The influence of the Maillard reaction on the immunogenic properties of food allergens [Elektronische Ressource] / von Anne Ilchmann

The influence of the Maillard reaction on the
immunogenic properties of food allergens



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften


vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main
von


Anne Ilchmann
aus Dresden












Frankfurt, 2010
(D 030)





Vom Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang
Goethe-Universität als Dissertation angenommen.


Dekan: Prof. Dr. H. Steinhilber



Gutachter: Prof. Dr. T. Dingermann
Prof. Dr. S. Vieths








Datum der Disputation: 23.04.2010















Meinen Eltern




Table of Contents

1 INTRODUCTION ........................................................................................................................... 1
1.1 Introduction to Allergy ........................................................................................................ 1
1.2 Mechanism of Type I Allergy ............................................................................................. 1
1.2.1 Allergens..................................................................................................................... 3
1.3 Food Allergy ....................................................................................................................... 3
1.4 Influence of thermal processing on food allergens ............................................................ 4
1.5 The Maillard reaction ......................................................................................................... 5
1.5.1 The Maillard reaction in food allergy .......................................................................... 6
1.5.2 Clinical relevance of the Maillard reaction .................................................................. 6
1.5.3 Influence of AGEs on DC function ............................................................................. 7
1.6 AGE binding receptors ....................................................................................................... 7
1.6.1 Macrophage scavenger receptors .............................................................................. 8
1.6.1.1 Class A Scavenger receptors ............................................................................. 8
1.6.1.1.1 Scavenger receptor class A type I and II ..................................................... 8
1.6.1.2 Class B Scav9
1.6.2 Receptor for advanced glycation end products (RAGE) .......................................... 10
1.6.3 Galectin-3 ................................................................................................................. 10
1.7 Model allergens used in this study .................................................................................. 11
1.7.1 Chicken Ovalbumin, a major allergen of egg white .................................................. 11
1.7.1.1 The Mannose receptor ...................................................................................... 12
1.7.2 Ara h 2, a major allergen of peanut .......................................................................... 12
1.8 Dendritic cells .................................................................................................................. 13
1.8.1 DC subsets ............................................................................................................... 13
1.9 Antigen uptake by dendritic cells ..................................................................................... 14
1.9.1 Pinocytosis and phagocytosis .................................................................................. 14
1.9.2 Receptor mediated endocytosis and the endocytotic pathway ................................ 15
1.10 Antigen processing by antigen presenting cells ............................................................ 16
1.10.1 MHC class I restricted presentation ....................................................................... 16
1.10.2 MHC class II restricted presentation ...................................................................... 16
1.11 Maturation of DCs .......................................................................................................... 17
1.12 T cells............................................................................................................................. 18
1.12.1 Activation of naïve T cells ...................................................................................... 18
+1.12.2 CD4 T cells ........................................................................................................... 19
1.12.2.1 Th1 cells .......................................................................................................... 19
1.12.2.2 Th2 ce20
1.12.2.3 Th17 cells ........................................................................................................ 20
1.12.2.4 Regulatory T cells ........................................................................................... 21
+1.12.3 CD8 T cells 21
1.13 B cells ............................................................................................................................ 22

2 AIMS OF THIS THESIS .............................................................................................................. 23

3 MATERIAL AND METHODS ...................................................................................................... 25
3.1 Material ............................................................................................................................ 25
3.1.1 Commonly used equipment...................................................................................... 25
3.1.2 Chemicals ............................................................................................................... 26
3.1.3 Buffers ...................................................................................................................... 26
3.1.4 Cell culture media and reagents .............................................................................. 26
3.1.5 Native and recombinant proteins ............................................................................. 27
3.1.6 Antibodies and reagents for immune detection ........................................................ 27
3.1.7 Mice .......................................................................................................................... 29
3.1.8 Oligonucleotides ....................................................................................................... 30
3.1.9 Software ................................................................................................................... 30
3.2 Methods ........................................................................................................................... 30
i



3.2.1 Molecular biological methods ................................................................................... 30
3.2.1.1 Total RNA isolation ........................................................................................... 30
3.2.1.2 Reverse transcriptase (RT) reaction ................................................................. 31
3.2.1.3 Polymerase chain reaction (PCR)..................................................................... 31
3.2.1.4 Agarose gel electrophoresis ............................................................................. 32
3.2.2 Biochemical methods ............................................................................................... 33
3.2.2.1 Preparation of proteins modified with AGEs ..................................................... 33
3.2.2.1.1 Preparation of AGE-OVA and AGE-BSA .................................................. 33
3.2.2.1.2 Preparation of AGE-rAra h 2 ..................................................................... 34
3.2.2.2 Measurement of the protein concentration using bicinchoninic acid (BCA) ..... 34
3.2.2.3 SDS-polyacrylamid gel electrophoresis (SDS-PAGE) ...................................... 34
3.2.2.4 Coomasie Brilliant blue protein stain ................................................................ 36
3.2.2.5 Structural analysis by circular dichroism spectroscopy .................................... 36
3.2.2.6 Labelling of proteins with Fluorescein isothiocyanate 37
3.2.3 Cell culture Methods .......................................................................