Study of cytokines in canine rheumatoid arthritis:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Berlin
Köster
2002
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| Ausgabe: | 1. Aufl. |
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XII, 134 S. Ill., graph. Darst. |
| ISBN: | 3895744530 |
Internformat
MARC
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
ABSTRACT IV
ZUSAMMENFASSUNG V
ACKNOWLEDGEMENTS VI
ABBREVIATIONS XI
J.O. INTRODUCTION 1
1.1. Epidemiology 3
1.2. Etiology 4
1.3.PATHOGENESIS 7
1.4. CL1N1CAL FEATURES 8
1.5. INVESTIGATIONS 10
1.5.1. Serological 10
1.5.2. Synovial analysis 10
1.5.3. Radiography 10
1.6. Treatment 11
1.6.1. Non-steroidal anti-inflammatory drugs (NSAID) //
1.6.2. Disease modifying antirheumatic drugs (DMARD) //
1.6.3. Immunomodulation 12
1.7. CANINE RHEUMATOID ARTHRITIS AS A POTENTIAL ANIMAL MODEL 12
1.8. CYTOKINES AND OTHER SOLUBLE MEDIATORS 1 3
1.8.1. Tumor Necrosis Faclor-a (TNF-a) 14
1.8.2. Interleukin-1 17
1.8.3. InterIeukin-4 19
1.8.4. Interleukin-6 22
1.8.5. lnterleukin-8 23
1.8.6. Interleukin 10 24
1.8.7. Interleukin 12 27
1.8.8. Interleukin 18 29
1.8.9. IFN-y. 30
1.8.10. Transforming Growth Factor-ßl (TGF-ßl) 31
vii
1.8.11. MetaUoproteinases 33
1.9. Rationale and Aim 34
2.0. MATERIALS AND METHODS 39
2.1. METHODS FOR THE ANALYSIS OF CYTOKINES 39
2.1.1. Processing of synovial fluid specimens 39
2.1.2. RNA extraction 39
2.1.3. Synthesis ofcDNA front RNA 39
2.1.4. Oligonucleolide primers for PCR amplification ofcanine cDNA 40
2.1.5. Polvmerase chain reaction (PCR) 42
2.1.6. Densitometric quantitalion 42
2.1.7. Bioassayfor the determination ofTNF-a. 43
2.1.8. FACScan analysis 44
2.1.9. Statistical analysis 44
2.2. METHODS FOR CLONING AND EXPRESSION OF CANINE IL-4 44
2.2.1. Stimulation ofPBMC 44
2.2.2. Preparation ofcDNA 45
2.2.3. 3 - and 5 - rapid amplification ofcDNA ends (RACE) cloning 45
2.2.4. Polvmerase chain reaction (PCR) amplification ofcanine IL-4 46
2.2.5. Restriction enzyme analysis 46
2.2.6. Dephosphorylation ofDNA 46
2.2.7. Ligation of DNA 47
2.2.8. Transformation ofcompetent cells with plasmid DNA 47
2.2.9. Expression plasmid and bacterial strain 48
2.2.10. Colony blot assay 48
2.2.11. Canine IL-4 production in E. coli 49
2.2.12. Solublization and refolding ofcanine IL-4 from inclusion granules 49
2.2.13. Ni-NTA resin purification 50
2.2.14. Canine IL-4 expression in COS-7 cells 50
2.2.15. IL-4 bioassay 51
2.2.16. Sodium dodecyl sulphatepolyacrlyamidegel electrophoresis (SDS-PAGE) 51
2.2.17. Western blotting 52
2.2.18. Mini prepartation 52
2.2.19. Maxi preparation 53
viii
2.2.20. Determination ofprotein concentration 53
2.2.21. Determination ofDNA concentration 54
2.2.22. Agarose electrophoresi 54
2.2.23. Purification ofDNAfrom agarose gel 54
2.2.24. Preparation of competent cells 55
2.3. Production of monoclonal antibodies (mAbs) tocanine IL-4 55
2.3.1 Enzyme HnkeJ immunosorbent assay (ELISA) 56
2.3.2. Purification ofmAb byprotein A affinity chromatography 56
2.4. Materials 58
2.4.1. Patients 55
2.4.2. Materialsfor cytokine analysis 58
2.4.3. Materials for the cloning and expression procedure 59
2.4.4. Materials for the production of monoclonal antibodies 59
2.5. Buffers and Solutions 59
2.5.1. General Solutions 59
2.5.2. Bacterial media and Solutions 59
2.5.3. Antibiotics 60
2.5.4. Agarose gel electrophoresis 60
2.5.5. SDS-Polyacrcylamide minigels 60
2.5.6. ELISA 62
2.5.7. Protein A affinity chromatography 62
2.5.8. Western blot 62
2.5.9. Colonyblot 63
2.6. Kits and Enzymes 64
2.7. Chemicals, Disposables and Media 64
2.8. Instruments 64
3.0. RESULTS 67
3.1. Cytokine Profile 68
3.1.1. Detection of proinflammatory cytokines 70
3.1.2. Expression ofT cell cytokines 76
3.1.3. Production of anti-inflammatory cytokines 78
3.1.4. Phenotypeof cells 80
3.2. Cloning and Expression of canine IL-4 82
ix
3.2.1. Motecular cloning ofcanine IL-4 82
3.2.2. Expression ofcanine IL-4 in E. coli 84
3.2.2. Expression ofcanine IL-4 in E. coli 85
3.2.3. Purification and refolding of inclusion body associated IL-4 87
3.2.4. Mammalian expression ofcanine IL-4 90
3.2.5. Biological activity of recombinant canine lL-4from E. coli and COS-7 teils 91
3.2.6. Produclion of anti-cIL4 monoclonal antibody and immunodeleclion 93
4.0. discussion 95
4.1. cytokine profile 96
4.2. Cloning and Expression of cIL-4 103
4.3. future prospects 109
5.0. REFERENCES 111
PUBLICATIONS 132
FORM THE PRESENT WORK 132
PREVIOUS PUBLICATIONS 132
LEBENSLAUF 134
X
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| indexdate | 2024-07-09T22:30:14Z |
| institution | BVB |
| isbn | 3895744530 |
| language | English |
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| physical | XII, 134 S. Ill., graph. Darst. |
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| spelling | Wondimu Abebe, Assefa Verfasser aut Study of cytokines in canine rheumatoid arthritis Assefa Wondimu Abebe 1. Aufl. Berlin Köster 2002 XII, 134 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zugl.: Berlin, Humboldt-Univ., Diss., 2002 (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=019911822&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wondimu Abebe, Assefa Study of cytokines in canine rheumatoid arthritis |
| subject_GND | (DE-588)4113937-9 |
| title | Study of cytokines in canine rheumatoid arthritis |
| title_auth | Study of cytokines in canine rheumatoid arthritis |
| title_exact_search | Study of cytokines in canine rheumatoid arthritis |
| title_full | Study of cytokines in canine rheumatoid arthritis Assefa Wondimu Abebe |
| title_fullStr | Study of cytokines in canine rheumatoid arthritis Assefa Wondimu Abebe |
| title_full_unstemmed | Study of cytokines in canine rheumatoid arthritis Assefa Wondimu Abebe |
| title_short | Study of cytokines in canine rheumatoid arthritis |
| title_sort | study of cytokines in canine rheumatoid arthritis |
| topic_facet | Hochschulschrift |
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