Malaria and tuberculosis together account for more than 2 million deaths worldwide each year. The present body of work examines interactions between these leading pathogens and cross-cutting themes in innate immunity to both diseases. Not only are malaria and tuberculosis important threats to public health in their own right, but malaria-tuberculosis co-infection appears to generate more severe pathology than either disease on its own, and malaria may exacerbate primary or re-activation tuberculosis (Chapter 2). Moreover, both diseases appear to share common host defense pathways, including CD36, a macrophage cell surface receptor important for innate immunity (Chapter 3). Biomarkers of host defense pathways common to both malaria and tuberculosis distinguish between clinical disease phenotypes and predict mortality in severe malaria (Chapters 4-7). Biomarker discovery led to the identification of angiopoitetin-2 (Ang-2) as a surrogate marker of disease severity in malaria and a potential therapeutic target. Nitric oxide, in addition to its antimycobacterial properties, is known to inhibit Ang-2 release from the endothelium, and is therefore hypothesized to improve outcomes in African children with severe malaria (Chapters 8 and 9). A broad range of methods are applied to address these diseases and their interactions, ranging from mammalian cell culture experiments in vitro, animal models of disease, analysis of human samples, and clinical epidemiology (randomized controlled trial). Translational aspects of this research are emphasized, outlining how advances in understanding of infectious disease pathogenesis can be applied to improved diagnosis, prognosis, and novel adjunctive therapies for two of the leading global infectious threats.

Diabetic nephropathy is the leading cause of end-stage renal disease, yet the mechanisms responsible for hyperglycemia-induced kidney injury have not been fully elucidated. Activation of the renin-angiotensin system and NADPH oxidase-dependent generation of reactive oxygen species are important mediators of chronic kidney disease. I first studied the effect of ACE2, an important enzyme in the renin-angiotensin system, in diabetic kidney injury in the Akita mouse and related the effect to angiotensin peptide and NADPH oxidase. I then demonstrated the interaction between Angiotensin II, the main substrate, and angiotensin-(1-7), the main product of ACE2, respectively, on cell signaling in mesangial cells to better understand the in vitro effect of ACE2. Finally I studied the effect of deletion of p47phox, a regulatory subunit of the NADPH oxidase, on initiation and progression of diabetic nephropathy in the Akita mouse and mesangial cell. Administration of human recombinant ACE2 decreased angiotensin II levels, increased angiotensin-(1-7) levels, normalized NADPH oxidase activity in the Akita mice, and ameliorated diabetes-induced kidney injury. In vitro, hrACE2 attenuated both high glucose and ANG II–induced oxidative stress and NADPH oxidase activity in mesangial cells. Ang-(1–7)-induced ERK1/2 phosphorylation in mesangial cells in a mas receptor-cAMP-PKA-dependent manner. This effect of ang-(1-7) on ERK1/2 phosphorylation is not mediated by AT1R, AT2R, epidermal growth factor or NADPH oxidase. Pre-treatment with Ang-(1-7) attenuated Ang II-induced NADPH oxidase activity and ERK1/2 activation also in a cAMP-PKA-dependent manner. Deletion of p47phox not only reduced diabetes-induced kidney injury but also reduced hyperglycemia by increasing pancreatic and circulating insulin concentrations. p47phox-/- mice exhibited improved glucose tolerance but modestly decreased insulin sensitivity. Deletion of p47phox attenuated high glucose-induced activation of NADPH oxidase and pro-fibrotic gene expression in mesangial cells. There was a positive correlation between p47phox and collagen Iα1 mRNA levels in renal biopsy samples from control subjects and subjects with diabetic nephropathy. The data generated in this thesis strongly suggest a protective role of ACE2, via Ang-(1-7), and a deleterious role of p47phox in diabetic nephropathy. Future therapeutic strategies should include enhancing ACE2 activity in the kidney and inhibiting p47phox-dependent activation of NADPH oxidase in both the kidney and the pancreas.

Background: Kidney stone disease is a common and often debilitating disorder, yet its pathophysiology is poorly understood. This dissertation studies predisposition to kidney stone formation from diurnal variation in physiochemical and physiologic properties of urine and in response to increased fluid intake. Methods: Urine volume, flow rate and constituents were measured in multiple timed specimens from healthy volunteers in a day. Further, subjects were asked to provide specimen over a period of increased fluid intake. Results: A 24-hour specimen missed significant periods of supersaturation in individual urine samples throughout the day. Despite a significant reduction in nocturnal urine flow rate, calcium concentration as well as urine pH and divalent phosphate remained unchanged. Finally, increased water intake did not dilute urine evenly. Conclusion: Mixing multiple urine samples obscures information about periods of increased calcium phosphate precipitation risk over 24 hours. Further, increased fluid intake does not uniformly provide risk protection.

Traumatic lumbar punctures (TLPs) in children with acute lymphoblastic leukemia are associated with a poorer prognosis. The objective of this study was to determine risk factors for TLPs using a retrospective cohort. We compared and contrasted three different regression methods for the analysis of repeated-measures data. In the multivariable model using generalized estimating equations, variables significantly associated with TLPs were age < l year or ≥ 10 years; body mass index percentile ≥ 95; platelet counts < 100 x 103/µL; fewer days since previous LP, and a preceding TLP. The same variables, with similar estimates and confidence-intervals, were identified by the random-effects model. In a fixed-effects model where each patient was used as their own control, days since prior LP and the effect of using image-guidance were significant. Random-effects and GEE lead to similar conclusions, whereas fixed-effects discards between-subject comparisons and leads to different estimates and interpretation of results.

Hip fracture trials have used a wide range of patient-reported outcomes (PRO) suggesting a lack of consensus among clinicians on what are considered the most relevant functional outcomes. We conducted a systematic review to identify the outcomes used in hip fracture randomized controlled trials (RCTs). We hypothesized that there had been an increase in numbers of PROs over time and the health status measure, SF-36, would be used the most. A database search and screening yielded 86 original trials. The mean Detsky score (and standard error) for quality was: 75.8% ± 1.76%. There was a trend in the increase of functional outcome; however, the SF-36 was used only in (10 out of 86) 11.6% trials. Both the ADL-Katz Index and HHS have lower respondent burden than the SF36 which may contribute to their frequent use in hip RCTs. There is a lack of applicable measures suitable for patients with dementia.

Introduction: Despite evidence in favour of cholecystectomy early during first presenting admission for most patients with acute calculous cholecystitis, variation in the timing of cholecystectomy remains evident worldwide. This dissertation characterizes the extent of variation within a large regional healthcare system, as well as addresses gaps in our current understanding of the clinical consequences and costs associated with early versus delayed cholecystectomy for acute cholecystitis. Methods: A population-based retrospective cohort of patients admitted emergently with acute cholecystitis was identified from administrative databases for the province of Ontario, Canada. First, the extent of variation across hospitals in the performance of early cholecystectomy (within 7 days of emergency department presentation) was characterized. Second, among patients discharged without cholecystectomy following index admission, the risk of recurrent gallstone symptoms over time was quantified. Third, operative outcomes of early cholecystectomy were compared to those of delayed cholecystectomy. Finally, a cost-utility analysis compared healthcare costs and quality-adjusted life-year gains associated with three management strategies for acute cholecystitis: early cholecystectomy, delayed cholecystectomy and watchful waiting, where cholecystectomy is performed urgently if recurrent gallstone symptoms arise. Results: The rate of early cholecystectomy varied widely across hospitals in Ontario (median rate 51%, interquartile range 25-71%), even after adjusting for patient characteristics (median odds ratio 3.7). Among patients discharged without cholecystectomy following an index cholecystitis admission, the probability of a gallstone-related emergency department visit or hospital admission was 19% by 12 weeks following discharge. Early cholecystectomy was associated with a lower risk of major bile duct injury (0.28%vs.0.53%, RR=0.53, 95%CI 0.31–0.90, p=0.025). No significant differences were observed in terms of open cholecystectomy (15%vs.14%, RR=1.07, 95%CI 0.99–1.16, p=0.10) or in conversion among laparoscopic cases (11%vs.10%, RR=1.02, 95%CI 0.93–1.13, p=0.68). Early cholecystectomy was on average less costly ($6,905 per person) and more effective (4.20 QALYs per person) than delayed cholecystectomy ($8,511; 4.18 QALYs per person) or watchful waiting ($7,274; 3.99 QALYs per person). Conclusions: Early cholecystectomy offers a benefit over delayed cholecystectomy in terms of major bile duct injury, mitigates the risk of recurrent symptoms, and is associated with the greatest QALY gains at the least cost.

The surgical management of renal masses involves either radical nephrectomy (RN) or partial nephrectomy (PN). The relationship between treatment choice and definitive outcomes of CKD are lacking. Our aim was to examine whether PN is associated with a lower risk of end-stage renal disease (ESRD) requiring renal replacement therapy (RRT). We performed a population-based, retrospective cohort study with data from administrative databases in the province of Ontario, Canada. We included individuals with renal cell carcinoma diagnosed between 1995 and 2010. Cox proportional hazards, propensity score, and competing risks models were used to assess the impact of treatment. PN compared to RN reduces the risk of ESRD in a modern cohort of patients (2003-2010). PN is associated with a lower risk of CKD, reduced cardiac morbidity, and improved overall survival. We provide further evidence for the benefit of PN compared to RN, particularly related to definitive outcomes of renal failure.

The surgical management of renal masses involves either radical nephrectomy (RN) or partial nephrectomy (PN). The relationship between treatment choice and definitive outcomes of CKD are lacking. Our aim was to examine whether PN is associated with a lower risk of end-stage renal disease (ESRD) requiring renal replacement therapy (RRT). We performed a population-based, retrospective cohort study with data from administrative databases in the province of Ontario, Canada. We included individuals with renal cell carcinoma diagnosed between 1995 and 2010. Cox proportional hazards, propensity score, and competing risks models were used to assess the impact of treatment. PN compared to RN reduces the risk of ESRD in a modern cohort of patients (2003-2010). PN is associated with a lower risk of CKD, reduced cardiac morbidity, and improved overall survival. We provide further evidence for the benefit of PN compared to RN, particularly related to definitive outcomes of renal failure.

BACKGROUND: Endothelial dysfunction is a principal player in the development of allograft vasculopathy and allograft failure. The hallmark of endothelial dysfunction is impaired nitric oxide bioavailability. Recent evidence implicates endothelin-1 as an integral component of endothelial dysfunction. Immunosuppressive drugs have also been associated with the development of graft vasculopathy. We speculated that endothelin-1 results in endothelial dysfunction by impairing nitric oxide homeostasis and is a player in hypoxia and reperfusion induced vasomotor injury. In addition, we hypothesized that endothelin-1 antagonism with bosentan will limit hypoxia and reperfusion injury and prevent immunosuppressive drug injury. METHODS: We utilized human saphenous vein endothelial cells to evaluate the effects of endothelin-1, hypoxia and reperfusion on endothelial function, protein kinase modulation and cell survival. We also employed a rodent model of chronic drug therapy to assess the effect of cyclosporine and rapamycin treatment on vasomotor function. We investigated the role of nitric oxide augmentation and bosentan in preventing hypoxia and reperfusion injury and in limiting immunosuppressive drug induced vasomotor dysfunction. RESULTS: Elevated endothelin-1 levels resulted in impaired nitric oxide release and endothelial function. The effects of endothelin-1 as well as hypoxia and reperfusion were mediated by altered protein kinase B and protein kinase C activity resulting in endothelial dysfunction. We revealed that endothelin-1 is a key player in hypoxia and reperfusion induced endothelial injury. The immunosuppressive drug cyclosporine induced vasomotor dysfunction while rapamycin preserved vessel homeostasis. Vasomotor dysfunction was characterized by impaired nitric oxide and endothelin-1 homeostasis. Bosentan limited the deleterious effects of endothelin-1, hypoxic injury, reperfusion injury and cyclosporine induced vasomotor impairment. CONCLUSIONS: Our study revealed that endothelin-1 exposure as well as hypoxia and reperfusion results in endothelial dysfunction by altering specific protein kinase C isoform activities and inhibiting protein kinase B. Cyclosporine induced vasomotor dysfunction was mediated by altered nitric oxide and endothelin-1 homeostasis while rapamycin was endothelial protective. Bosentan proved to be an effective therapy at preventing endothelin-1, hypoxia and reperfusion and cyclosporine induced endothelial dysfunction. Protein kinase C modulation as well as bosentan may prove to be NOVEL therapies to prevent endothelial injury during cardiac transplantation.

This thesis consisted of three major components: 1. A sample of randomized controlled trials of herbal medicines was collected and assessed with a recently developed extension of the CONSORT statement for herbal medicine trials. 2. A methodological review of proposed methods of assessing clinical heterogeneity in meta-analyses of randomized controlled trials, 3. The application of permutation based resampling in meta-regression of multiple covariates. An analysis of 406 RCTs of herbal medicine interventions revealed that these trials are regularly under reporting important aspects of the intervention. Next, the second project showed that there are many resources providing suggestions for investigating clinical heterogeneity in systematic reviews of controlled clinical trials and though there is minimal consensus some recommendations are common across sources. Finally, the third project found that permutation tests result in more conservative, larger, p-values potentially reducing the rate of false positive findings when exploring multiple covariates.