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“Auto-anti-IgE”: Naturally occurring IgG anti-IgE antibodies may inhibit allergen-induced basophil activation  Yih-Chih Chan, PhD, Faruk Ramadani, PhD,

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Presentation on theme: "“Auto-anti-IgE”: Naturally occurring IgG anti-IgE antibodies may inhibit allergen-induced basophil activation  Yih-Chih Chan, PhD, Faruk Ramadani, PhD,"— Presentation transcript:

1 “Auto-anti-IgE”: Naturally occurring IgG anti-IgE antibodies may inhibit allergen-induced basophil activation  Yih-Chih Chan, PhD, Faruk Ramadani, PhD, Alexandra F. Santos, MD, Prathap Pillai, MD, Line Ohm-Laursen, PhD, Clare E. Harper, MSc, Cailong Fang, PhD, Tihomir S. Dodev, PhD, Shih-Ying Wu, MSc, Sun Ying, PhD, Christopher J. Corrigan, PhD, Hannah J. Gould, PhD  Journal of Allergy and Clinical Immunology  Volume 134, Issue 6, Pages e4 (December 2014) DOI: /j.jaci Copyright © 2014 The Authors Terms and Conditions

2 Fig 1 Concentrations of IgG anti-IgE autoantibodies in sera from non-atopic controls, non-atopic asthmatic subjects, and atopic asthmatic subjects (A). Dotted line shows 95% confidence limit of the range in controls. Response of blood basophils to polyclonal anti-IgE in vitro (B). Basophil-activating activity of sera (C). Dotted line shows detection threshold. Samples marked # were used for later experiments. Bars represent the mean/SD of at least 3 independent experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

3 Fig 2 Concentration/response curve of blood basophils from a Der p 2–sensitized atopic donor to Der p 2 allergen in vitro (A). Response of the same basophils to Der p 2 30 ng/mL pre-incubated with all sera from Fig 1 normalized to baseline (pre-incubation of the cells with the donor's own serum) (B). Bars represent the mean/SD of at least 3 independent experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

4 Fig 3 Total IgG concentrations in 2 test sera ex vivo and following protein G depletion (A). IgE-binding proteins ex vivo and following depletion and isolation of IgE binding proteins (B). Effects of sera ex vivo and following IgG depletion (C) and removal and purification of IgE binding proteins (D) on Der p 2 (30 ng/mL)–induced basophil activation normalized to baseline (using the donor's own serum). Bars represent the mean/SD of 3 independent experiments. ∗P < .05 and ∗∗∗P < .001. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

5 Fig 4 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 < .01 and ∗∗∗P < .001. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

6 Fig E1 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 of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

7 Fig E2 Comparison of serum total IgE and IgG anti-IgE autoantibody concentrations in all study subjects (A). Spearman r = ; P > .5. Comparison of serum concentrations of IgG anti-IgE antibodies with ANA in all study subjects (B). Dotted line shows the threshold for a positive ANA result. Spearman r = − ; P > .5. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

8 Fig E3 Gating strategy to determine basophil activation by flow cytometry, comparing unstimulated and anti-IgE–stimulated PBMC. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

9 Fig E4 Response of blood basophils stimulated with Der p 2 (3 to 300 ng/mL), pre-incubated with 4 sera containing IgG anti-IgE autoantibodies and basophil-activating activity (A) and 3 sera containing IgG anti-IgE autoantibodies without basophil-activating activity normalized to baseline (pre-incubation of the cells with the donor's own serum) (B). Effects of the non-inhibitory serum NAA7 following the removal and purification of IgE binding proteins on Der p 2 (30 ng/mL)–induced basophil activation (C). Bars represent the mean/SD of 3 independent experiments. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2014 The Authors Terms and Conditions

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