Browsing by Subject "Allergens"

Now showing 1 - 5 of 5
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    ItemOpen Access
    Development of Data Analysis Methods and Applications for Proteome-Wide SPROX Measurements
    (2017) Ogburn, Ryenne

    Protein-ligand interactions can be detected and quantified using protein folding stability measurements. Thus, protein folding stability changes are closely linked to protein function and are an important biophysical measurement. Stability of Proteins from Rates of Oxidation (SPROX) is one approach for making proteome wide stability measurements on proteins. Previous SPROX data analysis strategies relied heavily on visual inspection of the data to identify protein stability changes. This created a bottleneck in the analysis and chances for human error. As part of this work, several new data analysis strategies were evaluated using data from previously reported ligand-binding studies. One strategy, the so-called difference analysis, was determined to be the data analysis strategy of choice as it minimized false positive and negatives. The difference analysis strategy was applied to identify protein targets of the breast cancer therapeutic tamoxifen (TAM) and its active metabolite 4-hydroxytamoxifen (4OHT) in yeast and protein targets of TAM and its most abundant metabolite n-desmethyl tamoxifen (NDT) in MCF-7 cell lysate. Several yeast protein targets of TAM and 4OHT were identified using SPROX, and they were subsequently validated using a pulse proteolysis strategy. The proteins in an MCF-7 cell line were probed for TAM- and NDT-induced stability changes using the SPROX in combination with two different quantitative proteomics strategies. Together the SPROX experiments on the proteins in an MCF-7 cell lysate enabled over 1000 proteins to be assayed for TAM- and NDT- induced protein stability changes. Ultimately, a total of 163 and 200 proteins with TAM- and NDT-induced stability changes were identified, respectively. A subset of 33 high confidence hits, including those identified using both proteomics strategies or those identified with multiples peptide probes, were assessed for experimental links to the ER using a STRING analysis. One high confidence protein hit, Y-box binding protein 1 (YBX1), was recently shown to bind the estrogen receptor, which is the known target of TAM. Preliminary results generated here using pulse proteolysis and a purified recombinant YBX1 protein construct suggest that YBX1 is a direct protein target of TAM. Proteins with altered expression levels with TAM and NDT treatment were also identified here. In total, 799 and 671 proteins were probed for TAM- and NDT- induced expression changes, respectively, and 49 and 30 proteins had altered expression. Out of the 49 and 30 proteins with TAM- and NDT- induced expression changes, 14 and 4 proteins had TAM- and NDT- induced altered stability, respectively. In addition to the above ligand-binding studies, SPROX was utilized to characterize the stability of the allergen containing proteomes of the European house dust mite, timothy grass pollen, and ragweed pollen. It was determined that the protein allergens in these proteomes were more stable and more abundant (based on transcriptomic data), than non-allergen proteins from these sources.

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    ItemOpen Access
    Exogenous leptin enhances markers of airway fibrosis in a mouse model of chronic allergic airways disease.
    (Respiratory research, 2022-05-24) Ihrie, Mark D; McQuade, Victoria L; Womble, Jack T; Hegde, Akhil; McCravy, Matthew S; Lacuesta, Cyrus Victor G; Tighe, Robert M; Que, Loretta G; Walker, Julia KL; Ingram, Jennifer L

    Background

    Asthma patients with comorbid obesity exhibit increased disease severity, in part, due to airway remodeling, which is also observed in mouse models of asthma and obesity. A mediator of remodeling that is increased in obesity is leptin. We hypothesized that in a mouse model of allergic airways disease, mice receiving exogenous leptin would display increased airway inflammation and fibrosis.

    Methods

    Five-week-old male and female C57BL/6J mice were challenged with intranasal house dust mite (HDM) allergen or saline 5 days per week for 6 weeks (n = 6-9 per sex, per group). Following each HDM exposure, mice received subcutaneous recombinant human leptin or saline. At 48 h after the final HDM challenge, lung mechanics were evaluated and the mice were sacrificed. Bronchoalveolar lavage was performed and differential cell counts were determined. Lung tissue was stained with Masson's trichrome, periodic acid-Schiff, and hematoxylin and eosin stains. Mouse lung fibroblasts were cultured, and whole lung mRNA was isolated.

    Results

    Leptin did not affect mouse body weight, but HDM+leptin increased baseline blood glucose. In mixed-sex groups, leptin increased mouse lung fibroblast invasiveness and increased lung Col1a1 mRNA expression. Total lung resistance and tissue damping were increased with HDM+leptin treatment, but not leptin or HDM alone. Female mice exhibited enhanced airway responsiveness to methacholine with HDM+leptin treatment, while leptin alone decreased total respiratory system resistance in male mice.

    Conclusions

    In HDM-induced allergic airways disease, administration of exogenous leptin to mice enhanced lung resistance and increased markers of fibrosis, with differing effects between males and females.
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    Particulate allergens potentiate allergic asthma in mice through sustained IgE-mediated mast cell activation.
    (2010) Jin, Cong
    In recent years, the incidence of allergic asthma as well as the severity of disease has rapidly increased worldwide. Numerous epidemiological studies have related the exacerbation of allergic asthma with exposure to increased ambient particles from air pollutants. However, the mechanism by which particulate allergens (pAg) exacerbate allergic asthma remains undefined. To evaluate this, we modeled environmental pAg induced allergic asthma by exposing mice to polystyrene beads coated with natural allergen extracts. Compared to equal amounts of soluble allergen extracts (sAg), pAg triggered markedly enhanced airway hyper-responsiveness and pulmonary eosinophilia in allergen sensitized mice. The cellular basis for this effect was determined to be mast cells (MCs), as both airway allergic responses were attenuated in MC deficient KitWsh/KitW-sh mice compared to MC reconstituted KitW-sh/KitW-sh mice. The divergent responses of MCs to pAg versus sAg were due to differences in the termination rate of IgE/FcεRI initiated signaling. Following ligation of sAg, IgE/FcεRI rapidly shuttled into a degradative endosome/lysosome pathway. However, following ligation by pAg, IgE/FcεRI migrated into lipid raft enriched compartments and subsequently failed to follow a degradative pathway, which resulted in a prolonged signaling and heightened synthesis of proinflammatory mediators. These observations highlight the overlooked contributions of the particulate nature of allergens and mast cell endocytic circuitry to the aggravation of allergic asthma.
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    Targeted HAS2 Expression Lessens Airway Responsiveness in Chronic Murine Allergic Airway Disease.
    (American journal of respiratory cell and molecular biology, 2017-12) Walker, Julia KL; Theriot, Barbara S; Ghio, Michael; Trempus, Carol S; Wong, Jordan E; McQuade, Victoria L; Liang, Jiurong; Jiang, Dianhua; Noble, Paul W; Garantziotis, Stavros; Kraft, Monica; Ingram, Jennifer L
    Hyaluronan (HA), a major component of the extracellular matrix, is secreted by airway structural cells. Airway fibroblasts in allergic asthma secrete elevated levels of HA in association with increased HA synthase 2 (HAS2) expression. Thus, we hypothesized that HA accumulation in the airway wall may contribute to airway remodeling and hyperresponsiveness in allergic airways disease. To examine this hypothesis, transgenic mice in which the α-smooth muscle actin (α-SMA) promoter drives HAS2 expression were generated. Mixed male and female α-SMA-HAS2 mice (HAS2+ mice, n = 16; HAS2- mice, n = 13) were sensitized via intraperitoneal injection and then chronically challenged with aerosolized ovalbumin (OVA) for 6 weeks. To test airway responsiveness, increasing doses of methacholine were delivered intravenously and airway resistance was measured using the forced oscillation technique. HA, cytokines, and cell types were analyzed in bronchoalveolar lavage fluid, serum, and whole lung homogenates. Lung sections were stained using antibodies specific for HA-binding protein (HABP) and α-SMA, as well as Masson's trichrome stain. Staining of lung tissue demonstrated significantly increased peribronchial HA, α-SMA, and collagen deposition in OVA-challenged α-SMA-HAS2+ mice compared with α-SMA-HAS2- mice. Unexpectedly, OVA-challenged α-SMA-HAS2+ mice displayed significantly reduced airway responsiveness to methacholine compared with similarly treated α-SMA-HAS2- mice. The total numbers of inflammatory cell types in the bronchoalveolar lavage fluid did not differ significantly between OVA-challenged α-SMA-HAS2+ mice and α-SMA-HAS2- mice. We conclude that allergen-challenged mice that overexpress HAS2 in myofibroblasts and smooth muscle cells develop increased airway fibrosis, which lessens airway hyperresponsiveness to bronchoconstrictors.
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    Vertical sleeve gastrectomy associates with airway hyperresponsiveness in a murine model of allergic airway disease and obesity.
    (Frontiers in endocrinology, 2023-01) Womble, Jack T; Ihrie, Mark D; McQuade, Victoria L; Hegde, Akhil; McCravy, Matthew S; Phatak, Sanat; Tighe, Robert M; Que, Loretta G; D'Alessio, David; Walker, Julia KL; Ingram, Jennifer L

    Introduction

    Asthma is a chronic airway inflammatory disease marked by airway inflammation, remodeling and hyperresponsiveness to allergens. Allergic asthma is normally well controlled through the use of beta-2-adrenergic agonists and inhaled corticosteroids; however, a subset of patients with comorbid obesity experience resistance to currently available therapeutics. Patients with asthma and comorbid obesity are also at a greater risk for severe disease, contributing to increased risk of hospitalization. Bariatric surgery improves asthma control and airway hyperresponsiveness in patients with asthma and comorbid obesity, however, the underlying mechanisms for these improvements remain to be elucidated. We hypothesized that vertical sleeve gastrectomy (VSG), a model of metabolic surgery in mice, would improve glucose tolerance and airway inflammation, resistance, and fibrosis induced by chronic allergen challenge and obesity.

    Methods

    Male C57BL/6J mice were fed a high fat diet (HFD) for 13 weeks with intermittent house dust mite (HDM) allergen administration to induce allergic asthma, or saline as control. At week 11, a subset of mice underwent VSG or Sham surgery with one week recovery. A separate group of mice did not undergo surgery. Mice were then challenged with HDM or saline along with concurrent HFD feeding for 1-1.5 weeks before measurement of lung mechanics and harvesting of tissues, both of which occurred 24 hours after the final HDM challenge. Systemic and pulmonary cytokine profiles, lung histology and gene expression were analyzed.

    Results

    High fat diet contributed to increased body weight, serum leptin levels and development of glucose intolerance for both HDM and saline treatment groups. When compared to saline-treated mice, HDM-challenged mice exhibited greater weight gain. VSG improved glucose tolerance in both saline and HDM-challenged mice. HDM-challenged VSG mice exhibited an increase in airway hyperresponsiveness to methacholine when compared to the non-surgery group.

    Discussion

    The data presented here indicate increased airway hyperresponsiveness in allergic mice undergoing bariatric surgery.