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Airway hyperresponsiveness (AHR) is a critical feature of wheezing and asthma in children, but the initiating immune mechanisms remain unconfirmed. We demonstrate that both recombinant interleukin-33 (rIL-33) and allergen [house dust mite (HDM) or Alternaria alternata] exposure from day 3 of life resulted in significantly increased pulmonary IL-13+CD4+ T cells, which were indispensable for the development of AHR. In contrast, adult mice had a predominance of pulmonary LinnegCD45+CD90+IL-13+ type 2 innate lymphoid cells (ILC2s) after administration of rIL-33. HDM exposure of neonatal IL-33 knockout (KO) mice still resulted in AHR. However, neonatal CD4creIL-13 KO mice (lacking IL-13+CD4+ T cells) exposed to allergen from day 3 of life were protected from AHR despite persistent pulmonary eosinophilia, elevated IL-33 levels, and IL-13+ ILCs. Moreover, neonatal mice were protected from AHR when inhaled Acinetobacter lwoffii (an environmental bacterial isolate found in cattle farms, which is known to protect from childhood asthma) was administered concurrent with HDM. A. lwoffii blocked the expansion of pulmonary IL-13+CD4+ T cells, whereas IL-13+ ILCs and IL-33 remained elevated. Administration of A. lwoffii mirrored the findings from the CD4creIL-13 KO mice, providing a translational approach for disease protection in early life. These data demonstrate that IL-13+CD4+ T cells, rather than IL-13+ ILCs or IL-33, are critical for inception of allergic AHR in early life.

Neuropeptide S receptor 1 (NPSR1, GPRA 154, GPRA) has been verified as a susceptibility gene for asthma and related phenotypes. The ligand for NPSR1, Neuropeptide S (NPS), activates signalling through NPSR1 and microarray analysis has identified Tenascin C (TNC) as a target gene of NPS-NPSR1 signalling. TNC has previously been implicated as a risk gene for asthma. We aimed therefore to study the genetic association of TNC in asthma- and allergy-related disorders as well as the biological and genetic interactions between NPSR1 and TNC. Regulation of TNC was investigated using NPS stimulated NPSR1 transfected cells. We genotyped 12 TNC SNPs in the cross-sectional PARSIFAL study (3113 children) and performed single SNP association, haplotype association and TNC and NPSR1 gene-gene interaction analyses. Our experimental results show NPS-dependent upregulation of TNC-mRNA. The genotyping results indicate single SNP and haplotype associations for several SNPs in TNC with the most significant association to rhinoconjunctivitis for a haplotype, with a frequency of 29% in cases (P = 0.0005). In asthma and atopic sensitization significant gene-gene interactions were found between TNC and NPSR1 SNPs, indicating that depending on the NPSR1 genotype, TNC can be associated with either an increased or a decreased risk of disease. We conclude that variations in TNC modifies, not only risk for asthma, but also for rhinoconjunctivitis. Furthermore, we show epistasis based on both a direct suggested regulatory effect and a genetic interaction between NPSR1 and TNC. These results suggest merging of previously independent pathways of importance in the development of asthma- and allergy-related traits

To the Editor: Complex diseases, including asthma, have genetic and environmental origins. Genome-wide association studies have identified multiple genes for the development of asthma, yet they only explain a limited proportion of asthma heritability. Interactions between genetic predisposition and exposure to passive smoking might explain in part the hidden heritability of childhood asthma. However, to date, this approach has not been reported for the discovery of interactions between genes and tobacco smoke exposure. We performed a genome-wide interaction study (GWIS) on childhood asthma to identify genes that interact with 2 well-known environmental risk factors for childhood-onset asthma: in utero and childhood tobacco smoke exposure. We meta-analyzed interaction results from 9 studies participating in the GABRIEL consortium1 including more than 6,000 subjects of European descent. We replicated our findings in 4 independent studies including more than 13,000 subjects. Childhood-onset asthma was defined as asthma diagnosed by a doctor before the age of 16 years, which is consistent with the definition in the GABRIEL consortium.1 In utero tobacco smoke exposure was defined as “exposure to maternal tobacco smoking at any time during pregnancy.” Childhood tobacco smoke exposure was defined as “exposure to passive tobacco smoking at any time from birth until 16 years of age.” Details on the number of subjects, the design of the individual studies, and outcome and exposure definitions are provided in Tables E1 to E4 in this article's Online Repository at www.jacionline.org. The effects of in utero tobacco smoke exposure and childhood tobacco smoke exposure were analyzed separately. All individual studies were analyzed by using a logistic regression model containing the genetic effect, the effect of tobacco smoke exposure, and an interaction term indicating the interaction between the genetic effect and tobacco smoke exposure. Further methodological considerations on GWISs and details on the statistical analyses are described in this article's Online Repository at www.jacionline.org. For in utero tobacco smoke exposure, the discovery genome-wide meta-analysis consisted of 2,654 cases and 3,073 control subjects derived from 7 studies (see Table E1). Overall, in utero tobacco smoke exposure increased the risk of childhood-onset asthma (see Fig E1 in this article's Online Repository at www.jacionline.org). A total of 536,705 single nucleotide polymorphisms (SNPs) were included in the interaction meta-analysis. Fig E2 in this article's Online Repository at www.jacionline.org shows the Manhattan plot. We identified 27 SNPs in the discovery sample with a P value of less than 10−4 based on the fixed effect model (Table I and see Table E5 in this article's Online Repository at www.jacionline.org). Findings did not reach genome-wide significance but were consistent over all studies included, and no significant heterogeneity across studies was present (P value Q-statistic < .05). Four of these SNPs on chromosome 10 were in high linkage disequilibrium with each other in the discovery meta-analysis (r2 = 0.82-0.96). The most prominent marker was located on chromosome 18 near EPB41L3 (Forest plot, see Fig E3 in this article's Online Repository at www.jacionline.org). Table E6 in this article's Online Repository at www.jacionline.org shows the associations in exposed and nonexposed subjects. EPB41L3 belongs to the protein 4.1 family of membrane-associated proteins, is involved in cell-cell junctions,2 and might play a role in apoptosis.3 The literature shows that in utero tobacco smoke exposure affects the expression of genes involved in biological processes, such as cell proliferation and apoptosis, and influences lung development of the child in general.4 Our data suggest that this effect of in utero smoke exposure might potentially occur through mechanisms involving EPB41L3 (see the additional text in this article's Online Repository). Table I Results of the GWIS of in utero tobacco smoke exposure and childhood-onset asthma For childhood tobacco smoke exposure, the discovery genome-wide meta-analysis consisted of 3,048 cases and 3,509 control subjects derived from 9 studies (see Table E1). Overall, childhood tobacco smoke exposure increased the risk of childhood-onset asthma (see Fig E1). A total of 538,233 SNPs were included in the interaction meta-analysis. Fig E4 in this article's Online Repository at www.jacionline.org shows the Manhattan plot. We identified 35 SNPs in the discovery sample with a P value of less than 10−4 based on the fixed effect model. Four of these SNPs were excluded because they showed heterogeneity, and the P value of the random effect was greater than 10−4. Findings did not reach genome-wide significance. Table II and Table E7 (see this article's Online Repository at www.jacionline.org) the results for the top SNPs. Seven SNPs on chromosome 5 (except rs2312164) were in high linkage disequilibrium with each other in the discovery studies (r2 = 0.83-1.00). Table II Results of the GWIS on childhood tobacco smoke exposure and childhood-onset asthma The most prominent marker was located on chromosome 6 in PACRG (parkin coregulated gene; Forest plot, see Fig E5 in this article's Online Repository at www.jacionline.org). Table E8 in this article's Online Repository at www.jacionline.org shows the associations in exposed and nonexposed subjects. PACRG is located next to and has an overlapping promoter region with parkin 2 (PARK2).5 The gene has been associated with leprosy and parkinsonian diseases and has an important role in motile cilia function and cilia morphogenesis.2,6 PACRG is relatively highly expressed in the trachea and nasal mucosa. Ciliary dysfunction might impair mucus clearance from the airways and has been shown to affect asthma severity. Our data suggest that changes in ciliary function particularly affect the development of asthma in children exposed to passive tobacco smoke. The genes that have been reported previously to interact with tobacco smoke exposure with respect to asthma development (ie, TNF,7 GSTP1,7 and ADAM338) were not among our most significant hits. This can be explained by the fact that the genetic variants in these candidate gene studies have a strong main effect on asthma development. Bouzigon et al9 showed a more pronounced effect of the 17q21 region on the development of early-onset asthma in children with early-life tobacco smoke exposure than in those without. The genetic effect of these markers in our GWIS showed a similar direction, but the interaction was not significant. This study on childhood asthma is the first hypothesis-free GWIS specifically aiming to identify SNPs that interact with tobacco smoke exposure in disease development. We found suggestive evidence for an interaction between rs8094633 on chromosome 18 near EPB41L3 and in utero tobacco smoke exposure and an interaction between rs1575472 on chromosome 6 in PACRG and childhood tobacco smoke exposure. The SNPs found have not been identified previously in general genome-wide association studies on childhood asthma. Interestingly, the SNPs interacting with in utero and childhood tobacco smoke exposure were different and were not involved in the same pathway (see Fig E6 in this article's Online Repository at www.jacionline.org). Interactions between these SNPs and tobacco smoke exposure in utero and in childhood might explain part of the missing heritability of asthma. Future research needs to confirm these findings and further unravel the biological pathways.

Long-term birth cohort studies are essential to understanding the life course and childhood predictors of allergy and the complex interplay between genes and the environment (including lifestyle and socioeconomic determinants). Over 100 cohorts focusing on asthma and allergy have been initiated in the world over the past 30 years. Since 2004, several research initiatives funded under the EU Framework Program for Research and Technological Development FP6-FP7 have attempted to identify, compare, and evaluate pooling data from existing European birth cohorts (GA2LEN: Global Allergy and European Network, FP6; ENRIECO: Environmental Health Risks in European Birth Cohorts, FP7; CHICOS: Developing a Child Cohort Research Strategy for Europe, FP7; MeDALL: Mechanisms of the Development of ALLergy, FP7). However, there is a general lack of knowledge about these initiatives and their potentials. The aim of this paper is to review current and past EU-funded projects in order to make a summary of their goals and achievements and to suggest future research needs of these European birth cohort networks. © 2012 S. Karger AG, Basel.

Background: Breast-feeding is protective against respiratory infections in early life. Given the co-evolutionary adaptations of humans and cattle, bovine milk might exert similar anti-infective effects in human infants. Objective: To study effects of consumption of raw and processed cow's milk on common infections in infants. Methods: The PASTURE birth cohort followed 983 infants from rural areas in Austria, Finland, France, Germany, and Switzerland, for the first year of life, covering 37,306 person-weeks. Consumption of different types of cow's milk and occurrence of rhinitis, respiratory tract infections, otitis, and fever were assessed by weekly health diaries. C-reactive protein levels were assessed using blood samples taken at 12 months. Results: When contrasted with ultra-heat treated milk, raw milk consumption was inversely associated with occurrence of rhinitis (adjusted odds ratio from longitudinal models [95% CI]: 0.71 [0.54-0.94]), respiratory tract infections (0.77 [0.59-0.99]), otitis (0.14 [0.05-0.42]), and fever (0.69 [0.47-1.01]). Boiled farm milk showed similar but weaker associations. Industrially processed pasteurized milk was inversely associated with fever. Raw farm milk consumption was inversely associated with C-reactive protein levels at 12 months (geometric means ratio [95% CI]: 0.66 [0.45-0.98]). Conclusions: Early life consumption of raw cow's milk reduced the risk of manifest respiratory infections and fever by about 30%. If the health hazards of raw milk could be overcome, the public health impact of minimally processed but pathogen-free milk might be enormous, given the high prevalence of respiratory infections in the first year of life and the associated direct and indirect costs.

Background: Numerous studies report a protective effect of farming against allergic diseases. Some specific underlying exposures contributing to this effect have recently been described in the GABRIEL survey. So far psycho social factors have not been included in these analyses. Methods: In order to assess the potential influence of health related quality of life (HRQOL) on the protective effect of farming 8259 school aged children from the European GABRIEL study answered questions concerning farming and allergic diseases as well as validated questions about HRQOL. Results: Farm children reported higher HRQOL than non farm children. However HRQOL did not modify the protective effect of farming against allergies. Children with allergic diseases reported significantly lower HRQOL scores suggesting that the higher HRQOL of farm children was in part explained by the lower prevalence of these diseases among farm children. Conclusion: Although farm children reported higher HRQOL scores than did non farm children HRQOL did not explain the protective effect of farming against allergic diseases. The relationship between allergic diseases and HRQOL is likely bidirectional and needs to be assessed prospectively.

International audience; The association between breastfeeding and wheezing, lung function and atopy was evaluated in the International Study of Asthma and Allergy in Childhood (ISAAC) Phase II. Cross-sectional studies were performed in 27 centres in 20 countries. Information on disease and exposure factors was collected by parental questionnaires. Data from 54,000 randomly selected school children (aged 8-12 yrs, 31,759 with skin prick testing) and a stratified subsample (n = 4,888) were used for testing the correlation of breastfeeding with bronchial hyperreactivity and lung function. Random effect models for meta-analysis were applied to calculate combined odds ratios (ORs). Any breastfeeding was associated with less wheeze both in affluent (adjusted OR (OR(adj)) 0.87, 95% confidence interval (CI) 0.78-0.97) and nonaffluent countries (OR(adj) 0.80, 95% CI 0.68-0.94). Further analyses revealed that this was true only for nonatopic wheeze in nonaffluent countries (OR(adj) 0.69, 95% CI 0.53-0.90). Breastfeeding was not associated with atopic wheeze and objective measures of allergy in both affluent and nonaffluent countries. In contrast, breastfeeding was associated with higher predicted forced expiratory volume in one second in affluent countries only (mean ratio 1.11, 95% CI 1.02-1.20). Breastfeeding is associated with protection against nonatopic wheeze, which becomes particularly evident in nonaffluent countries. Overall, breastfeeding was not related to any measure of allergy. These findings may explain some of the controversy regarding breastfeeding, since the direction of the association with breastfeeding depends on the predominating wheeze phenotype (e.g. atopic, nonatopic).

The number of allergens to be tested in order to identify sensitized patients is important in order to have the most cost-effective approach in epidemiological studies.To define the minimal number and the type of skin prick test (SPT) allergens required to identify a patient as sensitized using results of the new Pan-European GA(2)LEN skin prick test study.In a large Pan-European multicenter (17 centers in 14 countries) patient based study, a standardized panel of 18 allergens has been prick tested using a standardized procedure. Conditional approach allowed to determine the allergens selection.Among the 3034 patients involved, 1996 (68.2%) were sensitized to at least one allergen. Overall, eight allergens (grass pollen, Dermatophagoides pteronyssinus, birch pollen, cat dander, Artemisia, olive pollen, Blatella and Alternaria) allowed to identified more than 95% of sensitized subjects. However, differences were observed between countries, two allergens being sufficient for Switzerland (grass pollen and cat dander) as opposed to nine for France (grass pollen, Dermatophagoides pteronyssinus, olive pollen, cat dander, Blatella, cypress, dog dander, alder and [Artemisia or Alternaria]). According to country, up to 13 allergens were needed to identify all sensitized subjects.Eight to ten allergens allowed the identification of the majority of sensitized subjects. For clinical care of individual patients, the whole battery of 18 allergens is needed to appropriately assess sensitization across Europe.

Background: Childhood obesity and asthma are increasing worldwide. A possible link between the two conditions has been postulated. Methods: Cross-sectional studies of stratified random samples of 8-12-year-old children (n=10 652) (16 centres in affluent and 8 centres in non-affluent countries) used the standardized methodology of ISAAC Phase Two. Respiratory and allergic symptoms were ascertained by parental questionnaires. Tests for allergic disease were performed. Height and weight were measured, and overweight and obesity were defined according to international definitions. Prevalence rates and prevalence odds ratios were calculated. Results: Overweight (odds ratio=1.14, 95%-confidence interval: 0.98; 1.33) and obesity (odds ratio=1.67, 95%-confidence interval: 1.25; 2.21) were related to wheeze. The relationship was stronger in affluent than in non-affluent centres. Similar results were found for cough and phlegm, rhinitis and eczema but the associations were mostly driven by children with wheeze. There was a clear association of overweight and obesity with airways obstruction (change in FEV1/FVC, 20.90, 95%-confidence interval: 21.33%; 20.47%, for overweight and 22.46%, 95%-confidence interval: 23.84%; 21.07%, for obesity) whereas the results for the other objective markers, including atopy, were null. Conclusions: Our data from a large international child population confirm that there is a strong relation of body mass index with wheeze especially in affluent countries. Moreover, body mass index is associated with an objective marker of airways obstruction (FEV1/FVC) but no other objective markers of respiratory and allergic disorders. © 2014 Weinmayr et al.

Background: Within a large prospective study, the Global Asthma and Allergy European Network (GA2LEN) has collected skin prick test (SPT) data throughout Europe to make recommendations for SPT in clinical settings. Objective: To improve clinical interpretation of SPT results for inhalant allergens by providing quantitative decision points. Methods: The GA2LEN SPT study with 3068 valid data sets was used to investigate the relationship between SPT results and patient-reported clinical relevance for each of the 18 inhalant allergens as well as SPT wheal size and physician-diagnosed allergy (rhinitis, asthma, atopic dermatitis, food allergy). The effects of age, gender, and geographical area on SPT results were assessed. For each allergen, the wheal size in mm with an 80% positive predictive value (PPV) for being clinically relevant was calculated. Results: Depending on the allergen, from 40% (blatella) to 87-89% (grass, mites) of the positive SPT reactions (wheal size ≥ 3 mm) were associated with patient-reported clinical symptoms when exposed to the respective allergen. The risk of allergic symptoms increased significantly with larger wheal sizes for 17 of the 18 allergens tested. Children with positive SPT reactions had a smaller risk of sensitizations being clinically relevant compared with adults. The 80% PPV varied from 3 to 10 mm depending on the allergen. Conclusion: These 'reading keys' for 18 inhalant allergens can help interpret SPT results with respect to their clinical significance. A SPT form with the standard allergens including mm decision points for each allergen is offered for clinical use. © 2013 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.