Evaluating the Responses of Murine Epithelial Cells Lines to Nanoparticles
Andrij Holian, Mentor
Engineered nanomaterials (ENM) are a new class of materials that are being developed for a wide variety of uses ranging from medical to engineering applications and even many personal products. These materials have unique properties because they approach molecular size (< 100 nm in at least one dimension) and can be fabricated from many elements and chemically functionalized for specific applications. Consequently, the number of possible ENM becomes almost limitless. Because of their unique properties they can cause unexpected harmful biological responses. Many of these materials have been shown to cause respiratory inflammation and lung disease in animal models raising concerns in the scientific community for improved information on the potential adverse health effects. Because ENM are being developed at a rate that exceeds the ability to conduct comprehensive animal testing a major effort is underway to develop predictive in vitro testing. Our laboratory is a member of a national consortium to conduct in vitro and in vivo testing of ENM with an effort to study mechanisms of action and develop predictive models. A current need is to develop a suitable model of epithelial cells that would serve a predictive role. The summer project will be to test a series of epithelial cells using ENM under investigation in our laboratory to determine which cell line is the most appropriate and what properties of ENM cause adverse responses.
The student working on this project will develop expertise in the emerging field of nanotoxicology, cell culture, assays for cytotoxicity (necrosis and apoptosis), uptake of nanomaterials and various ELISA assays.
Mechanisms of Silica Uptake Leading to NLRP3 Inflammasome Activation
Teri Girtsman, Mentor
Macrophages are phagocytic cells responsible for clearing any foreign material in order to minimize inflammatory responses as part of normal innate host defense. However, some particles such as crystalline silica, asbestos and certain engineered nanomaterials (ENM) are taken into phagolysosomes where they cause lysis of the lysosome initiating an inflammatory response. The inflammatory response is caused by release of cathepsin B from the lysosome that leads to assembly of a multiprotein complex called the NLRP3 inflammasome resulting in activation of caspase-1. Caspase-1 cleaves pro-inflammatory cytokines (e.g., IL-1b) that are central to many acute and chronic diseases. It is unclear what mechanism the macrophage uses to internalize these particles leading to the sequence of inflammatory events described above. Macrophages utilize clathrin-mediated endocytosis, caveolae and phagocytic mechanisms for particle uptake including binding to scavenger receptors. This project will utilize bone marrow-derived macrophages from genetically modified mice to examine which internalization mechanisms are primarily responsible for activating the NLRP3 inflammasome.
The student working on this project will develop expertise in bone marrow-derived macrophage cultures, assays to measure particle uptake by macrophages, assessment of lysosomal lysis and inflammasome activation.
Wood Smoke-Induced Alterations to Immune System Development
Christopher Migliaccio, Mentor
Exposure to wood smoke particulate matter (WS-PM) can be from either periodic or chronic exposures. Exposure to environmental factors, such as WS, can have profound effects on the immune status of not only an individual, but on their offspring, as well. One study found a correlation between childhood asthma and exposures to biomass smoke. One of the contributors to WS toxicity is polycyclic aromatic hydrocarbons (PAH) that have been detected in both the particle- and the vapor-phases of smoke. Polycyclic aromatic hydrocarbons have recently been studied as a contributor, via changes in DNA methylation, to childhood asthma, a disease resulting from immune dysregulation. Studies have shown that hyper- or hypo-methylation of DNA due to maternal exposures can affect future risk for disease development. Because PAH are constituents of wood smoke (WS), and rural communities like those in Montana utilize wood as a major source of indoor heating, the present study is designed to assess the link between prenatal WS exposures and epigenetic alterations to respiratory immunity (i.e. asthma).
In this project, the student will learn animal exposure models, flow cytometry, and molecular biological techniques.
Biomarker of Asthma Severity
Tony Ward, Mentors
Symptoms and outcomes associated with childhood asthma are subject to high levels of variability dependent upon asthma phenotype and asthma control. Several NIH institutes that conduct asthma research formed an Asthma Biomarkers Subcommittee to propose standardized asthma outcomes measures. The measures recommended by the subcommittee are fairly consistent with similar efforts by the American Thoracic Society and the European Respiratory Society. Urinary leukotriene E4 (LTE4) is one of the biomarkers recommended as a supplemental outcome. LTE4 is a marker of cysteinyl leukotriene activity. Cysteinyl leukotrienes are eicosanoids produced by a variety of cells and associated with allergic inflammation. Urinary LTE4 increases with asthma exacerbastions. Mass spectroscopy is the preferred method for measuring LTE4.
Our current study of asthmatic children exposed to wood smoke includes several measures of asthma severity. We have also collected urine samples at up to 8 different time points for each of more than 100 asthmatic children. The summer intern will become familiar with GCMS techniques as they quantify urinary concentrations of LTE4 from archived samples. Results from LTE4 analyses will be compared to children’s home exposure data and indicators of asthma severity.
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