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Liver X receptor (LXR) is a member of the nuclear hormone receptor superfamily that is activated by hydroxylated cholesterol derivatives referred to as oxysterols. It has also been shown to play a crucial role in regulating cholesterol trafficking and lipid metabolism in liver and macrophages. Furthermore, LXR. has also been directly implicated in the reduction of atherosclerosis in several murine models of the disease by virtue of its ability to promote reverse cholesterol efllux from intima-resident lipid-loaded macrophages. While roles for LXR in monocyte biology have focused primarily on cholesterol trafficking, evidence for other functions for the receptor outside of its traditional role as a mediator of cholesterol homeostasis is lacking. Presented herein is evidence that LXR also serves as a mediator of cytokine expression. This work has shown that treatment of human peripheral blood monocytes or monocytic THP-1 cells with the LXR ligand 22(R)-hydroxycholesterol (22R-HC), in combination with 9-cis-retinoic acid (9cRA), a ligand for the LXR. heterodimerization partner retinoid X receptor (RXR), results in the specific induction of the potent pro-apoptotic and pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α.). Promoter analysis, inhibitor studies, and order-of-addition experiments demonstrated that TNF-α. induction by 22R-HC and 9cRA occurs by a novel two-step process. The initial step involves 22R-HC-dependent induction of TNF-α. mRNA, and intracellular accumulation of TNF-alpha protein, mediated by binding of LXRα/RXRα to an LXR response element at position -879 of the TNF-α promoter. Subsequent cell release of TNF-alpha protein occurs via a separable RXR-dependent step that requires de novo transcription and protein synthesis. Furthermore, the RXR-dependent secretory event can be mimicked by agents that induce monocytic differentiation like phorbol esters that culminate in RXR activation by a pathway that does not require exogenous ligand. In this context, RXR was also shown to be a down stream target of the protein kinase C (PKC) signal transduction cascade, that results in the activation of RXR and the induction of secretory factor(s) which facilitate secretion of LXR-derived TNF-α. These studies have provided evidence that should help to expand the currently known role for LXR in monocyte biology and have furthermore identified a new role for RXRs in promoting the secretion of soluble factors like cytokines. Furthermore, in light of reports that show LXR activity promotes a reduction in atherosclerosis, it stands to reason that this regulatory circuit of LXR-dependent production of TNF -α from monocytes would similarly contribute to the attenuation of atherosclerosis 𝘪𝘯 𝘷𝘪𝘷𝘰. / Thesis / Master of Science (MSc)

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.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease associated with high levels of tumour necrosis factor-alpha (TNF-a) in synovial fluid and synovial tissue (Saxne et al., 1989). Thalidomide is a proven inhibitor of the biological synthesis of TNF-a (Sampaio et al., 1991) and is believed to rely on this action for its suppression of the wasting of tissue which accompanies RA. Oral administration of thalidomide has proven to be effective in RA, but unacceptable side effects are easily provoked (Gutierrez-Rodriguez, 1984). Administration of thalidomide via the dermal route can down-regulate TNF-a production in and around the affected joint, and this without raising the systemic blood level to a problematical level. Based on thalidomide's physicochemical properties, it is unlikely that it can be delivered percutaneously at a dose required for RA. Therefore, we have embraced the idea of using N-alkyl analogues of thalidomide. The most important feature that an analogue of this compound might contribute is decreased crystallinity and increased lipophilicity. Ordinarily both these parameters should favour percutaneous delivery. The current study was primarily aimed at exploring the feasibility of percutaneous delivery of thalidomide and subsequently, three of its odd chain IV-alkyl analogues (methyl, propyl and pentyl) via physicochemical characterization and assessment of their innate abilities to diffuse through skin as an initial step towards developing a topical dosage form for the best compound. The biological activities, more specifically their potential to inhibit the production of TNF-a was determined for thalidomide and its N-alkyl analogues. In order to achieve the objectives, the study was undertaken by synthesizing and determining the physicochemical parameters of thalidomide and its N-alkyl analogues. A high level of crystallinity is expressed in the form of a high melting point and heat of fusion. This limits solubility itself, and thus also sets a limit on mass transfer across the skin. Generally, the greater a drug's innate tendency to dissolve, the more likely it is that the drug can be delivered at an appropriate rate across the skin (Ostrenga et al., 1971). Therefore, the melting points and heats of fusion were determined by differential scanning calorimetry. Aqueous solubility and the partition coefficient (relative solubility) are major determinants of a drug's dissolution, distribution and availability. N-octanollwater partition coefficients were determined at pH 6.4. Solubilities in water, a series of n-alcohols and mixed solvents were obtained, as well as the solubility parameters of the compounds in study. Secondly, in vitro permeation studies were performed from these solvents and vehicles using vertical Franz diffusion cells with human epidermal membranes. Thirdly, tumour necrosis factor-alpha (TNF-a) inhibition activities were assessed for thalidomide and its N-alkyl analogues. By adding a methyl group to the thalidomide structure, the melting point drops by over 100°C and, in this particular instance upon increasing the alkyl chain length to five -CH2- units the melting points decrease linearly. Heats of fusion decreased dramatically upon thalidomide's alkylation as well. Methylation of the thalidomide molecule enhanced the aqueous solubility 6-fold, but as the alkyl chain length is further extended from methyl to pentyl, the aqueous solubility decreased exponentially. The destabilization of the crystalline structure with increasing alkyl chain length led to an increase in lipophilicity and consequently an increase in solubility in nonpolar media. Log partition coefficients increased linearly with increasing alkyl chain length. Solubilities in a series of n-alcohols, methanol through dodecanol, were found to be in the order of pentyl > propyl > methyl > thalidomide. The N-alkyl analogues have more favourable physicochemical properties than thalidomide to be delivered percutaneously. The in vitro skin permeation data proved that the analogues can be delivered far easier than thalidomide itself. N-methyl thalidomide showed the highest steady-state flux through human skin from water, n-alcohols and combination vehicles. Thalidomide and its N-alkyl analogues were all active as TNF-a inhibitors. Finally, active as a TNF-a inhibitor, N-methyl thalidomide is the most promising candidate to be delivered percutaneously for treatment of rheumatoid arthritis, of those studied. / Thesis (PhD (Pharmaceutics))--PU for CHE, 1999.

Thesis (PhD (Pharmaceutics))--PU for CHE, 1999.