Publications by authors named "Ajay P Nayak"

35 Publications

Diacylglycerol Kinase Inhibition Reduces Airway Contraction by Negative Feedback Regulation of Gq-signaling.

Am J Respir Cell Mol Biol 2021 Jul 22. Epub 2021 Jul 22.

Thomas Jefferson University, 6559, Center for Translational Medicine, Philadelphia, Pennsylvania, United States;

Exaggerated airway smooth muscle (ASM) contraction regulated by the Gq family of G protein-coupled receptors (GPCRs) causes airway hyperresponsiveness (AHR) in asthma. Activation of Gq-coupled GPCRs leads to phospholipase C (PLC)-mediated generation of inositol triphosphate (IP3) and diacylglycerol (DAG). DAG signaling is terminated by the action of DAG kinase (DGK) that converts DAG into phosphatidic acid (PA). Our previous study demonstrated that DGKα and ζ isoform knockout mice are protected from the development of allergen-induced AHR. Here we aimed at determining the mechanism by which DGK regulates ASM contraction. Activity of DGK isoforms was inhibited in human ASM cells by siRNA-mediated knockdown of DGKα and ζwhile pharmacological inhibition was achieved by pan DGK inhibitor I (R59022). Effects of DGK inhibition on contractile agonist-induced activation of PLC and myosin light chain (MLC) kinase, elevation of IP3, and calcium levels were assessed. Further, we employed human precision-cut lung slices and assessed the role of DGK in agonist-induced bronchoconstriction. DGK inhibitor I attenuated histamine- and methacholine-induced bronchoconstriction. DGKα and ζ knockdown or pre-treatment with DGK inhibitor I resulted in attenuated agonist-induced phosphorylation of MLC and myosin light chain phosphatase in ASM cells. Further, DGK inhibition decreased Gq agonist-induced calcium elevation, generation of IP3, and increased histamine-induced production of PA. Finally, DGK inhibition or treatment with DAG analog resulted in attenuation of activation of PLC in human ASM cells. Our findings suggest that DGK inhibition perturbed the DAG:PA ratio resulting in inhibition of Gq-PLC activation in a negative feedback manner, resulting in protection against ASM contraction.
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http://dx.doi.org/10.1165/rcmb.2021-0106OCDOI Listing
July 2021

Cooperativity between β-agonists and c-Abl inhibitors in regulating airway smooth muscle relaxation.

FASEB J 2021 07;35(7):e21674

Department of Medicine, Pulmonary and Critical Care Medicine, Center for Translational Medicine, Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, PA, USA.

Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on β2 adrenoceptor agonists (β-agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with β-agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c-Abl). The cooperative effect of β-agonists (isoproterenol) and c-Abl inhibitors (GNF-5, or imatinib) on contractile agonist (methacholine, or histamine) -induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c-Abl inhibitors and β-agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c-Abl inhibitors and β-agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by β-agonists is near optimal and is not increased by c-Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both β-agonists and c-Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.
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http://dx.doi.org/10.1096/fj.202100154RDOI Listing
July 2021

Occupational Allergies to Cannabis.

J Allergy Clin Immunol Pract 2020 Nov - Dec;8(10):3331-3338

Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, Philadelphia, Pa; Jane & Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, Pa; Center for Translational Medicine, Thomas Jefferson University, Philadelphia, Pa. Electronic address:

Within the last decade there has been a significant expansion in access to cannabis for medicinal and adult nonmedical use in the United States and abroad. This has resulted in a rapidly growing and diverse workforce that is involved with the growth, cultivation, handling, and dispensing of the cannabis plant and its products. The objective of this review was to educate physicians on the complexities associated with the health effects of cannabis exposure, the nature of these exposures, and the future practical challenges of managing these in the context of allergic disease. We will detail the biological hazards related to typical modern cannabis industry operations that may potentially drive allergic sensitization in workers. We will highlight the limitations that have hindered the development of objective diagnostic measures that are essential in separating "true" cannabis allergies from nonspecific reactions/irritations that "mimic" allergy-like symptoms. Finally, we will discuss recent advances in the basic and translational scientific research that will aid the development of diagnostic tools and therapeutic standards to serve optimal management of cannabis allergies across the occupational spectrum.
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http://dx.doi.org/10.1016/j.jaip.2020.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837257PMC
May 2021

Therapeutic potential and challenges of bitter taste receptors on lung cells.

Curr Opin Pharmacol 2020 04 15;51:43-49. Epub 2020 Aug 15.

Center for Translational Medicine, Jane and Leonard Korman Lung Center, Thomas Jefferson University, Philadelphia, PA 19107, United States. Electronic address:

Airway smooth muscle (ASM) hyperresponsiveness and airway remodeling are pathological drivers of disease progression and mortality in asthma. Importantly, approximately 50% of affected individuals are unable to reliably manage disease symptoms using the current standard of care. Recently, T2Rs have been identified as a novel class of G protein-coupled receptors expressed in the airway that on activation can induce ASM relaxation and reduction in airway tone. Further, agonists of T2Rs may also remedy airway remodeling, which has been difficult to manage with currently available medications. In this review, we will discuss the recent developments in T2R biology and their role in cellular physiology (particularly ASM) and expand on the therapeutic potential of T2R agonists in treatment of asthma.
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http://dx.doi.org/10.1016/j.coph.2020.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530014PMC
April 2020

Species-specific evaluation of 2 patients with suspected crab allergy.

J Allergy Clin Immunol Pract 2021 01 18;9(1):511-513. Epub 2020 Jul 18.

Sidney Kimmel Medical College at Thomas Jefferson University, Jane and Leonard Korman Respiratory Institute, Division of Pulmonary, Allergy & Critical Care, Allergy & Immunology Section, Philadelphia, Pa.

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http://dx.doi.org/10.1016/j.jaip.2020.07.006DOI Listing
January 2021

The proton-sensing receptor ovarian cancer G-protein coupled receptor 1 (OGR1) in airway physiology and disease.

Curr Opin Pharmacol 2020 04 29;51:1-10. Epub 2020 Apr 29.

Department of Medicine, Division of Pulmonary and Critical Care Medicine, Center for Translational Medicine, Jane and Leonard Korman Lung Institute, Thomas Jefferson University, 1020 Locust St., Suite 543G JAH, Philadelphia, PA, 19107, United States. Electronic address:

Numerous G protein-coupled receptors (GPCRs) regulate multiple airway functions and play fundamental roles in normal and aberrant airway and lung physiology. Thus, GPCRs are prime candidates of targeting by disease therapeutics. The intriguing proton-sensing GPCR Ovarian cancer G-protein coupled receptor 1 (OGR1; aka GPR68) has recently been shown capable of regulating airway smooth muscle (ASM) contraction and proliferation. Although the study of OGR1 has been confounded by the fact that the proton is the presumed cognate ligand of OGR1, recent studies have begun to identify novel ligands and modulators capable of regulating the diverse signaling, and functional role of OGR1. Such studies offer hope for OGR1-targeting drugs as therapeutics for obstructive lung diseases such as asthma. Herein, we review the literature to date detailing the receptor biology and pharmacology of OGR1, receptor function in the airway, and describe the potential clinical utility of OGR1-modulating drugs.
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http://dx.doi.org/10.1016/j.coph.2020.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529780PMC
April 2020

Cannabis: An Emerging Occupational Allergen?

Ann Work Expo Health 2020 08;64(7):679-682

Department of Medicine, Center for Translational Medicine and Division of Pulmonary Allergy and Critical Care Medicine, Jane and Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, PA, USA.

Cannabis is the most commonly used psychoactive drug. In recent years, Cannabis access has expanded for both medicinal and non-medicinal has grown. This is also marked with an increasing number of individuals gaining employment in this emerging industry. In this article, we briefly discuss the health hazards associated with Cannabis exposure with an emphasis on the potential for allergic reactions in workers who handle and process Cannabis plant.
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http://dx.doi.org/10.1093/annweh/wxaa043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407602PMC
August 2020

Discovery of Human Signaling Systems: Pairing Peptides to G Protein-Coupled Receptors.

Cell 2019 10;179(4):895-908.e21

Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark. Electronic address:

The peptidergic system is the most abundant network of ligand-receptor-mediated signaling in humans. However, the physiological roles remain elusive for numerous peptides and more than 100 G protein-coupled receptors (GPCRs). Here we report the pairing of cognate peptides and receptors. Integrating comparative genomics across 313 species and bioinformatics on all protein sequences and structures of human class A GPCRs, we identify universal characteristics that uncover additional potential peptidergic signaling systems. Using three orthogonal biochemical assays, we pair 17 proposed endogenous ligands with five orphan GPCRs that are associated with diseases, including genetic, neoplastic, nervous and reproductive system disorders. We also identify additional peptides for nine receptors with recognized ligands and pathophysiological roles. This integrated computational and multifaceted experimental approach expands the peptide-GPCR network and opens the way for studies to elucidate the roles of these signaling systems in human physiology and disease. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2019.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838683PMC
October 2019

Inhalation of Fragments Induces Pulmonary Arterial Remodeling.

Am J Respir Cell Mol Biol 2020 05;62(5):563-576

Office of the Director, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.

is a fungal contaminant within the built environment and a respiratory health concern in the United States. The objective of this study was to characterize the mechanisms influencing pulmonary immune responses to repeatedly inhaled . Groups of B6C3F1/N mice repeatedly inhaled viable trichothecene-producing conidia (strain A or strain B), heat-inactivated conidia, or high-efficiency particulate absolute-filtered air twice per week for 4 and 13 weeks. Strain A was found to produce higher amounts of respirable fragments than strain B. Lung tissue, serum, and BAL fluid were collected at 24 and 48 hours after final exposure and processed for histology, flow cytometry, and RNA and proteomic analyses. At 4 weeks after exposure, a T-helper cell type 2-mediated response was observed. After 13 weeks, a mixed T-cell response was observed after exposure to strain A compared with a T-helper cell type 2-mediated response after strain B exposure. After exposure, both strains induced pulmonary arterial remodeling at 13 weeks; however, strain A-exposed mice progressed more quickly than strain B-exposed mice. BAL fluid was composed primarily of eosinophils, neutrophils, and macrophages. Both the immune response and the observed pulmonary arterial remodeling were supported by specific cellular, molecular, and proteomic profiles. The immunopathological responses occurred earlier in mice exposed to high fragment-producing strain A. The rather striking induction of pulmonary remodeling by appears to be related to the presence of fungal fragments during exposure.
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http://dx.doi.org/10.1165/rcmb.2019-0221OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263392PMC
May 2020

Bitter Taste Receptors: an Answer to Comprehensive Asthma Control?

Curr Allergy Asthma Rep 2019 09 5;19(10):48. Epub 2019 Sep 5.

Department of Medicine, Center for Translational Medicine and Division of Pulmonary, Allergy and Critical Care Medicine; and Jane & Leonard Korman Respiratory Institute, Thomas Jefferson University, Jefferson Alumni Hall, Room 543, 1020 Locust Street, Philadelphia, PA, 19107, USA.

Purpose Of Review: Asthma is marked by peculiar pathological features involving airway contraction, an impinging inflammation in the lungs, and an inexorably progressive remodeling of pulmonary architecture. Current medications for management of asthma exacerbations fail to optimally mitigate these pathologies, which is partly due to the intrinsic heterogeneity in the development and progression of asthma within different populations. In recent years, the discovery of the ectopic expression of TAS2Rs in extraoral tissues and different cell types, combined with significant strides in gaining mechanistic understanding into receptor signaling and function, has revealed the potential to target TAS2Rs for asthma relief.

Recent Findings: TAS2R activation leads to relaxation of airway smooth muscle cells and bronchodilation. In addition, findings from preclinical studies in murine model of asthma suggest that TAS2R agonists inhibit allergen-induced airway inflammation, remodeling, and hyperresponsiveness. In this review, we expand on the opportunity presented by TAS2Rs in the development of a comprehensive asthma treatment that overcomes the limitations set forth by current asthma therapeutics.
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http://dx.doi.org/10.1007/s11882-019-0876-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765386PMC
September 2019

Bitter Taste Receptors for Asthma Therapeutics.

Front Physiol 2019 16;10:884. Epub 2019 Jul 16.

Division of Pulmonary, Allergy and Critical Care Medicine, Center for Translational Medicine, Department of Medicine, Jane and Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, PA, United States.

Clinical management of asthma and chronic obstructive pulmonary disease (COPD) has primarily relied on the use of beta 2 adrenergic receptor agonists (bronchodilators) and corticosteroids, and more recently, monoclonal antibody therapies (biologics) targeting specific cytokines and their functions. Although these approaches provide relief from exacerbations, questions remain on their long-term efficacy and safety. Furthermore, current therapeutics do not address progressive airway remodeling (AR), a key pathological feature of severe obstructive lung disease. Strikingly, agonists of the bitter taste receptors (TAS2Rs) deliver robust bronchodilation, curtail allergen-induced inflammatory responses in the airways and regulate airway smooth muscle (ASM) cell proliferation and mitigate features of AR and in animal models. The scope of this review is to provide a comprehensive and systematic insight into our current understanding of TAS2Rs with an emphasis on the molecular events that ensue TAS2R activation in distinct airway cell types and expand on the pleiotropic effects of TAS2R targeting in mitigating various pathological features of obstructive lung diseases. Finally, we will discuss specific opportunities that could help the development of selective agonists for specific TAS2R subtypes in the treatment of asthma.
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http://dx.doi.org/10.3389/fphys.2019.00884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6647873PMC
July 2019

Glucocorticoids and Airway Smooth Muscle: A Few More Answers, Still More Questions.

Authors:
Ajay P Nayak

Am J Respir Cell Mol Biol 2019 Jul;61(1):9-10

1 Center for Translational Medicine and.

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http://dx.doi.org/10.1165/rcmb.2019-0089EDDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6604212PMC
July 2019

Regulation of ovarian cancer G protein-coupled receptor-1 expression and signaling.

Am J Physiol Lung Cell Mol Physiol 2019 05 6;316(5):L894-L902. Epub 2019 Feb 6.

Department of Medicine, Center for Translational Medicine and Division of Pulmonary, Allergy and Critical Care Medicine; and Jane & Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, Pennsylvania.

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a recently deorphanized G protein-coupled receptor shown to signal in response to low extracellular pH (↓pH) or certain benzodiazepines. The pleiotropic nature of OGR1 signaling in human airway smooth muscle (HASM) cells suggests that OGR1 is a potential therapeutic target for the management of obstructive lung diseases. However, the basic pharmacological and regulatory features of OGR1 remain poorly understood. We employed model systems of heterologously expressed [human embryonic kidney 293 (HEK293) cells] or endogenous (HASM) OGR1 to assess changes in expression, subcellular localization, and signaling capabilities following acute or chronic treatment with ↓pH or the benzodiazepines lorazepam and sulazepam. In HEK293 cells expressing OGR1, treatment with ↓pH and/or lorazepam, but not sulazepam, caused rapid OGR1 internalization. In HASM cells, acute treatment with ↓pH or benzodiazepines did not alter abundance of OGR1 mRNA; however, significant downregulation was observed following chronic treatment. Acute and chronic pretreatment of HASM cells with sulazepam or lorazepam resulted in receptor desensitization as demonstrated by reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) or p42/p44 upon rechallenge. Acid (acute but not chronic) pretreatment of HASM cells induced desensitization of OGR1-mediated VASP (but not p42/p44) phosphorylation. In contrast to a recent study reporting OGR1 upregulation and sensitization in cardiac tissue subject to ischemic/acidic insult, chronic OGR1 activation in multiple model systems did not increase OGR1 expression or signaling capacity. The ability to induce OGR1 internalization and desensitization was activator dependent, reflecting the ability of different activators to induce specific receptor confirmations and engagement of specific heterotrimeric G proteins.
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http://dx.doi.org/10.1152/ajplung.00426.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589584PMC
May 2019

Cooperativity of E-prostanoid receptor subtypes in regulating signaling and growth inhibition in human airway smooth muscle.

FASEB J 2019 04 2;33(4):4780-4789. Epub 2019 Jan 2.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Center for Translational Medicine, Jane and Leonard Korman Lung Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

Prostaglandin E2 (PGE) is produced in the airway during allergic lung inflammation and both promotes and inhibits features of asthma pathology. These mixed effects relate to 4 E-prostanoid (EP) receptor subtypes (EP1, 2, 3 and 4) expressed at different levels on different resident and infiltrating airway cells. Although studies have asserted both EP2 and EP4 expression in human airway smooth muscle (HASM), a recent study asserted EP4 to be the functionally dominant EP subtype in HASM. Herein, we employ recently-developed subtype-selective ligands to investigate singular or combined EP2 and EP4 receptor activation in regulating HASM signaling and proliferation. The subtype specificity of ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) was first demonstrated in human embryonic kidney 293 cells stably expressing different EP receptor subtypes. EP receptor knockdown and subtype-selective antagonists demonstrated EP2 and EP4 receptor responsiveness in HASM cells to the specific ONO compounds, whereas PGE appeared to preferentially signal via the EP4 receptor. Both singular EP2 and EP4 receptor agonists inhibited HASM proliferation, and combined EP2 and EP4 receptor agonism exhibited positive cooperativity in both chronic G-mediated signaling and inhibiting HASM proliferation. These findings suggest both EP2 and EP4 are functionally important in HASM, and their combined targeting optimally inhibits airway smooth muscle proliferation.-Michael, J. V. Gavrila, A., Nayak, A. P., Pera, T., Liberato, J. R., Polischak, S. R., Shah, S. D., Deshpande, D. A., Penn, R. B. Cooperativity of E-prostanoid receptor subtypes in regulating signaling and growth inhibition in human airway smooth muscle.
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http://dx.doi.org/10.1096/fj.201801959RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436661PMC
April 2019

New targets for resolution of airway remodeling in obstructive lung diseases.

F1000Res 2018 30;7. Epub 2018 May 30.

Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, USA.

Airway remodeling (AR) is a progressive pathological feature of the obstructive lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). The pathology manifests itself in the form of significant, progressive, and (to date) seemingly irreversible changes to distinct respiratory structural compartments. Consequently, AR correlates with disease severity and the gradual decline in pulmonary function associated with asthma and COPD. Although current asthma/COPD drugs manage airway contraction and inflammation, none of these effectively prevent or reverse features of AR. In this review, we provide a brief overview of the features and putative mechanisms affecting AR. We further discuss recently proposed strategies with promise for deterring or treating AR.
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http://dx.doi.org/10.12688/f1000research.14581.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5981194PMC
May 2018

Aspergillus fumigatus viability drives allergic responses to inhaled conidia.

Ann Allergy Asthma Immunol 2018 08 13;121(2):200-210.e2. Epub 2018 Apr 13.

Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia.

Background: Aspergillus fumigatus-induced allergic airway disease has been shown to involve conidial germination in vivo, but the immunological mechanisms remain uncharacterized.

Objective: A subchronic murine exposure model was used to examine the immunological mediators that are regulated in response to either culturable or nonculturable A fumigatus conidia.

Methods: Female B6C3F1/N mice were repeatedly dosed via inhalation with 1 × 105 viable or heat-inactivated conidia (HIC), twice per week for 13 weeks (26 exposures). Control mice inhaled high-efficiency particulate arrestor-filtered air. The influence of A fumigatus conidial germination on the pulmonary immunopathological outcomes was evaluated by flow cytometry analysis of cellular infiltration in the airways, assessment of lung messenger RNA expression, quantitative proteomics, and histopathology of whole lung tissue.

Results: Repeated inhalation of viable conidia, but not HIC, resulted in allergic inflammation marked by vascular remodeling, extensive eosinophilia, and accumulation of alternatively activated macrophages (AAMs) in the murine airways. More specifically, mice that inhaled viable conidia resulted in a mixed TH1 and TH2 (IL-13) cytokine response. Recruitment of eosinophils corresponded with increased Ccl11 transcripts. Furthermore, genes associated with M2 or alternatively activated macrophage polarization (eg, Arg1, Chil3, and Retnla) were significantly up-regulated in viable A fumigatus-exposed mice. In mice inhaling HIC, CD4+ T cells expressing IFN-γ (TH1) dominated the lymphocytic infiltration. Quantitative proteomics of the lung revealed metabolic reprogramming accompanied by mitochondrial dysfunction and endoplasmic reticulum stress stimulated by oxidative stress from repetitive microbial insult.

Conclusion: Our studies demonstrate that A fumigatus conidial viability in vivo is critical to the immunopathological presentation of chronic fungal allergic disease.
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http://dx.doi.org/10.1016/j.anai.2018.04.008DOI Listing
August 2018

Microbial hazards during harvesting and processing at an outdoor United States cannabis farm.

J Occup Environ Hyg 2018 05;15(5):430-440

d Office of the Director, Health Effects Laboratory Division, National Institute for Occupational Safety and Health , Centers for Disease Control and Prevention , Morgantown , West Virginia.

Cannabis cultivation is an emerging industry within the United States. Organic dust derived in part from naturally occurring microorganisms is known to cause byssinosis in the hemp industry. In this pilot study, bacteria and fungi encountered by workers at an outdoor cannabis farm that utilized organic practices were elucidated by 16 S ribosomal RNA (rRNA) and Internal Transcribed Spacer (ITS) region sequencing, respectively. Area (n = 14) and personal air samples (n = 12) were collected during harvesting and processing activities. 16 S rRNA and ITS regions of extracted bacterial and fungal genomic DNA were amplified and sequenced using Sanger sequencing. Bacterial sequencing resolved 1,077 sequences that were clustered into 639 operational taxonomic units (OTUs) and predominantly placed in the phylum, Actinobacteria (46%). Personal air samples revealed higher bacterial and Actinobacteria diversity compared to outdoor area samples collected within the facility (p < 0.05). A high degree of dissimilarity between bacteria was identified within and between samples. Fungal sequences (n = 985) were identified and predominantly clustered in the phylum Ascomycota (53%). Of the 216 fungal OTUs elucidated, the cannabis plant pathogenic species, Botrytis cinerea, was the most prevalent and accounted for 34% of all fungal sequences. The relative abundance of B. cinerea was highest in personal air samples (59%) compared to area samples collected in the drying room (19%), greenhouse (18%), and outdoor environment (6%). There was 49% sample similarity between fungi identified within personal air samples, but higher dissimilarity coefficients were observed within and between greenhouse, drying room, and outdoor area air samples. The results of this pilot study suggest that the cannabis farm workers are potentially exposed to Actinobacteria as well as the cannabis plant pathogen, B. cinerea during harvesting, bud-stripping, and hand-trimming processes.
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http://dx.doi.org/10.1080/15459624.2018.1432863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314012PMC
May 2018

Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.

FASEB J 2018 02 4;32(2):862-874. Epub 2018 Jan 4.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Center for Translational Medicine, Jane and Leonard Korman Lung Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

GPCRs have diverse signaling capabilities, based on their ability to assume various conformations. Moreover, it is now appreciated that certain ligands can promote distinct receptor conformations and thereby bias signaling toward a specific pathway to differentially affect cell function. The recently deorphanized G protein-coupled receptor OGR1 [ovarian cancer G protein-coupled receptor 1 ( GPR68)] exhibits diverse signaling events when stimulated by reductions in extracellular pH. We recently demonstrated airway smooth muscle cells transduce multiple signaling events, reflecting a diverse capacity to couple to multiple G proteins. Moreover, we recently discovered that the benzodiazepine lorazepam, more commonly recognized as an agonist of the γ-aminobutyric acid A (GABA) receptor, can function as an allosteric modulator of OGR1 and, similarly, can promote multiple signaling events. In this study, we demonstrated that different benzodiazepines exhibit a range of biases for OGR1, with sulazepam selectively activating the canonical Gs of the G protein signaling pathway, in heterologous expression systems, as well as in several primary cell types. These findings highlight the potential power of biased ligand pharmacology for manipulating receptor signaling qualitatively, to preferentially activate pathways that are therapeutically beneficial.-Pera, T., Deshpande, D. A., Ippolito, M., Wang, B., Gavrila, A., Michael, J. V., Nayak, A. P., Tompkins, E., Farrell, E., Kroeze, W. K., Roth, B. L., Panettieri, R. A. Jr Benovic, J. L., An, S. S., Dulin, N. O., Penn, R. B. Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.
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http://dx.doi.org/10.1096/fj.201700555RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5888400PMC
February 2018

Bitter Taste Receptor Agonists Mitigate Features of Allergic Asthma in Mice.

Sci Rep 2017 04 11;7:46166. Epub 2017 Apr 11.

Department of Medicine, Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA, 19107, USA.

Asthma is characterized by airway inflammation, mucus secretion, remodeling and hyperresponsiveness (AHR). Recent research has established the bronchodilatory effect of bitter taste receptor (TAS2R) agonists in various models. Comprehensive pre-clinical studies aimed at establishing effectiveness of TAS2R agonists in disease models are lacking. Here we aimed to determine the effect of TAS2R agonists on features of asthma. Further, we elucidated a mechanism by which TAS2R agonists mitigate features of asthma. Asthma was induced in mice using intranasal house dust mite or aerosol ova-albumin challenge, and chloroquine or quinine were tested in both prophylactic and treatment models. Allergen challenge resulted in airway inflammation as evidenced by increased immune cells infiltration and release of cytokines and chemokines in the lungs, which were significantly attenuated in TAS2R agonists treated mice. TAS2R agonists attenuated features of airway remodeling including smooth muscle mass, extracellular matrix deposition and pro-fibrotic signaling, and also prevented mucus accumulation and development of AHR in mice. Mechanistic studies using human neutrophils demonstrated that inhibition of immune cell chemotaxis is a key mechanism by which TAS2R agonists blocked allergic airway inflammation and exerted anti-asthma effects. Our comprehensive studies establish the effectiveness of TAS2R agonists in mitigating multiple features of allergic asthma.
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http://dx.doi.org/10.1038/srep46166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5387415PMC
April 2017

Topical application of the anti-microbial chemical triclosan induces immunomodulatory responses through the S100A8/A9-TLR4 pathway.

J Immunotoxicol 2017 12;14(1):50-59

a Allergy and Clinical Immunology Branch , National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention , Morgantown , WV , USA.

The anti-microbial compound triclosan is incorporated into numerous consumer products and is detectable in the urine of 75% of the general United States population. Recent epidemiological studies report positive associations with urinary triclosan levels and allergic disease. Although not sensitizing, earlier studies previously found that repeated topical application of triclosan augments the allergic response to ovalbumin (OVA) though a thymic stromal lymphopoietin (TSLP) pathway in mice. In the present study, early immunological effects following triclosan exposure were further evaluated following topical application in a murine model. These investigations revealed abundant expression of S100A8/A9, which reportedly acts as an endogenous ligand for Toll-like Receptor 4 (TLR4), in skin tissues and in infiltrating leukocytes during topical application of 0.75-3.0% triclosan. Expression of Tlr4 along with Tlr1, Tlr2 and Tlr6 increased in skin tissues over time with triclosan exposure; high levels of TLR4 were expressed on skin-infiltrating leukocytes. In vivo antibody blockade of the TLR4/MD-2 receptor complex impaired local inflammatory responses after four days, as evidenced by decreased Il6, Tnfα, S100a8, S100a9, Tlr1, Tlr2, Tlr4 and Tlr6 expression in the skin and decreased lymph node cellularity and production of IL-4 and IL-13 by lymph node T-cells. After nine days of triclosan exposure with TLR4/MD-2 blockade, impaired T-helper cell type 2 (T2) cytokine responses were sustained, but other early effects on skin and lymph node cellularity were lost; this suggested alternative ligands/receptors compensated for the loss of TLR4 signaling. Taken together, these data suggest the S100A8/A9-TLR4 pathway plays an early role in augmenting immunomodulatory responses with triclosan exposure and support a role for the innate immune system in chemical adjuvancy.
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http://dx.doi.org/10.1080/1547691X.2016.1258094DOI Listing
December 2017

Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin.

J Immunol Methods 2016 Apr 4;431:38-44. Epub 2016 Feb 4.

Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA.

Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb-TDI antigenicity was approximately 30% that of HSA-TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity.
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http://dx.doi.org/10.1016/j.jim.2016.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792703PMC
April 2016

Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy.

Appl Environ Microbiol 2015 Sep 19;81(17):5794-803. Epub 2015 Jun 19.

National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, Morgantown, West Virginia, USA.

Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-μm fragments, compared to 100% of >2-μm fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.
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http://dx.doi.org/10.1128/AEM.00929-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551229PMC
September 2015

Triclosan Induces Thymic Stromal Lymphopoietin in Skin Promoting Th2 Allergic Responses.

Toxicol Sci 2015 Sep 5;147(1):127-39. Epub 2015 Jun 5.

*Allergy and Clinical Immunology Branch, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia 26505;

Triclosan is an antimicrobial chemical incorporated into many personal, medical and household products. Approximately, 75% of the U.S. population has detectable levels of triclosan in their urine, and although it is not typically considered a contact sensitizer, recent studies have begun to link triclosan exposure with augmented allergic disease. We examined the effects of dermal triclosan exposure on the skin and lymph nodes of mice and in a human skin model to identify mechanisms for augmenting allergic responses. Triclosan (0%-3%) was applied topically at 24-h intervals to the ear pinnae of OVA-sensitized BALB/c mice. Skin and draining lymph nodes were evaluated for cellular responses and cytokine expression over time. The effects of triclosan (0%-0.75%) on cytokine expression in a human skin tissue model were also examined. Exposure to triclosan increased the expression of TSLP, IL-1β, and TNF-α in the skin with concomitant decreases in IL-25, IL-33, and IL-1α. Similar changes in TSLP, IL1B, and IL33 expression occurred in human skin. Topical application of triclosan also increased draining lymph node cellularity consisting of activated CD86(+)GL-7(+) B cells, CD80(+)CD86(+) dendritic cells, GATA-3(+)OX-40(+)IL-4(+)IL-13(+) Th2 cells and IL-17 A(+) CD4 T cells. In vivo antibody blockade of TSLP reduced skin irritation, IL-1β expression, lymph node cellularity, and Th2 responses augmented by triclosan. Repeated dermal exposure to triclosan induces TSLP expression in skin tissue as a potential mechanism for augmenting allergic responses.
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http://dx.doi.org/10.1093/toxsci/kfv113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734116PMC
September 2015

Production of a Chaetomium globosum enolase monoclonal antibody.

Monoclon Antib Immunodiagn Immunother 2014 Dec;33(6):428-37

Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health , Centers for Disease Control and Prevention, Morgantown, West Virginia.

Chaetomium globosum is a hydrophilic fungal species and a contaminant of water-damaged building materials in North America. Methods to detect Chaetomium species include subjective identification of ascospores, viable culture, or molecular-based detection methods. In this study, we describe the production and initial characterization of a monoclonal antibody (MAb) for C. globosum enolase. MAb 1C7, a murine IgG1 isotype MAb, was produced and reacted with recombinant C. globosum enolase (rCgEno) in an enzyme-linked immunosorbent assay and with a putative C. globosum enolase in a Western blot. Epitope mapping showed MAb 1C7 specific reactivity to an enolase decapeptide, LTYEELANLY, that is highly conserved within the fungal class Sordariomycetes. Cross-reactivity studies showed MAb 1C7 reactivity to C. atrobrunneum but not C. indicum. MAb 1C7 did not react with enolase from Aspergillus fumigatus, which is divergent in only two amino acids within this epitope. The results of this study suggest potential utility of MAb 1C7 in Western blot applications for the detection of Chaetomium and other Sordariomycetes species.
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http://dx.doi.org/10.1089/mab.2014.0042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278173PMC
December 2014

A murine inhalation model to characterize pulmonary exposure to dry Aspergillus fumigatus conidia.

PLoS One 2014 23;9(10):e109855. Epub 2014 Oct 23.

Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia, United States of America.

Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Δalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×105 twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8+ IL17+ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Δalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109855PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207673PMC
June 2015

Toluene diisocyanate (TDI) disposition and co-localization of immune cells in hair follicles.

Toxicol Sci 2014 Aug 5;140(2):327-37. Epub 2014 May 5.

Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia 26505.

Diisocyanates (dNCOs) are potent chemical allergens utilized in various industries. It has been proposed that skin exposure to dNCOs produces immune sensitization leading to work-related asthma and allergic disease. We examined dNCOs sensitization by using a dermal murine model of toluene diisocyanate (TDI) exposure to characterize the disposition of TDI in the skin, identify the predominant haptenated proteins, and discern the associated antigen uptake by dendritic cells. Ears of BALB/c mice were dosed once with TDI (0.1% or 4% v/v acetone). Ears and draining lymph nodes (DLNs) were excised at selected time points between 1 h and 15 days post-exposure and were processed for histological, immunohistochemical, and proteomic analyses. Monoclonal antibodies specific for TDI-haptenated protein (TDI-hp) and antibodies to various cell markers were utilized with confocal microscopy to determine co-localization patterns. Histopathological changes were observed following exposure in ear tissue of mice dosed with 4% TDI/acetone. Immunohistochemical staining demonstrated TDI-hp localization in the stratum corneum, hair follicles, and sebaceous glands. TDI-hp were co-localized with CD11b(+) (integrin αM/Mac-1), CD207(+) (langerin), and CD103(+) (integrin αE) cells in the hair follicles and in sebaceous glands. TDI-hp were also identified in the DLN 1 h post-exposure. Cytoskeletal and cuticular keratins along with mouse serum albumin were identified as major haptenated species in the skin. The results of this study demonstrate that the stratum corneum, hair follicles, and associated sebaceous glands in mice are dendritic cell accessible reservoirs for TDI-hp and thus identify a mechanism for immune recognition following epicutaneous exposure to TDI.
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http://dx.doi.org/10.1093/toxsci/kfu079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176049PMC
August 2014

A murine monoclonal antibody with broad specificity for occupationally relevant diisocyanates.

J Occup Environ Hyg 2014 ;11(2):101-10

a Allergy and Clinical Immunology Branch, Health Effects Laboratory Division , National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention , Morgantown , West Virginia.

Diisocyanates (dNCOs) used in industrial applications are well known low molecular weight allergens. Occupational exposure is associated with adverse health outcomes including allergic sensitization and occupational asthma. In this study, we report the production and initial characterization of a dNCO-hapten specific murine IgM monoclonal antibody (mAb). Female BALB/c mice were immunized intraperitoneally with 25 μg of 4,4'-methylene diphenyl diisocyanate (MDI)-keyhole limpet hemocyanin. Following six biweekly booster immunizations, splenocytes were recovered and fused to Sp2/0-Ag14 murine myeloma cell line for hybridoma production. Hybridomas were then screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against 40:1 4,4'-MDI- human serum albumin (HSA). mAb reactivity to dNCO-HSA conjugates and dNCO-HSA spiked human serum were characterized using a sandwich ELISA. One hybridoma produced a multimeric IgM mAb (15D4) that reacted with 4,4'-MDI-HSA. Sandwich ELISA analysis demonstrated comparable reactivity with other occupationally relevant dNCO-HSA adducts, including 2,4-toluene diisocyanate (TDI)-HSA, 2,6-TDI-HSA, and 1,6-hexamethylene diisocyanate (HDI)-HSA, but not other electrophilic chemical HSA conjugates. The limit of quantification (LOQ) of 4,4'-MDI-HSA, 2,4-TDI-HSA, 2,6-TDI-HSA, and 1,6-HDI-HSA sandwich ELISAs were 567.2, 172.7, 184.2, and 403.5 ng/mL (8.67, 2.60, 2.77, and 6.07 pmol/mL), respectively. In contrast, experiments using dNCO-supplemented human sera showed an increase in the detectable limit of the assay. A mAb has been produced that has potential utility for detecting mixed diisocyanate exposures in occupational environments. The mAb may have additional utility in the standardization of specific IgE detection immunoassays as well as chromatographic-mass spectrometric methods to enrich dNCO adducted HSA in the plasma of occupationally exposed workers.
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http://dx.doi.org/10.1080/15459624.2013.843783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624273PMC
August 2014

Aspergillus collagen-like genes (acl): identification, sequence polymorphism, and assessment for PCR-based pathogen detection.

Appl Environ Microbiol 2013 Dec 11;79(24):7882-95. Epub 2013 Oct 11.

Department of Microbiology, Immunology, and Cell Biology.

The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects.
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http://dx.doi.org/10.1128/AEM.02835-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837832PMC
December 2013

Pulmonary immune responses to Aspergillus fumigatus in an immunocompetent mouse model of repeated exposures.

J Immunotoxicol 2014 Apr-Jun;11(2):180-9. Epub 2013 Aug 6.

Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention , Morgantown, WV , USA .

Aspergillus fumigatus is a filamentous fungus that produces abundant pigmented conidia. Several fungal components have been identified as virulence factors, including melanin; however, the impact of these factors in a repeated exposure model resembling natural environmental exposures remains unknown. This study examined the role of fungal melanin in the stimulation of pulmonary immune responses using immunocompetent BALB/c mice in a multiple exposure model. It compared conidia from wild-type A. fumigatus to two melanin mutants of the same strain, Δarp2 (tan) or Δalb1 (white). Mass spectrometry-based analysis of conidial extracts demonstrated that there was little difference in the protein fingerprint profiles between the three strains. Field emission scanning electron microscopy demonstrated that the immunologically inert Rodlet A layer remained intact in melanin-deficient conidia. Thus, the primary difference between the strains was the extent of melanization. Histopathology indicated that each A. fumigatus strain induced lung inflammation, regardless of the extent of melanization. In mice exposed to Δalb1 conidia, an increase in airway eosinophils and a decrease in neutrophils and CD8(+) IL-17(+) (Tc17) cells were observed. Additionally, it was shown that melanin mutant conidia were more rapidly cleared from the lungs than wild-type conidia. These data suggest that the presence of fungal melanin may modulate the pulmonary immune response in a mouse model of repeated exposures to A. fumigatus conidia.
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http://dx.doi.org/10.3109/1547691X.2013.819054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604601PMC
December 2014

Characterization of Cannabis sativa allergens.

Ann Allergy Asthma Immunol 2013 Jul 22;111(1):32-7. Epub 2013 May 22.

Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, West Virginia 26505, USA.

Background: Allergic sensitization to Cannabis sativa is rarely reported, but the increasing consumption of marijuana has resulted in an increase in the number of individuals who become sensitized. To date, little is known about the causal allergens associated with C sativa.

Objective: To characterize marijuana allergens in different components of the C sativa plant using serum IgE from marijuana sensitized patients.

Methods: Serum samples from 23 patients with a positive skin prick test result to a crude C sativa extract were evaluated. IgE reactivity was variable between patients and C sativa extracts. IgE reactivity to C sativa proteins in Western blots was heterogeneous and ranged from 10 to 70 kDa. Putative allergens derived from 2-dimensional gels were identified.

Results: Prominent IgE reactive bands included a 23-kDa oxygen-evolving enhancer protein 2 and a 50-kDa protein identified to be the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. Additional proteins were identified in the proteomic analysis, including those from adenosine triphosphate synthase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and luminal binding protein (heat shock protein 70), suggesting these proteins are potential allergens. Deglycosylation studies helped refine protein allergen identification and demonstrated significant IgE antibodies against plant oligosaccharides that could help explain cross-reactivity.

Conclusion: Identification and characterization of allergens from C sativa may be helpful in further understanding allergic sensitization to this plant species.
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http://dx.doi.org/10.1016/j.anai.2013.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726218PMC
July 2013
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