Expression of the T cell regulatory molecule ICOS (CD278) and LICOS (CD275) on human blood cells [Elektronische Ressource] / vorgelegt von: Ionela Moanta

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Informations

• Publié par: ernst-moritz-arndt-universitat_greifswald
• Publié le: 01 janvier 2007
• Nombre de lectures: 120
• Langue: Deutsch
• Poids de l'ouvrage: 11 Mo

Extrait

Aus dem Institut für Immunologie und Transfusionsmedizin
(Direktorin Prof. Dr. Christine Schütt)
der Medizinischen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald
Expression of the T cell regulatory
molecule ICOS (CD278) and LICOS
(CD275) on human blood cells
Inaugural Dissertation
zur
Erlangung des akademischen Grades
Doktor der Naturwissenschaften in der Medizin (Dr. rer. med.)
der
Medizinischen Fakultät
der
Ernst-Moritz-Arndt-Universität
Greifswald
2006
vorgelegt von: Ionela Moanta
geb. am: 10.03.1976
in: Craiova, RumänienDekan: Prof. Dr. H. K. Kroemer
1. Gutachter: Frau Prof. Dr. B. Bröker (Greifswald)
2. Gutachter: Herr Prof. Dr. R. E. Schmidt (Hannover)
(3. Gutachter:) Frau PD Dr. E. Kauschke (Giessen)
Ort, Raum: G reifswald, Fleischmannstr. 6, Seminarraum 305
Tag der Disputation: 13.August 2007Table of Contents I
Table of Contents
1. Introduction...........................................................................................1
1.1 The innate immunity..............................................................................................................................1
1.2 The adaptive immunity .........................................................................................................................2
1.2.1 B-cells and T-cells..........................3
1.2.2 Th1 and Th2 cells4
1.2.3 T regulatory cells (T ) .........................................................................................................................5reg
1.3 T cell response.........................................................................................................................................6
1.3.1 TCR engagement – signal 1..................................................................................................................6
1.3.2 Costimulation – signal 2 .......................................................................................................................6
1.3.2.1 Costimulatory molecules...................6
1.3.2.1.1 The Inducible Costimulator (ICOS)...............................................................................................9
1.3.2.1.2 The Inducible Costimulator Ligand (LICOS)..............................................................................10
1.3.2.2 Functions of ICOS and LICOS........................................................................................................11
1.3.2.2.1 The role of ICOS and LICOS in T cell proliferation and cytokine production..........................11
1.3.2.2.2 The role of ICOS and LICOS in Th1 and Th2 responses ...........................................................12
1.3.2.2.3 The role of ICOS and LICOS in humoral response.....................................................................14
1.4 Sepsis – at the interface between innate and adaptive immunity ..................................................16
1.4.1 Trauma as risk factor for sepsis..........................................................................................................18
1.4.2 Mechanisms of immunosuppression in polytrauma and sepsis.........................................................19
1.4.2.1 Biased Th2 responses.......................................................................................................................19
1.4.2.2 Loss of antigen presentation and costimulatory capacity of monocytes........................................20
1.4.2.3 Other immunosuppressive mechanisms..........................................................................................21
1.4.3 Mechanisms of immunosuppression in stroke...................................................................................21
1.5 Aim of the study ...................................................................................................................................22
2. Material and Methods.........................................................................23
2.1 Materials................................................................................................................................................23
2.1.1 Laboratory equipment.........................................................................................................................23
2.1.2 Reagents ..............................................................................................................................................24
2.1.3 Consumables and kits....................26
2.1.4 Biological material..............................................................................................................................28
2.1.5 Plasmids..............................28
2.1.6 Enzymes ..............................................................................................................................................29
2.1.7 Culture media ......................................................................................................................................29
2.1.7.1 Media for bacteria.......................29
2.1.7.2 Media for cell lines and hybridoma.................................................................................................30
2.1.8 Buffers and stock solutions.................................................................................................................30
2.1.8.1 Molecular biology methods...............30Table of Contents II
2.1.8.2 Biochemical Methods ......................................................................................................................31
2.1.8.3 Cell culture methods ........................................................................................................................32
2.1.9 Antibodies ...........................................................................................................................................33
2.1.9.1 ELISA...............................................................................................................................................33
2.1.9.2 Western blot .....................................................................................................................................33
2.1.9.3 Flowcytometry .................................................................................................................................34
2.1.9.3.1 Labelled antibodies .......................................................................................................................34
2.1.9.3.2 Labelled isotype controls..............................................................................................................34
2.1.9.3.3 Primary and blocking antibodies..................................................................................................35
2.1.9.3.4 Secondary antibodies and conjugates...........................................................................................35
2.1.10 Fusion proteins..................................................................................................................................35
2.2 Methods .................................................................................................................................................36
2.2.1 Molecular biology methods ................................................................................................................36
2.2.1.1 Primers..............................................................................................................................................36
2.2.1.2 Polymerase chain reaction (PCR)....................................................................................................36
2.2.1.3 DNA gel electrophoresis..................37
2.2.1.4 Agarose gel extraction of DNA fragments .....................................................................................37
2.2.1.5 Quantitation of DNA........................................................................................................................37
2.2.1.6 Restriction digestion of DNA..........................................................................................................38
2.2.1.7 Ligation of DNA fragments..............................................................................................38
2.2.1.8 Storage of DNA and of bacteria strains ..........................................................................................39
2.2.1.9 Initiation of bacterial culture ...........................................................................................................39
2.2.1.10 Plasmid preparation from bacteria ................................................................................................39
2.2.1.10.1 Plasmid Mini Prep ......................................................................................................................39
2.2.1.10.2 Plasmid Maxi Prep39
2.2.1.11 Transformation of competent bacteria ..........................................................................................40
2.2.1.12 DNA sequencing............................................................................................................................40
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