Antigen specific depletion of autoreactive B lymphocytes in multiple sclerosis [Elektronische Ressource] / vorgelegt von Thomas Nachreiner

Tom Leahy

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2009
• Nombre de lectures: 28
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Antigen-specific depletion of autoreactive B
lymphocytes in Multiple Sclerosis




Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der
Rheinisch-Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften genehmigte Dissertation
vorgelegt von




Diplom Biologe
Thomas Nachreiner
aus Langerwehe




Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer
Universitätsprofessor Dr. rer. nat. Dr. rer. medic. Stefan Barth


Tag der mündlichen Prüfung: 16.03.2009



Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.
















You're only given a little spark of madness. You mustn't lose it.
- Robin Williams -

Index


1 Introduction ................................................................................................................ 1
1.1 Autoimmune diseases................................................................................................ 1
1.2 B cells in autoimmune diseases................................................................................ 4
1.3 Multiple sclerosis - overview, pathophysiology, therapy ........................................ 6
1.4 The myelin sheath and the demyelination process................................................ 10
1.5 Autoantigens ............................................................................................................ 12
1.5.1 Myelin Oligodendrocyte Glycoprotein (MOG) ................................................. 12
1.5.2 Proteolipid Protein (PLP)................................................................................ 13
1.5.3 Other antigenic determinants in MS ............................................................... 13
1.6 Experimental allergic encephalomyelitis (EAE) ..................................................... 15
1.7 Transgenic IgH mice ........................................................................................... 16 MOG
1.8 Immunotoxins........................................................................................................... 17
1.8.1 Immunotoxins for the depletion of autoreactive cells in MS ............................ 20
1.8.2 Expression of recombinant autoimmunotoxins ............................................... 21
1.9 Objectives ................................................................................................................. 22

2 Material and Methods............................................................................................... 25
2.1 Material...................................................................................................................... 25
2.1.1 Chemicals and Consumables......................................................................... 25
2.1.2 Media stock solutions and buffers .................................................................. 25
2.1.3 Standard buffer and media compositions ....................................................... 26
2.1.4 Kits................................................................................................................. 28
2.1.5 Buffers for protein work .................................................................................. 29
2.1.6 Antibodies and enzyme-labelled antibodies.................................................... 29
2.1.7 Bacterial strains.............................................................................................. 30
2.1.8 Eucaryotic cell lines........................................................................................ 30
2.1.9 Vector systems............................................................................................... 31

Index


2.1.10 Myelin protein clones...................................................................................... 33
2.2 Methods..................................................................................................................... 33
2.2.1 Polymerase Chain Reaction (PCR) ................................................................ 33
2.2.2 Synthetic oligonucleotides.............................................................................. 34
2.2.3 DNA cloning techniques ................................................................................. 35
2.2.4 Agarose gel electrophoresis ........................................................................... 36
2.2.5 Culturing of bacteria ....................................................................................... 37
2.2.6 Transformation of bacteria with plasmid DNA................................................. 37
2.2.7 Preparation of plasmid DNA from E. coli ........................................................ 38
2.2.8 DNA sequencing ............................................................................................ 38
2.2.9 Cell culture ..................................................................................................... 39
2.2.10 Protein analysis and immunological methods................................................. 40
2.2.11 Flow cytometry ............................................................................................... 45
2.2.12 Cell viability assays ........................................................................................ 46
2.2.13 Internalization studies..................................................................................... 47
2.2.14 Animal experiments........................................................................................ 48

3 Results ...................................................................................................................... 51
3.1 Design, expression and characterisation of autoimmunotoxins .......................... 51
3.1.1 Cloning of MOG fusion proteins for bacterial expression ................................ 51
3.1.2 Restriction analysis of pBM-MOG vector constructs....................................... 52
3.1.3 Expression and characterization of pBM-MOG-ETA’ and pBM-MOG ............. 53
3.1.4 Cloning of PLP fusion proteins for bacterial expression.................................. 55
3.1.5 Restriction analysis of pBM-PLP vector constructs......................................... 55
3.1.6 Expression and characterization of pBM-PLP-ETA’ and pBM-PLP................. 56
3.2 Design, expression and characterisation of GFP-fusions for imaging purposes 57
3.2.1 Cloning of GFP-ligand fusion proteins ............................................................ 58
3.2.2 Restriction analysis of pMS-L-eGFP plasmids................................................ 58
3.2.3 Expression and characterization of eGFP fusion proteins............................... 59
3.3 Native binding analysis of recombinant MOG and PLP proteins.......................... 60
3.3.1 Binding analyses by enzyme linked immunosorbant assay (ELISA) ............... 60


Index

3.3.2 Binding analyses by flow cytometry................................................................ 62
3.4 Internalization studies of eGFP-MOG on MOG-reactive hybridoma cells............. 67
3.5 Quantitative cell viability assays............................................................................. 69
3.5.1 Toxic in vitro activity of MOG-ETA’................................................................. 69
3.5.2 Toxic in vitro activity of PLP-ETA’................................................................... 71
3.5.3 FACS-based cell viability assay of MOG-ETA’ on IgH splenocytes ........... 73 MOG
3.6 Animal Experiments ................................................................................................. 74
3.6.1 In vivo targeting of MOG-reactive cells in IgH mice................................... 74 MOG
3.6.2 Evaluation of MOG-ETA’ autoimmunotoxin in an EAE mouse model.............. 76

4 Discussion ................................................................................................................ 79
4.1 Expression and purification of autoimmunotoxins................................................ 80
4.1.1 Purification and characterization of autoimmunotoxins ................................... 81
4.2 Eukaryotic expression of eGFP fusion proteins .................................................... 83
4.3 Functional binding characterization of autoimmunotoxins................................... 84
4.3.1 Binding analysis on antigen-specific hybridoma cell lines via ELISA .............. 85
4.3.2 Binding analysis on antigen-specific hybridoma cell lines via flow cytometry.. 85
4.3.3 Binding analysis o