Scott A Boitano
Publications
Exposure to arsenic in drinking water is associated with increased respiratory disease. Alpha-1 antitrypsin (AAT) protects the lung against tissue destruction. The objective of this study was to determine whether arsenic exposure is associated with changes in airway AAT concentration and whether this relationship is modified by selenium. A total of 55 subjects were evaluated in Ajo and Tucson, Arizona. Tap water and first morning void urine were analyzed for arsenic species, induced sputum for AAT and toenails for selenium and arsenic. Household tap-water arsenic, toenail arsenic and urinary inorganic arsenic and metabolites were significantly higher in Ajo (20.6±3.5 μg/l, 0.54±0.77 μg/g and 27.7±21.2 μg/l, respectively) than in Tucson (3.9±2.5 μg/l, 0.16±0.20 μg/g and 13.0±13.8 μg/l, respectively). In multivariable models, urinary monomethylarsonic acid (MMA) was negatively, and toenail selenium positively associated with sputum AAT (P=0.004 and P=0.002, respectively). In analyses stratified by town, these relationships remained significant only in Ajo, with the higher arsenic exposure. Reduction in AAT may be a means by which arsenic induces respiratory disease, and selenium may protect against this adverse effect.
BACKGROUND: Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. OBJECTIVES: We aimed to identify, isolate and characterize the trypsin-like proteinases in a German cockroach allergen extract used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50); (2) its amino acid sequence and (3) its ability to activate calcium signaling and/or ERK1/2 phosphorylation via PAR2. METHODS: Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signaling. FINDINGS: Each of the three serine proteinase-activity-based probe-labelled enzymes isolated were biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57 to 71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signaling via PAR2. CONCLUSIONS AND RELEVANCE: We have identified three distinct allergen proteinases from the German cockroach that may play different roles for allergen-sensitization in vivo via PAR2 and may represent attractive therapeutic targets for asthma.
Bordetella bronchiseptica can establish prolonged airway infection consistent with a highly developed ability to evade mammalian host immune responses. Upon initial interaction with the host upper respiratory tract mucosa, B. bronchiseptica are subjected to antimicrobial reactive nitrogen species (RNS) and reactive oxygen species (ROS), effector molecules of the innate immune system. However, the responses of B. bronchiseptica to redox species at physiologically relevant concentrations (nM-microM) have not been investigated. Using predicted physiological concentrations of nitric oxide (NO), superoxide and hydrogen peroxide (H2O2) on low numbers of CFU of B. bronchiseptica, all redox active species displayed dose-dependent antimicrobial activity. Susceptibility to individual redox active species was significantly increased upon introduction of a second species at subantimicrobial concentrations. An increased bacteriostatic activity of NO was observed relative to H2O2. The understanding of Bordetella responses to physiologically relevant levels of exogenous RNS and ROS will aid in defining the role of endogenous production of these molecules in host innate immunity against Bordetella and other respiratory pathogens.
Allergens are diverse proteins from mammals, birds, arthropods, plants, and fungi. Allergens associated with asthma (asthmagens) share a common protease activity that may directly impact respiratory epithelial biology and lead to symptoms of asthma. Alternaria alternata is a strong asthmagen in semiarid regions. We examined the impact of proteases from A. alternata on lung inflammation in vivo and on cleaving protease-activated receptor-2 (PAR(2)) in vitro. A. alternata filtrate applied to the airway in nonsensitized Balb/c mice induced a protease-dependent lung inflammation. Moreover, A. alternata filtrate applied to human bronchial epithelial cells (16HBE14o-) induced changes in intracellular Ca(2+) concentration ([Ca(2+)](i)), consistent with PAR(2) activation. These effects were blocked by heat inactivation or by serine protease inhibition of A. alternata filtrates, and mimicked by PAR(2) specific ligands SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2), but not the PAR(1)-specific ligand TFLLR-NH(2). Desensitization of PAR(2) in 16HBE14o- cells with 2-furoyl-LIGRLO-NH(2) or trypsin prevented A. alternata-induced [Ca(2+)](i) changes while desensitization of PAR(1), PAR(3), and PAR(4) with thrombin had no effect on A. alternata-induced Ca(2+) responses. Furthermore, the Ca(2+) response to A. alternata filtrates was dependent on PAR(2) expression in stably transfected HeLa cell models. These data demonstrate that A. alternata proteases act through PAR(2) to induce rapid increases in human airway epithelial [Ca(2+)](i) in vitro and cell recruitment in vivo. These responses are likely critical early steps in the development of allergic asthma.
Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.