With the recent advent of gene therapy, anti-apoptotic therapy has been receiving spotlight as a potential modality to inhibit the deterioration of pump function in the failing heart. We hypothesized that anti-apoptotic therapy using survivin gene delivery will 1) salvage H9c2 cells exposed to doxorubicin toxicity, and 2) ameliorate the progressive decline in left ventricular function in a rat model of doxorubicin-induced cardiomyopathy. The in vitro data suggested that survivin successfully prevented cell death under doxorubicin stress by both direct and indirect/paracrine mechanisms. Doxorubicin-treated animals developed progressive left ventricular dysfunction as evident by echocardiography and invasive pressure-volume loop analysis, which was prevented by ultrasound-mediated survivin plasmid delivery, but not empty plasmid delivery. Post-mortem analysis of myocardial tissue indicated a lowered apoptotic index in survivin-treated hearts, with evidence of decrease in interstitial fibrosis. In conclusion, survivin gene therapy was shown to ameliorate doxorubicin-induced cardiomyopathy, by decreasing apoptosis and preventing adverse remodeling.

This thesis investigated the role of CBF assessment in the management of stroke in children with sickle cell disease (SCD). It is divided into two parts. In the first part, a systematic review of CBF assessment using different imaging modalities in SCD children was designed. The prevalence of CBF abnormalities was found to be equal to or higher than those of structural MRI and transcranial Doppler (TCD) in SCD children who have not experienced stroke. Studies reviewed suggested CBF assessment in SCD could aid in addressing brain abnormalities at the tissue level. In the second part, the arterial spin labeling (ASL) technique was used to depict CBF abnormalities in SCD children. ASL demonstrated perfusion abnormalities that seem to remain invisible in TCD measurements; CBF interhemispheric asymmetries were associated with clinically silent infarctions with no corresponding flow velocity interhemispheric asymmetries assessed with TCD.

Activated coagulation Factor XII (FXIIa) elevates blood pressure (BP) acutely by stimulating adrenomedullary catecholamine (CA) release in Brown Norway (BN) bioassay rats. These effects are absent in kininogen-deficient BN Katholiek (BNK) bioassay rats, indicating that these FXIIa-induced responses require an intact kallikrein-kinin system (KKS). In three hypertensive anephric pediatric patients, ΔFXIIa concentrations tracked peri-dialytic ΔBP. We hypothesized that FXIIa exerts a vasoconstrictor pro-hypertensive action, via the KKS, particularly evident in chronic renal failure (CRF). In CRF patients (n=11) receiving conventional hemodialysis, mean plasma FXIIa concentrations were 3-fold (p<0.05) greater than in healthy controls. Although conversion from conventional to nocturnal hemodialysis did not change mean FXIIa concentrations there was intra-session variation within individuals, such that ΔFXIIa concentrations correlated with changes in mean arterial pressure (MAP, r=0.66, p=0.026) and total peripheral resistance (TPR, r=0.75, p=0.007). In normotensive BN rats, FXIIa infusion (85 ng/min/kg for 60 mins) increased MAP (10±1 mmHg), TPR (0.5±0.1 Units), and CA, whilst left-ventricular end-diastolic volume (LVEDV) and heart rate decreased (all p<0.05). After adrenalectomy, FXIIa infusion decreased MAP (5±1 mmHg), did not raise CA or induce sustained vasoconstriction, and caused a greater fall in LVEDV (all p<0.05). In the 5/6 nephrectomized (NX) rodent CRF model, MAP and TPR were significantly greater in BN NX (n=16) than in BNK NX (n=15) (147±4 vs. 133±2 mmHg, 2.8±0.2 vs. 2.3±0.2 Units; all p<0.05). Plasma FXIIa measured using our semi-quantitive ELISA was 3-fold higher in both BN NX and BNK NX than in controls (p<0.01), but only correlated with MAP (r=0.48, p=0.01) in the BN NX. Plasma CA were elevated in the BN NX (p<0.05) but not in BNK NX. Infusion of a specific FXIIa inhibitor into BN NX decreased MAP (-12 mmHg) and TPR (-0.5 Units) proportionally to baseline FXIIa (ΔMAP: r=-0.72, p=0.002; ΔTPR: r=-0.57, p=0.021), and plasma CA fell by 40-67% (all p<0.05). No such changes occurred in the BNK NX. In summary, a significant component of the hypertension of CRF can be attributed to FXIIa-induced vasoconstriction mediated via the KKS and stimulated CA release. In normal rats, FXIIa appears also to directly or indirectly decrease preload and heart rate.

Chronic kidney disease (CKD) and its cardiac complications represent a large and growing problem in Canada. The progression of CKD is driven by the activation of several final common pathways of injury, including fibrosis and oxidative stress. If left unchecked, these inter-connected processes lead to progressive damage and subsequent organ dysfunction. Current clinical therapies, consisting of aggressive blood pressure control and blockade of the renin-angiotensin system, fail to arrest this progressive injury in a significant number of patients. Early outgrowth cells (EOCs) represent a novel bone marrow-derived cell population that have been recently described to have tissue protective activity. In this work, we examined the effects of intravascular EOC infusion in two independent models of CKD, demonstrating potent anti-fibrotic renoprotective effects in the subtotally nephrectomized (SNX) rat, a well-established model of non-diabetic progressive CKD, and anti-fibrotic and anti-oxidant effects in the db/db mouse, a commonly used model of type 2 diabetic nephropathy. In the SNX rat, which is characterized by impaired cardiac relaxation reminiscent of a common and high risk clinical CKD phenotype, EOC infusion was also associated with improved cardiac structure and function. In both cases, infused EOCs were not retained in significant numbers within the diseased kidney or heart, but rather localized to distant organs such as the liver, spleen, and bone marrow. We further demonstrated that EOCs release soluble factors with anti-oxidant and anti-fibrotic activity in vitro, and that a cell-free preparation of EOC-derived factors can mimic the reno- and cardiac protective effects of the cells themselves when infused into the SNX rat. Taken together, our results demonstrate the therapeutic potential of an EOC-based strategy for the treatment of CKD and its cardiac complications, and provide the preclinical rationale for the design of clinical trials of EOC-based therapies for this devastating disease.

Pre-B Cell Colony-Enhancing Factor (PBEF) is a cytokine-like molecule that functions as a nicotinamide phosphoribosyl transferase (Nampt) in a salvage pathway of NAD biosynthesis. PBEF has well-characterized activity as an extracellular inflammatory mediator and has been proposed to signal through the insulin receptor (IR). As neutrophils are key effectors of the innate immune response to infection and injury, we hypothesized that PBEF promotes pro-inflammatory function in neutrophils and that these pro-inflammatory effects may occur through interactions with the neutrophil IR or through PBEF’s enzymatic Nampt activity. Our studies focused on two important facets of neutrophil inflammatory function: their ability to generate reactive oxygen species (ROS) and undergo constitutive apoptosis. We found that, although PBEF does not activate oxidative burst on its own, it primes for ROS generation through the NADPH oxidase. PBEF promotes membrane translocation of cytosolic NADPH oxidase subunits p40phox and p47phox, but not p67phox, induces p40phox phosphorylation and activates Rac. Priming, translocation and phosphorylation are dependent on activation of p38 and ERK mitogen activated protein kinases. PBEF priming of neutrophils occurs independent of its Nampt capacity or of interactions with IR. We next investigated the effects of PBEF on neutrophil constitutive apoptosis. Our lab previously established that extracellular PBEF delays neutrophil apoptosis. Accordingly, we next investigated the mechanism through which this delay was occurring. PBEF-induced delayed apoptosis was enhanced in the presence of Nampt substrates, and NAD alone could delay apoptosis to an extent comparable to PBEF. Delayed apoptosis was blocked by a Nampt inhibitor and was lacking when a mutated PBEF deficient in Nampt activity was utilized. The cell-surface NAD glycohydrolase, CD38, can convert NAD to cyclic ADP-ribose (cADPR). Blocking CD38 activity with a blocking antibody partially reversed the delay of apoptosis induced by PBEF in conjunction with its substrates, and delayed apoptosis could be achieved by addition of the CD38 product cADPR. Finally, we found that delayed apoptosis induced by PBEF did not involve IR. These results indicate that PBEF can prime for enhanced oxidative burst and delay apoptosis in neutrophils, and that these phenomena occur through distinct mechanisms.

Familial Pancreatic Cancer (FPC) is a cancer syndrome characterized by clustering of pancreatic cancer in families, but most FPC cases do not have a known genetic etiology. Understanding genetic predisposition to pancreatic cancer is important for improving screening as well as treatment. The central aim of this thesis is to identify candidate susceptibility genes for FPC, and I used three approaches of increasing resolution. First, based on a candidate-gene approach, I hypothesized that BRCA1 is inactivated by loss-of-heterozygosity in pancreatic adenocarcinoma of germline mutation carriers. I demonstrated that 5/7 pancreatic tumors from BRCA1-mutation carriers show LOH, compared to only 1/9 sporadic tumors, suggesting that BRCA1 inactivation is involved in tumorigenesis in germline mutation carriers. Second, I hypothesized that the germline genomes of FPC subjects differ in copy-number profile from healthy genomes, and that regions affected by rare deletions or duplications in FPC subjects overlap candidate tumor-suppressors or oncogenes. I found no significant difference in the global copy-number profile of FPC and control genomes, but I identified 93 copy-number variable genomic regions unique to FPC subjects, overlapping 88 genes of which several have functional roles in cancer development. I investigated one duplication to sequence the breakpoints, but I found that this duplication did not segregate with disease in the affected family. Third, I hypothesized that in a family with multiple pancreatic cancer patients, genes containing rare variants shared by the affected members constitute susceptibility genes. Using next-generation sequencing to capture most bases in coding regions of the genome, I interrogated the germline exome of three relatives who died of pancreatic cancer and a relative who is healthy at advanced age. I identified a short-list of nine candidate genes with unreported mutations shared by the three affected relatives and absent in the unaffected relative, of which a few had functional relevance to tumorigenesis. I performed Sanger sequencing to screen an unrelated cohort of approximately 70 FPC patients for mutations in the top two candidate genes, but I found no additional rare variants in those genes. In conclusion, I present a list of candidate FPC susceptibility genes for further validation and investigation in future studies.

Norfloxacin For Hepatopulmonary Syndrome: A Pilot Study of a Rare Disease Samir Gupta, Masters of Science, 2008 Graduate Department of Health Policy, Management and Evaluation University of Toronto Introduction: Hepatopulmonary Syndrome is a rare disease characterized by abnormal gas-exchange and a poor prognosis, with no known effective medical therapy. A rat model and preliminary human data suggest that this disease may be caused by intestinal bacterial overgrowth, systemic endotoxemia and increased nitric oxide. Methods: We conducted a pilot crossover randomized controlled trial of norfloxacin versus placebo over four weeks, in seven subjects with HPS or a milder condition called pre-HPS, with a primary outcome of alveolar-arterial oxygen gradient (AaDO2). Results: There was no trend toward improved AaDO2, this outcome and other intermediate outcomes were highly variable, and results suggested that a longer treatment course might be necessary. We identified multiple obstacles to recruitment. Conclusion: We believe that a full-scale study of norfloxacin therapy for HPS will require 1) a six-month therapeutic period, 2) more specific HPS diagnostic criteria for clinical and study populations, and 3) creative recruitment maneuvers.

Objective to systematically review randomized trials that compared methotrexate (MTX) monotherapy to MTX in combination with other non-biologic disease-modifying anti-rheumatic Drugs (DMARD) and to compare the performances of PubMed versus MEDLINE (Ovid®) and EMBASE. Methods We performed a systematic review of randomized trials comparing MTX alone and MTX in combination with other non-biologic DMARDs. Heterogeneity was investigated and explored. The performances of Pubmed and MEDLINE were evaluated. The EMBASE unique trials were identified and investigated. Results A total of 19 trials were included and grouped by the type of patients randomized. Trials in DMARD naive patients showed no significant advantage of the MTX combination versus monotherapy. The recall was 85% vs. 90% for Ovid and PubMed, respectively, while the precision and number-needed-to read of Ovid and Pubmed were comparable. Only 23% of trials were EMBASE unique trials Conclusions In DMARD naive patients, the balance of efficacy/toxicity favours MTX monotherapy.

An important mechanism involved in the pathogenesis of type 2 diabetes is elevation of plasma free fatty acids which induce insulin resistance and may impair both β-cell function and mass (β-cell lipotoxicity). The objective of my thesis was to investigate the role of oxidative stress in β-cell lipotoxicity, using in vivo, ex vivo, and in vitro models. I used in vivo models of 48h i.v. oleate or olive oil infusion in Wistar rats followed by hyperglycemic clamps, or islet secretion studies ex vivo, and in vitro models of 48h exposure to oleate in isolated islets. My first study showed that 48h oleate infusion decreased the insulin response to a hyperglycemic clamp, an effect prevented by coinfusion of the antioxidants N-acetylcysteine and taurine. Similar to the findings in vivo, 48h infusion of oleate decreased glucose stimulated insulin secretion (GSIS) ex vivo, and induced oxidative stress in isolated islets, effects prevented by coinfusion of the antioxidants N-acetylcysteine, taurine, or tempol. Islets exposed to oleate or palmitate showed a decreased insulin response to high glucose and increased levels of oxidative stress, effects prevented by taurine. Therefore, my data are the first demonstration that oxidative stress plays a role in the decrease in β-cell secretory function induced by prolonged exposure to FFA, in vitro and in vivo. My second study addressed downstream effects of oxidative stress involving inflammation. A 48h infusion of oleate or olive oil decreased β-cell function during a hyperglycemic clamp, an effect prevented by coinfusion of the IKKβ inhibitor salicylate. GSIS in isolated islets was impaired by olive oil or oleate and restored by salicylate. These results suggest a potential role for both oxidative stress and inflammation in lipid-induced β-cell dysfunction. My third study addressed downstream effects of oxidative stress involving β-cell insulin signalling. A 48h infusion of oleate or olive oil decreased β-cell function during a hyperglycemic clamp, an effect prevented by coinfusion of the tyrosine phosphatase inhibitor bisperoxovanadate. GSIS in isolated islets was impaired by olive oil or oleate and restored by bisperoxovanadate, suggesting a role of FFA in decreasing β-cell function by induction of β-cell insulin resistance.

Degenerative disease of the lumbar spine (DDLS) is a common condition for which surgery is beneficial in selected patients. Wide variation in surgical referral and rates of surgery has been observed contributing to unequal access to care. Our objectives were to examine (1) the variation in preferences for referral and surgery among surgeons, family physicians (FPs) and patients, (2) how FP referral practices compare with preferences and guideline recommendations, and (3) how the ‘enthusiasm’ of patients and physicians influence regional variation in surgical rates. We used conjoint analysis in a mailed survey to elicit preferences based on clinical vignettes from surgeons, FPs and patients. A Delphi expert panel provided consensus guideline recommendations for surgical referral to compare with actual FP referral practices. Rates of surgery for DDLS, obtained from Ontario hospital discharge data, were used to quantify regional variation and regression models assessed the relationship with patient and physician enthusiasm. We identified significant differences in preferences for referral and surgery between patients, FPs and surgeons. Surgeons placed high importance on leg-dominant symptoms while patients had high importance for quality of life symptoms (i.e. severity, duration, walking tolerance). Surgical referral practices were poorly predicted by individual FP preferences and guideline recommendations based on clinical factors alone. Variation in Ontario surgical rates was higher than that of hip or knee replacements and was highly associated with the enthusiasm of surgeons (p<0.008), rather than FPs or patients. By appreciating the variation in preferences between patients and physicians, and exploring other non-clinical factors that influence referrals, we may be able to improve the efficiency of referrals and enhance the shared decision making process. With an understanding of the influence that surgeons have in driving variation in surgical rates, further research may allow us to direct strategies to improve access and allow for a more equitable delivery of care for patients suffering from DDLS.