Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen:
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | German |
| Published: |
2007
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 102 S. Ill., graph. Darst. 21 cm |
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| 245 | 1 | 0 | |a Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen |c vorgelegt von Alexey Popov |
| 264 | 1 | |c 2007 | |
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Record in the Search Index
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| adam_text | Table of contents
Table of contents
Glossary 9
1. Introduction 11
1.1 Regulation of tolerance in the immune system 11
1.1.1 Tumor escape and tumor surveillance 11
1.1.2 Tumor-derived immunosuppressive factors 11
1.2 Dendritic cells 13
1.2.1 Dendritic cell subsets and their function in vivo 13
1.2.2 Generation of dendritic cells in vitro 14
1.2.3 Regulatory and tolerogenic dendritic cells 15
1.2.4 Assessment of dendritic cell s function by gene expression profiling 17
2. Aim of the study 19
3. Materials and Methods 20
3.1 Materials 20
3.1.1 Chemicals and reagents 20
3.1.2 Cytokines and soluble growth factors 21
3.1.3 Antibodies 21
3.1.4 Cell lines 22
3.1.5 Bacteria 22
3.1.6 Buffers 23
3.1.7 Cell culture media 24
3.2 Methods 24
3.2.1 Bacteria 24
3.2.2 Cell counting 24
3.2.3 Cell separation and cell subset enrichment 24
3.2.3.1 Purification of peripheral blood mononuclear cells with ficoll density
gradient 25
3.2.3.2 Enrichment of monocytes with Magnetically Activated Cell Sorting
(MACS) 25
3.2.3.3 Enrichment of dendritic cell subsets from peripheral blood with MACS.... 25
3.2.3.4 Separation of CD4* T cells with RosetteSep® 27
3.2.4 Assessment of cellular morphology by microscopy 27
3.2.5 Flow cytometry 27
3.2.5.1 Staining protocol for cell surface receptors 27
6
Table of contents
3.2.5.2 Intracellular staining of dendritic cells 28
3.2.5.3 Staining of T cells with CFSE 29
3.2.5.4 Assessment of cell viability 29
3.2.5.5 Data analysis 30
3.2.6 Culture of dendritic cells in vitro 30
3.2.6.1 Differentiation of dendritic cells from monocytes 30
3.2.6.2 Maturation of dendritic cells with cytokines 31
3.2.6.3 In vitro activation of primary dendritic cells 32
3.2.6.4 Infection of cells with Listeria monocytogenes 32
3.2.7 Cell proliferation assays 33
3.2.7.1 Stimulation of cells with artificial APCs 33
3.2.7.2 Mixed lymphocyte reaction 33
3.2.7.3 CTLL-2 assay 34
3.2.8 Gene expression profiling and PCR 34
3.2.8.1 Preparation of the cells and RNA purification 34
3.2.8.2 Microarray hybridization and quality control 34
3.2.8.3 Microarray data analysis 35
3.2.8.3 RT-PCR and quantitative real-time PCR 36
3.2.9 ELISA and EIA assays 36
3.2.10 Assessment of enzymatical activity of indoleamine 2,3 dioxygenase 37
3.2.10.1 Assessment of tryptophan levels by HPLC 37
3.2.10.2 Assessment of kynurenine levels by photometrical assay 38
3.2.11 Western Blotting 38
3.2.12 Statistical analysis 39
4. Results 40
4.1 Influence of prostaglandin E2 on dendritic cells morphology and phenotype. 40
4.1.1 Influence of prostaglandin E2 on DC morphology 40
4.1.2 Influence of prostaglandin E2 on the surface phenotype of DC and cytokine
production 41
4.2 Gene expression profiling identifies IDO and CD25 transcripts as targets of
PGE2 during DC maturation 42
4.3 Regulation of CD25 and IDO by PGE2 on protein level 44
4.3.1 PGE2 induces CD25 protein expression and secretion on maturing DC 44
4.3.2 Induction of IDO expression and enzymatic activity by PGE2 45
7
Table of contents
4.3.3 IDO and CD25 expression are retained after removal of PGE2 and
maturation signals 47
4.4 Influence of PGE2 on primary dendritic cells 48
4.5 Induction of IDO and CD25 on human DC during infection 50
4.5.1 IDO and CD25 are regulated on transcriptional level during infection 51
4.5.2 Expression of IDO and CD25 proteins on infected DC 54
4.5.3 TNF and PGE2 are induced prior IDO and CD25 induction 56
4.6 Functional implication of CD25 and IDO expression by DC 57
4.6.1 CD25* IDO+ DC generate an inhibitory environment for T cells 57
4.6.2 Influence of tryptophan-reduced environment on T-cell proliferation 58
4.6.3 Ablation of IL-2 signaling through surface and soluble CD25 as additional
factor of T-cell inhibition 60
4.7 Endogenous TNF predominantly controls the induction of regulatory DC
phenotype 62
4.8 Expression of IDO and CD25 by human DC in vivo 64
4.8.1 IDO and CD25 are co-expressed by peri-tumoral DC 64
4.8.2 IDO and CD25 expression characterize granuloma-forming regulatory DC
during advanced listeriosis 65
5. Discussion 67
5.1 Concomitant induction of IDO and CD25 in dendritic cells as a mechanism of
tumor immune escape 67
5.2 IDO+ CD25+ dendritic cells mediate tolerance during infection 69
5.3 Impact of the findings reported here for human disease 71
5.3.1 Implication of PGE2 effects on DC vaccination protocols 71
5.3.2 Role of IDO+ CD25* DC during infectious complications induced by anti-TNF
therapy 71
6. Summary 73
7. Zusammenfassung 74
8. References 76
9. Preliminary publications 100
10. Curriculum vitae 101
8
|
| adam_txt |
Table of contents
Table of contents
Glossary 9
1. Introduction 11
1.1 Regulation of tolerance in the immune system 11
1.1.1 Tumor escape and tumor surveillance 11
1.1.2 Tumor-derived immunosuppressive factors 11
1.2 Dendritic cells 13
1.2.1 Dendritic cell subsets and their function in vivo 13
1.2.2 Generation of dendritic cells in vitro 14
1.2.3 Regulatory and tolerogenic dendritic cells 15
1.2.4 Assessment of dendritic cell's function by gene expression profiling 17
2. Aim of the study 19
3. Materials and Methods 20
3.1 Materials 20
3.1.1 Chemicals and reagents 20
3.1.2 Cytokines and soluble growth factors 21
3.1.3 Antibodies 21
3.1.4 Cell lines 22
3.1.5 Bacteria 22
3.1.6 Buffers 23
3.1.7 Cell culture media 24
3.2 Methods 24
3.2.1 Bacteria 24
3.2.2 Cell counting 24
3.2.3 Cell separation and cell subset enrichment 24
3.2.3.1 Purification of peripheral blood mononuclear cells with ficoll density
gradient 25
3.2.3.2 Enrichment of monocytes with Magnetically Activated Cell Sorting
(MACS) 25
3.2.3.3 Enrichment of dendritic cell subsets from peripheral blood with MACS. 25
3.2.3.4 Separation of CD4* T cells with RosetteSep® 27
3.2.4 Assessment of cellular morphology by microscopy 27
3.2.5 Flow cytometry 27
3.2.5.1 Staining protocol for cell surface receptors 27
6
Table of contents
3.2.5.2 Intracellular staining of dendritic cells 28
3.2.5.3 Staining of T cells with CFSE 29
3.2.5.4 Assessment of cell viability 29
3.2.5.5 Data analysis 30
3.2.6 Culture of dendritic cells in vitro 30
3.2.6.1 Differentiation of dendritic cells from monocytes 30
3.2.6.2 Maturation of dendritic cells with cytokines 31
3.2.6.3 In vitro activation of primary dendritic cells 32
3.2.6.4 Infection of cells with Listeria monocytogenes 32
3.2.7 Cell proliferation assays 33
3.2.7.1 Stimulation of cells with artificial APCs 33
3.2.7.2 Mixed lymphocyte reaction 33
3.2.7.3 CTLL-2 assay 34
3.2.8 Gene expression profiling and PCR 34
3.2.8.1 Preparation of the cells and RNA purification 34
3.2.8.2 Microarray hybridization and quality control 34
3.2.8.3 Microarray data analysis 35
3.2.8.3 RT-PCR and quantitative real-time PCR 36
3.2.9 ELISA and EIA assays 36
3.2.10 Assessment of enzymatical activity of indoleamine 2,3 dioxygenase 37
3.2.10.1 Assessment of tryptophan levels by HPLC 37
3.2.10.2 Assessment of kynurenine levels by photometrical assay 38
3.2.11 Western Blotting 38
3.2.12 Statistical analysis 39
4. Results 40
4.1 Influence of prostaglandin E2 on dendritic cells morphology and phenotype. 40
4.1.1 Influence of prostaglandin E2 on DC morphology 40
4.1.2 Influence of prostaglandin E2 on the surface phenotype of DC and cytokine
production 41
4.2 Gene expression profiling identifies IDO and CD25 transcripts as targets of
PGE2 during DC maturation 42
4.3 Regulation of CD25 and IDO by PGE2 on protein level 44
4.3.1 PGE2 induces CD25 protein expression and secretion on maturing DC 44
4.3.2 Induction of IDO expression and enzymatic activity by PGE2 45
7
Table of contents
4.3.3 IDO and CD25 expression are retained after removal of PGE2 and
maturation signals 47
4.4 Influence of PGE2 on primary dendritic cells 48
4.5 Induction of IDO and CD25 on human DC during infection 50
4.5.1 IDO and CD25 are regulated on transcriptional level during infection 51
4.5.2 Expression of IDO and CD25 proteins on infected DC 54
4.5.3 TNF and PGE2 are induced prior IDO and CD25 induction 56
4.6 Functional implication of CD25 and IDO expression by DC 57
4.6.1 CD25* IDO+ DC generate an inhibitory environment for T cells 57
4.6.2 Influence of tryptophan-reduced environment on T-cell proliferation 58
4.6.3 Ablation of IL-2 signaling through surface and soluble CD25 as additional
factor of T-cell inhibition 60
4.7 Endogenous TNF predominantly controls the induction of regulatory DC
phenotype 62
4.8 Expression of IDO and CD25 by human DC in vivo 64
4.8.1 IDO and CD25 are co-expressed by peri-tumoral DC 64
4.8.2 IDO and CD25 expression characterize granuloma-forming regulatory DC
during advanced listeriosis 65
5. Discussion 67
5.1 Concomitant induction of IDO and CD25 in dendritic cells as a mechanism of
tumor immune escape 67
5.2 IDO+ CD25+ dendritic cells mediate tolerance during infection 69
5.3 Impact of the findings reported here for human disease 71
5.3.1 Implication of PGE2 effects on DC vaccination protocols 71
5.3.2 Role of IDO+ CD25* DC during infectious complications induced by anti-TNF
therapy 71
6. Summary 73
7. Zusammenfassung 74
8. References 76
9. Preliminary publications 100
10. Curriculum vitae 101
8 |
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| author | Popov, Alexey 1970- |
| author_GND | (DE-588)134046641 |
| author_facet | Popov, Alexey 1970- |
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| ctrlnum | (OCoLC)222333808 (DE-599)DNB987869841 |
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| dewey-ones | 616 - Diseases |
| dewey-raw | 616.079 |
| dewey-search | 616.079 |
| dewey-sort | 3616.079 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
| discipline_str_mv | Medizin |
| format | Thesis Book |
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| index_date | 2024-07-02T20:25:16Z |
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| spelling | Popov, Alexey 1970- Verfasser (DE-588)134046641 aut Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen vorgelegt von Alexey Popov 2007 102 S. Ill., graph. Darst. 21 cm txt rdacontent n rdamedia nc rdacarrier Köln, Univ., Diss., 2007 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016430208&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Popov, Alexey 1970- Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen |
| subject_GND | (DE-588)4113937-9 |
| title | Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen |
| title_auth | Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen |
| title_exact_search | Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen |
| title_exact_search_txtP | Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen |
| title_full | Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen vorgelegt von Alexey Popov |
| title_fullStr | Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen vorgelegt von Alexey Popov |
| title_full_unstemmed | Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen vorgelegt von Alexey Popov |
| title_short | Charakterisierung von Indoleamine 2,3-Dioxygenase-positiven CD25-positiven tolerogenen dendritischen Zellen |
| title_sort | charakterisierung von indoleamine 2 3 dioxygenase positiven cd25 positiven tolerogenen dendritischen zellen |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016430208&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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