Severe influenza, recognized as a clinical syndrome characterized by hyper-induction of pro-inflammatory cytokine production, results in approximately 250–500 thousand deaths annually worldwide. Current influenza research is focused on therapeutics to target the influenza virus or modulate influenza virus-induced inflammation as potential treatment options to improve clinical outcome in experimental influenza A (H1N1) virus infection. The goals of this work were: (1) to evaluate the utility of inhaled nitric oxide (iNO) for decreasing influenza virus production in the lungs, and (2) investigate the use of mesenchymal stromal (stem) cells (MSCs) for mitigating deleterious host responses to influenza infection. Here, we report that MSCs and iNO, administered alone either prophylactically or post-influenza virus infection, fail to modulate host inflammation, fail to improve acute lung injury, fail to dampen lung viral load, and fail to improve survival of infected mice.

HLA-G is a protein normally expressed during pregnancy, protecting the fetus from the maternal immune system. Previous studies have shown an association between HLA-G expression post-transplantation and lower incidences of organ rejection. To further examine this beneficial role, we conducted a prospective study following a cohort of heart transplant recipients for one year and measuring their plasma HLA-G levels at various time points. Preliminary analyses failed to reveal an association between HLA-G and various parameters of rejection and vasculopathy. However, we decided to examine the in vitro effects of HLA-G in a smooth muscle cell (SMC) migration assay and whether HLA-G can be modulated pharmacologically. We made the novel observations that purified HLA-G was capable of inhibiting migration of SMCs, a key event in the development of cardiac allograft vasculopathy. IL-10, an anti-inflammatory cytokine, was capable of upregulating HLA-G in a Jeg-3 cell line. The modulation of HLA-G may represent a strategy to protect again vasculopathy, which is a leading cause of morbidity and mortality in heart transplant recipients.

Intracerebral hemorrhage (ICH) results in secondary brain injury caused partially by blood and its metabolites. Survivors of ICH are often left with severe disabilities, therefore, decreasing the extent of this secondary injury may improve functional outcome of patients. Incubation of mouse brain slices with partially oxidized bilirubin, a neurotoxic blood breakdown product, caused a dose- and time-dependent decrease in axonal function, suggesting a reduced number of conducting myelinated axons. These effects did not occur when tissue was incubated with non-oxidized bilirubin. Injection of bilirubin into the corpus callosum of mice caused functional impairment of unmeylinated axons; however, immunohistochemical staining of the tissue showed evidence of structural damage to both oligodendrocytes and axons. This data provides evidence for functional and structural damage to white matter in the presence of partially oxidized bilirubin Therefore, diminishing the duration of presence of bilirubin and its oxidation in the brain warrants study as a means of decreasing secondary brain injury after ICH.

Introduction: Currently, there are no objective techniques to assess burn depth. An early assessment of burn depth would enable accurate management decisions, which would improve patient outcomes. Near infrared (NIR) technology has shown promise as a non-invasive monitor of oxygenation and perfusion, and its potential to assess the depth of burn injuries has been investigated clinically over the past five years. The purpose of the thesis was to determine the capacity of NIR technology to differentiate acute thermal injuries. Methods: Burn sites (n=5) and control sites (n=5) were created on the dorsum of sixteen animals with brass rods held at constant pressure and heated to 100°C and 37.5°C respectively. NIR data was collected from the burns and control sites pre-burn, immediately post-burn, and 1, 12, 24, 36, 48 and 96 hours after the burn injury. Biopsies of the burn and control sites were acquired at each time point and used to confirm the depth of injury. NIR data was processed for the content of water, oxy-, deoxy- and methemoglobin. Results: Oxyhemoglobin and total hemoglobin decreased as burn depth increased. The proportion of oxy- and deoxyhemoglobin to total hemoglobin showed that the ratio of oxy- to deoxyhemoglobin decreased as burn injury increased. Methemoglobin levels as a ratio of total hemoglobin also showed that as the severity of injury increased the proportion of methemoglobin also increased. Finally, superficial partial thickness injuries (3 s and 12 s) showed early peak levels of water, which rapidly declined towards baseline. The deep partial thickness injuries (20 s and 30 s) do not experience peak levels and retain water over the course of the experiment. The full thickness injuries water levels remain close or below baseline levels throughout the experiment. Conclusion: NIR spectroscopy could distinguish burn depth using water, oxy-, met- and total hemoglobin as separate entities. The presence of methemoglobin in the burn wounds is a novel finding that has not been described previously in burn literature.

Bladder obstruction (neurogenic or anatomic) induces strain injury in detrusor smooth muscle cells. Signaling via strain injury in other systems has been highly studied, while in bladder obstruction, it has been quite limited to a small number of pathways. In our study we have examined the effects of strain injury using a combination of in vivo, ex vivo and in vitro models, with the aim of understanding disease pathogenesis in the bladder. Using a combination of literature searches, phospho-protein screens and pathway analysis, we uncovered three pathways activated by mechanical strain, ERK, STAT3 and mTOR, with potential for changing not only the way we understand but also the way we treat obstructive myopathies of the bladder. We found that not only were these pathways activated in response to strain and distension injury of BSMC, but they were also responsible for proliferation and sometimes de-differentiation. Included herein are three chapters, published in 2006 and 2010, on the role of ERK, STAT3 and mTOR pathways in bladder smooth muscle cell proliferation and differentiation, 8 Appendices containing the first pages of other papers and reviews published during the course of my studies.

Previous studies reported that matrix protein supplementation (fibronectin/fibrinogen, FN/FG) of agarose gel microcapsules enhances survival and pulmonary retention of syngeneic rat multipotent stromal cells (MSCs). I hypothesized that additional supplementation of microcapsules with osteopontin (OPN) and transglutaminase 2 (TG2) would enhance cell survival, while stabilizing the provisional matrix. Using monomeric OPN or OPN polymerized with TG2, I examined human MSC adhesion, morphology, focal contact formation and apoptosis. Polymeric OPN induced greater adhesion than monomeric OPN (84.5±10.7 vs. 44.3±10.0cells/field), and also significantly enhanced focal contact formation (351.5±21.2 vs. 45.6±17.6 focal contact sites/cell) and cell spreading (2.7x103±0.20x103μm2 vs. 1.2x103±0.26x102μm2) while preserving MSC pluripotency. Microcapsules supplemented with FN/FG, polymeric OPN and TG2 demonstrated significantly less apoptotic cells than FN/FG microcapsules (14.0±2.34% vs. 28.2±3.22%). Reduced apoptosis was attributed to matrix stabilization by TG2 and the synergistic activity of matrix proteins. It is anticipated that this enhanced survival will maximize the therapeutic potential of MSCs.

Thrombosis is a vascular pathological process which frequently affects the veins of the lower limbs, and embolisation of thrombotic material is common. Thromboembolic disease is a common cause of death in Western societies, predominantly affecting the elderly. Numerous risk factors and co-morbidities have been identified. Safe and effective means of prophylaxis are available but are underutilised. Anticoagulant drugs are very effective in preventing thromboembolic disease, but their effects on the evolution of the thrombotic process remain poorly documented. These drugs are difficult to use, and treatment errors and treatment failures are common. The factors which determine the embolisation of deep vein thrombosis are poorly understood. When such embolisation occurs it is usually asymptomatic. Symptomatic pulmonary embolism presents in 3 general ways-pleuritic pain, shortness of breath, or collapse. Tachypnea is the commonest sign. Tests which are available to assist in the diagnosis of thromboembolic disease include the ventilation perfusion lung scan, ultrasound of the lower limbs, pulmonary angiography and echocardiography. The commonest investigation requested in this context is the lung scan. Although the results are often inconclusive, this is frequently the only specific investigation which is performed. The accurate interpretation of lung scans requires consideration of the pretest probability of pulmonary embolism. The D-dimer assay is another test which may be useful. The finding of a normal D-dimer level substantially reduces the probability of thromboembolic disease and may render a lung scan unnecessary. / Subscription resource available via Digital Dissertations only.

Thrombosis is a vascular pathological process which frequently affects the veins of the lower limbs, and embolisation of thrombotic material is common. Thromboembolic disease is a common cause of death in Western societies, predominantly affecting the elderly. Numerous risk factors and co-morbidities have been identified. Safe and effective means of prophylaxis are available but are underutilised. Anticoagulant drugs are very effective in preventing thromboembolic disease, but their effects on the evolution of the thrombotic process remain poorly documented. These drugs are difficult to use, and treatment errors and treatment failures are common. The factors which determine the embolisation of deep vein thrombosis are poorly understood. When such embolisation occurs it is usually asymptomatic. Symptomatic pulmonary embolism presents in 3 general ways-pleuritic pain, shortness of breath, or collapse. Tachypnea is the commonest sign. Tests which are available to assist in the diagnosis of thromboembolic disease include the ventilation perfusion lung scan, ultrasound of the lower limbs, pulmonary angiography and echocardiography. The commonest investigation requested in this context is the lung scan. Although the results are often inconclusive, this is frequently the only specific investigation which is performed. The accurate interpretation of lung scans requires consideration of the pretest probability of pulmonary embolism. The D-dimer assay is another test which may be useful. The finding of a normal D-dimer level substantially reduces the probability of thromboembolic disease and may render a lung scan unnecessary. / Subscription resource available via Digital Dissertations only.

Thrombosis is a vascular pathological process which frequently affects the veins of the lower limbs, and embolisation of thrombotic material is common. Thromboembolic disease is a common cause of death in Western societies, predominantly affecting the elderly. Numerous risk factors and co-morbidities have been identified. Safe and effective means of prophylaxis are available but are underutilised. Anticoagulant drugs are very effective in preventing thromboembolic disease, but their effects on the evolution of the thrombotic process remain poorly documented. These drugs are difficult to use, and treatment errors and treatment failures are common. The factors which determine the embolisation of deep vein thrombosis are poorly understood. When such embolisation occurs it is usually asymptomatic. Symptomatic pulmonary embolism presents in 3 general ways-pleuritic pain, shortness of breath, or collapse. Tachypnea is the commonest sign. Tests which are available to assist in the diagnosis of thromboembolic disease include the ventilation perfusion lung scan, ultrasound of the lower limbs, pulmonary angiography and echocardiography. The commonest investigation requested in this context is the lung scan. Although the results are often inconclusive, this is frequently the only specific investigation which is performed. The accurate interpretation of lung scans requires consideration of the pretest probability of pulmonary embolism. The D-dimer assay is another test which may be useful. The finding of a normal D-dimer level substantially reduces the probability of thromboembolic disease and may render a lung scan unnecessary. / Subscription resource available via Digital Dissertations only.

Thrombosis is a vascular pathological process which frequently affects the veins of the lower limbs, and embolisation of thrombotic material is common. Thromboembolic disease is a common cause of death in Western societies, predominantly affecting the elderly. Numerous risk factors and co-morbidities have been identified. Safe and effective means of prophylaxis are available but are underutilised. Anticoagulant drugs are very effective in preventing thromboembolic disease, but their effects on the evolution of the thrombotic process remain poorly documented. These drugs are difficult to use, and treatment errors and treatment failures are common. The factors which determine the embolisation of deep vein thrombosis are poorly understood. When such embolisation occurs it is usually asymptomatic. Symptomatic pulmonary embolism presents in 3 general ways-pleuritic pain, shortness of breath, or collapse. Tachypnea is the commonest sign. Tests which are available to assist in the diagnosis of thromboembolic disease include the ventilation perfusion lung scan, ultrasound of the lower limbs, pulmonary angiography and echocardiography. The commonest investigation requested in this context is the lung scan. Although the results are often inconclusive, this is frequently the only specific investigation which is performed. The accurate interpretation of lung scans requires consideration of the pretest probability of pulmonary embolism. The D-dimer assay is another test which may be useful. The finding of a normal D-dimer level substantially reduces the probability of thromboembolic disease and may render a lung scan unnecessary. / Subscription resource available via Digital Dissertations only.