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Publié par | julius-maximilians-universitat_wurzburg |
Publié le | 01 janvier 2007 |
Nombre de lectures | 17 |
Langue | English |
Poids de l'ouvrage | 1 Mo |
Extrait
INFLUENCE OF TRANSIENT B CELL DEPLETION ON
RECIRCULATING B CELLS AND PLASMA CELLS IN
RHEUMATOID ARTHRITIS
Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians- Universität Würzburg
Vorgelegt von
Arumugam Palanichamy
Tirunelveli, Indien
Würzburg, 2007
Eingereicht am: 30 July 2007
Mitglieder der Promotionskommission:
Vorsitzender: Prof. Dr. Martin. J. Müller
Gutachter: Prof. Dr. Hans-Peter Tony
Gutachter: Prof. Dr. Dr. h. c. mult. Jörg Hacker
Tag des Promotionskolloquims:
Doktorurkunde ausgehändigt am:
Contents
1 Introduction........................................................................................... 1
1.1 B cell developmental stages ...................................................................................... 1
1.2 Affinity maturation of B cells.................................................................................... 3
1.2.1 Somatic hypermutation of Ig genes........................................................................... 3
1.2.2 Gene conversion ........................................................................................................ 6
1.2.3 Class-switch recombination (CSR) 6
1.2.4 Activation induced cytidine deaminase (AID) - a regulator of B cell affinity
maturation.................................................................................................................. 7
1.3 Sites of Somatic hypermutation................................................................................. 9
1.3.1 Germinal centre – The site of somatic hypermutation .............................................. 9
1.3.2 Somatic hypermutations outside the germinal centre 10
1.4 Mutational hotspots ................................................................................................. 11
1.4.1 RGYW and WRCY motifs...................................................................................... 11
1.4.2 Expression of RGYW/WRCY motifs ..................................................................... 11
1.5 Replacement and silent mutations ........................................................................... 12
1.6 Rheumatoid arthritis ................................................................................................ 13
1.7 Role of B cell in health and disease......................................................................... 15
1.7.1 Functions of B cell in healthy subjects.................................................................... 15
1.7.2 Role of B cell in rheumatoid arthritis ...................................................................... 15
1.8 Human Ig- VH locus ............................................................................................... 18
1.8.1 Ig-VH4 gene rearrangements in healthy subject ..................................................... 18
1.8.2 Ig-VH4 gene usage in autoimmunity....................................................................... 18
1.9 B cells as therapeutic targets in autoimmune diseases ............................................ 19
1.9.1 B cell depletion........................................................................................................ 19
1.9.2 Inhibition of B cell survival..................................................................................... 19
1.9.3 Blockade of CD40 signalling in B cells .................................................................. 20
1.9.4 Suppression of cytokines......................................................................................... 20
1.10 Anti-CD20 therapy .................................................................................................. 21
1.10.1 Rituximab treatment – A novel B cell depletive therapy ........................................ 21
1.10.2 Human CD20 molecule ........................................................................................... 21
1.10.3 B cell subsets depleted by rituximab therapy .......................................................... 21
1.10.4 Mechanism of B cell depletion by rituximab therapy ............................................. 22
1.11 Rituximab therapy in treating rheumatoid arthritis ................................................. 23
2 Objectives of the project..................................................................... 24
3 Material and methods......................................................................... 26
3.1 Patients .................................................................................................................... 26
3.2 Ig analysis from total genomic DNA....................................................................... 27
3.2.1 Isolation of Peripheral blood mononuclear cells (PBMCs)..................................... 27
3.2.2 Extraction of total genomic DNA............................................................................ 27
3.3 Ig-VH4 gene amplification...................................................................................... 29
3.3.1 Oligonucleotide sequences 29
3.3.2 External amplification round................................................................................... 30
3.3.3 Internal amplification round .................................................................................... 31
3.4 PCR product isolation.............................................................................................. 32
3.5 Sub cloning of Ig-VH4 gene rearrangements.......................................................... 32
3.5.1 Polishing.................................................................................................................. 32
3.5.2 Subcloning and transformation................................................................................ 33
3.6 Liquid culture .......................................................................................................... 35
3.7 Plasmid DNA isolation............................................................................................ 36
3.8 Analysis of the transformants.................................................................................. 36
3.8.1 PCR on plasmid DNA ............................................................................................. 36
3.9 Sequencing .............................................................................................................. 37
3.9.1 Purification of sequencing reaction products .......................................................... 38
3.10 Analysis of Ig sequences ......................................................................................... 39
3.10.1 Sequence analysis software ..................................................................................... 39
3.11 Single cell RT-PCR ................................................................................................. 41
3.11.1 Cell preparation ....................................................................................................... 41
3.11.2 Preparation of lysis buffer ....................................................................................... 42
3.11.3 Cell sorting .............................................................................................................. 43
3.11.4 Upper reaction buffer .............................................................................................. 43
3.11.5 Synthesis of cDNA .................................................................................................. 44
3.11.6 Amplification of the VH4 genes.............................................................................. 44
3.11.7 Direct sequencing of Ig-VH4 genes ........................................................................ 45
3.12 Statistical analyses................................................................................................... 46
3.13 Materials .................................................................................................................. 46
4 Results 48
4.1 B cell regeneration 48
4.1.1 Identification of B cells in the periphery following rituximab therapy................... 48
4.1.2 Detection of B cells by PCR amplification of Ig genes........................................... 50
4.2 Clinical data............................................................................................................. 51
4.3 Kinetics of B cell subsets regeneration.................................................................... 52
4.4 Immunoglobulin analysis by total genomic DNA amplification ............................ 54
4.4.1 Analysis of Ig-VH4 gene rearrangement pre and post treatments .......................... 54
4.4.2 Comparison of mutational frequency of immunoglobulin genes 55
4.4.3 Grouping of Ig sequences on the basis of number of mutations ............................. 56
4.4.4 Phenotypic analysis of plasma cells pre and post treatment.................................... 59
4.4.5 Correlation of highly mutated sequences to the plasma cell fraction