Browsing by Author "Yipp, Bryan G."

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    Endotyping in ARDS: one step forward in precision medicine
    (2024-05-14) Côté, Andréanne; Lee, Chel H.; Metwaly, Sayed M.; Doig, Christopher J.; Andonegui, Graciela; Yipp, Bryan G.; Parhar, Ken K. S.; Winston, Brent W.
    Abstract Background The Berlin definition of acute respiratory distress syndrome (ARDS) includes only clinical characteristics. Understanding unique patient pathobiology may allow personalized treatment. We aimed to define and describe ARDS phenotypes/endotypes combining clinical and pathophysiologic parameters from a Canadian ARDS cohort. Methods A cohort of adult ARDS patients from multiple sites in Calgary, Canada, had plasma cytokine levels and clinical parameters measured in the first 24 h of ICU admission. We used a latent class model (LCM) to group the patients into several ARDS subgroups and identified the features differentiating those subgroups. We then discuss the subgroup effect on 30 day mortality. Results The LCM suggested three subgroups (n1 = 64, n2 = 86, and n3 = 30), and 23 out of 69 features made these subgroups distinct. The top five discriminating features were IL-8, IL-6, IL-10, TNF-a, and serum lactate. Mortality distinctively varied between subgroups. Individual clinical characteristics within the subgroup associated with mortality included mean PaO2/FiO2 ratio, pneumonia, platelet count, and bicarbonate negatively associated with mortality, while lactate, creatinine, shock, chronic kidney disease, vasopressor/ionotropic use, low GCS at admission, and sepsis were positively associated. IL-8 and Apache II were individual markers strongly associated with mortality (Area Under the Curve = 0.84). Perspective ARDS subgrouping using biomarkers and clinical characteristics is useful for categorizing a heterogeneous condition into several homogenous patient groups. This study found three ARDS subgroups using LCM; each subgroup has a different level of mortality. This model may also apply to developing further trial design, prognostication, and treatment selection.
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    Engineering of Gelatin Methacryloyl (GelMA) Hydrogels for Developing Biomimetic Tissue Constructs
    (2020-10-14) Janmaleki, Mohsen; Sanati Nezhad, Amir; Ungrin, Mark D.; Yipp, Bryan G.; Ronsky, Janet L.; Gaharwar, Akhilesh K.; Khoshnazar, Rahil; Lu, Qingye G.
    Three-dimensional (3D) cell culture offers a more physiologically-relevant context for disease modeling and drug screening. Hydrogel-based biomaterials support the long-term culture of cells in 3D and enhance memetic cell-cell and cell-matrix interactions. Herein, gelatin methacryloyl (GelMA) hydrogel, a well-known photo-crosslinkable hydrogel, was selected for engineering of different tissue constructs.First, the practicability of imprinting cell topography on GelMA hydrogel was investigated. A novel method was developed to fabricate cell-like niches over the hydrogel’s substrate, and its effects on cytocompatibility and drug susceptibility of breast cancer cells were studied. Second, GelMA hydrogel was tuned in terms of mechanical properties and porosity to facilitate in vitro myelination of dorsal root ganglia (DRG) neurons by Schwann cells (SCs). It was shown that the tuned GelMA enhanced single axonal generation (unlike collagen) and promoted DRGs’ interaction with SCs (unlike PDL). Third, the role of temperature on bioprintability of GelMA bioinks in a two-step crosslinking strategy was investigated. Lowering the temperature can enhance the physical gelation of GelMA and consequently improve filament formation. Results showed that the decrease in the temperature could improve the printability and shape fidelity of the deposited hydrogel, particularly at 15 °C. Time-dependent mechanical testing confirmed higher elastic properties of the collected hydrogel at the lower temperature.Fourth, a hydrogel-based 3D human intracranial aneurysm (IA) model was developed using liquid assisted injection molding. With clinically relevant dimensions and tuned fluidic and matrix properties, the essential endothelium was successfully lined inside the reconstructed IA over pre-cultured smooth muscle cells. Based on the characterized viscoelastic properties of the GelMA hydrogel and with the help of a fluid-structure interaction model, the capability of the IA construct model in predicting the response of the IA to different fluid flow profiles was demonstrated.Finally, using the techniques developed in this thesis, a new approach is suggested to fabricate a fully hydrogel-based platform for tissue engineering and organ-a-chip applications.
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    Enhanced 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 Stokes
    Up 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.
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    Investigating pulmonary vascular B cells
    (2019-04-26) Podstawka, John; Yipp, Bryan G.; Hirota, Simon Andrew; Kelly, Margaret Mary; Flannigan, Kyle L.
    The lung is a vital organ which is required for gas exchange. The anatomy of the lung itself, immune molecules, and leukocytes provide the host with protection from harmful debris and pathogenic stimuli. A component of pulmonary host defense is neutrophils which are found within the lung capillary network. Within the pulmonary capillaries, there is a population of neutrophils which remains adherent to the vascular endothelium for extended periods of time and exhibits marginated behaviour. While pulmonary vascular neutrophils are important in the acute immune response of the lung, they tend to exhibit an aged and pro-inflammatory phenotype. While inflammation within the lung is necessary for host defence and is a part of wound healing, cellular infiltration of the lung significantly compromises oxygen delivery and carbon dioxide removal. Therefore, pulmonary inflammation must be tightly controlled and regulated. We previously determined that pulmonary vascular B cells regulate neutrophils via CD18-mediated interactions, labelling them MHCII+AnnexinV+, and that in the absence of B cells, the lung will develop pathological fibrotic interstitial lung disease. In this body of work, we determined that pulmonary intravascular B cells can marginate which allows them to specialize in regulating lung neutrophils. These B cells exhibit a mature, naïve, conventional B2 cell phenotype, and engage in marginated behaviour which can be mediated by CD49e or via CXCR5/CXCL13. Blocking CD49e decreased the amount B cell-neutrophil interactions taking place within the lung capillaries, and providing exogenous CXCL13 or neutralizing endogenous CXCL13 would respectively increase and decrease the amount of interactions. Moreover, we demonstrated an intratracheal injection of CXCL13 increased the number of neutrophils which exhibited an MHCII+AnnexinV+ phenotype; indicating increased regulation via B cell interactions. Thus, we acquired novel insight into how lung intravascular B cells are engaging in prolonged interactions with lung neutrophils; interactions which were previously defined as regulatory and important for maintaining inflammatory homeostasis.
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    Mechanisms of vaccine protection in pneumococcal pneumonia
    (2018-06-13) Schubert, Courtney Lynn; Yipp, Bryan G.; Leigh, Richard A.; Peters, Nathan C.
    Despite the overwhelming success of vaccination in reducing mortality due to infectious diseases, it is unknown what makes some vaccines protective while others are not. Multiple vaccine candidates have failed clinical trial regardless of the production of neutralizing antibodies, therefore there is a need to establish correlates of protection that are not antibody production. We began investigating the Pnuemovax-23 vaccine which provides well characterized protection in both mice and humans, in an attempt to determine the mechanism of vaccine protection. Instead of finding that Pneumovax-23 vaccination induces disease resistance against Streptococcus pneumoniae pneumonia through eradication of the bacteria, we found that vaccinated mice survive the pneumococcal infection via disease tolerance. Therefore, we began investigating how the survival of vaccinated mice is independent of bacterial clearance from the lungs and the spleen. The role of neutrophils was first explored since there was still robust neutrophil recruitment during infection in vaccinated mice, and neutrophils are known to regulate B cells in the spleen and produce B cell growth factors. Ultimately, we determined that despite ample neutrophil recruitment during infection, neutrophils are not mediating this disease tolerance to infection in vaccinated mice. Instead, we turned to investigate whether B cells were required for disease tolerance, where we found B1 innate B cells to be required for vaccinated mice to survive the infection. CD19-/- mice are deficient in innate cells, however still have conventional B cells and neutrophil recruitment during infection. Therefore, we have found a novel mechanism of how vaccination protects against infection, through disease tolerance of the bacterial infection. With further research into the exact mechanism of B1 cells, these findings can potentially alter the future of vaccine development for bacterial pathogens that do not yet have a protective vaccine.
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    Regulation of cAMP-Dependent Gene Expression in Airway Epithelial Cells: A Transcriptional and Pharmacodynamic Analysis
    (2019-08-02) Yan, Dong; Giembycz, Mark A.; Slater, Donna M.; Yipp, Bryan G.; Hollenberg, Morley Donald
    Asthma and chronic obstructive pulmonary disease (COPD) are leading causes of death worldwide and their prevalence is predicted to increase over the next 20 years. Current treatments focus on providing symptomatic relief, with bronchodilators, and gaining disease control by suppressing inflammation. Long-acting β2-adrenoceptor agonists (LABAs) are effective bronchodilators that work by elevating cAMP in airway smooth muscle (ASM). Less appreciated and understood is the ability of cAMP to regulate gene expression. For example, previous studies have shown LABAs to protect ASM against pro-contractile stimuli by up-regulating RGS2, which attenuates signalling mediated through Gq-linked receptors. Based on these historical data, the first objective of the work reported here was to “mine” the LABA-regulated transcriptome for additional genes with disease-relevant functions. In the BEAS-2B airway epithelial cell line and in human primary bronchial epithelial cells (HBEC), the LABAs, indacaterol and salmeterol, promoted robust transcriptional responses. Functional annotation and gene ontology analyses identified genes with both anti-inflammatory and adverse effect potential. Moreover, there was significant overlap in gene expression changes between BEAS-2B and HBEC, and between the two LABAs. These observations suggested that pro- inflammatory gene expression changes could explain the known toxicity of chronic, β2- adrenoceptor agonist monotherapy initially reported in patients with asthma in the 1960s. Conversely, the expression of anti-inflammatory genes could help prevent exacerbations. Roflumilast is a phosphodiesterase (PDE) 4 inhibitor approved by the United States Food and Drug Administration as an ‘add-on’ therapy for patients with severe, bronchitic COPD with a history of frequent exacerbations. The second objective was to determine if inhibiting cAMP degradation could augment the expression of the LABA-regulated transcriptome and assess whether this could help explain the clinical activity of roflumilast. In BEAS-2B cells, roflumilast N-oxide (the active metabolite of roflumilast) augmented gene expression changes induced by a submaximal concentration of salmeterol, although the magnitude of the effect was dependent on the gene of interest. Further analysis established that this was because the sensitivity of the salmeterol-regulated transcriptome varied by a factor of 7.5-fold, which could be consistent with gene- dependent differences in regulation downstream of cAMP-dependent protein kinase. The final objective was to examine the pharmacology of GS-5759. This is a novel, bifunctional ligand composed a b2-adrenoceptor agonist linked to an analogue of the PDE4 inhibitor, GSK 256066, by a pent-1-yn-1ylbenzene spacer. Pharmacodynamic studies established that the PDE4 inhibitor pharmacophore produced a 35.5-fold increase in affinity for the b2-adrenoceptor relative to the monofunctional parent compound in the absence of any change in efficacy. However, this was not related to inhibition of PDE4 but to non-allosteric, ‘forced proximity’ binding, which predicts an increased in retention time of a compound near the orthosteric site of the receptor. In the context of obstructive lung diseases LABAs are established bronchodilators. The data presented in this thesis demonstrate that these drugs alone and in combination with a PDE4 inhibitor also have significant genomic actions. The possibility that gene expression changes occur in patients taking inhaled b2-adrenoceptor agonists and have functional consequences should be considered.
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    The Role of Lymph Node Subcapsular Sinus Macrophages in Limiting Tumor Progression through a B-cell Response
    (2019-07-10) Louie, Dante Alexander Patrick; Liao, Shan; von der Weid, Pierre Yves; Mahoney, Douglas J.; Yipp, Bryan G.
    Cancer immunotherapy targeting antitumor T-cells is demonstrated to be promising in treating several types of cancers. Adoptive T-cell transfer and immune checkpoint inhibitors are among the most successful immunotherapies today. However, these therapies are not effective in all patients and they can lead to devastating side effects in patients, including autoimmune diseases such as colitis. A better understanding of different aspects of cancer immunity can reveal additional targets for optimal cancer immunotherapy. One of these aspects is the tumor-draining lymph node - the first lymphoid organ that encounters tumor-derived antigens and generates the first wave of anti-tumor immunity. Recent publications have revealed that the activation of B-cells suppresses tumor growth in melanoma mice models. In my studies, using a C57BL/6 syngeneic E0771 breast cancer cell line, tumor-derived antigens were detected in the germinal center with CD169+ subcapsular sinus macrophage in the tumor-draining lymph node. The importance of B-cells was demonstrated by using B-cell deficient mice, which significantly suppressed tumor growth compared to wild-type mice. Transferring wild-type B-cells to B-cell deficient mice recovered tumor growth. To determine if subcapsular sinus macrophages regulate B-cell activity, I depleted the lymph node subcapsular sinus macrophages with clodronate liposomes. Tumor growth and germinal center formation were increased when in the macrophage-depleted tumor-draining lymph nodes, suggesting subcapsular sinus macrophages limit B-cell activation in the lymph node. Collectively, my results reveal subcapsular sinus macrophages limit B-cell activity to prevent tumor growth, which can be a potential target for future cancer immunotherapy development.
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    STING Activation as an Immunotherapeutic Strategy for Soft Tissue Sarcoma
    (2020-06-02) Marritt, Kayla Lynn; Monument, Michael J.; Jirik, Frank Robert; Krawetz, Roman J.; Yipp, Bryan G.
    Immunotherapy is an emerging field of cancer treatment that is transforming the management of numerous human cancers. However, there remains a substantial proportion of cancer subtypes that are unresponsive to many modern immunotherapy strategies. Soft tissue sarcomas (STS) are aggressive, connective-tissue derived solid cancers and are notoriously resistant to systemic therapies including immunotherapy. Immunologically, sarcomas are frequently characterized by a paucity of lymphocytic infiltrates, an immune suppressive microenvironment, and resistance to immunotherapies such as immune checkpoint inhibition and oncolytic viruses. Activation of the STING (stimulator of interferon genes) pathway can induce potent innate and adaptive anti-tumour immune responses within immunogenic solid tumours. However, this approach has never been tried in immune-inert sarcomas. Herein, STING activation in STS was analyzed to determine if STING activation could induce therapeutic anti-tumour effects and promote anti-tumour immunity. The long-term therapeutic responses of STING activation via 5,6-dimethylxanthenone-4-acetic acid (DMXAA) injection were assessed using a syngeneic murine model of undifferentiated pleomorphic sarcoma (UPS). Intratumoural DMXAA resulted in a durable cure in 50-60% of UPS-bearing mice. Flow cytometry was used to quantify immune infiltration after STING activation treatment and a higher proportion of CD8+/CD3+ cells was observed seven days post DMXAA treatment compared to the vehicle control treatment. Surviving mice all rejected UPS re-challenge in both the extremity and lung, and the therapeutic effects of DMXAA were mitigated by lymphocyte deficiency suggesting adaptive immunologic pathways are integral to the therapeutic response. This data suggests modulation of the STING pathway can elicit local and systemic anti-tumour immune responses in UPS and deserves further consideration as a novel local and systemic treatment for sarcomas.