Introduction: The objective of this study is to investigate the role of osteoblasts in the pathophysiology of premature suture fusion in infants. Methods: Bone and periosteal tissue from fused and patent cranial sutures and adjacent bone were harvested from infants undergoing surgery for craniosynostosis and used to develop primary osteoblast cell cultures. Dural tissue was obtained from neurosurgical procedures in order to generate an osteoblast-dural co-culture. Osteoblast proliferation, differentiation, mineralization, protein expression (Noggin, BMP3 and Runx2) and response to exogenous FGF2 stimulation were assessed. Results: Cell cultures demonstrated significant (p<0.05) regional variations in osteoblast proliferation, differentiation markers and in vitro bone nodule formation. The expression of anti-osteogenic molecules (Noggin and BMP3) was decreased in osteoblasts from fused suture regions. Conclusion: The creation of a pro-osteogenic environment through the decreased expression of anti-osteogenic signalling molecules and increased expression of osteogenic factors may be responsible for premature suture fusion in infants.

Subarachnoid hemorrhage (SAH) comprises 7% of all stroke cases, and is associated with a disproportionately high morbidity and mortality with few therapeutic options available. The goal of this project was to understand the mechanism of neurological deterioration after experimental SAH, with a focus on cerebral vasospasm and brain injury after SAH. We tested the hypothesis that endothelial nitric oxide synthase (eNOS) is upregulated and uncoupled after SA, resulting in exacerbated neurological injury in a mouse model of SAH. The project entailed the investigation of eNOS-dimer uncoupling, its association with oxidative and nitrosative stress in the brain parenchyma and finally its association with secondary complications after SAH. In our studies we demonstrated the crucial role eNOS plays in anti-microthromboembolism, anti-apoptosis and maintenance of physiological superoxide (O2-)/NO balance. This study suggests that SAH up-regulates and disrupts eNOS, producing peroxynitrite (OONO-) and other radicals that further exacerbate the oxidative insult and neurological injury.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease (CHD). The understanding of the genetics and inheritance of TOF is limited. Although about 15% of cases are associated with a 22q11.2 deletion, the majority have no known aetiology. Even in 22q11.2 Deletion Syndrome (22q11DS), factors that increase the likelihood of CHD expression are poorly understood. We aimed to determine the prevalence and phenotypes of CHD in relatives of adults with TOF. We also investigated the prevalence of CHD in relatives without a 22q11.2 deletion of individuals with 22q11DS. Offspring of patients with TOF had the greatest prevalence of CHD. Diverse cardiac phenotypes, including left heart obstructive lesions, were found in families. We also found that unaffected relatives of individuals with 22q11DS had a greater prevalence of complex CHD over population expectations, suggesting that modifier genetic factors may be involved in expression of CHD in 22q11DS.

BACKGROUND: MRI is increasingly being used for rectal cancer staging. The purpose of this study was to summarize published evidence to determine the accuracy of MRI for T-category, lymph node (LN) metastases, and circumferential resection margin (CRM) involvement in rectal cancer. METHODS: Sensitivity, specificity, and diagnostic odds ratios (DOR) were estimated using hierarchical summary receiver operating characteristics modeling and bivariate random effects modeling. RESULTS: MRI was more specific for CRM (94%, 95%CI 88-97) than for T-category (75%, 95%CI 68-80) and LN’s (71%, 95%CI 59-81) but was more sensitive for T-category (87%, 95%CI 81-92) than for CRM (77%, 95%CI 57-90) and LN’s (77%, 95%CI 69-84). DOR was higher for CRM (56.1, 95%CI 15.3-205.8) than for LN’s (8.3, 95%CI 4.6-14.7) and T-category (20.4, 95%CI 11.1-37.3). CONCLUSIONS: MRI has good accuracy for both CRM and T-category and should be considered for preoperative rectal cancer staging. In contrast, LN assessment is poor on MRI.

Normal endothelial cell function is controlled in part by a tightly regulated balance between angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and prothrombotic. Since an activated endothelial phenotype is seen in invasive group A streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis, we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore represent a shared final common pathway to endothelial activation as well as a clinically useful prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.

Background: There is an unacceptable delay in diagnosis of axial Spondyloarthritis (axSpA) especially in its early stages among patients with inflammatory bowel disease (IBD). Objective: to develop a sensible and reliable questionnaire to identify undetected axSpA among IBD patients. Methods: Candidate items for the questionnaire were selected on 3 domains (IBD, inflammatory back symptoms, and extra-axial features). Sensibility of the Toronto axSpA Questionnaire (TASQ) was assessed leading to drafting 18 items. Test-retest reliability study was conducted among 77 patients with established IBD and axSpA and kappa agreement coefficients were calculated for items. Results: The TASQ was developed using multiple steps of sensibility assessment resulting in 16 items. Kappa coefficients ranged from 0.81 to 1.00 for all items indicating almost perfect agreement. Conclusion: TASQ is a newly developed, sensible and reliable questionnaire that should facilitate identification and referral of IBD patients to rheumatologists and should avoid delay in diagnosis of axSpA.

Diffusion Tensor Imaging (DTI) can quantify tissue integrity in normal-appearing white matter (NAWM). NAWM abnormalities present at the earliest time point implicate neurodegeneration operative from the outset of multiple sclerosis (MS). DTI scans were obtained at first attacks from 6 children later diagnosed with MS and 6 children with monophasic demyelination, and from 6 controls, matched for age. DTI scans were also obtained from 22 children with established MS with clinical onset before age 12 years and compared to age-matched controls. Atlas- and tractography-based image processing methods were utilized. DTI metrics distinguished MS patients from patients with monophasic demyelination and from controls at the first attack. Differences in NAWM between children with established early-onset MS and controls were only notable when DTI was obtained in adolescence. DTI provides valuable insights into NAWM in children with MS, although in the youngest patients such changes may require time to develop.

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.