The modification of proteins by xenobiotic and endogenous electrophilic species produced in cells undergoing oxidative stress contributes to cellular toxicity and disease processes. Many xenobiotics are themselves reactive electrophiles; however non-reactive compounds may become reactive towards proteins and DNA following metabolism. Identifying actual sites of adduction on target proteins is critical for determining the structural and functional consequences associated with the modification. 1,4-benzoquinone (BQ) is a reactive quinone and environmental toxicant, formed from oxidative metabolism of benzene, an aromatic hydrocarbon found in gasoline and other fuels. Although environmental and occupational exposure to benzene is associated with the development of aplastic anemia and leukemia, the mechanism of toxicity remains elusive. Due to the electrophilic nature of BQ, it reacts with glutathione to form quinol-thioether (QT) conjugates that retain the ability to redox cycle between the reduced (HQ) and oxidized (BQ) forms. BQ and its QT metabolites are reactive, and can produce cellular necrosis through oxidative stress and protein modification. One further consequence of oxidative stress is the elevation of cellular membrane lipid peroxidation, resulting in the formation of reactive lipid-aldehydes such as 4-hydroxynonenal (4HNE). Adduction of critical amino acid residues in target bone marrow proteins by 4HNE and QTs following exposure to benzene could contribute to its hematotoxic effects. This dissertation builds upon the foundation of proteins targeted by electrophilic adduction by outlining techniques to pinpoint the specific amino acids targeted and furthermore predict the functional releavance of adduction. For the first time, protein targets of reactive endogenous lipid aldehydes are reported in the bone marrow of chemically treated rats. Furthermore, novel sites of adduction by aldehydes and benzene-glutathione conjugates are reported within functional regions of topoisomerase II. Inhibition of bone marrow DNA topoisomerase II by benzene metabolites is implicated as a potential mechanism of benzene-induced hematotoxicity and acute-myeloid leukemia. The strong inhibitory effect of these compounds on topoisomerase II activity suggests that their presence in the bone marrow may play a role in benzene-induced myelotoxicity.

It has been suggested that some adult bone marrow cells (BMC) can localize to the injured tissues and develop tissue-specific characteristics including those of the pulmonary epithelium. In Chapter 2 we show that the combination of mild airway injury as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype expressing Clara cell secretory protein (CCSP) and pro-surfactant protein-C. Bone marrow cells from transgenic mice expressing green fluorescent protein driven by the epithelial-specific cytokeratin-18 promoter were injected transtracheally into airway-injured wild-type recipients. BMC retention in the lung was observed to be at least 120 days following cell delivery with increasing transgene expression over time. The results indicate that targeted delivery of BMC can promote airway regeneration. Although bone marrow stem/progenitor cells can develop into lung epithelial cells, the specific subpopulation remains unknown. In Chapter 3 we identify a newly discovered population of murine and human BMC that express CCSP. These CCSP+ cells increase in the bone marrow and blood after airway injury and can be expanded in culture. CCSP+ cells are unique in that they express both hematopoietic and mesenchymal stromal cell markers and can give rise to various lung epithelial lineages in vitro. Importantly, bone marrow transplant of CCSP+ cells to CCSP knockout recipients confirms that bone marrow CCSP+ cells contribute to airway epithelium after airway injury. In Chapter 4 we enrich for a stem/progenitor cell population within the CCSP+ using the stem cell antigen (Sca)-1 as a marker. Here we identified a putative epithelial stem/progenitor cell that can be induced to differentiate into various lung epithelial cell lineages expressing markers exclusive to airway or alveolar epithelial cells when cultured under an air liquid interface. These cells also have self-renewal potential in vitro that can proliferate in vivo and repopulate the injured airway epithelium. This newly discovered epithelial-like cells may play a central role in the bone marrow contribution to lung repair and are exciting candidates for cell-based targeted therapy for treatment of lung diseases.

It has been suggested that some adult bone marrow cells (BMC) can localize to the injured tissues and develop tissue-specific characteristics including those of the pulmonary epithelium. In Chapter 2 we show that the combination of mild airway injury as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype expressing Clara cell secretory protein (CCSP) and pro-surfactant protein-C. Bone marrow cells from transgenic mice expressing green fluorescent protein driven by the epithelial-specific cytokeratin-18 promoter were injected transtracheally into airway-injured wild-type recipients. BMC retention in the lung was observed to be at least 120 days following cell delivery with increasing transgene expression over time. The results indicate that targeted delivery of BMC can promote airway regeneration. Although bone marrow stem/progenitor cells can develop into lung epithelial cells, the specific subpopulation remains unknown. In Chapter 3 we identify a newly discovered population of murine and human BMC that express CCSP. These CCSP+ cells increase in the bone marrow and blood after airway injury and can be expanded in culture. CCSP+ cells are unique in that they express both hematopoietic and mesenchymal stromal cell markers and can give rise to various lung epithelial lineages in vitro. Importantly, bone marrow transplant of CCSP+ cells to CCSP knockout recipients confirms that bone marrow CCSP+ cells contribute to airway epithelium after airway injury. In Chapter 4 we enrich for a stem/progenitor cell population within the CCSP+ using the stem cell antigen (Sca)-1 as a marker. Here we identified a putative epithelial stem/progenitor cell that can be induced to differentiate into various lung epithelial cell lineages expressing markers exclusive to airway or alveolar epithelial cells when cultured under an air liquid interface. These cells also have self-renewal potential in vitro that can proliferate in vivo and repopulate the injured airway epithelium. This newly discovered epithelial-like cells may play a central role in the bone marrow contribution to lung repair and are exciting candidates for cell-based targeted therapy for treatment of lung diseases.

Acute myeloid leukemia (AML) often remains refractory to current chemotherapy and transplantation approaches despite many advances in our understanding of mechanisms in leukemogenesis. The bone marrow “niche” or microenvironment, however, may be permissive to leukemia development and studying interactions between the microenvironment and leukemia cells may provide new insight for therapeutic advances. Mesenchymal stem cells (MSCs) are central to the development and maintenance of the bone marrow niche and have been shown to have important functional alterations derived from patients with different hematological disorders. The extent to which MSCs derived from AML patients are altered remains unclear. The aim of this study was to detect changes occurring in MSCs obtained from human bone marrow in patients with AML by comparing their function and gene expression pattern with normal age-matched controls. MSCs expanded from patients diagnosed with acute leukemia were observed to have heterogeneous morphological characteristics compared to the healthy controls. Immunohistochemistry and flow data confirmed the typical cell surface immunophenotype of CD90+ CD105+ CD73+ CD34- CD45-, although MSCs from two patients with AML revealed reduced surface expression of CD105 and CD90 antigens respectively. Differentiation assays demonstrated the potential of MSCs from AML patients and healthy donors to differentiate into bone, fat and cartilage. However, the ability of MSCs from AML samples to support hematopoietic function of CD34+ progenitors was found to be impaired while the key hematopoietic genes were found to be differentially expressed on AML-MSCs compared to nMSCs. These studies indicate that there exist differences in the biologic profile of MSCs from AML patients compared to MSCs derived from healthy donors. The results described in the thesis provide a formulation for additional studies that may allow us to identify new targets for improved treatment of AML.

No description available.

No description available.

Thesis (PhD in AppSc)--University of South Australia, 1996

Title page, contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The growth plate is a cartilaginous structure located at the proximal and distal ends of immature long bones, which contributes to longitudinal growth through the process of endochondral ossification. Cartilage has a limited ability to regenerate and in children, injury to the the growth plate can result in limb length discrepancies and angular deformity, due to formation of a bone bridge at the damaged site which disturbs structure and function of the growth plate. Current treatments of the abnormalities arising from growth plate arrest involve surgical correction once the deformities have manifested. To date, there is no biological based therapy for the repair of injured/damaged growth plate cartilage. Mesenchymal stem cells (MSC) are self renewable mulitpotential progenitor cells with the capacity to differentiate toward the chondrogenic lineage. Since their discovery, significant interest has been generated in the potential application of these cells for cartilage regeneration. In this study, the ability of autologous bone marrow mesenchymal stem cells to regenerate growth plate cartilage in a sheep model was examined. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1330837 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Circulating neutrophil activation occurs during cardiac surgery with extracorporeal circulation (ECC) and is implicated in the pathophysiology of inflammatory tissue injury and peri-operative organ dysfunction. However, neutrophil directed antiinflammatory strategies have failed to demonstrate consistent therapeutic benefit indicating that the nature and significance of peri-operative circulating neutrophil activation remains incompletely defined. In particular, conformational activation of the b2 integrin Mac-1 (CD11b/CD18), which is required for neutrophil adhesion competence and facilitation of effector functions, has not previously been investigated during cardiac surgery, and the relative contribution of cellular activation and bone marrow neutrophil recruitment to peri-operative changes in circulating neutrophil phenotype and function is unknown. A novel whole blood flow cytometric technique was used to analyze circulating neutrophil phenotype (total Mac-1, conformationally-active CD11b, CD10, CD16, L-selectin and P-selectin glycoprotein ligand-1) and function in cardiac surgery patients to characterize the nature of changes in Mac-1 expression and activation status, and the effects of relative neutrophil immaturity on circulating neutrophil phenotype and function. The effect of heparin, a known CD11b ligand, on Mac-1 epitope expression was also investigated. Circulating neutrophil numbers observed during ECC were mathematically modeled to determine the acute response of the bone marrow neutrophil reserve to an inflammatory stimulus. Plasma cytokine, chemokine and acute phase mediators were measured in cardiac and lung surgery patients to determine potential regulators of systemic neutrophil recruitment. Neutrophils newlyemergent from the bone marrow were characterized as CD10-/CD16low and exhibited distinct changes in cell surface markers and enhanced functional responses, relative to their more mature CD10+ counterparts. Conformational activation of CD11b occurred peri-operatively and provided a more sensitive measure of circulating neutrophil activation status than changes in total Mac-1 or L-selectin expression, although detection of Mac-1 epitopes was reduced in the presence of heparin. Modeling of circulating neutrophil numbers predicted that post-mitotic maturation time was acutely abbreviated by 8.4 hours during 71 minutes of ECC. Systemic chemokine release occurred with cardiac but not non-cardiac thoracic surgery indicating some specificity of the acute inflammatory response. These findings expand the understanding of peri-operative circulating neutrophil activation and recruitment, and identify potential therapeutic targets to limit neutrophil injurious potential during cardiac surgery with ECC.

Bibliography: leaves 152-223. / xxiii, 223, [137] leaves, [27] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Experiments were designed to identify and purify human bone marrow stromal precursor cells by positive immunoselection, based on the cell surface expression of the VCAM-1 and STRO-1 antigens. The data presented demonstrates a hierarchy of bone cell development in vitro. / Thesis (Ph.D.)--University of Adelaide, Dept. of Orthopaedics Surgery and Trauma, 1998