CD4+CD25+FoxP3+ regulatory T cells (Treg) possess the capacity to modulate both adaptive and innate immunity. Due to their suppressive nature, Treg cells have been studied and tested in a variety of scenarios in an attempt to ameliorate undesired immune responses. While graft versus host disease (GVHD) has in fact emerged as the first clinical application for human Treg cells (Riley et al. 2009), equally important are issues concerning hematopoietic engraftment and immune reconstitution. Currently, little is known about the effect(s) that regulatory T cells may exert outside the immune system in this context. Based on cytokine effector molecules they can produce we hypothesized that Treg cells could regulate hematopoietic phenomena. The studies portrayed in this dissertation demonstrate that Treg cells can differentially affect the colony forming activity of myeloid and erythroid progenitor cells. In-vitro as well as in-vivo findings demonstrate the ability of Tregs to inhibit and augment the differentiation of primitive and intermediate myeloid (interleukin (IL)-3 driven) and late erythroid (erythropoietin driven) hematopoietic progenitor cells, respectively. The inhibitory and enhancing affects appeared to be mediated by independent pathways, the former requiring cell-cell contact, major histocompatibility complex (MHC) class II expression on marrow cells and involving transforming growth factor beta (TGF-beta), whereas the latter required interleukin (IL)-9 and was not contact dependent. Strikingly, we observed that in addition to regulating hematopoietic activity in normal primary BM cells, Tregs were also capable of suppressing colony forming activity by the myelogenous leukemia cell line NFS-60. Furthermore, studies involving endogenous Treg manipulations in-situ (i.e. depletion of these cells) resulted in elevated overall myeloid colony activity (CFU-IL3) and diminished colony numbers of erythroid precursors (CFU-E) in recipients following BMT. Consistent with these results, it was found that upon co-transplant with limiting numbers of bone marrow cells, exogenously added Treg cells exert in-vivo regulation of myeloid and erythroid CFU activity during the initial weeks post-transplantation. This regulation of hematopoietic activity by freshly generated Tregs upon transplantation led to the elaboration of a second hypothesis; following lethal total body irradiation (TBI) the host microenvironment facilitates regulatory T cell activation/effector function. Substantial evidence has accumulated in support of this hypothesis, for example we demonstrate up-regulation of surface molecules such as GARP and CD150/SLAM, which have been previously reported as indicators of Treg activation following TCR signaling and co-stimulation, occurs in donor (reporter) Treg populations. Acquisition of an activated phenotype and hence of effector/modulatory function is consistent with the previous in-vivo observations, indicating that both recipient and donor Treg cells can influence hematopoietic progenitor cell activity post-transplant. Finally, the present studies may be of great relevance in the emerging field of Treg cell based immunotherapy for prevention and/or treatment of HSCT complications.

Chronic myelogenous leukemia is a myeloproliferative hematopoietic stem cell disease resulting from a reciprocal translocation that gives rise to BCR-ABL, a constitutively active tyrosine kinase. Imatinib and other tyrosine kinase inhibitors are currently standard therapy; however, point mutations often lead to drug resistance and disease relapse often occurs due to the persistence of quiescent leukemia stem cells that are shielded by stromal factors within the bone marrow microenvironment. In an effort to develop new therapies capable of eradicating these elusive cells, a novel approach has been proposed in which the biochemical properties of cancer cells are targeted. It has been established that one such property is oxidative stress due to the increased production of reactive oxygen species, which makes cancer cells especially dependent on their antioxidant systems to maintain redox homeostasis. Recent studies demonstrate that chaetocin, a mycotoxin produced by Chaetomium species fungi, possesses potent and specific antimyeloma activity due in part to its ability to inhibit thioredoxin reductase-1, a central oxidative stress remediation enzyme. In this study, the effectiveness of chaetocin against leukemia stem cells has been investigated using in vitro and in vivo murine chronic myelogenous leukemia models. Our results indicate that: chaetocin and imatinib function synergistically in decreasing cell viability, inducing apoptosis, and inhibiting the colony formation of chronic myelogenous leukemia cells in vitro; that chaetocin in combination with imatinib reduces leukemia stem cell frequency in vivo; that chaetocin increases intracellular reactive oxygen species levels; and that chaetocin does not disrupt the proliferation and differentiation of normal murine hematopoietic stem cells. Surprisingly, our results also show that while bone marrow stromal factors inhibit the activity of imatinib, they potentiate the activity of chaetocin, indicating that chaetocin could potentially be used to target leukemia stem cells within the bone marrow niche.

No description available.

Osteoporosis is defined as a skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk. Osteoporosis is well known increasing with age. The number and size of marrow adipocytes increase in a linear manner with age. Early histomorphometric observations suggested that the consequence of the adipose replacement of the marrow functional cell population was a cause of osteoporosis. The replacement of functional cells in the marrow by fat cells is common in several pathological study of osteoporosis. All these evidences clearly demonstrate the reciprocal relationship between osteoblast and adipocyte differentiation. The trans-differentiation of osteoblast to adipocyte is an important mechanism of pathogenesis of osteoporosis. Several reports have indicated that the long-term use of steroids could induce osteonecrosis and osteoporosis. Using a mouse pluripotent mesenchymal cell, D1, as a model, we have demonstrated that dexamethasone, a glucocorticoid, can induce adipogenesis. Peroxisome proliferator-activated receptors-£^ (PPAR£^) plays a critical role in glucose and lipid metabolism, macrophage function, and adipogenesis. It is a nuclear hormone receptor, activated through ligand binding, which results in allosteric changes in receptor conformation, recruitment of coactivators, assembly of a transcriptional complex, there regulates gene expression. Thiazolidinedione (TZD) is one of the agonist of PPAR£^ receptor which has been a medication for diabetic mellitus for years. Treatment with TZDs leads to selective accumulation of subcutaneous adipose tissue. We examined whether adipogenesis induction in D1 cells is initiated by activation of peroxisome proliferator-activated receptor-£^. The results revealed that pioglitazone induces adipogenesis in D1 cells in dosedependent manner and decreases alkaline phosphatase activity in D1 cells. Interestingly, this adipogenesis was not blocked by bisphenol A diglycidyl ether, a peroxisome proliferator-activated receptor-£^ antagonist. A peroxisome proliferator-activated receptor-£^-mediated reporter gene assay showed no response to pioglitazone. We then asked whether dexamethasone-induced adipogenesis can be repressed by mifepristone (RU486), an antagonist of glucocorticoid receptor. The results disclosed that mifepristone cannot counteract dexamethasone-induced adipogenesis, and mifepristone itself induced adipogenesis in D1 cells. Moreover, glucocorticoid receptor-mediated reporter gene assay was not responsive to dexamethasone or mifepristone. We concluded that the adipogenesis induced by pioglitazone and dexamethasone in D1 cells may not occur via a peroxisome proliferator-activated receptor-£^ and glucocorticoid receptor pathway. These results suggested that the adipogenesis induced by glucocorticoids and pioglitazone is directed by a multiple cell signaling pathway. Finally, data from microarray analysis confirmed this adipogenesis pathway, as several adipogenesis-related genes are highly provoked by DEX. We found that the expressions of several adipogenesis-related genes are highly provoked by this agent. Our studies suggest that the adipocyte conversion of bone marrow stromal cells may be the mechanism of bone loss caused by pioglitazone. Considering its widespread clinical use, the detrimental skeletal effects of pioglitazone should be closely monitored.

To examine the relationship between liver injury and the appearance of bone marrow derived hepatic cells we performed sex-mismatched bone marrow transplants in mice, with subsequent liver injury. Co-labeling for a marker of donor bone marrow origin and a marker of liver epithelial phenotype allowed us to identify rare marrow-derived hepatocytes at various time points following liver damage. The number of marrow-derived hepatocytes was low, however, and did not allow us to determine if liver-specific injury upregulated this process from baseline. We conclude that while marrow-derived hepatocytes are found, the low level of occurrence in this study makes it impossible to draw a clear temporal relationship between liver damage, recovery and the appearance of donor-derived cells. In addition, we cannot say whether liver-specific damage upregulates this phenomenon.

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references.

published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy