Browsing by Author "Muruve, Daniel A."
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Item Open Access Anticoagulant Related Nephropathy Induced by Dabigatran(2018-12-09) Sharfuddin, Nazia; Nourbakhsh, Mahra; Box, Alan; Benediktsson, Hallgrimur; Muruve, Daniel A.We describe a case of biopsy-proven dabigatran related nephropathy in a patient without underlying IgA nephropathy. To date, dabigatran related nephropathy was only reported in patients with concurrent or undiagnosed IgA nephropathy, suggesting that it may predispose patients to dabigatran associated injury. The patient is an 81-year-old woman with multiple medical comorbidities, including nonvalvular atrial fibrillation, who was anticoagulated with dabigatran. She presented to hospital with acute kidney injury in the setting of volume overload. Her estimated glomerular filtration rate decreased from a baseline of 57 mL/min/1.73 m2 to 4 mL/min/1.73 m2, necessitating hemodialysis. Renal ultrasound findings, fractional excretion of sodium, and urinalysis suggested acute kidney injury. Renal biopsy showed acute tubular injury, tubular red blood cell casts, and an absence of active glomerulonephritis, similar to the pathological findings of warfarin related nephropathy. A diagnosis of anticoagulant related nephropathy secondary to dabigatran was therefore established. This case demonstrates that dabigatran, like warfarin, may increase tubular bleeding risk in patients, irrespective of underlying kidney or glomerular disease.Item Open Access Application of immobilized ATP to the study of NLRP inflammasomes(2019-01-11) Liao, Kuo Chieh; Sandall, Christina F.; Carlson, David A.; Ulke-Lemée, Annegret; Platnich, Jaye; Hughes, Philip Floyd; Muruve, Daniel A.; Haystead, Timothy Arthur James; MacDonald, Justin AnthonyThe NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well-studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.Item Embargo Blunting Colitis by Targeting Gut Microbial Antigen-Loaded Antigen-Presenting Cells(2019-04-30) Hebbandi Nanjundappa, Roopa; Santamaria, Pere; McKay, Derek Mark; Muruve, Daniel A.The non-obese diabetic (NOD) mouse is commonly used for studying type-1 diabetes (T1D). Among many β-cell auto-antigens (Ags), the Santamaria lab has focused on understanding the role of the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) in T1D pathogenesis. In NOD mice, a highly-diabetogenic CD8+ T-cell population recognizing a H-2K-restricted IGRP206-214 epitope is found in the islets of pancreas. The deletional tolerance of these cells occurs independently of IGRP206-214/Kd recognition, particularly in the post-neonatal period when commensals actively colonize the gut. Hence, the first part of this thesis focused on identifying microbial epitope(s) similar to IGRP206-214 and determining the biological importance of such recognition in NOD mice and humans. Our findings suggested that an integrase protein expressed by several species of the genus Bacteroides contains a mimic for auto-Ag pancreatic -cell IGRP (residues 206-214; IGRP206-214). Bacteroides integrase promoted the recruitment of diabetogenic high-avidity IGRP206-214-specific 17.4-CD8+ T-cells into the gut and suppressed experimental colitis by targeting BacIYL36-44-presenting gut microbial Ag-loaded Ag-presenting cells (APCs). This suggests that Bacteroides integrase is a beneficial Ag for the host in suppressing colitis and that the suppression of pro-inflammatory APCs is beneficial in controlling inflammation in the gut. The Santamaria lab has shown that administration of disease-relevant peptide/major histocompatibility complex (pMHC II)-coated NPs in mice suppressed three different autoimmune diseases—T1D, experimental autoimmune encephalomyelitis (EAE: a model of MS) and experimental arthritis—without affecting systemic immunity, which resulted from the induction/expansion of disease-relevant Tr1-like CD4+ T-cells. The suppression of autoimmunity was mainly mediated by IL-10 and TGF- secretion by Tr1-like cells. By considering the importance of Tregs in the suppression of inflammatory bowel disease (IBD), In the second part of my thesis work, we developed pMHC II nanomedicines displaying peptides from prevalent gut microbial Ags (Bacteroides integrase, flagellin, Yidx and Fla-2) and tested their efficacy in murine models of colitis. We found that gut microbial Ag-relevant pMHC II-NPs induced/expanded cognate Tr1-like CD4+T-cells, resulting in the suppression of DSS-induced and T-cell transfer models of colitis without compromising mucosal immune response. The suppressive capacity of Tr1-like cells was mainly mediated by the secretion of IL-10 and TGF1 in the susceptible hosts.Item Open Access Characterizing GATA6+ cells in the kidney(2024-09-13) Belay, Sisay Getie; Muruve, Daniel A.; Chun, Justin; Pillai, Dylan R.Globally, acute kidney injury (AKI) and chronic kidney disease (CKD) cause an estimated 13 million and 700 million cases/year, and 1.7 million and 1.2 million deaths/year, respectively. Kidney disease is the result of many different insults. Kidney injury promotes the recruitment and proliferation of different leukocyte populations, including macrophages to the kidney that play a major role in disease pathogenesis. Recruitment of resolution and reparative macrophages induce anti-inflammatory responses and promote kidney repair, but the characteristics of these cell populations are not clearly defined. GATA6+ macrophages had been reported to facilitate tissue repair following injury in organs other than the kidney. However, whether GATA6+ macrophages are resolution and repair macrophages in the kidney is not known. Using immunofluorescence imaging, flow cytometry, and nCounter transcriptome techniques, we assessed GATA6+ cells in normal and diseased kidney of mouse and human while probing common tissue macrophage markers specifically CD206+ and CD163+ as a benchmark. We showed that both CD206+ and CD163+ macrophages were detectable in normal and diseased mouse and human kidney. In mouse, both CD206+ and CD163+ macrophages were upregulated in various kidney compartments especially during CKD. Whereas in human, CD206+ and CD163+ macrophages appeared to be resident in the kidney and upregulated in diseased states. Using the same technique, significant population of GATA6+ cells were identified in mouse and human kidney particularly during CKD. GATA6+ cells were mainly localized in the tubulointerstitial area of the kidney cortex. In mouse and human kidney, the majority of GATA6+ cells did not co-express common leukocyte/macrophage markers. Conversely, in mouse and human CKD, GATA6+ cells mostly co-expressed the stromal cell marker αSMA. Using nCounter transcriptome profile of mouse samples, GATA6+ cells represented a distinct non-immune cell population that expressed genes associated with stromal cell identity, inflammation regulation, angiogenesis and collagen biosynthesis. The role of GATA6+ cells in CKD will require further exploration to unravel specific pathological and/or repair mechanisms that may lead to improved management of kidney disease.Item Open Access Clostridium difficile toxin-induced intestinal injury is mediated by the inflammasome(2009) Ng, Jeffrey; Beck, Paul L.; Muruve, Daniel A.Item Open Access Exaggerated IL-15 and Altered Expression of foxp3+ Cell-Derived Cytokines Contribute to Enhanced Colitis in Nlrp3−/− Mice(2016-08-17) Hirota, Simon A.; Ueno, Aito; Tulk, Sarah E.; Becker, Helen M.; Schenck, L. Patrick; Potentier, Mireille S.; Li, Yan; Ghosh, Subrata; Muruve, Daniel A.; MacDonald, Justin A.; Beck, Paul L.The pathogenesis of Crohn’s disease (CD) involves defects in the innate immune system, impairing responses to microbes. Studies have revealed that mutations NLRP3 are associated with CD. We reported previously that Nlrp3−/− mice were more susceptible to colitis and exhibited reduced colonic IL-10 expression. In the current study, we sought to determine how the loss of NLRP3 might be altering the function of regulatory T cells, a major source of IL-10. Colitis was induced in wild-type (WT) and Nlrp3−/− mice by treatment with dextran sulphate sodium (DSS). Lamina propria (LP) cells were assessed by flow cytometry and cytokine expression was assessed. DSS-treated Nlrp3−/− mice exhibited increased numbers of colonic foxp3+ T cells that expressed significantly lower levels of IL-10 but increased IL-17. This was associated with increased expression of colonic IL-15 and increased surface expression of IL-15 on LP dendritic cells. Neutralizing IL-15 in Nlrp3−/− mice attenuated the severity of colitis, decreased the number of colonic foxp3+ cells, and reduced the colonic expression of IL-12p40 and IL-17. These data suggest that the NLRP3 inflammasome can regulate intestinal inflammation through noncanonical mechanisms, providing additional insight as to how NLRP3 variants may contribute to the pathogenesis of CD.Item Open Access Examining the Role of NLRP3 in Intestinal Homeostasis and Fibrosis(2018-07-24) Tjong, Jessica Ria; Beck, Paul L.; Muruve, Daniel A.; Hirota, Simon AndrewFibrosis is the most common cause of surgery in Crohn’s disease (CD) and the mechanisms of fibrosis in CD are not well understood. Nucleotide-binding oligomerization domain (NOD)-like receptors, including NLRP3, are cytosolic protein sensors involved in inflammatory pathways and implicated in CD pathogenesis. NLRP3 has been shown to drive fibrosis in various tissues, including the heart, kidney, lungs, liver and pancreas; however, its role in intestinal fibrosis is currently unknown. The main hypothesis of this project is that NLRP3 mediates intestinal fibrosis. Primary intestinal myofibroblasts isolated from Nlrp3-/- mice had reduced response to pro-fibrogenic cytokine TGFβ compared to wildtype (WT), with lower phosphorylation of downstream signalling protein Smad2, and decreased induction of connective tissue growth factor (CTGF), as well as decreased migration. Loss of NLRP3 in intestinal myofibroblasts also led to increased resistance to cell death during serum deprivation. In an in vivo model of chronic colitis, Nlrp3-/- mice had significantly worse disease and susceptibility compared to WT. However, both Nlrp3-/- and WT mice had similar levels of intestinal fibrosis. Relevance to the human disease was further highlighted by the finding that NLRP3 transcript levels in mucosal colonic biopsies from patients with CD were significantly increased, and increased expression correlated with inflammation. Overall, our data suggest that loss of NLRP3 leads to reduced response to TGFβ in colonic myofibroblasts, and that NLRP3 may potentiate TGFβ beta signalling in the gut. Further insights into the mechanisms of intestinal fibrosis will significantly impact the development of new tools that will help with assessing and treating patients with CD complications.Item Open Access Examining the Role of Non-Canonical NOD-like Receptors and Inflammasomes in Inflammation and Disease(2018-03-21) Platnich, Jaye Matthew; Muruve, Daniel A.; MacDonald, Justin Anthony; Power, Christopher; Duff, Henry J.; Peters, Nathan C.The NOD-like Receptors (NLRs) are a family of pattern recognition receptors known to regulate a variety of immune signaling pathways. A substantial portion of NLR research focuses on the pyrin domain-containing NLRP subfamily. The canonical NLRPs are inflammasome-forming proteins responsible for the activation of caspase-1 and the maturation and secretion of the pro-inflammatory cytokines IL-1β and IL-18. In contrast, the non-canonical inflammasome-independent NLRPs regulate a variety of other pathways, including MAPK and NF-κB, through the formation of non-inflammasome complexes. Interestingly, not all inflammasomes are nucleated by NLRPs. The recently characterized non-canonical caspase-4 (caspase-11 in mice) inflammasome is known to be a key driver of the innate immune response to intracellular pathogens (and the molecules associated with them), by triggering both inflammatory cell death and the activation of canonical inflammasomes. At the outset of this PhD work, the understanding of both non-inflammasome-forming NLRPs and the non-canonical caspase-4 inflammasome was poor and the studies were sparse. It was the goal of this thesis to characterize the expression, gene regulation, and function of the non-inflammasome-forming NLR protein NLRP6, both at the cellular and biochemical level. Furthermore, using a pathogen-associated molecular pattern (PAMP)-driven model of inflammation, we sought to elucidate the function of the non-canonical caspase-4 inflammasome, particularly as it pertains to the regulation of the canonical inflammasome and cell death. By studying the fundamental biology underlying these lesser-known mediators of the innate immune system, we hoped to better understand their contribution to the early immune response and their role in driving inflammatory disease with a view to, one day, ameliorating the condition of patients suffering from these afflictions through the development of targeted therapeutics.Item Open Access ING1 Impacts Ovarian Cancer by Altering EMT(2019-08-30) Yang, Yang; Riabowol, Karl T.; Bonni, Shirin; Rancourt, Derrick E.; Muruve, Daniel A.The INhibitor of Growth (ING) proteins are type II tumour suppressors that regulate epigenetic state and transcription by recruiting various chromatin-modifying complexes to chromatin. INGs are involved in multiple cellular processes such as DNA repair, apoptosis and cellular senescence. In this study, we investigated the potential role of ING1 in inhibiting the epithelial-mesenchymal transition (EMT) program that suppresses cancer metastasis and identifies the mechanism of how ING1 regulates gene transcription, as well as its clinical implication in epithelial ovarian cancer. Our analyses revealed that ING1 epigenetically regulates the transcription machinery of the EMT program through binding to the promoter region of Twist1, the critical gene encoding transcription factor (TF) that induce EMT. Subsequently, ING1 promoted the expression of epithelial cell markers, including E-cadherin, while suppressing the expression of mesenchymal markers such as N-cadherin. ING1 antagonized TGF-β or EGF induced EMT and inhibited cancer cell invasion and migration in a Twist1-dependent manner. Integration of ChIP-seq and RNA-seq data revealed the overall genomic binding characteristics of ING1 and its candidate target genes. Gene ontology (GO) and pathway analyses indicated that the genes targeted by ING1 were involved in diverse physiological processes and pathways, mostly associated with the EMT program. The bioinformatical analysis revealed the ING1 binding motif sequence, showing that ING1 recognized target genes through the TEAD family of transcription factors and their co-factor, YAP1, to further influence gene expression. Lastly, we found that high ING1 protein expression was associated with p16 and ARID1A levels and predicted better DSS in ovarian clear cell carcinoma (OCCC). Our data also suggested that high ING1/low N-cadherin expression predicts favourable disease-specific survival (DSS) in epithelial ovarian cancer. These data provide evidence that as a tumour suppressor, ING1 can impact ovarian cancer development through modulating the EMT process and play an essential role in regulating ovarian cancer chemoresistance. In summary, by downregulating the EMT-TF expression and hence and EMT program, ING1 suppresses tumour cell invasion and cancer metastasis. Global genomic profiling revealed new insights into ING1 biology and provided justification for further exploring ING1 functions. This study has also provided critical pre-clinical data that could help establish ING1 as a prognostic and therapeutic agent for ovarian cancer.Item Open Access Innate immunity to adeno- and adeno-associated virus vectors - Impact on functional apolioprotein AI gene transfer(2007) Zaiss, Anne Katherine; Wong, Norman C. W.; Muruve, Daniel A.Item Open Access Innate immunity to adeno- and adeno-associated virus vectors: impact on functional apolipoprotein AI gene transfer(2007) Zaiss, Anne Kathrin; Wong, Norman C. W.; Muruve, Daniel A.Item Open Access Map kinase activation increases bk polyomavirus replication and facilitates viral propagation in vitro(2011) Seamone, Mark E.; Muruve, Daniel A.