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

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105 Pages
English

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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 stDate 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 ..........................................................................................

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Published 01 January 2010
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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










III
Abbreviations
Abbreviations



2D Two Dimensional
AECA anti endothelial cell antibodies
antiARS antiaminoacyl tRNA synthetase antibodies
antiKu antiKu antibodies
antiMi2 antiMi2 antibodies
antiPMScl antiPMScl antibodies
antiSAE antismall ubiquitinlike modifier activating enzyme
antiSRP antiSignalRecognition particle antibodies
antiU1RNP antiU1ribonucleoprotein antibodies
antiU3RNP antiU3ribonucleoprotein antibodies
AP Alkaline Phosphatase;
DM Dermatomyositis
E.Coli Escherichia coli
EC Endothelial cell
ELISA Enzyme Linked Immunosorbent Assay
FITC Fluorescinisothiocyanate
HCMEC Human chorionic microvascular endothelial cells
HDMEC human dermal microvascular endothelial cells
HUVEC Human umbilical vascular endothelial cells
IFNγ Interferon gamma
IgG Immunoglobulin G
IIMs Idiopathic inflammatory myopathies
LEMS LambertEaton Myasthenic syndrome
LMP2 / 7 Low molecular mass polypeptide 2 / 7
MAAs myositisassociated autoantibodies
MCTD Mixed connective tissue diseases
MHC class I Major histocompatibility complex class I
III
Abbreviations
MSAs myositisspecific autoantibodies
mTOR mammalian target of rapamycin
OIM Other inflammatory myopathies
PBGD Porphobilinogen deaminase
PM Polymyositis
SEREX serological analysis of recombinant cDNA expression library
SERPA serological proteome analysis
SKMC Human primary skeletal muscle cells
SLE Systemic Lupus Erythematosus
TAP1 / 2 Transporter associated with antigen processing 1 / 2
TE671 Human rhabdomyosarcoma cells
WG Wegener’s granulomatosis
























IV
Introduction
1. Introduction
1.1. Immune system
The immune system is the body’s natural guardian against disease. It has evolved as a
way for us to defend ourselves from invading pathogenic organisms like bacteria,
parasites and viruses. It equals in complexity the intricacies of the brain and nervous
system, displays several remarkable characteristics which include distinction between
“self” and “nonself” and the ability to remember previous experiences and react
accordingly. Based on the type of response whether it is unspecific or specific, immune
system can be divided into innate and adaptive immune system respectively (Goldsby
et al. 2000; Janeway et al. 2001). These two systems are integrated and often interact
with each other (Hoebe et al. 2004).
Our first line of defence against any invasion from outside is the innate immune system,
which also includes a series of unspecific barriers that try to stop the invading
organisms. Our body surfaces are defended by epithelium, which provides a physical
barrier between the internal milieu and the external world that, contain pathogens.
Infections occur only when pathogen can colonize or pass through these barriers. The
low pH in the intestine and the presence of degrading enzymes also inhibit invading
pathogens. If a microorganism crosses the epithelial barrier and begins to replicate in
the tissues of host, in most cases it is immediately recognized by the mononuclear
phagocytes, or macrophages, that reside in these tissues (Fig. 1). While many cell types
are capable of endocytosis, only specialized cells like blood monocytes, tissue
macrophages and neutrophils are capable of phagocytosis. The next line of barrier also
includes the complement system, which consists of serum proteins that exist in from of
inactive proenzymes, which gets activated in response to different specific and non
specific immunologic mechanisms. Once activated, they lead to direct damage to the
cell membrane of the pathogenic microorganisms or make them more prone to
phagocytic uptake by binding to the pathogens (opsonisation).
1
Introduction

Figure 1. Major events in the inflammatory response. A bacterial infection causes tissue damage
with release of various vasoactive and chemotactic factors. These factors induce increased blood
flow to the area, increased capillary permeability, and an influx of white blood cells, including
phagocytes and lymphocytes, from the blood into the tissues. The serum proteins contained in
the exudate have antibacterial properties, and the phagocytes begin to engulf the bacteria.
(Goldsby et al. 2000).
As is evident from the very name of it, “adaptive immune system”, it has the ability to
adapt to the changes in the threat from the pathogenic microorganisms. Four attributes
that characterize the adaptive immune system are: specificity, diversity, memory and
self / nonself recognition. The antigenic specificity of the immune system permits it to
distinguish subtle differences among the antigens. The immune system is capable of
generating tremendous diversity in generating its recognition molecules, allowing it to
recognize billions of unique structure on foreign antigens (Fig. 2). Some of the cell types
of the adaptive immune system are long lived and these cells contribute to the
immunologic memory that makes it possible for the immune system to remember
foreign molecules that it has experienced before. The next time the same foreign
2