Longitudinal analyses of development of the immune system during the first five years of life in relation to lifestyle.

BACKGROUND: Changes in immune cell composition during the immunological window within the first years after birth are not fully understood, especially the effect that different lifestyles might have on immune cell functionality. METHODS: Peripheral blood mononuclear cells from mothers and their children at birth and at two anvd five years were analyzed by mass cytometry. Immune cell composition and functionality was analyzed according to family lifestyle (anthroposophic and non-anthroposophic). RESULTS: We found no significant differences in the proportions of major immune lineages between anthroposophic and non-anthroposophic children at each time point, but there were clear changes over time in the proportions of mononuclear leukocytes, especially in B-cells and T lymphocytes. Phenotypic distances between cord blood and maternal blood were high at birth but decreased sharply the first two years, indicating strong phenotypic convergence with maternal cells. We found that children exhibited similar stimulation responses at birth, but subsequently segregated into two discrete functional trajectories. Trajectory 1 was associated with a decrease in tumor necrosis factor alpha (TNFa) production by CD4+ T- and NK-cells, while Trajectory 2 depicted an increase in the production of IL-2 and interferon gamma (INFg) by T-cells. In both trajectories, there was an increase in IL-17A production by T-cells resulting in prominent differences at five years of age. CONCLUSIONS: This exploratory study suggests that leukocyte frequencies and cell phenotypes change with age in the same way across all children, while functional development follows one of two discrete trajectories that largely segregate by family lifestyle, supporting the hypothesis that early environmental exposures imprint immune cell function which may contribute to IgE sensitization. Our results also support that the first two years are critical for the environmental exposures to imprint the immune cells. Further studies with larger sample sizes are required to validate our findings.

Prevalence and Progression of Recurrent Abdominal Pain, From Early Childhood to Adolescence.

BACKGROUND & AIMS: Little is known about the natural history of childhood recurrent abdominal pain (RAP). We investigated the prevalence and progression of childhood RAP and its association with Rome III abdominal pain-related functional gastrointestinal disorders (AP-FGID) and irritable bowel syndrome (IBS) during adolescence. METHODS: We collected data from a prospective population-based birth cohort study of 4089 children, born from 1994 through 1996 in Sweden. We analyzed data from 2455 children with complete follow-up evaluation at ages 1, 2, 12, and 16 years and no parent-reported diagnoses of inflammatory bowel diseases or celiac disease at ages 12 or 16 years. A subpopulation of 2374 children who had answered questions based on the Rome III criteria at age 16 years was identified. We assessed RAP at 3 assessment points and defined it as parent-reported attacks of colic in early childhood (1-2 years) and as self-reported weekly abdominal pain at ages 12 years and 16 years. AP-FGID at age 16 years was defined according to the Rome III criteria. RESULTS: RAP was reported by 26.2% of children on at least 1 of 3 assessment points, of which 11.3% reported symptoms more than once. Children with RAP at 12 years had persistent symptoms at 16 years in 44.9% of cases and increased risks for RAP (relative risk, 2.2; 95% CI, 1.7-2.8), any AP-FGID (relative risk, 2.6; 95% CI, 1.9-3.6), and IBS (relative risk, 3.2; 95% CI, 2.0-5.1) at 16 years. Early childhood RAP was not associated significantly with any outcome. CONCLUSIONS: RAP affects many children from early childhood through age 16 years, but most children do not have persistent symptoms throughout childhood. RAP at age 12 years is a risk factor for RAP, any Rome III AP-FGID, and IBS, at age 16 years.

DNA Methylation Levels in Mononuclear Leukocytes from the Mother and Her Child Are Associated with IgE Sensitization to Allergens in Early Life.

DNA methylation changes may predispose becoming IgE-sensitized to allergens. We analyzed whether DNA methylation in peripheral blood mononuclear cells (PBMC) is associated with IgE sensitization at 5 years of age (5Y). DNA methylation was measured in 288 PBMC samples from 74 mother/child pairs from the birth cohort ALADDIN (Assessment of Lifestyle and Allergic Disease During INfancy) using the HumanMethylation450BeadChip (Illumina). PBMCs were obtained from the mothers during pregnancy and from their children in cord blood, at 2 years and 5Y. DNA methylation levels at each time point were compared between children with and without IgE sensitization to allergens at 5Y. For replication, CpG sites associated with IgE sensitization in ALADDIN were evaluated in whole blood DNA of 256 children, 4 years old, from the BAMSE (Swedish abbreviation for Children, Allergy, Milieu, Stockholm, Epidemiology) cohort. We found 34 differentially methylated regions (DMRs) associated with IgE sensitization to airborne allergens and 38 DMRs associated with sensitization to food allergens in children at 5Y (Sidak p ≤ 0.05). Genes associated with airborne sensitization were enriched in the pathway of endocytosis, while genes associated with food sensitization were enriched in focal adhesion, the bacterial invasion of epithelial cells, and leukocyte migration. Furthermore, 25 DMRs in maternal PBMCs were associated with IgE sensitization to airborne allergens in their children at 5Y, which were functionally annotated to the mTOR (mammalian Target of Rapamycin) signaling pathway. This study supports that DNA methylation is associated with IgE sensitization early in life and revealed new candidate genes for atopy. Moreover, our study provides evidence that maternal DNA methylation levels are associated with IgE sensitization in the child supporting early in utero effects on atopy predisposition.

Maternal allergen-specific IgG might protect the child against allergic sensitization.

BACKGROUND: Analysis of allergen-specific IgE responses in birth cohorts with microarrayed allergens has provided detailed information regarding the evolution of specific IgE responses in children. High-resolution data regarding early development of allergen-specific IgG are needed. OBJECTIVE: We sought to analyze IgG reactivity to microarrayed allergens in mothers during pregnancy, in cord blood samples, in breast milk, and in infants in the first years of life with the aim to investigate whether maternal allergen-specific IgG can protect against IgE sensitization in the offspring. METHODS: Plasma samples from mothers during the third trimester, cord blood, breast milk collected 2 months after delivery, and plasma samples from children at 6, 12, and 60 months of age were analyzed for IgG reactivity to 164 microarrayed allergens (ImmunoCAP ISAC technology) in 99 families of the Swedish birth cohort Assessment of Lifestyle and Allergic Disease During Infancy (ALADDIN). IgE sensitizations to microarrayed allergens were determined at 5 years of age in the children. RESULTS: Allergen-specific IgG reactivity profiles in mothers, cord blood, and breast milk were highly correlated. Maternal allergen-specific IgG persisted in some children at 6 months. Children's allergen-specific IgG production occurred at 6 months and reflected allergen exposure. Children who were IgE sensitized against an allergen at 5 years of age had significantly higher allergen-specific IgG levels than nonsensitized children. For all 164 tested allergens, children from mothers with increased (>30 ISAC standardized units) specific plasma IgG levels against an allergen had no IgE sensitizations against that allergen at 5 years of age. CONCLUSION: This is the first detailed analysis of the molecular IgG recognition profile in mothers and their children in early life. High allergen-specific IgG reactivity in the mother's plasma and breast milk and in cord blood seemed to protect against allergic sensitization at 5 years of age.

Relevance of low specific IgE levels to egg, milk and peanut in infancy.

BACKGROUND: IgE sensitization is usually associated with allergy-related diseases, but may also occur in asymptomatic individuals. The clinical importance of IgE antibody concentrations in the interval 0.1-0.34 kU/L in early life in relation to allergy development is poorly evaluated. OBJECTIVE: To assess the relevance of low specific IgE (s-IgE) to hen's egg, cow's milk and peanut at 6 months of age for development of sensitization and allergy-related disease during early childhood. METHODS: s-IgE concentrations to relevant allergens from blood samples taken at 6 months and 1, 2 and 5 years from children in the prospective ALADDIN cohort were divided into three categories: non-sensitized (<0.1 kU/L), low sensitized (0.1-0.34 kU/L) and sensitized (≥0.35 kU/L) and allergy-related disease assessed. RESULTS: A total of 372 children were included in this study. Compared with non-sensitized children at 6 months of age, children with low levels of allergen specific IgE (0.1-0.34 kU/L) to food allergens, especially to egg, at 6 months of age were associated with development of sensitization to aeroallergens at 5 years of age (10/14 [71%] vs 39/250 [15%]). In addition, children with low levels to egg or milk at 6 months were more often sensitized to the respective allergen at 1 year of age and, regarding low levels to egg, also to the development of eczema (6/18 [33%] vs 29/292 [10%]). CONCLUSION & CLINICAL RELEVANCE: IgE antibody concentrations in the interval 0.1-0.34 kU/L to food allergens in infancy seem to increase the probability of sensitization to aeroallergens and, regarding low levels to egg, also of eczema during early childhood. Thus, IgE levels during the first year of life, although below 0.35 kU/L, can provide additional allergy-related prognostic information.

Sublingual immunotherapy alters salivary IgA and systemic immune mediators in timothy allergic children.

BACKGROUND: Immunomodulatory effects of sublingual immunotherapy on systemic and mucosal mediators in allergic children are largely unexplored. The aim of this study was to investigate allergy-related cytokine and chemokine levels, as well as IgA-responses upon a 3-year treatment with timothy grass pollen sublingual immunotherapy in children with allergic rhinoconjunctivitis. METHODS: From children included in the GRAZAX® Asthma Prevention study, blood and saliva samples were analyzed at inclusion, after 3 years of treatment, and 2 years after treatment ending. By means of Luminex and ELISA methodologies, allergy-related cytokines and chemokines were measured in plasma samples and allergen-stimulated peripheral blood mononuclear cell supernatants. Furthermore, studies of total, secretory, and Phl p 1-specific salivary IgA antibodies were performed using the same methods. RESULTS: GRAZAX® -treated children exhibited significantly higher levels of Phl p 1-specific salivary IgA and serum IgG4 , along with significantly lower skin prick test positivity, after 3 years of treatment and 2 years after treatment cessation. Additionally, plasma levels of the Th1-associated chemokines CXCL10 and CXCL11 were significantly higher in treated than untreated children at these time points. Timothy-induced ratios of IL-5/IL-13 over IFN-γ were significantly decreased after 3 years with active treatment, as were symptoms of allergic rhinitis in terms of both severity and visual analogue scale scores. However, no consistent correlations were found between the clinical outcomes and immunologic parameters. CONCLUSION: Phleum pratense sublingual immunotherapy in grass pollen allergic children modulates the immune response in the oral mucosa as well as systemically-by increasing Th1-responses, decreasing Th2-responses, and inducing immunoregulatory responses-all signs of tolerance induction.

Placental inflammation, lifestyle, maternal and early child sensitisation to allergens - the assessment of lifestyle and allergic disease during infancy birth cohort.

AIM: To investigate (i) whether maternal sensitisation to allergens, and lifestyle can influence the risk of acute and chronic inflammation of the placenta, in the forms of chorioamnionitis and villitis, respectively, and (ii) whether these placental inflammations are associated with the outcome of sensitisation for the child during preschool age. METHODS: Placentas from term uncomplicated pregnancies (n = 275) in the assessment of lifestyle and allergic disease during infancy study were analysed for the presence of acute chorioamnionitis and chronic villitis. Stepwise logistic regression was performed to estimate the relative risk of placental inflammation in relation to maternal allergic sensitisation and lifestyle, and the association between placental inflammation and sensitisation of the child up to five years of age. RESULTS: Parity and delivery at home were independently associated with chorioamnionitis, home delivery only with the low grade. Maternal allergic sensitisation was associated with increased risk of villitis in the bivariable model, however, not in the multivariable model. No significant associations were detected between placental inflammation and the outcome of sensitisation to allergens at five years of age. CONCLUSION: Our data do not support the hypothesis that the increased risk for sensitisation of a child when the mother is allergic is mediated via placental inflammation.

Histone Acetylation of Immune Regulatory Genes in Human Placenta in Association with Maternal Intake of Olive Oil and Fish Consumption.

Maternal diet modifies epigenetic programming in offspring, a potentially critical factor in the immune dysregulation of modern societies. We previously found that prenatal fish oil supplementation affects neonatal T-cell histone acetylation of genes implicated in adaptive immunity including PRKCZ, IL13, and TBX21. In this study, we measured H3 and H4 histone acetylation levels by chromatin immunoprecipitation in 173 term placentas collected in the prospective birth cohort, ALADDIN, in which information on lifestyle and diet is thoroughly recorded. In anthroposophic families, regular olive oil usage during pregnancy was associated with increased H3 acetylation at FOXP3 (p = 0.004), IL10RA (p = 0.008), and IL7R (p = 0.007) promoters, which remained significant after adjustment by offspring gender. Furthermore, maternal fish consumption was associated with increased H4 acetylation at the CD14 gene in placentas of female offspring (p = 0.009). In conclusion, prenatal olive oil intake can affect placental histone acetylation in immune regulatory genes, confirming previously observed pro-acetylation effects of olive oil polyphenols. The association with fish consumption may implicate ω-3 polyunsaturated fatty acids present in fish oil. Altered histone acetylation in placentas from mothers who regularly include fish or olive oil in their diets could influence immune priming in the newborn.

Morning Salivary Cortisol in Young Children: Reference Values and the Effects of Age, Sex, and Acute Bronchiolitis.

OBJECTIVE: To identify morning salivary cortisol reference values in infancy and at 2 years of age and to investigate the influence of age, sex and acute bronchiolitis. STUDY DESIGN: In this South-East Norwegian cohort study, 308 children hospitalized with moderate to severe acute bronchiolitis in infancy in 2010-2011 were compared with 223 healthy controls included in 2012 by measuring morning salivary cortisol levels at inclusion and at 2 years of age. Samples were collected shortly after awakening after 6 am. The influences of age, sex, and acute bronchiolitis were assessed by regression analysis. RESULTS: In infancy, cortisol values were higher in acute bronchiolitis, with an age- and sex-adjusted weighted mean group difference of 13.9 nmol/L (95% CI 8.1-19.7; P < .0001). The median level in reference group was 23.7 nmol/L (95% CI 9.7-119.6). At 2 years of age, sex but not inclusion groups differed, with significantly higher values in girls. The weighted mean of all boys' cortisol levels was 32.4 nmol/L, (95% CI 30.5-34.3), and all girls' levels were 36.9 nmol/L (95% CI 34.7-39.2; P < .003). CONCLUSIONS: Salivary cortisol levels were higher at 2 years of age than in infancy in the reference group, were higher in girls than in boys at 2 years of age, and were higher in infants at the time of acute bronchiolitis than in healthy infants. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00817466.

Vaccination and allergy: EAACI position paper, practical aspects.

Immunization is highly effective in preventing infectious diseases and therefore an indispensable public health measure. Allergic patients deserve access to the same publicly recommended immunizations as non-allergic patients unless risks associated with vaccination outweigh the gains. Whereas the number of reported possible allergic reactions to vaccines is high, confirmed vaccine-triggered allergic reactions are rare. Anaphylaxis following vaccination is rare, affecting <1/100 000, but can occur in any patient. Some patient groups, notably those with a previous allergic reaction to a vaccine or its components, are at heightened risk of allergic reaction and require special precautions. Allergic reactions, however, may occur in patients without known risk factors and cannot be predicted by currently available tools. Unwarranted fear and uncertainty can result in incomplete vaccination coverage for children and adults with or without allergy. In addition to concerns about an allergic reaction to the vaccine itself, there is fear that routine childhood immunization may promote the development of allergic sensitization and disease. Thus, although there is no evidence that routine childhood immunization increases the risk of allergy development, such risks need to be discussed.

Irritable bowel syndrome and functional abdominal pain in five-year-old children are related to lifestyle.

AIM: Abdominal pain of functional origin is very common in childhood, and environmental factors are thought to be of aetiologic importance. The anthroposophic lifestyle has dietary and lifestyle characteristics that may influence child health, and this study aimed to assess the effect of such lifestyles on abdominal pain of functional origin. METHODS: A prospective Swedish lifestyle cohort (n = 470) was followed from birth to five years of age. Family lifestyles were characterised through questionnaires. Abdominal pain was defined as irritable bowel syndrome or functional abdominal pain according to the Rome III criteria and measured with parental questionnaires and interviews at the age of five. RESULTS: The prevalence of abdominal pain was 15%. Children were more likely to have abdominal pain at five years of age if their family had a partly anthroposophic lifestyle, with an adjusted odds ratio (OR) of 2.61 (95% CI 1.15-5.93), or an anthroposophic lifestyle, with an adjusted OR of 2.34 (95% CI 0.96-5.70). CONCLUSION: A family lifestyle with anthroposophic characteristics was associated with an increased risk of abdominal pain in five-year-old children. The mechanisms for this increase were unclear, but we speculate that there may have been different prerequisites for coping with stressors.

Anthroposophic lifestyle and salivary cortisol are associated with a lower risk of sensitization during childhood.

BACKGROUND: Infants from anthroposophic families have low cortisol levels and low risk of IgE-sensitization during first 2 years of life. Our aim was to study the impact of an anthroposophic lifestyle and cortisol levels at 6 months on allergy sensitization up to age 5 years. METHODS: A total of 507 families participated from maternal healthcare centers. Parental lifestyle was categorized as anthroposophic, partly anthroposophic, or non-anthroposophic. Blood samples for analyzes of sensitization were obtained from parents at inclusion and from children at 6, 12, 24, and 60 months. Salivary samples were collected at home at 6 months. RESULTS: Sensitization increased from 2.9% to 26.0% in the anthroposophic group, from 8.4% to 26.8% in the partly anthroposophic group, and from 19.1% to 44.1% in the non-anthroposophic group. Children from anthroposophic families had lower cortisol levels in the morning, afternoon, and evening. The odds ratio (OR) for anthroposophic lifestyle was always <1 and lowest at 12 months (OR, 0.10; 95% CI, 0.03-0.36). Adjusting for cortisol levels at 6 months increased these ORs at 12 and 24 months. At the same ages, ORs for sensitization were elevated also for cortisol levels at 6 months. Analyzes in children not sensitized at 6 months confirmed the cortisol-related risk of sensitization. CONCLUSIONS: Children from families with an anthroposophic lifestyle have lower risk than comparisons of developing sensitization up to 5 years. This risk is partially explained by low cortisol levels during infancy. High cortisol levels at 6 months predict sensitization up to 24 months.

Anthroposophic lifestyle influences the concentration of metals in placenta and cord blood.

Allergic diseases develop in genetically susceptible individuals in a complex interplay with the environment, usually early in life. We have previously shown that the anthroposophic lifestyle is associated with reduced risk of allergic disease in children, but details on the influencing environmental factors are largely unknown. This study aims to elucidate if anthroposophic lifestyle influences fetal exposure to selected toxic and essential elements. Randomly selected non-smoking mothers with (n=40) and without (n=40) anthroposophic lifestyle from the prospective birth cohort ALADDIN were included. Concentrations of 12 toxic and essential elements were analyzed in full term placentas and in the erythrocyte fractions of maternal peripheral blood and of umbilical cord blood, using inductively coupled plasma mass spectrometry. Cadmium concentrations in maternal blood and placenta were significantly higher in mothers with an anthroposophic lifestyle (p<0.001), while concentrations in cord blood were generally low, irrespective of lifestyle. Cobalt concentrations were higher in both maternal blood, placenta and cord blood in the anthroposophic group. Lead concentrations were higher in maternal blood and cord blood, but not placenta, of mothers with anthroposophic lifestyle. Analysis of covariance, including lifestyle, parity, maternal age, gestational age, vegetarian diet, use of herbal medicine and occupation in the model, showed that mainly the anthroposophic lifestyle was significantly associated with cadmium concentrations. In conclusion, women with an anthroposophic lifestyle had higher concentrations of cadmium, cobalt and lead concentrations. Cadmium concentrations might have been influenced by a diet rich in vegetables and/or low iron status of the mothers.

Herpesvirus infections and allergic sensitization in children of families with anthroposophic and non-anthroposophic lifestyle - the ALADDIN birth cohort.

BACKGROUND: Growing up in families with an anthroposophic lifestyle has been associated with reduced risk of allergic disease in children. The aim of this report was to assess whether children with this lifestyle are infected earlier with Epstein-Barr virus (EBV), which has been associated with reduced risk of allergic disease, and three other herpesviruses potentially involved in allergy development, namely Human herpesvirus 6 (HHV6), Human herpesvirus 7 (HHV7) and cytomegalovirus (CMV). METHODS: Within the ALADDIN (Assessment of Lifestyle and Allergic Disease During Infancy), birth cohort study 157 children were categorized according to lifestyle into anthroposophic and non-anthroposophic. IgG-levels for EBV, HHV6, HHV7 and CMV were determined in plasma samples collected at ages 12 and 24 months and from parents. IgE levels against seven common allergens were analyzed at 24 months. RESULTS: No significant differences in seroprevalence of EBV, HHV7 or CMV were detected at any age between the two lifestyle groups. The seroprevalence of HHV6 was significantly lower in the anthroposophic group at 24 months of age (74.6% vs. 87.5%, p-value 0.048). Further, no significant associations between allergic sensitization and seropositivity to any of the viruses were detected; however, an interaction effect of lifestyle could not be ruled out. CONCLUSIONS: Our results indicate that there is no strong influence of exposure to the anthroposophic lifestyle on the time for infection with EBV, HHV6, HHV7 or CMV. These infections can therefore not be assumed to be important factors in the allergy-protective effect of this lifestyle.

Parental Sense of Coherence in the first 2 years of life is not related to parental and child diurnal cortisol rhythm or proxies of anthroposophic lifestyle.

AIM: Sense of Coherence (SOC) is hypothesized to have direct physiological consequences on endocrine and immunological processes. In this study, we compare parental SOC scores from pregnancy in groups of infants and parents representing different lifestyles (anthroposophic, partly anthroposophic and nonanthroposophic). We also analyse whether these could predict cortisol levels of the parents and their infants at 6-24 months postpartum. METHODS: Parental SOC-13 was collected during the third trimester of pregnancy from a birth cohort of families with different lifestyles. Salivary samples were collected from the whole family when the child was 6 months (n = 210), 12 (n = 178) and 24 months of age (n = 149), and cortisol levels were analysed with radioimmunoassay technique. RESULTS: Sense of Coherence scores did not differ between the three lifestyle groups, and there were no correlations between SOC scores and salivary cortisol concentrations in separate analyses of mothers, fathers and children at any sampling age or at any sampling time during the day (morning, afternoon, bedtime). CONCLUSION: Sense of Coherence scores did not vary in parents with different lifestyles and were not associated with salivary cortisol levels in parents or in children.

DNA methylation levels within the CD14 promoter region are lower in placentas of mothers living on a farm.

BACKGROUND: Epigenetic regulation has been suggested to be a link between environmental intrauterine exposures and development of asthma and allergy. The placenta is an essential part of the intrauterine environment. We have previously found the innate immune receptor CD14 to be differentially expressed on the mRNA level in placentas in relation to lifestyle and parental allergen sensitization. We here hypothesized that the promoter region of CD14 may be subject to differential DNA methylation and therefore a link between intrauterine exposure and mRNA expression. METHODS: Ninety-four placentas from the ALADDIN (Assessment of Lifestyle and Allergic Disease During Infancy) study were investigated. We used methylation-sensitive high-resolution melting (MS-HRM) analysis to semi-quantitatively analyze the DNA methylation of the promoter region of CD14 in 36 placentas known to have different CD14 mRNA expression. EpiTYPER was used to validate the MS-HRM data and to analyze an additional 58 placentas selected on mothers living on a farm or not. RESULTS: MS-HRM analysis on 36 placenta samples revealed a relation between methylation of the CD14 promoter region with the level of CD14 mRNA expression. The MS-HRM and EpiTYPER data correlated highly significantly. EpiTYPER analysis of the additional 58 placentas demonstrated that DNA methylation in the CD14 promoter was significantly lower in placentas of mothers living on a farm compared with mothers not living on a farm. CONCLUSION: Our data suggest that epigenetic regulation of CD14 in placenta might be involved in the protective effect of 'living on a farm', with regard to allergy development.

Lifestyle and salivary cortisol at the age of 12 and 24 months.

AIM: To analyse salivary cortisol levels in 12- and 24-month-olds from families with an anthroposophic lifestyle and comparisons ('partly anthroposophic' and 'non-anthroposophic'). METHODS: Salivary samples were collected at child ages of 12 (n = 178) and 24 (n = 149) months. Cortisol was analysed with radioimmunoassay technique. RESULTS: Evening cortisol levels in children from anthroposophic families were lower than in comparisons at 12 months of age (geometric means: anthroposophic 1.7, partly anthroposophic 1.9, non-anthroposophic 3.6 nmol/L; p = 0.024) and at 24 months of age (1.1, 1.8 and 2.9 nmol/L, respectively; p = 0.002). At 24 months of age, similar differences were noted also for the afternoon levels (2.3, 3.3 and 3.9 nmol/L, respectively; p = 0.043). At age 12 months, the differences in the evening cortisol were statistically explained by a meat-free diet and at age 24 months by the anthroposophic lifestyle as such. The circadian variations were parallel in the three groups at age 12 and 24 months. No cortisol differences were observed between parents representing different lifestyles. CONCLUSIONS: An anthroposophic lifestyle is associated with low cortisol levels in the evening at age 12 and 24 months, at age 24 months also in the afternoon.