Michelle L. North

DNA Hypomethylation, Ambient Particulate Matter, and Increased Blood Pressure: Findings From Controlled Human Exposure Experiments

Background Short‐term exposures to fine (<2.5 μm aerodynamic diameter) ambient particulate‐matter (PM) have been related with increased blood pressure (BP) in controlled‐human exposure and community‐based studies. However, whether coarse (2.5 to 10 μm) PM exposure increases BP is uncertain. Recent observational studies have linked PM exposures with blood DNA hypomethylation, an epigenetic alteration that activates inflammatory and vascular responses. No experimental evidence is available to confirm those observational data and demonstrate the relations between PM, hypomethylation, and BP. Methods and Results We conducted a cross‐over trial of controlled‐human exposure to concentrated ambient particles (CAPs). Fifteen healthy adult participants were exposed for 130 minutes to fine CAPs, coarse CAPs, or HEPA‐filtered medical air (control) in randomized order with ≥2‐week washout. Repetitive‐element (Alu, long interspersed nuclear element‐1 [LINE‐1]) and candidate‐gene (TLR4, IL‐12, IL‐6, iNOS) blood methylation, systolic and diastolic BP were measured pre‐ and postexposure. After adjustment for multiple comparisons, fine CAPs exposure lowered Alu methylation (β‐standardized=−0.74, adjusted‐P=0.03); coarse CAPs exposure lowered TLR4 methylation (β‐standardized=−0.27, adjusted‐P=0.04). Both fine and coarse CAPs determined significantly increased systolic BP (β=2.53 mm Hg, P=0.001; β=1.56 mm Hg, P=0.03, respectively) and nonsignificantly increased diastolic BP (β=0.98 mm Hg, P=0.12; β=0.82 mm Hg, P=0.11, respectively). Decreased Alu and TLR4 methylation was associated with higher postexposure DBP (β‐standardized=0.41, P=0.04; and β‐standardized=0.84, P=0.02; respectively). Decreased TLR4 methylation was associated with higher postexposure SBP (β‐standardized=1.45, P=0.01). Conclusions Our findings provide novel evidence of effects of coarse PM on BP and confirm effects of fine PM. Our results provide the first experimental evidence of PM‐induced DNA hypomethylation and its correlation to BP.

Functionally important role for arginase 1 in the airway hyperresponsiveness of asthma.

l-Arginine metabolism by the arginase and nitric oxide (NO) synthase (NOS) families of enzymes is important in NO production, and imbalances between these pathways contribute to airway hyperresponsiveness (AHR) in asthma. To investigate the role of arginase isozymes (ARG1 and ARG2) in AHR, we determined the protein expression of ARG1, ARG2, the NOS isozymes, and other proteins involved in l-arginine metabolism in lung tissues from asthma patients and in acute (3-wk) and chronic (12-wk) murine models of ovalbumin-induced airway inflammation. Expression of ARG1 was increased in human asthma, whereas ARG2, NOS isoforms, and the other l-arginine-related proteins (i.e., cationic amino acid transporters 1 and 2, agmatinase, and ornithine decarboxylase) were unchanged. In the acute murine model of allergic airway inflammation, augmentation of ARG1 expression was similarly the most dramatic change in protein expression. However, ARG2, NOS1, NOS2, and agmatinase were also increased, whereas NOS3 expression was decreased. Arginase inhibition in vivo with nebulized S-(2-boronoethyl)-l-cysteine attenuated the methacholine responsiveness of the central airways in mice from the acute model. Further investigations in the chronic murine model revealed an expression profile that more closely paralleled the human asthma samples: only ARG1 expression was significantly increased. Interestingly, in the chronic mouse model, which generates a remodeling phenotype, arginase inhibition attenuated methacholine responsiveness of the central and peripheral airways. The similarity in arginase expression between human asthma and the chronic model and the attenuation of AHR after in vivo treatment with an arginase inhibitor suggest the potential for therapeutic modification of arginase activity in asthma.

Syk Associates with Clathrin and Mediates Phosphatidylinositol 3-Kinase Activation during Human Rhinovirus Internalization

Human rhinovirus (HRV) causes the common cold. The most common acute infection in humans, HRV is a leading cause of exacerbations of asthma and chronic obstruction pulmonary disease because of its ability to exacerbate airway inflammation by altering epithelial cell biology upon binding to its receptor, ICAM-1. ICAM-1 regulates not only viral entry and replication but also signaling pathways that lead to inflammatory mediator production. We recently demonstrated the Syk tyrosine kinase to be an important mediator of HRV-ICAM-1 signaling: Syk regulates replication-independent p38 MAPK activation and IL-8 expression. In leukocytes, Syk regulates receptor-mediated internalization via PI3K. Although PI3K has been shown to regulate HRV-induced IL-8 expression and clathrin-mediated endocytosis of HRV, the role of airway epithelial Syk in this signaling pathway is not known. We postulated that Syk regulates PI3K activation and HRV endocytosis in the airway epithelium. Using confocal microscopy and immunoprecipitation, we demonstrated recruitment of the normally cytosolic Syk to the plasma membrane upon HRV16-ICAM-1 binding, along with Syk-clathrin coassociation. Subsequent incubation at 37°C to permit internalization revealed redistribution of Syk to punctate structures resembling endosomes and colocalization with HRV16. Internalized HRV was not detected in cells overexpressing the kinase inactive SykK396R mutant, indicating that kinase activity was necessary for endocytosis. HRV-induced PI3K activation was dependent on Syk; Syk knockdown by small interfering RNA significantly decreased phosphorylation of the PI3K substrate Akt. Together, these data reveal Syk to be an important mediator of HRV endocytosis and HRV-induced PI3K activation.

Asymmetric Dimethylarginine Is Increased in Asthma

RATIONALE Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor that competes with L-arginine for binding to NOS. It has been suggested that ADMA contributes to inflammation, collagen deposition, nitrosative stress, and lung function in murine models. OBJECTIVES To test the hypothesis that ADMA is increased in asthma and that NOS inhibition by ADMA contributes to airways obstruction. METHODS We assessed alterations of L-arginine, ADMA, and symmetric dimethylarginine (SDMA) levels in a murine model of allergic airways inflammation using LC-tandem mass spectrometry. Based on the levels of ADMA observed in the murine model, we further tested the direct effects of nebulized inhaled ADMA on airways responsiveness in naive control mice. We also assessed alterations of L-arginine, ADMA, and SDMA in humans in adult lung specimens and sputum samples from pediatric patients with asthma. MEASUREMENTS AND MAIN RESULTS ADMA was increased in lungs from the murine model of allergic airways inflammation. Exogenous administration of ADMA to naive mice, at doses consistent with the levels observed in the allergically inflamed lungs, resulted in augmentation of the airways responsiveness to methacholine. ADMA levels were also increased in human asthma lungs and sputum samples. CONCLUSIONS ADMA levels are increased in asthma and contribute to NOS-related pathophysiology.

The role of epigenetics in the developmental origins of allergic disease.

OBJECTIVE To review current research findings in the field of epigenetics pertaining to the developmental origins of allergic disease. DATA SOURCES We examined original research and review articles identified from MEDLINE, OVID, and PubMed that addressed the topic of interest, using the search terms atopy, allergy, asthma, development, IgE, origins, and cord blood paired with epigenetic(s). Relevant references from each article were also procured for review. STUDY SELECTION Articles were selected based on their relevance to the contributory role of epigenetic modifications in asthma and other atopic diseases. RESULTS There is increasing evidence pointing to the influence of prenatal and early life exposures on the development of allergic disease. A growing body of literature supports the theory that transient environmental pressures can have permanent effects on gene regulation and expression through epigenetic mechanisms. Histone modifications have been associated with degree of bronchial hyperresponsiveness and corticosteroid resistance in asthma. Epigenetic mechanisms can operate independently in various cell types; recent studies have suggested a role in the differentiation of human T cells. Murine studies have revealed that a maternal diet rich in methyl donors can enhance susceptibility to allergic inflammation in the offspring, mediated through increased DNA methylation. Murine studies have also implicated epigenetically modified dendritic cells in the transmission of allergic risk from mothers to offspring. CONCLUSION The current literature offers exciting data to support a role for epigenetics in the development and persistence of asthma and allergic rhinitis. However, further human studies are necessary to explore these mechanisms and assess future clinical applicability.

Effects of phthalates on the development and expression of allergic disease and asthma

OBJECTIVE To review recent evidence relating phthalate exposures to allergies and asthma and to provide an overview for clinicians interested in the relevance of environmental health research to allergy and who may encounter patients with concerns about phthalates from media reports. DATA SOURCES PubMed, TOXLINE, and Web of Science were searched using the term phthalate(s) combined with the keywords allergy, asthma, atopy, and inflammation. STUDY SELECTIONS Articles were selected based on relevance to the goals of this review. Studies that involved humans were prioritized, including routes and levels of exposure, developmental and early-life exposures, immunotoxicity, and the development of allergic disease. RESULTS The general public and those with allergy are exposed to significant levels of phthalates via diet, pharmaceuticals, phthalate-containing products, and ambient indoor environment via air and dust. Intravenous exposures occur through medical equipment. Phthalates are metabolized and excreted quickly in the body with metabolites measured in urine. Phthalates, which are known endocrine disrupting compounds, have been associated with oxidative stress and alterations in cytokine expression. Metabolites in human urine, particularly of the higher-molecular-weight phthalates, have been associated with allergies and asthma in multiple studies. CONCLUSION Despite mounting evidence implicating phthalates, causation of allergic disease by these compounds cannot currently be established. In utero and early-life exposures and possible transgenerational effects are not well understood. However, considering the current evidence, reducing exposures to phthalates by avoiding processed and foods packaged and stored in plastics, personal care products with phthalates, polyvinyl chloride materials indoors, and reducing home dust is advised. Further longitudinal, molecular, and intervention studies are needed to understand the association between phthalates and allergic disease.

Environmental exposure unit: a sensitive, specific, and reproducible methodology for allergen challenge

INSTRUCTIONS Credit can now be obtained, free for a limited time, by reading the review article in this issue and completing all activity components. Please note the instructions listed below: Review the target audience, learning objectives and all disclosures. Complete the pre-test online at http://www.annallergy.org (click on the CME heading). Follow the online instructions to read the full version of the article; reflect on all content as to how itmay be applicable to your practice. Complete the post-test/evaluation and claim credit earned; at this time, you will have earned up to 1.0 AMA PRA Category 1 CreditTM. Please note that the minimum passing score on the post-test is 70%. Release Date: November 1, 2013 Expiration Date: October 31, 2015 Estimated Time to Complete: 60 minutes Target Audience: Physicians involved in providing patient care in the field of allergy/asthma/immunology Learning Objectives: At the conclusion of this activity, participants should be able to: Describe the Environmental Exposure Unit (EEU) methodology for controlled allergen challenge Discuss the technical aspects, study designs, and controlled variables in EEU studies that allow for the effective determination of onset of action and clinical efficacy of new anti-allergic treatments Accreditation: The American College of Allergy, Asthma & Immunology (ACAAI) is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. Designation: The American College of Allergy, Asthma& Immunology (ACAAI) designates this journal-based CME activity for amaximumof 1 AMA PRA Category 1 CreditTM. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Planning Committee Members: Anne K. Ellis, MD, MSc, FRCPC (Sr Author) Michael S. Tankersley, MD (CME Series Editor) Gailen D. Marshall, Jr, MD, PhD (Editor-in-Chief) Disclosure of Relevant Financial Relationships: A.K. Ellis has served as principal investigator for Circassia, Ltd., M.L. North, T. Walker, L.M. Steacy, M.S. Tankersley and G.D. Marshall have nothing to disclose. Reviewers and Education/Editorial staff have no relevant financial relationships to disclose. Astemizole has been withdrawn and is no longer available in the United States. Recognition of Commercial Support: This activity has not received external commercial support. Copyright Statement: 2013-2015 ACAAI. All rights reserved. CME Inquiries: Contact the American College of Allergy, Asthma & Immunology at education@acaai.org or 847-427-1200.

2-aminoimidazole amino acids as inhibitors of the binuclear manganese metalloenzyme human arginase I.

Arginase, a key metalloenzyme of the urea cycle that converts L-arginine into L-ornithine and urea, is presently considered a pharmaceutical target for the management of diseases associated with aberrant l-arginine homeostasis, such as asthma, cardiovascular diseases, and erectile dysfunction. We now report the design, synthesis, and evaluation of a series of 2-aminoimidazole amino acid inhibitors in which the 2-aminoimidazole moiety serves as a guanidine mimetic. These compounds represent a new class of arginase inhibitors. The most potent inhibitor identified in this study, 2-(S)-amino-5-(2-aminoimidazol-1-yl)pentanoic acid (A1P, 10), binds to human arginase I with K(d) = 2 microM and significantly attenuates airways hyperresponsiveness in a murine model of allergic airways inflammation. These findings suggest that 2-aminoimidazole amino acids represent new leads for the development of arginase inhibitors with promising pharmacological profiles.

Augmentation of arginase 1 expression by exposure to air pollution exacerbates the airways hyperresponsiveness in murine models of asthma

BackgroundArginase overexpression contributes to airways hyperresponsiveness (AHR) in asthma. Arginase expression is further augmented in cigarette smoking asthmatics, suggesting that it may be upregulated by environmental pollution. Thus, we hypothesize that arginase contributes to the exacerbation of respiratory symptoms following exposure to air pollution, and that pharmacologic inhibition of arginase would abrogate the pollution-induced AHR.MethodsTo investigate the role of arginase in the air pollution-induced exacerbation of airways responsiveness, we employed two murine models of allergic airways inflammation. Mice were sensitized to ovalbumin (OVA) and challenged with nebulized PBS (OVA/PBS) or OVA (OVA/OVA) for three consecutive days (sub-acute model) or 12 weeks (chronic model), which exhibit inflammatory cell influx and remodeling/AHR, respectively. Twenty-four hours after the final challenge, mice were exposed to concentrated ambient fine particles plus ozone (CAP+O3), or HEPA-filtered air (FA), for 4 hours. After the CAP+O3 exposures, mice underwent tracheal cannulation and were treated with an aerosolized arginase inhibitor (S-boronoethyl-L-cysteine; BEC) or vehicle, immediately before determination of respiratory function and methacholine-responsiveness using the flexiVent®. Lungs were then collected for comparison of arginase activity, protein expression, and immunohistochemical localization.ResultsCompared to FA, arginase activity was significantly augmented in the lungs of CAP+O3-exposed OVA/OVA mice in both the sub-acute and chronic models. Western blotting and immunohistochemical staining revealed that the increased activity was due to arginase 1 expression in the area surrounding the airways in both models. Arginase inhibition significantly reduced the CAP+O3-induced increase in AHR in both models.ConclusionsThis study demonstrates that arginase is upregulated following environmental exposures in murine models of asthma, and contributes to the pollution-induced exacerbation of airways responsiveness. Thus arginase may be a therapeutic target to protect susceptible populations against the adverse health effects of air pollution, such as fine particles and ozone, which are two of the major contributors to smog.

Comparative cardiopulmonary effects of size-fractionated airborne particulate matter

Context: Strong epidemiological evidence exists linking particulate matter (PM) exposures with hospital admissions of individuals for cardiopulmonary symptoms. The PM size is important in influencing the extent of infiltration into the respiratory tract and systemic circulation and directs the differential physiological impacts. Objective: To investigate the differential effects of the quasi-ultrafine (PM0.2), fine (PM0.15-2.5), and coarse PM (PM2.5-10) size fractions on pulmonary and cardiac function. Methods: Female BALB/c mice were exposed to HEPA-filtered laboratory air or concentrated coarse, fine, or quasi-ultrafine PM using Harvard Ambient Particle Concentrators in conjunction with our nose-only exposure system. These exposures were conducted as part of the “Health Effects of Aerosols in Toronto (HEAT)” campaign. Following a 4 h exposure, mice underwent assessment of respiratory function and recording of electrocardiograms using the flexiVent® system. Results: Exposure to coarse and fine PM resulted in a significant reduction in quasistatic compliance of the lung. Baseline total respiratory resistance and maximum responsiveness to methacholine were augmented after coarse PM exposures but were not affected by quasi-ultrafine PM exposures. In contrast, quasi-ultrafine PM alone had a significant effect on heart rate and in reducing heart rate variability. Conclusion: These findings indicate that coarse and fine PM influence lung function and airways responsiveness, while ultrafine PM can perturb cardiac function. This study supports the hypothesis that coarse and fine PM exerts its predominant physiologic effects at the site of deposition in the airways, whereas ultrafine PM likely crosses the alveolar epithelial barrier into the systemic circulation to affect cardiovascular function.