Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients.

BACKGROUND: Asthma is characterized by increased airway narrowing in response to nonspecific stimuli. The disorder is influenced by both environmental and genetic factors. Exosomes are nanosized vesicles of endosomal origin released from inflammatory and epithelial cells that have been implicated in asthma. In this study we characterized the microRNA (miRNA) content of exosomes in healthy control subjects and patients with mild intermittent asthma both at unprovoked baseline and in response to environmental challenge. OBJECTIVE: To investigate alterations in bronchoalveolar lavage fluid (BALF) exosomal miRNA profiles due to asthma, and following subway air exposure. METHODS: Exosomes were isolated from BALF from healthy control subjects (n = 10) and patients with mild intermittent asthma (n = 10) after subway and control exposures. Exosomal RNA was analyzed by using microarrays containing probes for 894 human miRNAs, and selected findings were validated with quantitative RT-PCR. Results were analyzed by using multivariate modeling. RESULTS: The presence of miRNAs was confirmed in exosomes from BALF of both asthmatic patients and healthy control subjects. Significant differences in BALF exosomal miRNA was detected for 24 miRNAs with a subset of 16 miRNAs, including members of the let-7 and miRNA-200 families, providing robust classification of patients with mild nonsymptomatic asthma from healthy subjects with 72% cross-validated predictive power (Q(2) = 0.72). In contrast, subway exposure did not cause any significant alterations in miRNA profiles. CONCLUSION: These studies demonstrate substantial differences in exosomal miRNA profiles between healthy subjects and patients with unprovoked, mild, stable asthma. These changes might be important in the inflammatory response leading to bronchial hyperresponsiveness and asthma.

The pulmonary deposition and retention of indium-111 labeled ultrafine carbon particles in healthy individuals.

CONTEXT: Particulate air pollution, for example, from ultrafine (UF) particles, has negative health effects. However, there is still limited knowledge regarding the fate of inhaled particles in the human body. OBJECTIVES: To describe the normal lung deposition and 1 week particle retention of indium-111 labeled UF carbon particles in healthy subjects. Additionally, the possibility to extend the follow-up period to 4 weeks was also investigated for one of the subjects. RESULTS: The cumulative pulmonary particle clearance 1 week post-administration, corrected for activity leaching and mucocilliary transport of activity deposited in the central airways, was 4.3 ± 8.5% (average ± standard deviation at group level), with marginal translocation of particles from lungs to blood, 0.3%. There was no observable elimination of particles from the body via urine. Seven days after exposure, the cumulated activity leaching was 3% (group level), which indicates a stable bonding between the particles and Indium-111. The volunteer followed for a total of 4 weeks, showed a cumulative decrease of activity retention in the lungs of 10.5%. After correction for activity leaching and clearance from central airway deposition, the estimated particle clearance was about 2%. CONCLUSIONS: No evidence for particle translocation from the lungs could be proven 7 days after exposure. It is possible to follow-up Indium-111 labeled UF carbon particles at least 1 month post-administration without increasing the administered activity.

Health effects of a subway environment in mild asthmatic volunteers.

Particle exposure is known to have negative health effects. In Stockholm the environment in the subway has been reported to have higher particle exposure levels, measured as PM(2.5) and PM(10), than roads with intense traffic in the inner city area. We have recently shown that healthy volunteers exposed to subway environment had statistically significant increase of fibrinogen and CD4 cells expressing regulatory T-cell marker CD25(bright)/FOXP3 in blood. The aim of the present study was to find out whether a more vulnerable population, asthmatics, would demonstrate similar or other changes in the lungs or in the peripheral blood. Sixteen mild asthmatics were exposed to a subway and a control environment for 2 h while being monitored by measurements of lung function, and inflammatory response in the lower airways evaluated by bronchoscopy and in peripheral blood. An attempt to standardize the exposures was done, by letting the volunteers alternate 15 min intervals of moderate exercise on a bicycle ergometer with 15 min of rest. We found a statistically significant increased frequency of CD4 cells expressing T-cell activation marker CD25 in bronchoalveolar lavage fluid, but no significant increase of regulatory T-cells in blood as was found in healthy volunteers. Our study shows that airway inflammatory responses after exposure in subway environment differ between asthmatic and healthy humans.

Asthmatics exhibit altered oxylipin profiles compared to healthy individuals after subway air exposure.

BACKGROUND: Asthma is a chronic inflammatory lung disease that causes significant morbidity and mortality worldwide. Air pollutants such as particulate matter (PM) and oxidants are important factors in causing exacerbations in asthmatics, and the source and composition of pollutants greatly affects pathological implications. OBJECTIVES: This randomized crossover study investigated responses of the respiratory system to Stockholm subway air in asthmatics and healthy individuals. Eicosanoids and other oxylipins were quantified in the distal lung to provide a measure of shifts in lipid mediators in association with exposure to subway air relative to ambient air. METHODS: Sixty-four oxylipins representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of bronchoalveolar lavage (BAL)-fluid. Validations through immunocytochemistry staining of BAL-cells were performed for 15-LOX-1, COX-1, COX-2 and peroxisome proliferator-activated receptor gamma (PPARγ). Multivariate statistics were employed to interrogate acquired oxylipin and immunocytochemistry data in combination with patient clinical information. RESULTS: Asthmatics and healthy individuals exhibited divergent oxylipin profiles following exposure to ambient and subway air. Significant changes were observed in 8 metabolites of linoleic- and α-linolenic acid synthesized via the 15-LOX pathway, and of the COX product prostaglandin E(2) (PGE(2)). Oxylipin levels were increased in healthy individuals following exposure to subway air, whereas asthmatics evidenced decreases or no change. CONCLUSIONS: Several of the altered oxylipins have known or suspected bronchoprotective or anti-inflammatory effects, suggesting a possible reduced anti-inflammatory response in asthmatics following exposure to subway air. These observations may have ramifications for sensitive subpopulations in urban areas.

¹¹¹Indium-labeled ultrafine carbon particles; a novel aerosol for pulmonary deposition and retention studies.

Continuous environmental or occupational exposure to airborne particulate pollution is believed to be a major hazard for human health. A technique to characterize their deposition and clearance from the lungs is fundamental to understand the underlying mechanisms behind their negative health effects. In this work, we describe a method for production and follow up of ultrafine carbon particles labeled with radioactive ¹¹¹Indium (¹¹¹In). The physicochemical and biological properties of the aerosol are described in terms of particle size and concentration, agglomeration rate, chemical bonding stability, and human lung deposition and retention. Preliminary in vivo data from a healthy human pilot exposure and 1-week follow up of the aerosol is presented. More than 98% of the generated aerosol was labeled with Indium and with particle sizes log normally distributed around 79 nm count median diameter. The aerosol showed good generation reproducibility and chemical stability, about 5% leaching 7 days after generation. During human inhalation, the particles were deposited in the alveolar space, with no central airways involvement. Seven days after exposure, the cumulative activity retention was 95.3%. Activity leaching tests from blood and urine samples confirmed that the observed clearance was explained by unbound activity, suggesting that there was no significant elimination of ultrafine particles. Compared to previously presented methods based on Technegas, ¹¹¹In-labelled ultrafine carbon particles allow for extended follow-up assessments of particulate pollution retention in healthy and diseased lungs.

Limited airway effects in mild asthmatics after exposure to air pollution in a road tunnel.

Ambient air pollution is a contributing factor to respiratory morbidity and mortality and asthmatics are a particularly vulnerable population. The aim of the study was to investigate whether acute exposure to traffic related air pollution in a road tunnel would increase bronchial responsiveness in mild asthmatics, and if this would be accompanied by increased measures of inflammatory markers in the airways assessed by nasal lavage (NAL) and induced sputum. Fourteen mild asthmatics (7 treated with inhaled corticosteroids) were exposed for 2 h in a road tunnel and a control environment, respectively, separated by at least 3 weeks. Symptoms and peak expiratory flow (PEF) were recorded. Seven hours following exposure sessions, subjects underwent measurements of fraction of exhaled nitric oxide (FENO), spirometry, and a bronchial provocation test. NAL, induced sputum and blood samples were collected. The median PM(2.5) and PM(10) levels during the exposure occasions in the road tunnel were 80 (range 41-93) µg/m(3) and 183 (72-213) µg/m(3) respectively. Irritative symptoms from the airways increased and PEF decreased after road tunnel exposure. Increased levels of IL-10, IL-12 and TNF-α were observed in NAL fluid from subjects without ongoing inhaled corticosteroid treatment. Forced expiratory volume in 1 s (FEV(1)) and the degree of bronchial responsiveness in asthmatics did not change significantly after tunnel exposure. We conclude that asthmatics exhibit increased symptoms, decreased PEF and signs of inflammatory response in the upper airways, after a 2 h road tunnel exposure. Our findings may further emphasize asthmatics as a vulnerable group to common air pollutants.

Work disability in rheumatoid arthritis--development over 15 years and evaluation of predictive factors over time.

OBJECTIVE: To investigate work disability rates over 15 years in an early rheumatoid arthritis (RA) cohort and to evaluate predictive factors during the course of the study. METHODS: All patients with early RA of working age (n = 148) were followed and treated at a team care unit. Mean disease duration at inclusion was 1 year. Work characteristics and disease-related variables were recorded annually. Logistic regression analyses were performed to identify predictors for work disability after 5, 10, and 15 years. RESULTS: Work disability rates were 28%, 35%, 39%, and 44% at study start and after 5, 10, and 15 years, respectively. Forty-seven patients reduced working hours and 34 changed work tasks during the study time. Older age, less education, heavy manual work, and much activity limitation assessed by Health Assessment Questionnaire (HAQ) were predictors of work disability. Demographics and work factors had best predictive value in the early phase, while HAQ was a strong predictor at all points in time. Odds ratios for baseline HAQ, 5 year HAQ, and 10 year HAQ were 6.3, 9.6, and 4.1 for work disability after 5, 10, and 15 years, respectively. CONCLUSION: The prevalence of work disability was 28% at inclusion. After 15 years' followup the prevalence was 44%, which is lower than previously reported. HAQ was the single prognostic factor with strong predictive value throughout the study.

History and results of the two inter-laboratory round robin endotoxin assay studies on cotton dust.

BACKGROUND: In the US cotton industry, airborne cotton dust levels are regulated, and other countries are moving to specify safety limits for airborne endotoxins. There is concern about potential respiratory health hazards associated with agricultural and other organic dusts. In laboratories, ranking which samples have high and low levels of endotoxin is usually in good agreement between laboratories. When different laboratories assay identical samples, the levels differ. The objective of this research was to evaluate the intra- and inter-laboratory variability for 13 laboratories measuring endotoxin in cotton dust. METHOD: Two inter-laboratory round robin endotoxin assay studies were conducted using cotton dust. In the first round robin, each laboratory used their normal in-house assay method and then used a common extraction protocol. In the second round robin, a common extraction protocol and endotoxin assay kit was used. RESULTS: The intra-laboratory results had small variations but inter-laboratory results had very high variations. The inter-laboratory results using a common extraction protocol showed reduced differences. Using the same extraction protocol and endotoxin assay kit, the intra-laboratory variation was small and inter-laboratory variation was reduced but not enough for inter-laboratory agreement. Most of the laboratories were able to discern between the high and low endotoxin concentration dusts. CONCLUSIONS: Standardization has reduced the differences in results between laboratories and possibly further standardization may bring closer inter-laboratory agreement.

Effects of 5-lipoxygenase inhibitor zileuton on airway responses to inhaled swine house dust in healthy subjects.

BACKGROUND: Inhalation of swine house dust induces acute airway inflammation and increased bronchial responsiveness in healthy subjects. OBJECTIVE: The aim of the study was to investigate whether 5-lipoxygenase products such as leukotrienes may have a role in this reaction. METHODS: Twenty-three healthy subjects were randomised into two groups receiving treatment with either zileuton (600 mg) or placebo four times a day. After 5 days of treatment, all subjects were exposed for 3h in a swine barn. Bronchial responsiveness, exhaled nitric oxide (NO), and mediators in nasal lavage (NAL), blood and urine were measured before and after the exposure. RESULTS: The exposure induced an increased bronchial responsiveness to methacholine in both groups with 2-3 doubling concentration steps, no significant difference between treatments. Leukotriene E(4) in urine increased significantly following exposure in the placebo group from 37.3 (29.1-45.6) (mean (95% confidence interval)) ng/mmol creatinine to 47.7 (36.3-59.0) ng/mmol creatinine (P<0.05), but not in the zileuton group. The post-exposure increase of LTB(4) levels in NAL fluid was totally abolished in the zileuton group (P<0.05 vs. the placebo). The levels of exhaled NO increased significantly (P<0.01), two-fold in both groups. The PGD(2) metabolite 9alpha, 11beta-PGF(2) increased in placebo-treated subjects (P<0.01; P<0.05 vs. zileuton), strengthening mast cell participation. Neutrophil counts and levels of IL-6 in peripheral blood increased in both groups, with a significantly larger increase in zileuton treated subjects (P<0.05 and P<0.001, respectively compared to placebo). CONCLUSIONS: Pre-treatment with clinically recommended doses of the 5-lipoxygenase inhibitor zileuton did not affect the increase of bronchial reactivity induced by swine dust exposure. The intervention totally abolished the LTB(4) release in NAL fluid, but only partially inhibited the formation of leukotrienes as monitored by urinary levels. The enhanced increase of neutrophils and IL-6 in peripheral blood in the zileuton group, suggests that inhibition of 5-lipoxygenase may have pro-inflammatory effects.

Interlaboratory evaluation of endotoxin analyses in agricultural dusts--comparison of LAL assay and mass spectrometry.

Endotoxin exposure is associated with wheeze and asthma morbidity, while early life exposure may reduce risk of allergy and asthma. Unfortunately, it is difficult to compare endotoxin results from different laboratories and environments. We undertook this study to determine if lipopolysaccharide (LPS) extraction efficiency could account for differences among laboratories. We generated and collected aerosols from chicken and swine barns, and corn processing. We randomly allocated side-by-side filter samples to five laboratories for Limulus assay of endotoxin. Lyophilized aliquots of filter extracts were analyzed for 3-hydroxy fatty acids (3-OHFAs) as a marker of LPS using gas chromatography-mass spectrometry. There were significant differences in endotoxin assay and GC-MS (LPS) results between laboratories for all dust types (p < 0.01). Patterns of differences between labs varied by dust type. Relationships between assay and GC/MS results also depended on dust type. The percentages of individual 3-OHFA chain lengths varied across labs (p < 0.0001) suggesting that each lab recovered a different fraction of the LPS available. The presence of large amounts of particle associated LPS and absence of a freezing thawing cycle were associated with lower correlations between LPS and bioactivity, consistent with an absence of Limulus response to cell-bound endotoxin. These data suggest that extraction methods affect endotoxin measurements. The LAL methods may be most suitable when comparing exposures within similar environments; GC-MS offers additional information helpful in optimizing sample treatment and extraction. GC-MS may be of use when comparing across heterogeneous environments and should be considered for inclusion in future studies of human health outcomes.

Gender-related long-term effects in adult rats by perinatal dietary ratio of n-6/n-3 fatty acids.

Epidemiological studies in humans have shown that perinatal nutrition affects health later in life. We have previously shown that the ratio of n-6 to n-3 polyunsaturated fatty acids (PUFA) in the maternal diet affects serum leptin levels and growth of the suckling pups. The aim of the present study was to investigate the long-term effects of various ratios of the dietary n-6 and n-3 PUFA during the perinatal period on serum leptin, insulin, and triacylglycerol, as well as body growth in the adult offspring. During late gestation and throughout lactation, rats were fed an isocaloric diet containing 7 wt% fat, either as linseed oil (n-3 diet), soybean oil (n-6/n-3 diet), or sunflower oil (n-6 diet). At 3 wk of age, the n-6/n-3 PUFA ratios in the serum phospholipids of the offspring were 2.5, 8.3, and 17.5, respectively. After weaning, all pups were given a standard chow. At the 28th postnatal wk, mean body weight and fasting insulin levels were significantly increased in the rats fed the n-6/n-3 diet perinatally compared with the other groups. The systolic blood pressure and serum triacylglycerol levels were only increased in adult male rats of the same group. These data suggest that the balance between n-6 and n-3 PUFA during perinatal development affects several metabolic parameters in adulthood, especially in the male animals.

Acute respiratory effects of exposure to ammonia on healthy persons.

OBJECTIVES: This study investigated the acute respiratory effects of low ammonia exposure on healthy persons. METHODS: Twelve healthy persons underwent sham or ammonia (5 and 25 ppm) exposure randomly in an exposure chamber on three occasions. The exposure duration was 3 hours, 1.5 hours resting (seated) and 1.5 hours exercising (50 W on a bicycle ergonometer). Symptoms were registered repeatedly before, during, and after the exposure on visual analogue scales. Bronchial responsiveness to methacholine, lung function, and exhaled nitric oxide (NO) were measured before and 7 hours after the exposure. In addition, nasal lavage was performed, and peripheral blood samples were drawn before and 7 hours after the exposure. RESULTS: All the symptom ratings increased significantly during 25-ppm ammonia exposure as compared with the control exposure. The cumulative dose of methacholine causing a 20% decrease in forced expiratory volume in 1 second was lower (<1 concentration step of methacholine) for the exposure than for a pretrial control challenge. However, no difference was found between the control and ammonia exposures (P=0.33). The ammonia exposure did not significantly influence lung function or the exhaled NO levels. The total cell or interleukin-8 concentration in nasal lavage fluid did not change. The total leucocyte concentration in peripheral blood increased significantly (P<0.001) after both the sham and ammonia exposure, mainly due to an increase in neutrophils (P<0.001). Ammonia exposure did not significantly alter complement factor 3b in plasma. CONCLUSIONS: During ammonia exposure in an exposure chamber, symptoms related to irritation and central nervous effects increase and are constant with no signs of adaptation. Ammonia inhalation does not cause detectable upper-airway inflammation or increased bronchial responsiveness to methacholine in healthy persons.

Partial protection by respirators on airways responses following exposure in a swine house.

BACKGROUND: Exposure to swine dust leads to an intense airway inflammation and increased bronchial responsiveness. The purpose of the present study was to evaluate the effect of a respiratory protection device during exposure in a swine house. METHODS: Twenty-two subjects, 11 with a respirator, were exposed. Symptoms, body temperature, nasal lavage, and a bronchial metacholine challenge were performed before and 7 hr after exposure. For exposure assessment a nasal sampler was evaluated. RESULTS: The subjects with a respirator showed an attenuated inflammatory nasal response. An increase in bronchial responsiveness was observed in both groups, significantly greater in the unprotected group. The use of respirators reduced endotoxin exposure by more than 90% (assessed by nasal samplers). CONCLUSION: The use of a respirator attenuated the inflammatory response compared with an unprotected group. The minor effect on bronchial responsiveness suggests that gases and/or ultrafine particles may also be important factors.

Comparison of endotoxin assays using agricultural dusts.

Endotoxins from gram-negative bacteria pose a significant respiratory hazard. Establishing dose-response relationships is problematic because there are no standard procedures for sampling and analysis. The goal of this study was to compare endotoxin analyses in six laboratories using Limulus-based assays for analysis of organic dusts from three agricultural environments: chicken barns, swine barns, and corn processing facilities. For each dust generation experiment 14 side-by-side air samples were collected on 37-mm glass fiber filters at flows of 1.8 L/min. Each laboratory was randomly allocated two filters from each of seven experiments per dust type. Three laboratories used the QCL-1000 endpoint assay, and three used the kinetic-QCL method. To eliminate variability among different lots, a single lot of Limulus amebocyte lysate for endpoint assays and one similar lot for kinetic assays was provided. Precision of assays performed within labs was very good, with pooled coefficients of variation for replicate samples ranging from 1 to 11% over all labs and all dust types. There were significant differences between laboratories for all three dust types (p < 0.01). The pattern of differences between labs varied by dust type. For chicken dust, labs using the endpoint method reported higher results than those using kinetic methods. For swine and corn dusts, labs using the kinetic method reported the highest endotoxin values. For chicken dust, results from all labs except A and B were highly correlated (r = 0.86-1.00). For swine dust, only labs B and E, and C and D were correlated. For corn, A, B, and D were significantly correlated with most other labs. In conclusion, statistical differences in performance between laboratories were apparent and may be related to the extraction and analytical methods. The results of this study will be useful for standardization of sampling and analysis of airborne endotoxin in agriculture.

Second inter-laboratory study comparing endotoxin assay results from cotton dust.

Previously, a large two-part inter-laboratory round robin endotoxin assay study was completed. This first study showed that when cotton dust samples, which are practically identical, are assayed for endotoxin that the intra- laboratory results had a very small variation while intra-laboratory results of the sample had a very high variation. In the first part of the study, each laboratory followed its own in-house assay protocol; but in the second part of the study, when the extraction protocol was standardized, the inter-laboratory results showed a lower variation, which suggested that with further standardization, further reduction of differences between laboratories might be achieved in order that results between laboratories would become more comparable. The results stimulated interest in extending the study to include cotton dust with two levels of endotoxin, standardization of the extraction protocol, and using the same assay kit from the same production lot. The results of this second round robin endotoxin assay study indicate that differences between laboratories are still high, but most of the laboratories could discern the cotton dusts with the different levels of endotoxin.

Measuring health related quality of life in patients with rheumatoid arthritis--reliability, validity, and responsiveness of a Swedish version of RAQoL.

OBJECTIVE: To adapt the Rheumatoid Arthritis Quality of Life (RAQoL) questionnaire for Swedish patients and evaluate psychometric properties in a prospective study. METHODS: Reliability was assessed in 61 patients filling in RAQoL two times with one week's interval. 114 patients completed RAQoL and Nottingham Health Profile (NHP) on 2-3 occasions 6 months apart. Validity was evaluated comparing RAQoL-scores to disease-related variables and NHP subscales. Standardized response mean was applied to calculate responsiveness with the RA-related variables as external indicators of change. RESULTS: Test-retest reliability was high and internal consistency sufficient. RAQoL correlated as expected to NHP section scores. In a multivariate model the Stanford Health Assessment Questionnaire disability index (HAQ) and general health could explain 40% and disease activity measures 13% of the variance of RAQoL. Correlations between change scores of clinical variables and RAQoL and NHP were weak but positive. Standardized response means regarding change of disease activity, HAQ, and general health were small but in the same range for both RAQoL and NHP. CONCLUSION: The Swedish RAQoL had similar measurement properties as the original version. However, responsiveness regarding condition specific measures was not better than for the generic instrument NHP.

Airways inflammation after exposure in a swine confinement building during cleaning procedure.

BACKGROUND: Healthy volunteers exposed for 3 hr during weighing of pigs develop an airway inflammation characterized by a massive influx of neutrophilic granulocytes in the upper and lower airways and increased bronchial responsiveness to methacholine. The purpose of the present study was to investigate health effects from exposure during cleaning of the swine confinement building and to evaluate the effect of a respiratory protection device. METHODS: Sixteen subjects were exposed for 3 hr during cleaning of a swine confinement room with a high-pressure cleaner. Seven out of sixteen subjects were equipped with a mask during exposure. RESULTS: The bronchial responsiveness increased in all subjects following exposure, significantly more in the group exposed without a mask (P < 0.05). The cell concentration (mainly neutrophilic granulocytes) in nasal lavage fluid as well as the concentration of interleukin-8, increased significantly only in those subjects exposed without a respiratory protection device. In peripheral blood, an increase of neutrophilic granulocytes was observed in both groups, although it was significantly higher in the group without mask (P < 0.05). The inhalable dust level was 0.94 (0.74 - 1.55) mg/m(3) and respirable dust 0.56 (0.51-0.63) mg/m(3). CONCLUSION: Exposure to dust aerosols during the cleaning of the interior of a swine confinement building induces increased bronchial responsiveness and an acute inflammatory reaction in the upper airways. The use of a mask attenuated but did not abolish the inflammatory response. This suggests that gases and/or ultrafine particles in this environment could be important factors in the development of increased bronchial responsiveness.