Influence of transient B cell depletion on recirculating B cells and plasma cells in rheumatoid arthritis [Elektronische Ressource] / vorgelegt von Arumugam Palanichamy
120 pages
English

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Influence of transient B cell depletion on recirculating B cells and plasma cells in rheumatoid arthritis [Elektronische Ressource] / vorgelegt von Arumugam Palanichamy

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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........................................................................................... 11.1 B cell developmental stages ...................................................................................... 1 1.2 Affinity maturation of B cells.................................................................................... 3 1.2.1 Somatic hypermutation of Ig genes........................................................................... 31.2.2 Gene conversion ........................................................................................................ 61.2.3 Class-switch recombination (CSR) 61.2.4 Activation induced cytidine deaminase (AID) - a regulator of B cell affinity maturation..........................................................................

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Publié par
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

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