Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection [Elektronische Ressource] / von Martin Holdener

Sujets

• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2008
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 44 Mo

Extrait

Breaking tolerance to the natural
human liver autoantigen cytochrome
P450 2D6 by virus infection
Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften
Vorgelegt beim Fachbereich
Chemische und Pharmazeutische Wissenschaften
der Johann Wolfgang Goethe- Universität
in Frankfurt am Main
von
Martin Holdener
aus Luzern

Frankfurt am Main, 2008
D30
vom Fachbereich
Chemische und Pharmazeutische Wissenschaften der
Johann Wolfgang Goethe-Universitä tals Dissertation angenommen.
Dekan: Prof. Dr. D. Steinhilber
1. Gutachter: Prof. Dr. Th. Dingermann
2. Gutachter: PD Dr. U. Christen
Datum der Disputation: 16.7.2009
1To my family
21 Table of Contents
1 TABLE OF CONTENTS.............................................. .............................................................3
2 SUMMARY..................................................... ....5...............................................................
3 ZUSAMMENFASSUNG.............................................. .............................................................6
4 LIST OF ABBREVIATIONS............................................ .......................................................10
5 LIST OF FIGURES.............................................1.2 ..................................................................
6 LIST OF TABLES................................................1 .2................................................................
7 INTRODUCTION..................................................1.3 .................................................................
7. 1 OVERVIEW OF THEIM MUNE SYSTEM............................ ........................................................13
7.1.1 Innate Immune syste.m............................................... ............................................13
7.1.2 Adaptive Immune system .............................................. ..........................................14
7. 2 TOLERANCE....................................... ................................................................................17
7.2.1 Generation of Tolerance.............................................. ............................................18
7.2.2 Breaking of tolerance – induction of autoimmune disease................. ..................19
7. 3 THE LIVER........................................ ..................................................................................25
7.3.1 Anatomy and function of the liver..................................... ......................................25
7.3.2 Liver immunology.................................................... .................................................26
7.3.3 Liver diseases...................................................... .....................................................28
7.3.4 Autoimmune liver diseases............................................. .........................................28
8 AIM OF THE STUDY............................................ .................................................................36
8. 1 STRATEGY........................................ .................................................................................36
8.1. 1 The CYP2D6 humanized mice........................................... .....................................36
8.1. 2 Adenoviruses........................................................ ....................................................38
8. 2 WORKING HYPOTHESIS .................................. .....................................................................39
9 RESULTS.......................................................... ......4.1...............................................................
9. 1 INTRODUCTION OF THEC YP2D6 MOUSE MODEL FOR AUTOIMMUNE HEPATIT.IS....... ............41
9.1.1 Route of infection................................................... ..................................................41
9.1.2 Protein expression in the liver......................................... ........................................42
9.1.3 Infection of mice with adenovirus results in transient hepatic damage..... ..........43
9.1.4 Infection with Ad-2D6 results in severe and persistent liver damage....... ...........45
9. 2 PATHOGENIC MECHANISM IN THCE YP2D6 MOUSE MODEL FOR AUTOIMMUNE HEPATIT.IS.. ..54
9.2.1 B cell tolerance to liver-specific self antigen is broken after Ad-2D6 infection... 54
9.2.2 Anti-CYP2D6 antibodies in Ad-2D6-infected mice and sera from AIH-2 patients
recognize an identical immunodominant epitope.......................... .......................58
39.2.3 Virus infection is essential for persistent liver damage but not autoantibody
production............................................................ ....................................................61
9.2.4 T cell tolerance to liver-specific self antigen is broken after Ad-2D6 infection ...62
10 DISCUSSION..................................................6. 7..................................................................
10. 1BREAKING TOLERANCE TO THE NATURAL HUMAN LIVER AUTOANTIGCENY P2D6 BY VIRUS
INFECTION............................................. ..........................................................................67
10. 2MOUSE MODELS FOR HUMAN AUTOIMMUNE HEPATIT.IS.................... .................................67
10. 3LESSONS LEARNED FROM ANIMAL MODELS FORAIH ....................... ..................................69
10. 4 THE CYP2D6 ANIMAL MODEL FORA IH............................. ...............................................71
10. 5 ADVANTAGES OF THEC YP2D6 MOUSE MODEL......................... ........................................73
10. 6 THE CYP2D6 MOUSE MODEL AND THE ETIOLOGY OFAIH .................... .............................74
10. 7 THE CYP2D6 MODEL AND PATHOGENIC MECHANISM OFAI H.................. ..........................77
10. 8POSSIBILITIES OF THEC YP2D6 MOUSE MODEL......................... .......................................81
11 APPENDIX...................................................... .84................................................................
12 MATERIAL AND METHODS........................................ ......................................................85
12. 1SOURCE OFM ATERIALS ..................................... ..............................................................85
12.1.1 Plastic ware........................................................... ...................................................85
12.1.2 Chemicals............................................................ ....................................................85
12.1.3 Composition of buffers, solutions and culture media...................... .....................87
12.1.4 Enzymes and proteins.................................................. ...........................................89
12.1.5 Nucleotides and nucleic acids.......................................... .....................................89
12.1.6 Antibodies............................................................ ....................................................90
12.1.7 Kits..................................................................91. .........................................................
12.1.8 Human Sera.......................................................... ...................................................91
12.1.9 Cell lines............................................................. ......................................................91
12.1.10 Viruses............................................................... .......................................................91
12.1.11 Mice strains........................................................... ...................................................92
12. 2METHODS............................................. ...........................................................................92
12.2.1 Cell biological methods............................................... ...........................................92
12.2.2 Molecular biological methods........................................... ...................................102
12.2.3 Experiments with mice.................................................. ........................................107
13 REFERENCES:................................................09.. .................................................................1
14 ACKNOWLEDGEMENTS........................................... .......................................................118
15 CURRICULUM VITAE............................................... ........................................................119
16 PUBLICATIONS................................................1. ................................................................12

42 Summary
Autoimmune hepatitis (AIH) is a chronci liver disease of unknown etiology,
characterized by a loss oftolerance against hepatocytes leading to the
progressive destruction of hepatic parenchyma and cirrhosis. Clinical signs
for AIH are interface hepatitis andp ortal plasma cell infiltration,
hypergammaglobulinemia, and autoantibodies. Based on serological markers
AIH is defined in subtypes. The hallmark of AIH type 2 are type 1 liver/kidney
microsomal autoantibodies (LKM-1), whereas AIH type 1 is characterized by
the presence of anti-nuclear (ANA) and/or anti-smooth mus