An approach to the asthma-protective farm effect by geocoding: Good farms and better farms.

BACKGROUND: The highly consistent association of growing up on a farm with a reduced asthma risk has so far been attributed to direct farm exposure. In contrast, geographic determinants of the larger environment have never been assessed. In this study, the effects of proximity to farms and environmental variables in relation to the residential address on asthma and atopy were assessed. METHODS: Addresses of 2265 children of the Bavarian arm of the GABRIELA study were converted into geocodes. Proximity to the nearest cow farm was calculated, and environmental characteristics were derived from satellite data or terrestrial monitoring. Bacterial diversity in mattress dust samples was assessed in 501 children by sequencing of the 16S rRNA amplicons. Logistic regression models were used to calculate associations between outcomes and exposure variables. RESULTS: Asthma and atopy were inversely associated with the presence of a farm within a radius of maximum 100 m. The environmental variables greenness, tree cover, soil sealing, altitude, air pollution differed not only between farm and non-farm children but also between farm children with and without another farm nearby. The latter distinction revealed strong associations with characteristics of traditional farms including a broader diversity of microbial exposure, which mainly contributed to the protective effect on asthma. In non-farm children, the protective effect of a farm nearby was completely explained by consumption of farm milk. CONCLUSIONS: Clustering of farms within a neighborhood of 100 m is strongly associated with the protective effect on asthma and may represent a more traditional style of farming with broader microbial exposure.

A switch in regulatory T cells through farm exposure during immune maturation in childhood.

BACKGROUND: Farm exposure protects against development of allergies early in life. At 4.5 years, protection against asthma by farm-milk exposure was partially mediated by regulatory T cells (Tregs). The aim of this study was to investigate the critical time window of the 'asthma-protective' farm effect via Tregs during childhood immune maturation. METHODS: Tregs were assessed longitudinally at 4.5 and 6 years in 111 children (56 farm and 55 reference children) from the PASTURE/EFRAIM birth cohort (flow cytometry). Peripheral blood mononuclear cells were cultured unstimulated (U), with phorbol 12-myristate 13-acetate/ionomycin (PI) or lipopolysaccharide (LPS), and stained for Tregs (CD4+ CD25high FOXP3upper20% ). mRNA expression of Treg/Th1/Th2/Th17-associated cell markers was measured ex vivo. Suppressive capacity of Tregs on effector cells and cytokines was assessed. Detailed questionnaires assessing farm exposures and clinical phenotypes from birth until age 6 years were answered by the parents. RESULTS: Treg percentage before and after stimulation and FOXP3mRNA expression ex vivo decreased from age 4.5 to 6 years (P(U,LPS) < 0.001; P(PI) = 0.051; P(FOXP3) < 0.001). High vs low farm-milk and animal-stable exposure was associated with decreased LPS-stimulated Treg percentage at age 6 years (P(LPS) = 0.045). Elevated LPS-stimulated-Treg percentage at age 6 was associated with increased risk of asthma (aOR = 11.29, CI: 0.96-132.28, P = 0.053). Tregs from asthmatics vs nonasthmatics suppressed IFN-γ (P = 0.015) and IL-9 (P = 0.023) less efficiently. mRNA expression of Th1/Th2/Th17-associated cell markers decreased between 4.5 and 6 years (P < 0.001). CONCLUSIONS: Tregs at the age of 6 years were decreased with farm exposure and increased within asthmatics, opposite to age 4.5 years. This immunological switch defines a critical 'time window' for Treg-mediated asthma protection via environmental exposure before age 6 years.

Environmental and mucosal microbiota and their role in childhood asthma.

BACKGROUND: High microbial diversity in the environment has been associated with lower asthma risk, particularly in children exposed to farming. It remains unclear whether this effect operates through an altered microbiome of the mucosal surfaces of the airways. METHODS: DNA from mattress dust and nasal samples of 86 school age children was analyzed by 454 pyrosequencing of the 16S rRNA gene fragments. Based on operational taxonomic units (OTUs), bacterial diversity and composition were related to farm exposure and asthma status. RESULTS: Farm exposure was positively associated with bacterial diversity in mattress dust samples as determined by richness (P = 8.1 × 10-6 ) and Shannon index (P = 1.3 × 10-5 ). Despite considerable agreement of richness between mattress and nasal samples, the association of richness with farming in nasal samples was restricted to a high gradient of farm exposure, that is, exposure to cows and straw vs no exposure at all. In mattress dust, the genera Clostridium, Facklamia, an unclassified genus within the family of Ruminococcaceae, and six OTUs were positively associated with farming. Asthma was inversely associated with richness [aOR = 0.48 (0.22-1.02)] and Shannon index [aOR = 0.41 (0.21-0.83)] in mattress dust and to a lower extent in nasal samples [richness aOR 0.63 = (0.38-1.06), Shannon index aOR = 0.66 (0.39-1.12)]. CONCLUSION: The stronger inverse association of asthma with bacterial diversity in mattress dust as compared to nasal samples suggests microbial involvement beyond mere colonization of the upper airways. Whether inhalation of metabolites of environmental bacteria contributes to this phenomenon should be the focus of future research.

Soluble immunoglobulin A in breast milk is inversely associated with atopic dermatitis at early age: the PASTURE cohort study.

BACKGROUND: The role of breastfeeding for the development of atopic diseases in childhood is contradictory. This might be due to differences in the composition of breast milk and levels of antimicrobial and anti-inflammatory components. OBJECTIVE: The objective of this study was to examine whether levels of total immunoglobulin A (IgA) or transforming growth factor-ß1 (TGF-ß1) in breast milk were associated with the risk of developing atopic dermatitis (AD), atopic sensitization or asthma at early age taking breastfeeding duration into account. METHODS: The birth cohort study PASTURE conducted in Finland, France, Germany and Switzerland provided 610 breast milk samples collected 2 months after delivery in which soluble IgA (sIgA) and TGF-ß1 levels were measured by ELISA. Duration of breastfeeding was assessed using weekly food frequency diaries from month 3 to month 12. Data on environmental factors, AD and asthma were collected by questionnaires from pregnancy up to age 6. Atopic status was defined by specific IgE levels in blood collected at the ages of 4 and 6 years. Multivariate logistic regression models were used for statistical analysis. RESULTS: Soluble IgA and TGF-ß1 levels in breast milk differed between countries, and sIgA levels were associated with environmental factors related to microbial load, for example, contact to farm animals or cats during pregnancy, but not with raw milk consumption. sIgA levels were inversely associated with AD up to the of age 2 years (P-value for adjusted linear trend: 0.005), independent of breastfeeding duration. The dose of sIgA ingested in the first year of life was associated with reduced risk of AD up to the age of 2 (aOR, 95% CI: 0.74; 0.55-0.99) and 4 years (0.73; 0.55-0.96). No clear associations between sIgA and atopy or asthma up to age 6 were observed. TGF-ß1 showed no consistent association with any investigated health outcome. CONCLUSION AND CLINICAL RELEVANCE: IgA in breast milk might protect against the development of AD.

Serum vitamin E concentrations at 1 year and risk of atopy, atopic dermatitis, wheezing, and asthma in childhood: the PASTURE study.

BACKGROUND: Prospective studies investigating the role of serum vitamin E concentrations during early life in the development of childhood allergies and asthma are limited. OBJECTIVE: 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. METHODS: 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). RESULTS: 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). CONCLUSIONS: 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.

Genome-wide association study of body mass index in 23 000 individuals with and without asthma.

BACKGROUND: Both asthma and obesity are complex disorders that are influenced by environmental and genetic factors. Shared genetic factors between asthma and obesity have been proposed to partly explain epidemiological findings of co-morbidity between these conditions. OBJECTIVE: To identify genetic variants that are associated with body mass index (BMI) in asthmatic children and adults, and to evaluate if there are differences between the genetics of BMI in asthmatics and healthy individuals. METHODS: In total, 19 studies contributed with genome-wide analysis study (GWAS) data from more than 23 000 individuals with predominantly European descent, of whom 8165 are asthmatics. RESULTS: We report associations between several DENND1B variants (P = 2.2 × 10(-7) for rs4915551) on chromosome 1q31 and BMI from a meta-analysis of GWAS data using 2691 asthmatic children (screening data). The top DENND1B single nucleotide polymorphisms(SNPs) were next evaluated in seven independent replication data sets comprising 2014 asthmatics, and rs4915551 was nominally replicated (P < 0.05) in two of the seven studies and of borderline significance in one (P = 0.059). However, strong evidence of effect heterogeneity was observed and overall, the association between rs4915551 and BMI was not significant in the total replication data set, P = 0.71. Using a random effects model, BMI was overall estimated to increase by 0.30 kg/m(2) (P = 0.01 for combined screening and replication data sets, N = 4705) per additional G allele of this DENND1BSNP. FTO was confirmed as an important gene for adult and childhood BMI regardless of asthma status. CONCLUSIONS AND CLINICAL RELEVANCE: DENND1B was recently identified as an asthma susceptibility gene in a GWAS on children, and here, we find evidence that DENND1B variants may also be associated with BMI in asthmatic children. However, the association was overall not replicated in the independent data sets and the heterogeneous effect of DENND1B points to complex associations with the studied diseases that deserve further study.

Farm exposure and time trends in early childhood may influence DNA methylation in genes related to asthma and allergy.

BACKGROUND: Genetic susceptibility and environmental influences are important contributors to the development of asthma and atopic diseases. Epigenetic mechanisms may facilitate gene by environment interactions in these diseases. METHODS: We studied the rural birth cohort PASTURE (Protection against allergy: study in rural environments) to investigate (a) whether epigenetic patterns in asthma candidate genes are influenced by farm exposure in general, (b) change over the first years of life, and (c) whether these changes may contribute to the development of asthma. DNA was extracted from cord blood and whole blood collected at the age of 4.5 years in 46 samples per time point. DNA methylation in 23 regions in ten candidate genes (ORMDL1, ORMDL2, ORMDL3, CHI3L1, RAD50, IL13, IL4, STAT6, FOXP3, and RUNX3) was assessed by pyrosequencing, and differences between strata were analyzed by nonparametric Wilcoxon-Mann-Whitney tests. RESULTS: In cord blood, regions in ORMDL1 and STAT6 were hypomethylated in DNA from farmers' as compared to nonfarmers' children, while regions in RAD50 and IL13 were hypermethylated (lowest P-value (STAT6) = 0.001). Changes in methylation over time occurred in 15 gene regions (lowest P-value (IL13) = 1.57*10(-8)). Interestingly, these differences clustered in the genes highly associated with asthma (ORMDL family) and IgE regulation (RAD50, IL13, and IL4), but not in the T-regulatory genes (FOXP3, RUNX3). CONCLUSIONS: In this first pilot study, DNA methylation patterns change significantly in early childhood in specific asthma- and allergy-related genes in peripheral blood cells, and early exposure to farm environment seems to influence methylation patterns in distinct genes.

Asthma and allergies: is the farming environment (still) protective in Poland? The GABRIEL Advanced Studies.

BACKGROUND: Evidence exists that a farming environment in childhood may provide protection against atopic respiratory disease. In the GABRIEL project based in Poland and Alpine regions of Germany, Austria and Switzerland, we aimed to assess whether a farming environment in childhood is protective against allergic diseases in Poland and whether specific exposures explain any protective effect. METHODS: In rural Poland, 23 331 families of schoolchildren completed a questionnaire enquiring into farming practices and allergic diseases (Phase I). A subsample (n = 2586) participated in Phase II involving a more detailed questionnaire on specific farm exposures with objective measures of atopy. RESULTS: Farming differed between Poland and the Alpine centres; in the latter, cattle farming was prevalent, whereas in Poland 18% of village farms kept ≥1 cow and 34% kept ≥1 pig. Polish children in villages had lower prevalences of asthma and hay fever than children from towns, and in the Phase II population, farm children had a reduced risk of atopy measured by IgE (aOR = 0.72, 95% CI 0.57, 0.91) and skin prick test (aOR = 0.65, 95% CI 0.50, 0.86). Early-life contact with grain was inversely related to the risk of atopy measured by IgE (aOR = 0.66, 95% CI 0.47, 0.92) and appeared to explain part of the farming effect. CONCLUSION: While farming in Poland differed from that in the Alpine areas as did the exposure-response associations, we found in communities engaged in small-scale, mixed farming, there was a protective farming effect against objective measures of atopy potentially related to contact with grain or associated farm activities.

Exposure to microbial agents in house dust and wheezing, atopic dermatitis and atopic sensitization in early childhood: a birth cohort study in rural areas.

BACKGROUND: Early-life exposure to environmental microbial agents may be associated with development of wheezing and allergic diseases. OBJECTIVE: To assess the association of microbial exposure in rural homes with the risk of asthma, wheezing, atopic dermatitis and sensitization. METHODS: Birth cohorts of rural children (n = 1133), half from farmer families, were followed up from birth to 2 years of age by questionnaires in five European centres. Endotoxin and extracellular polysaccharides (EPS) of Penicillium and Aspergillus spp. were determined from living room floor and mother's mattress dust samples collected at 2 months of age. Specific IgE against 19 allergens was measured at 1 year of age. Discrete-time hazard models, generalized estimations equations (GEE) and logistic regression were used for statistical analyses. RESULTS: The incidence of asthma was inversely associated with the amount of dust (adjusted odds ratio (aOR) 0.73, 95% CI 0.58-0.93) and the loads (units/m(2)) of EPS (aOR 0.75, 95% CI 0.55-1.04) and endotoxin (aOR 0.79, 95% CI 0.60-1.05) in the mother's mattress. Similar associations were seen with wheezing and with living room floor dust. The microbial markers were highly correlated and their effects could not be clearly separated. The inverse associations were seen especially among non-farmers. The risk of sensitization to inhalant allergens increased with increasing endotoxin exposure from mattress dust. No associations were observed with concentrations (units/g) or with atopic dermatitis. CONCLUSION AND CLINICAL RELEVANCE: The amount and microbial content of house dust were inversely associated with asthma and wheezing, but due to high correlations between microbial agents and amount of dust, it was not possible to disentangle their individual effects. New ways to better measure and represent exposure to environmental microbes, including indexes of biodiversity, are needed especially among farmers.

Short communication: appropriate and alternative methods to determine viable bacterial counts in cow milk samples.

Farm milk consumption is reported to be inversely related to the development of asthma and atopy in children and it has been hypothesized that microorganisms in milk might contribute to this protective effect. The GABRIEL study was designed to investigate this hypothesis in a large population of European children, calling for a rapid alternative to classical culture techniques to determine bacteriological properties of milk samples. One objective was to evaluate 2 different rapid methods to determine bacteriological properties in a large number of cow milk samples collected under field conditions. BactoScan (Foss Analytical, Hillerød, Denmark), an automated standard flow cytometric method utilized for routine testing of milk quality, and TEMPO (bioMérieux, Marcy l'Etoile, France), an automated most-probable-number method, were used to assess the total viable bacterial count in farm and commercial milk samples. Both methods were compared with standard plate count method and each other. Measurements based on the TEMPO method were in good agreement with the standard plate count method and showed reliable results, whereas BactoScan results did not correlate with standard plate count measurements and yielded higher bacteria counts in heat-treated milk samples compared with raw milk samples. Most likely, these discrepant results were due to inferences with staining reactions and detection of bacteria in heat-treated milk samples. We conclude that, in contrast to the routinely used BactoScan method, the TEMPO method is an inexpensive and rapid alternative to standard culture methods suitable to assess total bacterial counts in processed and raw milk samples.

Can farm milk consumption prevent allergic diseases?

Cow's milk is an important part of human diet and a source of food allergy for some individuals. Medical guidance strongly discourages consumption of raw milk because of the known health risk associated with pathogenic bacteria present in unpasteurized milk. Despite these risks there is a growing body of epidemiological evidence suggesting that consumption of unprocessed cow's milk does not increase but rather decreases the risk of asthma, hay fever and atopic sensitisation. The article reviews the epidemiological literature and discusses components of unprocessed milk potentially responsible for this protection. It focuses on the role of bacteria in raw milk, the fatty acid profile, whey proteins and finally the role of allergens in milk. Although the epidemiological evidence consistently suggest a protective role of unprocessed cow's milk consumption on the development of asthma, hay fever and atopic sensitization the underlying mechanisms are not yet understood and the consumption of raw milk cannot be recommended as a preventive measure for allergic diseases.

Exposure to moulds and actinomycetes in Alpine farms: a nested environmental study of the PASTURE cohort.

Several studies have suggested that children exposed to a farm environment are protected against allergies and asthma. The present work is an environmental study nested within the PASTURE cohort and includes 97 farmers and 74 non-farmers in three regions of the Alpine Arc (Switzerland, France and Germany). The objectives were to determine and compare the fungi and actinomycetes present in farming and non-farming environments (children's bedrooms and cowsheds), and to identify the agricultural practices associated with an increase in airborne fungi and actinomycetes in cowsheds. Air samples were collected by air pump and were analysed by culture and by direct counting of spores on membranes. During their stay in bedrooms, children living on farms were exposed to significantly greater amounts of Absidia spp., Eurotium spp., Cladosporium spp., Penicillium spp. and mesophilic actinomycetes than children who did not live on farms. Depending on the season, the levels of moulds, yeasts and actinomycetes were from 14 to 82 times higher in cowsheds before feeding the cattle than in children's bedrooms, and from 12 to 464 times higher in cowsheds after feeding than in children's bedrooms. Feeding cattle in cowsheds was associated with a significant peak in airborne moulds and actinomycetes, and this peak was higher in winter than in summer. Silage distribution was associated with low amounts of moulds and actinomycetes. Other significant agricultural factors were the type of cowshed, cowshed volume, method of food distribution to cattle and use of fresh grass. An assessment of the microbiological diversity on farms and in children's rooms may help to determine the factors protecting children from asthma and atopic diseases.

Maternal vitamin D intake during pregnancy increases gene expression of ILT3 and ILT4 in cord blood.

BACKGROUND: Recent studies indicate that prenatal vitamin D intake may protect against the development of atopic diseases in young children. Vitamin D has been shown to induce tolerogenic antigen-presenting cells such as dendritic cells. Whether the allergy-protective potential of prenatal vitamin D is mediated through such mechanisms is, however, unknown. OBJECTIVE: To evaluate the association between prenatal vitamin D supplementation and tolerogenic antigen-presenting cells in cord blood (CB) as determined by mRNA measurement of immunoglobulin-like transcripts (ILT)3 and ILT4. METHODS: A prospective multi-centre birth cohort was established in rural areas of five European countries. Information on maternal exposures including vitamin D intake was collected by questionnaires during pregnancy. The gene expression of ILT3 and ILT4 was analysed by real-time PCR in the CB of 927 children. Maternal vitamin D supplementation was assessed in Finland and France (n=349). RESULTS: Maternal vitamin D supplementation during pregnancy was associated with an increase in the gene expression of ILT3 (P=0.012) and ILT4 (P<0.001). This association remained significant for ILT4 (P=0.020) and showed a positive trend for the gene expression of ILT3 (P=0.059) after multivariate analysis controlling for various confounders. CONCLUSIONS: Vitamin D supplementation during pregnancy may increase the mRNA levels of ILT3 and ILT4 in CB. This finding may point towards an early induction of tolerogenic immune responses by maternal vitamin D intake.

Evaluation of a physical activity promotion program in primary care.

BACKGROUND: Physical inactivity increases the risk of many chronic disorders. It is not clear which strategies are the most appropriate to enable people to adopt a more active lifestyle. Randomized controlled trials have found that brief advice from GPs supported by written material had a significant positive effect on patient's physical activity. The pilot project 'Move for Health and the Environment' translated this evidence into a program suitable for the real-life situation of busy practices. The aim of this study was to evaluate the change in physical activity level of the participating patients 1 year after the intervention. METHODS: Patients aged 16-65 years completed a screening questionnaire before consultation with their physician. Insufficiently active patients were offered an information leaflet and a voucher for a physical activity counselling session. One year later, all inactive patients and a random selection of the active were re-contacted and invited to answer identical questions. RESULTS: A total of 1239 (73.9%) returned the follow-up questionnaire. In all, 37.3% of the formerly inactive patients met the threshold of sufficient activity at follow-up, whereas 20.3% of the previously active no longer did. Formerly inactive patients reported an increase of 58.8 minutes/week of moderate and 34.6 minutes/week of vigorous activity and spending more time walking and cycling. Formerly active patients reported less time spent in moderate activities. CONCLUSIONS: Systematic counselling in primary care encouraged insufficiently active patients to adopt a more active lifestyle. Yet it became evident that active patients also need counselling to maintain their activity levels.

High levels of grass pollen inside European dairy farms: a role for the allergy-protective effects of environment?

BACKGROUND: There is evidence of an allergy protective effect in children raised on farm. It has been assumed that microbial exposure may confer this protection. However in farm, little attention has been given to the pollen level and to concomitant microbiological exposure, and indoor pollen concentrations have never been precisely quantified. METHODS: The kinetics of pollen in dairy farms have been studied in a pilot study (n = 9), and exposure in a sub-sample of the ongoing European birth cohort PASTURE (n = 106). Measurements of viable microorganisms and pollen were performed in air samples. To identify factors that modulate the pollen concentration multivariate regression analyses were run. RESULTS: Indoor pollen (95% of Poaceae fragments and grains) were significantly higher in winter than in summer (P = 0.001) and ranged between 858 to 11 265 counts/m(3) during feeding in winter, thus exceeding typical outdoor levels during the pollen season. Geometric mean in French farms was significantly higher than in German and Swiss farms (7 534, 992 and 1 079 count/m(3), respectively). The presence of a ventilation system and loose housing systems significantly reduced indoor pollen levels. This pollen concentration rise after feeding was accompanied by an increase in fungal and actinomycetal levels, whereas the concentration of bacteria was not associated with feeding. CONCLUSIONS: Farmers and their children who attend cowsheds during the feeding sessions are exposed perennially to high pollen concentrations. It might be speculated that the combined permanent exposure to microbes from livestock and grass pollen may initiate tolerance in children living on a farm.

Weight-bearing bones are more sensitive to physical exercise in boys than in girls during pre- and early puberty: a cross-sectional study.

UNLABELLED: We carried out a cross-section study of the sex-specific relationship between bone mineral content and physical activity at sites with different loading in pre- and early pubertal girls and boys. There was significant sensitivity of bone mineral content of the hip to physical exercise in boys, but not in girls. BACKGROUND: Since little is known whether there are sex differences in sensitivity of bone to loading, we investigated sex differences in the cross-sectional association between measures of physical activity (PA) and bone mass and size in pre- and early pubertal children of both sexes. METHODS: We measured bone mineral content/density (BMC/BMD) and fat-free mass (FFM) in 269 6- to 13-year-old children from randomly selected schools by dual-energy X-ray absorptiometry. Physical activity (PA) was measured by accelerometers and lower extremity strength by a jump-and-reach test. RESULTS: Boys (n = 128) had higher hip and total body BMC and BMD, higher FFM, higher muscle strength and were more physically active than girls (n = 141). Total hip BMC was positively associated with time spent in total and vigorous PA in boys (r = 0.20-0.33, p < 0.01), but not in girls (r = 0.02-0.04, p = ns), even after adjusting for FFM and strength. While boys and girls in the lowest tertile of vigorous PA (22 min/day) did not differ in hip BMC (15.62 vs 15.52 g), boys in the highest tertile (72 min/day) had significantly higher values than the corresponding girls (16.84 vs 15.71 g, p < 0.05). CONCLUSIONS: Sex differences in BMC during pre- and early puberty may be related to a different sensitivity of bone to physical loading, irrespective of muscle mass.

Gene expression measurements in the context of epidemiological studies.

BACKGROUND: Gene expression measurements became an attractive tool to assess biological responses in epidemiological studies. However, collection of blood samples poses various technical problems. We used gene expression data from two epidemiological studies to evaluate differences between sampling methods, comparability of two methods for measuring RNA levels and stability of RNA samples over time. METHODS: For the PARSIFAL study, PBLC of 1155 children were collected using EDTA tubes in two countries. In the PASTURE study, tubes containing RNA-stabilizing solutions (PAXgene) Blood RNA Tubes; PreAnalytiX) were used to collect cord blood leucocytes of 982 children in five countries. Real-time PCR (conventional single tube assay and high-throughput low density arrays) was used to quantify expression of various innate immunity genes. In 77 PARSIFAL samples, gene expression was measured repeatedly during prolonged storage. RESULTS: In PARSIFAL (EDTA tubes) the median RNA yield after extraction significantly differed between the two centres (70 and 34 ng/microl). Collecting blood into an RNA-stabilizing solution markedly reduced differences in RNA yield in PASTURE (range of medians 91-107 ng/microl). The agreement [Spearman rank correlation (r)] between repeated measurements of gene expression decreased with increasing storage time [e.g., for CD14: r (first/second measurement) = 0.35; r (first/third measurement) = 0.03]. RNA levels measured with either the conventional method or low-density arrays were comparable (r > 0.9). CONCLUSION: Collecting blood samples into tubes containing an RNA-stabilizing solution increases RNA yield and reduces its variability. Long-term storage of samples may lead to RNA degradation, requiring special attention in longitudinal studies.

Specific IgE to allergens in cord blood is associated with maternal immunity to Toxoplasma gondii and rubella virus.

BACKGROUND: Various studies have found reduced prevalences of atopic sensitization and atopic diseases in children previously exposed to infections or living conditions with a high microbial burden, such as the farming environment. OBJECTIVE: We sought to determine the relationships of cord blood immunoglobulin E (IgE) with maternal health conditions before and during pregnancy. METHODS: Pregnant women living in rural areas in five European countries were recruited in the third trimester of pregnancy. Information on maternal health during pregnancy was collected from maternity records and by questionnaires (n = 497). Specific IgE for inhalant and food allergens was assessed in cord blood and peripheral blood samples of the mothers. RESULTS: Inverse associations of cord blood IgE to seasonal allergens with positive maternal records for Toxoplasma gondii (adjusted odds ratio = 0.37 [0.17-0.81]) and rubella virus (adjusted odds ratio = 0.35 [0.13-0.96]) were found. The previously described effect of prenatal farm exposure on IgE to seasonal allergens was partly confounded by a positive maternal record for T. gondii. The number of maternal siblings, maternal contact to cats during pregnancy or during her first year of life, predicted a positive maternal record for T. gondii. CONCLUSIONS: Maternal immunity to T. gondii and rubella may impact on atopic sensitization in the fetus. A positive T. gondii record explained the previously identified effect of prenatal farm exposure on IgE to seasonal allergens only to a minor extent.