Differences in autophagy marker levels at birth in preterm vs. term infants.

BACKGROUND: Preterm infants are susceptible to oxidative stress and prone to respiratory diseases. Autophagy is an important defense mechanism against oxidative-stress-induced cell damage and involved in lung development and respiratory morbidity. We hypothesized that autophagy marker levels differ between preterm and term infants. METHODS: In the prospective Basel-Bern Infant Lung Development (BILD) birth cohort we compared cord blood levels of macroautophagy (Beclin-1, LC3B), selective autophagy (p62) and regulation of autophagy (SIRT1) in 64 preterm and 453 term infants. RESULTS: Beclin-1 and LC3B did not differ between preterm and term infants. However, p62 was higher (0.37, 95% confidence interval (CI) 0.05;0.69 in log2-transformed level, p = 0.025, padj = 0.050) and SIRT1 lower in preterm infants (-0.55, 95% CI -0.78;-0.31 in log2-transformed level, padj < 0.001). Furthermore, p62 decreased (padj-value for smoothing function was 0.018) and SIRT1 increased (0.10, 95% CI 0.07;0.13 in log2-transformed level, padj < 0.001) with increasing gestational age. CONCLUSION: Our findings suggest differential levels of key autophagy markers between preterm and term infants. This adds to the knowledge of the sparsely studied field of autophagy mechanisms in preterm infants and might be linked to impaired oxidative stress response, preterm birth, impaired lung development and higher susceptibility to respiratory morbidity in preterm infants. IMPACT: To the best of our knowledge, this is the first study to investigate autophagy marker levels between human preterm and term infants in a large population-based sample in cord blood plasma This study demonstrates differential levels of key autophagy markers in preterm compared to term infants and an association with gestational age This may be linked to impaired oxidative stress response or developmental aspects and provide bases for future studies investigating the association with respiratory morbidity.

Increased Impact of Air Pollution on Lung Function in Preterm versus Term Infants: The BILD Study.

Rationale: Infants born prematurely have impaired capacity to deal with oxidative stress shortly after birth. Objectives: We hypothesize that the relative impact of exposure to air pollution on lung function is higher in preterm than in term infants. Methods: In the prospective BILD (Basel-Bern Infant Lung Development) birth cohort of 254 preterm and 517 term infants, we investigated associations of particulate matter ⩽10 µm in aerodynamic diameter (PM10) and nitrogen dioxide with lung function at 44 weeks' postconceptional age and exhaled markers of inflammation and oxidative stress response (fractional exhaled nitric oxide [FeNO]) in an explorative hypothesis-driven study design. Multilevel mixed-effects models were used and adjusted for known confounders. Measurements and Main Results: Significant associations of PM10 during the second trimester of pregnancy with lung function and FeNO were found in term and preterm infants. Importantly, we observed stronger positive associations in preterm infants (born 32-36 wk), with an increase of 184.9 (95% confidence interval [CI], 79.1-290.7) ml/min [Formula: see text]e per 10-µg/m3 increase in PM10, than in term infants (75.3; 95% CI, 19.7-130.8 ml/min) (pprematurity × PM10 interaction = 0.04, after multiple comparison adjustment padj = 0.09). Associations of PM10 and FeNO differed between moderate to late preterm (3.4; 95% CI, -0.1 to 6.8 ppb) and term (-0.3; 95% CI, -1.5 to 0.9 ppb) infants, and the interaction with prematurity was significant (pprematurity × PM10 interaction = 0.006, padj = 0.036). Conclusions: Preterm infants showed significantly higher susceptibility even to low to moderate prenatal air pollution exposure than term infants, leading to increased impairment of postnatal lung function. FeNO results further elucidate differences in inflammatory/oxidative stress response when comparing preterm infants with term infants.

Pollen exposure is associated with risk of respiratory symptoms during the first year of life.

BACKGROUND: Pollen exposure is associated with respiratory symptoms in children and adults. However, the association of pollen exposure with respiratory symptoms during infancy, a particularly vulnerable period, remains unclear. We examined whether pollen exposure is associated with respiratory symptoms in infants and whether maternal atopy, infant's sex or air pollution modifies this association. METHODS: We investigated 14,874 observations from 401 healthy infants of a prospective birth cohort. The association between pollen exposure and respiratory symptoms, assessed in weekly telephone interviews, was evaluated using generalized additive mixed models (GAMMs). Effect modification by maternal atopy, infant's sex, and air pollution (NO2 , PM2.5 ) was assessed with interaction terms. RESULTS: Per infant, 37 ± 2 (mean ± SD) respiratory symptom scores were assessed during the analysis period (January through September). Pollen exposure was associated with increased respiratory symptoms during the daytime (RR [95% CI] per 10% pollen/m3 : combined 1.006 [1.002, 1.009]; tree 1.005 [1.002, 1.008]; grass 1.009 [1.000, 1.23]) and nighttime (combined 1.003 [0.999, 1.007]; tree 1.003 [0.999, 1.007]; grass 1.014 [1.004, 1.024]). While there was no effect modification by maternal atopy and infant's sex, a complex crossover interaction between combined pollen and PM2.5 was found (p-value 0.003). CONCLUSION: Even as early as during the first year of life, pollen exposure was associated with an increased risk of respiratory symptoms, independent of maternal atopy and infant's sex. Because infancy is a particularly vulnerable period for lung development, the identified adverse effect of pollen exposure may be relevant for the evolvement of chronic childhood asthma.

Maternal asthma is associated with reduced lung function in male infants in a combined analysis of the BLT and BILD cohorts.

RATIONALE: Asthma in pregnancy is associated with respiratory diseases in the offspring. OBJECTIVE: To investigate if maternal asthma is associated with lung function in early life. METHODS: Data on lung function measured at 5-6 weeks of age were combined from two large birth cohorts: the Bern Infant Lung Development (BILD) and the Australian Breathing for Life Trial (BLT) birth cohorts conducted at three study sites (Bern, Switzerland; Newcastle and Sydney, Australia). The main outcome variable was time to reach peak tidal expiratory flow as a percentage of total expiratory time(tPTEF:tE%). Bayesian linear hierarchical regression analyses controlling for study site as random effect were performed to estimate the effect of maternal asthma on the main outcome, adjusting for sex, birth order, breast feeding, weight gain and gestational age. In separate adjusted Bayesian models an interaction between maternal asthma and sex was investigated by including an interaction term. MEASUREMENTS AND MAIN RESULTS: All 406 BLT infants were born to mothers with asthma in pregnancy, while 193 of the 213 (91%) BILD infants were born to mothers without asthma. A significant interaction between maternal asthma and male sex was negatively associated with tPTEF:tE% (intercept 37.5; estimate: -3.5; 95% credible interval -6.8 to -0.1). Comparing the model posterior probabilities provided decisive evidence in favour of an interaction between maternal asthma and male sex (Bayes factor 33.5). CONCLUSIONS: Maternal asthma is associated with lower lung function in male babies, which may have lifelong implications on their lung function trajectories and future risk of wheezing and asthma.

Associations of air pollution and greenness with the nasal microbiota of healthy infants: A longitudinal study.

BACKGROUND: Air pollution and greenness are associated with short- and long-term respiratory health in children but the underlying mechanisms are only scarcely investigated. The nasal microbiota during the first year of life has been shown to be associated with respiratory tract infections and asthma development. Thus, an interplay between greenness, air pollution and the early nasal microbiota may contribute to short- and long-term respiratory health. We aimed to examine associations between fine particulate matter (PM2.5), nitrogen dioxide (NO2) and greenness with the nasal microbiota of healthy infants during the first year of life in a European context with low-to-moderate air pollution levels. METHODS: Microbiota characterization was performed using 16 S rRNA pyrosequencing of 846 nasal swabs collected fortnightly from 47 healthy infants of the prospective Basel-Bern Infant Lung Development (BILD) cohort. We investigated the association of satellite-based greenness and an 8-day-average exposure to air pollution (PM2.5, NO2) with the nasal microbiota during the first year of life. Exposures were individually estimated with novel spatial-temporal models incorporating satellite data. Generalized additive mixed models adjusted for known confounders and considering the autoregressive correlation structure of the data were used for analysis. RESULTS: Mean (SD) PM2.5 level was 17.1 (3.8 µg/m3) and mean (SD) NO2 level was 19.7 (7.9 µg/m3). Increased PM2.5 and increased NO2 were associated with reduced within-subject Ruzicka dissimilarity (PM2.5: per 1 µg/m3 -0.004, 95% CI -0.008, -0.001; NO2: per 1 µg/m3 -0.004, 95% CI -0.007, -0.001). Whole microbial community comparison with nonmetric multidimensional scaling revealed distinct microbiota profiles for different PM2.5 exposure levels. Increased NO2 was additionally associated with reduced abundance of Corynebacteriaceae (per 1 µg/m3: -0.027, 95% CI -0.053, -0.001). No associations were found between greenness and the nasal microbiota. CONCLUSION: Air pollution was associated with Ruzicka dissimilarity and relative abundance of Corynebacteriaceae. This suggests that even low-to-moderate exposure to air pollution may impact the nasal microbiota during the first year of life. Our results will be useful for future studies assessing the clinical relevance of air-pollution-induced alterations of the nasal microbiota with subsequent respiratory disease development.

6q12 and 11p14 variants are associated with postnatal exhaled nitric oxide levels and respiratory symptoms.

BACKGROUND: Exhaled nitric oxide (eNO) is a biomarker of airway inflammation and seems to precede respiratory symptoms, such as asthma, in childhood. Identifying genetic determinants of postnatal eNO levels might aid in unraveling the role of eNO in epithelial function or airway inflammation and disease. OBJECTIVE: We sought to identify genetic determinants of early postnatal eNO levels and subsequent respiratory symptoms during the first year of life. METHODS: Within a population-based birth cohort, eNO levels were measured in healthy term infants aged 5 weeks during quiet tidal breathing in unsedated sleep. We assessed associations of single nucleotide polymorphisms with eNO levels in a genome-wide association study and subsequent symptoms of lower respiratory tract infections during the first year of life and asked whether this was modified by prenatal and early-life environmental factors. RESULTS: We identified thus far unknown determinants of infant eNO levels: rs208515 (P = 3.3 × 10-8), which is located at 6q12, probably acting in "trans" and explaining 10.3% of eNO level variance, and rs1441519 (P = 1.6 × 10-6), which is located at 11p14, potentially affecting nitric oxide synthase 3 (NOS3) expression, as shown by means of in vitro functional analyses. Moreover, the 6q12 locus was inversely associated with subsequent respiratory symptoms (P < .05) and time to recovery after first respiratory symptoms during the first year of life (P < .05). CONCLUSION: The identification of novel genetic determinants of infant eNO levels might implicate that postnatal eNO metabolism in healthy infants before first viral infections and sensitization is related to mechanisms other than those associated with asthma, atopy, or increased risk thereof later in life.

Effects of Breastfeeding on Respiratory Symptoms in Infancy.

OBJECTIVE: To assess the impact of potential risk factors on the development of respiratory symptoms and their specific modification by breastfeeding in infants in the first year of life. STUDY DESIGN: We prospectively studied 436 healthy term infants from the Bern-Basel Infant Lung Development cohort. The breastfeeding status, and incidence and severity of respiratory symptoms (score) were assessed weekly by telephone interview during the first year of life. Risk factors (eg, pre- and postnatal smoking exposure, mode of delivery, gestational age, maternal atopy, and number of older siblings) were obtained using standardized questionnaires. Weekly measurements of particulate matter <10 µg were provided by local monitoring stations. The associations were investigated using generalized additive mixed model with quasi Poisson distribution. RESULTS: Breastfeeding reduced the incidence and severity of the respiratory symptom score mainly in the first 27 weeks of life (risk ratio 0.70; 95% CI 0.55-0.88). We found a protective effect of breastfeeding in girls but not in boys. During the first 27 weeks of life, breastfeeding attenuated the effects of maternal smoking during pregnancy, gestational age, and cesarean delivery on respiratory symptoms. There was no evidence for an interaction between breastfeeding and maternal atopy, number of older siblings, child care attendance, or particulate matter <10 µg. CONCLUSIONS: This study shows the risk-specific effect of breastfeeding on respiratory symptoms in early life using the comprehensive time-series approach.

CHI3L1 polymorphisms, cord blood YKL-40 levels and later asthma development.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in chitinase 3-like 1 (CHI3L1), the gene encoding YKL-40, and increased serum YKL-40 levels are associated with severe forms of asthma. It has never been addressed whether SNPs in CHI3L1 and cord blood YKL-40 levels could already serve as potential biomarkers for milder forms of asthma. We assessed in an unselected population whether SNPs in CHI3L1 and cord blood YKL-40 levels at birth are associated with respiratory symptoms, lung function changes, asthma, and atopy. METHODS: In a prospective birth cohort of healthy term-born neonates (n = 260), we studied CHI3L1 polymorphisms, and measured cord blood YKL-40 levels by ELISA in (n = 170) infants. Lung function was performed at 5 weeks and 6 years. Respiratory health during the first year of life was assessed weekly by telephone interviews. Diagnosis of asthma and allergic sensitisation was assessed at 6 years (n = 142). RESULTS: The SNP rs10399805 was significantly associated with asthma at 6 years. The odds ratio for asthma was 4.5 (95 % CI 1.59-12.94) per T-allele. This finding was unchanged when adjusting for cord blood YKL-40 levels. There was no significant association for cord blood YKL-40 levels and asthma. SNPs in CHI3L1 and cord blood YKL-40 were not associated with lung function measurements at 5 weeks and 6 years, respiratory symptoms in the first year, and allergic sensitisation at 6 years. CONCLUSION: Genetic variation in CHI3L1 might be related to the development of milder forms of asthma. Larger studies are warranted to establish the role of YKL-40 in that pathway.

Optimized algorithm for speed-of-sound-based infant sulfur hexafluoride multiple-breath washout measurements.

INTRODUCTION: Major methodological issues with the existing algorithm (WBreath) used for the analysis of speed-of-sound-based infant sulfur hexafluoride (SF6) multiple-breath washout (MBW) measurements lead to implausible results and complicate the comparison between different age groups and centers. METHODS: We developed OASIS-a novel algorithm to analyze speed-of-sound-based infant SF6 MBW measurements. This algorithm uses known context of the measurements to replace the dependence of WBreath on model input parameters. We validated the functional residual capacity (FRC) measurement accuracy of this new algorithm in vitro, and investigated its use in existing infant MBW data sets from different infant cohorts from Switzerland and South Africa. RESULTS: In vitro, OASIS managed to outperform WBreath at FRC measurement accuracy, lowering mean (SD) absolute error from 5.1 (3.2) % to 2.1 (1.6) % across volumes relevant for the infant age range, in variable temperature, respiratory rate, tidal volume and ventilation inhomogeneity conditions. We showed that changes in the input parameters to WBreath had a major impact on MBW results, a methodological drawback which does not exist in the new algorithm. OASIS produced more plausible results than WBreath in longitudinal tracking of lung clearance index (LCI), provided improved measurement stability in LCI over time, and improved comparability between centers. DISCUSSION: This new algorithm represents a meaningful advance in obtaining results from a legacy system of lung function measurement by allowing a single method to analyze measurements from different age groups and centers.

TOLLIP and MUC5B modulate the effect of ambient NO2 on respiratory symptoms in infancy.

BACKGROUND: Current knowledge suggests that the gene region containing MUC5B and TOLLIP plays a role in airway defence and airway inflammation, and hence respiratory disease. It is also known that exposure to air pollution increases susceptibility to respiratory disease. We aimed to study whether the effect of air pollutants on the immune response and respiratory symptoms in infants may be modified by polymorphisms in MUC5B and TOLLIP genes. METHODS: 359 healthy term infants from the prospective Basel-Bern Infant Lung Development (BILD) birth cohort were included in the study. The main outcome was the score of weekly assessed respiratory symptoms in the first year of life. Using the candidate gene approach, we selected 10 single nucleotide polymorphisms (SNPs) from the MUC5B and TOLLIP regions. Nitrogen dioxide (NO2) and particulate matter ≤10 µm in aerodynamic diameter (PM10) exposure was estimated on a weekly basis. We used generalised additive mixed models adjusted for known covariates. To validate our results in vitro, cells from a lung epithelial cell line were downregulated in TOLLIP expression and exposed to diesel particulate matter (DPM) and polyinosinic-polycytidylic acid. RESULTS: Significant interaction was observed between modelled air pollution (weekly NO2 exposure) and 5 SNPs within MUC5B and TOLLIP genes regarding respiratory symptoms as outcome: E.g., infants carrying minor alleles of rs5744034, rs3793965 and rs3750920 (all TOLLIP) had an increased risk of respiratory symptoms with increasing NO2 exposure. In vitro experiments showed that cells downregulated for TOLLIP react differently to environmental pollutant exposure with DPM and viral stimulation. CONCLUSION: Our findings suggest that the effect of air pollution on respiratory symptoms in infancy may be influenced by the genotype of specific SNPs from the MUC5B and TOLLIP regions. For validation of the findings, we provided in vitro evidence for the interaction of TOLLIP with air pollution.

Antibiotics in pregnancy influence nasal microbiome and respiratory morbidity in infancy.

Background: The effects of prenatal antibiotic exposure on respiratory morbidity in infancy and the involved mechanisms are still poorly understood. We aimed to examine whether prenatal antibiotic exposure in the third trimester is associated with nasal microbiome and respiratory morbidity in infancy and at school age, and whether this association with respiratory morbidity is mediated by the nasal microbiome. Methods: We performed 16S ribosomal RNA gene sequencing (regions V3-V4) on nasal swabs obtained from 296 healthy term infants from the prospective Basel-Bern birth cohort (BILD) at age 4-6 weeks. Information about antibiotic exposure was derived from birth records and standardised interviews. Respiratory symptoms were assessed by weekly telephone interviews in the first year of life and a clinical visit at age 6 years. Structural equation modelling was used to test direct and indirect associations accounting for known risk factors. Results: α-Diversity indices were lower in infants with antibiotic exposure compared to nonexposed infants (e.g. Shannon index p-value 0.006). Prenatal antibiotic exposure was also associated with a higher risk of any, as well as severe, respiratory symptoms in the first year of life (risk ratio 1.38, 95% CI 1.03-1.84; adjusted p-value (padj)=0.032 and risk ratio 1.75, 95% CI 1.02-2.97; padj=0.041, respectively), but not with wheeze or atopy in childhood. However, we found no indirect mediating effect of nasal microbiome explaining these clinical symptoms. Conclusion: Prenatal antibiotic exposure was associated with lower diversity of nasal microbiome in infancy and, independently of microbiome, with respiratory morbidity in infancy, but not with symptoms later in life.

Correction to: Lung functional development and asthma trajectories.

The original version of this article unfortunately contained a mistake. The given names and family names of all authors were switched in the original publication.

Lung functional development and asthma trajectories.

Early life environmental risk factors are associated with chronic respiratory morbidity in child- and adulthood. A possible mechanism for this sustained effect is their influence on early life lung functional growth and development, a susceptible phase of rapid lung growth with increased plasticity. We summarize evidence of hereditary and environmental ante-, peri-, and early postnatal factors on lung functional development, such as air pollution, tobacco exposure, nutrition, intrauterine growth retardation, prematurity, early life infections, microbiome, and allergies and their effect on lung functional trajectories. While some of the factors (e.g., prematurity) directly impair lung growth, the influence of many environmental factors is mediated through inflammatory processes (e.g., recurrent infections or oxidative stress). The timing and nature of these influences and their impact result in degrees of impaired maximal lung functional capacity in early adulthood; and they potentially impact future long-term respiratory morbidity such as chronic asthma or chronic obstructive airway disease (COPD). We discuss possibilities to prevent or modify such early abnormal lung functional growth trajectories and the need for future studies and prevention programs.

Effect of breastfeeding duration on lung function, respiratory symptoms and allergic diseases in school-age children.

BACKGROUND: A positive effect of breastfeeding on lung function has been demonstrated in cohorts of children with asthma or risk for asthma. We assessed the impact of breastfeeding on lung function and symptoms at the age of 6 years in an unselected, healthy birth cohort. METHODS: We prospectively studied healthy term infants from the Bern-Basel Infant Lung Development (BILD) cohort from birth up to 6 years. Any breastfeeding was assessed by weekly phone calls during the first year of life. Risk factors (eg, smoking exposure, parental history of allergic conditions, and education) were obtained using standardized questionnaires. The primary outcomes were lung function parameters measured at 6 years of age by spirometry forced expiratory volume in 1 second, body plethysmography (functional residual capacity [FRCpleth ], the total lung capacity [TLCpleth ], and the effective respiratory airway resistance [Reff ]) and fractional exhaled nitric oxide (FeNO). Secondary outcomes included ever wheeze (between birth and 6 years), wheeze in the past 12 months, asthma, presence of allergic conditions, atopic dermatitis, rhinitis, and positive skin prick test at the age of 6 years. RESULTS: In 377 children the mean breastfeeding duration was 36 weeks (SD 14.4). We found no association of breastfeeding duration with obstructive or restrictive lung function and FeNO. After adjustment for confounders, we found no associations of breastfeeding duration with respiratory symptoms or the presence of allergic conditions. CONCLUSION: This study found no evidence of an association between breastfeeding and comprehensive lung function in unselected healthy children with long-term breastfeeding. Our findings do not support the hypothesis that the duration of breastfeeding has a direct impact on lung function in a healthy population with low asthmatic risk.

Exposure to moderate air pollution and associations with lung function at school-age: A birth cohort study.

BACKGROUND: Adverse effects of higher air pollution levels before and after birth on subsequent lung function are often reported in the literature. We assessed whether low-to-moderate levels of air pollution during preschool-age impact upon lung function at school-age. METHODS: In a prospective birth cohort of 304 healthy term-born infants, 232 (79%) completed lung function at follow-up at six years. Using spatial-temporal models, levels of individual air pollution (nitrogen dioxide (NO2) and ozone (O3), particulate matter with a diameter <10 µm (PM10)) were estimated for the time windows pregnancy, first up to the sixth year of life separately, and birth until follow-up at six years. Time window means were compared to World Health Organization (WHO) guideline limits. Associations of exposure windows with spirometry and body plethysmography indices were analyzed using regression models, adjusting for potential confounders. For subgroup analysis, air pollution exposure was categorized into quartiles (four groups of 52 children). RESULTS: Mean NO2 level from birth until follow-up was [mean (range)] [11.8 (4.9 to 35.9 µg/m3)], which is almost 4-times lower than the WHO suggested limit of 40 µg/m3. In the whole population, increased air pollution levels from birth until follow-up were associated with reduced lung function at six years. In the subgroup analysis, the 52 children exposed to NO2 levels from the highest quartile during pregnancy, the first and second years of life and from birth until follow-up, had a significant decrease in forced expiratory volume in 1 s (FEV1). Per interquartile range increase of NO2, FEV1 decreased by [z-score change (95% confidence interval)] [-1.07 (-1.67 to -0.47)], [-1.02 (-1.66 to -0.39)], [-0.51 (-0.86 to -0.17)] and [-0.80 (-1.33 to -0.27)], respectively. Air pollution exposure during pregnancy and childhood resulted in a non-significant decrease in lung volume at six years, as assessed by functional residual capacity measured by body plethysmography (FRCpleth). CONCLUSION: Our results suggest that exposure to higher NO2 levels, which are still much lower than WHO guideline limits, especially during the sensitive period of early lung development, may be associated with reduced lung function at school-age. These findings support the concept of age and dose-dependent pollution effects on lung function in healthy school-aged children and underline the importance of pollution reduction measures.

Response of cord blood cells to environmental, hereditary and perinatal factors: A prospective birth cohort study.

BACKGROUND: Many studies investigating the impact of individual risk factors on cord blood immune cell counts may be biased given that cord blood composition is influenced by a multitude of factors. The aim of this study was to comprehensively investigate the relative impact of environmental, hereditary and perinatal factors on cord blood cells. METHODS: In 295 neonates from the prospective Basel-Bern Infant Lung Development Cohort, we performed complete blood counts and fluorescence-activated cell sorting scans of umbilical cord blood. The associations between risk factors and cord blood cells were assessed using multivariable linear regressions. RESULTS: The multivariable regression analysis showed that an increase per 10µg/m3 of the average nitrogen dioxide 14 days before birth was associated with a decrease in leukocyte (6.7%, 95% CI:-12.1,-1.0) and monocyte counts (11.6%, 95% CI:-19.6,-2.8). Maternal smoking during pregnancy was associated with significantly lower cord blood cell counts in multiple cell populations. Moreover, we observed sex differences regarding eosinophilic granulocytes and plasmacytoid dendritic cells. Finally, significantly increased numbers of cord blood cells were observed in infants exposed to perinatal stress. Cesarean section seems to play a significant role in Th1/Th2 balance. CONCLUSIONS: Our results suggest that all three: environmental, hereditary and perinatal factors play a significant role in the composition of cord blood cells at birth, and it is important to adjust for all of these factors in cord blood studies. In particular, perinatal circumstances seem to influence immune balance, which could have far reaching consequences in the development of immune mediated diseases.

Dynamics of respiratory symptoms during infancy and associations with wheezing at school age.

Children with frequent respiratory symptoms in infancy have an increased risk for later wheezing, but the association with symptom dynamics is unknown. We developed an observer-independent method to characterise symptom dynamics and tested their association with subsequent respiratory morbidity. In this birth-cohort of healthy neonates, we prospectively assessed weekly respiratory symptoms during infancy, resulting in a time series of 52 symptom scores. For each infant, we calculated the transition probability between two consecutive symptom scores. We used these transition probabilities to construct a Markov matrix, which characterised symptom dynamics quantitatively using an entropy parameter. Using this parameter, we determined phenotypes by hierarchical clustering. We then studied the association between phenotypes and wheezing at 6 years. In 322 children with complete data for symptom scores during infancy (16 864 observations), we identified three dynamic phenotypes. Compared to the low-risk phenotype, the high-risk phenotype, defined by the highest entropy parameter, was associated with an increased risk of wheezing (odds ratio (OR) 3.01, 95% CI 1.15-7.88) at 6 years. In this phenotype, infants were more often male (64%) and had been exposed to environmental tobacco smoke (31%). In addition, more infants had siblings (67%) and attended childcare (38%). We describe a novel method to objectively characterise dynamics of respiratory symptoms in infancy, which helps identify abnormal clinical susceptibility and recovery patterns of infant airways associated with persistent wheezing.