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.

Objective: To develop a score for hematoma expansion prediction, identify predictors of outcome, and validated existing prognostic scores in acute intracerebral hemorrhage (ICH). Methods: Data were obtained from a prospective multicenter ICH cohort. Patients were evaluated with computed tomography (CT) angiography, 24-hour CT, and 3-month outcome. Multivariable regression identified predictors of 24-hour expansion and 3-month outcome and a score was developed for expansion prediction. Existing scores were evaluated in the dataset. Results: The PREDICT Hematoma Expansion Score predicts 24-hour expansion based on number of spots, stroke severity, time from onset, and coagulopathy (c-statistic: 0.803). Prediction was improved over spot sign presence (p<0.001) and clinical variables alone (p=0.002). Existing scores for hematoma expansion and clinical outcome demonstrated reduced discrimination compared with the original reports. Conclusion: The PREDICT Hematoma Expansion Score may aid patient selection for future interventions aimed at reducing expansion however external validation is required. Existing scores demonstrated reduced discrimination.

In search of thyroid carcinoma biomarkers, proteins secreted by thyroid cancer cell lines, papillary-derived TPC-1 and anaplastic-derived CAL62, were analyzed using liquid chromatography-tandem mass spectrometry. Of forty six high-confidence identifications, six proteins were considered for verification in thyroid cancer patients’ tissues and blood. The localization of two proteins, nucleolin and prothymosin-alpha (PTMA), was confirmed in TPC-1 and CAL62 by confocal microscopy and immunohistochemically in xenografts of TPC-1 cells and human thyroid carcinomas. Increased nuclear and cytoplasmic expression of PTMA was observed in anaplastic carcinomas compared to normal thyroid tissues, papillary and poorly differentiated carcinomas. Importantly, six proteins were detected in thyroid cancer patients’ sera, warranting future analysis to confirm their potential as blood-based thyroid cancer markers. Herein we demonstrate the ability of secretome analysis of thyroid cancer cell lines to identify proteins that may be studied for application in management of thyroid carcinomas upon future validation.

In search of thyroid carcinoma biomarkers, proteins secreted by thyroid cancer cell lines, papillary-derived TPC-1 and anaplastic-derived CAL62, were analyzed using liquid chromatography-tandem mass spectrometry. Of forty six high-confidence identifications, six proteins were considered for verification in thyroid cancer patients’ tissues and blood. The localization of two proteins, nucleolin and prothymosin-alpha (PTMA), was confirmed in TPC-1 and CAL62 by confocal microscopy and immunohistochemically in xenografts of TPC-1 cells and human thyroid carcinomas. Increased nuclear and cytoplasmic expression of PTMA was observed in anaplastic carcinomas compared to normal thyroid tissues, papillary and poorly differentiated carcinomas. Importantly, six proteins were detected in thyroid cancer patients’ sera, warranting future analysis to confirm their potential as blood-based thyroid cancer markers. Herein we demonstrate the ability of secretome analysis of thyroid cancer cell lines to identify proteins that may be studied for application in management of thyroid carcinomas upon future validation.

Objective: To develop a score for hematoma expansion prediction, identify predictors of outcome, and validated existing prognostic scores in acute intracerebral hemorrhage (ICH). Methods: Data were obtained from a prospective multicenter ICH cohort. Patients were evaluated with computed tomography (CT) angiography, 24-hour CT, and 3-month outcome. Multivariable regression identified predictors of 24-hour expansion and 3-month outcome and a score was developed for expansion prediction. Existing scores were evaluated in the dataset. Results: The PREDICT Hematoma Expansion Score predicts 24-hour expansion based on number of spots, stroke severity, time from onset, and coagulopathy (c-statistic: 0.803). Prediction was improved over spot sign presence (p<0.001) and clinical variables alone (p=0.002). Existing scores for hematoma expansion and clinical outcome demonstrated reduced discrimination compared with the original reports. Conclusion: The PREDICT Hematoma Expansion Score may aid patient selection for future interventions aimed at reducing expansion however external validation is required. Existing scores demonstrated reduced discrimination.

Both CD200 and its receptor(s), CD200R(s), are type I membrane glycoproteins belonging to the immunoglobulin (Ig) supergene family. CD200:CD200R(s) interaction manipulates host immunity in multiple models, including those exploring allograft rejection, autoimmune disease, tumor development, spontaneous fetal loss, infection/inflammation, and virus infection. The studies described in this thesis were focused on investigation possible mechanism(s) involved in CD200-mediated regulation, using transgenic mice over-expressing CD200, and exploring models of skin allograft rejection and LPS-induced abortion in mice. A Tet-on system was chosen to create CD200tg mice (rtTA CD200tg animal line), in which transgenic expression of CD200 is induced by the presence of doxycycline (Dox-treated mice). Splenocytes from Dox-treated transgenic mice, used as either responder cells or stimulator cells in mixed leukocyte cultures, showed antigen-specific suppressed lymphocyte proliferation and induction of CTL. Although enhanced survival of skin allografts was achieved in Dox-treated transplanted CD200tg mice (BALB/c to BL/6 Tg), all grafts were rejected by 28 days post transplantation (see chapter 2). A superior “second generation” Tet-on system was used to create a new CD200tg animal referred to as rtTA2s-M2 CD200tg mice. Transgenic overexpression of CD200 in this mouse was stably induced at much lower Dox concentrations, with less (or no) “leaky” expression of the transgene in the absence of Dox. Using these mice in an LPS-induced murine abortion model, transgenic expression of CD200 was found to reduce the LPS-induced abortion rate from ~49% to 6% (see chapter 3). Long term increased survival of grafted tissues (of both cardiac and skin allografts) was achieved using the rtTA2s-M2 CD200tg mice as recipients. To explore the potential molecular mechanism(s) involved in this allograft tolerance, a commercial microarray kit focusing on detecting altered expression of genes related with T-cell anergy/tolerance was used to investigate the gene expression profile in grafted tissue of mice with transgenic expression of CD200. Expression of genes associated with Foxp3+ regulatory T-populations (Foxp3, CTLA4 and GITR) and type 2 cytokine genes showed increased expression in CD200tg recipients. With particular note in regards to Foxp3+ regulatory T cells, expression of the gene encoding chemokine receptor CCR4, reported to play a key role in attracting Foxp3+ regulatory T cells to grafted tissues and DLNs, was found to be increased in Dox-treated CD200tg recipients, along with genes encoding chemokines CCL22/17, the ligands for CCR4. Immunochemistry staining also showed increased numbers of Foxp3+ cells in both grafted skin tissues and the DLNs of transplant at day 14 post transplantation. Using CCR4-shRNA lentivirus administered to Dox-treated CD200tg recipients to block expression of CCR4, the transgene-induced increased presence of regulatory T cell populations in grafted tissues and DLNs was attenuated, along with loss of enhanced skin graft survival and the histological appearance of graft acceptance (see chapter 4). These data provide support for a model suggesting that altered migration of Treg mediated through a CCR4:CCL17/22 pathway is an important mechanism underlying increased allograft acceptance following CD200tg expression.

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.

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.

Juvenile GM2 gangliosidosis (jGM2) is a group of inherited neurodegenerative diseases caused by deficiency of lysosomal β-hexosaminidase A (Hex A) resulting in GM2 ganglioside accumulation in brain. Like many other lysosomal storage diseases (LSDs), no specific treatment currently exists. In order to establish clinical outcomes for the investigation of potential therapies for jGM2, I collected comprehensive information on the natural history of the condition by studying retrospective and prospectively a cohort of 21 patients with the disease, and reviewing previously published reports of 134 patients. Several symptoms at disease onset, symptom latencies, and the survival curve were described. Genotype-phenotype correlations and neuroradiological findings were also studied. Based on pre-clinical results in animal models, we studied substrate reduction therapy (SRT), with miglustat, in a phase I/II clinical trial to assess its pharmacokinetics (PK), safety, tolerability in infantile and jGM2. Miglustat showed a PK profile similar to the one found in adult patients. The drug was found to be safe and well-tolerated in patients with jGM2, with diarrhea and weight loss being the most common drug-related adverse events. The analysis of efficacy showed that SRT was unable to arrest the full neurological progression of the condition; however, relative stabilization of cognitive function was noted, which was consistent with brain MRI findings. Because of the limited efficacy obtained with SRT, enzyme-enhancement therapy was considered to be an attractive alternative therapy for the late onset forms of GM2 gangliosidosis. Screening of a FDA-approved library of approved therapeutic compounds resulted in the identification of pyrimethamine, as a potential pharmacological chaperone for mutant forms of Hex A. Relative enhancements of enzyme activity and protein levels were observed in patient cells treated with therapeutic concentrations of drug. Applying the same principles, ambroxol was identified as a potential PC for mutant glucocerebrosidase (GCC), the lysosomal enzyme that when deficient causes Gaucher disease (GD). Significant increases of residual mutant GCC were observed in cultured patients cells with type 1 GD. In conclusion, principles developed in the course of studies on jGM2 were shown to be useful for the investigation of novel small-molecule therapies for LSDs, associated with significant neurodegeneration.

Both CD200 and its receptor(s), CD200R(s), are type I membrane glycoproteins belonging to the immunoglobulin (Ig) supergene family. CD200:CD200R(s) interaction manipulates host immunity in multiple models, including those exploring allograft rejection, autoimmune disease, tumor development, spontaneous fetal loss, infection/inflammation, and virus infection. The studies described in this thesis were focused on investigation possible mechanism(s) involved in CD200-mediated regulation, using transgenic mice over-expressing CD200, and exploring models of skin allograft rejection and LPS-induced abortion in mice. A Tet-on system was chosen to create CD200tg mice (rtTA CD200tg animal line), in which transgenic expression of CD200 is induced by the presence of doxycycline (Dox-treated mice). Splenocytes from Dox-treated transgenic mice, used as either responder cells or stimulator cells in mixed leukocyte cultures, showed antigen-specific suppressed lymphocyte proliferation and induction of CTL. Although enhanced survival of skin allografts was achieved in Dox-treated transplanted CD200tg mice (BALB/c to BL/6 Tg), all grafts were rejected by 28 days post transplantation (see chapter 2). A superior “second generation” Tet-on system was used to create a new CD200tg animal referred to as rtTA2s-M2 CD200tg mice. Transgenic overexpression of CD200 in this mouse was stably induced at much lower Dox concentrations, with less (or no) “leaky” expression of the transgene in the absence of Dox. Using these mice in an LPS-induced murine abortion model, transgenic expression of CD200 was found to reduce the LPS-induced abortion rate from ~49% to 6% (see chapter 3). Long term increased survival of grafted tissues (of both cardiac and skin allografts) was achieved using the rtTA2s-M2 CD200tg mice as recipients. To explore the potential molecular mechanism(s) involved in this allograft tolerance, a commercial microarray kit focusing on detecting altered expression of genes related with T-cell anergy/tolerance was used to investigate the gene expression profile in grafted tissue of mice with transgenic expression of CD200. Expression of genes associated with Foxp3+ regulatory T-populations (Foxp3, CTLA4 and GITR) and type 2 cytokine genes showed increased expression in CD200tg recipients. With particular note in regards to Foxp3+ regulatory T cells, expression of the gene encoding chemokine receptor CCR4, reported to play a key role in attracting Foxp3+ regulatory T cells to grafted tissues and DLNs, was found to be increased in Dox-treated CD200tg recipients, along with genes encoding chemokines CCL22/17, the ligands for CCR4. Immunochemistry staining also showed increased numbers of Foxp3+ cells in both grafted skin tissues and the DLNs of transplant at day 14 post transplantation. Using CCR4-shRNA lentivirus administered to Dox-treated CD200tg recipients to block expression of CCR4, the transgene-induced increased presence of regulatory T cell populations in grafted tissues and DLNs was attenuated, along with loss of enhanced skin graft survival and the histological appearance of graft acceptance (see chapter 4). These data provide support for a model suggesting that altered migration of Treg mediated through a CCR4:CCL17/22 pathway is an important mechanism underlying increased allograft acceptance following CD200tg expression.