Nervana Metwali

Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children

BACKGROUND The Amish and Hutterites are U.S. agricultural populations whose lifestyles are remarkably similar in many respects but whose farming practices, in particular, are distinct; the former follow traditional farming practices whereas the latter use industrialized farming practices. The populations also show striking disparities in the prevalence of asthma, and little is known about the immune responses underlying these disparities. METHODS We studied environmental exposures, genetic ancestry, and immune profiles among 60 Amish and Hutterite children, measuring levels of allergens and endotoxins and assessing the microbiome composition of indoor dust samples. Whole blood was collected to measure serum IgE levels, cytokine responses, and gene expression, and peripheral-blood leukocytes were phenotyped with flow cytometry. The effects of dust extracts obtained from Amish and Hutterite homes on immune and airway responses were assessed in a murine model of experimental allergic asthma. RESULTS Despite the similar genetic ancestries and lifestyles of Amish and Hutterite children, the prevalence of asthma and allergic sensitization was 4 and 6 times as low in the Amish, whereas median endotoxin levels in Amish house dust was 6.8 times as high. Differences in microbial composition were also observed in dust samples from Amish and Hutterite homes. Profound differences in the proportions, phenotypes, and functions of innate immune cells were also found between the two groups of children. In a mouse model of experimental allergic asthma, the intranasal instillation of dust extracts from Amish but not Hutterite homes significantly inhibited airway hyperreactivity and eosinophilia. These protective effects were abrogated in mice that were deficient in MyD88 and Trif, molecules that are critical in innate immune signaling. CONCLUSIONS The results of our studies in humans and mice indicate that the Amish environment provides protection against asthma by engaging and shaping the innate immune response. (Funded by the National Institutes of Health and others.).

Airborne endotoxin concentrations in homes burning biomass fuel

Background About half of the world’s population is exposed to smoke from burning biomass fuels at home. The high airborne particulate levels in these homes and the health burden of exposure to this smoke are well described. Burning unprocessed biological material such as wood and dried animal dung may also produce high indoor endotoxin concentrations. Objective In this study we measured airborne endotoxin levels in homes burning different biomass fuels. Methods Air sampling was carried out in homes burning wood or dried animal dung in Nepal (n = 31) and wood, charcoal, or crop residues in Malawi (n = 38). Filters were analyzed for endotoxin content expressed as airborne endotoxin concentration and endotoxin per mass of airborne particulate. Results Airborne endotoxin concentrations were high. Averaged over 24 hr in Malawian homes, median concentrations of total inhalable endotoxin were 24 endotoxin units (EU)/m3 in charcoal-burning homes and 40 EU/m3 in wood-burning homes. Short cooking-time samples collected in Nepal produced median values of 43 EU/m3 in wood-burning homes and 365 EU/m3 in dung-burning homes, suggesting increasing endotoxin levels with decreasing energy levels in unprocessed solid fuels. Conclusions Airborne endotoxin concentrations in homes burning biomass fuels are orders of magnitude higher than those found in homes in developed countries where endotoxin exposure has been linked to respiratory illness in children. There is a need for work to identify the determinants of these high concentrations, interventions to reduce exposure, and health studies to examine the effects of these sustained, near-occupational levels of exposure experienced from early life.

Modulation of reactive oxygen species by Rac1 or catalase prevents asbestos-induced pulmonary fibrosis

The release of reactive oxygen species (ROS) and cytokines by alveolar macrophages has been demonstrated in asbestos-induced pulmonary fibrosis, but the mechanism linking alveolar macrophages to the pathogenesis is not known. The GTPase Rac1 is a second messenger that plays an important role in host defense. In this study, we demonstrate that Rac1 null mice are protected from asbestos-induced pulmonary fibrosis, as determined by histological and biochemical analysis. We hypothesized that Rac1 induced pulmonary fibrosis via generation of ROS. Asbestos increased TNF-alpha and ROS in a Rac1-dependent manner. TNF-alpha was elevated only 1 day after exposure, whereas ROS generation progressively increased in bronchoalveolar lavage cells obtained from wild-type (WT) mice. To determine whether ROS generation contributed to pulmonary fibrosis, we overexpressed catalase in WT monocytes and observed a decrease in ROS generation in vitro. More importantly, administration of catalase to WT mice attenuated the development of fibrosis in vivo. For the first time, these results demonstrate that Rac1 plays a crucial role in asbestos-induced pulmonary fibrosis. Moreover, it suggests that a simple intervention may be useful to prevent progression of the disease.

Evaluation of the Limulus Amebocyte Lysate and Recombinant Factor C Assays for Assessment of Airborne Endotoxin

ABSTRACT As a potent inflammatory agent, endotoxin is a key analyte of interest for studies of lung ailments in domestic environments and occupational settings with organic dust. A relatively unexplored advance in endotoxin exposure assessment is the use of recombinant factor C (rFC) from the Limulus pathway in a fluorometric assay. In this study, we compared airborne endotoxin concentrations in laboratory- and field-collected parallel air samples using the kinetic Limulus amebocyte lysate (LAL) assay and the rFC assay. Air sampling was performed using paired Institute of Occupational Medicine (IOM) samplers, Button samplers, closed-face cassettes, and cyclone samplers. Field sampling was performed in 10 livestock production facilities, including those housing swine, chicken, turkey, dairy cows, cattle, and horses. Laboratory sampling was performed in exposure chambers using resuspended airborne dust collected in five livestock facilities. Paired samples were extracted in pyrogen-free water with 0.05% Tween 20 and analyzed using LAL and rFC assays. In 402 field sample pairs there was excellent agreement between endotoxin concentrations determined by LAL and rFC (r = 0.93; P < 0.0001). In 510 laboratory sample pairs there was also excellent agreement between the two assays (r = 0.86; P < 0.0001). Correlations for subgroups of facility or dust type ranged from 0.65 to 0.96. Mixed-model analysis of variance (ANOVA) for the field studies showed significant interactions of facility-sampler and facility-assay. rFC/LAL ratios of the geometric means were 0.9 to 1.14 for the samplers (not significantly different from 1.0). The data from this study demonstrate that the LAL assay and the rFC assay return similar estimates of exposure in livestock facilities. Both methods provided suitable lower limits of detection such that all but 19 of 1,824 samples were quantifiable.

Endotoxin Exposure: Predictors and Prevalence of Associated Asthma Outcomes in the United States

RATIONALE Inhaled endotoxin induces airway inflammation and is an established risk factor for asthma. The 2005-2006 National Health and Nutrition Examination Survey included measures of endotoxin and allergens in homes as well as specific IgE to inhalant allergens. OBJECTIVES To understand the relationships between endotoxin exposure, asthma outcomes, and sensitization status for 15 aeroallergens in a nationally representative sample. METHODS Participants were administered questionnaires in their homes. Reservoir dust was vacuum sampled to generate composite bedding and bedroom floor samples. We analyzed 7,450 National Health and Nutrition Examination Survey dust and quality assurance samples for their endotoxin content using extreme quality assurance measures. Data for 6,963 subjects were available, making this the largest study of endotoxin exposure to date. Log-transformed endotoxin concentrations were analyzed using logistic models and forward stepwise linear regression. Analyses were weighted to provide national prevalence estimates and unbiased variances. MEASUREMENTS AND MAIN RESULTS Endotoxin exposure was significantly associated with wheeze in the past 12 months, wheeze during exercise, doctor and/or emergency room visits for wheeze, and use of prescription medications for wheeze. Models adjusted for age, sex, race and/or ethnicity, and poverty-to-income ratio and stratified by allergy status showed that these relationships were not dependent upon sensitization status but were worsened among those living in poverty. Significant predictors of higher endotoxin exposures were lower family income; Hispanic ethnicity; participant age; dog(s), cat(s), cockroaches, and/or smoker(s) in the home; and carpeted floors. CONCLUSIONS In this U.S. nationwide representative sample, higher endotoxin exposure was significantly associated with measures of wheeze, with no observed protective effect regardless of sensitization status.

Assessment of airborne exposures and health in flooded homes undergoing renovation.

UNLABELLED In June 2008, the Cedar River crested flooding more than 5000 Cedar Rapids homes. Residents whose homes were flooded were invited to participate in this study. Household assessments and resident interviews were conducted between November 2008 and April 2009. We characterized exposures and symptoms experienced by individuals inhabiting 73 flood-damaged homes. Active air sampling and passive electrostatic dust collectors were used to assess exposures to culturable mold, culturable bacteria, fungal spores, inhalable particulate matter (iPM), endotoxin, glucans, allergens, lead, asbestos, radon, carbon dioxide, and carbon monoxide. Wall moisture levels and relative humidity were also measured. Exposures and questionnaire-based health assessments were compared at two levels of remediation, in-progress and completed. Homes with remediation in-progress (N = 24), as compared to the completed homes (N = 49), had significantly higher airborne concentrations of mold, bacteria, iPM, endotoxin, and glucan. Residents of in-progress homes had a significantly higher prevalence of doctor-diagnosed allergies (adjusted OR = 3.08; 95% CI: 1.05, 9.02) and all residents had elevated prevalence of self-reported wheeze (adjusted OR = 3.77; 95% CI: 2.06, 6.92) and prescription medication use for breathing problems (adjusted OR = 1.38; 95% CI: 1.01, 1.88) after the flood as compared to before. Proper post-flood remediation led to improved air quality and lower exposures among residents living in flooded homes. PRACTICAL IMPLICATIONS The number and severity of floods is on the rise, and health departments need evidence-based information to advise homeowners on recovery after such disasters. Our study suggests that proper remediation of flood-damaged homes can reduce bioaerosols to acceptable levels but exposures are significantly increased while remediation is in-progress leading to an increased burden of allergy and allergic rhinitis.

beta-(1,3)-Glucan exposure assessment by passive airborne dust sampling and new sensitive immunoassays.

ABSTRACT Associations between house dust-associated β-(1,3)-glucan exposure and airway inflammatory reactions have been reported, while such exposures in early childhood have been suggested to protect against asthma and wheezing. Most epidemiological studies have used reservoir dust samples and an inhibition enzyme immunoassay (EIA) for β-(1,3)-glucan exposure assessment. The objective of this study was to develop inexpensive but highly sensitive enzyme immunoassays to measure airborne β-(1,3)-glucans in low-exposure environments, like homes. Specificities of available anti-β-(1,3)-glucan antibodies were defined by direct and inhibition experiments. Three suitable antibody combinations were selected for sandwich EIAs. β-(1,3)-Glucans in passive airborne dust collected with an electrostatic dust fall collector (EDC) and floor dust from seven homes were measured with the three EIAs. Floor dust samples were additionally analyzed in the inhibition EIA. The sandwich EIAs were sensitive enough for airborne glucan measurement and showed different specificities for commercial glucans, while the β-(1,3)-glucan levels in house dust samples correlated strongly. The feasibility of measuring glucans in airborne dust with the recently introduced EDC method was further investigated by selecting the most suitable of the three EIAs to measure and compare β-(1,3)-glucan levels in the EDC and in floor and actively collected airborne dust samples of the previously performed EDC validation study. The EDC β-(1,3)-glucan levels correlated moderately with β-(1,3)-glucans in actively collected airborne dust and floor dust samples, while the glucan levels in the airborne dust and floor dust samples did not correlate. The combination of the newly developed β-(1,3)-glucan sandwich EIA with EDC sampling now allows assessment in large-scale population studies of exposure to airborne β-(1,3)-glucans in homes or other low-exposure environments.

A Multi-Center Ring Trial of Allergen Analysis using Fluorescent Multiplex Array Technology

BACKGROUND Consistent performance of allergen assays is essential to ensure reproducibility of exposure assessments for investigations of asthma and occupational allergic disease. This study evaluated intra- and inter-laboratory reproducibility of a fluorescent multiplex array, which simultaneously measures eight indoor allergens in a single reaction well. METHODS A multi-center study was performed in nine laboratories in the US and Europe to determine the inter-laboratory variability of an 8-plex array for dust mite, cat, dog, rat, mouse and cockroach allergens. Aliquots of 151 dust extract samples were sent to participating centers and analyzed by each laboratory on three separate occasions. Agreement within and between laboratories was calculated by the concordance correlation coefficient (CCC). RESULTS Results were obtained for over 32,000 individual allergen measurements. Levels covered a wide range for all allergens from below the lower limit of detection (LLOD = 0.1-9.8 ng/ml) to higher than 6800 ng/ml for all allergens except Mus m 1, which was up to 1700 ng/ml. Results were reproducible within as well as between laboratories. Within laboratories, 94% of CCC were ≥ 0.90, and 80% of intra-laboratory results fell within a 10% coefficient of variance (CV%). Results between laboratories also showed highly significant positive correlations for all allergens (~0.95, p<0.001). Overall means of results were comparable, and inter-laboratory CV% for all allergens except Rat n 1 ranged between 17.6% and 26.6%. CONCLUSION The data indicate that performance criteria for fluorescent multiplex array technology are reproducible within and between laboratories. Multiplex technology provides standardized and consistent allergen measurements that will streamline environmental exposure assessments in allergic disease.

Fungal Exposure, Atopy, and Asthma Exacerbations in Puerto Rican Children

BACKGROUND Glucan is a component of the fungal cell wall that is used as a marker of fungal exposure. Little is known about indoor glucan, atopy, and asthma exacerbations among children living in tropical environments such as Puerto Rico. Our objective was to examine whether glucan exposure is associated with degree of atopy or visits to the emergency department (ED)/urgent care for asthma in Puerto Rican children. METHODS This was a cross-sectional study of 317 children aged 6 to 14 years with (cases, n = 160) and without (control subjects, n = 157) asthma in San Juan, Puerto Rico. Our primary outcomes were the number of positive skin tests to allergens (range, 0-15) and (in cases only) having had at least one visit to the ED/urgent care for asthma in the prior year. Levels of glucan, endotoxin, peptidoglycan, and five allergens (Der p 1, Bla g 2, Fel d 1, Can f 1, and Mus m 1) were measured in samples of house dust. Linear or logistic regression was used for the multivariate analysis. MEASUREMENTS AND MAIN RESULTS In a multivariate analysis adjusting for case-control status, mouse allergen, and other covariates, children exposed to glucan levels in the second and third quartiles had approximately two more positive skin tests than those in the lowest quartile (P < 0.01 in both instances). Among children with asthma, exposure to the highest quartile of glucan was associated with nearly ninefold greater odds of one or more visits to the ED/urgent care for asthma (95% confidence interval for adjusted odds ratio, 2.7-28.4; P < 0.001). CONCLUSIONS Our results suggest that indoor fungal exposure leads to an increased degree of atopy and visits to the ED/urgent care for asthma in Puerto Rican children.

Passive monitors to measure hydrogen sulfide near concentrated animal feeding operations

Hydrogen sulfide (H2S) is one of many airborne pollutants emitted by concentrated animal feeding operations (CAFOs). However, few studies have characterized ambient H2S levels near these facilities, largely due to the lack of low-cost, reliable, and easily transportable instrumentation available to researchers. We determined intermediate environmental H2S exposure near CAFOs using Radiello passive monitors. First, a laboratory study was performed to determine the accuracy of the device. Next, a total of eight passive H2S monitors were deployed bi-weekly in close proximity (<40 m) to a medium-sized swine confinement for seven months in order to determine the temporal and spatial variability of H2S. Finally, we measured H2S concentrations across two rural Iowa counties to characterize ambient exposure near thirteen CAFOs and two schools. The value of the temperature-adjusted H2S passive diffusion rate provided by the supplier was 29% larger than the 24 h rate determined experimentally. Concentrations of H2S measured near the medium-sized confinement were varied and ranged from 0.2 to 48.6 ppb depending on the sampling period and proximity to a lagoon on the property. Two-week concentrations near the schools were low (<1 ppb), while concentrations near the thirteen CAFOs ranged from 0.1 to 42.9 ppb. The passive monitors were effective in measuring H2S concentrations near a swine CAFO as long as they were exposed for a sufficient period of time (two weeks). Radiello passive monitors are a promising new device in measuring intermediate H2S exposure in rural populations. Measured values in excess of an Iowa state limit of 30 ppb (24 h average) suggest that enforcement actions are needed to mitigate H2S migration from swine CAFOs.