The mouse cholera toxin model for evaluation of allergenicity
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1 The mouse cholera toxin model for evaluation of allergenicity Joost Smit, ILSI 2012
2 Of mice and men.(mestas, J Immunol 2004; 172:2731) Mouse Human Neutrophils in blood 10-25% 50-70% Lymphocytes in blood 75-90% 30-50% Predominant T cells in skin and gut γδ TCR αβ TCR FcαRI absent present FcγRIIA absent present IgG classes IgA, IgD, IgE, IgG1 IgA, IgA2, IgD, IgE, IgG1 IgG2a/b/c, IgG3, IgM IgG2, IgG3, IgG4, IgM Th expression of IL-10 Th2 Th1 and Th2 IL-13 effect on B cells none IgE switch Epithelial cells present Ag to CD4+ no Yes Immunological tissues investigated Lung, gut, spleen, thymus Blood lymph nodes, etc, etc..
3
4 A peanut food allergy model Sensitisation Challenge Peanut extract (PE) + PE i.g. Mast cell Sacrifice Cholera toxin i.g. degranulation PE i.p./i.v. Anaphylaxis -Antibodies -T cell PE i.d. Ear swelling responses
5 A peanut food allergy model: Parameters - Cytokine production after restimulation of MLN and spleen cells - Flow cytometry of isolated cell populations of MLN, Intestinal epithelial lymphocytes (IEL), Lamina Propria lymphocytes (LPL), Peyer s Patches (PP) - Antibody measurements of allergen-specific IgG1, IgG2a, IgE, IgA, IgM - Histological analysis of the intestine or lymphoid organs - Mast cell degranulation in vivo (histamine, MMCP-I release) - Functional parameters: Gut permeability, airway reactivity - Anaphylaxis: temperature and anaphylaxis score - Ear swelling after intra-dermal challenge
6 A peanut food allergy model: determinants (dirty little secrets.) - Allergen used (peanut, whey, ß-lactoglobulin, etc..) - Dose of allergen during sensitization and challenge - Dose of CT (10, 15, 20µg) - Unofficial adjuvants: Alcohol, NSAIDs, high protein dose (!)
7 A peanut food allergy model: determinants (dirty little secrets.) - Allergen used (peanut, whey, ß-lactoglobulin, etc..) - Dose of allergen during sensitization and challenge - Dose of CT (10, 15, 20µg) - Unofficial adjuvants: Alcohol, NSAIDs, high protein dose (!)
8 A peanut food allergy model: determinants (dirty little secrets.) - Allergen used (peanut, whey, ß-lactoglobulin, etc..) - Dose of allergen during sensitization and challenge - Dose of CT (10, 15, 20µg) - Unofficial adjuvants: Alcohol, NSAIDs, high protein dose (!) - Matrix: Lipids, sugars, aggregated proteins - Contamination of protein with endotoxin: e.g. ß-lactoglobulin - (Microbial) status of the mouse housing - Mouse strain
9 A (mouse) model for food allergy - Mechanisms of (food) allergy - Risk assessment of (novel) proteins: - Allergenicity - Availability of proteins in vivo -Diagnostics of allergy: -New biomarkers of disease -New therapeutic opportunities: -Medicines -Immunotherapy (antibodies) -Allergen immunotherapy
10 A (mouse) model for food allergy - Mechanisms of (food) allergy - Risk assessment of (novel) proteins: - Allergenicity - Availability of proteins in vivo -Diagnostics of allergy: -New biomarkers of disease -New therapeutic opportunities: -Medicines -Immunotherapy (antibodies) -Allergen immunotherapy
11 A peanut food allergy model Sensitisation Challenge mg Peanut extract (PE) + PE i.g. Mast cell Sacrifice 15 µg Cholera toxin i.g. degranulation PE i.p. Anaphylaxis -Antibodies -T cell PE i.d. Ear swelling responses
12 A peanut food allergy model in 3 mouse strains C3H/HeOuJ C57BL/6 BALB/c Antibodies: IgE IgG IgG2a ++ IgG2c - Cytokines: Th1 (IFNy) Th2(IL-5, IL-13) +/ Smit JJ, Plos One, 2012
13 Mast cell degranulation i.g. challenge i.p. challenge MMCP-I (pg/ml) * * MMCP-I (pg/ml) * * 0 0 C3H Cont C3H PE C57Bl/6 Cont C57Bl/6 PE BALB/c Cont BALB/c PE C3H Cont C3H PE C57Bl/6 Cont C57Bl/6 PE BALB/c Cont BALB/c PE Smit JJ, Plos One, 2012
14 Peanut-induced systemic anaphylaxis 38 Temperature Temperature ( C) * * # * Time after challenge (m) C57BL/6 Cont C57BL/6 PE BALB/c Cont BALB/c PE C3H Cont C3H PE Smit JJ, Plos One, 2012
15 A peanut food allergy model in 3 mouse strains C3H/HeOuJ C57BL/6 BALB/c Antibodies: IgE IgG IgG2a ++ IgG2c - Cytokines: Th2(IL-5, IL-13) +/ Mucosal mast cell degranulation Anaphylaxis Ear swelling Smit JJ, Plos One, 2012
16 Classic Pathway Alternative Pathway IgG Allergen IgG FcγRIII IgE FcεR FcγRIIb FcγRIIb FcγRIII Mast cell Macrophage Histamine + PAF Smooth muscle Vascular endothelium PAF Anaphylaxis Finkelman 2007
17 Mast cells and peanut-induced anaphylaxis IgE Temperature ( C) * IgE (AU) C57BL/6 C57BL/6 W-sh/W-sh C57BL/6 cont C57BL/6 PE Time after challenge (m) C57BL/6 W-sh/W-sh cont C57BL/6 W-sh/W-sh PE 90 Smit JJ, Plos One, 2012
18 Food allergy, more than mast cells and IgE? - Anaphylaxis can occur in the absence of mucosal mast cell degranulation (MMCP-I) in C57BL/6 mice But. - Mast cells are essential for peanut-induced anaphylaxis in C57BL/6 mice, are these connective tissue mast cells? - Components of the alternative pathway such as macrophages and PAF do play a role in peanut induced anaphylaxis, but only in C57BL/6 mice (Smit JJ, Plos One, 2011)
19 A (mouse) model for food allergy - Mechanisms of (food) allergy - Risk assessment of (novel) proteins: - Allergenicity - Availability of proteins in vivo -Diagnostics of allergy: -New biomarkers of disease -New therapeutic opportunities: -Medicines -Immunotherapy (antibodies) -Allergen immunotherapy
20 IgE responses allergens in the CT model Bowman CC, Tox Sci 2008
21 A (mouse) model for food allergy - Mechanisms of (food) allergy - Risk assessment of (novel) proteins: - Allergenicity - Availability of proteins in vivo -Diagnostics of allergy: -New biomarkers of disease -New therapeutic opportunities: -Medicines -Immunotherapy (antibodies) -Allergen immunotherapy
22 Immunotherapy in food allergy Sensitisation S.c. therapy Challenge PE+ CT PE PE PE PE PE i.g. s.c i.g. i.p. i.g. i.p. Anaphylaxis
23 Efficacy of immunotherapy IgG1 (AU*10 5 ) Control No IT 0.03 PE 0.1 PE IgG1 IgG2a (AU*10 4 ) Control No IT 0.03 PE 0.1 PE IgG2a IgE (AU*10 3 ) Control No IT 0.03 PE 0.1 PE IgE
24 Efficacy of immunotherapy Temperature d92 Temperature ( C) W W Time after challenge (m) 90 * * # # 100 Cont No IT 0.03 PE 0.1 PE W - On heating pad
25 A (mouse) model for food allergy - Mechanisms of (food) allergy - Risk assessment of (novel) proteins: - Allergenicity - Availability of proteins in vivo -Diagnostics of allergy: -New biomarkers of disease -New therapeutic opportunities: -Medicines -Immunotherapy (antibodies) -Allergen immunotherapy
26 Acknowledgements Manon van Roest Karina Willemsen Marianne Bol-Schoenmakers Ine Hassing Daniëlle Fiechter Rob Bleumink Raymond Pieters Faculty of Veterinary Medicine Dietmar Zaiss Maarten Pennings Els van Hoffen Jolanda van Bilsen André Penninks Stef Koppelman Hanneke van der Kleij
Heterogeneous responses and cross reactivity between the major peanut allergens Ara h 1, 2,3 and 6 in a mouse model for peanut allergy
Smit et al. Clinical and Translational Allergy (2015) 5:13 DOI 10.1186/s13601-015-0056-9 RESEARCH Open Access Heterogeneous responses and cross reactivity between the major peanut allergens Ara h 1, 2,3
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