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.

A polymorphism in the TH 2 locus control region is associated with changes in DNA methylation and gene expression.

BACKGROUND: Genomewide association and epigenetic studies found a region within the RAD50 gene on chromosome 5q31 to be associated with total serum IgE levels and asthma. In mice, this region harbors a locus control region for nearby TH 2 cytokines, which is characterized by four Rad50 DNase I hypersensitive sites (RHS4-7). Among these, RHS7 seems to have the strongest impact on TH 2 differentiation. We investigated whether within the human homolog of RHS7, functional polymorphisms exist, which could affect DNA methylation or gene expression in the 5q31 locus and might have an influence on asthma status or IgE regulation. METHODS: The human RHS7 region was fine mapped using 1000 genomes database information. In silico analysis and electrophoretic mobility shift assays were used to assess SNP function. Allele-specific effects on DNA methylation were evaluated in cord blood (n = 73) and at age of 4.5 years (n = 61) by pyrosequencing. Allele-specific effects on RAD50, IL4, and IL13 expression were analyzed in 100 subjects. Associations with asthma and IgE levels were investigated in the MAGICS/ISAAC II population (n = 1145). RESULTS: Polymorphism rs2240032 in the RHS7 region is suggestive of allele-specific transcription factor binding, affects methylation of the IL13 promoter region and influences RAD50 and IL4 expression (lowest P = 0.0027). It is also associated with total serum IgE levels (P = 0.0227). CONCLUSION: A functional relevant polymorphism in the TH 2 locus control region, equivalent to RHS7 in mice, affects DNA methylation and gene expression within 5q31 and influences total serum IgE on the population level.

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.

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.

The effect of BDNF gene variants on asthma in German children.

BACKGROUND: Allergic inflammation can trigger neuronal dysfunction and structural changes in the airways and the skin. Levels of brain-derived neurotrophic factor (BDNF) are strongly up regulated at the location of allergic inflammation. AIM: We systematically investigated whether polymorphisms in the BDNF gene influence the development or severity of asthma and atopic diseases. METHODS: The BDNF gene was screened for mutations in 80 chromosomes. Genotyping of six BDNF tagging polymorphisms was performed in a cross-sectional study population of 3099 children from Dresden and Munich (age 9-11 years, ISAAC II). Furthermore, polymorphisms were also investigated in an additional 655 asthma cases analysed with a random sample of 767 children selected from ISAAC II. Associations were calculated via chi-square test and anova using SAS Genetics and spss. RESULTS: We identified nine polymorphisms with minor allele frequency >or=0.03, one of them leading to an amino acid change from Valine to Methionine. In the cross-sectional study population, no significant association was found with asthma or any atopic disease. However, when more severe asthma cases from the MAGIC study were analysed, significant asthma effects were observed with rs6265 (odds ratio 1.37, 95% confidence interval 1.14-1.64, P = 0.001), rs11030101 (OR 0.82, 95%CI 0.70-0.95, P = 0.009) and rs11030100 (OR 1.19, 95%CI 1.00-1.42, P = 0.05). CONCLUSIONS: As in previous studies, effects of BDNF polymorphisms on asthma remain controversial. The data may suggest that BDNF polymorphisms contribute to severe forms of asthma.

Genetic variation in CRTh2 influences development of allergic phenotypes.

BACKGROUND: Allergic disorders are characterized by an increase in the Th2 cytokines IL-4, IL-5 and IL-13, produced primarily by Th2 cells. These cells are marked by the expression of CRTh2 (chemoattractant receptor-homologous molecule expressed on Th2 cells), a receptor for prostaglandin D(2). As genetic variation plays a significant role in the predisposition for allergic disorders, we investigated the influence of single nucleotide polymorphisms (SNPs) in CRTh2. METHODS: In a large study population of German children (n = 4264) from the International Study of Asthma and Allergy in Children (ISAAC II), six polymorphisms in CRTh2 were genotyped. Statistical analyses were performed using single SNP and haplotype analyses. RESULTS: Uncorrected associations among -6373G>A, +1431G>C and +1538A>G were observed with a number of allergic phenotypes (P < 0.05). After correction, association between +1431C and specific IgE to food allergens remained significant (P = 0.04). Associations of haplotype (H)3 (containing +1538G) with reduced risk for asthma and H2 (containing +1431C) with increased risk for specific IgE to food allergens also remained significant after correction for multiple testing (P = 0.004). CONCLUSIONS: Genetic variation within CRTh2 modifies the development of allergic sensitization and asthma in a population of German children.

Rare TLR2 mutations reduce TLR2 receptor function and can increase atopy risk.

BACKGROUND: Common genetic variations in toll-like receptor 2 (TLR2), an innate pathogen recognition receptor, may influence the development of atopic diseases. So far, very little is known about the role of rare TLR2 mutations in these diseases. OBJECTIVE: We investigated the functional properties of six rare amino acid changes in TLR2 (and one amino acid change in a TLR2 pseudogene) and studied their effect on atopic sensitization and disease. METHODS: We identified rare TLR2 mutations leading to amino acid changes from databases. Functional effects of TLR2 variants were analyzed by NF-kappaB-dependent luciferase reporter assay and interleukin-8 enzyme linked immunosorbent assay in vitro. The frequency of these mutations was determined in a random sample of the general population (n = 368). Association with atopic diseases were studied in a cross sectional German study population (n = 3099). RESULTS: Three out of six mutations in the TLR2 gene altered receptor activity in vitro. Out of these, only the minor allele of R753Q occurred reasonably frequent in the German population (minor allele frequency 3%). The risk to develop atopy increased by 50% in carriers of the 753Q allele (P = 0.021) and total (P = 0.040) as well as allergen specific serum IgE levels (P = 0.011) were significantly elevated. CONCLUSION: The rare but functionally relevant mutation R753Q in TLR2 may significantly affect common conditions such as atopic sensitization in the general population.

IL15 gene variants are not associated with asthma and atopy.

BACKGROUND: Interleukin 15 (IL15) promotes activation and proliferation of CD8+ T cells and enhances the differentiation into Th2 cells. A previous study described five polymorphisms in the IL15 gene to be associated with asthma in a haplotype analysis. AIM: We selected HapMap tagging single nucleotide polymorphisms (SNPs) from IL15 to systematically investigate these IL15 associations in a large population-based sample. METHODS: Genotyping of seven IL15 SNPs was performed using MALDI-TOF MS in a cross-sectional study population of 3099 children from Dresden or Munich (age 9-11 years). All children were phenotyped by standardized and validated protocols for atopic phenotypes. Effects of single SNPs and haplotypes were studied using sas 9.1.3 and haploview. Equivalence tests were performed to prove the significance of negative results. RESULTS: Neither single IL15 polymorphisms nor haplotype analyses showed associations with asthma or atopy after correction for multiple testing. CONCLUSION: These results do not confirm previous case-control studies and suggest that IL15 gene variants do not play an important role in the development for asthma or other atopic disorders.

STAT1 gene variations, IgE regulation and atopy.

BACKGROUND: Signal transducer and activator of transcription 1 (STAT1), an intracellular signal transducer and activator of transcription centrally involved in many inflammatory pathways, was recently suggested to play an important role in allergy related immune responses. AIM: Thus, we investigated the effect of polymorphisms in the STAT1 gene on the development of atopic sensitization and allergic diseases. METHODS: Haplotype tagging single nucleotide polymorphisms (SNPs) previously described in the STAT1 gene were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology in a cross-sectional study population of 3099 German children recruited and phenotyped by the International Study of Asthma and Allergy in Childhood, phase II (ISAAC II). Effects of single SNPs and haplotypes were studied using SAS/Genetics and Haploview. RESULTS: The polymorphism C39134A (rs3771300), located in a potentially cis acting regulatory element in STAT1 intron 24, was inversely related to atopy measured by skin prick test, total and specific serum IgE levels while no effect on atopic disease risk was observed. CONCLUSION: Our results indicate that STAT1 SNP C39134A may protect from atopic sensitization. Because of its location in a highly conserved noncoding sequence near a putative GATA3 binding site, this polymorphism represents an interesting target for further studies.

Polymorphisms in eosinophil pathway genes, asthma and atopy.

BACKGROUND: Eosinophilic inflammation is considered to play an important role in the development as well as in the perpetuation of asthma. As eosinophil production and survival is under genetic control we investigated whether polymorphisms in eosinophil regulation pathway genes (IL-3, IL-5, GM-CSF and their respective enhancers and receptors) may influence the development of atopic diseases. METHODS: In two large study populations of children, the German part of the International Study of Asthma and Allergy in Childhood (ISAAC II) and the German Multicentre Atopy Study (MAS), 3099 and 824 children, seven polymorphisms previously associated with the development of atopic diseases were genotyped: two in and around the GM-CSF gene (Ile117Thr and T3085G), one in IL-3 (Pro27Ser), in IL-5 (C-746T), and in the IL-5 high affinity receptor chain IL-5R (G-80A) and two in the common receptor chain CSFR2b for IL-3, IL-5, and GM-CSF (Asp312Asn and Glu249Gln). Statistical analyses were performed using chi-squared tests and variance analyses. Gene by gene interactions were evaluated in logistic regression models. RESULTS: The T allele at position -746 in the IL-5 gene was significantly protective for atopy in the ISAAC II population (P = 0.006). Furthermore, the risk for atopic asthma was decreased in carriers of the T allele (P = 0.036) and evidence for interaction with the enhancer polymorphism 3085 bp 3' of GM-CSF was detected. CONCLUSIONS: IL-5 C-746T influenced atopic outcomes and showed evidence for gene by gene interaction. No significant associations were found with all other tested polymorphisms in the eosinophil regulation pathway after correction for multiple testing.

Genetic association of the human corticotropin releasing hormone receptor 1 (CRHR1) with binge drinking and alcohol intake patterns in two independent samples.

To investigate the role of the corticotropin releasing hormone receptor 1 (CRHR1) in patterns of human alcohol drinking and its potential contribution to alcohol dependence, we analysed two independent samples: a sample of adolescents, which consisted of individuals from the 'Mannheim Study of Risk Children' (MARC), who had little previous exposure to alcohol, and a sample of alcohol-dependent adults, who met DSM-IV criteria of alcohol dependence. Following determination of allelic frequencies of 14 polymorphisms of the CRHR1 gene, two haplotype tagging (ht)SNPs discriminating between haplotypes with a frequency of > or =0.7% were identified. Both samples were genotyped and systematically examined for association with the htSNPs of CRHR1. In the adolescent sample, significant group differences between genotypes were observed in binge drinking, lifetime prevalence of alcohol intake and lifetime prevalence of drunkenness. The sample of adult alcohol-dependent patients showed association of CRHR1 with high amount of drinking. This is the first time that an association of CRHR1 with specific patterns of alcohol consumption has been reported. Our findings support results from animal models, suggesting an importance of CRHR1 in integrating gene-environment effects in alcohol use disorders.

Effects of major depression, aging and gender upon calculated diurnal free plasma cortisol concentrations: a re-evaluation study.

Depression, aging and female gender are associated with increased diurnal concentrations of total plasma cortisol. For the physical effects of hypercortisolemia, however, it is generally assumed that free rather than total plasma cortisol concentrations are of importance. Herein, we report a mathematical approach to determine free plasma cortisol concentrations on the basis of total cortisol, corticosteroid binding-globulin (CBG) and albumin plasma concentrations. This approach was used to re-evaluate two sets of data in order to estimate the effect of depression as well as the effect of aging and gender upon free plasma cortisol concentrations. Comparing male depressed patients with healthy controls, we found 24-hour free cortisol minima (MIN: 4.1 +/- 1.8 vs. 1.6 +/- 1.1 nmol/l, p < 0.0001), mean (MEAN: 25.5 +/- 6.7 vs. 10.4 +/- 2.7 nmol/l, p < 0.0001) and maximal (MAX: 85.3 +/- 23.3 vs. 45.2 +/- 15. 8 nmol/l, p < 0.0001) concentrations to be significantly increased in depressed patients. In general, the impact of depression upon total plasma cortisol were not only maintained, but stronger regarding free plasma cortisol. Also, age was associated with free plasma cortisol MIN (F1,30= 10.8, p < 0.003) and free plasma cortisol MEAN (F1,30 = 8.9, p < 0.006). All effects of age upon total plasma cortisol were generally also found in free plasma cortisol, though with less impact. No effect of gender upon any of the given free plasma cortisol outcome variables was found. Taken together, our re-evaluation clearly shows not only depression but also aging to be associated with increases in free plasma cortisol concentrations. This finding is in line with the observation that in both conditions medical problems triggered and/or maintained by glucocorticoids (e.g. osteoporosis) are frequently seen.

Influence of gallic acid esters on drug-metabolizing enzymes of rat liver.

The effect of three antioxidants, propyl, octyl and dodecyl gallate, on hepatic drug metabolism in male rats was studied in vivo and in vitro. When fed at a dietary concentration of 1% for 14 days, only dodecyl gallate increased relative liver weight. Cytochrome P-450 content was not influenced, but a slight increase in cytochrome b5 content was observed after the feeding of propyl gallate. Monooxygenase activity (benzo[a]pyrene-hydroxylase and ethoxycoumarin-deethylase activities) was not affected by propyl or octyl gallate, but a significant decrease in benzo[a]pyrene-hydroxylase activity was apparent in rats fed dodecyl gallate. Study of benzo[a]pyrene-metabolite formation in liver microsome preparations from control and propyl gallate-treated rats showed an overall decrease in metabolite production following gallate treatment, the decrease being statistically significant for the formation of the 9,10-dihydrodiol. Epoxide-hydratase activity was enhanced by a factor of 1.5 in rats fed propyl gallate; glutathione-transferase activity was unaffected. In vitro, the gallates proved to be potent inhibitors of ethoxycoumarin deethylation in liver microsomes from untreated and phenobarbital-treated rats; however, when cytochrome P-448 had been induced by pretreatment with 3-methylcholanthrene, ethoxycoumarin deethylase was less sensitive to the inhibitory action of the gallates.