Ng, Sin Man. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 96-104). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Allogeneic haematopoietic stem cell transplantation (HSCT) has been used successfully to treat children and adults with high-risk or relapsed hematopoietic malignancies, marrow failure syndromes, and hereditary immunodeficiency disorders. When initially developed, allogeneic HSCT was conceived as a method of rescuing patients from the toxic side effects of dose-intensive chemoradiotherapy. Due to transplant-related toxicities, the application of myeloablative allogeneic HSCT has been limited to younger patients without organ dysfunctions. Since the early 1990s many groups of investigators have explored strategies using less intensive preparative regimens that would allow engraftment of hematopoietic progenitor cells from either identical or non-identical donors. These reduced-intensity conditioning (RIC) regimens result in less tissue damage, less inflammatory cytokine secretion, and possibly lower rates of graft-versus-host disease (GVHD) and non-relapse mortality (NRM). Such non-myeloablative approach, or "mini-transplant", has been suggested to benefit older patients as well as in conditions in which traditional myeloablative conditioning regimens are associated with high rates of non-relapse mortality. / Allogeneic HSCT is the only curative therapy for many patients with myeloid malignancies or myelodysplastic syndrome (MDS). The development of reduced-intensity preparative regimens may allow the extension of this form of treatment to older and patients with coexisting medical illness. On the other hand, relapse after transplantation remains the most important cause of treatment failure in patients with refractory acute myeloid leukemia (AML) or MDS, and is associated with poor survival. Evaluation of prognostic factors may help to improve the results of myeloablative and RIC allogeneic HSCT in this group of patients. Furthermore, the impact of comorbidities on outcomes of RIC allogeneic HSCT in this group of patients with refractory AML or MDS needs to be defined. / The application of embryonic and adult stem cells in regenerative and reparative therapies of non-hematopoietic diseases is emerging rapidly. Human umbilical cord blood (UCB) is a rich source of hematopoietic stem cells and mesenchymal progenitor cells. Although clinical experience to date with UCB has focused on hematological application, early preclinical studies support the hypothesis that multipotential stem cells derived from UCB exhibit functional characteristics similar to that observed in adult marrow-derived stem cells in mediating vascular and organ regenerative capabilities. However, the application of these preclinical findings in clinical setting needs to be further studied. Mini-transplant of human UCB may be an effective approach to repair organ damage in patients with non-hematological diseases. / Wong Siu Ming Raymond. / Adviser: Joseph J.Y. Sung. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 187-223). / 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.

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.

Septic shock is a leading cause of death in intensive care units. As part of the septic process, the endothelium becomes activated and propagates the septic condition. It has become evident that reactive oxygen species (ROS) are involved in the signaling of mediators of sepsis, such as tumor necrosis factor-alpha (TNF-alpha) and the lipopolysaccharide coating of gram-negative bacteria (LPS). An important source of these ROS is NADPH oxidase, which is a ubiquitously expressed enzyme complex that also exists in endothelial cells (EC). We showed that O2- from NADPH oxidase was important for LPS, as well as TNF-alpha, induction of two markers of an activated endothelium, interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVEC). / Expression of a gene can be increased by a rise in transcription as well as post-transcriptional changes, such as mRNA stability modifications. We assessed the role of NADPH oxidase in this process and found a complex interaction. Although LPS increases IL-8 transcription, it also destabilizes IL-8 mRNA in a p38 and extracellular signal-regulated kinase (ERK) MAPK dependent manner, which was only evident after blocking NADPH oxidase. This regulation involved the mRNA de-stabilizing factor tristetraprolin (TTP). In contrast, TNF-alpha enhanced the stability of IL-8, IL-6 and ICAM-1 mRNA in a p38 MAPK dependent, but NADPH oxidase independent manner. Furthermore, LPS did not have an effect on mRNA stability of IL-6 or ICAM-1 in our system. Thus, we conclude from our studies that the NAPDH oxidase is important for the induction of inflammatory molecules in LPS and TNF-alpha treated EC and is also involved in mRNA stability regulation of these molecules in a signal and gene specific fashion.

The activity of dihydrofolate reductase, a key enzyme in the de novo biosynthesis of thymidylate, purines, and some amino acids, had previously been found to be increased in a methotrexate-resistant mouse leukemia (L5178Y) cell line as compared to the activity in the parental methotrexate-sensitive cell line. The increased activity was composed of two forms of the enzyme, one of which, form 2, was highly insensitive to inhibition by methotrexate. Both forms were purified to near homogeneity and using the antibodies prepared against them, it could be demonstrated that the two forms are antigenically distinct. The increased dihydrofolate reductase activity present in the methotrexate resistant cells resulted from an overproduction of both forms of the enzyme due to the presence of abundant mRNA coding for these enzymes. An increase in the dihydrofolate reductase gene copy number could be demonstrated in the resistant cells. mRNA coding for form 1 and form 2 enzymes was greatly enriched by polysome immunoprecipitation and complimentary DNA (cDNA) was synthesized in vitro from these enriched mRNA molecules. Evidence was found for the presence of methotrexate-insensitive forms of dihydroflate reductase in the blast cells of three out of eight acute myelogenous leukemia patients, and in two (distinct from the above) of the eight patients the activity was significantly increased. In contrast to the overproduction of dihydrofolate reductase protein in the methotrexate-resistant mouse cell line, increased enzyme activity in a methotrexate-resistant human promyelocytic leukemia (HL-60) cell line could not be correlated with an increase in the enzyme protein. Furthermore, the amounts of dihydrofolate reductase mRNA and gene-dosages were similar in the parental metho-trexate-sensitive and methotrexate-resistant cells. The enzyme from the resistant cells differed significantly in some of its physical and kinetic properties from that present in the parental cells. An increase in dihydrofolate reductase activity resulting from a modification of the enzyme rather than gene amplification has not to date been reported in the literature and may present a novel mechanism of resistance to methotrexate. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

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The increase in ASMC mass is a major structural change described in airway remodeling in asthma. This increase has been attributed to ASMC hyperplasia and hypertrophy. The distance between ASMC and the epithelium is reduced suggesting expansion of the muscle bundle towards the epithelium. Recent studies have suggested a role of epithelial derived chemokines in ASMC migration toward the epithelium. We hypothesized that chemokines (Eotaxin, RANTES, MIP-1alpha and IL-8) can directly influence ASMC mass by increasing the rate of proliferation or enhancing survival. ASMCs were exposed to different concentrations of eotaxin, RANTES, IL-8 or MIP-1alpha. To test for proliferation, stimulated ASMC were pulsed with 3H-thymidine or stained with BrdU and then analyzed with flow cytometry. Apoptosis was measured using Annexin V and flow cytometry. Expression of phosphorylated p42/p44 and MAPKinases was assessed by Western analysis. In a concentration-dependent manner, chemokines such as Eotaxin, RANTES, IL-8 and MIP-lalpha increased ASMCs 3H-thymidine incorporation and DNA synthesis. Eotaxin, RANTES and IL-8 decreased the number of apoptotic ASMCs compared to the matched controls. A significant increase in phosphorylated p42/p44 MAPKs was seen after treating ASMCs with RANTES and eotaxin. We conclude that chemokines might contribute to airway remodeling by increasing the number of ASMCs.

No description available.

A confocal scanning laser ophthalmoscope (CSLO) was used to image the axonal and dendritic aborizations of RGCs in the Thy-1 YFP mice. With quantitative analysis of cell body area, axon diameter, dendritic field, number of terminal branches, total dendritic branch length, branching complexity, symmetry and distance from the optic disc, the morphologies of RGCs and the patterns of axonal and dendritic degeneration were analyzed. After optic nerve crush, RGC damage was observed prospectively to begin with progressive dendritic shrinkage, followed by loss of the axon and the cell body. Similar pattern of RGC degeneration was observed after 90 minutes of retinal ischemia although no morphological changes were detected when the duration of ischemia was shortened to 30 minutes. The rate of dendritic shrinkage was variable and estimated on average 2.0% per day and 11.7% per day with linear mixed modeling, after optic nerve crush and retinal ischemic injury, respectively. RGCs with a larger dendritic field had a slower rate of dendritic shrinkage. / In summary, we demonstrated that dendritic shrinkage could be evident even before axonal degeneration after optic nerve crush and retinal ischemic injury. We have established a methodology for in vivo and direct visualization of RGCs and retinal microglia, which could provide reliable and early markers for neuronal damage. Measuring the rate of dendritic shrinkage and tracking the longitudinal activation of microglia would provide new paradigms to study the mechanism of neurodegenerative diseases and offer new insights in testing novel therapies for neuroprotection. / Progressive neuronal cell death and microglial activation are the key pathological features in most neurodegenerative diseases. While investigating the longitudinal profiles of neuronal degeneration and microglial activation is pertinent to understanding disease mechanism and developing treatment, analyzing progressive changes has been obfuscated by the lack of a non-invasive approach that allows long term, serial monitoring of individual neuronal and microglial cells. Because of the clear optical media in the eye, direct visualization of the retinal ganglion cells (RGCs) and microglia is possible with high resolution in vivo imaging technique. In this study, we developed experimental models to visualize and characterize the cellular morphology of RGCs and retinal microglia in vivo in the Thy-1 YFP and the CX3CR1 +/GFP transgenic mice, described the patterns of axonal and dendritic shrinkage of RGCs, discerned the dynamic profile of microglial activation and investigated the relationship between RGC survival and microglial activation after optic nerve crush and retinal ischemic injury induced by acute elevation of intraocular pressure. / The longitudinal profile of microglial activation was investigated by imaging the CX3CR1GFP/+ transgenic mice with the CSLO. Activation of retinal microglia was characterized with an increase in cell number reaching a peak at a week after optic nerve crush and retinal ischemic injury, which was followed by a gradual decline falling near to the baseline at the 4 th week. The activation of retinal microglia was proportional to the severity of injury. The number of RGCs survival at 4 weeks post-injury was significantly associated with the number of activated retinal microglia. / Li, Zhiwei. / Adviser: Leung Kai Shun. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 50-66). / 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.