Aus der Klinischen Forschergruppe für Rheumatologie, Abteilung Rheumatologie und Klinische Immunologie, Medizinische Universittsklinik der Albert-Ludwigs-Universitt Freiburg
Molecular defects in B lymphocytes from patients with CVIDsyndrome
Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultt für Biologie der Albert-Ludwigs-Universitt Freiburg i. Br.
vorgelegt von Marcus von Hornung geb. in München 2002
Gewidmet meinen Eltern
Dekan der Fakultt für Biologie: Prof. Dr. Hans Kleinig Leiter der Arbeit und Referent: PD. Dr. Hermann Eibel Koreferent: Prof. Dr. Michael Reth Tag der Verkündigung: 10 Februar 2003
Contents 1. 1.1 1.2 1.3 1.4 1.4.1 1.4.2 1.4.2.1 1.4.2.2 1.4.2.3 1.4.2.4 1.5 1.5.1 1.5.2 1.5.3 1.5.4 1.5.5 1.5.6 2 3 3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.1.7 3.1.8 Introduction The immune system The adaptive immune system Histology of lymph node tissue B cell activation in secondary lymphoid tissues Pre-follicular B cell activation The germinal center reaction Inside the follicle Affinity maturation occurs in the light zone Immunoglobulin class switching Generation of memory and plasma B cells during the germinal center reaction Common variable immunodeficiency (CVID) Epidemiology Clinical Manifestations Autoimmune diseases Lymphoproliferative disorders Granulomatous Disease Immunologic features Aims of this thesis Material and Methods Material Antibodies Chemicals and consumable material Reagents for immunohistochemistry Buffers and solutions Cell culture reagents Reagents for isolation of human B and T cells from peripheral blood Eukaryotic cell lines and bacterial strains Mice 24 25 25 21 22 22 23 24 12 14 15 15 16 16 17 17 19 1 1 2 4 4 6 7 9 10
Contents 3.1.9 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.2.7 3.2.8 3.2.9 3.2.10 3.2.11 3.2.12 3.2.13 3.2.14 3.2.14.1 3.2.14.2 3.2.15 3.2.16 3.2.17 3.2.18 3.2.18.1 3.2.18.2 3.2.18.3 3.2.18.4 3.2.18.5 3.2.18.6 3.2.18.7 Plasmids Methods Restriction digestion of plasmid DNA Isolation and purification of DNA fragments from agarose gels Ligation of DNA fragments Preparation of competent bacteria Transformation of competent bacteria Screening for recombinants Transient transfection of eukaryotic cells Limiting dilution Fluorescence activated cell sorting (FACS) Isolation of human peripheral blood mononuclear cells (PBMNC) Isolation of human B- and T cells from PBMNC Co-culture of human B cells with 293T CD40L cells Proliferation assay The EL-4 system Preparation of T cell-macrophage supernatant Stimulation of peripheral blood B cells in the EL-4 system ELISA for IgM and IgG Extraction of cells from secondary lymphoid tissue organs Reconstitution of Rag2/yc with human CD19+ B cells and CD4+ T cells Immunohistochemical methods Preparation of tissue samples for cryosections Preparation of frozen section slides Fixation of tissue sections for immunohistochemistry Blocking of endogenous peroxidase activity Detection of first antigen Detection of second antigen Embedding and documentation 31 32 32 32 33 33 33 34 34 28 29 29 29 30 30 31 31 31 25 26 26 26 27 27 27 28 28 25

下一页