Allergic Polysensitization Clusters: Newly Recognized Severity Marker in Urban Asthmatic Adults.

INTRODUCTION: While reliable, quantitative in vitro testing for sensitivity to aeroallergens has been available for decades, such information has largely been ignored in clustering analyses of asthma. Our aim is to explore allergic polysensitization as a possible marker of asthma severity and, as such, to be considered as an integral marker in future asthma clustering analyses. METHODS: We constructed a database of sensitizations to the 25 aeroallergens in our geographic area (zone 1, Northeastern US) using the ImmunoCAP® in vitro assay. We used the Scikit-Learn® machine learning library for model-based clustering to identify allergic polysensitization clusters. Clusters were compared for differences in common office-based clinical markers of asthma. RESULTS: The database consisted of 509 patients. Unbiased machine learning identified ten clusters of increasing allergic polysensitization of varying sizes (n = 1-339) characterized by significant increases in mean serum immunoglobulin E (p < 0.001), peripheral blood eosinophil count (p < 0.001), and DLCO (p = 0.02). There was a significant decline in mean age at presentation (p < 0.001), FEV1/FVC (p = 0.01), and FEF25-75 (p = 0.002) with increasing allergic polysensitization. Finally, we identified two divergent paths for the poly-atopic march, one driven by perennial and the other by seasonal allergens. CONCLUSION: This pilot study showed that allergic polysensitization, using readily available qualitative and quantitative in vitro sensitization data, largely ignored in cluster analyses to date, may add further clinical precision in cluster analyses of asthma. We suggest the methods used here can be applied and tested using larger databases and aeroallergens present in diverse geographic regions.

Enhancement of Mast Cell Degranulation Mediated by Purinergic Receptors' Activation and PI3K Type δ.

Mast cells express multiple metabotropic purinergic P2Y receptor (P2YR) subtypes. Few studies have evaluated their role in human mast cell (HMC) allergic response as quantified by degranulation induced by cross-linking the high-affinity IgE receptor (FcεRI). We have previously shown that extracellular nucleotides modify the FcεRI activation-dependent degranulation in HMCs derived from human lungs, but the mechanism of this action has not been fully delineated. This study was undertaken to determine the mechanism of activation of P2YRs on the degranulation of HMCs and elucidate the specific postreceptor pathways involved. Sensitized LAD2 cells, a human-derived mast cell line, were subjected to a weak allergic stimulation (WAS) using a low concentration of Ag in the absence and presence of P2YR agonists. Only the metabotropic purinergic P2Y11 receptor (P2Y11R) agonist, adenosine 5'-(3-thio)triphosphate (ATPγS), enhanced WAS-induced degranulation resulting in a net 7-fold increase in release (n = 4; p < 0.01). None of the P2YR agonists tested, including high concentrations of ATPγS (1000 µM), enhanced WAS-induced intracellular Ca2+ mobilization, an essential component of activated FcεRI-induced degranulation. Both a PI3K inhibitor and the relevant gene knockout decreased the ATPγS-induced enhancement. The effect of ATPγS was associated with enhanced phosphorylation of PI3K type δ and protein kinase B, but not the phosphoinositide-dependent kinase-1. The effects of ATPγS were dose dependently inhibited by NF157, a P2Y11R antagonist. To our knowledge, these data indicate for the first time that P2YR is linked to enhancement of allergic degranulation in HMC via the PI3K/protein kinase B pathway.

Adenosine 5'-triphosphate's role in bradycardia and syncope associated with pulmonary embolism.

Adenosine 5'-triphiosphate (ATP) is released from cells under physiologic and pathophysiologic conditions. Extracellular ATP acts as an autocrine and paracrine agent affecting various cell types by activating cell surface P2 receptors (P2R), which include trans-cell membrane cationic channels, P2XR, and G protein coupled receptors, P2YR. We have previously shown that ATP stimulates vagal afferent nerve terminals in the lungs by activating P2X2/3R. This action could lead to bronchoconstriction, cough and the local release of pro-inflammatory neuropeptides. In addition, ATP markedly enhances the IgE-dependent histamine release from human lung mast cells. Thus, we have proposed for the first time that extracellular ATP plays a mechanistic role in pulmonary pathophysiology in general and chronic obstructive pulmonary disease (COPD), and acute bronchoconstriction in asthma in particular. The present review examines whether ATP could also play a role in bradycardia and syncope in a subset of patients with pulmonary embolism.

Improving Asthma Outcomes During Pregnancy in Underserved Communities.

It is known that poor asthma control is common in pregnancy, and asthma in general disproportionally affects underserved communities. However, there is a paucity of data examining strategies to improve asthma control specifically among pregnant women from vulnerable populations. Identified barriers to optimal asthma care in other underserved groups include health literacy, financial constraints, cultural differences, and poor environmental controls. These deficiencies may also be targets for multimodal interventions geared toward improving asthma outcomes for underserved women during pregnancy.

Degranulation of human mast cells: modulation by P2 receptors' agonists.

Since the late 1970s, there has been an alarming increase in the incidence of asthma and its morbidity and mortality. Acute obstruction and inflammation of allergic asthmatic airways are frequently caused by inhalation of exogenous substances such as allergens cross-linking IgE receptors expressed on the surface of the human lung mast cells (HLMC). The degree of constriction of human airways produced by identical amounts of inhaled allergens may vary from day to day and even hour to hour. Endogenous factors in the human mast cell (HMC)'s microenvironment during allergen exposure may markedly modulate the degranulation response. An increase in allergic responsiveness may significantly enhance bronchoconstriction and breathlessness. This review focuses on the role that the ubiquitous endogenous purine nucleotide, extracellular adenosine 5'-triphosphate (ATP), which is a component of the damage-associated molecular patterns, plays in mast cells' physiology. ATP activates P2 purinergic cell-surface receptors (P2R) to trigger signaling cascades resulting in heightened inflammatory responses. ATP is the most potent enhancer of IgE-mediated HLMC degranulation described to date. Current knowledge of ATP as it relates to targeted receptor(s) on HMC along with most recent studies exploring HMC post-receptor activation pathways are discussed. In addition, the reviewed studies may explain why brief, minimal exposures to allergens (e.g., dust, cat, mouse, and grass) can unpredictably lead to intense clinical reactions. Furthermore, potential therapeutic approaches targeting ATP-related enhancement of allergic reactions are presented.

Improving Asthma Symptoms Among Inner-City Women During Pregnancy: A Prospective Cohort Intervention.

BACKGROUND: It is unknown how active asthma management influences symptom control among inner-city pregnant women who have unique exposures and socioeconomic limitations affecting their care. OBJECTIVE: To assess the impact of an integrated subspecialty intervention composed of education and monitoring on asthma control among underserved women in an antenatal clinic setting. METHODS: We conducted a prospective cohort study of pregnant asthmatic patients participating in a subspecialty clinic integrated into routine prenatal care. We compared baseline characteristics and objective measurements of asthma control between women at an initial visit and those who were evaluated in at least one follow-up. For follow-up, we measured symptom control at successive visits and the incidence of asthma-related complications. RESULTS: Among 85 women enrolled, 53 (62.4%) returned for at least one follow-up visit. Mean baseline Asthma Control Test scores were similarly low (≤19) between groups (one or more follow-up and no follow-up), as were self-administered Asthma Quality of Life Questionnaire scores (<4.7). A total of 72 women had inadequate asthma control resulting in step-up therapy after the initial visit (84.7%). There was a significant increase in ACT scores between the initial and first follow-up visits. For those with an intervening self-administered Asthma Quality of Life Questionnaire, there was also a significant increase by 1.39 ± 0.67 (P = .0003). CONCLUSIONS: We found that uncontrolled asthma is common among urban women seeking routine obstetric care. Our results suggest that even one interventional visit can result in significant improvement in asthma control. Further investigation into mechanisms for optimizing treatment strategies may improve the quality of asthma care during pregnancy in this underserved population.

Primary immune deficiency diseases as unrecognized causes of chronic respiratory disease.

BACKGROUND: More than half of all primary immune deficiency diseases (PIDD) affect antibody production and are well known as causes of recurrent sinusitis and lung infections. Chronic and recurrent infections of the upper and/or lower airways can contribute to inflammatory and obstructive processes in the lower airways which are initially reversible and considered "asthma", but can eventually cause irreversible remodeling and chronic obstructive pulmonary disease (COPD). Conversely, several lines of evidence suggest that many patients who present with a diagnosis of asthma have an increased incidence of infection, suggesting underlying host-defense defects. Asthma and respiratory infections in the first decades of life are recognized as risk factors for development of COPD, but when patients present with COPD as adults, underlying primary immune deficiency disease may be unrecognized. MAIN FINDINGS AND CONCLUSIONS: Detection of PIDD as a potentially treatable underlying contributor to recurrent/acute exacerbations and morbidity of COPD, and provision of immunoglobulin (Ig) G replacement therapy, when appropriate, may decrease the progression of COPD. Decreasing the severity and rate of exacerbations and admissions should improve the quality of life and longevity of an important subset of patients with COPD, while decreasing costs. Major steps toward achieving these goals include developing a high index of suspicion, more frequent use and appropriate interpretation of screening tests such as quantitative immunoglobulins and vaccine responses, and prompt institution of IgG replacement therapy when antibody deficiency has been diagnosed.

IgE Receptor-Mediated Histamine Release in Human Lung Mast Cells: Modulation by Purinergic Receptor Ligands.

BACKGROUND: Mast cells derived from human lungs (HLMCs) express multiple G-protein coupled purinergic receptors (P2YR) and the so-called α-ketoglutarate receptor GPR99, which is homologous to P2YR. The role of the P2YR of HLMC is not clear. Thus, the aim of the present study was to determine the effects of purinergic and purine-related compounds on allergic histamine release (HR) in HLMCs. METHODS: FcεRI-mediated HR was quantified in primary culture of HLMC (cHLMC). The effects of the FcεRI-mediated allergic stimulation on the expression of GPR99 were also determined. RESULTS: Adenosine produced a dual effect on HR: enhancement and marked inhibition at low and high concentrations, respectively. Adenosine 5'-monophosphate (AMP) did not affect FcεRI-mediated HR. However, the non-hydrolysable AMP analog, adenosine-5'-O-thiomonophosphate (AMP-S), concentration dependently inhibited the FcεRI-mediated HR without any enhancement. At high concentrations, α-ketoglutarate moderately inhibited FcεRI-mediated HR. However, inhibitions by AMP-S and α-ketoglutarate of HR were dissimilar in the inhibitory manner (IC50 and Hill slope) on histamine release by allergic stimulation. CONCLUSIONS: cHLMC express functional GPR99 receptor that is up-regulated following allergic stimulation. Responsiveness to AMP-S is the first indication that cHLMC express P2YR, the activation of which can inhibit FcεRI-mediated HR.

Extracellular Adenosine 5'-Triphosphate in Obstructive Airway Diseases.

In recent years, numerous studies have generated data supporting the hypothesis that extracellular adenosine 5'-triphosphate (ATP) plays a major role in obstructive airway diseases. Studies in animal models and human subjects have shown that increased amounts of extracellular ATP are found in the lungs of patients with COPD and asthma and that ATP has effects on multiple cell types in the lungs, resulting in increased inflammation, induction of bronchoconstriction, and cough. These effects of ATP are mediated by cell surface P2 purinergic receptors and involve other endogenous inflammatory agents. Recent clinical trials reported promising treatment with P2X3R antagonists for the alleviation of chronic cough. The purpose of this review was to describe these studies and outline some of the remaining questions, as well as the potential clinical implications, associated with the pharmacologic manipulation of ATP signaling in the lungs.

Rationale for specific allergen testing of patients with asthma in the clinical pulmonary office setting.

Asthma is a chronic inflammatory disease that affects an estimated 25 million people in the United States. In 70% to 90% of cases, asthma is associated with IgE-mediated mechanisms, which have proved central to allergen-induced inflammation in preclinical and clinical models. The importance of IgE levels in patients with moderate to severe asthma has been confirmed in randomized controlled studies with a targeted IgE blocker. Advances in laboratory methods to detect and quantify allergen-specific IgE antibodies have allowed for a quick-and-easy diagnosis of allergic IgE-mediated sensitivities in the office. Pulmonologists tend to order in vitro tests to measure allergen-specific IgE rather than to perform allergen skin testing, which is seen as the purview of allergists. This article reviews the importance of allergen testing in patients with asthma­whether by skin testing or by in vitro methods­and highlights the advantages, limitations, and interpretation of results derived from each method. Additionally, this article includes suggested documentation and administrative details for physician reporting in the office setting.

Hypoxia enhances differentiation of mouse embryonic stem cells into definitive endoderm and distal lung cells.

We investigated the effects of hypoxia on spontaneous (SP)- and activin A (AA)-induced definitive endoderm (DE) differentiation of mouse embryonic stem cells (mESCs) and their subsequent differentiation into distal pulmonary epithelial cells. SP differentiation for 6 days of mESCs toward endoderm at hypoxia of 1% O2, but not at 3% or 21% (normoxia), increased the expression of Sox17 and Foxa2 by 31- and 63-fold above maintenance culture, respectively. Treatment of mESCs with 20 ng/mL AA for 6 days under hypoxia further increased the expression of DE marker genes Sox17, Foxa2, and Cxcr4 by 501-, 1,483-, and 126-fold above maintenance cultures, respectively. Transient exposure to hypoxia, as short as 24 h, was sufficient to enhance AA-induced endoderm formation. The involvement of hypoxia-inducible factor (HIF)-1α and reactive oxygen species (ROS) in the AA-induced endoderm enrichment was assessed using HIF-1α(-/-) mESCs and the ROS scavenger N-acetylcysteine (NAC). Under SP conditions, HIF-1α(-/-) mESCs failed to increase the expression of endodermal marker genes but rather shifted toward ectoderm. Hypoxia induced only a marginal potentiation of AA-induced endoderm differentiation in HIF-1α(-/-) mESCs. Treatment of mESCs with AA and NAC led to a dose-dependent decrease in Sox17 and Foxa2 expression. In addition, the duration of exposure to hypoxia in the course of a recently reported lung differentiation protocol resulted in differentially enhanced expression of distal lung epithelial cell marker genes aquaporin 5 (Aqp5), surfactant protein C (Sftpc), and secretoglobin 1a1 (Scgb1a1) for alveolar epithelium type I, type II, and club cells, respectively. Our study is the first to show the effects of in vitro hypoxia on efficient formation of DE and lung lineages. We suggest that the extent of hypoxia and careful timing may be important components of in vitro differentiation bioprocesses for the differential generation of distal lung epithelial cells from pluripotent progenitors.

Heterogeneous mixed-lineage differentiation of mouse embryonic stem cells induced by conditioned media from a549 cells.

Conditioned media (CM) of transformed cells, such as the human lung-derived A549 cells, is a useful tool for directing differentiation of embryonic stem cells (ESCs). Previous work indicates that A549-CM induced pulmonary differentiation of mouse ESCs (mESCs). In this study, we compared the effects of A549-CM treatment on the differentiation of mESCs organized in monolayer or embryoid bodies. We analyzed the cultures treated with A549-CM using specific lineage markers by quantitative polymerase chain reaction (qPCR) and lineage-focused PCR arrays and demonstrated heterogeneous CM-induced differentiation. We then constructed bioinformatics-based gene networks to establish correlations between the upregulated lineage-specific genes and proteins in the A549-CM identified by proteomic analysis. Network analysis supported the phenotypic and genotypic heterogeneic differentiation of mESCs into multiple cell lineages via enriched stemness, cardiovascular, neuronal, and lung development gene ontologies (GOs). The significant enrichment toward lung ontologies was specific for treatment with A549-CM, but not CM of liver (HepG2) and pancreas (Capan-1) cells. Based on network analysis, we identified laminin alpha5, prosaposin, lamin A/C, dickkopf homolog 1, clusterin, and calreticulin as the most relevant proteins related to the enrichment of lung GOs. We validated the effects of laminin isoforms on mESC differentiation in vitro and found enriched differential induction of surfactant protein gene expression. Our data suggest that A549-CM can be used for identifying secreted proteins for the heterogeneous mixed-lineage differentiation of mESCs toward a variety of lung-relevant cells. Such a heterogeneous cell population will be required for the in vitro generation of complex lung tissue and mixed cell populations for regenerative pulmonary therapy.

Enhanced reseeding of decellularized rodent lungs with mouse embryonic stem cells.

Repopulation of decellularized lung scaffolds (DLS) is limited due to alterations in the repertoire and ratios of the residual extracellular matrix (ECM) proteins, characterized by e.g., the retention of type I collagen and loss of glycoproteins. We hypothesized that pre-treatment of decellularized matrices with defined ECM proteins, which match the repertoire of integrin receptors expressed by the cells to be seeded (e.g., embryonic stem cells) can increase the efficacy of the reseeding process. To test this hypothesis, we first determined the integrin receptors profile of mouse embryonic stem cells (mESCs). Mouse ESCs express α3, α5, α6, α9 and ß1, but not α1, α2 and α4 integrin subunits, as established by Western blotting and adhesion to laminin and fibronectin, but not to collagens type I and IV. Reseeding of DLS with mESCs was inefficient (6.9 ± 0.5%), but was significantly enhanced (2.3 ± 0.1 fold) by pre-treating the scaffolds with media conditioned by A549 human lung adenocarcinoma cells, which we found to contain ∼5 µg/ml laminin. Furthermore, pre-treatment with A549-conditioned media resulted in a significantly more uniform distribution of the seeded mESCs throughout the engineered organ as compared to untreated DLS. Our study may advance whole lung engineering by stressing the importance of matching the integrin receptor repertoire of the seeded cells and the cell binding motifs of DLS.

Successful treatment of chylous ascites secondary to sarcoidosis with methotrexate.

A 39-year-old African American woman with sarcoidosis developed chylous ascites. Computed tomography (CT) scan showed extensive thoracic, mesenteric, and retroperitoneal lymphadenopathy. Initial therapy with corticosteroids was unsuccessful in managing ascites and she needed frequent large-volume paracentesis for symptomatic relief. Methotrexate was effective in treating the condition and the patient has remained symptom-free for > 2 years. Chylous ascites secondary to sarcoidosis is extremely rare and this is the first reported case of successful treatment with methotrexate.

Obstructive sleep apnea syndrome and asthma: the role of continuous positive airway pressure treatment.

OBJECTIVE: To review the concept of a possible link between asthma and obstructive sleep apnea syndrome (OSAS) and the impact on asthma symptoms of treatment of OSAS with continuous positive airway pressure (CPAP) in patients with both conditions. DATA SOURCES: The Ovid, MEDLINE, and PubMed databases from 1950 to the present were searched for relevant articles regarding a possible relationship between asthma and OSAS and the effectiveness of CPAP in treating OSAS. STUDY SELECTION: Articles describing pathophysiologic conditions occurring in OSAS that may be linked to asthma pathogenesis were used for this review. RESULTS: The data suggest that OSAS is an independent risk factor for asthma exacerbations. CPAP has been shown in prospective clinical studies to have a positive impact on asthma outcome in patients with concomitant OSAS. Ameliorative mechanisms of treatment with CPAP include mechanical and neuromechanical effects, gastroesophageal acid reflux suppression, local and systemic anti-inflammatory effects (including suppression of increased serum levels of inflammatory cytokines, chemokines, and vascular endothelial growth factor), cardiac function improvements, leptin level suppression, weight reduction, and sleep restoration. CONCLUSIONS: Asthma and OSAS are increasingly troublesome public health issues. Mounting evidence implicates OSAS as a risk factor for asthma exacerbations, thereby linking these 2 major epidemics. We describe potential mechanisms whereby CPAP, the first line of therapy for OSAS, might modify airway smooth muscle function and asthma control in patients with both disorders. Despite the ever-increasing population of patients with both disorders, large, prospective, randomized controlled studies are necessary to more fully evaluate CPAP and asthma outcomes.

c-Kit immunophenotyping and metalloproteinase expression profiles of mast cells in interstitial lung diseases.

Diverse interstitial lung diseases (ILD) demonstrate mesenchymal infiltration by an abundance of activated mast cells whose role in parenchymal fibrogenesis remains unclear. Since mast cells differentiate in a dynamic, tissue-specific manner via signals transduced by c-Kit receptor, we examined the effect of ILD microenvironments on c-Kit expression and metalloproteinase phenotypes of mesenchymal mast cell populations. Immunohistochemical and flow cytometric analyses characterized surface expression of c-Kit on mast cells in tissues obtained from patients with idiopathic pulmonary fibrosis, systemic sclerosis, sarcoidosis, and lymphangioleiomyomatosis, thus identifying a unique immunophenotype not shared by normal lung mast cells. Isolation of c-Kit+/FcepsilonRI+/CD34- mast cells via immunocytometric sorting of heterogeneous cell populations from mechanically disaggregated lung tissues permitted analysis of gene expression patterns by two-step real-time polymerase chain reaction. Transcriptional profiling identified expression of c-Kit and the neutral serine proteases, tryptase and chymase, establishing the identity of sorted populations as mature mast cells. Mast cells harvested from ILD tissues demonstrated characteristic metalloproteinase phenotypes which included expression of matrix metalloproteinase (MMP)-1 and a disintegrin and metalloproteinase (ADAM)-9, -10, and -17. Immunohistochemical co-localization guided by gene profiling data confirmed expression of chymase, MMP-1, and ADAM-17 protein in subpopulations of mast cells in remodelling interstitium. Gene profiling of harvested mast cells also showed increased transcript copy numbers for TNFalpha and CC chemokine receptor 2, which play critical roles in lung injury. We conclude that ILD microenvironments induce unique c-Kit receptor and metalloproteinase mast cell phenotypes.