Browsing by Author "Buret, Andre"
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Item Restricted Activation of latent Escherichia coli virulence factors by Campylobacter jejuni(2014-07-11) Reti, Kristen; Buret, AndreCampylobacter jejuni is the most common cause of bacterial gastroenteritis, and while typically self-limiting, C. jejuni infections are associated with post-infectious intestinal disorders, including inflammatory bowel disease and irritable bowel syndrome. This study sought to determine if C. jejuni induces virulence in commensal, non-invasive E. coli. Expression of adhesin, flagella, hemolysin, biofilm formation, and antibiotic resistance genes were increased in E. coli upon exposure to C. jejuni-conditioned media. Atomic force microscopy demonstrated E. coli was more adherent to human colonic epithelial cells when exposed to C. jejuni-conditioned media. C. jejuni and C. jejuni-conditioned media also induced E. coli flagella expression. In vitro, this altered E. coli phenotype disrupted TLR4 expression and induced IL-8 secretion. These data suggest C. jejuni and C. jejuni-conditioned media can induce virulence in non-invasive, commensal E. coli, and this contributes to host inflammation. These results provide a novel mechanism for post-infectious complications.Item Open Access Anti-inflammatory Properties of Retinoids and Retinoid Derivatives in Innate Immune Cells(2013-09-06) Duquette, Stephanie; Buret, AndreClearance of apoptotic neutrophils following infection is critical for the resolution of inflammation. Despite demonstrating immunomodulatory properties, the effects of retinoids in neutrophils in the context of an inflammatory response remain unknown. The objective of this project was to evaluate the immunomodulatory properties of retinoic acid (RA) and oxidatively transformed carotene-β (OxC-β) in a model of Mannheimia haemolytica-induced bovine respiratory disease, which is characterized by severe inflammation. Results in vitro and in vivo demonstrate that both RA and OxC-β induce apoptosis in bovine neutrophils and that this phenomenon promotes efferocytosis in bovine macrophages. These effects occur in the absence of antimicrobial properties and any alteration in neutrophil function. The induction of neutrophil apoptosis and their subsequent phagocytic clearance by macrophages are key mechanisms that promote the resolution of inflammation following infection. As such, RA and OxC-β may represent novel nutraceuticals that confer anti-inflammatory benefits following infection.Item Open Access Assessment of the Rat Tapeworm Hymenolepis diminuta and its Antigens in the Treatment of DNBS Colitis(2015) Campbell, Alec; McKay, Derek; Rioux, Kevin; Buret, Andre; McCafferty, Donna-Marie; Chadee, KhrisandrethThe concept of helminth therapy for autoinflammatory disorders is well-established. We utilized the H. diminuta-mouse model system to explore novel therapeutic options. We hypothesized that an optimal treatment regimen for H. diminuta existed which would be ideal for further study. Mice were infected with H. diminuta, or injected with one of two H. diminuta protein extracts, and the impact of this on dinitrobenzene sulphonic acid (DNBS)-induced colitis assessed. We found infection with a single H. diminuta significantly protected mice from DNBS. A single intraperitoneal injection of H. diminuta soluble products was also protective. Selective degradation of H. diminuta soluble products revealed a glycosylated, heat-stable immunomodulator. Finally, treatment with H. diminuta excretory-secretory products was effective at blocking DNBS. We conclude that infection with H. diminuta could be developed therapeutically for IBD and that further studies investigating the mechanism of H. diminuta’s immunomodulation should focus on the study of its excretory-secretory products.Item Open Access Colonic Responses to Swine Dysentery in Pigs(2022-04) Fodor, Cristina; Cobo, Eduardo; Buret, Andre; Morck, DouglasSwine dysentery (SD) is an enteric infectious disease of grower-finisher pigs caused by the bacterium Brachyspira hyodysenteriae. The disease causes bloody diarrhea and mortality, but not all infected pigs are clinically affected. Experimental infection of pigs with this pathogen rarely produces bloody diarrhea with 100% success rate. To determine whether colonization with B. hyodysenteriae is sufficient for SD pathogenesis, deeper knowledge of colonic host defenses is required. This thesis hypothesizes that colonic defenses drive SD pathogenesis and clinical presentation, then tests this hypothesis by assessing histological grade of colitis, neutrophil and macrophage defenses, and mucus and microbiome composition in pigs experimentally challenged with B. hyodysenteriae. This thesis shows that some challenged pigs were not colonized with B. hyodysenteriae. Others were infected but did not show clinical signs of disease (non-diseased), while most were both infected and clinically affected (diseased). Diseased pigs were the only group to show bloody diarrhea, high pathogen load, histological colitis, colonic neutrophil infiltration, and mucus hypersecretion. These changes were not present in B. hyodysenteriae-infected pigs that did not develop bloody diarrhea. Furthermore, diseased pigs showed dysbiosis with alterations of colonic microbiome composition. Thus, we concluded that clinical signs of disease resulted from defects in colonic microbiome, neutrophil and mucus barrier defenses in the colon. Manipulation of these host defenses may be a promising new treatment strategy for SD. Immunomodulatory cathelicidin peptides, such as the murine cathelicidin CRAMP, are attractive candidates for new SD treatments. Here, we showed that a single intraperitoneal injection of CRAMP was well tolerated in uninfected pigs, and the animals remained clinically healthy. CRAMP treatment decreased neutrophil influx near the colonic epithelium and altered mucin glycosylation patterns by mobilizing sialylated mucins to colonic lumen. Future testing of doses and routes of administration of CRAMP in porcine infection models would determine the potential of this peptide to boost colonic defenses against B. hyodysenteriae. This thesis brings new insight into intestinal innate immune responses during SD, thus laying the groundwork for development of cathelicidin-based treatments against this increasingly antibiotic-resistant disease.Item Open Access The EGF Receptor and HER2 Participate in TNF-α-Dependent MAPK Activation and IL-8 Secretion in Intestinal Epithelial Cells(Hindawi Publishing Corporation, 2012-07-24) Jijon, Humberto B.; Buret, Andre; Hirota, Christina L.; Hollenberg, Morley D.; Beck, Paul L.Item Open Access Enteric Tuft Cell Responses and Host-Microbial Dynamics During Giardia muris Infection(2024-09-18) Sosnowski, Olivia Stefania; Buret, Andre; Hirota, Simon; McKay, DerekEnteric tuft cells constitute an important aspect of the host response to intestinal parasites. Tuft cells detect and respond to enteric parasites, secreting molecules such as IL-25, leading to the expansion of tuft and goblet cells to ultimately promote parasite clearance. The intestinal protozoan parasite Giardia disrupts the intestinal environment and leads to impaired barrier function. Infection is also associated with altered microbiota composition and function. This study characterised the tuft cell response in the murine small intestine after G. muris infection, identifying tuft and goblet cell hyperplasia during the clearance phase (day 21). G. muris infected mice also displayed increased expression of genes defining secretory epithelial cells (Aoth1), tuft cell-related genes (Dclk1, Tas2r130, Tas2r), and goblet cell-related genes (Muc2, Klf4) during early infection (day 4 and day 11). Increases in cell number and many of the changes in gene expression during G. muris infection were observed at both the site of active Giardia colonization (jejunum) and more distally (ileum). Tuft cell-deficient (Pou2f3-/-) mice had a lower trophozoite burden compared to wild-type (C57Bl/6) mice during early infection, demonstrating that tuft cells help facilitate G. muris colonization. To investigate other contributors to the tuft cell-Giardia crosstalk, the microbiota was considered since microbial derived products can modify tuft cell functions, Giardia can cause microbiota dysbiosis, and the microbiota can influence Giardia colonization. A fecal microbiota transplantation (FMT) using microbiota from naïve C57Bl/6 or Pou2f3-/- donors revealed that FMT from Pou2f3-/- donors prior to G. muris infection resulted in a lower parasite load than in mice that received FMT from C57Bl/6 donors, illustrating that the microbiota influences G. muris colonization. Upon evaluating if Giardia-modified microbiota (GMM) could elicit a tuft cell response, transplantation with small intestinal GMM 11 days after infection did not lead to changes in tuft or goblet cell number, thus the mechanism through which Giardia engages tuft cells requires further exploration. Collectively, this study characterises the tuft cell response to Giardia, identifies a novel role for tuft cells in promoting Giardia colonization, and provides insights into how the microbiota contributes to the dynamics between enteric tuft cells and parasitic infections.Item Open Access Epithelial Immunomodulation by Giardia(2015-09-25) Cotton, James; Buret, AndreGiardia duodenalis (syn. G. intestinalis, G. lamblia) is a non-invasive, protozoan parasite of the upper small intestine of animals, including humans. Despite parasite loads exceeding 106 trophozoites per cm of small intestine, the intestinal mucosa in the majority of the Giardia-infected individuals are devoid of signs of overt inflammation. Human studies also suggest Giardia infections are capable of modulating the development of diarrheal disease in their host via unknown mechanisms. The first part of this study focused on whether Giardia infections were capable of attenuating neutrophil (PMN) accumulation in different experimental models of inflammation. Accumulation of PMNs is an archetypal event during many acute intestinal inflammatory responses contributing to the development of diarrheal disease. Our findings demonstrated Giardia infections attenuate granulocyte infiltration in an experimental model of colitis, in an isolate-dependent manner. Giardia infections also decrease tissue expression of mediators associated with PMN recruitment during in vivo colitis. Similar results were observed when Giardia trophozoites were incubated ex vivo with inflamed mucosal biopsy tissues collected from the descending colon of patients with Crohn’s disease. The second part of this study focused on identifying mechanisms by which Giardia trophozoites attenuate PMN accumulation. The intestinal epithelium is a single layer of polarized cells separating the external environment from underlying host tissues. In response to pro-inflammatory stimuli, this structure releases the PMN chemokine interleukin-8 (CXCL8) that recruits PMNs to the basolateral membrane of the intestinal epithelium. We demonstrated that Giardia trophozoites attenuate CXCL8 secretion from ex vivo small intestinal mucosal biopsy tissues and in vitro Caco-2 monolayers, following administration of IL-1β or Salmonella sp.; this involved parasite-mediated degradation of CXCL8 and attenuation of CXCL8- and C5a-induced PMN chemotaxis. Genetic assemblages of Giardia capable of infecting humans contain unique cathepsin proteases and Giardia trophozoties release cathepsin proteases into supernatants. Inhibition of Giardia cathepsin B proteases prevented parasite-mediated degradation of CXCL8 and attenuation of CXCL8 and C5a-induced PMN chemotaxis. Our findings demonstrate that Giardia infections attenuate PMN accumulation in an in vivo model of colitis. Research done in vitro highlights a role for Giardia cathepsin B proteases in degrading CXCL8 and attenuating CXCL8-induced PMN chemotaxis.Item Open Access Genetic Manipulation of Bacteroides for Transient Colonization of Germ-free Mice(2022-04) Dong, Sharon; Geuking, Markus; Harrison, Joe; Hirota, Simon; Buret, AndreHuman and murine hosts have a dynamic relationship with the microbes that colonize their bodily surfaces. Shifts in composition of the community of microbes, or their function, are implicated in many chronic and/or inflammatory conditions. There are currently gaps in understanding whether these shifts cause disease phenotypes or are artefacts of changed host conditions due to disease. The ability to tease apart these dynamics by generating bacterial tools that limited bacterial exposure of germ-free mice to a certain time-period was explored in this thesis. I target genes in synthesis pathways of two uniquely-bacterial amino acids, meso-diaminopimelic acid (mDAP) and D-Alanine (D-Ala) in a species of interest, Bacteroides thetaiotaomicron. These amino acids are incorporated into the peptidoglycan, or cell wall, of most bacteria, like B. thetaiotaomicron. This study builds on concepts from a previously developed E. coli strain called HA107, where disruption of these two synthesis pathways generated an auxotrophic mutant that grew if supplemented with said amino acids, however, did not proliferate and persist in germ-free murine gastrointestinal tracts. I designed deletion alleles that would exchange with the functional allele of target genes, and transferred the allelic exchange vector, pExchange, containing the deletion allele into B. thetaiotaomicron through conjugation. The double and triple gene deletion mutants failed to grow when not supplemented with mDAP and D-Ala. This mutation strategy may be adapted to genetically manipulate other notable Bacteroides species. The generated auxotrophic mutants from this study will be useful tools in answering questions regarding how timing or dosage of bacterial exposure affect health or disease outcomes in germ-free mice.Item Open Access Giardia Cathepsins and Their Role in Intestinal Disease(2014-08-08) Bhargava, Amol; Buret, AndreGiardia duodenalis, a non-invasive protozoan parasite of the upper small intestine of mammals, including humans, closely associates with intestinal epithelial cells. The pathophysiology of giardiasis includes intestinal barrier dysfunction and cytoskeletal injury; however, the mechanisms or parasitic factors involved remain unclear. The Giardia genome contains genes for cathepsin-like cysteine proteases; however, their roles are unknown. Using an in vitro model for studying host-parasite interactions, we illustrated that G. duodenalis trophozoites contain and release cathepsin B/L-like cysteine proteases. While cathepsin-like cysteine proteases are not involved in the tight junctional disruption caused by G. duodenalis, such proteases cleaved and disrupted cytoskeletal villin. This disruption of villin was sustained over time, at least in part, by host MLCK. Overall, this study establishes a reliable model for studying roles of parasitic cysteine proteases during host-parasite interactions. Further understanding of these proteases may pave the way for therapeutic development.Item Open Access Leishmania donovani Cathepsin B: Impact on Proteins and ncRNAs of Small Extracellular Vesicles, and on Infection Metabolism(2023-02-17) dos Santos Meira, Camila; Gedamu, Lashitew; Buret, Andre; Muench, Douglas; Claire Arrieta, Marie; Jardim, ArmandoLeishmaniasis is a spectrum of chronic diseases caused by protozoan parasites of the Leishmania genus. These parasites can circumvent immune defenses and survive in phagolysosomes of host cells by disrupting signaling processes and cellular functions. Leishmania virulence factors including papain-like cysteine proteases, have been extensively implicated in immune evasion and parasite differentiation. The disruption of the cathepsin B-like cysteine protease of L. donovani was found to attenuate infectivity and induce proteome remodeling in these parasites, affecting the expression of metabolic enzymes and proteins released in extracellular vesicles (EVs). Here, the effects of L. donovani cathepsin B on EV-derived proteins and small non-coding RNAs (ncRNAs), and on metabolism of infected macrophages were investigated. L. donovani cathepsin B wild type (WT), cathepsin B knockout (KO), and episomally complemented knockout (CM) parasites were used to generate comparative proteomic and transcriptomic profiles of small EVs (sEVs), and of metabolites in the spent media of infected U937 cells. LC-MS/MS semi-quantitative analyses revealed the cathepsin B-induced modulation of diverse elements in sEVs, including metabolic enzymes, calpain cysteine proteases, kinases, and translation-related components. Protein-protein networks were remarkable in sEVs and cathepsin B-induced alterations in their proteomes seemed to differ from those previously observed in the parasites, indicating selective packaging of protein cargo in these vesicles. The exploration of the Illumina sequenced small RNAs (18-30nt) from sEVs showed striking abundances of tRNA, rRNA and snoRNA fragments originating from a small subset of genes and with suspected regulatory functions. Moreover, ncRNA genes were differentially expressed in WT and CM sEVs, while KO sEVs exhibited significant expression of coding genes. The bioinformatics predictions of miRNA-like and other regulatory elements for non-annotated transcripts identified in sEVs suggested potential non-canonical regulatory mechanisms in Leishmania. The mass-spectrometry-based metabolomics study indicated extensive differences in the metabolism of macrophages infected with L. donovani WT, KO and CM, especially in nucleotide, energy and carbon metabolisms. Several disturbances appeared to correlate with cathepsin B-induced modulation of metabolic enzymes. Overall, the data suggest the participation of cathepsin B in protein and RNA sorting/packaging into Leishmania EVs and demonstrate the potential mechanisms by which this protease affects host-parasite interaction.Item Open Access Microbiome-Mediated Disruption of Mucin Glycosylation and Mechanisms of Protease Activated Receptor-Dependent Modulation of Mucin Gene Expression During Giardia spp. Infection(2022-09-26) Fekete, Elena; Buret, Andre; Chadee, Kris; Gedamu, Lashitew; Hollenberg, MorleyDisruption of the intestinal mucus gel barrier has a significant impact on intestinal health and homeostasis. Mucus barrier alterations have been associated with dysbiosis, increased intestinal permeability, and increased susceptibility to intestinal infection. Giardia duodenalis is a leading cause of diarrheal disease in humans and has been linked to development of post-infectious functional gastrointestinal disorders. Giardia infection is associated with intestinal barrier dysfunction and altered composition and function of the commensal microbiome, both of which are predicted to contribute to acute and chronic disease manifestations. This study reveals new mechanisms by which Giardia and its secreted cysteine proteases can modulate goblet cell activity and the properties of the intestinal mucus barrier. Alterations to the glycosylation of intestinal mucins and the expression of mucin and mucin-associated glycosyltransferase genes were found to be time-dependent, appearing as early as day 2 post-infection, and persisting in mice that had cleared the infection. Alterations to glycan abundance and glycosyltransferase gene expression were observed not only in the small intestine, at the site of parasite colonization, but also in the distal colon, where no trophozoites are present. Many of these alterations were microbiome-dependent and were recapitulated in mice transplanted with small intestinal bacteria collected from Giardia infected mice. Alterations were also similar between the mouse-specific isolate Giardia muris, and the zoonotic isolate Giardia duodenalis GS/M. In human cell cultures, alterations to mucin gene expression were found to be dependent both on Giardia cysteine protease activity and on protease-activated receptor-2 signaling. Isolate-dependent effects on mucin gene expression correlated both with cysteine protease activity levels and with the cleavage of PAR2. MUC2 mucin gene upregulation was dependent on intracellular calcium mobilization and on activation of the ERK1/2 mitogen activated protein kinase pathway, indicating that Giardia proteases can cleave PAR2 at a tethered ligand activation site. The present findings demonstrate novel mechanisms by which Giardia alters the intestinal mucus barrier, potentially facilitating pathogen translocation and contributing to acute, chronic, and post-infectious disease manifestations.Item Open Access Modulation of Human Intestinal Microbiota Composition and Structure by Campylobacter jejuni(2015-05-01) Akierman, Sarah; Buret, AndreCampylobacter jejuni (C. jejuni), one of the most common causes of human bacterial gastroenteritis in both the developed and developing worlds, has been found to contribute to post-infectious symptoms in patients with inflammatory bowel disease (IBD). While the postulated causes of IBD are various, evidence suggests a role of modulation of the intestinal microbiota in the pathophysiology. There is currently a lack of knowledge as to what extent the exposure to enteropathogens may lead to gastrointestinal microbiota dysbiosis by the promotion of an inappropriate immunological response, leading to the development of IBD-like symptoms. This study investigated the interaction between C. jejuni and the commensal microbiota of the descending colon, in a Calgary Biofilm Device (CBD) model with human mucosa biopsy samples. Terminal restriction fragment length polymorphism (T-RFLP) studies demonstrated that anaerobic C. jejuni co-incubation leads to a loss of microbiota diversity, a decrease in the relative abundance of the unknown bacteria and an increase in the relative abundance of the phyla Firmicutes. Microscopy depicts that C. jejuni co-incubation decreases microbiota biofilm thickness and decreases the extracellular matrix. Whether the loss of extracellular matrix occurs along the surface of the biofilm or throughout the biofilm requires further investigation. These novel findings contribute to our understanding of C. jejuni pathogenesis, specifically C. jejuni-microbiota modulation, and may play a role in post-Campylobacterosis flares in patients with IBD.Item Open Access Molecular, Pathogenesis and Immunological Studies of the Canadian Delmarva (DMV/1639) Infectious Bronchitis Virus (IBV) Variant(2022-09-16) Hassan, Mohamed Saleh Hussein; Careem, Faizal; Cork, Susan; Coffin, Carla; Goldsmith, Dayna; van der Meer, Franciscus; Buret, Andre; Gallardo, RodrigoInfectious bronchitis virus (IBV) is a gammacoronavirus that infects chickens leading to economic losses globally. Although the disease caused by IBV is known as infectious bronchitis (IB), the virus also replicates and induces lesions in the renal, reproductive, and gastrointestinal systems of chickens. The control of IB relies mainly on using live attenuated and inactivated vaccines. The IBV Delmarva (DMV)/1639 variant has impacted the layer flocks in Eastern Canada over the past few years. The affected flocks showed a high incidence of false layer syndrome (FLS). The studies conducted in this thesis focused on three major areas: 1) molecular characterization of IBV DMV/1639 isolates obtained from clinical cases in Eastern Canada, 2) investigating the pathogenesis of the Canadian IBV DMV/1639 variant and the host responses to this virus in chickens, and 3) evaluating the protective efficacy of the existing IB vaccines against the infection with this virus in chickens. Following whole genome sequencing (WGS), phylogenetic and recombination analyses showed that the Canadian IBV DMV/1639 strain is a chimeric virus derived from a Connecticut (Conn) vaccine-like strain, a 4/91 vaccine-like strain, and one strain that is yet-unidentified. An experimental challenge of 1-day-old specific-pathogen-free (SPF) chicks showed that this virus has a wide tissue tropism and induces marked cystic lesions in the oviduct of growing pullets. A significant drop in egg production, accompanied by characteristic gross and microscopic lesions in the reproductive organs, followed the experimental challenge of SPF chickens at peak of egg production. Infected chickens showed significant recruitments of KUL01+ macrophages and CD4+ and CD8+ T cell subsets in the oviduct tissues. In addition, anti-IBV antibodies were detected systemically and locally in the oviduct washes. Heterologous IB vaccines that are commercially available in Canada protected laying SPF chickens against a drop in egg production induced by the Canadian IBV DMV/1639 strain. Vaccinated chickens had lower viral replication and mild reproductive tract lesions. Overall, the knowledge generated in this thesis contributed to the understanding of IBV-host interactions including virus evolution, pathogenesis, host responses, and vaccination-based control.Item Open Access The Role of Extracellular Vesicles in Giardia Microbiota Interactions(2021-06-16) Siddiq, Affan; Buret, Andre; McKay, Derek; Gedamu, LashitewGiardia duodenalis is one of the most common cause of parasitic driven diarrheal diseases worldwide with over 200 million cases. Limited evidence indicates that the parasite is capable of shedding extracellular vesicles (EVs) which have been implicated in pathogenesis. In this work, we characterized Giardia EVs using state-of-the-art methods such as nanoparticle track analysis, transmission electron microscopy, proteomics, and small RNA sequencing. The effects of Giardia-derived EVs on the growth and behavior of commensal bacteria were examined. Our results indicate that Giardia produces EVs with characteristic morphology and these EVs contained virulence factors, and a variety of small RNA molecules including miRNAs. The EVs were shown to affect the growth and motility of commensal bacteria by exhibiting bacteriostatic effects, increasing bacterial motility, promoting bacterial adhesion to epithelial cells and decreasing their ability to form biofilms. The effects of EVs on bacterial motility, adhesion and biofilm forming ability were found to be, in part, mediated by the RNA content of EVs. Taken together, these findings highlight the novel role of Giardia derived EVs in mediating trans-kingdom crosstalk with the commensal bacteria and contribute to our understanding of EVs in Giardia pathogenesis.Item Open Access The Effects of Giardia duodenalis on the Intestinal Mucus Layer(2017) Amat, Christina; Buret, Andre; Chadee, KrisThe intestinal mucus layer is a highly dynamic interface that protects the intestinal epithelium and underlying tissues against noxious stimuli in the external environment. Alterations to the mucus matrix or the mucus-secreting goblet cells can result in impaired barrier function, lending to translocation of luminal antigens and opportunistic pathogens. Giardia duodenalis - a cosmopolitan parasite that colonizes the small intestine – is one of the leading causes of infectious diarrhea and a major contributor to post-infectious functional gastrointestinal disorders. This study reveals a complex interaction at the surface of intestinal epithelial cells, between G. duodenalis and the intestinal mucus layer. Here, we reveal mechanisms whereby G. duodenalis evades and disrupts the first line of host defense, the mucus layer, by degrading mucin-2 (MUC2), depleting mucin stores, and inducing differential gene expression in the small and large intestine. Infection in Muc2-/- mice demonstrated the importance of a functional mucus layer, as mice devoid of intestinal mucus had elevated trophozoite colonization in the small intestine and suffered significant weight loss. Wild-type mice infected with G. duodenalis had a thinner mucus layer and demonstrated differential mucin gene expression, including an increase in Muc2 and de novo expression of Muc5ac in the colon. Human colonic biopsies exposed to G. duodenalis exhibited a reduction of mucus stores. In vitro, G. duodenalis depleted mucins from human LS174T goblet cells and induced elevated expression of MUC2 mRNA. Mucin depletion was dependent on protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) cell-signaling. Overall, the effects of G. duodenalis on the intestinal mucus barrier resulted in impaired barrier function, observed by the translocation of bacterial species into the extra-intestinal organs of infected mice. Importantly, the broad-spectrum cysteine protease inhibitor E64 prevented mucus degradation, mucin depletion, and the increase in MUC2 gene expression. This study demonstrates a critical role for Giardia’s cysteine proteases in pathogenesis, and describes how Giardia’s disruptions of the mucus barrier facilitate bacterial translocation that may contribute to the onset and propagation of disease.Item Open Access The EGF Receptor and HER2 Participate in TNF-α-Dependent MAPK Activation and IL-8 Secretion in Intestinal Epithelial Cells(2012-09-05) Jijon, Humberto B.; Buret, Andre; Hirota, Christina L.; Hollenberg, Morley D.; Beck, Paul L.TNF-alpha activates multiple mitogen-activated protein kinase (MAPK) cascades in intestinal epithelial cells (IECs) leading to the secretion of interleukin 8 (IL-8), a neutrophil chemoattractant and an angiogenic factor with tumor promoting properties. As the epidermal growth factor receptor (EGFR) is a known transducer of proliferative signals and a potent activator of MAPKs, we hypothesized that the EGFR participates in TNF-dependent MAPK activation and IL-8 secretion by intestinal epithelial cells (IECs). We show that the EGFR is tyrosine-phosphorylated following treatment of IECs (HT-29 and IEC-6) with TNF-alpha. This requires EGFR autophosphorylation as it was blocked by the EGFR kinase inhibitor AG1478. Autophosphorylation was also inhibited by both a Src-kinase inhibitor and the metalloproteinase inhibitor batimastat. TNF treatment of IECs resulted in the accumulation of soluble TGF-alpha; treatment of IECs with batimastat suppressed TGF-alpha release and immunoneutralization of TGF-alpha resulted in decreased EGFR and ERK phosphorylations. TNF-alpha treatment of IECs resulted in an association between EGFR and HER2 and inhibition of HER2 using a specific inhibitor AG879 in combination with AG1478-suppressed TNF-alpha-dependent ERK phosphorylation and IL-8 release. Downregulation of HER2 via siRNA resulted in a significant decrease in ERK phosphorylation and a 50% reduction in IL-8 secretion.Item Open Access The Immune Modulatory Effects of Tylvalosin in Porcine Neutrophils and Macrophages in vitro(2017) Moges, Ruth; Buret, Andre; Morck, Douglas; Storey, DouglasTylavlosin (TYL), a veterinary macrolide antibiotic, has been reported to have superior efficacy treating bacterial infections of the respiratory tract in swine. This study aims to characterize potential pro-resolving of tylvalosin benefits in a porcine model by elucidating its effects, in isolated neutrophils and monocyte-derived macrophages from piglets. Our findings indicate that TYL increases porcine neutrophil and macrophage apoptosis in a dose-dependent and time-dependent manners, without affecting levels of necrosis. TYL also modulates mediators of inflammation, by increasing pro-resolving lipid mediators (LXA4 and RvD1) in neutrophils and down regulating the pro- inflammatory mediators in stimulated neutrophils (LTB4) and in stimulated macrophages (CXCL8 and IL-1α). Together, these findings demonstrate that tylvalosin has immuno- modulating properties in vitro, including the induction of leukocyte apoptosis, the inhibition of pro-inflammatory cytokines and lipids mediators, and the induction of pro- resolving lipid mediators. Future studies using live piglets will help determine whether and how these effects may translate into anti-inflammatory benefits in the content of inflammatory disease within the lung.Item Open Access The Impact of Giardia duodenalis on Human Intestinal Microbiota Biofilms: A Role in Chronic Post-infectious Gastrointestinal Disease Development?(2015-09-04) Beatty, Jennifer; Buret, AndreIrritable Bowel Syndrome (IBS) represents a common cause of altered gastrointestinal function seen in individuals throughout the developed world. Despite its prevalence, the pathophysiological mechanisms underlying IBS remain incompletely characterized. Numerous reports indicate post-infectious IBS (PI-IBS)-symptoms in upwards of 36% of patients previously subject to acute enteric infections. Distinct differences in the species distribution of the microbiota of IBS patients are well documented. On the other hand, disruptions in the microbiota structure, which normally exists as a series of biofilm communities, have yet to be analyzed in the context of IBS pathophysiology. Considering PI-IBS, it is inherently important to examine the possibility that disease pathogenesis may be determined by a pathogen-mediated disruption in the homeostatic function of the resident intestinal microbiota. Giardia duodenalis infections (giardiasis) represent a highly prevalent cause of acute diarrheal disease in symptomatic patients, worldwide. Recent outbreaks in Italy and Norway have elucidated a link between inititial Giardia infection, and subsequent development of PI-IBS symptoms in many patients. Giardia represents a novel model that can be used to study the complex polymicrobial interplay that occurs between a pathogen and the resident intestinal microbiota, while also providing insight into the mechanisms thorugh which chronic gastrointestinal disturbances, such as PI-IBS, are initiated. Additionally, much of our understanding of the role of the microbiota in PI-IBS has come from studies focusing on populations collected from fecal samples, or single species, and therefore not entirely representative of the host microbiota composition and biofilm structure. Consequently, an in vitro model system that encompasses the complex community structure, and species distribution of the mucosal mibrobiota, used in concert with an infection model through which poly-microbial interactions can be assessed, represents an important tool in understading the early initiation events underlying PI-IBS development. This work employed the use of a novel model system, representative of the human resident intestinal microbiota, to characterize the impact of G. duodenalis on intestinal microbiota biofilms. Collectively, our findings illustrate the consequences of polymicrobial interactions that occur during acute enteric infection, and offer insight into the mechanisms leading to post-infectious chronic gastrointestinal disorders, such as PI-IBS.Item Open Access The Role of the Tight Junction in the Serine Protease-Mediated Increase in Epithelial Barrier Function(2017) Ronaghan, Natalie; MacNaughton, Wallace; Hollenberg, Morley; McKay, Derek; Buret, Andre; McCole, DeclanOne of the main contributors to intestinal epithelial barrier function is the composition of the tight junction (TJ). In inflammatory bowel disease (IBD), inflammatory mediators induce disruption of the TJ and may contribute to pathogenesis. The apical addition of the serine proteases trypsin or matriptase to intestinal epithelial cell lines induces a rapid and sustained increase in transepithelial electrical resistance (TER). However, the mechanism behind this effect is incompletely understood. Determining how barrier function is modulated by serine proteases may provide further avenues to maintain barrier function and remission in IBD. It was hypothesized that serine proteases induce an increase in barrier function through modification of the TJ, and that treatment could reverse barrier disruption. Using the intestinal epithelial SCBN cell line, it was found that the response to serine proteases was independent of protease-activated receptor 2 (PAR2) and myosin regulatory light chain (MLC) phosphorylation, and mass spectrometry did not reveal a target that was being cleaved by serine proteases. Serine proteases did not enhance the recovery of barrier function after calcium depletion, and the barrier had to recover sufficiently for trypsin or matriptase to have an effect. Treatment by TNFα and IFNγ induced a concentration-dependent decrease in barrier function and prevented the increase in TER by serine proteases. Therefore, a functional tight junction is needed for the serine protease-induced increase in TER. Knockdown of occludin by siRNA prevented the increase in TER in response to serine proteases, and the membrane mobility of occludin was increased following trypsin treatment, as assessed by fluorescence recovery after photobleaching (FRAP). Thus, occludin is a key TJ protein in the serine protease mediated increase in TER. This work demonstrates that although serine proteases may not enhance barrier function in epithelia that are already disrupted, understanding the mechanisms by which they modify the junction could be used as targets for potential therapies to maintain remission.