Many pathological conditions, including renal disease, are associated with oxidative stress. 2,3,5-tris(Glutathion-S-yl)hydroquinone (TGHQ), a potent nephrotoxic and nephrocarcinogenic metabolite of benzene and hydroquinone, generates reactive oxygen species (ROS), can cause DNA strand breaks, and the subsequent activation of DNA repair proteins, including poly(ADP-ribose) polymerase (PARP)-1. Under robust oxidative damage, PARP-1 is hyper-activated, which causes elevations in intracellular calcium concentrations (iCa²⁺), NAD⁺ and ATP depletion, and ultimately necrotic cell death. The role of Ca²⁺ in PARP-dependent necrotic cell death remains unclear. We therefore sought to determine the relationship between Ca²⁺ and PARP-1 during TGHQ-induced necrotic cell death in human renal proximal tubule epithelial cells (HK-2). Extracellular Ca²⁺ is responsible for coupling PARP-1 activation to increases in iCa²⁺ during TGHQ-induced cell death. Moreover, organelles such as the endoplasmic reticulum and the mitochondria, which contain intracellular Ca²⁺ stores play no role in increases of iCa²⁺. PARP-1 inhibition attenuates increases in iCa²⁺ induced by TGHQ, and treatment with 2-aminoethoxydiphenyl borate (2-APB), a store-operated Ca²⁺ channel (SOC) inhibitor, restored cell viability, levels of NAD⁺, and attenuated PAR protein-ribosylation (PARylation). Concurrent with SOC activation having a direct effect on PARP-1 activity, and PARP-1 inhibition attenuating increases in iCa²⁺, the results suggest that PARP-1 and SOCs are coupled during TGHQ-induced cell death. We also explored the relationship between SOC activation and PARP-1 downstream of PARP-1 activity. Poly(ADP-ribose)glycohydrolase (PARG), which catalyzes the degradation of PARs to yield free ADP-ribose (ADPR), is known to activate SOCs. Interestingly, siRNA knockdown of PARG modestly increased PAR ribosylation, but did not restore cell viability in the presence of TGHQ, indicating that free ADPR is not responsible for SOC activation in HK-2 cells. Overall, our results suggest that PARP-1 and Ca²⁺ are coupled through SOC entry, and that this relationship may involve alternative PAR-mediated signaling that leads to necrotic cell death. To further elucidate the role of PAR polymers in response to TGHQ, we determined the cellular co-localization of PAR by immunofluorescent staining. PAR polymers originally co-localized in the nucleus, and in the cytosol at later time points. Immunoprecipitation with a pADPr antibody and further analysis via mass spectrometry revealed PARylation of many stress-related proteins and Ca²⁺-related proteins upon TGHQ treatment. We therefore speculate that cytosolic PAR may cause downstream signaling, PARylating proteins that activate store-operated Ca²⁺ entry either directly through Ca²⁺-related proteins or PARylation of stress-related proteins. Thus, PARylation of proteins may contribute to increases in iCa²⁺ concentrations, leading to PARP-1-dependent necrotic cell death. Our studies provide new insight into PARP-mediated necrotic cell death. Ca²⁺ is coupled to PARP-1 hyperactivation through SOCs, where iCa²⁺ increases are independent of PARG activity, demonstrating a novel signaling pathway for PARP-dependent necrotic cell death.

The human melanocortin receptors (hMCRs) play a fundamental role in human behavior such as satiety, feeding, sexual and more. A set of SHU9119 peptide derivatives were studied for their structure-activity relationships. These peptides contained a sequential a-N-methylation amino acid scan.A second set of peptide derivatives intended to be used to create TNF-a; inhibition, via the melanocortin receptors. These peptides were shown to bind to all of the hMCR receptors, and only exhibit cAMP stimulation at hMC1R/hMC5R.The data from both of the sets of compounds illustrate that small changes in the stereochemistry of the SH9119 and TNF-a; derivatives cause drastic changes in the binding and the agonistic/antagonist properties of the compounds.This thesis determined the effect that hMC1R mutations have on the binding and cAMP response of well characterized ligands. This study ruled out 9 different residues for being the required for the cAMP response of the hMC1R.

Mechanisms of cell death extend beyond the simple apoptosis/necrosis relationship to include regulated modes of cell death that do not readily fit either of the classic descriptors. One such mechanism of cell death involves poly(ADP-ribose)polymerase-1 (PARP-1)-mediated cell death. 2,3,5-Tris(Glutathion-S-yl)-hydroquinone (TGHQ), a reactive oxygen species (ROS) generating nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, causes necrotic renal cell death, the basis for which is unclear. We therefore investigated TGHQ-mediated cell death in human renal proximal tubule epithelial HK-2 cells. TGHQ induced ROS generation, DNA strand breaks, hyperactivation of PARP-1, rapid depletion of nicotinamide adenine dinucleotide (NAD), elevations in intracellular Ca2+ concentrations, loss of mitochondrial membrane potential, and subsequent necrotic cell death. Interestingly, PARP-1 hyperactivation was not accompanied by the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARP-dependent cell death. Inhibition of PARP-1 with PJ34 blocked TGHQ-mediated accumulation of poly(ADP-ribose) polymers, NAD consumption, and the consequent necrotic cell death. However, HK-2 cell death was only delayed by PJ34, and cell death remained necrotic in nature. In contrast, chelation of intracellular Ca2+ with BAPTA-AM completely abrogated TGHQ-induced necrotic cell death. Ca2+ chelation not only prevented the collapse in the mitochondrial potential but also attenuated PARP-1 hyperactivation. Conversely, inhibition of PARP-1 modulated TGHQ-mediated changes in Ca2+ homeostasis. Moreover, TGHQ caused a sequential oxidation of peroxiredoxin III (PrxIII), a protein considered the primary antioxidant defense within mitochondria. Thus, TGHQ induced two acidic shifts in PrxIII, with both pI shifted spots representing oxidized forms of PrxIII. Transient expression of a dominant negative version of PrxIII resulted in a significant increase in TGHQ-induced cytotoxicity, whereas overexpression of wild-type PrxIII significantly attenuated cytotoxicity. Our studies provide new insights into PARP-1-mediated necrotic cell death. Changes in intracellular Ca2+ concentrations appear to couple PARP-1-hyperactivation to subsequent cell death, but in the absence of AIF release from mitochondria. NAD depletion, mitochondrial membrane depolarization, Ca2+-mediated calpain activation, and PrxIII oxidation, all contribute to TGHQ-driven ROS-mediated necrotic cell death.

Due to its escalating prevalence, obesity is becoming a leading cause of morbidity and mortality worldwide. Obesity is a complex health problem accompanied by metabolic abnormalities and low-grade inflammation that increases the risk for developing comorbidities including type 2 diabetes. Recent evidence supports a role for fat (adipose) tissue derived factors, called adipokines, in the development of obesity and obesity-related metabolic pathologies. Chemerin is an adipokine that mediates immune and metabolic effects through the chemokine-like receptor 1 (CMKLR1). Chemerin is secreted as an inactive proform, prochemerin, which subsequently undergoes enzymatic cleavage into multiple chemerin products that differentially activate CMKLR1. Multiple studies have reported elevated total chemerin (a combination of prochemerin and various chemerin products) in obese humans suggesting chemerin involvement in obesity pathophysiology. However, the observational nature of these human studies have restricted them from identifying specific forms of chemerin that are elevated in obesity and the mechanisms that govern them. Herein, I have reported that the levels of both serum total chemerin and chemerin products capable of activating CMKLR1 are elevated in obese mice and in wild type mice following treatment with an obesity-associated inflammatory mediator tumor necrosis factor-? (TNF?). Likewise, cultured adipocytes produced active chemerin under basal conditions and highly active chemerin following TNF? treatment as measured by CMKLR1 activation. The current belief is that prochemerin circulates through blood primed for activation by immune and fibrinolytic enzymes present within injured tissues. My results challenge this theory, identifying adipocytes as cells alone produce and proteolytically activate chemerin. Under basal conditions, a balance between activating serine proteases and deactivating aminopeptidases governed the amount of CMKLR1-activating chemerin formed by adipocytes. Treatment of adipocytes with TNF? elevated the levels of serine proteases elastase and tryptase, which cumulatively shifted the proteolytic balance toward the production of chemerin products that highly activate CMKLR1. Taken together, my results are the first to identify that local TNF? triggers increased adipocyte production of chemerin providing an explanation for the elevated concentrations of chemerin in obese animals and humans. Furthermore, adipocyte processing represents a novel mechanism that likely governs the amount and type of circulating chemerin in obesity.

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Antigen induced activation of Th1 cells in the peripheral blood leads to elevated production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that have been directly linked to disease induction and neuropathic pain. It was hypothesized that following antigenic induction, cytokines gain access to the spinal cord and participate in direct cellular interaction with dorsal horn neurons. Using an animal model of MS, we show that TNF-α gene and protein expression in the dorsal root ganglia (DRG) and spinal cord tissue is increased in the active group. In addition, our findings show TNF-α mRNA expression in the dorsal root entry point. Therefore, our results support the hypothesis that antigen induced DRG derived TNF-α can transport to the spinal cord via the dorsal roots and is involved in the underlying pathogenesis of MS induced neuropathic pain.

No description available.

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Antigen induced activation of Th1 cells in the peripheral blood leads to elevated production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that have been directly linked to disease induction and neuropathic pain. It was hypothesized that following antigenic induction, cytokines gain access to the spinal cord and participate in direct cellular interaction with dorsal horn neurons. Using an animal model of MS, we show that TNF-α gene and protein expression in the dorsal root ganglia (DRG) and spinal cord tissue is increased in the active group. In addition, our findings show TNF-α mRNA expression in the dorsal root entry point. Therefore, our results support the hypothesis that antigen induced DRG derived TNF-α can transport to the spinal cord via the dorsal roots and is involved in the underlying pathogenesis of MS induced neuropathic pain.

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.

No description available.

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.