Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2008
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| Online-Zugang: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-10161 http://d-nb.info/989951367/34 Inhaltsverzeichnis |
| Beschreibung: | 173 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols |c von Ilka Knippertz |
| 264 | 1 | |c 2008 | |
| 300 | |a 173 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
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| 502 | |a Erlangen-Nürnberg, Univ., Diss., 2008 | ||
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Datensatz im Suchindex
| _version_ | 1804137812583251968 |
|---|---|
| adam_text | 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 ChlP-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 56
4.2.2.1.1. Isolation of small plasmids ( 12kb) 56
4.2.2.1.2. Isolation of large plasmids ( 12 kb) 56
4.2.2.1.3. Isolation of bacterial artificial chromosome (BAC)- ONA
(Alkaline lysis) 57
4.2.2.1.4. Isolation of human chromosomal DNA from cells 58
4.2.2.1.5. Isolation of total DNA from tissue 58
4.2.2.1.6. Preparation of DNA fragments from PCR reactions and
enzymatic digestions 59
4.2.2.1.7. Preparation of DNA fragments from gel electrophoresis 59
4.2.2.2. Preparation of DNA for ChlP-chip microarray 59
4.2.2.3. Isolation of total RNA from animal cells 60
4.2.2.4. Nucleic acid quantification 60
4.2.2.5. Separation of DNA or RNA using agarose gel electrophoresis 61
4.2.2.6. Pulse-field gel electrophoresis (PFGE) 61
4.2.2.7. Reverse transcription 61
4.2.2.8. Polymerase chain reaction (PCR) 62
4.2.2.9. Two-step PCR 63
4.2.2.10. Quantitative real time- PCR (qPCR) 64
4.2.2.11. Cloning of DNA fragments or PCR products 65
4.2.2.11.1. Dephosphorylation of cleaved DNA 65
4.2.2.11.2. Conversion of DNA overhangs 65
4.2.2.11.3. Ligation 65
4.2.2.12. Sequencing of DNA 66
4.2.3. Protein biochemistry methods 66
4.2.3.1. Preparation of whole cell extracts for SDS-PAGE 66
4.2.3.2. Bradford assay 66
4.2.2.3. Denaturing (SDS) discontinuous polyacrylamide gel
electrophoresis (PAGE): Laemmli method 67
4.2.2.4. Western blotting 67
4.2.4. Cell culture 68
4.2.4.1. Generation of DC 68
4.2.4.2. Cryopreservation of primary cells 68
4.2.4.3. Cryopreservation of cell lines 69
4.2.5. Heat shock 69
4.2.5.1. Heat shock of DC 69
4.2.5.2. Heat shock of cell lines 69
4.2.6. Flow cytometric analysis (FACS) 70
4.2.7. Cytometric bead array (CBA) 70
4.2.8. Transient transfection methods and luciferase reporter assay 70
4.2.8.1. Transfection of DNA with the DEAE-Dextran method 70
4.2.8.2. Lipofection of DNA with Lipofektamin™ (Invitrogen) or
Lipofektamin™ PLUS reagent (Invitrogen) 71
4.2.8.3. Transfection of RNA with Transmessenger transfection
reagent (Qiagen) 72
4.2.8.4. Luciferase reporter assay 72
4.2.9. Cytokine enzyme linked immunosorbent assay (ELISA) 72
4.2.10. Transwell migration assay 73
4.2.11. Cytotoxic T cell induction assay 74
4.2.12. Tetramer staining and phenotyping of antigen-specific
CD8T cells 74
4.2.13. Recombinant adenoviruses 75
4.2.13.1. Cloning of plasmids containing the recombinant adenoviral
genome 75
4.2.13.2. Preparation of recombinant adenoviruses 75
4.2.13.3. Determination of the physical particle concentration 76
4.2.13.4. Determination of the infectious particle concentration 76
4.2.13.5. Adenoviral transduction of cells 77
5. Results 78
5.1. Generation of human dendritic cells that simultaneously secrete
IL-12 and have migratory capacity mediated by adenoviral gene
transfer of human CD40L in combination with IFN-y treatment 78
5.1.1. Transduction of moDC with Ad5hCD40L leads to an intracellular,
but not to a surface expression of the CD40L protein 79
5.1.2. Transduction with Ad5hCD40L induces partial maturation of iDC 81
5.1.3. Timing of IFN-y-treatment after transduction of iDC with
Ad5hCD40L influences IL-12p70-expression 83
5.1.4. Kinetics of CD40L- and IL-12p70 expression after modulation
of DC with Ad5hCD40L and IFN-y 84
5.1.5. Dendritic cells transduced with Ad5hCD40L and stimulated with
IFN-y are able to simultaneously migrate and secrete IL-12p70 85
5.1.6. moDC modified with Ad5hCD40L in combination with IFN-y
efficiently prime naive CD8* T cells in vitro 90
i
5.2. Analysis of the human CD83 promoter for cell type- as well as
status-specificity and development of a promoter-responsive
system for multiple transgene expression in human dendritic cells 91
5.2.1. Approaches to identify CD83 promoter sequences which are
specifically active in mature human dendritic cells 91
5.2.1.1. Analysis of up-/downstream or internal gene fragments of the human
CD83 gene in order to identify additional regulatory elements 92
5.2.1.2. Generation of CD83-BAC transgenic animals 94
5.2.1.3. ChlP-chip™ microarray analysis of the human CD83-gene 96
5.2.2. Development of a vector system for the expression of multiple
therapeutic transgenes 99
5.2.2.1. Development of the Multiple Transgene Switch system 100
5.2.2.1.1. A hsp70B promoter fragment can be efficiently induced by heat
shock in HeLa cells 100
5.2.2.1.2. Specific expression of the tumor antigen MelanA, the
Th1 -cytokine IL-12 and the survival protein Bcl-xL under the
control of the hsp70B promoter in HeLa cells 102
5.2.2.1.3. Generation of a plasmid containing a triple hsp70B -bclxL-hsp70B -
MelA-hsp70B -IL12 cassette (p3xhsp70B ) 104
5.2.2.1.4. HeLa cells lipofected with p3xhsp70B express MelanA, Bcl-xL
and IL-12 specifically after transduction with mHSFi or after
exposure to heat 104
5.2.2.1.5. The multiple switch system is functional in Ad vectors after
transduction of DC 106
5.2.2.1.6. DC transduced with Ad5-3xhsp70B are efficient in priming
naive CD8* T cells in vitro 107
5.2.2.2. Effects on DC caused by the overexpression of HSF1 109
5.2.2.2.1. Upregulation of heat shock proteins 40,70A and 70B
in moDC after transduction with Ad5mHSF1 109
5.2.2.2.2. Ad5mHSF1 only minimally affects MC-induced upregulation
of DC-surface maturation markers 111
5.2.2.2.3. DC transduced with Ad5mHSF1 showed enhanced secretion
of the anti-inflammatory cytokine IL-10 and the pro-inflammatory
cytokine TNF-a 112
5.3. Mild thermal stress modulates dendritic cell function resulting in
an enhanced capacity to prime naive CD8+T cells to differentiate
into CTL in vitro 114
5.3.1. moDC exposed to heat show an enhanced expression of hsp70A.... 114
5.3.2. Heat treatment induces an upregulation of maturation markers
on the surface of moDC 115
5.3.3. Mild thermal stress induces the simultaneous upregulation of the
anti-inflammatory cytokine IL-10 and the pro-inflammatory
cytokine TNF-a 117
5.3.4. Stimulation by mild thermal heat does not influence the migratory
capacity of moDC in vitro 118
5.3.5. Heat treatment enhances the capacity of human moDC to prime
naive CD8+ T cells to differentiate into melanoma antigen-specific
CTL in vitro 119
6. Discussion 121
6.1. Modification of human monocyte-derived DC by adenoviral gene
transfer of CD40L 121
6.1.1. After transduction of DC with Ad5hCD40L, CD40L-protein is
expressed intracellularly but not on the cell surface 121
6.1.2. Efficacious induction of IL-12p70-production is dependent on the
time point of IFN-v-addition 123
6.1.3. Linking migratory- and IL-12 secreting capacity of moDC 123
6.1.4. Improved priming of naive autologous CD8* T cells to develop
into MelanA antigen-specific CTL by DC treated with both
Ad5hCD40L and IFN-y 126
6.1.5. Possible benefits for clinical applications 126
6.1.6. Future developments 128
6.2. Identification of transcriptional regulatory sequences of the human
CD83gene 129
6.2.1. Analysis of CD83 genomic fragments 129
6.2.2. Generation of transgenic animals to study the human CD83
promoter in vivo 130
6.2.3. Analysis of the human CD83 gene chromatin activation status 131
6.3. Controlled induction of multiple therapeutic transgenes by
mHSF1 using the hsp70B*promoter 132
6.3.1. The pGL3-3xhsp70B vector system 133
6.3.2. Ad5-3xhsp70B -mediated gene transfer in human
monocyte-derived dendritic cells 134
6.3.3. Influence of overexpression of HSF1 on DC 136
6.3.4. Future prospects 136
6.4. Modulation of human monocyte-derived dendritic cells by
physiologically relevant thermal stress 137
6.4.1. Thermal regulation of DC maturation 138
6.4.2. Thermal regulation of cytokine release 138
6.4.3. Thermal regulation of migration 139
6.4.4. Thermal regulation of DC-mediated T cell stimulation 140
6.4.5. Challenges for clinical development 141
6.5. Concluding remarks 142
7. Literature 143
Acknowledgements/ Danksagung
Curriculum Vitae
|
| adam_txt |
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 ChlP-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 56
4.2.2.1.1. Isolation of small plasmids ( 12kb) 56
4.2.2.1.2. Isolation of large plasmids ( 12 kb) 56
4.2.2.1.3. Isolation of bacterial artificial chromosome (BAC)- ONA
(Alkaline lysis) 57
4.2.2.1.4. Isolation of human chromosomal DNA from cells 58
4.2.2.1.5. Isolation of total DNA from tissue 58
4.2.2.1.6. Preparation of DNA fragments from PCR reactions and
enzymatic digestions 59
4.2.2.1.7. Preparation of DNA fragments from gel electrophoresis 59
4.2.2.2. Preparation of DNA for ChlP-chip microarray 59
4.2.2.3. Isolation of total RNA from animal cells 60
4.2.2.4. Nucleic acid quantification 60
4.2.2.5. Separation of DNA or RNA using agarose gel electrophoresis 61
4.2.2.6. Pulse-field gel electrophoresis (PFGE) 61
4.2.2.7. Reverse transcription 61
4.2.2.8. Polymerase chain reaction (PCR) 62
4.2.2.9. Two-step PCR 63
4.2.2.10. Quantitative real time- PCR (qPCR) 64
4.2.2.11. Cloning of DNA fragments or PCR products 65
4.2.2.11.1. Dephosphorylation of cleaved DNA 65
4.2.2.11.2. Conversion of DNA overhangs 65
4.2.2.11.3. Ligation 65
4.2.2.12. Sequencing of DNA 66
4.2.3. Protein biochemistry methods 66
4.2.3.1. Preparation of whole cell extracts for SDS-PAGE 66
4.2.3.2. Bradford assay 66
4.2.2.3. Denaturing (SDS) discontinuous polyacrylamide gel
electrophoresis (PAGE): Laemmli method 67
4.2.2.4. Western blotting 67
4.2.4. Cell culture 68
4.2.4.1. Generation of DC 68
4.2.4.2. Cryopreservation of primary cells 68
4.2.4.3. Cryopreservation of cell lines 69
4.2.5. Heat shock 69
4.2.5.1. Heat shock of DC 69
4.2.5.2. Heat shock of cell lines 69
4.2.6. Flow cytometric analysis (FACS) 70
4.2.7. Cytometric bead array (CBA) 70
4.2.8. Transient transfection methods and luciferase reporter assay 70
4.2.8.1. Transfection of DNA with the DEAE-Dextran method 70
4.2.8.2. Lipofection of DNA with Lipofektamin™ (Invitrogen) or
Lipofektamin™ PLUS reagent (Invitrogen) 71
4.2.8.3. Transfection of RNA with Transmessenger transfection
reagent (Qiagen) 72
4.2.8.4. Luciferase reporter assay 72
4.2.9. Cytokine enzyme linked immunosorbent assay (ELISA) 72
4.2.10. Transwell migration assay 73
4.2.11. Cytotoxic T cell induction assay 74
4.2.12. Tetramer staining and phenotyping of antigen-specific
CD8T cells 74
4.2.13. Recombinant adenoviruses 75
4.2.13.1. Cloning of plasmids containing the recombinant adenoviral
genome 75
4.2.13.2. Preparation of recombinant adenoviruses 75
4.2.13.3. Determination of the physical particle concentration 76
4.2.13.4. Determination of the infectious particle concentration 76
4.2.13.5. Adenoviral transduction of cells 77
5. Results 78
5.1. Generation of human dendritic cells that simultaneously secrete
IL-12 and have migratory capacity mediated by adenoviral gene
transfer of human CD40L in combination with IFN-y treatment 78
5.1.1. Transduction of moDC with Ad5hCD40L leads to an intracellular,
but not to a surface expression of the CD40L protein 79
5.1.2. Transduction with Ad5hCD40L induces partial maturation of iDC 81
5.1.3. Timing of IFN-y-treatment after transduction of iDC with
Ad5hCD40L influences IL-12p70-expression 83
5.1.4. Kinetics of CD40L- and IL-12p70 expression after modulation
of DC with Ad5hCD40L and IFN-y 84
5.1.5. Dendritic cells transduced with Ad5hCD40L and stimulated with
IFN-y are able to simultaneously migrate and secrete IL-12p70 85
5.1.6. moDC modified with Ad5hCD40L in combination with IFN-y
efficiently prime naive CD8* T cells in vitro 90
i
5.2. Analysis of the human CD83 promoter for cell type- as well as
status-specificity and development of a promoter-responsive
system for multiple transgene expression in human dendritic cells 91
5.2.1. Approaches to identify CD83 promoter sequences which are
specifically active in mature human dendritic cells 91
5.2.1.1. Analysis of up-/downstream or internal gene fragments of the human
CD83 gene in order to identify additional regulatory elements 92
5.2.1.2. Generation of CD83-BAC transgenic animals 94
5.2.1.3. ChlP-chip™ microarray analysis of the human CD83-gene 96
5.2.2. Development of a vector system for the expression of multiple
therapeutic transgenes 99
5.2.2.1. Development of the Multiple Transgene Switch system 100
5.2.2.1.1. A hsp70B" promoter fragment can be efficiently induced by heat
shock in HeLa cells 100
5.2.2.1.2. Specific expression of the tumor antigen MelanA, the
Th1 -cytokine IL-12 and the survival protein Bcl-xL under the
control of the hsp70B" promoter in HeLa cells 102
5.2.2.1.3. Generation of a plasmid containing a triple hsp70B"-bclxL-hsp70B"-
MelA-hsp70B'-IL12 cassette (p3xhsp70B") 104
5.2.2.1.4. HeLa cells lipofected with p3xhsp70B' express MelanA, Bcl-xL
and IL-12 specifically after transduction with mHSFi or after
exposure to heat 104
5.2.2.1.5. The multiple switch system is functional in Ad vectors after
transduction of DC 106
5.2.2.1.6. DC transduced with Ad5-3xhsp70B'are efficient in priming
naive CD8* T cells in vitro 107
5.2.2.2. Effects on DC caused by the overexpression of HSF1 109
5.2.2.2.1. Upregulation of heat shock proteins 40,70A and 70B"
in moDC after transduction with Ad5mHSF1 109
5.2.2.2.2. Ad5mHSF1 only minimally affects MC-induced upregulation
of DC-surface maturation markers 111
5.2.2.2.3. DC transduced with Ad5mHSF1 showed enhanced secretion
of the anti-inflammatory cytokine IL-10 and the pro-inflammatory
cytokine TNF-a 112
5.3. Mild thermal stress modulates dendritic cell function resulting in
an enhanced capacity to prime naive CD8+T cells to differentiate
into CTL in vitro 114
5.3.1. moDC exposed to heat show an enhanced expression of hsp70A. 114
5.3.2. Heat treatment induces an upregulation of maturation markers
on the surface of moDC 115
5.3.3. Mild thermal stress induces the simultaneous upregulation of the
anti-inflammatory cytokine IL-10 and the pro-inflammatory
cytokine TNF-a 117
5.3.4. Stimulation by mild thermal heat does not influence the migratory
capacity of moDC in vitro 118
5.3.5. Heat treatment enhances the capacity of human moDC to prime
naive CD8+ T cells to differentiate into melanoma antigen-specific
CTL in vitro 119
6. Discussion 121
6.1. Modification of human monocyte-derived DC by adenoviral gene
transfer of CD40L 121
6.1.1. After transduction of DC with Ad5hCD40L, CD40L-protein is
expressed intracellularly but not on the cell surface 121
6.1.2. Efficacious induction of IL-12p70-production is dependent on the
time point of IFN-v-addition 123
6.1.3. Linking migratory- and IL-12 secreting capacity of moDC 123
6.1.4. Improved priming of naive autologous CD8* T cells to develop
into MelanA antigen-specific CTL by DC treated with both
Ad5hCD40L and IFN-y 126
6.1.5. Possible benefits for clinical applications 126
6.1.6. Future developments 128
6.2. Identification of transcriptional regulatory sequences of the human
CD83gene 129
6.2.1. Analysis of CD83 genomic fragments 129
6.2.2. Generation of transgenic animals to study the human CD83
promoter in vivo 130
6.2.3. Analysis of the human CD83 gene chromatin activation status 131
6.3. Controlled induction of multiple therapeutic transgenes by
mHSF1 using the hsp70B*promoter 132
6.3.1. The pGL3-3xhsp70B" vector system 133
6.3.2. Ad5-3xhsp70B"-mediated gene transfer in human
monocyte-derived dendritic cells 134
6.3.3. Influence of overexpression of HSF1 on DC 136
6.3.4. Future prospects 136
6.4. Modulation of human monocyte-derived dendritic cells by
physiologically relevant thermal stress 137
6.4.1. Thermal regulation of DC maturation 138
6.4.2. Thermal regulation of cytokine release 138
6.4.3. Thermal regulation of migration 139
6.4.4. Thermal regulation of DC-mediated T cell stimulation 140
6.4.5. Challenges for clinical development 141
6.5. Concluding remarks 142
7. Literature 143
Acknowledgements/ Danksagung
Curriculum Vitae |
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| author | Knippertz, Ilka 1977- |
| author_GND | (DE-588)136037135 |
| author_facet | Knippertz, Ilka 1977- |
| author_role | aut |
| author_sort | Knippertz, Ilka 1977- |
| author_variant | i k ik |
| building | Verbundindex |
| bvnumber | BV023420259 |
| classification_rvk | XH 3904 XH 5587 |
| collection | ebook |
| ctrlnum | (OCoLC)436305530 (DE-599)BVBBV023420259 |
| dewey-full | 616.994 615.3 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 616 - Diseases 615 - Pharmacology and therapeutics |
| dewey-raw | 616.994 615.3 |
| dewey-search | 616.994 615.3 |
| dewey-sort | 3616.994 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
| discipline_str_mv | Medizin |
| format | Thesis Book |
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| id | DE-604.BV023420259 |
| illustrated | Illustrated |
| index_date | 2024-07-02T21:30:48Z |
| indexdate | 2024-07-09T21:18:15Z |
| institution | BVB |
| language | English |
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| physical | 173 S. Ill., graph. Darst. |
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| spelling | Knippertz, Ilka 1977- Verfasser (DE-588)136037135 aut Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols von Ilka Knippertz 2008 173 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Erlangen-Nürnberg, Univ., Diss., 2008 Langzeitarchivierung Nationalbibliothek gewährleistet Krebsbekämpfung (DE-588)4165563-1 gnd rswk-swf Dendritische Zelle (DE-588)4313846-9 gnd rswk-swf Immuntherapie (DE-588)4026640-0 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Krebsbekämpfung (DE-588)4165563-1 s Immuntherapie (DE-588)4026640-0 s Dendritische Zelle (DE-588)4313846-9 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:29-opus-10161 https://open.fau.de/handle/openfau/680 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-10161 Resolvingsystem http://d-nb.info/989951367/34 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016602710&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Knippertz, Ilka 1977- Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols Krebsbekämpfung (DE-588)4165563-1 gnd Dendritische Zelle (DE-588)4313846-9 gnd Immuntherapie (DE-588)4026640-0 gnd |
| subject_GND | (DE-588)4165563-1 (DE-588)4313846-9 (DE-588)4026640-0 (DE-588)4113937-9 |
| title | Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols |
| title_auth | Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols |
| title_exact_search | Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols |
| title_exact_search_txtP | Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols |
| title_full | Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols von Ilka Knippertz |
| title_fullStr | Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols von Ilka Knippertz |
| title_full_unstemmed | Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols von Ilka Knippertz |
| title_short | Genetic and physical modification of human monocyte-derived dendritic cells in order to improve vaccination protocols |
| title_sort | genetic and physical modification of human monocyte derived dendritic cells in order to improve vaccination protocols |
| topic | Krebsbekämpfung (DE-588)4165563-1 gnd Dendritische Zelle (DE-588)4313846-9 gnd Immuntherapie (DE-588)4026640-0 gnd |
| topic_facet | Krebsbekämpfung Dendritische Zelle Immuntherapie Hochschulschrift |
| url | https://open.fau.de/handle/openfau/680 https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-10161 http://d-nb.info/989951367/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016602710&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT knippertzilka geneticandphysicalmodificationofhumanmonocytederiveddendriticcellsinordertoimprovevaccinationprotocols |