Mast cell progenitors originate from the bone marrow and migrate to the lungs via the blood. During maturation, these cells acquire granules that contain a potent array of bronchoconstrictive mediators. The number of pulmonary mast cells is augmented in asthmatic patients and in mice with allergic airway inflammation, possibly contributing to airway hyperreactivity. An increase in mast cells is likely due to an increased recruitment of committed mast cell progenitors from the blood. However, until now a committed mast cell progenitor population has not been found in adult peripheral blood. We isolated Lin- c-kithi ST2+ integrin β7hi CD16/32hi progenitors from murine blood and showed that these cells were committed to the mast cell lineage. Based on the expression of FcεRI, these cells were less mature in Th1-prone C57BL/6 mice than in Th2-prone BALB/c mice. Asthma is associated with elevated levels of IgE. Upon exposure to allergens, IgE immune complexes are formed. In a mouse model of allergic airway inflammation, we showed that intranasal administration of IgE immune complexes to antigen-sensitized mice resulted in an increased number of mast cell progenitors compared with antigen administration alone. The increase in mast cell progenitors was independent of the low-affinity IgE receptor CD23. Rather, signaling through the common FcRγ-chain was required to enhance the number of lung mast cell progenitors. Signaling through FcεRI was likely responsible for the increase. However a role for FcγRIV could not be excluded. CD11c+ cells, such as dendritic cells, are important for antigen sensitization. In a mouse model of allergic airway inflammation, these cells are also important for the development of airway hyperreactivity, eosinophilia and Th2 cytokine production in response to antigen challenge. We showed that CD11c+ cells are critical for the recruitment of lung mast cell progenitors and the subsequent increase in mast cells. These CD11c+ cells were needed for the upregulation of endothelial vascular cell adhesion molecule-1 (VCAM-1), which is a prerequisite for the antigen-induced recruitment of lung mast cell progenitors.

Yip Kwok Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 231-260). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Acknowledgements --- p.vi / Publications --- p.vii / Abbreviations --- p.viii / Contents --- p.xi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Mast cells --- p.2 / Chapter 1.2. --- "Mast cell origin, growth and development" --- p.2 / Chapter 1.2.1. --- Stem cell factor --- p.4 / Chapter 1.2.2. --- Interleukins --- p.6 / Chapter 1.3. --- Mast ceII heterogeneity --- p.7 / Chapter 1.4. --- Mast ceII mediators --- p.9 / Chapter 1.4.1. --- Pre-Synthesized mediators --- p.9 / Chapter 1.4.1.1. --- Histamine --- p.10 / Chapter 1.4.1.2. --- Protease --- p.11 / Chapter 1.4.2. --- Newly synthesized mediators --- p.13 / Chapter 1.4.2.1. --- Prostanoid --- p.14 / Chapter 1.4.2.2. --- Cysteinyl Leukotriene --- p.15 / Chapter 1.4.3. --- Mast cell-derived cytokines and growth factors --- p.16 / Chapter 1.5. --- Mast cell activation --- p.17 / Chapter 1.5.1. --- FceRI-dependent mast cell activation --- p.18 / Chapter 1.5.1.1. --- FceRI and IgE aggregation --- p.19 / Chapter 1.5.1.2. --- Protein-tyrosine kinase activation --- p.21 / Chapter 1.5.1.3. --- Phospholipase activation and calcium ion mobilization --- p.22 / Chapter 1.5.1.4. --- GTPase and MAPK activation --- p.24 / Chapter 1.5.2. --- Non-immunogical mast cell activation --- p.26 / Chapter 1.6. --- Roles of mast cell in inflammatory disease --- p.27 / Chapter 1.7. --- Nitric oxide --- p.28 / Chapter 1.8. --- Nitric oxide synthase --- p.30 / Chapter 1.9. --- Nitric oxide signaling in cellular level --- p.31 / Chapter 1.9.1. --- Direct effects of NO --- p.32 / Chapter 1.9.2. --- Indirect effects of NO --- p.34 / Chapter 1.10. --- Mast cell and nitric oxide --- p.35 / Chapter 1.11. --- Aim of Study --- p.37 / Chapter 2. --- Materials and Methods --- p.43 / Chapter 2.1. --- Material --- p.44 / Chapter 2.1.1. --- Human buffy coat for mast cell culture --- p.44 / Chapter 2.1.2. --- Materials for cell isolation and cell counting --- p.44 / Chapter 2.1.3. --- Materials for mast cell culture --- p.45 / Chapter 2.1.4. --- Material for buffers --- p.45 / Chapter 2.1.5. --- Materials for cytospin and May-Griinwald-Giemsa staining --- p.46 / Chapter 2.1.6. --- Materials for immunocytochemical staining --- p.46 / Chapter 2.1.7. --- Mast cell secretagogues --- p.47 / Chapter 2.1.8. --- Nitric oxide donors --- p.47 / Chapter 2.1.9. --- Soluble Guanylyl Cyclase activators and cGMP analogues --- p.47 / Chapter 2.1.10. --- Drugs involved in NO-sGC-cGMP pathway --- p.48 / Chapter 2.1.11. --- Materials for histamine assay --- p.48 / Chapter 2.1.12. --- Materials for Enzyme Immunosorbent Assay (EIA) --- p.49 / Chapter 2.1.13. --- Pro-inflammatory cytokines --- p.49 / Chapter 2.1.14. --- Materials for RNA extraction and RT-PCR --- p.49 / Chapter 2.1.15. --- Materials for Immunofluorescence staining --- p.50 / Chapter 2.1.16. --- Anti-asthmatic compounds --- p.51 / Chapter 2.1.17. --- Buffer and stock solution --- p.51 / Chapter 2.1.17.1. --- Buffer ingredients --- p.51 / Chapter 2.1.17.2. --- Stock solution --- p.52 / Chapter 2.2. --- Methods --- p.52 / Chapter 2.2.1. --- CD34+ cell isolation from human buffy coat --- p.52 / Chapter 2.2.2. --- CD34+ cell culture --- p.53 / Chapter 2.2.3. --- Human mast cell line (HMC-1 cells) culture --- p.54 / Chapter 2.2.4. --- Mast cell heterogeneity identification --- p.54 / Chapter 2.2.4.1. --- Cell smear preparation --- p.54 / Chapter 2.2.4.2. --- May-Gruwald-Giemsa staining --- p.55 / Chapter 2.2.4.3. --- Immunocytochemical staining --- p.55 / Chapter 2.2.5. --- Histamine release and measurement --- p.56 / Chapter 2.2.5.1. --- Histamine release --- p.56 / Chapter 2.2.5.2. --- Spectroflurometric determination of histamine content --- p.57 / Chapter 2.2.5.3. --- Calculation of histamine level --- p.57 / Chapter 2.2.6. --- Prostaglandin D2 (PGD2) measurement --- p.58 / Chapter 2.2.6.1. --- PGD2 production --- p.58 / Chapter 2.2.6.2. --- EIA methods for PGD2 measurement --- p.58 / Chapter 2.2.6.3. --- Calculation of PGD2 concentration --- p.59 / Chapter 2.2.7. --- Cysteinyl Leukotrienes (Cys-LTs) measurement --- p.59 / Chapter 2.2.7.1. --- Cys-LTs production --- p.59 / Chapter 2.2.7.2. --- EIA methods for Cys-LTs measurement --- p.60 / Chapter 2.2.7.3. --- Calculation of Cys-LTs concentration --- p.60 / Chapter 2.2.8. --- Tumor necrosis factor-alpha (TNF-α) measurement --- p.61 / Chapter 2.2.8.1. --- TNF-α production --- p.61 / Chapter 2.2.8.2. --- EIA methods for TNF-α measurement --- p.61 / Chapter 2.2.8.3. --- Calculation of TNF-α concentration --- p.62 / Chapter 2.2.9. --- Interleukin-8 (IL-8) measurement --- p.62 / Chapter 2.2.9.1. --- IL-8 production --- p.62 / Chapter 2.2.9.2. --- ELISA for IL-8 measurement --- p.62 / Chapter 2.2.9.3. --- Calculation of IL-8 concentration --- p.63 / Chapter 2.2.10. --- Data presentation --- p.63 / Chapter 2.2.11. --- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.64 / Chapter 2.2.11.1. --- RNA extraction --- p.64 / Chapter 2.2.11.2. --- Reverse Transcriptase reaction for cDNA synthesis --- p.65 / Chapter 2.2.11.3. --- Polymerase Chain Reaction --- p.66 / Chapter 2.2.11.4. --- Agarose Gel Electrophoresis --- p.67 / Chapter 2.2.11.5. --- Data representation in RT-PCR experiment --- p.67 / Chapter 2.2.12. --- Immunofluorescence staining --- p.67 / Chapter 2.2.12.1. --- Cell smear preparation --- p.68 / Chapter 2.2.12.2. --- Immunofluorescence staining --- p.68 / Chapter 2.3. --- Statistical analysis --- p.69 / Chapter 3. --- Effect of Nitric Oxide Donors on Mast Cell Activation --- p.70 / Chapter 3.1. --- Introduction --- p.71 / Chapter 3.1.1. --- Mechanisms of NO release from NO donors --- p.71 / Chapter 3.1.2. --- Experimental aims --- p.77 / Chapter 3.2. --- Materials and methods --- p.77 / Chapter 3.3. --- Results --- p.78 / Chapter 3.3.1. --- Development of mast cells from buffy coat --- p.78 / Chapter 3.3.2. --- Morphological features of cultured mast cells --- p.78 / Chapter 3.3.3. --- Phenotype of cultured mast cells --- p.79 / Chapter 3.3.4. --- Effects of NO donors on immunologically stimulated mediators release --- p.79 / Chapter 3.3.4.1. --- SIN-1 and NOR-3 --- p.80 / Chapter 3.3.4.2. --- SNP and SNAP --- p.80 / Chapter 3.3.4.3. --- Diazeniumdiolates (NONOates) --- p.80 / Chapter 3.3.5. --- Effects of NO scavenger on NO donors mediated inhibition of immunologically stimulated mediators release --- p.82 / Chapter 3.3.6. --- Discussion --- p.83 / Chapter 4. --- Interaction between NO donors and pharmacological agentsin modulating mast cell activation --- p.123 / Chapter 4.1. --- Introduction --- p.124 / Chapter 4.1.1. --- Modulators of NO-sGC-cGMP pathway --- p.125 / Chapter 4.1.2. --- Anti-asthmatic compounds --- p.128 / Chapter 4.1.3. --- Experimental aims --- p.130 / Chapter 4.2. --- Materials and methods --- p.131 / Chapter 4.3. --- Results --- p.132 / Chapter 4.3.1. --- Effect of sGC activators on immunologically stimulated histamine release and the inhibitory action of DEA/NO --- p.132 / Chapter 4.3.2. --- Effect of cGMP analog on immunologically stimulated histamine release --- p.133 / Chapter 4.3.3. --- "Effects of the sGC inhibitor, ODQ, on DEA/NO induced inhibition on immunologically stimulated mediators release" --- p.134 / Chapter 4.3.4. --- Effects of anti-oxidants on the actions of NO donors in modulating immunologically stimulated mediators release --- p.134 / Chapter 4.3.5. --- The effects of NO donors on salbutamol mediated inhibition of immunologically stimulated histamine release from human mast cells --- p.135 / Chapter 4.3.6. --- The effects of NO donors on theophylline mediated inhibition of immunologically stimulated histamine release from human mast cells --- p.136 / Chapter 4.3.7. --- The effects of NO donors and DSCG on immunologically stimulated histamine release from human mast cells --- p.137 / Chapter 4.4. --- Discussion --- p.137 / Chapter 4.5. --- Further studies --- p.150 / Chapter 5. --- Human mast cells as a source of nitric oxide --- p.178 / Chapter 5.1. --- Introduction --- p.179 / Chapter 5.1.1. --- Nitric oxide synthases expression in mast cell --- p.180 / Chapter 5.1.2. --- Modulation of NOS expression --- p.182 / Chapter 5.1.3. --- Experimental aims --- p.186 / Chapter 5.2. --- Materials and methods --- p.186 / Chapter 5.3. --- Results --- p.187 / Chapter 5.3.1. --- NOS expression in human mast cell-line HMC-1 --- p.187 / Chapter 5.3.1.1. --- Basal --- p.187 / Chapter 5.3.1.2. --- Effect of cytokines --- p.188 / Chapter 5.3.2. --- NOS expression in cultured CD34+ derived human mast cells --- p.189 / Chapter 5.3.2.1. --- Basal --- p.189 / Chapter 5.3.2.2. --- Effect of cytokines --- p.189 / Chapter 5.3.2.3. --- Effect ofIgE and anti-IgE --- p.190 / Chapter 5.4. --- Discussion --- p.191 / Chapter 5.5. --- Further studies --- p.200 / Chapter 6. --- Conclusion --- p.218 / Chapter 7. --- References --- p.230

Wong Lai Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 162-173). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgements --- p.vi / Publications --- p.vii / Abbreviations --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Historical Background --- p.2 / Chapter 1.2. --- Heterogeneity of mast cells --- p.3 / Chapter 1.3. --- Mast cell mediators --- p.5 / Chapter 1.3.1. --- Performed and granule associated mediators --- p.5 / Chapter 1.3.2. --- Newly synthesized mediators --- p.8 / Chapter 1.3.3. --- Cytokines --- p.10 / Chapter 1.4. --- Mast cell activation --- p.10 / Chapter 1.4.1. --- Aggregation of IgE Receptors (FcεRI) --- p.10 / Chapter 1.4.2. --- Activation of Phospholipase C --- p.11 / Chapter 1.4.3. --- Activation of Adenylate cyclase --- p.13 / Chapter 1.5. --- Adenosine --- p.14 / Chapter 1.5.1. --- Adenosine receptors --- p.14 / Chapter 1.5.2. --- Selective agonists and antagonists --- p.17 / Chapter 1.5.3. --- Physiological and pathological roles of adenosine --- p.20 / Chapter 1.6. --- Role of adenosine receptors in mast cell activation --- p.21 / Chapter 1.7. --- Aims of the study --- p.23 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1. --- Materials --- p.31 / Chapter 2.1.1. --- Mast cells secretagogues --- p.31 / Chapter 2.1.2. --- Anti-allergic compounds --- p.31 / Chapter 2.1.3. --- Adenosine receptor agonists and antagonists --- p.31 / Chapter 2.1.4. --- Materials for buffers --- p.32 / Chapter 2.1.5. --- Materials for rat sensitization --- p.32 / Chapter 2.1.6. --- Materials for histamine assay --- p.33 / Chapter 2.1.7. --- Miscellaneous --- p.33 / Chapter 2.2. --- Buffers and stock solutions --- p.34 / Chapter 2.2.1 --- Buffer ingredients --- p.34 / Chapter 2.2.2 --- Stock solutions --- p.34 / Chapter 2.3. --- Source of mast cells --- p.35 / Chapter 2.3.1. --- Animals --- p.35 / Chapter 2.3.2. --- Sensitization of animals --- p.35 / Chapter 2.3.3. --- Isolation of rat peritoneal mast cells --- p.35 / Chapter 2.3.4. --- Mast cells purification --- p.36 / Chapter 2.3.5. --- Cell counting --- p.36 / Chapter 2.4. --- General protocol for histamine release --- p.37 / Chapter 2.4.1. --- Histamine assay --- p.37 / Chapter 2.4.2. --- Antagonist studies --- p.38 / Chapter 2.4.3. --- Determination of histamine contents --- p.38 / Chapter 2.4.4. --- Calculation of histamine levels --- p.39 / Chapter 2.5. --- Statistical analysis --- p.40 / Chapter Chapter 3 --- "Effects of adenosine, adenosine deaminase and adenosine receptor agonists on mast cell activation" --- p.42 / Chapter 3.1. --- Introduction --- p.43 / Chapter 3.2. --- Materials and methods --- p.44 / Chapter 3.3. --- Results --- p.45 / Chapter 3.3.1. --- Effects of adenosine on anti-IgE induced histamine release in HEPES buffer --- p.45 / Chapter 3.3.2. --- Effects of NECA on anti-IgE induced histamine release in HEPES buffer --- p.46 / Chapter 3.3.3. --- Effects of CCPA on anti-IgE induced histamine release in HEPES buffer --- p.47 / Chapter 3.3.4. --- Effects of CPA on anti-IgE induced histamine release in HEPES buffer --- p.47 / Chapter 3.3.5. --- Effects of CGS21680 on anti-IgE induced histamine release in HEPES buffer --- p.48 / Chapter 3.3.6. --- Effects of Cl-MECA on anti-IgE induced histamine release in HEPES buffer --- p.49 / Chapter 3.3.7. --- Effects of adenosine deaminase on anti-IgE induced histamine release from rat peritoneal mast cells --- p.50 / Chapter 3.3.8. --- Effects of NECA on anti-IgE induced histamine release with and without adenosine deaminase --- p.50 / Chapter 3.3.9. --- Effects of Cl-MECA on anti-IgE induced histamine release with and without adenosine deaminase --- p.53 / Chapter 3.3.10. --- Effects of CV1808 on anti-IgE induced histamine release in HEPES buffer --- p.55 / Chapter 3.4. --- Discussion --- p.76 / Chapter 3.5. --- Conclusion --- p.83 / Chapter Chapter 4 --- Effects of adenosine receptor antagonists on mast cell activation --- p.88 / Chapter 4.1. --- Introduction --- p.89 / Chapter 4.2. --- Materials and methods --- p.90 / Chapter 4.3. --- Results --- p.91 / Chapter 4.3.1. --- Effects of A1 receptor antagonist DPCPX on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.91 / Chapter 4.3.2. --- Effects of A2A receptor antagonist ZM241385 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.91 / Chapter 4.3.3. --- Effects of A2B receptor antagonist MRS 1706 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.92 / Chapter 4.3.4. --- Effects of A3 receptor antagonist VUF5574 on modulations of anti-IgE induced histamine release by adenosine receptor agonists --- p.93 / Chapter 4.3.5. --- Further characterization of adenosine mediated potentiation of anti-IgE histamine release using VUF5574 and ZM241385 --- p.93 / Chapter 4.3.6. --- Effects of theophylline on anti-IgE induced percentage potentiation in HEPES buffer --- p.95 / Chapter 4.4. --- Discussion --- p.130 / Chapter 4.5. --- Conclusion --- p.135 / Chapter Chapter 5 --- Further characterization of the effects of adenosine on mast cells --- p.138 / Chapter 5.1. --- Introduction --- p.139 / Chapter 5.2. --- Materials and methods --- p.141 / Chapter 5.3. --- Results --- p.142 / Chapter 5.3.1. --- Effects of anti-IgE induced histamine release in calcium free and HEPES buffers --- p.142 / Chapter 5.3.2. --- Effects of adenosine on anti-IgE induced histamine release in calcium free buffer --- p.143 / Chapter 5.3.3. --- Effects of adenosine deaminase on compound48/80 induced histamine release from rat peritoneal mast cells --- p.143 / Chapter 5.3.4. --- Effects of adenosine on compound 48/80 induced histamine release in HEPES buffer --- p.144 / Chapter 5.3.5. --- Effects of adenosine deaminase on A23187 induced histamine release from rat peritoneal mast cells --- p.144 / Chapter 5.3.6. --- Effects of adenosine on calcium ionophore A23187 induced histamine release in HEPES buffer --- p.145 / Chapter 5.3.7. --- Effects of adenosine receptor antagonists on inosine mediated enhancement of anti-IgE induced histamine release --- p.145 / Chapter 5.4. --- Discussion --- p.157 / Chapter 5.5. --- Conclusion --- p.160 / References --- p.162

Mast cells are pivotal effector cells in the pathogenesis of allergic and inflammatory diseases. Activation of FcepsilonRI in mast cells by antigen initiates a complex series of biochemical events leading to the release and synthesis of myriads of chemical mediators and cytokines. Adenosine is an endogenous nucleoside formed from cleavage of AMP by the enzyme 5'-nucleotidase. It exerts modulating effects in a large number of cellular systems by acting through four distinct subtypes of adenosine receptors (A1, A 2A, A2B and A3) which belong to the G-protein-coupled receptor (GPCR) family. Increasing evidence have been provided to show that adenosine plays a role in the pathophysiology of asthma through a mast cell dependent mechanism. / Pharmacological studies using specific adenosine agonists and antagonists revealed that A1 receptor was responsible for the potentiating effect of adenosine with the involvement of the pertussis toxin-sensitive Galphai-protein. Conversely, inhibition of HCMC activation was mediated by A2B receptor and was accompanied by the elevation of cAMP level suggesting the participation of Galphas-protein. / Taken together, the current studies explored the dual effect of adenosine on human mast cells activation which enhanced our understanding of adenosine receptor biology. The effectiveness of adenosine in modulating the important mast cell activation pathways definitely facilitates the rational exploitation of these receptors as therapeutic targets that could be converted into clinical benefits for asthmatic patients. / To better characterize the effect of adenosine on human mast cell under asthmatic environment, we incubated HCMC under different inflammatory condition found in asthmatic, including toll-like receptor (TLR) ligands and inflammatory cytokines. Functional studies on mediator release from HCMC indicated that out of all tested substances, Peptidoglycan (PGN) pre-incubation enhanced the IL-8 synthesis from HCMC in response to low concentration of adenosine (10-9--10-7 M). / We also investigated the action of adenosine on key signal transduction pathways involved in mast cells activation. Study on intracellular calcium concentration ([Ca2+]i) revealed that low concentration of adenosine (10-8 M) through activation of PI3Kgamma significantly enhanced Ca2+ influx. In contrast, high concentration of adenosine at 10-4 M substantially inhibited [Ca2+] i in response to anti-IgE. Furthermore, investigation on intracellular signaling molecules provided evidence that adenosine at concentrations over 10-6 M does-dependently inhibited the immunoglobulin (IgE)-dependent activation of ERK, JNK or NF-kappaB pathways, whereas enhancement of IkappaBalpha was found on low concentration of adenosine. The above observation help to justify the dual action of adenosine on anti-IgE-induced mediators release from HCMC. Our investigation further suggested that adenosine may inhibit HCMC activation through a novel cAMP-dependent, but PKA- and EPAC-independent, signaling pathway. / We generated human cultured mast cells (HCMC) from human buffy coat and confirmed the expression of all adenosine receptor subtypes in them. We showed that adenosine alone did not induce HCMC degranulation and cytokine release. However, adenosine and the non-selective agonist, 5'-N-Ethylcarbox-amidoadenosine (NECA), produced a biphasic response on anti-IgE induced mast cell activation. An enhancement of HCMC activation was observed with low concentrations of adenosine and NECA (10-9--10-7 M), whereas a predominant inhibitory action was observed at concentrations higher than 10-6 M. / Yip, Kwok Ho. / Adviser: Alaster H.Y. Lau. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 237-263). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lee, Hoi Ying. / "September 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 171-189). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgement --- p.v / Publications --- p.vi / Abbreviations --- p.vii / Table of Content --- p.x / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Human mast cells and its activation --- p.1 / Chapter 1.2 --- Role of mast cells in inflammation --- p.2 / Chapter 1.3 --- Mast cell heterogeneity --- p.5 / Chapter 1.4 --- Interaction of bone and immune system --- p.1 / Chapter 1.5 --- Introduction of bone system --- p.8 / Chapter 1.6 --- Bone remodeling --- p.9 / Chapter 1.7 --- Regulation of bone remodeling --- p.10 / Chapter 1.8 --- Introduction of Osteoporosis --- p.12 / Chapter 1.9 --- Pathophysiology of osteoporosis --- p.13 / Chapter 1.10 --- Pharmacological interventions in osteoporosis --- p.14 / Chapter 1.11 --- Involvement of mast cells in bone metabolism --- p.18 / Chapter 1.12 --- Aim of study --- p.20 / Chapter 2 --- Materials and Methods --- p.27 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Human mast cells culture --- p.34 / Chapter 2.2.2 --- Human mast cells characterization --- p.35 / Chapter 2.2.3 --- Histamine release assay --- p.36 / Chapter 2.2.4 --- Immunofluorescence staining of estrogen receptors --- p.37 / Chapter 2.2.5 --- Reverse Transcriptase Polymerase Chain Reaction --- p.37 / Chapter 2.2.6 --- TNF measurement --- p.38 / Chapter 2.2.7 --- Calcium mobilization studies of mast cells --- p.38 / Chapter 2.2.8 --- Statistical analysis --- p.39 / Chapter 3 --- Effects of estrogen and selective estrogen receptor modulators (SERMs) on mediators release from human mast cells --- p.41 / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Materials and methods --- p.50 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Characterization of human mast cells --- p.51 / Chapter 3.3.2 --- Effect of estrogen on mediator release from human mast cells --- p.52 / Chapter 3.3.2.1 --- Basal histamine release after treatment of estrogen --- p.52 / Chapter 3.3.2.2 --- Histamine release induced by immunological stimulus --- p.52 / Chapter 3.3.2.3 --- Histamine release induced by chemical secretagogues --- p.54 / Chapter 3.3.3 --- Effect of selective estrogen receptor modulators (SERMs) on mast cell activity --- p.54 / Chapter 3.3.3.1 --- Basal histamine release after SERMs treatment --- p.54 / Chapter 3.3.3.2 --- Histamine release induced by immunological stimulus --- p.55 / Chapter 3.3.3.3 --- Histamine release induced by chemical secretagogues --- p.57 / Chapter 3.3.4 --- Effect of estradiol on TNF-α release from human mast cells --- p.57 / Chapter 3.3.5 --- Effect of SERMs on TNE-α release from human mast cells --- p.58 / Chapter 3.3.6 --- Expression of estrogen receptors on human mast cells --- p.59 / Chapter 3.3.6.1 --- Expression of estrogen receptor after treatment of estradiol --- p.59 / Chapter 3.3.7 --- Expression of various bone remodeling molecules on human mast cells --- p.60 / Chapter 3.3.7.1 --- Expression of bone remodeling molecule after treatment of estradiol --- p.61 / Chapter 3.4 --- Discussion --- p.63 / Chapter 4 --- Effects of anti-osteoporosis Chinese herbal medicines on activity of human mast cells --- p.98 / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.2 --- Materials and methods --- p.103 / Chapter 4.3 --- Results --- p.104 / Chapter 4.3.1 --- Effect of the anti-osteoporosis Chinese herbal formulation ELP on histamine release from human mast cells --- p.104 / Chapter 4.3.1.1 --- Histamine release induced by immunological stimulus --- p.104 / Chapter 4.3.1.2 --- Histamine release induced by chemical secretagogues --- p.105 / Chapter 4.3.2 --- Effect of Herba Epimedii (HEP) on histamine release from human mast cells --- p.105 / Chapter 4.3.2.1 --- Histamine release induced by immunological stimulus --- p.106 / Chapter 4.3.2.2 --- Histamine release induced by chemical secretagogues --- p.106 / Chapter 4.3.3 --- Effect of Fructus Ligustri Lucidi (FLL) on histamine release from human mast cells --- p.107 / Chapter 4.3.3.1 --- Histamine release induced by immunological stimulus --- p.107 / Chapter 4.3.3.2 --- Histamine release induced by chemical secretagogues --- p.107 / Chapter 4.3.4 --- Effect of Fructus Psoraleae (FP) on histamine release from human mast cells --- p.108 / Chapter 4.3.4.1 --- Histamine release induced by immunological stimulus --- p.108 / Chapter 4.3.4.2 --- Histamine release induced by chemical secretagogues --- p.109 / Chapter 4.3.5 --- Effect of various partitions from solvent extraction of HEP on histamine release from human mast cells --- p.109 / Chapter 4.3.5.1 --- Histamine release induced by immunological stimulus --- p.110 / Chapter 4.3.5.2 --- Histamine release induced by chemical secretagogue --- p.111 / Chapter 4.3.6 --- Effect of various partitions from solvent extraction of FLL on histamine release from human mast cells --- p.112 / Chapter 4.3.6.1 --- Histamine release induced by immunological stimulus --- p.113 / Chapter 4.3.6.2 --- Histamine release induced by chemical secretagogue --- p.114 / Chapter 4.3.7 --- Effect of ELP and its herbal constituents on the production of cytokine from human mast cells --- p.115 / Chapter 4.3.8 --- Modulation in calcium mobilization in activated human mast cell by ELP and its herbal constituents --- p.117 / Chapter 4.4 --- Discussion --- p.119 / Chapter 5 --- General discussion --- p.163 / Reference --- p.171

In autoimmune diseases such as rheumatoid arthritis (RA), self-reactive antibodies are present at high levels, which contributes to disease pathogenesis. The antibodies mediate their effect predominantly by binding to Fc gamma receptors (FcγR) on various leukocytes, such as monocytes, macrophages and mast cells, where FcγR ligation leads to cell activation. In this thesis the role of FcγR in RA was investigated. We could, for the first time, demonstrate an increased expression of the inhibitory FcγRIIb in RA synovial tissue, while this receptor as well as FcγRI were almost absent in healthy synovial tissue. The enhanced FcγRI expression in RA synovia was reduced by one intraarticular injection of glucocorticoids, indicating that FcγRI participates in the joint inflammation. Interestingly, RA patients with an ongoing joint inflammation exhibited blood monocytes with immune compromised features, such as decreased FcγR binding of IgG1-IC and reduced TNF production. These effects were associated with high levels of auto-antibodies in the patients, implying that the monocyte FcγR are saturated with IgG. In order to investigate whether soluble FcγR could be used as a therapy in arthritis, we injected human soluble FcγR into mice with collagen-induced arthritis (CIA). The soluble FcγR reduced the levels of pathogenic IgG anti-collagen type II (CII) antibodies, arthritis severity and pro-inflammatory cytokines. Thus, suggesting that soluble FcγR may represent a novel therapeutic agent in RA. We also studied the disease-aggravating role of mast cells in arthritis by investigating mouse mast cell protease-4 (mMCP-4) in CIA. We found that mMCP-4 deficient mice displayed a reduced IgG anti-CII response and reduced arthritis severity. This indicates a role for mMCP-4 in adaptive immunity. In conclusion, these data demonstrate that IgG occupancy of FcγR and mast cell secretion of mMCP-4 play vital roles in the development of autoimmune arthritis.

<p>Mast cells, best known for their pathologic role in allergy, have recently been shown to have key roles in the initiation of adaptive immune responses. These cells are located throughout the body just beneath barriers separating host from environment, possess multiple pathogen recognition systems, and store large quantities of fully active inflammatory mediators. These key features make them uniquely situated to act as sentinels of immunity, releasing the very earliest alarm signals when a pathogen is present. As a testament to the importance of these cells, mast cell-deficient mice have suboptimal immune responses, and mast cell activators can act as potent adjuvants for experimental immunizations. Specifically, mast cells have been shown to enhance the number of naive lymphocytes in infection site-draining lymph nodes, and to encourage the migration of dendritic cells to responding lymph nodes.</p><p>Although infections usually occur at peripheral sites, adaptive immune responses are initiated in distant lymph nodes. Despite the distance, signals from the site of infection result in dramatic, rapid reorganization of the node, including massive recruitment of naive lymphocytes from the circulation and extensive vascular restructuring to accommodate the increase in size. How such signals reach the lymph node is not well understood.</p><p>When mast cells degranulate, in addition to releasing soluble mediators such as histamine, they expel large, stable, insoluble particles composed primarily of heparin and cationic proteins. The work presented herein demonstrates that these particles act as extracellular chaperones for inflammatory mediators, protecting them from dilution into the interstitial space, degradation, and interaction with non-target host cells and molecules. The data show clearly that mast cells release such particles, that they are highly stable, that they contain tumor necrosis factor (a critically important immunomodulator), and that they can traffic from peripheral sites to draining lymph nodes via lymphatic vessels. Furthermore, extensive biochemical characterization of purified mast cell-derived particles was performed. Finally, evidence is presented that such particles can elicit lymph node enlargement, an infection-associated phenomenon that favors the development of adaptive immunity, by delivering peripheral TNF to draining lymph nodes. </p><p>This signaling concept, that particles may chaperone signals between distant sites, also has important implications for adjuvant design. The evidence presented here shows that encapsulation of TNF into synthetic particles similar to mast cell-derived particles greatly enhances its potency for eliciting lymph node enlargement, an indication that adaptive immunity may be improved. This delivery system should ensure that more adjuvant arrives in the draining lymph node intact, where it would lead to changes favorable to the development of the immune response. Such a system would also facilitate the delivery of multi-component adjuvants that would act synergistically at the level of the lymph node when gradually released from microparticle carriers. An additional advantage of microparticle encapsulation is that vaccine formulations of this type may require much lower doses of expensive antigen and adjuvants.</p><p>The delivery of inflammatory mediators to lymph nodes during immune responses may be an important general feature of host defense. Although the action of mediators of peripheral origin on draining lymph nodes has been described before, this is the first demonstration of a specific adaptation to deliver such mediators. Not only is the characterization of mast cell-derived particles important to basic immunology, but mimicking this adaptation may also lead to improved therapeutics.</p> / Dissertation

Measurements of the neutron emission, resulting from nuclear fusion reactions between the hydrogen isotopes deuterium and tritium, can provide a wealth of information on the confinement properties of fusion plasmas and how these are affected by Magneto-Hydro-Dynamic (MHD) instabilities. This thesis describes work aimed to develop neutron measurement techniques for nuclear fusion plasma experiments, specifically regarding the performance and design of collimated neutron flux monitors (neutron cameras) for the Mega Ampere Spherical Tokamak, MAST, and for MAST Upgrade. The first part of the thesis focuses on the characterization of a prototype neutron camera installed at MAST and provides an account of the very first measurements of the neutron emissivity along its collimated fields of view. It is shown that the camera has sufficient temporal and spatial resolution to measure the effect of MHD instabilities on the neutron emissivity. The neutron camera fulfils the requirement on the measurements of the neutron count rate profile with less than 10 % statistical uncertainty in a time resolution of 1 ms. The instrument's more rudimentary capabilities to provide information on the neutron energy distribution are also presented and discussed. The encouraging results obtained with the prototype neutron camera show the potential of a collimated neutron flux monitor at MAST and suggest that an upgraded instrument for MAST Upgrade will provide crucial information on fast ions behavior and other relevant physics issues. The design of such an upgraded instrument for MAST Upgrade is discussed in the second part of the thesis. Two design options are explored, one consisting of two collimator arrays in the horizontal direction, another more traditional design with lines-of-sight in the poloidal cross section plane. On the basis of the experience gained with the prototype neutron camera and on the exploratory design and estimated performance for the upgraded camera presented here, a conceptual design of a neutron camera upgrade is proposed.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine, Department of Medicine. Title from pdf file main screen (viewed on November 1, 2009). Includes bibliographical references.