Devic mouse: a spontaneous double transgenic mouse model of human opticospinal multiple sclerosis and autoimmune T-B-cell cooperation [Elektronische Ressource] / vorgelegt von Gurumoorthy Krishnamoorthy

Gurumoorthy Krishnamoorthy

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Devic mouse: a spontaneous double-transgenic mouse
model of human opticospinal multiple sclerosis and
autoimmune T- B cell cooperation








Dissertation

zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer. nat.)
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München







vorgelegt von
Gurumoorthy Krishnamoorthy
India



Munich, June 2006

Hiermit erkläre ich, dass ich die vorliegende Dissertation mit Ausnahme der
Immunhistochemie selbständig und ohne unerlaubte Hilfe habe.

Ich habe weder anderweitig versucht, eine Dissertation oder Teile einer Dissertation
einzureichen beziehungsweise einer Prüfungskommission vorzulegen, noch eine
Doktorprüfung durchzuführen.




Munich, June 2006












Dissertation eingereicht: 12-06-06
Tag der mündlichen Prüfung:

Erstgutachter: Prof. Dr. Elisabeth Weiß
Zweitgutachter: PD Dr. Christine Falk

Acknowledgements
At the outset, I would like to express my deep appreciation and gratitude to Prof. Dr.
Hartmut Wekerle for offering me a graduate fellowship that enabled me to work in
his department. I would like to thank him for his critical comments and continuous
support. While instilling a tough scientific attitude in me, he was also able to maintain
a relaxed atmosphere in the lab, which made it not only stimulating to work there but
also fun.

I am also grateful to Dr. Andreas Holz who supervised this thesis. As my supervisor,
he constantly supported, encouraged, and taught me how to focus my research. His
constructive comments and constant encouragement brought out the best in me.
Our hour-long discussions have sharpened my thinking about scientific problems.

My special thanks to Prof. Dr. Hans Lassmann for the excellent histological analysis.

I would like to thank Bernadette Pöllinger for her helpfulness and patient open ear
for my problems. The list of my indebtedness to her is endless, especially for her
help in correcting my thesis.

My sincere thanks to Irene Arnold-Ammer for her excellent technical assistance. She
also helped to maintain the relaxed atmosphere in the lab with her sense of humor.

I am grateful to Prof. Dr. Elisabeth Weiß for agreeing to be an examiner of my thesis.
I wish to extend my thanks to PD Dr. Christine Falk, Prof. Dr. George Boyan, Prof.
Dr. Harry MacWilliams, Prof. Dr. Heinrich Jung and Prof. Dr. Benedikt Grothe for
evaluating my thesis.

I thank Dr. Anjali (IISc, Bangalore) and my dear friends Raghu, Madhavi, and Rajesh
for helping me to come to Germany.

My life would have been very difficult without my friends Sham, Sona, to mention
only a few.

Special thanks go to my wife Jeeva for her love and care.

Last, but not least, I thank my family. No words can describe the care, guidance,
love, understanding, and support they tirelessly gave me.


TABLE OF CONTENTS
1. SUMMARY ............................................................................................................ 1
2.1 Multiple sclerosis (MS) ..............................................................................................................3
2.1.1 Definition ...............................................................................................................................3
2.1.2 Clinical course.......................................................................................................................4
2.1.3 Pathology ..............................................................................................................................5
2.2 Devic’s neuromyelitis optica (NMO): a variant of classic MS................................................6
2.3 Disease etiology.........................................................................................................................8
2.3.1 Genetic factors............8
2.3.2 Environmental factors.......9
2.4 Target autoantigens in MS and EAE ......................................................................................11
2.4.1 Myelin basic protein (MBP).................................................................................................12
2.4.2 Proteolipid protein (PLP).....................................................................................................13
2.4.3 Myelin oligodendrocyte glycoprotein (MOG) ......................................................................13
2.4.4 Other myelin and non-myelin antigens ...............................................................................15
2.5 Immune responses in the pathogenesis of MS.....................................................................16
2.6 Major players in MS .................................................................................................................18
+2.6.1 CD4 T Cells .......................................................................................................................18
+2.6.2 CD819
2.6.3 B cells and antibodies.........................................................................................................19
2.7 Animal models of multiple sclerosis......................................................................................20
2.7.1 Mouse models.....................................................................................................................21
2.7.1.1 Induced models ..........................................................................................................................22
2.7.1.2 Transgenic models .....................................................................................................................23
2.7.1.3 Novel model of T and B cell interaction ......................................................................................25
3. OBJECTIVES 27
4. MATERIALS AND METHODS ............................................................................ 28
4.1. Materials...................................................................................................................................28
4.1.1 Antibodies ...........................................................................................................................28
4.1.2 Buffers and reagents...........................................................................................................30
4.1.3 Primers................................................................................................................................32
4. 2 Methods....................................................................................................................................34
4.2.1 Animals.............34
4.2.2 Genotyping...........34
4.2.3 Disease scoring ..................................................................................................................35
4.2.4 Antigens.............36
4.2.5 Immunization of animals .....................................................................................................36
4.2.6 Serum collection .................................................................................................................36
4.2.7 Isolation of CNS mononuclear cells....................................................................................36
4.2.8 Preparation of PBMCs ........................................................................................................37
4.2.9 Cell purification ...................................................................................................................37
4.2.10 CFSE labeling of lymphocytes..........................................................................................37
4.2.11 In vitro proliferation assay.................................................................................................37
4.2.12 Bone marrow chimera..38
4.2.13 Adoptive transfer...............................................................................................................38
4.2.14 Histological analysis..........................................................................................................38
4.2.15 Immunohistochemistry......................................................................................................39
4.2.16 Flow cytometry..................................................................................................................39
4.2.17 Hybridoma generation..40
4.2.18 Antibody purification .........................................................................................................40
4.2.19 Enzyme linked immunosorbent assay (ELISA) ................................................................40
4.2.20 Enzyme linked immunospot (ELISPOT) assay.................................................................41

4.2.21 Quantitative real-time TaqMan PCR analysis...................................................................41
4.2.22 Statistical analysis...........................................................................................