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Background and Aims Acute Pancreatitis is an inflammatory disorder of varied aetiology and outcome. Tumour necrosis factor (TNF) and interleukin-10 are important mediators of disease pathogenesis. To investigate if the TNF and IL-10 gene loci influence susceptibility to and severity of acute pancreatitis, 135 patients with acute pancreatitis, ethnically matched normal controls, and alcoholics without pancreatic disease were studied. Methods Aetiology was classified as being secondary to alcohol, gallstones, or idiopathic. Patients were stratified into groups according to disease severity by assigning an organ failure score. Three TNF microsatellite loci (TNFa, TNFb, and TNFc), the -308 polymorphism within the TNF gene, the IL-10.G microsatellite locus, and 3 hi-allelic polymorphisms in the 5' flanking region of the IL-l 0 gene were typed using the polymerase chain reaction. Results There was no difference in allelic frequency of any of the cytokine gene loci between groups stratified according to disease severity. When patients were stratified according to aetiology of disease there was a decrease in the frequency of the TNFa2 allele in those patients with alcoholic acute pancreatitis compared to controls (14.3 vs. 35.5%, χ²=7.24, p=0.007). There was also a reduction in the frequency of the IL-10.Gl3 allele in patients with alcoholic pancreatitis compared to controls (4.8 vs. 21.3%, χ² =6.46, p=0.011). Data is also presented showing that a number of haplotypes exist as well as linkage disequilibrium across all 4 loci of the IL-10 gene, which contrasts with findings from previous work. The 3 locus haplotypes GCC and ATA are in strongest linkage disequilibrium, as is the microsatellite allele G9 and -1117.A and G9 with the 3-locus haplotype ATA. Conclusions This work has identified an allele within the TNF gene locus, and an allele within the IL-1 0.G locus which have different frequencies in patients with alcohol induced acute pancreatitis compared to other aetiologies. This finding may in part explain individuals' differing susceptibility to the development of acute pancreatitis after excessive alcohol consumption. Haplotypes not previously described exist across the IL-10 locus.

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Kawasaki disease (KD) is a multisystem vasculitis that results in localized coronary artery elastin breakdown and aneurysm formation. It is the leading cause of acquired heart disease of children in North America. Despite conventional treatment, a significant proportion of patients continue to develop coronary sequelae. The mechanisms of arterial aneurysm formation in KD are not known. Using a murine model of KD, Lactobacillus casei cell wall extract-induced coronary arteritis, the processes leading to coronary aneurysm formation were examined. Vessel damage occurred as a result of the increased enzymatic activity of the elastase, matrix metalloproteinase (MMP)-9. MMP-9 protein and activity levels were elevated in the heart post-disease induction. Expression and activity were specific for and localized to inflamed coronary arteries. The pro-inflammatory cytokine, tumour necrosis factor (TNF)-α, was required for increasing local MMP-9 expression. Importantly, MMP-9-deficient animals had a significantly reduced incidence of elastin breakdown. Furthermore, in a cohort of KD patients, serum MMP-9 did not correlate with coronary outcome, highlighting the importance of local expression of this elastase. Intravenous immunoglobulin (IVIG) and aspirin/salicylate are therapeutic agents in current use for the treatment of KD, though their exact mechanisms of action in KD are not known. The biologic effects of IVIG and salicylate on critical stages of disease development were examined. IVIG and salicylate had differential effects on TNF-α expression, with therapeutic concentrations of IVIG inhibiting, and salicylate inducing, TNF-α expression leading to an indirect modulation of MMP-9 expression. Interestingly, TNF-α expression and MMP-9 activity were both directly inhibited by the metal-chelating drug doxycycline. Treatment of affected mice with doxycycline significantly improved coronary outcome. Inhibiting both the inflammatory response as well as the downstream effects of inflammation were of therapeutic value in this model of KD. These results taken together demonstrate the importance of MMP-9 in the pathogenesis of coronary artery aneurysms in KD. Targeting MMP activity holds the promise of transforming KD from the leading cause of acquired heart disease to a self-limited febrile illness.

Kawasaki disease (KD) is a multisystem vasculitis that results in localized coronary artery elastin breakdown and aneurysm formation. It is the leading cause of acquired heart disease of children in North America. Despite conventional treatment, a significant proportion of patients continue to develop coronary sequelae. The mechanisms of arterial aneurysm formation in KD are not known. Using a murine model of KD, Lactobacillus casei cell wall extract-induced coronary arteritis, the processes leading to coronary aneurysm formation were examined. Vessel damage occurred as a result of the increased enzymatic activity of the elastase, matrix metalloproteinase (MMP)-9. MMP-9 protein and activity levels were elevated in the heart post-disease induction. Expression and activity were specific for and localized to inflamed coronary arteries. The pro-inflammatory cytokine, tumour necrosis factor (TNF)-α, was required for increasing local MMP-9 expression. Importantly, MMP-9-deficient animals had a significantly reduced incidence of elastin breakdown. Furthermore, in a cohort of KD patients, serum MMP-9 did not correlate with coronary outcome, highlighting the importance of local expression of this elastase. Intravenous immunoglobulin (IVIG) and aspirin/salicylate are therapeutic agents in current use for the treatment of KD, though their exact mechanisms of action in KD are not known. The biologic effects of IVIG and salicylate on critical stages of disease development were examined. IVIG and salicylate had differential effects on TNF-α expression, with therapeutic concentrations of IVIG inhibiting, and salicylate inducing, TNF-α expression leading to an indirect modulation of MMP-9 expression. Interestingly, TNF-α expression and MMP-9 activity were both directly inhibited by the metal-chelating drug doxycycline. Treatment of affected mice with doxycycline significantly improved coronary outcome. Inhibiting both the inflammatory response as well as the downstream effects of inflammation were of therapeutic value in this model of KD. These results taken together demonstrate the importance of MMP-9 in the pathogenesis of coronary artery aneurysms in KD. Targeting MMP activity holds the promise of transforming KD from the leading cause of acquired heart disease to a self-limited febrile illness.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, predominantly experienced by children in sub-Saharan Africa. Patients with CM are comatose and often convulse, develop retinal haemorrhages and motor abnormalities. Recent histological studies on brain tissue obtained from patients who have died from CM have identified heterogeneity in brain pathology. As a result, CM is considered to be a complex disease that may be comprised of a number of syndromes. Patients admitted to hospital with CM are treated with anti-malaria drugs; however, even in the best equipped hospitals, a large number of CM patients die within the first 24-hours following hospital admission before the anti-malarial treatment can have an effect. For this reason, it is critical that the mechanisms leading to CM are elucidated in order to develop effective adjunct therapies. Experimental cerebral malaria (ECM) caused by P. berghei ANKA (PbA) infection of susceptible mice displays many features of human CM. A key feature of this model is the pivotal role of the host immune response in pathogenesis, particularly the involvement of T cells. Evidence, predominantly from ECM studies, suggests that tumour necrosis factor (TNF) superfamily (TNFSF) members play critical roles in the immunopathology associated with CM. The first hypothesis investigated in this thesis was that key immune response pathways contribute to the development of CM and, despite the heterogeneity observed between CM patients, common pathways exist that may be targeted to prevent CM. The second hypothesis tested was that members of the TNF superfamily modulate the immune response to infection and are involved in the development of pathology observed in severe malaria (SM). In order to investigate the above hypotheses, three projects were carried out. First, we examined the great heterogeneity in brain expression profiles between ECM-susceptible CBA/CaH (CBA) and C57BL/6 (B6) mice at the peak of disease, as well as the significant differences in circulating cytokine expression and expansion of microglia in brain tissue. We found that, despite these differences, common therapeutic and preventative strategies existed to disrupt the development of ECM in the two ECM-susceptible mouse strains. Second, studies in ECM mice have identified T cells and TNFSF members, TNF and lymphotoxin (LT)-a, as critical mediators of ECM pathology. We extend these studies to examine the role of the TNFSF member LIGHT in ECM. Specific blockade of LIGHT signalling through its receptor, LTβR, in PbA-infected B6 mice abrogated the hallmark features of ECM brain pathology and improved the control of parasite growth. Importantly, specific blockade of LIGHT-LTβR signalling caused the expansion of splenic monocytes and an overall enhanced capacity to remove and process antigen during infection. Together, this study discovered a novel pathogenic role for LIGHT and LTβR in ECM and identified this TNF family receptor-ligand interaction as a potential target for therapeutic intervention in SM. Finally, we investigated the role of LTa in human SM and, more specifically, CM. We tested whether the polymorphisms within the gene encoding LTa (LTA) were associated with susceptibility to SM in Papuan Highland children and adults who had migrated from an area without malaria pressure to a region where malaria is endemic. Despite a lack of association between single nucleotide polymorphisms (SNPs) in the LTA/TNF locus and susceptibility to SM in Papuan Highland children and adults, we found a significant association between a SNP in the LTa-related gene encoding galactin-2 (LGALS2) and susceptibility to CM in children, but not adults in this study population. Interestingly, no association was found between this SNP and susceptibility to CM in Tanzanian children originating from and living in a malaria endemic region. These results suggest that there may be differences in the mechanisms leading to CM in adults and children, as well as between individuals from malaria endemic and non-endemic areas. Together, the findings outlined in this thesis are important to both the understanding of the underlying mechanisms leading to CM and to the development of improved interventions and adjunct therapies.

The presence of inflammation in the progression of fatty liver disease induced by fasting was determined in mink. Tumour necrosis factor alpha (TNF-?), and monocyte chemoattractant protein 1 (MCP-1) liver mRNA levels were quantified by real-time PCR. Mink fasted for 5 and 7 days had significantly higher levels of TNF-? and MCP-1 liver mRNA, compared to mink fasted for 0, 1, and 3 days. Mink fasted for 7 days, but re-fed for 28 days had the lowest mRNA levels of both TNF-?, and MCP-1 demonstrating the liver’s ability to restore homeostasis post-fasting. TNF-? mRNA levels were correlated with MCP-1 liver mRNA and liver fat percent. To confirm the physical presence of inflammation, slides stained with haematoxylin and eosin were analyzed for bile ducts resulting in no significant differences. Results indicate that elevated MCP-1 and TNF-? expression are associated with fasting induced fatty liver in mink.