Environmental estrogen exposures alter molecular signaling in immune cells that promote the development of childhood asthma.

Environmental estrogens (EEs) are associated with an increased prevalence of asthma. These epigenetic alterations of the immune cells may explain the multigenerational effects on asthma development. We hypothesized that exposure to immune cells enhances allergic sensitization by initiating signaling in these cells. Human T cell lines (TIB-152, CCL-119) were exposed to varying concentrations of estradiol, bisphenol A, bisphenol S, or bisphenol A + estradiol. H3K27me3, phosphorylations of EZH2 (pEZH2), AKT (pAKT), and phosphatidylinositide 3-kinase (pPI3K) were assessed. pAKT and pPI3K were decreased in response to some of the concentrations of these exposures in both cell lines. It is likely that EEs exposure to immune cells is one of the factors in the increase in the prevalence of asthma.

Genotypic and phenotypic variability of 22q11.2 microdeletions - an institutional experience.

Patients with chromosome 22q11.2 deletion syndromes classically present with variable cardiac defects, parathyroid and thyroid gland hypoplasia, immunodeficiency and velopharyngeal insufficiency, developmental delay, intellectual disability, cognitive impairment, and psychiatric disorders. New technologies including chromosome microarray have identified smaller deletions in the 22q11.2 region. An increasing number of studies have reported patients presenting with various features harboring smaller 22q11.2 deletions, suggesting a need to better elucidate 22q11.2 deletions and their phenotypic contributions so that clinicians may better guide prognosis for families. We identified 16 pediatric patients at our institution harboring various 22q11.2 deletions detected by chromosomal microarray and report their clinical presentations. Findings include various neurodevelopmental delays with the most common one being attention deficit hyperactivity disorder (ADHD), one reported case of infant lethality, four cases of preterm birth, one case with dual diagnoses of 22q11.2 microdeletion and Down syndrome. We examined potential genotypic contributions of the deleted regions.

Design of peptides with high affinity binding to a monoclonal antibody as a basis for immunotherapy.

About half of the US population is sensitized to one or more allergens, as found by a National Health and Nutrition Examination Survey (NHANES). The most common treatment for seasonal allergic responses is the daily use of oral antihistamines, which can control some of the symptoms, but are not effective for nasal congestion, and can be debilitating in many patients. Peptide immunotherapy is a promising new approach to treat allergic airway diseases. The small size of the immunogens cannot lead to an unwanted allergic reaction in sensitized patients, and the production of peptides with sufficient amounts for immunotherapy is time- and cost-effective. However, it is not known what peptides are the most effective for an immunotherapy of allergens. We previously produced a unique monoclonal antibody (mAb) E58, which can inhibit the binding of multiple groups of mAbs and human IgEs from patients affected by the major group 1 allergens of ragweed (Amb a 1) and conifer pollens (Jun a 1, Cup s 1, and Cry j 1). Here, we demonstrated that a combined approach, starting from two linear E58 epitopes of the tree pollen allergen Jun a 1 and the ragweed pollen allergen Amb a 1, and residue modifications suggested by molecular docking calculations and peptide design could identify a large number of high affinity binding peptides. We propose that this combined experimental and computational approach by structural analysis of linear IgE epitopes and peptide design, can lead to potential new candidates for peptide immunotherapy.

Bisphenol A Activates an Innate Viral Immune Response Pathway.

Bisphenol A (BPA) is a ubiquitous component in the manufacturing of plastic. It is commonly found in food and beverage containers. Because of its broad exposure and evidence that it may act as an estrogen-like molecule, many have studied its potential effects. For example, epidemiological studies have found an association between in utero BPA exposure and onset of childhood asthma. Our previous work suggested BPA treated mice induced asthma-like symptoms in both mothers and their pups. In order to better understand theconsequences of BPA exposure and potential mechanisms, we used a proteomics approach. Using both CD4+ T cells from an in vivo model of BPA exposure and an in vitro epithelial cell model, we identified activation of both innate and adaptive immune signaling following BPA exposure. Furthermore, our proteomic results from our multigenerational mouse model study implicates aberrant immune activation across several generations. We propose the following; BPA can active an innate viral immune response by upregulating a probable palmitoyltransferase ZDHHC1, and its binding partner stimulator of interferon-gamma (STING). It also has additional histone epigenetic perturbations, suggesting a role for epigenetic inheritance of these immune perturbations.

Pollen food allergy syndrome to tomato in mountain cedar pollen hypersensitivity.

BACKGROUND: Mountain cedar pollen is recognized as a major cause of seasonal hypersensitivity in the US. We describe here that a subgroup of these patients also suffer from pollen food allergy syndrome (PFAS). OBJECTIVE: We performed this study to determine the frequency of PFAS among patients with mountain cedar hypersensitivity. METHODS: We performed mail-out/telephone surveys of 800 mountain cedar-sensitive patients in Austin, TX. The subjects for this survey were selected by telephone screening, and skin and serologic testing. We performed immunoblot inhibition assay and mass spectrometry (MS) to identify the allergens that cause PFAS. RESULTS: Of the 28 patients with suspected food allergies, 15 had clinical manifestations of PFAS. Eleven of them had positive skin tests to tomato, six to banana, and one to apple. The subjects with PFAS have stronger cutaneous and in vitro reactivity to cedar pollen. The intensities of the tomato and banana reactivity were correlated with the cedar reactivity. The results of the ImmunoCAP inhibition experiments demonstrated a strong cross-reactivity between IgE antibodies to cedar pollen and fruits. This suggested that their primary sensitization was to cedar pollen, since absorption with cedar pollen extract strongly inhibited reactivity to each of the fruits, while the absorption with tomato extract did not significantly inhibit IgE binding to cedar extract. We determined that polygalacturonase 2 A (PG2 A) in tomato is the cause of PFAS. CONCLUSION: This is the first report of a PFAS in patients with mountain cedar pollinosis. Sensitivity to tomato, banana, and apple should be considered in cedar-sensitive patients.

Molecular patterns in the isotype-specific antibody responses to the major cedar aeroallergen Jun a 1.

Most studies of the immune responses in allergic rhinitis have focused on IgE antibodies to mixtures of allergenic proteins. Based on our previous studies of the major mountain cedar allergen Jun a 1, we sought to describe a broader assessment of the humoral immune responses to a single, dominant allergen, in three groups of allergic subjects, all of whom had similarly exposures to the whole cedar pollen. The major outcomes of this study was that, with the onset of allergic rhinitis symptoms, and after treatment with immunotherapy, serum IgE and IgG (but not IgA) antibodies to Jun a 1 increased. Interestingly, both IgE and IgG4 antibodies to the Jun a 1 allergen were strongly focused on its conformation epitopes. These IgG antibodies to conformationalstructures may be a useful marker of the therapeutic response to immunotherapy.

A single mouse monoclonal antibody, E58 modulates multiple IgE epitopes on group 1 cedar pollen allergens.

We recently described a dominant role for conformational epitopes on the group 1 allergen of the mountain cedar (Juniperus ashei, Cupressaceae), Jun a 1, in pollen hypersensitivity in South Central U.S.A. Since these epitopes are surface exposed and are likely to be flexible, they may be susceptible to molecular or physical perturbations. This may make Jun a 1 a potential target for new forms of therapy for cedar pollinosis. Here, we describe a mouse monoclonal antibody, termed E58, which binds to the group 1 allergens of the cedar pollens from three highly populated regions of the world (central U.S.A., France and Japan). Upon binding to these allergens, E58 strongly reduces the binding of patient's IgE antibodies to these dominant allergens. This characteristic of E58, and potentially other similar antibodies, suggests an opportunity to develop preventative or therapeutic agents that may inhibit cedar pollen sensitization or prevent their allergic reactions.

Choosing wisely: adherence by physicians to recommended use of spirometry in the diagnosis and management of adult asthma.

PURPOSE: The National Asthma Education and Prevention Program (NAEPP) and the American Thoracic Society provide guidelines stating that physicians should use spirometry in the diagnosis and management of asthma. The aim of this study was to evaluate the trends, over a 10-year period, in the utilization of spirometry in patients newly diagnosed with asthma. We hypothesized that spirometry use would increase in physicians who care for asthma patients, especially since 2007, when the revised NAEPP guidelines were published. METHODS: This retrospective cohort analysis of spirometry use in subjects newly diagnosed with asthma used a privately insured adult population for the years 2002-2011. Our primary outcome of interest was spirometry performed within a year (± 365 days) of the initial date of asthma diagnosis. We also examined the type of asthma medications prescribed. RESULTS: In all, 134,208 patients were found to have a diagnosis of asthma. Only 47.6% had spirometry performed within 1 year of diagnosis. Younger patients, males, and those residing in the Northeast were more likely to receive spirometry. Spirometry use began to decline in 2007. Patients cared for by specialists were more likely to receive spirometry than those cared for by primary care physicians; 80.1% vs 23.3%, respectively. Lastly, even without spirometry, a significant portion of patients (78.3%) was prescribed asthma drugs. CONCLUSIONS: Our study suggests that spirometry is underutilized in newly diagnosed asthma patients. Moreover, the use of controller medications in those diagnosed with asthma without spirometry remains high.

Multi-well plate immunoassays for measuring signaling protein activations/deactivations and membrane vs. intracellular receptor levels.

We developed fixed-cell multi-well plate immunoassays that increase the throughput and ease of quantification for questions formerly assessed by immunoblot scanning. The assays make use of the now abundant antibodies designed to recognize receptor subtypes and posttranslationally modified signaling proteins. By optimizing permeabilization and fixation conditions, mainly based on specific cell types, the assay can be adapted to the study of many different antigens of importance to hormonal and neurotransmitter signaling scenarios.

Epitope mapping with membrane-bound synthetic overlapping peptides.

Epitope mapping with synthetic overlapping peptides is used for identifying epitopes of monoclonal antibodies (mAbs) and antibodies from patient sera (Midoro-Horiuti et al. Mol Immunol 43:509-518, 2006; Ivanciuc et al. J Agric Food Chem 51:4830-4837, 2003; Midoro-Horiuti et al. Mol Immunol 40:555-562, 2003; Wang et al. J Allergy Clin Immunol 125:695-702, 702.e1-702.e6, 2010). When the mAbs recognize epitopes that are also recognized by patients of interest, they may be useful as surrogates for patient antibodies.

Epitope mapping with random phage display library.

Random phage display library is used to map conformational as well as linear epitopes. These libraries are available in varying lengths and with circularization. We provide here a protocol conveying our experience using a commercially available peptide phage display library, which in our hands provides good results.

Fetal exposure to bisphenol A as a risk factor for the development of childhood asthma: an animal model study.

BACKGROUND: The prevalence of asthma in industrialized countries has been increasing dramatically and asthma is now the most common chronic disease of children in the United States. The rapidity of the increase strongly suggests that changes in environmental exposures are the likely cause of this epidemic. Further, the early onset of allergic manifestations suggests that these exposures may act on the prenatal development of the immune system. We have focused on the potential effects of bisphenol A (BPA), a chemical pollutant with one of the largest productions, on the development of childhood asthma. We have reported that perinatal BPA exposure promotes the development of allergic asthma in a mouse model. The current study was designed to identify a critical period of BPA exposure and to begin elucidating the mechanisms for this susceptibility. METHODS: Female BALB/c mice received 10 micro g/ml BPA in their drinking water from one week before pregnancy until the end of the study. Some of the pups were transferred in the first 48 h of life from their BPA-loaded mother to an unexposed mother, or vice versa. Half of the pups were sensitized with a low dose of the experimental allergen ovalbumin (OVA), the rest received PBS as an unsensitized controls. On day 22, the pups were challenged by inhalations of ovalbumin or PBS followed by quantification of eosinophils in and hyperreactivity of their airways, major indicators of experimental asthma in this classical mouse model. Hepatic expression of two isoforms of UDP-glucuronosyltransferase (Ugt) was quantified by quantitative RT-PCR at various ages. RESULTS: Pups exposed to BPA in utero and through breast milk, or in utero only, displayed an asthma phenotype in response to their "suboptimal" allergic sensitization, whereas, pups only exposed to BPA postnatally from breast milk, did not. The expression of Ugt2b1, an isoform related to BPA clearance in rats, was not detectable in mouse fetuses and newborn pups, but increased by day 5 and approached adult levels by day 25. CONCLUSIONS: Prenatal exposures that produce environmentally relevant burdens of BPA, followed by postnatal allergic sensitization and challenges, promote the development of experimental allergic asthma. Delayed expression of BPA-metabolizing enzymes may explain, at least in part, the enhanced fetal susceptibility to this common environmental contaminant.

Validation of a phage display and computational algorithm by mapping a conformational epitope of Bla g 2.

BACKGROUND: Bla g 2, one of the major cockroach allergens, induces a strong IgE response against conformational epitopes, and on reexposure, sensitized individuals often display symptoms of allergic rhinitis and asthma. The aim of the current study was to perform a test of the efficacy of a modified phage display screening, characterization of selected phages and an automated algorithm, EpiSearch, in locating an important conformational epitope. METHODS: The monoclonal antibody 7C11, which partially inhibits the binding of patient IgE antibodies to Bla g 2, was used to screen a random peptide phage library. After 3 rounds of panning, 32 phage clones were isolated and the amino acid sequences of their peptides were determined. The relative affinity and specificity of the binding of these peptides to 7C11 were tested in ELISAs. The amino acid composition of these peptides was then matched with clusters of residues on the surface of the 3-dimensional (3D) structure of Bla g 2, using our EpiSearch algorithm. RESULTS: The amino acid sequences of the peptides on selected phages differed at only one position, occupied by 1 of 2 negatively charged residues. The two 12-mer sequences bound to 7C11 with similar avidity and specificity. There was good concordance between the residues in the 3D clusters identified from our phage display/computational method with the co-crystal structural analysis. CONCLUSION: Conformational epitopes may be mapped through screening of clones from random peptide phage display libraries and EpiSearch.

Molecular cloning and expression of Cro s 1: an occupational allergen from saffron pollen (Crocus sativus).

BACKGROUND: The cultivation of saffron is expanding through the southeast of Iran, and allergy to saffron pollen occurs in workers involved in processing this plant. We aimed to clone, sequence and express a major allergen involved in saffron pollen allergy, and to compare the recombinant with the natural allergen. METHODS: The N-terminal amino acid sequence of Cro s 1, an allergen from saffron pollen, was determined after immunoblotting. The cDNA encoding for this allergen was cloned by PCR utilizing a primer based on the N-terminal amino acid sequence. Recombinant Cro s 1 (rCro s 1) was expressed as a soluble protein in Pichia pastoris and purified to homogeneity by gel filtration. Inhibition of IgE binding to rCro s 1 by pollen extract was analyzed by ELISA. SECTION TITLE: The allergen Cro s 1 was identified from saffron pollen extracts and cloned by PCR. Cro s 1 cDNA defined an acidic polypeptide with homology to pollen proteins from Chenopodium album and Ligastrum vulgaris. The rCro s 1 was expressed in P. pastoris at 28 mg/l. Saffron pollen extract inhibited the binding of patient serum IgE to rCro s 1. CONCLUSION: We identified and cloned the first Crocus sativus pollen allergen. rCro s 1 cDNA shows a very high homology with Che a 1, the major allergen of lamb's-quarter, Chenopodium album, Caryophyllales, pollen (97%). Cro s 1 is a useful tool for specific diagnosis and structural studies of occupational allergy to saffron.

Mountain cedar pollen induces IgE-independent mast cell degranulation, IL-4 production, and intracellular reactive oxygen species generation.

Cedar pollens cause severe allergic disease throughout the world. We have previously characterized allergenic pollen glycoproteins from mountain cedar (Juniperus ashei) that bind to allergen-specific immunoglobulin E (IgE). In the present report, we investigated an alternative pathway of mast cell activation by mountain cedar pollen extract through IgE-independent mechanisms. We show that mountain cedar pollen directly induces mast cell serotonin and IL-4 release and enhances release induced by IgE cross-linking. Concomitant with mediator release, high levels of intracellular reactive oxygen species (ROS) were generated, and both ROS and serotonin release were inhibited by anti-oxidants. These findings suggest that alternative mechanisms exist whereby pollen exposure enhances allergic inflammatory mediator release through mechanisms that involve ROS. These mechanisms have the potential for enhancing the allergenic potency of pollens.

An Allergen Portrait Gallery: Representative Structures and an Overview of IgE Binding Surfaces.

Recent progress in the biochemical classification and structural determination of allergens and allergen-antibody complexes has enhanced our understanding of the molecular determinants of allergenicity. Databases of allergens and their epitopes have facilitated the clustering of allergens according to their sequences and, more recently, their structures. Groups of similar sequences are identified for allergenic proteins from diverse sources, and all allergens are classified into a limited number of protein structural families. A gallery of experimental structures selected from the protein classes with the largest number of allergens demonstrate the structural diversity of the allergen universe. Further comparison of these structures and identification of areas that are different from innocuous proteins within the same protein family can be used to identify features specific to known allergens. Experimental and computational results related to the determination of IgE binding surfaces and methods to define allergen-specific motifs are highlighted.

Inhaled insulin forms toxic pulmonary amyloid aggregates.

It is well known that interfaces, such as polar-nonpolar or liquid-air, play a key role in triggering protein aggregation in vitro, in particular the aggregation of peptides and proteins with the predisposition of misfolding and aggregation. Here we show that the interface present in the lungs predisposes the lungs to form aggregation of inhaled insulin. Insulin inhalers were introduced, and a large number of diabetic patients have used them. Although inhalers were safe and effective, decreases in pulmonary capacity have been reported in response to inhaled insulin. We hypothesize that the lung air-tissue interface provides a template for the aggregation of inhaled insulin. Our studies were designed to investigate the harmful potential that inhaled insulin has in pulmonary tissue in vivo, through an amyloid formation mechanism. Our data demonstrate that inhaled insulin rapidly forms amyloid in the lungs causing a significant reduction in pulmonary air flow. Our studies exemplify the importance that interfaces play in protein aggregation in vivo, illustrating the potential aggregation of inhaled proteins and the formation of amyloid deposits in the lungs. These insulin deposits resemble the amyloid structures implicated in protein misfolding disorders, such as Alzheimer's and Parkinson's diseases, and could as well be deleterious in nature.

Plant-expressed recombinant mountain cedar allergen Jun a 1 is allergenic and has limited pectate lyase activity.

BACKGROUND: Mountain cedar (Juniperus ashei) pollen commonly causes a winter time allergic rhinitis in the central USA. Jun a 1 is the dominant allergenic protein, but biologically active recombinant Jun a 1 has not been successfully expressed, despite numerous attempts with several expression systems. METHOD: Jun a 1 cDNA was inserted into a tobacco mosaic virus vector and transferred to Agrobacterium tumefaciens. Bacteria were syringe-inoculated into leaves of Nicotiana benthamiana (agroinoculation). The interstitial (apoplastic) fluid containing Jun a 1 was isolated. The recombinant protein was analyzed by SDS-PAGE, N-terminal sequencing and MALDI-TOF to confirm identity. Immunogenicity was examined with IgE from allergic patient's sera, mouse monoclonal anti-Jun a 1 antibodies, IgE-binding inhibition and by degranulation of RBL SX-38 cells sensitized with sera from allergic patients. Pectate lyase activity was assayed by capillary zone electrophoresis and mass spectrometry analysis. RESULTS: Recombinant Jun a 1 was recovered in good quantity (100 µg/g leaf material), was confirmed as Jun a 1, bound IgE from sera from cedar hypersensitive patients and inhibited IgE binding to native Jun a 1. Jun a 1 mutants were created and their pectate lyase activity quantified. For the first time, Jun a 1 pectate lyase activity was demonstrated, which may explain the necrosis seen on host plants, which was similar to that of control plants expressing banana pectate lyase. CONCLUSIONS: A means of producing recombinant Jun a 1 is now available for structure/function studies and potentially for diagnostic and therapeutic uses.

Maternal bisphenol a exposure promotes the development of experimental asthma in mouse pups.

BACKGROUND: We recently reported that various environmental estrogens induce mast cell degranulation and enhance IgE-mediated release of allergic mediators in vitro. OBJECTIVES: We hypothesized that environmental estrogens would enhance allergic sensitization as well as bronchial inflammation and responsiveness. To test this hypothesis, we exposed fetal and neonatal mice to the common environmental estrogen bisphenol A (BPA) via maternal loading and assessed the pups' response to allergic sensitization and bronchial challenge. METHODS: Female BALB/c mice received 10 microg/mL BPA in their drinking water from 1 week before impregnation to the end of the study. Neonatal mice were given a single 5 microg intraperitoneal dose of ovalbumin (OVA) with aluminum hydroxide on postnatal day 4 and 3% OVA by nebulization for 10 min on days 13, 14, and 15. Forty-eight hours after the last nebulization, we assessed serum IgE antibodies to OVA by enzyme-linked immunosorbent assay (ELISA) and airway inflammation and hyperresponsiveness by enumerating eosinophils in bronchoalveolar lavage fluid, whole-body barometric plethysmography, and a forced oscillation technique. RESULTS: Neonates from BPA-exposed mothers responded to this "suboptimal" sensitization with higher serum IgE anti-OVA concentrations compared with those from unexposed mothers (p < 0.05), and eosinophilic inflammation in their airways was significantly greater. Airway responsiveness of the OVA-sensitized neonates from BPA-treated mothers was enhanced compared with those from unexposed mothers (p < 0.05). CONCLUSIONS: Perinatal exposure to BPA enhances allergic sensitization and bronchial inflammation and responsiveness in a susceptible animal model of asthma.