The role of TNF receptor type 2 on myeloid cells in sepsis: functional analysis
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| Format: | Thesis Book |
| Language: | English |
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2010
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| Online Access: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-170908 Inhaltsverzeichnis |
| Physical Description: | XVI, 135 Bl. Ill., graph. Darst. |
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| adam_text | IMAGE 1
THE ROLE OF TNF RECEPTOR TYPE 2 ON
MYELOID CELLS IN SEPSIS - FUNCTIONAL
ANALYSIS
DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR.
RER. NAT.) DER NATURWISSENSCHAFTLICHEN FAKULTAET III - BIOLOGIE UND
VORKLINISCHE MEDIZIN -
DER UNIVERSITAET REGENSBURG
VORGELEGT VON
JOHANNES POLZ
AUS PFAFFENHOFEN AN DER HM
JULI 2010
BIBLIOGRAFISCHE INFORMATIONEN HTTP://D-NB.INFO/1008152749
DIGITALISIERT DURCH
IMAGE 2
INDEX
INDEX OF CONTENT
1 INTRODUCTION 1
1.1 PREAMBLE 1
1.2 THE TNF / TNF RECEPTOR SUPERFAMILY 1
1.2.1 STRUCTURE OF TNF 2
1.2.2 FUNCTIONS OF TNF 3
1.2.3 REGULATION OF TNF RECEPTORS 3
1.2.3.1 SIGNALING OF TNFR1 4
1.2.3.2 SIGNALING OF TNFR2 5
1.2.4 REVERSE SIGNALING OF TNFR2 VIA MEMBRANE-BOUND TNF 5
1.2.5 TNF INHIBITOR FUNCTION OF SOLUBLE TNFR2 6
1.2.6 ANTI-TNF THERAPY 7
1.2.7 AFFINITY OF HUMAN AND MOUSE TNF FOR MOUSE TNF RECEPTORS 7
1.2.8 TNFR2 + SYSTEMS 7
1.2.9 PHENOTYPE OF TNFR2 KNOCKOUT MICE (TNFR2 V) 9
1.2.10 ANTI-MOUSE TNFR2 MONOCLONAL ANTIBODIES (MAB) 9
1.3 SEPSIS AND IMMUNOPARALYSIS 10
1.4 MYELOID CELLS 12
1.4.1 MACROPHAGES 13
1.4.1.1 HISTORICAL BACKGROUND 13
1.4.1.2 MONOCYTE-DERIVED MACROPHAGES 13
1.4.2 BIOLOGICAL RELEVANCE OF MACROPHAGES 14
1.4.3 INTERLEUKIN 6 16
1.4.4 REGULATION OF THE INOS EXPRESSION AND NO SIGNALING 16
1.4.5 DIFFERENT TYPES OF MYELOID CELLS USED IN THIS STUDY 17
1.4.5.1 PERITONEAL EXUDATE CELLS (PEC) 17
1.4.5.2 CD11B*SPLENOCYTES 17
1.4.5.3 BONE MARROW-DERIVED DENDRITIC CELLS (BMDC) 17
1.4.6 MYELOID-DERIVED SUPPRESSOR CELLS (MDSC) 17
1.5 AIM OF THE THESIS 21
2 MATERIALS AND METHODS 23
2.1 MATERIALS 23
2.1.1 INSTRUMENTATION 23
2.1.2 CONSUMABLES 24
2.1.3 CHEMICALS AND REAGENTS 25
IMAGE 3
INDEX
2.1.4 ANTIBODIES 27
2.1.5 ELISAKITS 29
2.1.6 BUFFERS AND SOLUTIONS 30
2.1.7 KITS 31
2.1.8 OLIGONUCLEOTIDES 32
2.1.9 PLASMIDS 33
2.1.10 CELL CULTURE MEDIA 34
2.1.11 MOUSE STRAINS 34
2.1.12 EUKARYOTIC CELL LINES 35
2.1.13 SOFTWARE AND INTERNET RESOURCES 36
2.2 MOLECULAR BIOLOGY 36
2.2.1 WORKING WITH DNA 36
2.2.1.1 SEQUENCING OF PLASMID DNA 36
2.2.2 WORKING WITH RNA 36
2.2.2.1 RNA ISOLATION 36
2.2.2.2 RNA CONCENTRATION DETERMINATION USING A PHOTOMETER 37
2.2.2.3 CDNA SYNTHESIS 37
2.2.2.4 QUANTITATIVE REAL-TIME PCR 38
2.2.2.4.1 PRIMER DESIGN 39
2.2.2.4.2 QUANTITATIVE REAL-TIME PCR SETUP 39
2.2.2.4.3 QUANTITATIVE REAL-TIME PCR PROGRAM 40
2.2.2.4.4 DATA INTERPRETATION 40
2.2.3 WORKING WITH PROTEINS 41
2.2.3.1 EXPRESSION OF RECOMBINANT PROTEINS IN DROSOPHILA DS-2 CELLS 41
2.2.3.2 MEASURING OF PROTEIN CONCENTRATIONS 41
2.2.3.3 SDS-PAGE 41
2.2.3.4 COOMASSIE STAINING 41
2.2.3.5 WESTERN BLOT 42
2.2.3.6 ELISA 42
2.2.3.6.1 ELISA FOR THE DETECTION OF MOUSE SERUM IGG TITERS 43
2.2.3.6.2 ELISA FOR THE DETECTION OF IGG FROM HYBRIDOMA SUPEMATANTS 43
2.2.3.6.3 ELISA FOR THE DETECTION OF TNF, TNFR2, AND IL-6 44
2.2.3.7 DETECTION OF NO - GRIESS REAGENT 44
2.2.3.8 BIOLOGICAL ASSAY FOR TNF DETECTION - L-929 1 CELL KILL 44
2.2.3.9 VIABILITY ASSAY USING MTT 45
IMAGE 4
INDEX
2.2.3.10 PURIFICATION OF V5HIS-TAGGED PROTEINS 45
2.2.3.11 PURIFICATION OF HUMAN IGG-TAGGED PROTEINS 46
2.2.3.12 PURIFICATION OF IGG FROM HYBRIDOMA SUPEMATANTS 46
2.3 CELL-BIOLOGICAL METHODS 47
2.3.1 CELL CULTURE CONDITIONS 47
2.3.2 CRYO PRESERVATION OF CELLS - FREEZING AND THAWING 47
2.3.3 DETERMINATION OF CELL NUMBERS 47
2.3.4 STIMULATION OF CELLS 48
2.3.5 GENERATION OF GM-CSF-CONTAINING SUPERNATANT 48
2.3.6 STABLE TRANSFECTION OF EUKARYOTIC CELLS USING DOTAP 48
2.3.7 STABLE RETROVIRAL TRANSDUCTION OF EUKARYOTIC CELLS 48
2.3.8 CYTOSPIN 49
2.3.9 DIFFERENTIAL STAINING 49
2.3.10 FLOW CYTOMETRY 49
2.3.11 FACS ARIA CELL SEPARATION 50
2.3.12 MACS CELL SEPARATION 50
2.3.13 BRDU STAINING 51
2.3.14 COMBINED ANNEXIN V / 7-AAD STAINING 52
2.4 METHODS USING MICE 52
2.4.1 HOUSING OF ANIMALS 52
2.4.2 ANESTHESIA 53
2.4.3 CECAL LIGATION AND PUNCTURE 53
2.4.4 SPLEEN CELL PREPARATION 53
2.4.5 PERITONEAL EXUDATE CELL PREPARATION 54
2.4.6 BONE MARROW-DERIVED DENDRITIC CELL GENERATION 54
2.4.7 GENERATION OF BONE MARROW CHIMERIC MICE 55
2.5 MONOCLONAL ANTI-TNFR2 ANTIBODY PRODUCTION 56
2.5.1 SPECIES 56
2.5.2 VACCINATION 56
2.5.3 FUSION 57
2.5.4 DETECTION OF POSITIVE HYBRIDOMA CLONES 58
2.5.5 SUBCLONING 58
2.5.6 GENERATION OF SUPEMATANTS 59
2.5.7 PROTEIN G PURIFICATION OF MONOCLONAL ANTIBODIES 59
2.5.8 FUNCTIONAL CHARACTERIZATION OF MONOCLONAL ANTI-TNFR2 ANTIBODY 59
IMAGE 5
INDEX
2.5.8.1 DETERMINATION OF THE IGG ISOTYPES 59
2.5.8.2 ELISA FOR ANTI TNFR2 ANTIBODY CHARACTERIZATION 59
2.5.8.3 WESTERN BLOT FOR ANTI-TNFR2 ANTIBODY CHARACTERIZATION 59
2.5.8.4 FLOW CYTOMETRY FOR ANTI-TNFR2 ANTIBODY CHARACTERIZATION 60
2.5.8.5 TNFR2 ACTIVATION OR INHIBITION ASSAY FOR ANTI-TNFR2 MAB 60
2.6 STATISTICAL CALCULATIONS 62
3 RESULTS 63
3.1 INOS MRNA EXPRESSION AND NITRIC OXIDE (NO) PRODUCTION OF TNFR2 +
MYELOID CELLS. 63
3.1.1 CD11 B* CD11C SPLENOCYTES AFTER CLP 63
3.1.2 PERITONEAL EXUDATE CELLS (PEC) 65
3.1.3 BONE MARROW-DERIVED DENDRITIC CELLS (BMDC) 66
3.2 THE ROLE OF MDSC FOR THE TNFR2 + PHENOTYPE IN MYELOID CELLS 67
3.2.1 MDSC IN CD11B* SPLENOCYTES 67
3.2.1.1 RELATIVE PROPORTION OF MDSC 67
3.2.1.2 NITRIC OXIDE (NO) PRODUCTION OF MDSC 68
3.2.2 MDSC POPULATION IN BONE MARROW-DERIVED DENDRITIC CELLS (BMDC) 69
3.2.3 CD11 B* CELLS AND MDSC IN BONE MARROW 69
3.2.3.1 DIFFERENTIATION AND DEVELOPMENT OF BMDC 71
3.2.3.2 FREQUENCY OF MDSC 71
3.2.3.3 NITRIC OXIDE (NO) PRODUCTION OF MDSC 72
3.2.4 ARG1 EXPRESSION OF BMDC AND MDSC 73
3.3 PHENOTYPES OF TNFR2 V BONE MARROW-DERIVED DENDRITIC CELLS (BMDC) 74
3.3.1 NON-STIMULATED BMDC 74
3.3.1.1 CELL NUMBERS IN BMDC CULTURES 74
3.3.1.2 FREQUENCY OF CELLS EXPRESSING ACTIVATION MARKERS (MHCII* CD80*
CD86*) IN
BMDC CULTURES 75
3.3.1.3 FREQUENCY OF MDSC IN BMDC CULTURES 76
3.3.1.4 PROLIFERATION IN BMDC CULTURES 77
3.3.1.5 CELL DEATH IN BMDC CULTURES 77
3.3.1.6 TNF CONCENTRATIONS IN BMDC CULTURES 78
3.3.1.7 TNFR2 CONCENTRATIONS IN BMDC CULTURES 78
3.3.2 STIMULATED BMDC CULTURES 79
3.3.2.1 NO PRODUCTION CAPACITY IN TNFR2 + BMDC CULTURES 79
3.3.2.2 IL-6 PRODUCTION CAPACITY IN BMDC CULTURES 80
3.3.2.3 STNF CONCENTRATIONS IN TNFR2 7 BMDC CULTURES 81
IMAGE 6
INDEX
3.3.2.4 STNRF2 CONCENTRATIONS IN BMDC CULTURES 81
3.3.3 MIXED AND NON-STIMULATED BMDC CULTURES 82
3.3.3.1 CELL PROPORTIONS IN MIXED BMDC CULTURES 82
3.3.3.2 FREQUENCY OF CELLS EXPRESSING ACTIVATION MARKERS (MHCH* CD80*
CD86*) IN
MIXED BMDC CULTURES 83
3.3.3.3 FREQUENCY OF MDSC IN MIXED BMDC CULTURES 84
3.3.3.4 PROLIFERATION OF MIXED BMDC CULTURES 85
3.3.3.5 CELL DEATH IN MIXED BMDC CULTURES 86
3.3.4 MIXED BMDC CULTURES, SORTED AND STIMULATED 86
3.3.4.1 NO PRODUCTION IN MIXED BMDC CULTURES 86
3.3.4.2 IL-6 PRODUCTION IN MIXED BMDC CULTURES 87
3.4 BONE MARROW CHIMERIC MICE 88
3.4.1 RECONSTITUTION 88
3.4.2 PEC CELL DISTRIBUTION AND NO PRODUCTION 89
3.4.3 BMDC FROM BM CHIMERIC MICE 89
3.4.3.1 FREQUENCY OF CELLS EXPRESSING ACTIVATION MARKERS (MHCH* CD80*
CD86*) IN
BMDC CULTURES FROM BM CHIMERIC MICE 89
3.4.3.2 NITRIC OXIDE (NO) PRODUCTION OF BMDC FROM BM CHIMERIC MICE 90
3.4.3.3 IL-6 PRODUCTION OF BMDC CULTURES FROM BM CHIMERIC MICE 91
3.4.3.4 STNF CONCENTRATIONS IN BMDC CULTURES FROM BM CHIMERIC MICE 91
3.4.3.5 STNFR2 CONCENTRATIONS IN BMDC CULTURES FROM BM CHIMERIC MICE 92
3.5 GENERATION OF MOUSE ANTI-MOUSE TNFR2 MAB 93
3.5.1 CLONING OF RECOMBINANT TNFR2ED-HULGG AND TNFR2ED-V5HIS-TAGGED
PROTEINS93
3.5.2 EXPRESSION OF TNFR2ED-HULGG AND TNFR2ED-V5HIS-TAGGED PROTEINS 94
3.5.3 TEST FOR BIOLOGICAL ACTIVITY OF TNFR2ED-HULGG / V5HIS CONSTRUCTS
95
3.5.4 IMMUNIZATION OF TNFR2^ MICE AND TEST OF SERUM WER 96
3.5.5 FUSION AND CHARACTERIZATION OF MOUSE ANTI-MOUSE TNFR2 MAB 96
3.5.5.1 FUSION 96
3.5.5.2 ELISA 97
3.5.5.3 ISOTYPE TEST 98
3.5.5.4 SDS-PAGE 98
3.5.5.5 WESTERN BLOT 98
3.5.5.6 FLOW CYTOMETRY 99
3.5.5.7 TRANSDUCOEON OF WIRBEL CELLS WITH TNFR1/2ED-HUFASID 100
3.5.5.8 TEST FOR AGONISTIC PROPERTIES 103
IMAGE 7
INDEX
3.5.5.9 TEST FOR ANTAGONISTIC PROPERTIES 104
4 DISCUSSION 107
4.1 CHARACTERIZATION OF CD11B* CELLS OF TNFR2 +MICE 107
4.1.1 SPLENOCYTES IN THE ANIMAL MODEL OF CLP 107
4.1.2 FUNCTIONAL CHARACTERIZATION OF MDSC 108
4.1.3 INOS MRNA EXPRESSION AND NO PRODUCTION 109
4.1.4 CHARACTERIZATION OF BMDC 111
4.1.4.1 BM CHIMERIC MICE 115
4.1.4.2 BMDCFROM MIXED CULTURES 117
4.2 MOUSE ANTI-MOUSE TNFR2 MAB WITH AGONISTIC OR ANTAGONISTIC PROPERTIES
119
5 CONCLUSION 121
6 REFERENCES 123
7 APPENDIX 133
8 ACKNOWLEDGMENTS 134
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| publishDateSort | 2010 |
| record_format | marc |
| spelling | Polz, Johannes Verfasser aut The role of TNF receptor type 2 on myeloid cells in sepsis functional analysis vorgelegt von Johannes Polz 2010 XVI, 135 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Regensburg, Univ., Diss., 2010 Tumor-Nekrose-Faktor (DE-588)4202932-6 gnd rswk-swf Rezeptor (DE-588)4049722-7 gnd rswk-swf Makrophage (DE-588)4193041-1 gnd rswk-swf Sepsis (DE-588)4181014-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Sepsis (DE-588)4181014-4 s Makrophage (DE-588)4193041-1 s Tumor-Nekrose-Faktor (DE-588)4202932-6 s Rezeptor (DE-588)4049722-7 s b DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:355-epub-170908 http://epub.uni-regensburg.de/17090/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-170908 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020660038&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Polz, Johannes The role of TNF receptor type 2 on myeloid cells in sepsis functional analysis Tumor-Nekrose-Faktor (DE-588)4202932-6 gnd Rezeptor (DE-588)4049722-7 gnd Makrophage (DE-588)4193041-1 gnd Sepsis (DE-588)4181014-4 gnd |
| subject_GND | (DE-588)4202932-6 (DE-588)4049722-7 (DE-588)4193041-1 (DE-588)4181014-4 (DE-588)4113937-9 |
| title | The role of TNF receptor type 2 on myeloid cells in sepsis functional analysis |
| title_auth | The role of TNF receptor type 2 on myeloid cells in sepsis functional analysis |
| title_exact_search | The role of TNF receptor type 2 on myeloid cells in sepsis functional analysis |
| title_full | The role of TNF receptor type 2 on myeloid cells in sepsis functional analysis vorgelegt von Johannes Polz |
| title_fullStr | The role of TNF receptor type 2 on myeloid cells in sepsis functional analysis vorgelegt von Johannes Polz |
| title_full_unstemmed | The role of TNF receptor type 2 on myeloid cells in sepsis functional analysis vorgelegt von Johannes Polz |
| title_short | The role of TNF receptor type 2 on myeloid cells in sepsis |
| title_sort | the role of tnf receptor type 2 on myeloid cells in sepsis functional analysis |
| title_sub | functional analysis |
| topic | Tumor-Nekrose-Faktor (DE-588)4202932-6 gnd Rezeptor (DE-588)4049722-7 gnd Makrophage (DE-588)4193041-1 gnd Sepsis (DE-588)4181014-4 gnd |
| topic_facet | Tumor-Nekrose-Faktor Rezeptor Makrophage Sepsis Hochschulschrift |
| url | http://epub.uni-regensburg.de/17090/ https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-170908 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020660038&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT polzjohannes theroleoftnfreceptortype2onmyeloidcellsinsepsisfunctionalanalysis |