by Li Pok Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 94-114). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abbreviations --- p.II / Abstract --- p.V / 摘要 --- p.VIII / Publications --- p.XI / Table of contents --- p.XII / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Human Th lymphocytes and their immunopathogenic roles --- p.1 / Chapter 1.1.1 --- Characteristics of Th lymphocytes --- p.1 / Chapter 1.1.2 --- Migration and activation --- p.1 / Chapter 1.1.3 --- Th cell differentiation --- p.2 / Chapter 1.1.4 --- Pathological roles --- p.4 / Chapter 1.2 --- Cytokines as modulator in Th lymphocyte activation --- p.6 / Chapter 1.2.1 --- IL-18 --- p.6 / Chapter 1.2.2 --- IL-25 --- p.7 / Chapter 1.3 --- Surface marker expression in Th lymphocytes --- p.8 / Chapter 1.3.1 --- Adhesion molecules --- p.8 / Chapter 1.3.2 --- Cytokine and chemokine receptors --- p.9 / Chapter 1.3.3 --- Costimulatory molecules --- p.11 / Chapter 1.4 --- Cytokine and chemokine release from Th lymphocytes / Chapter 1.4.1 --- Thl cytokines --- p.13 / Chapter 1.4.2 --- Th2 cytokines --- p.14 / Chapter 1.4.3 --- Chemokines --- p.15 / Chapter 1.5 --- Intracellular signaling pathways in Th lymphocytes --- p.19 / Chapter 1.5.1 --- p38 MAPK pathway --- p.19 / Chapter 1.5.2 --- ERK pathway --- p.20 / Chapter 1.5.3 --- JNK pathway --- p.20 / Chapter 1.5.4 --- NF- k B pathway --- p.21 / Chapter 1.6 --- Pharmacological intervention of signaling pathways --- p.22 / Chapter 1.7 --- Aims and scope of the study --- p.24 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.26 / Chapter 2.1.1 --- Blood samples --- p.26 / Chapter 2.1.2 --- Media and reagents for cell culture --- p.26 / Chapter 2.1.3 --- Antibodies for costimulation of Th cells --- p.28 / Chapter 2.1.4 --- Recombinant human cytokines --- p.28 / Chapter 2.1.5 --- "Signaling pathway inhibitors: SB203580, PD98035, SP600125 and BAY117082" --- p.28 / Chapter 2.1.6 --- Monoclonal antibodies and reagents for immunofluorescent staining --- p.29 / Chapter 2.1.7 --- Reagents and buffers for the purification of human Th lymphocytes --- p.31 / Chapter 2.1.8 --- Reagents and buffers for protein array --- p.32 / Chapter 2.1.9 --- Reagents and buffers for Thl/2 cytokine and chemokine detection --- p.32 / Chapter 2.1.10 --- Reagents and buffers for protein extraction --- p.32 / Chapter 2.1.11 --- Reagents and buffers for SDS-polyacrylamide gel electrophoresis --- p.33 / Chapter 2.1.12 --- Reagents and buffers for Western blot analysis --- p.35 / Chapter 2.1.13 --- Reagents and buffers for non-radioactive electromobility shift assay (EMSA) --- p.37 / Chapter 2.1.14 --- Reagents and buffers for cell viability and proliferation assay --- p.39 / Chapter 2.1.15 --- Reagent kit for endotoxin level assay --- p.39 / Chapter 2.1.16 --- Other reagent kits --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Purification of human Th lymphocytes and cell culture --- p.41 / Chapter 2.2.2 --- Measurement of total and allergen-specific IgE concentrations --- p.41 / Chapter 2.2.3 --- Immunophenotyping of cells by flow cytometry --- p.42 / Chapter 2.2.4 --- Protein array --- p.42 / Chapter 2.2.5 --- Quantitative analysis of cytokines and chemokines by flow cytometry --- p.43 / Chapter 2.2.6 --- Quantitative analysis of IFN-γ by ELISA --- p.43 / Chapter 2.2.7 --- SDS-PAGE --- p.44 / Chapter 2.2.8 --- Western blot analysis --- p.44 / Chapter 2.2.9 --- EMSA / gel shift assay --- p.45 / Chapter 2.2.10 --- MTT assay --- p.46 / Chapter 2.2.11 --- Cell proliferation assay --- p.46 / Chapter 2.2.12 --- Endotoxin level assay --- p.47 / Chapter 2.2.13 --- Statistical analysis --- p.47 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effects of IL-18 and IL-25 on the induction of Thl/2 cytokine and chemokine release from costimulated Th lymphocytes --- p.48 / Chapter 3.1.1 --- IL-18 and IL-25 could up-regulate the protein expression of cytokines and chemokines --- p.48 / Chapter 3.1.2 --- IL-18 but not IL-25 induced the release of IFN-γ and TNF-α --- p.48 / Chapter 3.1.3 --- "IL-18 and IL-25 induced the release of IL-5, IL-6 and IL-10" --- p.49 / Chapter 3.1.4 --- "IL-18 induced the release of IP-10, MIG, RANTES, MlP-lα and IL-8" --- p.49 / Chapter 3.1.5 --- "IL-25 induced the release of IP-10, MIG and RANTES" --- p.49 / Chapter 3.1.6 --- IL-18 and IL-25 did not enhance the proliferation of costimulated Th cells --- p.49 / Chapter 3.2 --- "Effects of IL-18 and IL-25 on the activation of p38 MAPK, ERK, JNK and NF- k B" --- p.58 / Chapter 3.2.1 --- "Costimulation with or without IL-18 and IL-25 could activate p38 MAPK, ERK and JNK" --- p.58 / Chapter 3.2.2 --- Costimulation with or without IL-18 and IL-25 could induce NF- k B activity --- p.58 / Chapter 3.3 --- Effects of inhibitors on the IL-18 and IL-25-induced release of Thl/2 cytokines and chemokines --- p.63 / Chapter 3.3.1 --- "Optimal dosage of SB203580, PD98035, SP600125 and BAY117082" --- p.63 / Chapter 3.3.2 --- "SB203580, PD98035 and BAY 117082 but not SP600125 suppressed the IL-18 and IL-25-induced release of Thl/2 cytokines" --- p.63 / Chapter 3.3.3 --- SP600125 suppressed the IL-18 and IL-25-induced release of chemokines --- p.64 / Chapter 3.4 --- Effects of inhibitors on the cell surface expression of IL-18 and IL-25 receptors --- p.72 / Chapter 3.4.1 --- "SB203580, PD98035, BAY 117082 but not SP600125 could suppress IL-18 receptor on costimulated Th cells" --- p.72 / Chapter 3.4.2 --- "SB203580, SP600125, PD98035 and BAY 117082 could not suppress IL-25 receptor on costimulated Th cells" --- p.72 / Chapter 3.5 --- Effects of costimulation on the expression of cell surface markers on Th lymphocytes --- p.75 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Effects of IL-18 and IL-25 on the release of Th1/2 cytokines and chemokines --- p.80 / Chapter 4.2 --- "Regulation of Thl/2 cytokines and chemokines through intracellular p38 MAPK, ERK, JNKand NF-kB" --- p.83 / Chapter 4.3 --- Effects of costimulation on different surface markers in Th cells --- p.87 / Chapter 4.4 --- Concluding remarks and future perspectives --- p.90 / References --- p.94