Effects of established allergen sensitization on immune and airway responses after secondary allergen sensitization Katharina Blumchen, MD, Kerstin Gerhold,

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Effects of established allergen sensitization on immune and airway responses after secondary allergen sensitization Katharina Blumchen, MD, Kerstin Gerhold, MD, Marcus Schwede, MD, Bodo Niggemann, MD, Anzhela Avagyan, MD, Anna-Maria Dittrich, MD, Birgit Wagner, PhD, Heimo Breiteneder, PhD, Eckard Hamelmann, MD Journal of Allergy and Clinical Immunology Volume 118, Issue 3, Pages 615-621 (September 2006) DOI: 10.1016/j.jaci.2006.04.054 Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 1 Experimental protocol. Mice were immunized with either ovalbumin or latex as a primary sensitization followed by a secondary systemic sensitization with the other allergen (OVA/Latex, Latex/OVA). Single-sensitized mice received a sham sensitization followed by sensitization with either latex (PBS/Latex) or ovalbumin (PBS/OVA). A control group was purely sham-sensitized (PBS/PBS). All animals received airway challenges with the second allergen. in, Intranasal; ip, intraperitoneal; Neb, via nebulization. Journal of Allergy and Clinical Immunology 2006 118, 615-621DOI: (10.1016/j.jaci.2006.04.054) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 2 Total IgE. Experimental protocol as indicated in Fig 1. Mice were bled on day 72, and serum total IgE was determined by ELISA. Double-sensitized vs single-sensitized mice: PBS/Latex vs OVA/Latex, PBS/OVA vs Latex/OVA, ∗∗P < .01, ∗P < .05. Sham-sensitized vs allergen-sensitized mice: PBS/PBS vs PBS/Latex or PBS/OVA, ++P < .01. Journal of Allergy and Clinical Immunology 2006 118, 615-621DOI: (10.1016/j.jaci.2006.04.054) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 3 Mitogen-induced cytokine production by spleen MNCs. Experimental protocol as indicated in Fig 1. Splenic MNCs were obtained on day 72 and restimulated in vitro with concanavalin A. Cytokine production in supernatants was measured by ELISA: IL-4 (A), IL-5 (B), and IFN-γ (C). Single-sensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, PBS/OVA vs Latex/OVA, ∗∗P < .01, ∗P < .05. Sham-sensitized vs allergen-sensitized mice: PBS/PBS vs PBS/Latex or PBS/OVA, ++P < .01. Journal of Allergy and Clinical Immunology 2006 118, 615-621DOI: (10.1016/j.jaci.2006.04.054) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 4 Latex-specific immunoglobulin production. Experimental protocol as indicated in Fig 1. Mice were bled on day 72. Latex-specific IgE (A), IgG1 (B), and IgG2a (C) were evaluated by ELISA in measuring immunoglobulin production specific for Hev b 3, a major latex allergen. Levels are documented in OD units. Single-sensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, ∗P < .05. Sham-sensitized vs single-sensitized mice: PBS/PBS vs PBS/Latex, ++P < .01, +P < .05. Journal of Allergy and Clinical Immunology 2006 118, 615-621DOI: (10.1016/j.jaci.2006.04.054) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 5 Ovalbumin-specific immunoglobulin production. Experimental protocol as indicated in Fig 1. Mice were bled on day 72. Ovalbumin-specific IgE (A), IgG1 (B), and IgG2a (C) were measured by ELISA and compared with known hyperimmune standards (EU/mL). Single-sensitized vs double-sensitized mice: PBS/OVA vs Latex/OVA, ∗∗P < .01. Purely sham-sensitized animals (PBS/PBS) showed OVA-IgE, IgG1, and IgG2a below detection limits. Journal of Allergy and Clinical Immunology 2006 118, 615-621DOI: (10.1016/j.jaci.2006.04.054) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 6 Bronchoalveolar lavage. Experimental design as indicated in Fig 1. On day 72, two days after last airway challenge, lungs were lavaged, and differential cell counts in bronchoalveolar lavage fluids were obtained. Total cell counts, eosinophil counts, and lymphocyte counts are presented. Single-sensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, ∗P < .05. Sham-sensitized vs allergen-sensitized mice: PBS/PBS vs PBS/Latex or OVA/Latex, ++P < .01. Journal of Allergy and Clinical Immunology 2006 118, 615-621DOI: (10.1016/j.jaci.2006.04.054) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 7 Airway reactivity. Experimental design as indicated in Fig 1. On day 71, lung function to inhaled methacholine was measured by whole-body plethysmography. The dosage of methacholine increasing AR by 200% (PC 200) or 500% (PC 500) was calculated for each animal. A, AR of mice that received OVA as primary and Latex as secondary sensitization agent (OVA/Latex). B, AR of mice that received the reversed protocol (Latex/OVA). Single-sensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, PBS/OVA vs Latex/OVA, ∗∗P < .01, ∗P < .05. Sham-sensitized vs allergen-sensitized mice: PBS/PBS vs PBS/Latex, PBS/OVA or double-sensitized mice, +P < .05. MCh, Methacholine. Journal of Allergy and Clinical Immunology 2006 118, 615-621DOI: (10.1016/j.jaci.2006.04.054) Copyright © 2006 American Academy of Allergy, Asthma and Immunology Terms and Conditions