Chronic Obstructive Pulmonary Disease (COPD) is a common lung disease characterized by progressive and irreversible airway obstruction, and mainly caused by a chronic exposure to lung irritants. As of 2010, 384 million people suffered from COPD worldwide. It is widely accepted that a chronic inflammatory response is integral to COPD pathogenesis and linked to disease progression. The cellular mediators and molecular mechanisms of COPD-associated inflammation are not completely understood and are difficult to emulate in animal models, which hinders the development of better treatments. In this study, experimental COPD and its associated inflammation were induced in mice using a 4-week protocol involving intranasal administration of LPS and elastase. Model validation on wild-type mice yielded COPD-like disease judging from flow cytometric analyses with and pulmonary function testing. After 4 weeks of exposure to LPS and elastase, mice developed classic aspects of COPD such an increase in lung-infiltrating cells, (e.g. neutrophils, CD4+and CD8+ T-cells). Acute inflammation in the form of substantial neutrophilia was due to the last LPS administration, whereas the observed eosinophilia and elevated counts of mast cell populations, CD4+ and CD8+ T-cells were due to the cumulative effects of LPS and elastase. The nature of COPD-associated inflammation in mast cell deficient mice was investigated in two experiments. Our first experiment suggested a mild protective role of mast cells, a finding not reproduced in the second experiment possibly due to expired elastase. Our study suggests that mast cells are not required for COPD-associated inflammation.

Expt. 1 evaluated effects of 0 or 10 ppm CAP in the starter phase (d 1-16) on chicks challenged with SE on d of age. Therapeutic inclusion of 10ppm CAP increased (P < 0.05) L/S and ceca positives. In Expt. 2, capsaicin oleoresin (CO) was included in finisher diets (d 30-37) at 0, 5, or 20 ppm with SE challenge on day 31. Inclusion of 5 ppm CO increased (P < 0.05) ceca SE positives and demonstrated 1.05 and 1.39-log fewer SE cfu at CO concentration of 5 or 20 ppm, respectively. A linear decrease (P < 0.05) in lamina propria thickness of SE challenged birds was observed with increased CO. Expt. 3 evaluated prophylactic CO treatment at 0, 5, or 20 ppm in starter, grower, and finisher diets for resistance to SE or ST challenge on d 14 or 29. With challenge on d 14, 5 ppm CO reduced ceca (P<0.005) SE positives and 1.1-log fewer SE cfu. Likewise, 20 ppm CO reduced (P < 0.05) SE ceca positives. Salmonella typhimurium isolation rate was reduced (P<0.05) with 5 ppm CO, and ST cfu were reduced 1.4-log with 5 ppm CO compared to 20 ppm. Lamina propria thickness increased (P < 0.05) linearly as CO concentration increased. With d 29 challenge birds fed 5 ppm CO exhibited 1.08-log fewer SE cfu, and 20 ppm CO reduced L/S positives (P < 0.025) for SE and resulted in 1.39-log fewer SE cfu. Lamina propria thickness decreased with 5 ppm CO and SE or ST challenge compared to non-challenged birds fed 5 ppm (P < 0.0005). An increase was observed in ST or SE, birds fed 20 ppm CO compared to non-challenged, birds fed 20 ppm CO (P < 0.01). No differences were observed in mast cell number in either Expt. 2 or 3. These data provide evidence that prophylactic or therapeutic dietary CAP differentially affect broiler susceptibility to Salmonella and prophylactic administration may provide non-antibiotic means to reduce Salmonella in broilers. / Master of Science

No description available.

Pui Lan Wong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (Leaves 109-122). / Abstract --- p.i / Acknowledgements --- p.iii / Chapter Chapter1 --- Introduction / Chapter 1.1 --- A general introduction on mast cells --- p.1 / Chapter 1.2 --- Activation of mast cells --- p.6 / Chapter 1.3 --- Mediators of mast cells --- p.18 / Chapter 1.4 --- Usage of β-adrenoceptor agonists in asthma therapy --- p.26 / Chapter 1.5 --- Aim of this study --- p.32 / Chapter Chapter2 --- Materials and methods / Chapter 2.1 --- Chemicals --- p.42 / Chapter 2.2 --- Buffers and stock solutions --- p.43 / Chapter 2.3 --- Source of mast cells --- p.45 / Chapter 2.4 --- Animal sensitization --- p.45 / Chapter 2.5 --- Isolation of mast cells --- p.46 / Chapter 2.6 --- Procedure for the investigation of the effects of adrenoceptor agonists on histamine release from mast cells --- p.48 / Chapter 2.7 --- Procedure for the investigation of propranolol antagonism --- p.49 / Chapter 2.8 --- Histamine assay --- p.50 / Chapter 2.9 --- Data analysis --- p.50 / Chapter Chapter3 --- Results / Chapter 3.1 --- Establishment of experimental conditions --- p.53 / Chapter 3.2 --- The effects of β-agonists on immunologically induced histamine release from guinea pig lung mast cells --- p.54 / Chapter 3.3 --- The effects of β-agonists and two anti-allergic drugs on immunologically induced histamine release from guinea pig lung mast cells --- p.56 / Chapter 3.4 --- The effects of β2-agonists on histamine release induced by non-immunological agents from guinea pig lung mast cells --- p.56 / Chapter 3.5 --- Antagonism by propranolol on the effects of β2-agonists on histamine release from guinea pig lung mast cells --- p.57 / Chapter 3.6 --- The effects of β2-agonists on immunologically induced histamine release from rat peritoneal mast cells --- p.58 / Chapter 3.7 --- The effects of β2-agonists on immunologically induced histamine release from human lung mast cells --- p.58 / Chapter 3.8 --- "Comparison of the effects of β2-agonists on immunologically induced histamine release from mast cells isolated from the rat peritoneum, the guinea pig lung and the human lung" --- p.59 / Chapter Chapter4 --- Discussion / Chapter 4.1 --- The effects of β-agonists on immunologically induced histamine release from guinea pig lung mast cells --- p.89 / Chapter 4.2 --- The effects of β2-agonists and two anti-allergic drugs on immunologically induced histamine release from guinea pig lung mast cells --- p.97 / Chapter 4.3 --- The effects of novel β2-agonists on histamine release induced by non-immunological agents from guinea pig lung mast cells --- p.99 / Chapter 4.4 --- The study of propranolol --- p.100 / Chapter 4.5 --- The heterogeneity of mast cells --- p.103 / Chapter Chapter5 --- General conclusion --- p.107 / References --- p.109

by Tsang Chi Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 137-155). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 撮要 --- p.ix / Publications --- p.xi / Table of contents --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Human mast cells and their pathological roles in inflammation --- p.1 / Chapter 1.1.1 --- Morphology of mast cells --- p.1 / Chapter 1.1.2 --- Mediators of mast cells --- p.1 / Chapter 1.1.3 --- Migration and activation --- p.3 / Chapter 1.1.4 --- Pathological roles of mast cells --- p.3 / Chapter 1.1.5 --- Human mast cell-1 (HMC-1) --- p.5 / Chapter 1.2 --- Cytokines as stimulator of mast cells in inflammation --- p.7 / Chapter 1.2.1 --- SCF --- p.7 / Chapter 1.2.2 --- TNF-α --- p.8 / Chapter 1.2.3 --- IL-13 --- p.8 / Chapter 1.2.4 --- IL-18 --- p.9 / Chapter 1.2.5 --- IL-25 --- p.9 / Chapter 1.3 --- Interaction of mast cells with inflammatory cells through adhesion molecules and chemokines --- p.11 / Chapter 1.3.1 --- Adhesion molecules on mast cells --- p.11 / Chapter 1.3.2 --- Chemokines released by mast cells --- p.12 / Chapter 1.4 --- Intracellular signaling pathways in mast cells --- p.16 / Chapter 1.4.1 --- p38-MAPK pathway --- p.16 / Chapter 1.4.2 --- ERK pathway --- p.17 / Chapter 1.4.3 --- NF-kB Pathway --- p.18 / Chapter 1.4.3 --- Cross-talking of pathways --- p.18 / Chapter 1.5 --- Signal transduction pathways and pharmacological intervention --- p.23 / Chapter 1.6 --- Traditional Chinese Medicine and pharmacological intervention --- p.25 / Chapter 1.6.1 --- Anti-allergic effects of traditional Chinese Medicine --- p.25 / Chapter 1.6.2 --- Anti-asthmatic effects of a newly developed Wheeze-Relief Formula --- p.26 / Chapter 1.7 --- Aims and scope of the study --- p.30 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- HMC-1 cell Line --- p.32 / Chapter 2.1.2 --- Media and reagents for cell culture --- p.32 / Chapter 2.1.3 --- Recombinant human cytokines --- p.33 / Chapter 2.1.4 --- "Signal transduction pathway inhibitors: PD98035, SB203580 and BAY 117082" --- p.34 / Chapter 2.1.5 --- Monoclonal antibodies and reagents for immunofluorescent staining --- p.34 / Chapter 2.1.6 --- Reagents and buffers for chemokine detection --- p.35 / Chapter 2.1.7 --- Reagents and buffers for total RNA extraction --- p.36 / Chapter 2.1.8 --- Reagents and buffers for reverse transcription 一 polymerase chain reaction (RT-PCR) --- p.37 / Chapter 2.1.9 --- Reagents and buffers for protein extraction --- p.40 / Chapter 2.1.10 --- Reagents and buffers for detection of activated signaling pathways --- p.41 / Chapter 2.1.11 --- Reagents and buffers for agarose gel electrophoresis --- p.42 / Chapter 2.1.12 --- Reagents and buffers for SDS-polyacrylamide gel electrophoresis (PAGE) --- p.43 / Chapter 2.1.13 --- Reagents and buffers for Western blot analysis --- p.45 / Chapter 2.1.14 --- Reagents and buffers for cDNA expression array analysis --- p.47 / Chapter 2.1.15 --- Reagents and buffers for cell viability and proliferation assay --- p.48 / Chapter 2.1.16 --- Reagent kit for endotoxin level assay --- p.49 / Chapter 2.2 --- Methods --- p.49 / Chapter 2.2.1 --- HMC-1 cell cultures --- p.49 / Chapter 2.2.2 --- Flow cytometry of cell surface expression of ICAM-1 and ICAM-3 --- p.50 / Chapter 2.2.3 --- Total cellular RNA extraction --- p.50 / Chapter 2.2.4 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) --- p.51 / Chapter 2.2.5 --- Agarose gel electrophoresis --- p.51 / Chapter 2.2.6 --- "Quantitative analysis of IL-8, IP-10,MCP-1 and RANTES" --- p.52 / Chapter 2.2.7 --- Quantitative analysis of 1-309 and MIP-1β --- p.52 / Chapter 2.2.8 --- Detection of phosphorylated-ERX and phosphorylated-p38 MAPK --- p.53 / Chapter 2.2.9 --- Detection of NF-kB activity --- p.53 / Chapter 2.2.10 --- Detection of phosphorylated-ATF-2 --- p.53 / Chapter 2.2.11 --- Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) --- p.54 / Chapter 2.2.12 --- Western blot analysis --- p.54 / Chapter 2.2.13 --- MTT assay --- p.55 / Chapter 2.2.14 --- Cell proliferation assay --- p.55 / Chapter 2.2.15 --- Hot water extraction of TCM --- p.56 / Chapter 2.2.16 --- Endotoxin level assay --- p.56 / Chapter 2.2.17 --- cDNA expression array analysis --- p.57 / Chapter 2.2.18 --- Statistical analysis --- p.57 / Chapter Chapter 3 --- Results / Chapter 3.1 --- The effects of cytokines on the expression of ICAM-1 and ICAM-3 on HMC-1 --- p.59 / Chapter 3.1.1. --- "SCF, TNF-α and IL-13 up-regulated ICAM-1 but not ICAM-3 expression on HMC-1 cells" --- p.59 / Chapter 3.1.2. --- "SCF, TNF-α and IL-13 up-regulated the mRNA expression of ICAM-1" --- p.59 / Chapter 3.1.3 --- "The combined treatment of SCF and TNF-α, and SCF and IL-13 showed synergistic and additive effect on ICAM-1 expression respectively" --- p.60 / Chapter 3.1.4 --- Synergistic up-regulation of ICAM-1 expression in combined treatment of SCF and TNF-α was dose-dependently enhanced by SCF --- p.60 / Chapter 3.2 --- "The effects of cytokines on the release of IL-8, IP-10, MCP-1, RANTES, 1-309 and MIP-1β from HMC-1 cells" --- p.66 / Chapter 3.2.1 --- "SCF induced the release of IL-8, MCP-1, RANTES, 1-309 and MIP-1β" --- p.66 / Chapter 3.2.2 --- "TNF-a induced the release of IL-8, IP-10, MCP-1, RANTES and 1-309" --- p.66 / Chapter 3.2.3 --- SCF and TNF-α did not enhance the proliferation rate of HMC-1 --- p.66 / Chapter 3.3 --- "The effect of SCF and TNF-α on the activation of ERK, p38 MAPK and NK-kB" --- p.71 / Chapter 3.3.1 --- SCF activated ERK but not p38 MAPK and NF-kB --- p.71 / Chapter 3.3.2 --- TNF-α activated p38 MAPK and NF-kB but not ERK --- p.71 / Chapter 3.4 --- The effect of inhibitors on the SCF and TNF-a-induced release of chemokines --- p.76 / Chapter 3.4.1 --- "The optimal dose of PD98059, SB203580 and BAY117082" --- p.76 / Chapter 3.4.2 --- "PD98059 suppressed the SCF induced IL-8, MCP-1, RANTES, 1-309 and MIP-1β release from HMC-1 cells" --- p.76 / Chapter 3.4.3 --- SB203580 and BAY117082 differentially suppressed the TNF-α induced chemokine release from HMC-1 cells --- p.77 / Chapter 3.5 --- The effect of inhibitors on the SCF and TNF-a-induced upregulation of ICAM-1 --- p.83 / Chapter 3.5.1 --- BAY117082 but not SB203580 suppressed the TNF-α-induced ICAM-1 expression --- p.83 / Chapter 3.5.2 --- PD98059 and BAY 117082 suppressed the combined treatment of SCF and TNF-α induced ICAM-1 expression --- p.83 / Chapter 3.6 --- "Effect of inhibitors on TNF-α and SCF-induced ERK, p38 MAPK and NF-kB activities in HMC-1 cells." --- p.85 / Chapter 3.6.1 --- PD98059 suppressed the SCF-induced activity of ERK --- p.85 / Chapter 3.6.2 --- SB203580 and BAY117082 suppressed the TNF-α induced p38 MAPKand NF-kB activity respectively --- p.85 / Chapter 3.6.3 --- PD98059 suppressed the enhanced NF-kB activity after the combined treatment of SCF and TNF-α for 18 hours --- p.86 / Chapter 3.7 --- Effect of TNF-α and SCF on the gene expression profile of inflammatory cytokines and receptors of HMC-1 cells. --- p.90 / Chapter 3.8 --- The effects of TCM on the SCF-induced 1-309 and MCP-1 from HMC-1 cells --- p.95 / Chapter 3.8.1 --- "Endotoxin level of Radix astragali, Radix Scutellariae, Radix stemonae, Bulbus Fritillariae cirrhosae and Cordyceps sinensis" --- p.95 / Chapter 3.8.2 --- The effects of TCM on the proliferation rate of HMC-1 cells --- p.95 / Chapter 3.9.3 --- The effects of TCM on the SCF-induced release of 1-309 from HMC-1 cells --- p.96 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Involvement of adhesion molecules and chemokines in mast cell-mediated immunological events --- p.107 / Chapter 4.2 --- HMC-1 as the in vitro mast cell model adapted in my project --- p.108 / Chapter 4.3 --- The effect of cytokines on the expression of ICAM-1 and ICAM-3 in HMC-1 cells --- p.109 / Chapter 4.4 --- The effect of cytokines on the release of chemokines in HMC-1 cells --- p.111 / Chapter 4.5 --- "The regulation of ICAM-1, IL-8, IP-10, MCP-1, RANTES, 1-309 and MIP-1β through p-38 MAPK, ERK and NF-kB signaling pathways in HMC-1 cells" --- p.115 / Chapter 4.6 --- Further characterization of HMC-1 cells using cDNA array --- p.119 / Chapter 4.7 --- Investigating the in vitro anti-allergic activities of a newly developed Wheeze-relief formula using cytokine-activated HMC-1 cells --- p.128 / Chapter 4.8 --- Concluding remarks and future prospective --- p.132 / References --- p.137 / Appendix --- p.156

by Grant Richardson Stenton. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 259-287). / Abstract --- p.i / Acknowledgements --- p.iii / Publications --- p.iv / Abbreviations --- p.v / Contents --- p.vii / Chapter Chapter 1 --- Introduction / Chapter 1 1.1. --- Historical Background --- p.2 / Chapter 1.2. --- Origin and distribution of mast cells --- p.2 / Chapter 1.3. --- Mast cell heterogeneity --- p.3 / Chapter 1.4. --- Mast cell mediators --- p.5 / Chapter 1.4.1. --- Preformed mast cell mediators --- p.6 / Chapter 1.4.2. --- Newly synthesised mast cell mediators --- p.7 / Chapter 1.5. --- Mast cell activation --- p.11 / Chapter 1.5.1. --- Antigenic pathway of mast cell activation --- p.11 / Chapter 1.5.1.1. --- Antigen binding and receptor aggregation --- p.12 / Chapter 1.5.1.2. --- Early events following FcεRI aggregation --- p.13 / Chapter 1.5.1.3. --- Antigenic induction of mast cell second messenger production --- p.15 / Chapter 1.5.1.4. --- Phospholipase C activation and mast cells --- p.16 / Chapter 1.5.1.5. --- Phospholipase A2 activation and mast cells --- p.17 / Chapter 1.5.1.6. --- Intracellular calcium and mast cells --- p.18 / Chapter 1.5.1.7. --- Calcium and calmodulin --- p.21 / Chapter 1.5.1.8. --- Adenylate cyclase activation and mast cells --- p.21 / Chapter 1.5.2. --- Non-antigenic pathway of mast cell activation --- p.22 / Chapter 1.6. --- Aims of the study --- p.25 / Chapter 1.6.1. --- Diuretics --- p.26 / Chapter 1.6.2. --- Histamine receptor directed compounds --- p.27 / Chapter 1.6.3. --- Cyclo-oxygenase inhibitors --- p.28 / Chapter 1.6.4. --- Immunosuppressive compounds --- p.29 / Chapter Chapter 2 --- Materials and Methods --- p.31 / Chapter 2.1. --- Materials and methods --- p.32 / Chapter 2.1.1. --- Secretagogues --- p.32 / Chapter 2.1.2. --- Anti-allergic compounds --- p.32 / Chapter 2.1.3. --- Diuretics --- p.32 / Chapter 2.1.4. --- Immunosuppressants --- p.33 / Chapter 2.1.5. --- Histamine agonists and antagonists --- p.33 / Chapter 2.1.6. --- Cyclo-oxygenase inhibitors --- p.33 / Chapter 2.1.7. --- Materials for buffers --- p.34 / Chapter 2.1.8. --- Materials for rat sensitization --- p.34 / Chapter 2.1.9. --- Materials for histamine assay --- p.35 / Chapter 2.1.10. --- Materials for calcium measurement --- p.35 / Chapter 2.1.11. --- Materials for prostaglandin D2 measurement --- p.35 / Chapter 2.1.12. --- Materials for leukotriene C4 measurement --- p.36 / Chapter 2.1.13. --- Materials for cyclic AMP measurement --- p.36 / Chapter 2.1.14. --- Miscellaneous --- p.36 / Chapter 2.2. --- Buffers and stock solutions --- p.37 / Chapter 2.2.1. --- Buffer ingredients --- p.37 / Chapter 2.2.2. --- Stock solutions --- p.38 / Chapter 2.3. --- Animals and cell isolation --- p.39 / Chapter 2.3.1. --- Animals --- p.39 / Chapter 2.3.2. --- Sensitization of animals --- p.39 / Chapter 2.3.3. --- Cell isolation --- p.40 / Chapter 2.3.4. --- Cell washing and purification --- p.41 / Chapter 2.3.5. --- Preparation of cells for counting --- p.42 / Chapter 2.3.6. --- Cell counting on a haemocytometer --- p.42 / Chapter 2.4. --- General protocol for histamine release and histamine measurement --- p.43 / Chapter 2.4.1. --- Histamine release --- p.43 / Chapter 2.4.2. --- Spectroflurometric determination of histamine contents --- p.44 / Chapter 2.4.3. --- Calculation of histamine levels --- p.45 / Chapter 2.5. --- Protocol for cellular calcium measurement --- p.47 / Chapter 2.5.1. --- 45Ca2+ influx measurement --- p.47 / Chapter 2.5.2. --- Calculation of 45Ca2+ influx --- p.48 / Chapter 2.5.3. --- Fura-2 fluorescence measurement of intracellular calcium --- p.48 / Chapter 2.5.4. --- Fura-2 cell loading --- p.48 / Chapter 2.5.5. --- Fura-2 fluorescence parameters --- p.49 / Chapter 2.5.6. --- Calculation of basal calcium levels --- p.50 / Chapter 2.6. --- Protocol for prostaglandin D2 (PGD2) measurement --- p.52 / Chapter 2.6.1. --- PGD2 production --- p.52 / Chapter 2.6.2. --- Enzyme Immunosorbent Assay (EIA) method of PGD2 measurement --- p.52 / Chapter 2.6.3. --- Calculation of (EIA) PGD2 production --- p.53 / Chapter 2.6.4. --- Radio Immunosorbent Assay (RIA) method of PGD2 measurement --- p.53 / Chapter 2.6.5. --- Calculation of (RIA) PGD2 concentration --- p.54 / Chapter 2.7. --- Protocol for leukotriene C4 (LTC4) measurement --- p.54 / Chapter 2.7.1. --- LTC4 production --- p.54 / Chapter 2.7.2. --- Enzyme Immunosorbent Assay (EIA) method of LTC4 measurement --- p.55 / Chapter 2.7.3. --- Calculation of (EIA) LTC4 concentration --- p.55 / Chapter 2.8. --- Protocol for cyclic adenosine monophosphate (cAMP) measurement --- p.56 / Chapter 2.8.1. --- cAMP production --- p.56 / Chapter 2.8.2. --- Radio Immunosorbent Assay (RIA) method of cAMP measurement --- p.56 / Chapter 2.8.3. --- Calculation of cAMP concentration --- p.57 / Chapter 2.9. --- Statistical analysis --- p.57 / Chapter Chapter 3 --- "Frusemide, Bumetanide and DSCG" --- p.58 / Chapter 3.1. --- Introduction --- p.59 / Chapter 3.1.1. --- Frusemide and bumetanide as loop diuretics --- p.59 / Chapter 3.1.2. --- Effects of frusemide and bumetanide on the airways --- p.59 / Chapter 3.1.3. --- Effects of frusemide on mast cells --- p.60 / Chapter 3.1.4. --- Experimental aims --- p.61 / Chapter 3.2. --- Materials and methods --- p.62 / Chapter 3.3. --- Results --- p.63 / Chapter 3.3.1 --- "Effects of frusemide, bumetanide and DSCG on immunologically induced histamine release from rat peritoneal mast cells" --- p.63 / Chapter 3.3.2. --- "Effects of frusemide, bumetanide and DSCG on compound 48/80 induced histamine release from rat peritoneal mast cells" --- p.64 / Chapter 3.3.3. --- "Effects of frusemide, bumetanide and DSCG on compound 48/80 induced histamine release from rat peritoneal mast cells suspended in calcium free buffer" --- p.65 / Chapter 3.3.4. --- "Effects of frusemide, bumetanide and DSCG on ionophore A23187 and thapsigargin induced histamine release from rat peritoneal mast cells" --- p.65 / Chapter 3.3.5. --- Cross-tachyphylaxis effects of frusemide and bumetanide --- p.66 / Chapter 3.3.6. --- Effects of DSCG on the inhibition of anaphylactic histamine release due to frusemide --- p.67 / Chapter 3.3.7. --- Effects of frusemide and DSCG on immunologically and non-immunologically induced 45Ca2+ uptake --- p.67 / Chapter 3.3.8. --- Effects of frusemide and DSCG on immunologically and non-immunologically induced changes in the free intracellular calcium concentration of rat peritoneal mast cells --- p.68 / Chapter 3.3.9. --- Effects of frusemide and bumetanide on the spontaneous and secretagogue induced PGD2 production from rat peritoneal mast cells --- p.69 / Chapter 3.3.10. --- Effects of frusemide and DSCG on cellular cAMP levels --- p.70 / Chapter 3.4. --- Discussion --- p.101 / Chapter 3.5. --- Summary --- p.111 / Chapter 3.6. --- Conclusion --- p.114 / Chapter 3.7. --- Future studies --- p.114 / Chapter Chapter 4 --- Histamine Receptor Directed Compounds --- p.115 / Chapter 4.1. --- Introduction --- p.116 / Chapter 4.1.1. --- Histamine receptor subtypes --- p.116 / Chapter 4.1.2. --- Histamine effects on the airways --- p.117 / Chapter 4.2. --- Signal transduction mechanisms --- p.118 / Chapter 4.2.1. --- H1-receptors --- p.118 / Chapter 4.2.2. --- H2-receptors --- p.119 / Chapter 4.2.3. --- H3-receptors --- p.120 / Chapter 4.3. --- Histamine receptors and mast cells --- p.120 / Chapter 4.3.1. --- Effects of histamine agonists and antagonists on mast cells --- p.120 / Chapter 4.3.2. --- Experimental aims --- p.122 / Chapter 4.3.3. --- Materials and methods --- p.123 / Chapter 4.4. --- Results --- p.123 / Chapter 4.4.1. --- Effects of the test compounds on the spontaneous histamine release from rat peritoneal mast cells --- p.123 / Chapter 4.4.2. --- Effects of the test compounds on anti-IgE induced histamine release from rat peritoneal mast cells --- p.125 / Chapter 4.4.3. --- Effects of the test compounds on compound 48/80 induced histamine release from rat peritoneal mast cells --- p.126 / Chapter 4.4.4. --- Effects of the test compounds on anti-IgE and compound 48/80induced histamine release from rat peritoneal mast cells in calcium free buffer --- p.126 / Chapter 4.4.5. --- Effects of the test compounds on ionophore A23187 induced histamine release from rat peritoneal mast cells --- p.127 / Chapter 4.4.6. --- "Effects of histamine antagonists on dimaprit, imetit and impromidine induced histamine release from rat peritoneal mast cells" --- p.128 / Chapter 4.4.7. --- "Effects of anti-IgE, dimaprit and imetit on PGD2 production from rat peritoneal mast cells" --- p.128 / Chapter 4.4.8. --- "Effects of benzalkonium chloride (BAC) on dimaprit, imetit, compound 48/80 and anti-IgE induced histamine release from rat peritoneal mast cells" --- p.129 / Chapter 4.4.9. --- "Effects of pertussis toxin on dimaprit, imetit, compound 48/80and anti-IgE induced histamine release from rat peritoneal mast cells" --- p.129 / Chapter 4.4.10. --- "Effects of dimaprit, imetit, compound 48/80 and anti-IgE on the free intracellular calcium concentration of rat peritoneal mast cells" --- p.130 / Chapter 4.5. --- Discussion --- p.171 / Chapter 4.5.1. --- The possible existence of histamine receptors on rat peritoneal mast cells --- p.171 / Chapter 4.5.2. --- "Possible mechanism of action for the histamine releasing actions of dimaprit, imetit and impromidine on rat peritoneal mast cells" --- p.174 / Chapter 4.6. --- Conclusion --- p.181 / Chapter 4.7. --- Future studies --- p.182 / Chapter Chapter 5 --- Cyclo-oxygenase Inhibitors --- p.184 / Chapter 5.1. --- Introduction --- p.185 / Chapter 5.1.1. --- Cyclo-oxygenase isozymes --- p.185 / Chapter 5.1.2. --- Cyclo-oxygenase inhibitors and mast cells --- p.186 / Chapter 5.1.3. --- Experimental aims --- p.190 / Chapter 5.2. --- Materials and methods --- p.190 / Chapter 5.3. --- Results --- p.191 / Chapter 5.3.1. --- Effects of cyclo-oxygenase inhibitors on immunologically and non-immunologically induced histamine release from rat peritoneal mast cells - --- p.191 / Chapter 5.3.2. --- Effects of cyclo-oxygenase inhibitors on immunologically and non-immunologically induced PGD2 production from rat peritoneal mast cells --- p.192 / Chapter 5.3.3. --- Effects of cyclo-oxygenase inhibitors on immunologically and non-immunologically induced LTC4 production from rat peritoneal mast cells --- p.192 / Chapter 5.3.4. --- Effects of cyclo-oxygenase inhibitors on immunologically and non-immunologically induced 45Ca uptake by rat peritoneal mast cells --- p.193 / Chapter 5.4. --- Discussion --- p.221 / Chapter 5.5. --- Summary and Conclusion --- p.225 / Chapter Chapter 6 --- Immunosuppressive Drugs --- p.228 / Chapter 6.1. --- Introduction --- p.229 / Chapter 6.1.1. --- CsA and FK506 binding proteins --- p.230 / Chapter 6.1.2. --- Distribution of CyPA and FKBP12 --- p.231 / Chapter 6.1.3 --- Mechanism of immunosuppression --- p.232 / Chapter 6.1.4. --- The role of calcineurin in IL-2 promoter induction --- p.233 / Chapter 6.2. --- Immunosuppressive agents and mast cells --- p.234 / Chapter 6.2.1. --- Introduction --- p.234 / Chapter 6.2.2. --- CsA and FK506 inhibit mast cell cytokine production --- p.235 / Chapter 6.2.3. --- "CsA mediated inhibition of mediator release from, and calcium uptake by mast cells and basophils" --- p.236 / Chapter 6.2.4. --- Inhibition of mediator release from mast cells and basophils by FK506 --- p.239 / Chapter 6.2.5. --- Aim of this study --- p.240 / Chapter 6.2.6. --- Materials and methods --- p.241 / Chapter 6.3. --- Results --- p.241 / Chapter 6.3.1. --- Effects of CsA and FK506 on immunologically and non-immunologically induced histamine release from rat peritoneal mast cells --- p.241 / Chapter 6.3.2. --- Effects of CsA and FK506 on immunologically and non-immunologically induced PGD2 production from rat peritoneal mast cells --- p.242 / Chapter 6.3.3. --- Effects of CsA and FK506 on immunologically and non-immunologically induced 45Ca uptake by rat peritoneal mast cells --- p.243 / Chapter 6.4. --- Discussion --- p.254 / Chapter 6.4.1. --- "Effects of CsA on histamine release from, and 45Ca uptake by rat peritoneal mast cells, following immunological and non-immunological activation" --- p.254 / Chapter 6.4.2. --- Effects of CsA on PGD2 production from rat peritoneal mast cells --- p.256 / Chapter 6.4.3. --- "Effects of FK506 on histamine release from, and 45Ca uptake by rat peritoneal mast cells, following immunological and non-immunological activation" --- p.256 / Chapter 6.4.4. --- Effects of FK506 on immunological PGD2 production from rat peritoneal mast cells --- p.257 / Chapter 6.5. --- Summary --- p.257' / Chapter 6.6. --- Future work --- p.258 / References

Mast cells have been implicated in the pathogenesis of both atopic and non-atopic asthma. Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a mast cell-restricted guanine nucleotide exchange factor and diacylglycerol (DAG)/ phorbol ester receptor whose function has not been deduced. RT-PCR analysis of 40 asthmatic patients and 40 non-asthmatic controls demonstrated a higher hRasGRP4 mRNA expression in a subgroup of the asthmatics. A RasGRP4-defective variant of the human mast cell line HMC-1 was used to create stable clones expressing green fluorescent protein-labeled human RasGRP4 for monitoring the movement of this signaling protein inside mast cells before and after exposure to phorbol-12-myristate 13-acetate (PMA) and for evaluating the protein???s ability to control the development, phenotype, and function of mast cells. Transcript-profiling approaches revealed hRasGRP4 constitutively regulates the expression of numerous genes in the HMC-1 cell line. For example, expression of hRasGRP4 in HMC-1 cells substantially decreased GATA-1 levels without altering GATA-2 levels, suggesting that hRasGRP4 regulates mast cell commitment of multipotential progenitors in part by controlling the intracellular levels of at least one lineage-dependent transcription factor for hematopoietic cells. hRasGRP4 resided primarily in the cytosol before HMC-1 cells were stimulated with PMA. After exposure to PMA, hRasGRP4 translocated to the inner leaflet of the cell???s plasma membrane and then to perinuclear and Golgi compartments. Extracellular signal-regulated kinases 1 and 2 were activated during this translocation process, and the PMA-treated cells transiently increased their expression of the transcripts encoding the interleukin 13 receptor IL-13R??2 and numerous other proteins. The accumulated data in our mast cell model suggest hRasGRP4 translocates to various intracellular compartments via its DAG/PMA-binding domain to regulate those signaling pathways that allow mast cells to respond quickly to changes in their tissue microenvironments.

Nitric oxide (NO) is a short-lived free radical that plays a critical role in the regulation of cellular signalling. Mast cell (MC) derived NO and exogenous NO regulate MC activities including the inhibition of MC degranulation. At a molecular level the intermediate metabolites of NO modify protein structure and function through several mechanisms including protein tyrosine nitration. To begin to elucidate the molecular mechanisms underlying the effects of NO in MC, we investigated protein tyrosine nitration in human mast cell lines HMC-1 and LAD2 treated with the NO donor S-nitrosoglutathione (SNOG). Using two dimensional gel western blot analysis with an anti-nitrotyrosine antibody together with mass spectroscopy we identified aldolase A, an enzyme of the glycolytic pathway, as a target for tyrosine nitration in MC. S-nitrosoglutathione treatment also reduced the Vmax of aldolase in HMC-1 and LAD2. Nuclear magnetic resonance (NMR) analysis showed that despite these changes in activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was altered. Elevated levels of lactate and pyruvate suggested that SNOG treatment enhanced glycolysis. Reduced aldolase activity was associated with increased intracellular levels of its substrate, fructose-1,6-bisphosphate (FBP). Interestingly, FBP inhibited IgE-mediated MC degranulation and intracellular Ca2+ levels in LAD2 cells. In addition to aldolase, 15-hydroxy prostaglandin dehydrogenase (PGDH), a critical enzyme in the metabolism of PGE2, was identified as a prominent target for tyrosine nitration in LAD2 cells. Thus for the first time we report evidence of protein tyrosine nitration in human MC lines and identify aldolase A as a prominent target in HMC-1 and LAD2; and PGDH in LAD2 cells. The post translational nitration of aldolase A and PGDH may be important pathways that regulate MC phenotype and function. / Experimental Medicine

Mast cells (MCs), normally classified into connective tissue MCs and mucosal MCs, are highly granulated cells found in the interface between the interior and the exterior environment of our body, e.g. skin, airways and gastro-intestinal tract. They react to bacteria, parasites, viruses, and allergens by degranulation and release of premade and newly synthesized inflammatory mediators. The MC-proteases (tryptases, chymases and carboxypeptidase A), histamine and serglycin (SG) proteoglycans are premade mediators. Among these, SG is also expressed in a variety of other immune and non-immune cells. Heparin and chondroitin sulphate glycosaminoglycan chains confer highly negative charge to SG, by which MC-proteases are retained in secretory granules. Deletion of SG cause impaired packing and storage of most MC-proteases. During challenge with Toxoplasma gondii the SG-deficient mice showed significant lower inflammatory cytokine levels in comparison to wild-type mice. Results were consistently similar in vitro, bringing forward the importance of SG in inflammatory cytokine and innate immune responses towards T. gondii. Infection with Trichinella spiralis in SG-/- mice caused increased intestinal enteropathy, a tendency of delayed worm expulsion and increased larval burden in the muscle tissue as compared to wild-type animals. An altered TH2 cytokine response was also observed, and all these effects were not repaired by wild-type MC reconstitution of the SG-/- mice. Altogether, our results suggest that SG is important for tissue homeostasis, and that SG expressing cells seem capable of switching from a SG-dependent storage mode to a SG-independent secretory mode upon infection. The chymase (MCPT4) expressed by connective tissue MC has been implicated to have a protective role during infection and in limiting inflammation. We explored a protective role by inducing T. gondii infection in the Mcpt4-null mice, and found MCPT4-mediated recruitment of neutrophils and eosinophils via control of cytokine signaling. Endogenous proteins “alarmins” released by dead cells can trigger tissue and cell damage. We conclusively show that chymase efficiently degrades Hsp70 both in vitro and in vivo and that the degradation of other alarmins, e.g. HMGB1, biglycan and IL-33 may also depend on chymase.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 水野紘樹氏の博士論文として提出された