Genetic and physical modification of human monocyte derived dendritic cells in order to improve vaccination protocols [Elektronische Ressource] / vorgelegt von Ilka Knippertz

Ilka Kreimendahl

Sujets

• Gesundheit

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Genetic and physical modification of human
monocyte-derived dendritic cells in order to improve
vaccination protocols






Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur

Erlangung des Doktorgrades






vorgelegt von
Ilka Knippertz
aus Mönchengladbach Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten
der Universität Erlangen-Nürnberg












Tag der mündlichen Prüfung: 01.07.2008

Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch

Erstberichterstatter: Prof. Dr. Lars Nitschke

Zweitberichterstatter: Prof. Dr. Eckhart Kämpgen Table of contents

Abbreviations

1. Summary - English .......................................................................................... 1
- German ......................................................................................... 3

2. Introduction.......................................................................................... 5

2.1. The biology of dendritic cells ................................................................ 5
2.1.1. Dendritic cell subsets ............................................................................ 5
2.1.2. Intimate link between antigen capture/-processing, maturation,
activation and migration of DC.............................................................. 7
2.1.2.1. Antigen capture ..................................................................................... 7
2.1.2.2. Antigen processing ................................................................................ 7
2.1.2.3. Maturation of DC ................................................................................... 9
2.1.2.4. The cell surface molecule CD83.......................................................... 10
2.1.2.4.1. Functions of membrane bound CD83 and soluble CD83 .................... 10
2.1.2.4.2. The CD83 promoter............................................................................. 11
2.1.2.5. Activation of DC................................................................................... 12
2.1.2.5.1. Innate immunity activation signals ....................................................... 12
2.1.2.5.2. Adaptive immunity activation signals ................................................... 13
2.1.2.6. Migration of DC.................................................................................... 14
2.1.3. Control of the type of T cell response by DC ....................................... 15
2.1.4. The role of heat shock proteins in DC-mediated immunity .................. 17
2.1.4.1. The human heat shock protein 70 family............................................. 18
2.1.4.2. Regulation of the heat shock response................................................ 20
2.1.4.3. The hsp-APC interaction as an inducer of adaptive and innate
immune events .................................................................................... 21
2.1.4.4. Heat shock factor 1-independent activation of dendritic cells by
heat shock ........................................................................................... 22
2.1.5. DC in cancer immunotherapy .............................................................. 23
2.1.5.1. Immune escape mechanisms of cancer .............................................. 24
2.1.5.2. Genetic modification of DC for therapeutic vaccination ....................... 25
2.2. Adenovirus and its use as a vector for cancer gene therapy and
genetic DC-mediated vaccination ........................................................ 26
2.2.1. Adenoviruses and gene therapy.......................................................... 26
2.2.2. Virus structure ..................................................................................... 27
2.2.3. The viral life cycle ................................................................................ 28
2.2.3.1 Binding and entry................................................................................. 28
2.2.3.2. Expression of viral genes..................................................................... 29
2.2.3.3. Virus assembly and release................................................................. 30
2.2.4. Use of adenovirus vectors in gene therapy.......................................... 31
2.2.4.1. Targeting of adenovirus vectors .......................................................... 31
2.2.4.2. Adenovirus vectors .............................................................................. 32
2.2.4.2.1. First- and second-generation adenovirus vectors................................ 32
2.2.4.2.2. Helper-dependent (gutless; high capacity) adenovirus vectors ........... 33
2.2.4.3. DC-based adenoviral vaccines in gene therapy .................................. 33

3. Tasks .................................................................................................. 36

4. Material and Methods........................................................................ 38

4.1. Material................................................................................................ 38
4.1.1. Chemicals............................................................................................ 38
4.1.2. Buffers and cell culture media ............................................................. 39
4.1.2.1. General buffers.................................................................................... 39
4.1.2.2. Reagents for transfection of cells ........................................................ 40
4.1.2.3. ELISA buffer ........................................................................................ 41
4.1.2.4. Buffer for SDS-PAGE and Western blotting......................................... 41
4.1.2.5. Buffer for ChIP-chip microarray ........................................................... 42
4.1.2.6. Cell lines and cell culture media .......................................................... 43
4.1.2.7. Further cell media................................................................................ 44
4.1.3. Weight markers for gel electrophoresis ............................................... 45
4.1.3.1. Weight markers for DNA gel electrophoresis....................................... 45
4.1.3.2. Weight marker for protein gel electrophoresis ..................................... 45
4.1.4. Bacteria ............................................................................................... 46
4.1.5. Plasmid vectors ................................................................................... 46 4.1.6. Primers ................................................................................................ 46
4.1.6.1. Primers used for screening of human- and adenoviral genome .......... 47
4.1.6.2. Primers used for screening of BAC-transgenic mice ........................... 47
4.1.6.3. Primers used for Real Time PCR ........................................................ 48
4.1.6.4. Primers used for cloning...................................................................... 48
4.1.6.4.1. Primers used for cloning of CD83 promoter sequences ...................... 48
4.1.6.4.2. Further primers used for cloning from the human genome.................. 50
4.1.7. Adenoviruses....................................................................................... 50
4.1.8. Human cytokines ................................................................................. 51
4.1.9. Antibodies............................................................................................ 51
4.1.9.1. Antibodies used for FACS ................................................................... 52
4.1.9.2. Antibodies used for ELISA................................................................... 53
4.1.9.3. Antibodies used for Western blotting and immunoprecipitation ........... 53
4.1.9.4. Antibodies used for MACS................................................................... 53
4.1.10. Purchased kits for RNA and DNA purification...................................... 54
4.1.11. Mice..................................................................................................... 54

4.2. Methods............................................................................................... 54
4.2.1. Generation of transformation-competent bacteria and
transformation...................................................................................... 54
4.2.1.1. Generation of chemical-competent E.coli and transformation
by heat................................................................................................. 54
4.2.1.2. Generation of electro-competent E. coli and transformation by
electroporation..................................................................................... 55
4.2.2. Molecular biology methods.................................................................. 56
4.2.2.1. Isolation of DNA..........................................................