Surface binding autoantibodies and their functional effects in dermatomyositis and polymyositis [Elektronische Ressource] / by Pratibha Singh

Singh Vijay, Prahibha

Sujets

• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2010
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Surface binding autoantibodies and their functional effects in
dermatomyositis and polymyositis







Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfilment of the requirements
For the PhDDegree
of the Faculties of Veterinary Medicine and Medicine
of the JustusLiebig University Giessen, Germany


by
Pratibha Singh
from
Allahabad, India
Giessen 2010















From the Department of Neurology of the
Justus Liebig University Giessen



First Supervisor and Committee Member: Prof. Dr. Franz Blaes
Second Supervisor: Prof. Dr. Christiane Herden
Chairman of the examination panel: Prof. Dr. Wolfgang Kummer
Examiner: Prof. Dr. Stefan Arnhold





st
Date of the Doctoral Defense: 21 September, 2010.






















Dedicated to my grand mother and my parents.................











Declaration


I declare that the present thesis is my original work and that it has not been previously
presented in this or any other university for any degree. I have also abided by the
principles of good scientific conduct laid down in the charter of the Justus Liebig
University of Giessen in carrying out the investigations described in the dissertation.





















Index

INDEX

INDEX ............................................................................................................................. II
ABBREVIATIONS.......................................................................................................... III
1. INTRODUCTION......................................................................................................... 1
1.1. Immune system...............................................................................................................................1
1.2. Autoimmunity .................................................................................................................................4
1.3. Idiopathic inflammatory myopathies/ myositis .............................................................................7
1.3.1. Clinical features .........................................................................................................................7
1.3.2. Extramuscular manifestations ....................................................................................................9
1.3.3. Epidemiology........................................................................................................................... 12
1.3.4. Etiology ................................................................................................................................... 14
1.3.5. Pathogenesis........................................................................................................................... 14
1.3.6. Treatment................................................................................................................................ 21
2. AIM........................................................................................................................ 22
3. PATIENTS ............................................................................................................ 23
4. MATERIALS ......................................................................................................... 24
4.1. Chemicals and Solutions ............................................................................................................. 24
4.2. Consumables ............................................................................................................................... 25
4.3. Instruments................................................................................................................................... 26
4.4. Molecular biology and biochemistry kits.................................................................................... 27
4.5. Buffers and Solutions................................................................................................................... 27
4.6. Media and solutions for cell culture ............................................................................................ 30
4.7. Cell lines........................................................................................................................................ 31
4.8. Antibodies..................................................................................................................................... 31
4.9. Primers.......................................................................................................................................... 32
4.10. Software ...................................................................................................................................... 32
5. METHODS ................................................................................................................ 33
5.1. Cell culture.................................................................................................................................... 33
5.2. Flow cytometry ............................................................................................................................. 33
5.2.1. Autoantibody detection by flow cytometry................................................................................ 33
5.2.2. MHC Class I detection ............................................................................................................. 34
5.3. RNA isolation, RT8PCR, and Real time PCR ............................................................................... 34
5.4. Cytotoxicity assay ........................................................................................................................ 35
5.5. Calcium imaging ........................................................................................................................... 35
II
Index
5.5. Protein extraction ......................................................................................................................... 36
5.5. ONE8DIMENSIONAL GEL ELECTROPHORESIS (18DE).................................. 37
5.6. Two8dimensional gel electrophoresis (28DE)....................................................................... 37
5.7. Protein identification by peptide mass fingerprinting........................................................... 38
5.8. Statistical Analysis ................................................................................................................. 38
6. RESULTS ............................................................................................................. 40
6.1. Presence of surface binding autoantobodies........................................................................ 40
6.2. Western blotting of autoantibodies with muscle cell extract................................................ 47
6.3. Identification of autoantigens in muscle cells....................................................................... 48
6.4. Cytotoxic effect of autoantibodies ......................................................................................... 50
6.5. Effect of autoantibodies on endothelial calcium flux............................................................ 53
6.6. Effects of statins on muscles cells ........................................................................................ 54
6.6.1. Statins enhance IFNγinduced MHC class I expression in TE671 but not in SKMC .......... 54
6.6.2. TAP and LMP expression ................................................................................................. 60
7. DISCUSSION........................................................................................................ 62
7.1. Surface binding autoantibodies................................................................................................... 62
7.2. Proteomic approach to identify autoantigens ............................................................................. 64
7.3. Functional effects of autoantibodies ........................................................................................... 66
7.4. Skeletal muscle cell MHC I expression in response to statin treatment .................................... 68
8. SUMMARY............................................................................................................ 74
9. ZUSAMMENFASSUNG........................................................................................ 75
10. REFERENCES...................................................................................................... 77
ACKNOWLEDGEMENTS............................................................................................. 96
PUBLICATIONS ........................................................................................................... 97