Browsing by Author "Jenne, Craig N."
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- ItemEmbargoAntigen-specific CD4+ T-B cell interplay induces a robust, polyreactive systemic immunoglobulin response to commensal bacteremia.(2020-03-04) Koegler, Mia Elizabeth; Geuking, Markus B.; Peters, Nathan C.; Hirota, Simon Andrew; Jenne, Craig N.The impact of cognate CD4+ T cell help on the systemic antibody response during commensal bacteremia was assessed in detail in this thesis. To specifically evaluate cognate T cell-B cell interactions, we utilized a genetically modified commensal E. coli strain that expressed gp61, an additional T helper epitope, in its outer membrane ompC protein (E. coli ompC_gp61). Germ-free mice that were systemically primed with E. coli ompC_gp61 produced a significantly more robust E. coli-specific antibody response than mice that received the corresponding wild-type (WT) E. coli strain. The observed antibody response to E. coli ompC_gp61 appeared to be MHC II haplotype-dependent, as this phenomenon was reproducible in C57BL/6 mice (I-Ab) but not in BALB/c mice (I-Ad and I-Ed). Furthermore, mice adoptively transferred with gp61-specific SMARTA CD4+ T cells and later challenged with E. coli ompC_gp61 produced significantly more E. coli-specific IgM than recipient mice that were primed with WT E. coli. This finding suggests that the proportion of antigen-specific CD4+ T cells present during systemic immune priming may impact on class switch recombination and IgM+ memory formation. Finally, increasing gut microbiota complexity resulted in lower E. coli-specific antibody titers compared to germ-free mice in response to E. coli ompC_gp61 priming. However, non-primed mice with a more complex gut microbiota had higher total serum antibodies than their germ-free counterparts. Collectively, these data suggest that cognate CD4+ T cell help during commensal-induced bacteremia can orchestrate a potent, commensal-specific, and polyreactive antibody response. These findings shed new light on the systemic humoral immune response to bacteremia and could potentially be exploited to develop more effective and personalized vaccine strategies.
- ItemOpen AccessCytokines and Chemokines in Pediatric Appendicitis: A Multiplex Analysis of Inflammatory Protein Mediators(2019-02-21) Naqvi, S. Ali; Thompson, Graham C.; Joffe, Ari R.; Blackwood, Jaime; Martin, Dori-Ann; Brindle, Mary; Barkema, Herman W.; Jenne, Craig N.Objectives. We aimed to demonstrate the potential of precision medicine to describe the inflammatory landscape present in children with suspected appendicitis. Our primary objective was to determine levels of seven inflammatory protein mediators previously associated with intra-abdominal inflammation (C-reactive protein—CRP, procalcitonin—PCT, interleukin-6 (IL), IL-8, IL-10, monocyte chemoattractant protein-1—MCP-1, and serum amyloid A—SAA) in a cohort of children with suspected appendicitis. Subsequently, using a multiplex proteomics approach, we examined an expansive array of novel candidate cytokine and chemokines within this population. Methods. We performed a secondary analysis of targeted proteomics data from Alberta Sepsis Network studies. Plasma mediator levels, analyzed by Luminex multiplex assays, were evaluated in children aged 5-17 years with nonappendicitis abdominal pain (NAAP), acute appendicitis (AA), and nonappendicitis sepsis (NAS). We used multivariate regression analysis to evaluate the seven target proteins, followed by decision tree and heat mapping analyses for all proteins evaluated. Results. 185 children were included: 83 with NAAP, 79 AA, and 23 NAS. Plasma levels of IL-6, CRP, MCP-1, PCT, and SAA were significantly different in children with AA compared to those with NAAP (). Expansive proteomic analysis demonstrated 6 patterns in inflammatory mediator profiles based on severity of illness. A decision tree incorporating the proteins CRP, ferritin, SAA, regulated on activation normal T-cell expressed and secreted (RANTES), monokine induced by gamma interferon (MIG), and PCT demonstrated excellent specificity (0.920) and negative predictive value (0.882) for children with appendicitis. Conclusions. Multiplex proteomic analyses described the inflammatory landscape of children presenting to the ED with suspected appendicitis. We have demonstrated the feasibility of this approach to identify potential novel candidate cytokines/chemokine patterns associated with a specific illness (appendicitis) amongst those with a broad ED presentation (abdominal pain). This approach can be modelled for future research initiatives in pediatric emergency medicine.
- ItemOpen AccessThe Development of Immunoreagents to Detect for a Cellular Immune Response(2019-08-20) Ma, Crystal Wai Yin; De Buck, Jeroen M.; Jenne, Craig N.; Santamaria, Pere; van Marle, GuidoCell mediated immunity (CMI) is the adaptive T-cell-mediated defense mechanism against pathogens. Oftentimes, by assessing lymphocyte counts and/or cytokine profiles, the presence of an infection can be detected. While current diagnostic tests (e.g. interferon gamma release assay, delayed type hypersensitivity test) exist, many are not widely adopted due to the requirement of specific equipment for quantification, time restraints and/or false negative results. As such, the discovery of new biomarkers that can accurately detect and quantify a T-cell mediated, antigen specific immune response would be very valuable. We aimed to explore cellular immune responses (CIRs) through the direct detection of antigen specific Major Histocompatibility Complex II - T Cell Receptor (MHC II-TCR) interactions. We anticipated to create bioreagents that could enumerate TCR:MHC interactions, thereby giving possible insights of a previously activated CMI by targeting effector T-cells. To do this, superantigens (SAgs) (here: TSST-1) were split and fused to a reporter protein (YFP), allowing for specific TCR-pMHC interactions on antigen presenting cells (APCs) to be detected through a fluorescent marker. In the presence of a cognate pMHCII:TCR interaction, our split superantigens would be able to bind and reassemble, thereby quantifying the interaction using fluorescence. To further detect for sensitized T-cells, we engineered peptide-recombinant T cell receptor ligands (pRTLs) to be displayed on the surface of fluorescent E. coli using a pAIDA1 expression system. This system is intended to mimic the expression of MHC II on APCs. Interactions between the T-cells and pRTLs on the surface the E. coli can therefore be visualized using microscopy followed by flow cytometry analysis, whereby clustering of GFP-bacteria around effector T-cells would arise as a result of successful peptide display. Our results indicated no binding occurred between the individual C-domain of TSST-1 and T-cells. It was also revealed that our designed pRTL that were expressed on the surface of E.coli were unable to bind to T-cells, possibly due to complications with expressing the pRTL using a bacterial surface expression vector. Here, we describe the methodology of our approach, including construct design, protein production and analysis. We also explore existing technologies,and suggest future approaches and considerations that can be made to supplement the current research we have performed in this project. In conclusion, our developed biomarkers did not successfully bind to T-cells as we had anticipated, however, we did develop valuable insight regarding the use of SAg and pRTLs are potential bioreagents for the detection of a cellular immune response.
- ItemOpen AccessEnhanced Induction of Epithelial-Derived IL-17C Following Bacteria and Rhinovirus Exposure(2019-05-15) Jamieson, Kyla Carol; Proud, David; Jenne, Craig N.; Hirota, Simon Andrew; Yipp, Bryan G.; McCoy, Kathy D.; Peebles, Ray StokesUp to 80% of Chronic Obstructive Pulmonary Disease (COPD) exacerbations are associated with bacterial and/or viral pathogens, with bacteria-virus co-infections detected in up to 25% of exacerbations. These co-infections are associated with increased symptoms, increased systemic inflammation, longer hospital stays, and increased risk of hospital re-admission. Human rhinovirus (HRV) is the most common viral pathogens detected, while non-typeable Haemophilus influenzae (NTHI) and Pseudomonas aeruginosa (PAO) are among the most common bacterial pathogens identified. The airway epithelium is the first line of defence against these pathogens and responds by releasing proinflammatory cytokines and anti-microbial peptides. Interleukin (IL)-17C is a novel pro-inflammatory cytokine that is typically released from epithelial cells in response to bacteria, viral, or fungal pathogens, and in response to pro-inflammatory cytokines such as TNFα and IL-1β. In this thesis, we performed the first study to assess the involvement and functional role of IL-17C in bacteria-rhinovirus co-infections in human bronchial epithelial cells (HBECs). Bacteria-rhinovirus co-exposure for 24 hours induced significant, and synergistic, IL-17C gene expression and protein release. Synergistic IL-17C release was dependent on viral replication recognition sensors, RIG-I and MDA5, as well as NF-κB and p38 signalling. In an autocrine/paracrine manner, IL-17C acted on the airway epithelium to induce CXCL1, CXCL2, TFRC, and NFKBIZ gene expression, to induce CXCL1 protein release, and to promote HBEC-induced neutrophil recruitment. To assess how IL-17C is involved in the clinical context of COPD, HBECs were obtained via bronchial brushings from non-smokers, smokers with normal lung function, and patients with physician-diagnosed COPD and these cells were exposed to NTHI and HRV-1A concurrently. Interestingly, in response to concurrent NTHI and HRV-1A exposure, HBECs from COPD patients released significantly more IL-17C than cells from either non-smokers or healthy smokers, and HBECs from healthy smokers released significantly less IL-17C than non-smokers. Further, acute cigarette smoke extract exposure significantly reduced microbial-induced IL-17C release from cells from normal subjects. Using a morphologically-relevant well-differentiated HBEC model, IL-17C was predominantly released basolaterally, from apical cells, in response to HRV in a dose-, time-, and replication-dependent manner. High doses of NTHI could also induce basolateral IL-17C, however synergy was no longer achieved with NTHI+HRV-1A co-infections. Similar to monolayer culture, IL-17C acted on basal cells to induce significant basolateral release of CXCL1, providing physiological relevance for subsequent neutrophil recruitment. These results suggest that IL-17C acts to induce CXCL1 release and promote neutrophil recruitment to the site of bacteria or rhinovirus respiratory infections, however, this response is exaggerated in epithelial cells from COPD patients.
- ItemOpen AccessExploration of innate immune response during infectious bovine digital dermatitis and the evaluation of topical therapeutic treatment(2018-09-18) Watts, Kaitlyn; Cobo, Eduardo R.; Barkema, Herman W.; De Buck, Jeroen M.; Jenne, Craig N.Digital dermatitis (DD) is a frequently occurring infectious disease amongst dairy cattle associated with ulcerative and necrotizing lesions. Due to the associated pain and lameness, DD is a recognized animal welfare problem and has economic implications associated with decreased milk production, lower reproduction rates, and premature culling. DD is of polymicrobial etiology, with the main causative agent identified as belonging to the Treponema genus. Current treatments include topical application of antibiotics such as oxytetracycline or foot baths containing caustic chemicals; however clinical cure rates remain highly variable. In this thesis cattle with DD were monitored to explore the skin innate immune response. An exhaustive description of the inflammatory response during disease progression and a novel description of bovine host defence peptides (HDPs) and their contribution to disease resolution are found herein. It was observed that active DD was characterized by necrotic tissue populated with neutrophils and elevated Cxcl-8 and Tlr4 expression. Tracheal antimicrobial peptide (Tap) was vastly increased in active lesions and key for the resolution of DD. An in vitro model utilizing human keratinocytes showed pro-inflammatory cytokines are released in the absence of living treponemes through Tlr2 signaling and that secretory treponeme products induced cathelicidins. The ability to manipulate inflammatory reactions via treatment with vitamin D3 in DD was compared to the commonly-used oxytetracycline. A cohort of cattle with M2 were topically treated with vitamin D3 against powdered oxytetracycline for 5 days. Although vitamin D3 did elevated Tap expression, lesions and inflammatory markers remained unchanged. In contrast, oxytetracycline reduced neutrophil chemoattractant Cxcl-8 while Tlr2 remained elevated. Histologic assessment evidenced scab formation. Taken together, this thesis established the skin innate response and role of host defence peptides (Tap) during DD and supported oxytetracycline as a treatment, providing lesion resolution and aiding in bacterial elimination.
- ItemOpen AccessHigh Mobility Group Box-1 Protein and Outcomes in Critically Ill Surgical Patients Requiring Open Abdominal Management(2017-02-14) Malig, Michelle S.; Jenne, Craig N.; Ball, Chad G.; Roberts, Derek J.; Xiao, Zhengwen; Kirkpatrick, Andrew W.Background. Previous studies assessing various cytokines in the critically ill/injured have been uninformative in terms of translating to clinical care management. Animal abdominal sepsis work suggests that enhanced intraperitoneal (IP) clearance of Damage-Associated Molecular Patterns (DAMPs) improves outcome. Thus measuring the responses of DAMPs offers alternate potential insights and a representative DAMP, High Mobility Group Box-1 protein (HMGB-1), was considered. While IP biomediators are being recognized in critical illness/trauma, HMGB-1 behaviour has not been examined in open abdomen (OA) management. Methods. A modified protocol for HMGB-1 detection was used to examine plasma/IP fluid samples from 44 critically ill/injured OA patients enrolled in a randomized controlled trial comparing two negative pressure peritoneal therapies (NPPT): Active NPPT (ANPPT) and Barker’s Vacuum Pack NPPT (BVP). Samples were collected and analyzed at the time of laparotomy and at 24 and 48 hours after. Results. There were no statistically significant differences in survivor versus nonsurvivor HMGB-1 plasma or IP concentrations at baseline, 24 hours, or 48 hours. However, plasma HMGB-1 levels tended to increase continuously in the BVP cohort. Conclusions. HMGB-1 appeared to behave differently between NPPT cohorts. Further studies are needed to elucidate the relationship of HMGB-1 and outcomes in septic/injured patients.
- ItemOpen AccessImmunoglobulin repertoire development and diversification in sheep(2005) Jenne, Craig N.; Reynolds, John D.
- ItemEmbargoKupffer Cells In Action: Understanding The Mechanisms Of Hepadnaviral Persistence(2023-06) Al-Yasiri, Layla; Coffin, Carla S.; Jenne, Craig N.; van der Meer, FrankHepatitis B virus (HBV) is the prototypical member of Hepadnaviridae family, and a causative agent of liver disease. Despite the availability of a vaccine, an estimated 1.5 million new individuals become infected each year. HBV can establish persistent infection in hepatocytes, which can disrupt hepatic homeostasis. Kupffer cells (KCs) are liver-resident macrophages that act as the first of line of defense against infectious agents. Woodchuck hepatitis virus (WHV) is a member of Hepadnaviridae. North American woodchucks (Marmota monax) infected with WHV present a natural immunocompetent model and demonstrate comparable infection outcomes and progression to hepatocellular carcinoma. We hypothesized that KC absence in acute viral hepatitis leads to chronicity by impacting virus processing and immune responses. To investigate this, we sought to uncover the early host-virus interactions in acute WHV infection. We designed and optimized in-house WHV and woodchuck-specific assays to assess intrahepatic and systemic virus presence following WHV challenge (<24 hours) and infection (6 weeks). An intravital imaging protocol was developed to capture real-time events in a living woodchuck using purified and fluorescently labelled WHV. Clodronate liposome-mediated depletion of KCs in woodchucks was performed to evaluate pre-acute challenge and acute infection in either the presence or absence of KCs. Intravital imaging of woodchucks challenged with WHV showed first appearance of virus in the liver within 30 seconds and capture by KCs within 5-10 minutes. In addition, depletion of KCs did not impact the localization of WHV during the initial hour or 24 hours of challenge. Assessment of WHV markers, serology and liver histology revealed differences in viral and biochemical markers between the KC-depleted and non-depleted animals over the initial 6 weeks. KC depletion, prior to acute WHV infection, resulted in faster onset of acute hepatitis (elevated liver enzymes) and greater suppression of WHV DNA (in circulation and intrahepatically). However, there was no difference in WHV DNA suppression between KC-depleted and non-depleted cohorts after 6 weeks p.i, as all assayed woodchucks achieved suppression of plasma WHV DNA. This may suggest that KCs normally operate under tolerogenic conditions to prevent hepatocyte damage and hepatitis, allowing for greater viral replication and immune evasion.