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Papers by Elizabeth Schwantes

Investigative Ophthalmology & Visual Science, 2011

Investigative Ophthalmology & Visual Science, 2013

Eosinophils and their catabolic mediators are major features in the pathogenesis of allergic conj... more Eosinophils and their catabolic mediators are major features in the pathogenesis of allergic conjunctivitis, contributing to corneal damage in chronic disease. The prostaglandin, PGD 2 , which is released from activated mast cells, has recently been shown to promote eosinophil chemotaxis. The purpose of this study was to further examine the effects of PGD 2 on activation of eosinophil pro-inflammatory processes (chemotaxis, degranulation, phosphorylation of spleen tyrosine kinase [Syk] and survival/viability). Methods: Human purified peripheral blood eosinophils were obtained using negative immunomagnetic bead selection. For most experiments, PGD 2 was added at concentrations ranging from 0.1-1000 nM. For chemotaxis, eosinophils were added to the top compartment of 5.0 µm Transwell chambers, with either media or PGD 2 added to the bottom for 1 hr after which eosinophils migrating to the bottom compartment were counted. For degranulation, eosinophils were stimulated with media or PGD 2 for 4 hrs and supernates were harvested and evaluated for eosinophil derived neurotoxin using commercial ELISA. Flow cytometry was used for evaluation of Syk-phosphorylation, and trypan blue exclusion was used for evaluation of survival. Results: Stimulation of eosinophils with PGD 2 promoted chemotaxis (1.0-100 nM, n=6 subjects, p<0.05), and trended toward promoting degranulation (10, 100 nm, n=4 subjects, p=0.1) and phosphorylation of Syk (100 nM, n=2 subjects), but not survival or viability (n=3 subjects). Conclusions: PGD 2-mediated activation of eosinophils could play a role in eosinophil mediated processes in allergic conjunctivitis and, thus, presents a potential target for therapeutic intervention..

$Research paper thumbnail of Elevated fractional exhaled nitric oxide and blood eosinophil counts are associated with a 17q21 asthma risk allele in adult subjects$

Journal of asthma and allergy, 2018

Genome-wide association studies identified single-nucleotide polymorphisms (SNPs) at the 17q21 lo... more Genome-wide association studies identified single-nucleotide polymorphisms (SNPs) at the 17q21 locus conferring increased risk for childhood-onset asthma. Little is known about how these SNPs impact adult asthma patients. We sought to examine an adult population for associations between rs7216389 (17q21-associated SNP) and features of asthma including fractional exhaled nitric oxide (FeNO), eosinophil counts, and age of asthma onset. Subjects were genotyped at SNP rs7216389. The geometric mean of FeNO measurements and peripheral blood eosinophil counts from 2008 to 2015 were collected. Demographics and medical history were collected including self-reported allergy diagnoses and age of asthma onset. Eosinophils, monocytes, and peripheral blood mononuclear cells (PBMCs) were isolated for the examination of ORMDL3 expression. FeNO levels from 157 genotyped subjects (31CC, 72CT, and 54TT) and peripheral eosinophil counts from 252 genotyped subjects (46CC, 122CT, and 84TT) were analyzed....

Clinical & Experimental Allergy, 2017

Background-Children with risk alleles at the 17q21 genetic locus who wheeze during rhinovirus ill... more Background-Children with risk alleles at the 17q21 genetic locus who wheeze during rhinovirus illnesses have a greatly increased likelihood of developing childhood asthma. In mice, overexpression of the 17q21 gene ORMDL3 leads to airway remodeling and hyperresponsiveness. However, the mechanisms by which ORMDL3 predisposes to asthma are unclear. Previous studies have suggested that ORMDL3 induces endoplasmic reticulum (ER) stress and production of the type I interferon (IFN) regulated chemokine CXCL10. Objective-The purpose of this study was to determine the relationship between ORMDL3 and rhinovirus-induced ER stress and type I IFN in human leukocytes. Methods-ER stress was monitored by measuring HSPA5, CHOP and spliced XBP1 gene expression, and type I IFN by measuring IFNB1 (IFN-β) and CXCL10 expression in human cell lines and primary leukocytes following treatment with rhinovirus. Requirements for cell contact and specific cell type in ORMDL3 induction were examined by transwell assay and depletion experiments, respectively. Finally, the effects of 17q21 genotype on the expression of ORMDL3, IFNB1, and ER stress genes were assessed. Results-THP-1 monocytes overexpressing ORMDL3 responded to rhinovirus with increased IFNB1 and HSPA5. Rhinovirus-induced ORMDL3 expression in primary leukocytes required cell

Journal of Allergy and Clinical Immunology, 2017

RATIONALE: Monitoring disease activity in eosinophilic esophagitis is challenging as there is a l... more RATIONALE: Monitoring disease activity in eosinophilic esophagitis is challenging as there is a lack of validated tools to replace invasive procedure-based endoscopy and pathology evaluation. We aim to identify a profile of eosinophil surface biomarkers that will correlate with eosinophil activation and disease activity in eosinophilic esophagitis. Previous studies have demonstrated that expression of eotaxin in esophageal tissue is a prominent mediator for the recruitment and activation of eosinophils in eosinophilic esophagitis. METHODS: Peripheral blood eosinophils purified by density centrifugation and negative selection using AutoMACS were stimulated with eotaxin. In an effort to identify biomarkers that correlate with EoE disease activity, previously identified eosinophil surface proteins associated with activation or allergic diseases were selected for analysis by flow cytometry. This pilot experiment was designed to concurrently examine biomarkers including: alphaL, alphaM, beta1, and beta2 integrins, activated beta1 integrin (monitored by mAb N29), CCR3, CD40, CD44, CD66b, CRTH2 and P-selectin glycoprotein ligand-1 (PSGL-1) on eosinophils, as well as alphaIIb integrin and cell-surface-associated P-selectin as markers of associated platelets. RESULTS: Eotaxin stimulation of purified eosinophils upregulated surface protein expression of beta2, alphaM and alphaL integrins, and CD66b; while downregulating CRTH2, CCR3, and N29 signals. No major changes in the expression of the other biomarkers were observed. CONCLUSIONS: Eotaxin stimulation of eosinophils results in the upregulation or downregulation of several surface biomarkers. We expect that the modulation of the surface expression of these biomarkers will be observed and will correspond to changes in eosinophilic esophagitis disease activity.

Annals of allergy, asthma & immunology : official publication of the American College of Allergy, Asthma, & Immunology, Nov 30, 2016

Journal of Proteome Research, 2016

A system-wide understanding of biological processes requires a comprehensive knowledge of the pro... more A system-wide understanding of biological processes requires a comprehensive knowledge of the proteins in the biological system. The eosinophil is a type of granulocytic leukocyte specified early in hematopoietic differentiation that participates in barrier defense, innate immunity, and allergic disease. The proteome of the eosinophil is largely unannotated with under 500 proteins identified. We now report a map of the nonstimulated peripheral blood eosinophil proteome assembled using two-dimensional liquid chromatography coupled with high-resolution mass spectrometry. Our analysis yielded 100,892 unique peptides mapping to 7,086 protein groups representing 6,813 genes as well as 4,802 site-specific phosphorylation events. We account for the contribution of

Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 2014

Asthma exacerbations contribute to significant morbidity, mortality and healthcare utilization. F... more Asthma exacerbations contribute to significant morbidity, mortality and healthcare utilization. Furthermore, viral infections are associated with asthma exacerbations by mechanisms that are not fully understood. The aim of this analysis was to determine whether cytokine patterns in patients with colds could identify risks for subsequent asthma exacerbations. We analysed cytokine levels in nasal lavage fluid (NLF) in 59 subjects (46 with asthma) with acute upper respiratory symptoms and after symptomatic resolution. Analyte choice was based on potential relevance to asthma exacerbations: antiviral (IFN-α, IFN-β, IFN-γ, IFN-λ1, IP-10, TRAIL), cell recruiting (G-CSF, IL-1β, IL-8, MCP-1, MCP-3, TNF-α), polarizing (CXCL13, IL-10, IL-13, IL-17, TSLP), and injury remodelling (fibronectin, IL-33, MMP-9, VEGF). The overall cytokine response induced during viral infections was not different between asthmatic and non-asthmatic individuals for a wide array of cytokines. However, mean levels of ...

Proceedings of the National Academy of Sciences, 2014

Significance Asthma is a chronic inflammatory disorder that is notoriously difficult to diagnose,... more Significance Asthma is a chronic inflammatory disorder that is notoriously difficult to diagnose, characterize, and properly treat with tests that are available to clinicians today. Therefore, clinicians would benefit from additional tools that objectively characterize asthma in patients. In this work, we describe a handheld microfluidic device that discriminates asthma from allergic rhinitis patients based on neutrophil function—an inflammatory cell that has been implicated in the pathogenesis of asthma. The device can sort neutrophils from a drop of whole blood within minutes, and was used in a clinical setting to characterize asthmatic and nonasthmatic patients. This technology provides a new tool for clinicians to characterize asthma based on cellular function.

Journal of Allergy and Clinical Immunology, 2008

Journal of Allergy and Clinical Immunology, 2011

ABSTRACT Background / Purpose: The conjunctival epithelium forms a barrier that isolates the eye ... more ABSTRACT Background / Purpose: The conjunctival epithelium forms a barrier that isolates the eye from the external environment and regulates the active and passive movement of macromolecules, bacterial components, allergens and cells through both paracellular and transcellular pathways. Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine that plays a critical role in allergic diseases and has recently been shown to affect respiratory epithelial barrier function. The purpose of this study was to examine the effect of TSLP on human conjunctival epithelial cell (HCE) barrier function and mediator release. Main conclusion: Studies with primary human conjunctival epithelial cells showed mRNA expression of both Thymic Stromal Lymphopoietin (TSLP) and thymic stromal derived lymphopoietin receptor (TSLPR), which was inducible by TNFa, IFNg, and IL-1B and surface expression of TSLPR.Studies with IOBA-NHC epithelial cells showed that TSLP enhanced barrier function via a TSLPR- specific mechanism; however TSLP did not promote the release of TNFa and IL-8 under these conditions.

Journal of Allergy and Clinical Immunology, 2012

RATIONALE: We hypothesized that incubation of Dendritic Cells (DC) with tolerogenic factors may i... more RATIONALE: We hypothesized that incubation of Dendritic Cells (DC) with tolerogenic factors may induce tolerogenic function artificially in order to use these cells as research and therapeutic tools. METHODS: several combinations of compounds were tested for their effect on the tolerogenic function of DC. The capacity of these cells to induce deletion of antigen-specific cells and induce the differentiation of na€ ıve CD4+ T cells (Tn) into Treg was surveyed in vitro as well as their capacity to ameliorate autoimmune conditions in vivo. RESULTS: mouse splenic and bone marrow-derived DC and human monocyte-derived DC treated in vitro with rapamycin and TGFb induce Treg differentiation. The resulting adaptive Treg (aTreg) are phenotypically and functionally equivalent to natural-occurring Treg (nTreg) in vitro. These induced-tolerogenic DC (itDC) cotreated with toll-like receptor (TLR) agonists remain tolerogenic, required direct itDC-Tn contact, TCR stimulation, but no cell division or IL-6, IL-10, TGFb, IDO1, FasL or EBI3 to stimulate Treg differentiation. Further, a marked reduction in the numbers of Tn in the first two days could be observed previous to Treg differentiation. Using the Experimental Autoimmune Encephalomyelitis model (EAE) we demonstrate that infusions of itDC blocked the progress of the disease while significantly diminishing the number of effector T cells and increasing Treg. CONCLUSIONS: itDC can shape the repertoire of antigen specific T cells through the elimination of antigen specific cells and/or the induction aTreg that suppress autoimmune responses in vivo.

Journal of Allergy and Clinical Immunology, 2007

In contrast to the well accepted mechanism of corticosteroid action at the genomic level, rapid n... more In contrast to the well accepted mechanism of corticosteroid action at the genomic level, rapid non-genomic effects of corticosteroids are poorly defined. In this study we investigated the fast effects of corticosteroids on the function of opsonin and adhesion receptors on human eosinophils. METHODS: The effect of short-term treatment (<309) with dexamethasone on adhesion, migration and Fc-receptor activation was investigated on freshly isolated eosinophils from blood of healthy donors. RESULTS: Short-term corticosteroid treatment of eosinophils (<309) with dexamethasone did not influence cell surface CD11b/CD18 expression, adhesion and/or chemokinesis. In contrast, incubation with dexamethasone resulted in a rapid increase in binding of IgA-beads, but not IgG-beads, to human eosinophils showing that dexamethasone can upregulate the activation of FcaRI (CD89), specifically (n58 experiments; p<0.005). This priming response by dexamethasone was dose dependent and optimal between 10-8 M to 10-6 M (n 5 3). Furthermore, IgA-binding studies with pharmacological inhibitors showed that dexamethasone-induced FcaRI is mediated by the glucocorticoid receptor (n 5 4; p<0.05) and involves the p38 MAP-kinase signalling pathway (n53; p<0.05). CONCLUSIONS: This work demonstrates that short-term treatment with corticosteroids selectively primes the functionality of the FcaR on eosinophils by activation of the p38 MAP-kinase signalling pathway. This occurred with no effect on expression of adhesion molecules, on adhesion and migration and on the respiratory burst. These data underscore the importance of non-genomic mechanisms involved in corticosteroid-induced modulation of eosinophil function in mucosal immunity.

Journal of Allergy and Clinical Immunology, 2010

RATIONALE: Given the presence of thymic stromal lymphopoietin (TSLP) and eosinophils as prominent... more RATIONALE: Given the presence of thymic stromal lymphopoietin (TSLP) and eosinophils as prominent components of allergic inflammation, we sought to determine whether eosinophils express the TSLP receptor a-chain (TSLPR) and respond directly to TSLP stimulation. METHODS: Eosinophil mRNA expression for TSLPR was examined by real-time quantitative PCR of purified human peripheral blood eosinophils treated with cytokines in various combinations, including IL-3, IL-5, GM-CSF, and TNFa. Immunoblotting for TSLPR protein expression was performed on eosinophil lysates with a goat anti-TSLPR antibody. Flow cytometry with a goat anti-TSLPR antibody was used to detect surface TSLPR expression. Eosinophils were stimulated with human recombinant TSLP at various concentrations for 4 hours and supernatants were analyzed by ELISA for degranulation of eosinophil derived neurotoxin (EDN). RESULTS: Expression of mRNA and cell surface TSLPR was upregulated by stimulation with TNFa and IL-5 family cytokines. TSLPR protein was also detectable by Western blotting of eosinophil lysates. Stimulation of eosinophils with TSLP resulted in phosphorylation of the STAT5 transcription factor. In addition, eosinophil stimulation with TSLP resulted in degranulation of eosinophil derived neurotoxin (EDN) at levels comparable to IL-5. Furthermore, the TSLP-stimulated eosinophil degranulation was inhibited by a functional blocking antibody to the TSLPR. CONCLUSIONS: This study demonstrates a role for eosinophils as a target of the allergic inflammatory cytokine, TSLP. Thus, in allergic inflammation, multiple pathways can serve to activate eosinophils, perhaps in a synergistic manner.

Immunity & Ageing, 2010

Chest, 2008

Background-Aging results in changes in immune cell function which have been described for T-cells... more Background-Aging results in changes in immune cell function which have been described for T-cells, macrophage, neutrophils, and dendritic cells, but not yet for eosinophils. We sought to define age-related changes in eosinophil function and their potential implications for asthma. Methods. We recruited human subjects with asthma in two age groups, a younger group (20-40 years old) and older group (55-80 years old). Lung function, induced sputum, and peripheral blood were obtained from each subject. Eosinophils isolated from the peripheral blood were examined for in vitro functional activities including degranulation, superoxide anion production, adhesion, and chemotaxis. Results-Eosinophil degranulation in response to IL-5 stimulation was significantly decreased in the older group (p=0.025). Eosinophil production of superoxide anions in response to phorbol myristate acetate was lower in the older group, but did not achieve statistical significance (p=0.097). Eosinophil adhesion, eosinophil chemotaxis, lung function, and the percentage of sputum eosinophils were similar in the two groups. Conclusion-Airway eosinophilia is comparable in younger and older asthma subjects. However, there are age-related changes in peripheral blood eosinophil "effector" functions. Diseases such as asthma, in which eosinophils are thought to play a pathophysiological role, may exhibit important clinical differences in the elderly due to age-related changes in inflammatory cell function that affect the manifestations of the disease and/or responsiveness to specific classes of medications.