages (hFcεRI − ) was visualized by confocal microscopy 30 min. after bead internalization. Yellow arrows point to ingested beads. TLR9 + phagosomes were quantified ( n = 75 phagosomes per/group from three independent experiments). Data are presented as mean ± s.d. (c) Human pDCs were incubated with DNA+IgE D for 30 min. at 4°C (0 min) and then transferred to 37°C for 120 min. (120 min.). DNA+IgE D (green) and LAMP1 (red) intracellular localization was visualized by confocal microscopy and frames from one representative experiment out of three are shown. BF = bright field. Scale bars, 5μm. (d) Human pDCs were incubated for 5 h at 37°C in the presence of DNA+IgE D or untreated (ut). IRF7 intracellular localization was visualized by immunostaining using confocal microscopy. Frames from one representative experiment out of three are shown. (e) pDCs were stimulated with DNA+IgE D in the presence of 0.05 or 0.5 μg/ml of TLR9 oligodeoxynucleotide (ODN) inhibitor (TLR9 inhib) or control ODN (Control). After 16 h, IFN-α was measured by ELISA in supernatants. Data are presented as mean ± s.e.m. from three independent. * P

    Article Snippet: For the TLR9 inhibition assay, TLR9 inhibitor ODN TTAGGG or control ODN for TTAGGG (both from Invivogen) were added to the cells at concentrations specified in the figure legend.

    Techniques: Flow Cytometry, Cytometry, Expressing, Confocal Microscopy, Incubation, Immunostaining, Inhibition, Enzyme-linked Immunosorbent Assay

    dsDNA-specific IgE enhance pDC interferon responses through increased DNA phagocytosis (a) Serum concentration of dsDNA-specific IgG and dsDNA-specific IgE in SLE patients that tested positive for dsDNA-specific IgE ( n = 98). Mean values were 88.0 μg/ml for DNA-IgG and 4.8 μg/ml for DNA-IgE. Mean ± s.e.m are represented in red. (b) IFN-α in supernatants of pDCs stimulated with DNA-ICs containing a fixed amount of IgG D (10 μg/ml) and DNA (0.5 μg/ml) and increasing concentrations (as indicated in x-axis) of IgE D for 16 h. The ratio IgE D /IgG D in the immune complexes for each of the condition is indicated in red above each bar. Data are presented as fold increase over pDCs treated with ICs containing IgG D and DNA (red dotted line) and are mean ± s.e.m. of three independent experiments. (c) IFN-α in supernatants of pDCs treated with by biotinylated RNP (RNP) and biotinylated IgG, IgE or IgG + IgE in the presence of streptavidin to form RNA-containing immune complexes. Cells were incubated with the ICs for 16 h. Data are presented as mean ± s.e.m. of four independent experiments. (d) IFN-α in supernatant of pDCs treated with similar RNA immune complexes containing a fixed amount of IgG (10 μg/ml) and DNA (0.5 μg/ml) and increasing concentrations of IgE as indicated. The ratio IgE D /IgG D in the immune complexes for each of the condition is indicated in red above each bar. Data are represented as fold increase over the IFN-α obtained from cells treated with RNA-ICs containing IgG only (red dotted line). Data are presented as mean ± s.e.m. of five independent experiments. (e) Internalized DNA in pDCs treated with DNA-ICs. Representative frames from three independent experiments are shown. Scale bar = 5.0 μm. (f) Engulfment of DNA was quantified by FACS and the percentage of cells that were positive for phagocyted DNA is represented. Data are presented as mean ± s.e.m. from seven independent experiments. (g) DNA-ICs containing the indicated IgE D /IgG D ratio were fed to pDCs. Percentage of pDCs positive for engulfed DNA was measured by FACS. Data represent fold increase over cells incubated with DNA+ IgG D (open circle) and are presented as mean ± s.e.m. of five independent experiments. * P

    Journal: Nature immunology

    Article Title: Self-reactive IgE exacerbates interferon responses associated with autoimmunity

    doi: 10.1038/ni.3326

    Figure Lengend Snippet: dsDNA-specific IgE enhance pDC interferon responses through increased DNA phagocytosis (a) Serum concentration of dsDNA-specific IgG and dsDNA-specific IgE in SLE patients that tested positive for dsDNA-specific IgE ( n = 98). Mean values were 88.0 μg/ml for DNA-IgG and 4.8 μg/ml for DNA-IgE. Mean ± s.e.m are represented in red. (b) IFN-α in supernatants of pDCs stimulated with DNA-ICs containing a fixed amount of IgG D (10 μg/ml) and DNA (0.5 μg/ml) and increasing concentrations (as indicated in x-axis) of IgE D for 16 h. The ratio IgE D /IgG D in the immune complexes for each of the condition is indicated in red above each bar. Data are presented as fold increase over pDCs treated with ICs containing IgG D and DNA (red dotted line) and are mean ± s.e.m. of three independent experiments. (c) IFN-α in supernatants of pDCs treated with by biotinylated RNP (RNP) and biotinylated IgG, IgE or IgG + IgE in the presence of streptavidin to form RNA-containing immune complexes. Cells were incubated with the ICs for 16 h. Data are presented as mean ± s.e.m. of four independent experiments. (d) IFN-α in supernatant of pDCs treated with similar RNA immune complexes containing a fixed amount of IgG (10 μg/ml) and DNA (0.5 μg/ml) and increasing concentrations of IgE as indicated. The ratio IgE D /IgG D in the immune complexes for each of the condition is indicated in red above each bar. Data are represented as fold increase over the IFN-α obtained from cells treated with RNA-ICs containing IgG only (red dotted line). Data are presented as mean ± s.e.m. of five independent experiments. (e) Internalized DNA in pDCs treated with DNA-ICs. Representative frames from three independent experiments are shown. Scale bar = 5.0 μm. (f) Engulfment of DNA was quantified by FACS and the percentage of cells that were positive for phagocyted DNA is represented. Data are presented as mean ± s.e.m. from seven independent experiments. (g) DNA-ICs containing the indicated IgE D /IgG D ratio were fed to pDCs. Percentage of pDCs positive for engulfed DNA was measured by FACS. Data represent fold increase over cells incubated with DNA+ IgG D (open circle) and are presented as mean ± s.e.m. of five independent experiments. * P

    Article Snippet: For the stimulation of pDCs with RNA-containing ICs, cells were incubated with 2.0 μg/ml of biotinylated U1 snRNP (Arotec Diagnostics) + 0.5 μg/ml streptavidin (Sigma-Aldrich), alone or in combination with biotinylated human IgG or biotinylated IgE (Medimmune LLC), or both immunoglobulin isotypes, as indicated in the figure legend, for 16 h. snRNP and antibodies were biotinylated using the Biotin-XX Microscale Protein Labeling Kit (Life Technologies).

    Techniques: Concentration Assay, Incubation, FACS

    Representative standard concentration curve for the IgG anti-IgE ELISA using omalizumab (A) . Comparison of the capacity of IgE-specific IgG autoantibodies to bind to “free” IgE (dark bars) and FcεRI-bound IgE (white bars) # analyte below detection limit of assay. Bars show mean/SD of 3 experiments using duplicate samples (B) . SDS-PAGE showing the purified IgG anti-IgE from serum compared with recombinant IgE and IgG (C) . Representative standard concentration curve for the IgG anti-FcεRI-bound ELISA using anti-IgE purified from human sera (D) .

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: “Auto-anti-IgE”: Naturally occurring IgG anti-IgE antibodies may inhibit allergen-induced basophil activation

    doi: 10.1016/j.jaci.2014.06.029

    Figure Lengend Snippet: Representative standard concentration curve for the IgG anti-IgE ELISA using omalizumab (A) . Comparison of the capacity of IgE-specific IgG autoantibodies to bind to “free” IgE (dark bars) and FcεRI-bound IgE (white bars) # analyte below detection limit of assay. Bars show mean/SD of 3 experiments using duplicate samples (B) . SDS-PAGE showing the purified IgG anti-IgE from serum compared with recombinant IgE and IgG (C) . Representative standard concentration curve for the IgG anti-FcεRI-bound ELISA using anti-IgE purified from human sera (D) .

    Article Snippet: Allergen binding to IgE is inhibited by IgG anti-IgE antibody containing sera To begin to address the mechanisms of inhibition of allergen-induced basophil activation by the inhibitory sera NAA16 and AA12, we used the rat basophilic cell line RBL-SX38, which expresses surface human FcεRI, and recombinant IgE specific for the timothy grass allergen Phl p 7 .

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, SDS Page, Purification, Recombinant

    Binding of recombinant Phl p 7 -specific IgE to FcεRI on RBL-SX38 cells (A) . Phl p 7 binding following further incubation with specific allergen (representative of 3 independent experiments) (B) . Changes in surface-bound IgE (C) and surface-bound Phl p 7 (D) on RBL-SX38 cells pre-incubated with recombinant, anti- Phl p 7 IgE test sera then Phl p 7 compared with no serum control. Bars represent the mean/SD of 3 independent experiments. ∗∗ P

    Journal: The Journal of Allergy and Clinical Immunology

    Article Title: “Auto-anti-IgE”: Naturally occurring IgG anti-IgE antibodies may inhibit allergen-induced basophil activation

    doi: 10.1016/j.jaci.2014.06.029

    Figure Lengend Snippet: Binding of recombinant Phl p 7 -specific IgE to FcεRI on RBL-SX38 cells (A) . Phl p 7 binding following further incubation with specific allergen (representative of 3 independent experiments) (B) . Changes in surface-bound IgE (C) and surface-bound Phl p 7 (D) on RBL-SX38 cells pre-incubated with recombinant, anti- Phl p 7 IgE test sera then Phl p 7 compared with no serum control. Bars represent the mean/SD of 3 independent experiments. ∗∗ P

    Article Snippet: Allergen binding to IgE is inhibited by IgG anti-IgE antibody containing sera To begin to address the mechanisms of inhibition of allergen-induced basophil activation by the inhibitory sera NAA16 and AA12, we used the rat basophilic cell line RBL-SX38, which expresses surface human FcεRI, and recombinant IgE specific for the timothy grass allergen Phl p 7 .

    Techniques: Binding Assay, Recombinant, Incubation

    Effect of the synthetic CCD blocker. Results for patients with anti-CCD IgE antibodies and one without such cross-reactive IgEs, as obtained on the Mediwiss test strip without ( n ) and with ( i ) an inhibitor. The left strips contained inhalant allergens (P, positive control; CCD, mixture of bromelain, horseradish peroxidase and ascorbate oxidase). Vegetable and animal foods as well as bromelain, horseradish peroxidase and ascorbate oxidase were applied on the right strip as CCD indicators. The symptoms of the CCD-positive patient were confined to perennial nasal congestion.

    Journal: Allergologie Select

    Article Title: Inhibition of cross-reactive carbohydrate determinants (CCDs) enhances the accuracy of in vitro allergy diagnosis

    doi: 10.5414/ALX01638E

    Figure Lengend Snippet: Effect of the synthetic CCD blocker. Results for patients with anti-CCD IgE antibodies and one without such cross-reactive IgEs, as obtained on the Mediwiss test strip without ( n ) and with ( i ) an inhibitor. The left strips contained inhalant allergens (P, positive control; CCD, mixture of bromelain, horseradish peroxidase and ascorbate oxidase). Vegetable and animal foods as well as bromelain, horseradish peroxidase and ascorbate oxidase were applied on the right strip as CCD indicators. The symptoms of the CCD-positive patient were confined to perennial nasal congestion.

    Article Snippet: In 3 patients (7%) the inhibition failed (CCD reduced; IgE antibodies to specific allergens unchanged).

    Techniques: Stripping Membranes, Positive Control