Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type I (NF1) is a common genetic disease that affects over 200,000 patients in North America, Europe, and Japan. Individuals with NF1 display a wide variety of pathologies; importantly, 15-40% of NF1 patients are affected by plexiform neurofibromas. Neurofibromas are complex tumors consisting of tumorgenic Schwann cells surrounded by endothelial cells, fibroblasts, and inflammatory mast cells. These peripheral nerve sheath tumors contribute significantly to the morbidity and mortality associated with NF1. Currently, no medical therapies exist for treating neurofibromas. Recent evidence indicates that the hematopoietic tumor microenvironment carries out a crucial function in the formation of plexiform neurofibromas. Neurofibromatosis is the result of mutations at the NF1 locus, which encodes the GTPase activating protein neurofibromin. Neurofibromin is a negative regulator of the proto-oncogene Ras. Ras hyperactivation is the molecular basis of NF1 associated phenotypes, and it has been demonstrated that restoration of Ras signaling to wild type levels can correct NF1 associated phenotypes in vitro and in vivo. In keeping with the long term goal of detecting potential molecular targets for medical therapies to treat human plexiform neurofibromas, we have identified the kinase Pak1 as a possible downstream intermediary of Ras signaling in NF1 deficient cells. Studies described here utilized murine genetic models to study the effects of genetic inactivation of Pak1 on molecular signaling and cellular functions related to neurofibromas. We demonstrate that inactivation of Pak1 leads to correction of SCF mediated gain-in-function phenotypes seen in Nf1 haploinsufficient mast cells, in vivo and in vitro. However, by using a conditional Nf1 knockout mouse that is a reliable model of plexiform neurofibroma formation, we shown that loss of Pak1 alone in the hematopoeitic compartement is not sufficient to prevent neurofibroma formation. Additionally, we describe a key role for Pak1 in regulating PDGF and TGF-β mediated fibroblast functions, in vitro and in vivo. These studies provide insight into the causes of debilitating tumors related to a common genetic disease, and this research could potentially lead to the development of medical therapies for these tumors, increasing the quality of life for tens of thousands of affected individuals each year.

Indiana University-Purdue University Indianapolis (IUPUI) / Stem cell factor (SCF) mediated c-Kit signaling, and downstream activation of Phosphatidylinositol-3 Kinase (PI-3K) is critical for multiple biological effects mediated by mast cells. Mast cells express multiple regulatory subunits of PI-3Kinase, including p85α, p85β, p50α and p55α. In the present study, we have examined the relationship between p85α and p85β subunit in mast cell development and show that loss of p85α in mast cell progenitors impairs their growth, maturation and survival whereas loss of p85β enhances this process. To further delineate the mechanism (s) by which p85α provides specificity to mast cell biology, we compared the amino acid sequences between p85α and p85β subunits. The two isoforms share significant structural homology in the two SH2 domains, but show significant differences in the N-terminal SH3 domain as well as the BCR homology domain. To determine whether the c-Kit induced reduction in growth of mast cells is contributed via the N-terminal SH3 or the BCR homology domain, we cloned and expressed the shorter splice variant p50α, and various truncated mutant versions of p85α in p85α deficient mast cells. We demonstrate both invitro and invivo that while the SH3 and the BH domains of p85 are dispensable for mast cell maturation; they are essential for normal growth and survival. In contrary to existing dogma on redundant functional role of PI-3K regulatory subunits, this study proves that p85α and p85β regulatory subunits of PI-3K have unique roles in mast cell development. We prove that p85α deficiency impairs the expression of multiple growth, survival and maturation related genes whereas p85β deficiency inhibits c-Kit receptor internalization and degradation. This novel finding on negative role of p85β in mast cell development has significant clinical implication, as this knowledge could be used to develop treatments for mast-cell-associated leukemia and mastocytosis.

Background and Research Aim: Stress affects the immune system, which influences host physiology. Mast cells have been associated with several stress-induced changes in gut physiology. Mast cells also have the potential to influence the brain and behaviour. We investigated how mast cells influenced the body, brain, and behaviour during stress. Methodology: We investigated the behaviour of mast cell deficient animals and deficient animals that received whole bone marrow (WBM) transplants. We also studied the effects of mast cell stabilization during stress on changes in gut motility, via ex vivo. recordings of intestinal segments, and brain, via behavioural measurements and flow cytometry analysis of proinflammatory monocyte trafficking to the brain. Results: Mast cell deficiency leads to several behavioural changes related to activity level, exploration, and sociability. Furthermore, deficient animals that received WBM transplants demonstrated social and anxiety-like behaviour that differed from their deficient counterparts. Mast cell stabilization during stress prevented many of the stress-induced changes in gut motility commonly observed in the intestine. Mast cell stabilization during stress also prevented proinflammatory monocyte trafficking to the brain and was associated with reduced anxiety-like behaviour. Conclusion: Our findings support the role of mast cells in baseline behaviour, suggesting the presence of mast cells is needed for normal social and anxiety-related functioning. We also found that mast cell activation contributes to stress-induced intestinal dysmotility, suggesting that mast cells should be a target for interventions of stress-related gut motility disorders, such as irritable bowel syndrome. Lastly, our findings on the role of mast cells in monocyte trafficking and anxiety adds to our knowledge of neuroimmune interactions during stress and supports a potential role for mast cells in anxiety-related mood disorders, where stabilization of mast cells during stressful events may be of benefit. / Thesis / Master of Science (MSc) / Stress affects the immune system, which influences the body. Mast cells of the immune system are involved in several stress-induced changes in the body. They can also influence the brain and behaviour. We investigated how mast cells influence the changes that occur in the gut, brain, and behaviour during stress. Using mouse models, we prevented mast cells from activating during stress and looked at this effect on gut movement, changes in brain chemistry, and behaviour. We also compared the behaviour of mast cell deficient mice and deficient mice that gained mast cells. We found that by preventing mast cells from activating during stress, we can prevent several stress-associated changes in gut movement, brain chemistry, and anxiety behaviour. We also found that mast cells affect anxiety and social behaviour. These results suggest that mast cells impact the body, brain chemistry, and behaviour in stress and non-stress conditions.

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

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.

Kainate receptor knockout mice have unique differences within their immune system. They exhibit an attenuated TH2 branch, while maintaining a robust TH1 response. Specifically, blocking the formation of functional kainate receptors affects mast cells and their related pathologies. While they seem to develop and activate normally in vivo and in vitro, KAR KO mast cells release more inflammatory mediators upon degranulation. These mice experience severe anaphylactic shock due to two compounding abnormalities. First, KAR KO mast cells release significantly more histamine in vivo upon IgE-mediated activation. Second, the animals over-respond to exogenous histamine with drastic temperature drops compared to WT. This report shows that the kainate receptor plays an important role in mast cell-mediated immune responses.