Bone marrow-derived progenitor cell populations have been implicated in tissue regeneration and also in human disease pathology. This thesis investigated the hypothesis that Clara Cell Secretory Protein positive (CCSP+) epithelial-like progenitor cells and circulating fibrocyte numbers are altered in human lung disease and injury, and aimed to determine the predictive value of these cell profiles. It was found that cystic fibrosis patients have an increased number of CCSP+ cells in their bone marrow and peripheral blood, while patients with bronchiolitis obliterans syndrome (BOS) have a decreased number. In addition, BOS and pulmonary fibrosis patients have increased circulating fibrocytes. In response to ischemia reperfusion injury, an increase in CCSP+ cells in the peripheral blood was found at 24 hrs following lung transplant. Lastly, in patients studied greater than 1-year from transplant, those diagnosed with BOS had a higher number of fibrocytes and a loss of CCSP+ peripheral blood cells when compared to patients with stable lung function, with increased fibrocytes being associated with time post-transplant. In these patients, the ratio of fibrocytes-to-CCSP+ cells was predictive of lung function. Multiplex protein arrays were used to investigate corresponding patient plasma, aiming to elucidate key mediators of progenitor cell recruitment. While differences in various cytokines were found between end-stage diseases, a specific relationship between Stem Cell Growth Factor- and CCSP+ cells was identified and between Monocyte Chemotactic Protein-1 and fibrocytes. Conversely, response of CCSP+ cells following transplant appears to be mediated by known mobilizing factors SDF-1 and GM-CSF. Interestingly, in patients followed long-term after transplant, MCP-1 was associated with the number of CCSP+ cells, while SDF-1 correlated with fibrocyte numbers. These observations suggest common pathways acting on both populations that may be altered by the microenvironment, and may further suggest a common origin. This work contributes important information regarding changes in lung progenitor cells and their association with human disease and tissue repair, which could ultimately support future directions that directly advance therapy and improve patient care.

This thesis explores aspects pertinent to the phenotypic classification of paediatric patients with inflammatory bowel disease (IBD). In the current era it has never been more important to have rigourous phenotypic classification to facilitate genotype-phenotype correlation studies as well as to optimize design of clinical trials of emerging therapies, where frequently response may differ according to phenotype of disease. The first study examined the reliability of the Montreal Classification for classifying paediatric IBD patients. This is the first study exploring the reliability of phenotypic classification in a paediatric population. The reliability of assigning an overall diagnosis of type of IBD was good, but not excellent. Amongst Crohn’s disease patients, reliability of assigning disease behaviour was excellent, while the reliability of assigning disease location categories varied from fair to good. The percentage agreement when describing disease extent for ulcerative colitis was high. The second study described the evolution of disease phenotype in a cohort of paediatric IBD patients. Similar to observations in adult-onset IBD, disease location was found to be relatively stable, while Crohn’s disease behaviour evolves from an inflammatory to a stricturing and/or penetrating phenotype in 20% of patients by 5 years of follow-up. The final study explored the association between 2 polymorphisms in the NOD2 gene with the requirement for intestinal resection (a surrogate marker of complicated disease) in models that included and excluded disease duration. Although no difference was found, this may have been influenced by data quality, which was suboptimal. In conclusion, this thesis has demonstrated that imprecision exists in the phenotyping of paediatric IBD patients, in whom phenotypic characteristics evolve over time. It is pertinent that disease duration be considered in any study attempting to make phenotypic correlations.

BACKGROUND: We compared hernia recurrence rates in patients undergoing primary elective inguinal hernia repair at general hospitals with the Shouldice Hospital in Ontario, Canada. METHODS: We conducted an administrative data analysis of persons who underwent inguinal hernia repair in Ontario, Canada from 1993-2007. Risk of recurrent hernia repair was estimated according to hospital type and volume, using Cox proportional-hazards regression models. RESULTS: Recurrence risk in the lowest volume quartile was 5.7%, compared to 3.9% at high volume general hospitals and 1.1% at the Shouldice hospital. Compared to persons who had surgery at the lowest volume hospitals, hernia recurrence among Shouldice Hospital patients was substantially lower after adjustment for confounding variables (hazard ratio 0.18, CI (0.16 to 0.19), P <0.001). CONCLUSIONS: Persons who had elective primary inguinal hernia repair at the Shouldice Hospital had a substantially lower risk of recurrence than those treated at general hospitals, including high volume general hospitals.

Bone marrow-derived progenitor cell populations have been implicated in tissue regeneration and also in human disease pathology. This thesis investigated the hypothesis that Clara Cell Secretory Protein positive (CCSP+) epithelial-like progenitor cells and circulating fibrocyte numbers are altered in human lung disease and injury, and aimed to determine the predictive value of these cell profiles. It was found that cystic fibrosis patients have an increased number of CCSP+ cells in their bone marrow and peripheral blood, while patients with bronchiolitis obliterans syndrome (BOS) have a decreased number. In addition, BOS and pulmonary fibrosis patients have increased circulating fibrocytes. In response to ischemia reperfusion injury, an increase in CCSP+ cells in the peripheral blood was found at 24 hrs following lung transplant. Lastly, in patients studied greater than 1-year from transplant, those diagnosed with BOS had a higher number of fibrocytes and a loss of CCSP+ peripheral blood cells when compared to patients with stable lung function, with increased fibrocytes being associated with time post-transplant. In these patients, the ratio of fibrocytes-to-CCSP+ cells was predictive of lung function. Multiplex protein arrays were used to investigate corresponding patient plasma, aiming to elucidate key mediators of progenitor cell recruitment. While differences in various cytokines were found between end-stage diseases, a specific relationship between Stem Cell Growth Factor- and CCSP+ cells was identified and between Monocyte Chemotactic Protein-1 and fibrocytes. Conversely, response of CCSP+ cells following transplant appears to be mediated by known mobilizing factors SDF-1 and GM-CSF. Interestingly, in patients followed long-term after transplant, MCP-1 was associated with the number of CCSP+ cells, while SDF-1 correlated with fibrocyte numbers. These observations suggest common pathways acting on both populations that may be altered by the microenvironment, and may further suggest a common origin. This work contributes important information regarding changes in lung progenitor cells and their association with human disease and tissue repair, which could ultimately support future directions that directly advance therapy and improve patient care.

This thesis explores aspects pertinent to the phenotypic classification of paediatric patients with inflammatory bowel disease (IBD). In the current era it has never been more important to have rigourous phenotypic classification to facilitate genotype-phenotype correlation studies as well as to optimize design of clinical trials of emerging therapies, where frequently response may differ according to phenotype of disease. The first study examined the reliability of the Montreal Classification for classifying paediatric IBD patients. This is the first study exploring the reliability of phenotypic classification in a paediatric population. The reliability of assigning an overall diagnosis of type of IBD was good, but not excellent. Amongst Crohn’s disease patients, reliability of assigning disease behaviour was excellent, while the reliability of assigning disease location categories varied from fair to good. The percentage agreement when describing disease extent for ulcerative colitis was high. The second study described the evolution of disease phenotype in a cohort of paediatric IBD patients. Similar to observations in adult-onset IBD, disease location was found to be relatively stable, while Crohn’s disease behaviour evolves from an inflammatory to a stricturing and/or penetrating phenotype in 20% of patients by 5 years of follow-up. The final study explored the association between 2 polymorphisms in the NOD2 gene with the requirement for intestinal resection (a surrogate marker of complicated disease) in models that included and excluded disease duration. Although no difference was found, this may have been influenced by data quality, which was suboptimal. In conclusion, this thesis has demonstrated that imprecision exists in the phenotyping of paediatric IBD patients, in whom phenotypic characteristics evolve over time. It is pertinent that disease duration be considered in any study attempting to make phenotypic correlations.

BACKGROUND: We compared hernia recurrence rates in patients undergoing primary elective inguinal hernia repair at general hospitals with the Shouldice Hospital in Ontario, Canada. METHODS: We conducted an administrative data analysis of persons who underwent inguinal hernia repair in Ontario, Canada from 1993-2007. Risk of recurrent hernia repair was estimated according to hospital type and volume, using Cox proportional-hazards regression models. RESULTS: Recurrence risk in the lowest volume quartile was 5.7%, compared to 3.9% at high volume general hospitals and 1.1% at the Shouldice hospital. Compared to persons who had surgery at the lowest volume hospitals, hernia recurrence among Shouldice Hospital patients was substantially lower after adjustment for confounding variables (hazard ratio 0.18, CI (0.16 to 0.19), P <0.001). CONCLUSIONS: Persons who had elective primary inguinal hernia repair at the Shouldice Hospital had a substantially lower risk of recurrence than those treated at general hospitals, including high volume general hospitals.

The brain is a complex dynamic and self-organizing system. Normal brain function emerges from synchronized neuronal firing between local neurons which are integrated into large scale networks via white matter tracts. Normal brain function and consciousness arise from the continual integration and dissolution of neuronal networks, and this fluctuation in synchronization is termed variability. Brain electrical activity is recorded as local field potentials using electroencephalography (EEG). The phase synchrony and variability of EEG waveforms can be quantified. The healthy brain exhibits a relatively low degree of phase synchrony and a high degree of variability. Clinicians are interested in using a complex system approach to brain function to provide dynamic information on neuronal physiology and pathology not available by other evaluation methods. A common challenge in paediatric critical care is evaluation of the comatose child post brain injury. Coma and medical interventions confound the clinical examination making monitoring and prognostication of outcome difficult. Brain cells and white matter tracts are disrupted post injury altering the phase synchrony between neuronal networks. It is proposed in this thesis that the estimation of the variability in EEG phase synchrony can evaluate paediatric brain function. The EEG recordings of normal children and patients in coma post brain injury are used, in a series of studies, to test the main hypothesis that slow EEG wave brain states associated with brain injury have higher magnitudes of EEG phase synchrony and lower variability values than those of EEG waves associated with consciousness. Further, the effects of age, brain development brain and the effect of a conscious slow wave EEG state (hyperventilation) on phase synchrony and variability are evaluated. Results of the studies showed that EEG phase synchrony is increased in all slow wave states and is highest in comatose children with poor neurological outcome. Younger children’s brains have higher phase synchrony than older children. The variability of the EEG phase synchrony differentiates between the awake (higher values) and unconscious states (lower values). Physiologic models underlying EEG phase synchrony are discussed. The EEG phase synchrony and variability measures provide new insight into paediatric brain function.

Due to its significant morbidity, alternatives to autogenous bone grafting are required. Recent research has focused on application of growth factors, including bone morphogenetic proteins (BMPs). Two recombinant human BMP (rhBMP) containing bioimplants, Infuse® (rhBMP-2) and OP-1® (rhBMP-7), are approved for human application. Objective: to provide a direct comparison of the two approved rhBMP containing bioimplants in their clinically available forms. Methods: Activity of rhBMP-2 and -7 were tested using the C2C12 cell based assay comparing alkaline phosphatase (ALP) activity. Activity of Infuse® or OP-1® bioimplants containing 52.5µg of rhBMP-2 or rhBMP-7 were compared using a mouse muscle pouch assay and analyzed with micro CT (mCT) and histology. Results: in vitro: rhBMP-2 induced greater ALP production than rhBMP-7 at different time points. In vivo: OP-1® induced greater bone volume than Infuse® of equivalent quality based on mCT. Conclusions: In the clinically available form, OP-1® induced greater bone volume than Infuse®.

Bisphosphonate-related osteonecrosis of the jaws (BRONJ) occurs subsequent to intravenous and oral bisphosphonate exposure in a small subset of patients. Evidence of concurrent bacterial colonization at sites of bone necrosis, previous reports of neutrophil-related complications in some patients taking bisphosphonates along with perturbed neutrophil function in bisphosphonate-treated mice suggests an innate immune role in the development of bisphosphonate-related osteonecrosis of the jaws. This study investigates neutrophil function in BRONJ patients to determine if neutrophil functional defects may serve as a potential biomarker for BRONJ susceptibility. Patients with BRONJ and patients beginning intravenous pamidronate were studied. Eighteen patients with BRONJ and five patients beginning pamidronate therapy provided oral and blood neutrophil samples. Neutrophils from the population of patients with bisphosphonate-related osteonecrosis of the jaws and from those post-pamidronate treatment showed lower reactive-oxygen species production. These data suggest that a compromise in neutrophil function may be a potential biomarker for BRONJ susceptibility.

Spontaneous subarachnoid hemorrhage (SAH) is a form of hemorrhagic stroke that accounts for approximately 7% of all strokes worldwide. Recently, researchers have gained insight into some possible genetic influences involved in the response to SAH. The goal of this study was to investigate the potential contribution of different mouse genetic backgrounds to brain injury after SAH. SAH was induced in 7 inbred strains of mice, and the degree of large artery vasospasm and brain injury was assessed. After 48 hours, SAH mice showed a significant reduction in middle cerebral artery diameter and increased neuronal injury in the cerebral cortex compared to sham controls. The degree of vasospasm and brain injury varied across strains. This data suggests that vasospasm and neuronal injury may not correlate, and that different genetic factors may influence each one. Future investigations may provide invaluable insight into the causes of these inter-strain differences and potential genetic contributors.