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Introduction: Environmental exposure to mites and fungi has been proposed to critically contribute to the development of IgE-mediated asthma. A common denominator of such organisms is chitin. Human chitinases have been reported to be upregulated by interleukin-13 secreted in the context of Th2-type immune responses and to induce asthma. We assessed whether chitin-containing components induced chitinases in an innate immune-dependent way and whether this results in bronchial hyperresponsiveness. Materials and Methods: Monocyte/macrophage cell lines were stimulated with chitin-containing or bacterial components in vitro. Chitinase activity in the supernatant and the expression of the chitotriosidase gene were measured by enzyme assay and quantitative PCR, respectively. Non-sensitized mice were stimulated with chitin-containing components intranasally, and a chitinase inhibitor was administered intraperitoneally. As markers for inflammation leukocytes were counted in the bronchoalveolar lavage (BAL) fluid, and airway hyperresponsiveness was assessed via methacholine challenge. Results: We found both whole chitin-containing dust mites as well as the fungal cell wall component zymosan A but not endotoxin-induced chitinase activity and chitotriosidase gene expression in vitro. The intranasal application of zymosan A into mice led to the induction of chitinase activity in the BAL fluid and to bronchial hyperresponsiveness, which could be reduced by applying the chitinase inhibitor allosamidin. Discussion: We propose that environmental exposure to mites and fungi leads to the induction of chitinase, which in turn favors the development of bronchial hyperreactivity in an IgE-independent manner.

BACKGROUND: Allergies are a serious public health issue, and prevalences are rising worldwide. The role of antibiotics in the development of allergies has repeatedly been discussed, as results remain inconsistent. The aim of this study was to investigate the association between pre- and post-natal antibiotic exposure and subsequent development of allergies (atopic dermatitis, food allergy, asthma, atopic sensitization and allergic rhinitis). METHODS: A total of 1080 children who participated in a European birth cohort study (PASTURE) were included in this analysis. Data on antibiotic exposure during pregnancy and/or first year of life and allergic diseases were collected by questionnaires from pregnancy up to 6 years of age and analysed by performing logistic regressions. To take into account reverse causation, we included models, where children with diagnosis or symptoms of the respective disease in the first year of life were excluded. RESULTS: Antibiotic exposure in utero was significantly and positively associated with atopic dermatitis and food allergy. The strongest effect was on diseases with onset within the first year of life (for atopic dermatitis: aOR 1.66, 95% CI 1.11-2.48 and for food allergy: aOR 3.01, 95% CI 1.22-7.47). Antibiotics in the first year of life were positively associated with atopic dermatitis up to 4 years (aOR 2.73, 95% CI 1.66-4.49) and also suggested a dose-response relationship. A tendency was observed with asthma between 3 and 6 years (aOR 1.65, 95% CI 0.95-2.86). CONCLUSIONS: Our findings show positive associations between exposure to antibiotics and allergies, mainly atopic dermatitis and food allergy within the first year of life, after prenatal exposure, and atopic dermatitis and asthma after post-natal exposure to antibiotics in children born in rural settings.

International audience; Background The effect of exposure to micro-organisms on allergic diseases has been well studied. The protective effect of early food diversity against allergic diseases was previously shown in the PASTURE cohort study. The consumption of cheese, a food potentially rich in microbial diversity, deserves further examination. We aimed to evaluate whether cheese consumption is associated with allergic diseases. Methods In the PASTURE study (birth cohort in 5 European countries), data on feeding practices, environmental factors and allergic diseases were collected by questionnaires from birth to 6 years (N=931). Cheese consumption at 18 months of age was quantified in terms of frequency and diversity (i.e., number of consumed types among 6 types: hard pressed, semi-pressed, soft, blue, fresh cheese, from the farm). Multiple logistic regressions were performed to evaluate the effect of cheese consumption on atopic dermatitis (AD), food allergy (FA), allergic rhinitis, asthma and atopic sensitization at 6 years after adjustment for confounders of atopy. Results Cheese consumption (vs. non-consumption) had a significant protective effect on AD (OR=0.51 [0.29-0.90], p=0.02) and FA (OR=0.32, [0.15-0.71], p=0.004), but no effect on atopic sensitization, allergic rhinitis and asthma at 6 years. This effect on AD and FA may be related to the diversity of consumed cheeses (OR=0.64 [0.48-0.85] per cheese type, p=0.002; OR=0.55 [0.33-0.92], p=0.02, respectively). Conclusions Although reverse causality cannot totally be ruled out, cheese diversity at 18 months had a protective effect against AD and FA at 6 years in addition to the protective effect of diversity of other foods.

Growing up on a farm is associated with an asthma-protective effect, but the mechanisms underlying this effect are largely unknown. In the Protection against Allergy: Study in Rural Environments (PASTURE) birth cohort, we modeled maturation using 16S rRNA sequence data of the human gut microbiome in infants from 2 to 12 months of age. The estimated microbiome age (EMA) in 12-month-old infants was associated with previous farm exposure (beta = 0.27 (0.12-0.43), P = 0.001, n = 618) and reduced risk of asthma at school age (odds ratio (OR) = 0.72 (0.56-0.93), P = 0.011). EMA mediated the protective farm effect by 19%. In a nested case-control sample (n = 138), we found inverse associations of asthma with the measured level of fecal butyrate (OR = 0.28 (0.09-0.91), P = 0.034), bacterial taxa that predict butyrate production (OR = 0.38 (0.17-0.84), P = 0.017) and the relative abundance of the gene encoding butyryl-coenzyme A (CoA):acetate-CoA-transferase, a major enzyme in butyrate metabolism (OR = 0.43 (0.19-0.97), P = 0.042). The gut microbiome may contribute to asthma protection through metabolites, supporting the concept of a gut-lung axis in humans.Growing up in the rich microbial environment of a farm strongly influences the maturation of the gut microbiome in the first year of life, which helps protect against the development of asthma in children.

Background: Childhood asthma is often preceded by early wheeze. Usually, wheezing episodes are recorded retrospectively, which may induce recall bias. Aims and objectives: The aim of this study was to investigate true-positive recall of parent-reported wheeze at 1year of age, its determinants, and its implications for asthma and lung function at 6years of age. Methods: The PASTURE (Protection Against Allergy—Study in Rural Environments) study followed 880 children from rural areas in 5 European countries from birth to age 6years. Wheeze symptoms in the first year were asked weekly. At age 6, parent-reported asthma diagnosis was ascertained and lung function measurements were conducted. Correct parental recall of wheeze episodes at the end of the first year was assessed for associations with lung function, asthma, and the asthma risk locus on chromosome 17q21. Results: Parents correctly recalled wheeze after the first year in 54% of wheezers. This true-positive recall was determined by number of episodes, timing of the last wheeze episode, and parental asthma. Independently from these determinants, true-positive recall predicted asthma at age 6years (odds ratio 4.54, 95% confidence interval (CI) [1.75-14.16]) and impaired lung function (β=−0.62, 95% CI [−1.12; −0.13], P-value=.02). Associations were stronger in children with asthma risk SNPs on chromosome 17q21. Conclusion: Correct parental recall of wheezing episodes may reflect clinical relevance of early wheeze and its impact on subsequent asthma and lung function impairment. Questions tailored to parental perception of wheezing episodes may further enhance asthma prediction.

BACKGROUND Multiple breath washout (MBW) is an attractive test to assess ventilation inhomogeneity, a marker of peripheral lung disease. Systematic research on MBW feasibility in preschool children is scanty. OBJECTIVES We assessed feasibility of nitrogen MBW in children aged 3-7 years in a clinical setting applying current ERS/ATS consensus. METHODS Sixty-two children with asthma were enrolled in the Swiss rehabilitation center Hochgebirgsklinik Davos without MBW experience and prior to consensus release. Age range was 3.1-6.7 years. All children were naive to MBW, received training, and then aimed for triplicate nitrogen MBW within 20 min. We afterward assessed the number of MBW attempts and successful tests according to consensus asking for three technically acceptable trials with functional residual capacity (FRC) measurements within 25% of median FRC. Secondly, factors related to success rate and FRC variability were assessed. RESULTS Out of 205 MBW attempts in 62 children, 103 trials (50%) in 51 children were successful. Only 15 children (24%) achieved three valid trials as suggested by consensus. At least two valid trials were obtained in 37 children (60%). Age was positively correlated with success rate. FRC variability was inversely correlated with variability of tidal volume. CONCLUSIONS MBW was only feasible in one quarter of inexperienced children

Background: Childhood asthma prevalence is significantly greater in urban areas compared with rural/farm environments. Murine studies have shown that TNF-alpha-induced protein 3 (TNFAIP3; A20), an anti-inflammatory regulator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) signaling, mediates environmentally induced asthma protection. Objective: We aimed to determine the role of TNFAIP3 for asthma development in childhood and the immunomodulatory effects of environmental factors. Methods: In a representative selection of 250 of 2168 children from 2 prospective birth cohorts and 2 cross-sectional studies, we analyzed blood cells of healthy and asthmatic children from urban and rural/farm environments from Europe and China. PBMCs were stimulated ex vivo with dust from "asthma-protective'' farms or LPS. NF-kappa B signaling-related gene and protein expression was assessed in PBMCs and multiplex gene expression assays (NanoString Technologies) in isolated dendritic cells of schoolchildren and in cord blood mononuclear cells from newborns. Results: Anti-inflammatory TNFAIP3 gene and protein expression was consistently decreased, whereas proinflammatory Toll-like receptor 4 expression was increased in urban asthmatic patients (P

Background Dietary changes are suggested to play a role in the increasing prevalence of allergic diseases and asthma. Short-chain fatty acids (SCFAs) are metabolites present in certain foods and are produced by microbes in the gut following fermentation of fibers. SCFAs have been shown to have anti-inflammatory properties in animal models. Our objective was to investigate the potential role of SCFAs in the prevention of allergy and asthma. Methods We analyzed SCFA levels by high-performance liquid chromatography (HPLC) in fecal samples from 301 one-year-old children from a birth cohort and examined their association with early life exposures, especially diet, and allergy and asthma later in life. Data on exposures and allergic diseases were collected by questionnaires. In addition, we treated mice with SCFAs to examine their effect on allergic airway inflammation. Results Significant associations between the levels of SCFAs and the infant's diet were identified. Children with the highest levels of butyrate and propionate (≥95th percentile) in feces at the age of one year had significantly less atopic sensitization and were less likely to have asthma between 3 and 6 years. Children with the highest levels of butyrate were also less likely to have a reported diagnosis of food allergy or allergic rhinitis. Oral administration of SCFAs to mice significantly reduced the severity of allergic airway inflammation. Conclusion Our results suggest that strategies to increase SCFA levels could be a new dietary preventive option for allergic diseases in children.

Asthma prevalence has increased in epidemic proportions with urbanization, but growing up on traditional farms offers protection even today

Allergies are usually referred to as type I hypersensitivity reactions against innocuous environmental antigens, characterized by a Th2/IgE-dominated inflammation. They can manifest themselves in various organs, such as skin, gastrointestinal and respiratory tract, and comprise diseases as diverse as allergic rhinitis and conjunctivitis, bronchial asthma, oral allergy syndrome, food allergy, urticaria and atopic eczema, but also anaphylactic shock. Within the last decades, there was a significant global increase in allergy prevalence, which has been mostly attributed to changes in environment and lifestyle. But which, among all factors discussed, are the most relevant, and what are the mechanisms by which these factors promote or prevent the development of allergic diseases? To answer this, it is necessary to go back to the two key questions that have occupied allergy researchers for the last decades: Firstly, what makes an allergen an allergen? Secondly, why are more and more individuals affected? Within the last decade, we have made considerable progress in answering these questions. This review gives an overview over scientific progress in the field, summarizes latest findings and points out future prospective and research needs.

RationaleAsthma is characterised by inflammation and reversible airway obstruction. However, these features are not always closely related. Fluctuations of daily lung function contain information on asthma phenotypes, exacerbation risk and response to long-acting β-agonists.ObjectivesIn search of subgroups of asthmatic participants with specific lung functional features, we developed and validated a novel clustering approach to asthma phenotyping, which exploits the information contained within the fluctuating behaviour of twice-daily lung function measurements.MethodsForced expiratory volume during the first second (FEV1) and peak expiratory flow (PEF) were prospectively measured over 4 weeks in 696 healthy and asthmatic school children (Protection Against Allergy – Study in Rural Environments (PASTURE)/EFRAIM cohort), and over 1 year in 138 asthmatic adults with mild-to-moderate or severe asthma (Pan-European Longitudinal Assessment of Clinical Course and BIOmarkers in Severe Chronic AIRway Disease (BIOAIR) cohort). Using enrichment analysis, we explored whether the method identifies clinically meaningful, distinct clusters of participants with different lung functional fluctuation patterns.Measurements and main resultsIn the PASTURE/EFRAIM dataset, we found four distinct clusters. Two clusters were enriched in children with well-known clinical characteristics of asthma. In cluster 3, children from a farming environment predominated, whereas cluster 4 mainly consisted of healthy controls. About 79% of cluster 3 carried the asthma-risk allele rs7216389 of the17q21locus. In the BIOAIR dataset, we found two distinct clusters clearly discriminating between individuals with mild-to-moderate and severe asthma.ConclusionsOur method identified dynamic functional asthma and healthy phenotypes, partly independent of atopy and inflammation but related to genetic markers on the17q21locus. The method can be used for disease phenotyping and possibly endotyping. It may identify participants with specific functional abnormalities, potentially needing a different therapeutic approach.

Background: Rehabilitational programs at moderate altitude (1500-2500m) showed improvement of lung function and reduction in airways inflammation in asthmatic adults. Allergen avoidance was postulated as the major cause of these improvements. Methods: Spirometries of 344 and fractional exhaled nitric oxide measurements (FeNO) of 124 asthmatic children and adolescents, staying in a rehabilitation hospital in Davos (1590m) with at least 14days between admission and discharge, were analyzed in association with atopic sensitization (skin-prick testing and/or specific IgE), level of asthma control, and inhalative corticosteroid (ICS) dose. Results: Pulmonary conditions improved significantly on average during the sojourn. Uncontrolled asthmatics benefited most with an absolute increase in predicted FEV 1 , MEF 25 , and MEF 75 of 7.7%, 9.9%, and 12.7%, respectively (P < .001). FeNO decreased by 36.9ppb for uncontrolled, by 26.9ppb for partly controlled, and by 11.8ppb for controlled asthmatics. In uncontrolled subjects, pulmonary improvement was comparable between patients with and without house dust mites (HDM) sensitization. Pulmonary improvements of pollen-sensitized patients were not dependent on the season of the sojourn. For the group with constant ICS level, the absolute increase in FEV 1 was 4.9% (P < .001) with a FeNO decreased by 32.7ppb (P < .001). When the ICS dose was elevated by one GINA level, the absolute increase in FEV 1 was slightly higher (6.6%, P < .001), with a FeNO decrease of 31.4ppb (P < .001). Conclusion: Inpatient rehabilitation at moderate altitude improved pulmonary conditions in asthmatic children and adolescents independent of sensitization status to HDM or pollen. A positive effect was also observed in patients without change in medication.

Background: Phenotypes of childhood-onset asthma are characterized by distinct trajectories and functional features. For atopy, definition of phenotypes during childhood is less clear. Objective: We sought to define phenotypes of atopic sensitization over the first 6 years of life using a latent class analysis (LCA) integrating 3 dimensions of atopy: allergen specificity, time course, and levels of specific IgE (sIgE). Methods: Phenotypes were defined by means of LCA in 680 children of the Multizentrische Allergiestudie (MAS) and 766 children of the Protection against allergy: Study in Rural Environments (PASTURE) birth cohorts and compared with classical nondisjunctive definitions of seasonal, perennial, and food sensitization with respect to atopic diseases and lung function. Cytokine levels were measured in the PASTURE cohort. Results: The LCA classified predominantly by type and multiplicity of sensitization (food vs inhalant), allergen combinations, and sIgE levels. Latent classes were related to atopic disease manifestations with higher sensitivity and specificity than the classical definitions. LCA detected consistently in both cohorts a distinct group of children with severe atopy characterized by high seasonal sIgE levels and a strong propensity for asthma; hay fever; eczema; and impaired lung function, also in children without an established asthma diagnosis. Severe atopy was associated with an increased IL-5/IFN-gamma ratio. A path analysis among sensitized children revealed that among all features of severe atopy, only excessive sIgE production early in life affected asthma risk. Conclusions: LCA revealed a set of benign, symptomatic, and severe atopy phenotypes. The severe phenotype emerged as a latent condition with signs of a dysbalanced immune response. It determined high asthma risk through excessive sIgE production and directly affected impaired lung function.

Recurrent upper or lower respiratory symptoms, possibly allergy-related, are very frequent in childhood. It is therefore important that physicians involved in the primary care of these children have an accurate initial diagnostic tool available. In this study, we investigated the value of an in vitro diagnostic device testing 10 common allergens, the ImmunoCAP Rapid Wheeze/Rhinitis Child, for the primary evaluation of allergy. Children with non-infectious upper or lower respiratory symptoms possibly related to allergy were recruited in the primary health care setting of private practices of physician trained in immunology/allergology. The investigators carried out their usual diagnostic work-up including IgE tests, and the ImmunoCAP Rapid test was performed with capillary whole blood in a blinded way to the investigator. The investigators' conclusions on major triggering allergens were compared to the ImmunoCAP Rapid test results. In the whole patient population (n = 185), the sensitivity of the ImmunoCAP Rapid test for unveiling allergic disease was 92% (95% CI: 86-96%) and the specificity 97% (95% CI: 86-100%). Current guidelines for allergy diagnosis suggest screening children with recurrent, moderate/severe diseases for allergies. For children with asthma falling into these categories, sensitivity was 100% (95% CI: 88-100%) and specificity 100% (95% CI: 69-100%); for children with moderate and severe rhinitis sensitivity was 93% (95% CI: 86-97%) and the specificity 100% (95% CI: 79-100%). The ImmunoCAP Rapid test is an accurate test, in particular with regard to high specificity, for diagnosing allergy in children with recurrent respiratory diseases in primary care settings.

Background Consumption of farm milk in early life is associated with less asthma and allergies. Objective We hypothesized that genetic variation in the innate immunity receptor CD14 might modify the association between farm milk consumption and asthma and atopy. Methods Questionnaire data, serum IgE levels, and genotypes for 4 single nucleotide polymorphisms in CD14 were assessed in farmers' and nonfarmers' children from 2 European populations (Allergy and Endotoxin study, n=576; Prevention of Allergy Risk factors for Sensitization in children related to Farming and Anthroposophic Lifestyle study, n=1539). In a subsample (n = 222) CD14 gene expression was measured in peripheral blood leukocytes. The effects of farm milk and CD14 genotypes on asthma, allergies, and CD14 expression and their interactions were investigated. Results We found a significant interaction between genetic variation in CD14/−1721 and farm milk consumption. Adjusted odds ratios for the association between farm milk and asthma varied between the genotypes: AA, 0.18 (95% CI, 0.07-0.47); AG, 0.47 (95% CI, 0.26-0.86); and GG, 0.98 (95% CI, 0.46-2.08). Similar patterns were observed for symptoms of allergic rhinoconjunctivitis and pollen sensitization. CD14/−1721 also modified the association between farm milk and CD14 gene expression (adjusted geometric means ratios: AA, 1.61 (95% CI, 0.98-2.66); AG, 1.11 (95% CI, 0.71-1.72); and GG, 0.76 (95% CI, 0.39-1.48). Conclusion The protective effect of farm milk consumption on allergic diseases is stronger in children carrying the A allele in CD14/−1721 than in children homozygous for the G allele. This might be mediated through farm milk–induced upregulated CD14 gene expression. Clinical implications Our results support the hypothesis that the inverse association between farm milk consumption and allergic diseases is mediated by CD14 -activated innate immune mechanisms.

Background In recent years, studies have shown a protective effect of being raised in a farm environment on the development of hay fever and atopic sensitization. Inconsistent data on the relation of farming to asthma and wheeze have raised some doubt about a true protective effect. Objective We sought to study the differential effects of farm-associated exposures on specific asthma-related health outcomes. Methods The cross-sectional Prevention of Allergy Risk Factors for Sensitization in Children Related to Farming and Anthroposophic Lifestyle study included 8263 school-age children from rural areas in 5 European countries. Information on farm-related exposures and health outcomes was obtained by using questionnaires. In subsamples allergen-specific IgE and RNA expression of CD14 and Toll-like receptor genes were measured, and dust from children's mattresses was evaluated for microbial components. Results Inverse relations with a diagnosis of asthma were found for pig keeping (odds ratio [OR], 0.57; 95% CI, 0.38-0.86), farm milk consumption (OR, 0.77; 95% CI, 0.60-0.99), frequent stay in animal sheds (OR, 0.71; 95% CI, 0.54-0.95), child's involvement in haying (OR, 0.56; 95% CI, 0.38-0.81), and use of silage (OR, 0.55; 95% CI, 0.31-0.98; for nonatopic asthma) and in Germany for agriculture (OR, 0.34; 95% CI, 0.22-0.53). Protective factors were related with higher expression levels of genes of the innate immunity. Potential risk factors for asthma and wheeze were also identified in the farm milieu. Levels of endotoxin and extracellular polysaccharides were related to the health outcomes independently of the farm exposures. Conclusions The protective effect of being raised in a farm environment was ascribed to distinct exposures. Clinical implications The development of atopic sensitization and atopic and nonatopic asthma is most likely determined by different environmental factors, possibly reflecting distinct pathomechanisms.

Summary Background Dietary interventions as a means for atopy prevention attract great interest. Some studies in rural environments claimed an inverse association between consumption of farm-produced dairy products and the prevalence of allergic diseases, but current evidence is controversial. Objective To investigate whether consumption of farm-produced products is associated with a lower prevalence of asthma and allergy when compared with shop-purchased products. Methods Cross sectional multi-centre study (PARSIFAL) including 14 893 children aged 5–13 years from five European countries (2823 from farm families and 4606 attending Steiner Schools as well as 5440 farm reference and 2024 Steiner reference children). A detailed questionnaire including a dietary component was completed and allergen-specific IgE was measured in serum. Results Farm milk consumption ever in life showed a statistically significant inverse association with asthma: covariate adjusted odds ratio (aOR) 0.74 [95% confidence interval (CI) 0.61–0.88], rhinoconjunctivitis: aOR 0.56 (0.43–0.73) and sensitization to pollen and the food mix fx5 (cut-off level of 3.5 kU/L): aOR 0.67 (0.47–0.96) and aOR 0.42 (0.19–0.92), respectively, and sensitization to horse dander: aOR 0.50 (95% CI 0.28–0.87). The associations were observed in all four subpopulations and independent of farm-related co-exposures. Other farm-produced products were not independently related to any allergy-related health outcome. Conclusion Our results indicate that consumption of farm milk may offer protection against asthma and allergy. A deepened understanding of the relevant protective components of farm milk and a better insight into the biological mechanisms underlying this association are warranted as a basis for the development of a safe product for prevention.

Growing up on a farm protects from childhood asthma and early wheeze. Virus-triggered wheeze in infancy predicts asthma in individuals with a genetic asthma risk associated with chromosome 17q21.To test environmental determinants of infections and wheeze in the first year of life, potential modifications of these associations by 17q21, and the implications for different trajectories of wheeze.We followed 983 children in rural areas of Europe from birth until age 6 years. Symptoms of wheeze, rhinitis, fever, and environmental exposures were documented with weekly diaries during year 1. Asthma at age 6 was defined as ever having a reported doctor's diagnosis. Single-nucleotide polymorphisms related to ORMDL3 (rs8076131) and GSDMB (rs7216389, rs2290400) at 17q21 were genotyped.Early wheeze was positively associated with presence of older siblings among carriers of known asthma risk alleles at 17q21 (e.g., rs8076131) (adjusted odds ratio [aOR], 1.53; 95% confidence interval [CI], 1.16-2.01). Exposure to farm animal sheds was inversely related to wheeze (aOR, 0.44; 95% CI, 0.33-0.60). Both effects were similarly observed in children with transient wheeze up to age 3 years without subsequent development of asthma (aOR, 1.71 [95% CI, 1.09-2.67]; and aOR, 0.48 [95% CI, 0.30-0.76], respectively).These findings suggest that the chromosome 17q21 locus relates to episodes of acute airway obstruction common to both transient wheeze and asthma. The previously identified asthma risk alleles are the ones susceptible to environmental influences. Thus, this gene-environment interaction reveals two faces of 17q21: The same genotype constitutes genetic risk and allows for environmental protection, thereby providing options for prospective prevention strategies.

Introduction: Clustering approaches to asthma phenotyping using clinical parameters have facilitated the identification of various asthma phenotypes. However, several challenges remain unresolved: 1) The characterizing parameters are only assessed at a single point in time, yielding only “snapshot” phenotypes. 2) Some of the biomarkers utilized in previous studies can be difficult to obtain in a clinical setting. 3) Available clinical and biomarker data are often incomplete. Objective: To develop a clustering approach to asthma phenotyping solely based on low-cost and readily obtainable biomarkers measured during a pre-established time window that systematically copes with incomplete data sets. Methods: The EFRAIM/PASTURE study is a cohort of asthmatic and healthy children. At age 6, N=604 children were subject to twice daily PEF and FEV1 measurements during 4 weeks. We compared the individual PEF and FEV1 distributions among all children using a metric assessing differences in the mean value, and in the magnitude and frequency of fluctuations around the mean. Using hierarchical clustering we identified clusters of children sharing similar distributions. We assessed the stability of the identified clusters upon random data removal. Results: Tolerable levels of missing data were found via cluster stability analysis. We clinically validated the method within the cohort by means of enrichment analysis of clinical characteristics. We found a good agreement between the most relevant features of some of the clusters identified and well-known clinical characteristics of asthma. Conclusion: Our method may be useful in the context of personalized asthma care.

Background: Growing up on a farm is inversely related to childhood asthma. The asthma risk locus 17q21 modifies the effects of smoking and early viral infections on subsequent asthma. Aims and objectives: To determine whether wheeze or farm exposure before age 1 year predict asthma at age 6 years; whether farm exposure affects wheezing during year 1; and whether these relationships are modified by the 17q21 locus. Methods: We followed 983 children in rural areas of Austria, Finland, France, Germany, and Switzerland from birth until age 6 years. Symptoms and exposures during year 1 were recorded with weekly diaries. Questionnaires were administered between pregnancy and 6 years. Wheezing during year 1 was stratified by a concomitant cold. Asthma at age 6 was defined as reported doctor9s diagnosis, or wheezy bronchitis more than once. SNPs in genes ORMDL3 and GSDMB at locus 17q21 were assessed in cord blood. Results: Asthma at age 6 was predicted by wheeze without a cold (aOR, 95%CI: 2.67, 1.47-4.85) and wheeze with a cold (2.24, 1.33-3.77). The association of wheeze with a cold was modified by SNPs at locus 17q21 (gene-environment interaction: P=0.001) . Animal shed exposure during year 1 was inversely related to asthma (0.55, 0.31-0.94). Siblings (carriers of allele A in rs8076131: 1.57, 1.20-2.06) and animal shed exposure (allele A: 0.45, 0.34-0.61) were associated with wheeze before age 1 among carriers of asthma risk alleles. Conclusions: Environmental exposures such as siblings or exposure to animal sheds affect wheezing largely in carriers of known asthma risk alleles. The 17q21 locus might be related both to environmental susceptibility and to progress from wheeze towards asthma.

Living on farms has repeatedly been shown to protect children from asthma and allergies. A major factor involved in this effect is consumption of unprocessed cow9s milk directly obtained from a farm (Loss, G. et al. JACI 2011;128:766-773). In contrast to farm milk, industrially processed cow9s milk is subjected to heat-treatment and alteration of fat fraction and fat globule size. Aim of this analysis was to assess whether the composition of fatty acids (FA) differed between unprocessed farm milk and industrially processed milk and, if so, whether this contributed to the protective effect of farm milk on asthma. The PASTURE study followed 934 children living in rural areas in 5 European countries from birth to age 6 years. During this time, their milk consumption was assessed by questionnaires. Samples of the usually consumed milk were taken at the age of 4.5 years. Doctor-diagnosed asthma was parent-reported at age 6 years. In a random sample of 35 asthma cases and 49 controls, the content of 46 FA was quantified. Proportions of omega-3 FA were highest in raw farm milk and lowest in UHT milk. The risk of asthma was reduced when consuming raw farm milk as compared to shop milk (OR=0.31 [0.14-0.69]). Part of the effect was explained by the higher fat content of farm milk (OR high vs. low fat 0.59 [0.33-1.05]). Half of this effect was attributable to the contents of omega-3 FA (CIE=56%), which had significant inverse association with asthma after adjustment for milk fat content (OR=0.2 [0.04-0.91]). The asthma-protective effect of unprocessed farm milk is partially attributable to omega-3-FA, which counterbalance the synthesis of pro-inflammatory leukotriens and prostaglandins.

BackgroundGut microbiota and intestinal inflammation regulate the development of immune-mediated diseases, such as allergies. Fecal calprotectin is a biomarker of intestinal inflammation.ObjectiveWe evaluated the association of early-age fecal calprotectin levels to the later development of allergic diseases in children from farming and non-farming environments and further studied the effect of gut microbiota on the fecal calprotectin levels.MethodsFecal calprotectin was measured from 758 infants participating in the PASTURE study at the age of 2months using the ELISA method. Serum-specific IgE levels were measured at 6years of age. Data of environmental factors, doctor-diagnosed atopic dermatitis (AD) and asthma were collected by questionnaire. Multivariate logistic regression models were used for analysis. The composition of fecal microbiota was analysed in a subgroup of 120 infants with 16S rRNA pyrosequencing. The effect of Escherichia coli lipopolysaccharide (LPS) on in vitro monocyte IL-10 secretion was studied by flow cytometry.ResultsThe infants with high fecal calprotectin levels at 2months, that is above the 90th percentile, had an increased risk of developing AD and asthma/asthmatic bronchitis by the age of 6years (aOR 2.02 (1.06-3.85) and 2.41 (1.25-4.64), respectively). High fecal calprotectin levels correlated negatively with fecal Escherichia. LPS from E.coli stimulated production of IL-10 in monocytes.Conclusion and Clinical RelevanceHigh degree intestinal inflammation at 2months of age, detected as high fecal calprotectin, predicted asthma and AD by the age of 6years and was linked to low abundance of fecal Escherichia. Impaired IL-10 activation due to the lack of colonization with E.coli could explain the intestinal inflammation associated high fecal calprotectin and later risk of asthma and AD. Our results have implications for the design of probiotic treatments and suggest that early intestinal colonization has long-term health effects.

Allergic diseases are influenced by both genes and environment. A 70-kb haplotype block in the G protein-coupled receptor for asthma susceptibility gene (GPR154; alias GPRA) on chromosome 7p was recently identified to influence susceptibility to asthma and elevated total serum IgE levels in adults.To assess the impact of GPR154 on childhood allergic disease, including allergic sensitization, asthma, and rhinoconjunctivitis, in study populations with diverse environmental backgrounds.We studied farm children, Steiner school children, and two reference groups from five Western European countries in the cross-sectional PARSIFAL (Prevention of Allergy Risk factors for Sensitization In children related to Farming and Anthroposophic Lifestyle) study and a sample of children from the Swedish birth cohort study BAMSE. DNA samples from 3,113 PARSIFAL and 800 BAMSE children were genotyped for 7 GPR154 polymorphisms and haplotypes were inferred. The proportions of alleles and haplotypes (H1-H7) were compared in affected children with their healthy counterparts.Data indicate a global association of the haplotype block to sensitization (allergen-specific serum IgEor = 0.35 kU/L, p = 0.022), with significant haplotype-specific associations for H1, H5, and H6. Haplotypes H1 and H5 were also significantly associated with childhood allergic asthma (p = 0.045 and p = 0.023, respectively), and H5 to asthma regardless of sensitization. A broader involvement of GPR154 in allergic diseases was further supported in allergic rhinoconjunctivitis (H3: p = 0.046). The associated haplotypes could be allocated into risk (H5/H6) and nonrisk (H1/H3) groups, a pattern supported by allelic association of single nucleotide polymorphisms (SNPs) rs324384 and rs324396.Our results indicate that polymorphisms and haplotypes in the haplotype block of GPR154 are associated with asthma, rhinoconjunctivitis, and sensitization in European children.

Asthma, atopic dermatitis, and atopic rhinoconjunctivitis are three distinct conditions, which are characterized by the production of specific immunoglobulin E (IgE) against common, ubiquitous allergens such as house dust mites, pollen, or animal dander. Once sensitized, re-exposure to allergen induces an inflammatory reaction of the affected organ: the bronchial system, the nasal and ocular mucosa, or the skin, respectively. At present, atopic diseases are the most common chronic illnesses in children living in the industrialized world. In general, asthma and atopy rates are higher in affluent, Western countries with a high degree of industrialization than in developing countries with a large, rural population. In a worldwide study in approximately 500,000 school-aged children from 56 countries, the highest prevalence of asthma was found in the United Kingdom, New Zealand, and Australia, and most developing countries had comparatively low prevalence rates. Within European countries, a West–East gradient in the prevalence of childhood asthma is present, which became apparent in studies shortly after the fall of the communist East. In the Baltic area of Northern Europe, Swedish children had a higher prevalence of atopic sensitization and asthma than did those from Poland and Estonia [1, 2]. Likewise, the prevalence of asthma, allergic rhinitis, and atopic sensitization was significantly higher in West Germany than in the East [3], a genetically homogeneous study population that had lived under very different economic and environmental circumstances for 40 years. In the years since unification, however, atopic sensitization as well as atopic rhinitis have increased significantly in East German children, almost to levels found in Western Germany, and levels of asthma were still low [4]. As most children in the communist system grew up in day-care centers from an early age, an intriguing explanation for these findings was that the early exposures to infections and microbial matter in day-care centers may have influenced the susceptibility to atopic diseases.

In early life, the innate immune system can recognize both viable and nonviable parts of microorganisms. Immune activation may direct the immune response, thus conferring tolerance to allergens such as animal dander or tree and grass pollen.Parents of children who were 6 to 13 years of age and were living in rural areas of Germany, Austria, or Switzerland where there were both farming and nonfarming households completed a standardized questionnaire on asthma and hay fever. Blood samples were obtained from the children and tested for atopic sensitization; peripheral-blood leukocytes were also harvested from the samples for testing. The levels of endotoxin in the bedding used by these children were examined in relation to clinical findings and to the cytokine-production profiles of peripheral-blood leukocytes that had been stimulated with lipopolysaccharide and staphylococcal enterotoxin B. Complete data were available for 812 children.Endotoxin levels in samples of dust from the child's mattress were inversely related to the occurrence of hay fever, atopic asthma, and atopic sensitization. Nonatopic wheeze was not significantly associated with the endotoxin level. Cytokine production by leukocytes (production of tumor necrosis factor alpha, interferon-gamma, interleukin-10, and interleukin-12) was inversely related to the endotoxin level in the bedding, indicating a marked down-regulation of immune responses in exposed children.A subject's environmental exposure to endotoxin may have a crucial role in the development of tolerance to ubiquitous allergens found in natural environments.

The aim of the study was to determine the differences in nitrite, in the exhaled breath condensates of healthy children and those children with asthma, cystic fibrosis (CF) and nonasthmatic, episodic cough. Breath condensates were obtained from 66 children (43 males:23 females, 3.1-16 yrs) and included 29 asthmatics, 12 clinically stable CF patients, 12 children with cough but not asthma and 13 healthy volunteers. The collected condensate was assayed colourimetrically using the Griess reaction to determine nitrite concentrations. Patients with CF (median: 5-95% percentiles; 2.02: 0.43-6.37 microM) or asthma (2.10: 0.63-5.45 microM) had significantly higher levels of nitrite compared to healthy subjects (0.41: 0.13-1.83 microM; p

International audience; BACKGROUND: European cross-sectional studies have suggested that prenatal and postnatal farm exposure decreases the risk of allergic diseases in childhood. Underlying immunologic mechanisms are still not understood but might be modulated by immune-regulatory cells early in life, such as regulatory T (Treg) cells. OBJECTIVE: We sought to assess whether Treg cells from 4.5-year-old children from the Protection against Allergy: Study in Rural Environments birth cohort study are critical in the atopy and asthma-protective effect of farm exposure and which specific exposures might be relevant. METHODS: From 1133 children, 298 children were included in this study (149 farm and 149 reference children). Detailed questionnaires until 4 years of age assessed farming exposures over time. Treg cells were characterized as upper 20% CD4(+)CD25(+) forkhead box protein 3 (FOXP3)(+) (intracellular) in PBMCs before and after stimulation (with phorbol 12-myristate 13-acetate/ionomycin or LPS), and FOXP3 demethylation was assessed. Atopic sensitization was defined by specific IgE measurements; asthma was defined by a doctor's diagnosis. RESULTS: Treg cells were significantly increased in farm-exposed children after phorbol 12-myristate 13-acetate/ionomycin and LPS stimulation. Exposure to farm milk was defined as a relevant independent farm-related exposure supported by higher FOXP3 demethylation. Treg cell (upper 20% CD4(+)CD25(+), FOXP3(+) T cells) numbers were significantly negatively associated with doctor-diagnosed asthma (LPS stimulated: adjusted odds ratio, 0.26; 95% CI, 0.08-0.88) and perennial IgE (unstimulated: adjusted odds ratio, 0.21; 95% CI, 0.08-0.59). Protection against asthma by farm milk exposure was partially mediated by Treg cells. CONCLUSIONS: Farm milk exposure was associated with increased Treg cell numbers on stimulation in 4.5-year-old children and might induce a regulatory phenotype early in life, potentially contributing to a protective effect for the development of childhood allergic diseases.

Prospective studies investigating the role of serum vitamin E concentrations during early life in the development of childhood allergies and asthma are limited.; To study the associations between serum vitamin E concentrations at first year of life and longitudinal development of atopy, atopic dermatitis, wheeze, and asthma up to 6 years of age.; The setting was the PASTURE study, a multicenter prospective birth cohort study in five European rural settings. Children of 1133 mothers recruited during pregnancy were followed from birth with measurement of serum vitamin E levels at year 1 and repeated assessments of serum immunoglobulin E antibodies (year 1, 4.5, 6), atopic dermatitis, wheezing symptoms, and asthma (year 1, 1.5, 2, 3, 4, 5, 6).; At 6 years of age, 66% and 82% of the original 1133 subjects underwent blood test for IgE and answered the questionnaire, respectively. We did not observe any statistically significant associations between serum vitamin E concentrations at year 1 and the endpoints, but borderline inverse associations between alpha tocopherol and wheezing without cold (OR 0.45, 95% CI 0.19-1.09) and any wheezing symptom (OR 0.52, 95% CI 0.27-1.02).; Serum vitamin E concentrations at year 1 were not associated with allergies or asthma by 6 years of age. While further prospective studies with repeated assessments of vitamin E during early life may clarify its putative role in the development of the diseases, it is also possible that the antioxidant hypothesis in the development of allergies and asthma does not hold.

International audience; BACKGROUND: Microbial exposure may induce low-grade inflammation at an early age and decrease the risk of allergic diseases, as suggested by the hygiene hypothesis. We examined the associations between low-grade inflammation and the development of allergic sensitization, atopic dermatitis (AD), and asthma at the age of 4.5 yr. METHODS: We studied 636 children participating in the PASTURE study in Finland, Germany, Austria, France, and Switzerland. Data of environmental factors, doctor-diagnosed AD, and asthma were collected by questionnaire. The serum high-sensitivity C-reactive protein (CRP) values were measured at the age of 1 yr, and serum-specific IgE concentrations (sIgE) at the age of one and 4.5 yr. Analyses were made by logistic regression analysis. RESULTS: The risk of allergic sensitization at the age of 4.5 yr was decreased in children who had increased CRP levels at the age of 1 yr (level in the highest vs. lowest quartile: aOR 0.48, 95% CI 0.24-0.95; p = 0.014). The risk of AD and asthma was not significantly related to CRP. CONCLUSION: The findings confirm that elevated levels of CRP at early age showed association with decreased allergic sensitization later in life. Our results suggest that poor inflammatory response could predispose for IgE sensitization.

Background: Living on a farm has repeatedly been shown to protect children from asthma and allergies. A major factor involved in this effect is consumption of unprocessed cow's milk obtained directly from a farm. However, this phenomenon has never been shown in a longitudinal design, and the responsible milk components are still unknown. Objectives: We sought to assess the asthma-protective effect of unprocessed cow's milk consumption in a birth cohort and to determine whether the differences in the fatty acid (FA) composition of unprocessed farm milk and industrially processed milk contributed to this effect. Methods: The Protection Against Allergy-Study in Rural Environments (PASTURE) study followed 1133 children living in rural areas in 5 European countries from birth to age 6 years. In 934 children milk consumption was assessed by using yearly questionnaires, and samples of the ``usually'' consumed milk and serum samples of the children were collected at age 4 years. Doctor-diagnosed asthma was parent reported at age 6 years. In a nested case-control study of 35 asthmatic and 49 nonasthmatic children, 42 FAs were quantified in milk samples. Results: The risk of asthma at 6 years of age was reduced by previous consumption of unprocessed farm milk compared with shop milk (adjusted odds ratio for consumption at 4 years, 0.26; 95% CI,0.10-0.67). Part of the effect was explained by the higher fat content of farm milk, particularly the higher levels of omega-3 polyunsaturated FAs (adjusted odds ratio, 0.29; 95% CI,0.11-0.81). Conclusion: Continuous farm milk consumption in childhood protects against asthma at school age partially by means of higher intake of omega-3 polyunsaturated FAs, which are precursors of anti-inflammatory mediators.

The dramatic increase in the incidence and severity of allergy and asthma has been proposed to be linked with an altered exposure to, and colonization by, micro-organisms, particularly early in life. However, other lifestyle factors such as diet and physical activity are also thought to be important, and it is likely that multiple environmental factors with currently unrecognized interactions contribute to the atopic state. This review will focus on the potential role of microbial metabolites in immunoregulatory functions and highlights the known molecular mechanisms, which may mediate the interactions between diet, microbiota, and protection from allergy and asthma.

Inpatient pulmonary rehabilitation programs have evolved from tuberculosis sanatoriums to modern medical centres providing standardized comprehensive care in a multidiciplinatory environment. Goals of rehabilitation programs for children and adolescents include restoration of professional activity, improvement of health condition, compliance and disease management as well as restoration of quality of life. Eligibility for an intervention is assessed by defined social and medical criteria. Comprehensive pulmonary rehabilitation programs provide a wide range of health care recourses, including diagnostic procedures, specific medical care, educational interventions and a multiprofessional team. Paediatric rehabilitation programs for chronic respiratory diseases, such as asthma or cystic fibrosis, have been shown to reduce symptoms, increase aerobic fitness and physical strength, improve pulmonary function and inflammation and enhance compliance, self-management, quality of life and psychological symptoms. Regional climatic effects have demonstrated an additional positive effect on the rehabilitation outcome. In addition, first evidence suggests an overall reduction of health care costs.

Human Th2 cytokines (interleukins 4 and 13) induce co-expression of IgE and IgG4 through sequential switching. The regulation of IgG4 responses and the role of these responses in the pathogenesis of allergy have not been characterized. We are addressing these issues by comparing and contrasting the expression of allergen-specific IgE and IgG4 in a population of European children thoroughly defined for lifestyle, environmental exposures and allergic phenotypes. The current analysis focused exclusively on children from non-farming families (n=493) in order to avoid potential effects of exposure to microbial products abundant in farming environments. We found that allergens induce Th2-mediated IgG4 and/or IgE responses in the majority of the population. Approximately two-thirds of the children had allergen-specific IgG4 but not IgE, only a minority had both IgG4 and IgE, only a few were negative for both, and virtually none had only IgE. The prevalence of asthma and hay fever was dramatically higher in children with high IgG4 and IgE compared to children who only mounted IgG4 or low IgG4 and IgE responses. These results appear to recapitulate different stages of in vivo Th2-dependent sequential switching from IgG4 to IgE. These patterns of Th2-induced antibody responses may warrant a redefinition of the notion of allergen sensitization.

Background There is increasing evidence that environmental exposures determining childhood illnesses operate early in life. Prenatal exposure to a farming environment through the mother might also play an important role. Objective We sought to investigate the role of maternal exposures to environments rich in microbial compounds for the development of atopic sensitization, asthma, and corresponding alterations in the innate immune system in offspring. Methods In the children of the cross-sectional Prevention of Allergy Risk Factors for Sensitization in Children Related to Farming and Anthroposophic Life Style study, asthma and atopy were assessed by means of standardized questionnaires (n = 8263) and serum IgE measurements (n = 2086). In a subsample (n = 322) gene expression of Toll-like receptors (TLR2 and TLR4) and CD14 was assessed. Maternal exposures were defined through questionnaire information. Results Both atopic sensitization (adjusted odds ratio, 0.58; 95% CI, 0.39-0.86) and the gene expression of receptors of innate immunity were strongly determined by maternal exposure to stables during pregnancy, whereas current exposures had much weaker or no effects. A dose-response relation was found between the extent of upregulation of these genes and the number of different farm animal species the mother had encountered in her pregnancy. Each additional farm animal species increased the expression of TLR2, TLR4, and CD14 by a factor of 1.16 (95% CI, 1.07-1.26), 1.12 (95% CI, 1.04-1.2), and 1.10 (95% CI, 1.03-1.23), respectively. Conclusion Maternal exposure to an environment rich in microbial compounds might protect against the development of atopic sensitization and lead to upregulation of receptors of the innate immune system. The underlying mechanisms potentially operating through the intrauterine milieu or epigenetic inheritance await further elucidation. Clinical implications When assessing risk factors of allergies in an infant's medical history, attention must also be paid to environmental exposures affecting the mother.

Background Pediatric respiratory allergies are increasing problem in Armenia being underestimated according to official reports. “Allergic Sensitization and Diseases in Armenian Children” study has been conducted to assess the prevailing sensitizations to inhalant allergens of Armenia using the standardized panel and method of PEP study (Pan-European standard Skin prick test study) conducted by Global Allergy and Asthma European Network (GA2LEN).

Background Exhaled nitric oxide (FeNO) is a well described marker of airway inflammation in asthma and is also known to increase after chronic exposure to inhaled allergens. It is not known whether monitoring FeNO could be useful during food challenges to detect early or subclinical reactions. Methods Forty children aged 3 to 16 years undergoing an allergen-food challenge at two centres were prospectively recruited for this study. FeNO was assessed before and repeatedly after the food-challenge. Results Data were obtained from a total of 53 challenges (16 positive, 37 negative) and were compared between the two groups. Half of the patients with a positive food challenge exhibited clinical upper respiratory symptoms. The FeNO significantly decreased in 7 of 16 patients with a positive challenge test within 60 to 90 minutes after the first symptoms of an allergic reaction. Conclusion Our results show a significant decrease in FeNO after a positive food challenge suggesting involvement of the lower airways despite absence of clinical and functional changes of lower airways. Prospective blinded studies are needed to confirm these results.