Survivors of subarachnoid hemorrhage (SAH) often have learning and memory deficits. This study tested the hypothesis that SAH in rats is associated with similar deficits and that they are due to neuronal injury in the hippocampus. SAH was induced in rats. Behaviour was investigated in the Morris water maze and brain injury by microscopy. Rats with SAH had deficits in spatial learning and working memory and had significantly more fluoro-Jade- and TUNEL-positive neurons in the hippocampus, cerebral cortex and cerebellum. Microthromboemboli in microvessels were more frequent in brains of rats with SAH and deficits there was vasospasm of the anterior and middle cerebral arteries. The amount of cell death in the hippocampus did not appear to be sufficient to cause the observed in the Morris water maze. This suggests that other factors such as dysfunction of neurotransmission or other pathology in hippocampal pathways might contribute to the impairment.

Severe influenza, recognized as a clinical syndrome characterized by hyper-induction of pro-inflammatory cytokine production, results in approximately 250–500 thousand deaths annually worldwide. Current influenza research is focused on therapeutics to target the influenza virus or modulate influenza virus-induced inflammation as potential treatment options to improve clinical outcome in experimental influenza A (H1N1) virus infection. The goals of this work were: (1) to evaluate the utility of inhaled nitric oxide (iNO) for decreasing influenza virus production in the lungs, and (2) investigate the use of mesenchymal stromal (stem) cells (MSCs) for mitigating deleterious host responses to influenza infection. Here, we report that MSCs and iNO, administered alone either prophylactically or post-influenza virus infection, fail to modulate host inflammation, fail to improve acute lung injury, fail to dampen lung viral load, and fail to improve survival of infected mice.

HLA-G is a protein normally expressed during pregnancy, protecting the fetus from the maternal immune system. Previous studies have shown an association between HLA-G expression post-transplantation and lower incidences of organ rejection. To further examine this beneficial role, we conducted a prospective study following a cohort of heart transplant recipients for one year and measuring their plasma HLA-G levels at various time points. Preliminary analyses failed to reveal an association between HLA-G and various parameters of rejection and vasculopathy. However, we decided to examine the in vitro effects of HLA-G in a smooth muscle cell (SMC) migration assay and whether HLA-G can be modulated pharmacologically. We made the novel observations that purified HLA-G was capable of inhibiting migration of SMCs, a key event in the development of cardiac allograft vasculopathy. IL-10, an anti-inflammatory cytokine, was capable of upregulating HLA-G in a Jeg-3 cell line. The modulation of HLA-G may represent a strategy to protect again vasculopathy, which is a leading cause of morbidity and mortality in heart transplant recipients.

Intracerebral hemorrhage (ICH) results in secondary brain injury caused partially by blood and its metabolites. Survivors of ICH are often left with severe disabilities, therefore, decreasing the extent of this secondary injury may improve functional outcome of patients. Incubation of mouse brain slices with partially oxidized bilirubin, a neurotoxic blood breakdown product, caused a dose- and time-dependent decrease in axonal function, suggesting a reduced number of conducting myelinated axons. These effects did not occur when tissue was incubated with non-oxidized bilirubin. Injection of bilirubin into the corpus callosum of mice caused functional impairment of unmeylinated axons; however, immunohistochemical staining of the tissue showed evidence of structural damage to both oligodendrocytes and axons. This data provides evidence for functional and structural damage to white matter in the presence of partially oxidized bilirubin Therefore, diminishing the duration of presence of bilirubin and its oxidation in the brain warrants study as a means of decreasing secondary brain injury after ICH.

Spinal cord injury (SCI) is a debilitating condition accompanied by motor and sensory deficits and a reduced quality of life. Current treatment options are limited and are associated with variable efficacy and a risk of adverse effects. The pathophysiology of SCI is initiated by a primary mechanical insult to the spinal cord, followed by a complex series of deleterious events known as secondary injury. Secondary injury processes include free radical formation, glutamate excitotoxicity, inflammation and cell death. Apoptotic cell death in particular plays a key role in the secondary injury processes and exacerbates tissue degradation and loss of function. The role of Fas-mediated apoptosis in SCI pathophysiology is poorly defined in the literature to date. Correlative evidence suggests that this form of cell death is delayed and occurs in white matter adjacent to sites of primary damage. The cellular and temporal mechanisms of Fas-mediated apoptosis following experimental SCI were evaluated using a clinically relevant clip compression SCI model in the rat. Furthermore, therapeutic manipulation of Fas activation using a soluble form of the Fas receptor (sFasR) was carried out to establish the efficacy and clinical relevance of targeting this aspect of secondary injury. This work shows that Fas-mediated apoptosis is an important contributor to secondary SCI pathology. Oligodendrocytes are targeted by this form of cell death in a delayed fashion post-injury, providing an opportunity for therapeutic intervention. Intrathecal administration of sFasR following SCI reduced post-traumatic apoptosis, improved cell survival, enhanced tissue preservation and resulted in an improved motor recovery. Administration of sFasR was effectively delayed by up to 24 hours post-injury, however a shorter delay of 8 hours post-injury was most efficacious. A surprising result emerged from this work. Delayed intrathecal administration of IgG following SCI showed significant efficacy in both cellular and tissue level outcomes, as well as at the functional level. Fas-mediated apoptosis is an important aspect of secondary SCI pathophysiology and is an attractive therapeutic target. The beneficial outcomes of manipulating Fas activation using sFasR provide further evidence for this. Future work will refine this treatment strategy, bringing it into the SCI patient population.

Mechanisms to augment the cellular function and mass of beta-cells may be effective means of treating type 2 diabetes. Important in the physiological control of beta-cell function and nutrient disposal are factors released from gut enteroendocrine cells during nutrient digestion. In enteroendocrine L-cells, post-translational processing of proglucagon gives rise to a number of proglucagon-derived peptides. One such peptide, glucagon-like peptide-1 (GLP-1), acts via its own receptor (GLP-1R) to stimulate beta-cell insulin secretion, proliferation and survival. Another, oxyntomodulin (OXM), weakly activates the GLP-1R and inhibits food intake in a GLP-1R-dependent manner in rodents, which led us to hypothesize that OXM modulates GLP-1R-dependent glucoregulation. While OXM did not mimic the inhibitory effect of GLP-1 on gastric emptying in mice, OXM stimulated insulin secretion, beta-cell survival and improved glucose tolerance in a GLP-1R-dependent manner. In a similar manner to GLP-1, glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K-cells, physiologically stimulates insulin secretion via a distinct GIP receptor (GIPR) in beta-cells. Beyond the beta-cell, GIP and GLP-1 appear to exert divergent actions for the control of glucose homeostasis. Moreover, I illustrate that physiological and pharmacological GLP-1R signalling may be comparatively more important for the preservation of beta-cell mass and glucose homeostasis in murine streptozotocin-induced diabetes. Lastly, studies in rodents and humans have showed that metformin increases circulating levels of GLP-1, leading us to hypothesize that GIP and GLP-1 may be involved in the glucoregulatory effects of metformin. Interestingly, transcripts for the Glp1r and Gipr were significantly increased within islets of metformin-treated mice, and metformin treatment enhanced the sensitivity of cultured beta-cells to GIP and GLP-1. In summary, these studies illustrate mechanisms by which enteroendocrine peptides compare and contrast with respect to beta-cell survival and function and the control of glucose homeostasis.

Ankylosing spondylitis (AS) is associated with HLA-B*2704 and B*2705 but not with HLA-B*2706 and B*2709. Genome wide studies recently identified ERAP1 as an important genetic association in AS and could be the missing link in the pathogenesis of AS. I studied the implications of the two known actions of ERAP1 on AS pathogenesis. For assessing the peptide trimming function, surface HLA-B27 and MHC-I free heavy chain (FHC) expression on peripheral blood mononuclear cells of AS patients were studied. Subsequently, in an in vitro system of C1R cells expressing different AS-associated and AS-neutral HLA-B27 subtypes, I studied the effect of ERAP1 suppression on HLA-B27 and FHC expression. To assess the cytokine receptor shedding function, I studied serum cytokine receptor level variation with ERAP1 polymorphisms and its relationship to disease activity in AS patients. Finally, I studied the effect of variants of ERAP1 and other members of the antigen presentation machinery on radiographic severity in AS patients. AS patients with the major allele of the ERAP1 rs27044 polymorphism had higher FHC expression on monocytes. In C1R cells ERAP1 suppression led to an increase in intracellular FHC (IC-FHC) and B27-peptide complexes identified by a special MARB4 antibody, but only in C1R cells expressing the AS-associated subtypes HLA-B*2704 and B*2705. ERAP1 variants had no effect on serum cytokine receptor levels. Baseline radiographic severity was associated with ERAP1 polymorphism in univariate analysis only. LMP2 variants were associated with baseline radiographic severity in multivariate analysis. ERAP1 affects peptide presentation and FHC formation by HLA-B27 and could be the missing link in the pathogenesis of AS. ERAP1 through its differential HLA-B27 subtype interaction could explain why certain subtypes of HLA-B27 are associated with AS while others are not. Larger studies are required to look closely at the effect of ERAP1 on radiographic severity and progression in AS.

The function of skeletal adaptation to mechanical load is to adjust the amount and distribution of bone tissue (geometry); such that stresses experienced within the bone are kept within certain physiological limits and fractures are prevented. Genetic, environmental or hormonal factors may cause heterogeneity in this adaptive response, altering geometry and consequently fragility. The purpose of this thesis was to explore the skeletal response to load in vivo, by evaluating stress at the hip under three different conditions: FRACTURE (Study 1), DIABETES (Study 2) and ESTROGEN deficiency (STUDY 3). We studied women 25 years of age or older who participated in the Canadian Multicentre Osteoporosis Study and had available Hip Structure Analysis (HSA) data from baseline dual energy x-ray absorptiometry (DXA) scans. Women were categorized according to fracture status (fracture or no fracture), diabetes status (diabetes or no diabetes) and estrogen use (current users or never users). We computed stress (megapascals=MPa) at the infero-medial margin of the femoral neck in a one-legged iii stance using a 2-D engineering beam analysis. We used linear regression to determine associations between femoral neck stress and each categorical variable. Study 1 (n=2168) demonstrated higher stresses in postmenopausal women with fractures compared to women without fractures (10.57 ± 2.19 vs. 10.30 ± 2.03 MPa; p=0.0031). Study 2 (n=3665) demonstrated higher stresses in women with Type 2 Diabetes Mellitus compared to non-diabetic women (10.98 ± 2.33 vs. 10.57 ± 2.20 MPa; p=0.0194). Study 3 (n=2447) demonstrated higher stresses in postmenopausal women not on estrogen than in premenopausal women (10.66 ± 2.14 vs. 10.09 ± 2.01 MPa; p<0.0001), but no differences in stresses between postmenopausal women on estrogen and premenopausal women (10.16 ± 2.00 vs. 10.09 ± 2.01 MPa; p=0.6102). Since stress is an indicator of underlying geometry, and geometry should be adapted to prevalent loads, higher stress indicates weaker geometry and suggests an impaired modeling response in these three conditions. Compromised modeling has important clinical implications in terms of treatment selection, as individuals with reduced load sensitivity may respond best to metabolic agents that would improve modeling responses to load stimuli.

This study is a prospective longitudinal outcome study investigating the presence of response shift in disease and generic functional outcome measures in 105 patients undergoing spinal surgery. The then-test method which compares pre-test scores to retrospective pre-test scores was used to quantitate response shift. There was a statistically significant response shift for the Oswestry Disability Index (ODI) (p=0.001) and the Short Form-36-PCS (p=0.078). At three months, seventy-two percent of patients exhibited a response shift with the ODI. Fifty-six and 21 percent of patients exhibited a response shift with the SF-36 physical and mental component scores respectively. When accounting for response shift and using the minimal clinically important difference, the success rate of the surgery at 3 months increased by 20 percent. The presence of response shift has implications for the measurement properties of standard spinal surgery outcome measures including the effect size of treatment and the number of responders to treatment.

Survivors of subarachnoid hemorrhage (SAH) often have learning and memory deficits. This study tested the hypothesis that SAH in rats is associated with similar deficits and that they are due to neuronal injury in the hippocampus. SAH was induced in rats. Behaviour was investigated in the Morris water maze and brain injury by microscopy. Rats with SAH had deficits in spatial learning and working memory and had significantly more fluoro-Jade- and TUNEL-positive neurons in the hippocampus, cerebral cortex and cerebellum. Microthromboemboli in microvessels were more frequent in brains of rats with SAH and deficits there was vasospasm of the anterior and middle cerebral arteries. The amount of cell death in the hippocampus did not appear to be sufficient to cause the observed in the Morris water maze. This suggests that other factors such as dysfunction of neurotransmission or other pathology in hippocampal pathways might contribute to the impairment.