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Informations
Publié par | humboldt-universitat_zu_berlin |
Publié le | 01 janvier 2009 |
Nombre de lectures | 32 |
Langue | Deutsch |
Poids de l'ouvrage | 2 Mo |
Extrait
Mycobacterium tuberculosis-specific T-cell responses
in latent infection and active disease
DISSERTATION
zur Erlangung des akademischen Grades
Doctor rerum naturalium
(Dr. rer. nat.)
eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt Universität zu Berlin
von
Diplom-Biologe
Sebastian D. Schuck
geboren am 22.05.1981 in Recklinghausen
Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Christoph Markschies
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Lutz-Helmut Schön
Gutachter/-innen: 1. Prof. Dr. Hans-Dieter Volk
2. Prof. Dr. Stefan Kaufmann
3. Prof. Florian Kern
Tag der mündlichen Prüfung: 13. März 2009
Content
1 ABBREVIATIONS...................................................................................................................................1
2 ABSTRACT............................................................................................................................................4
3 INTRODUCTION....................................................................................................................................6
3.1 THE IMMUNE SYSTEM............................................................................................................................. 6
3.1.1 Innate immune responses................................................................................................................. 6
3.1.2 Adaptive immune responses............................................................................................................. 9
3.2 TUBERCULOSIS....................................................................................................................................14
3.2.1 History and modern weapons......................................................................................................... 15
3.2.2 The immune response against M. tuberculosis.............................................................................. 16
3.2.3 Granuloma formation..................................................................................................................... 20
3.2.4 Latency versus active disease 22
3.2.5 Vaccines and biomarkers................................................................................................................ 25
4 AIMS OF THIS STUDY..........................................................................................................................29
5 MATERIAL AND METHODS..................................................................................................................31
5.1 MATERIAL..........................................................................................................................................31
5.1.1 Human subjects..............................................................................................................................31
5.1.1 Proteins and peptides..................................................................................................................... 31
5.1.1 M. tuberculosis / M. bovis strains................................................................................................... 32
5.1.1 Equipment......................................................................................................................................32
5.1.1 Kits..................................................................................................................................................32
5.1.2 Antibodies..................33
5.1.3 Other material.........34
5.1.4 Buffers and solutions 35
5.1.5 Software.........................................................................................................................................36
5.1.6 Web resources..........37
5.2 METHODS..........................................................................................................................................37
5.2.1 Cell culture for intracellular cytokine analysis................................................................................ 37
5.2.2 Cytokine analysis in the culture supernatant by ELISA................................................................... 38
35.2.3 Proliferation analysis by [ H]‐Thymidine incorporation..................................................................38
5.2.4 In vitro killing assay........................................................................................................................ 39
5.2.5 Bio‐Plex Assay.................................................................................................................................40
5.2.6 Lysate generation...........................................................................................................................40
5.2.7 Bradford assay................................................................................................................................41
5.2.8 Gel Filtration...................................................................................................................................41
5.2.9 Analysis procedures and statistics.................................................................................................. 41
5.2.10 Discrimination analysis............................................................................................................. 42
6 RESULTS.............................................................................................................................................43
6.1 IDENTIFICATION OF RELEVANT M. TUBERCULOSIS ANTIGENS.........................................................................43
6.1.1 Fractionated M. tuberculosis lysate................................................................................................ 44
6.2 LATENCY‐ASSOCIATED ANTIGENS 49
6.2.1 Assay optimization and candidate identification........................................................................... 51
6.2.2 Clinical study of selected candidates.............................................................................................. 59
6.2.3 Immunogenic epitopes within Rv3407............................................................................................ 62
6.2.4 T‐cell response under drug treatment............................................................................................ 64
6.2.5 Childhood tuberculosis....................................................................................................................66
6.3 CHARACTERIZATION OF T‐CELL SUBSETS...................................................................................................67
6.3.1 Cytokine profiles.............................................................................................................................67
6.3.2 Effect of interleukin 7...................................................................................................................... 73
6.4 FUNCTIONAL IN VITRO ASSAYS................................................................................................................ 76
6.4.1 Methodology..................................................................................................................................76
6.4.2 Functional properties of different T‐cell populations...................................................................... 79
7 DISCUSSION.......................................................................................................................................88
7.1 ANTIGEN IDENTIFICATION 88
7.2 LATENCY‐ASSOCIATED ANTIGENS............................................................................................................. 91
7.3 CHARACTERIZATION OF T‐CELL SUBSETS.................................................................................................100
7.4 FUNCTIONAL IN VITRO ASSAYS.............................................................................................................. 102
8 REFERENCES................107
9 APPENDIX.........................................................................................................................................119
9.1 SUPPLIER LIST...................................................................................................................................119
9.2 HLA PHENOTYPING............................................................................................................................ 121
9.3 ZUSAMMENFASSUNG......................................................................................................................... 122
9.4 PUBLIKATIONEN.124