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Background: Vitamin D (25(OH)D) deficiency has been implicated as a possible risk factor for asthma development, but studies at selected time points measuring 25(OH)D levels during childhood have yielded conflicting findings. Prospective studies tracking 25(OH)D levels during the initiation phase of asthma in early childhood have not been reported. Objective: We sought to elucidate relationships between 25(OH)D levels from birth to age 10 years and susceptibility to allergic sensitization, respiratory tract infections, and asthma. Methods: Asthma-, allergy-, and respiratory tract infection-associated phenotypes (including pathogen identification) were characterized in a high-risk birth cohort. Plasma 25(OH)D concentrations were quantified at birth and at clinical follow-ups at the ages of 0.5, 1, 2, 3, 4, 5, and 10 years, and relationships with clinical outcomes were examined. Results: Cross-sectional analyses demonstrated inverse associations between 25(OH)D concentrations and the risk for concurrent sensitization at age 0.5, 2, and 3 years, and mixed-effects regression demonstrated inverse longitudinal associations of 25(OH)D levels with both sensitization and eczema. Multivariate regression modeling suggested that the number of 25(OH)D-deficient follow-ups was positively associated with risk for asthma/wheeze, eczema, and sensitization at 10 years; adjustment for sensitization (particularly by 2 years) in the asthma/wheeze models reduced 25(OH)D associations with these latter outcomes. 25(OH)D levels were also inversely associated with early nasopharyngeal colonization with . Streptococcus species and age of first febrile lower respiratory illness, both of which are known asthma risk factors. Conclusion: 25(OH)D deficiency in early childhood is associated with increased risk for persistent asthma, potentially through modulating susceptibility to early allergic sensitization, upper respiratory tract colonization with bacterial pathogens, or both. These relationshi

Radiation damage is a major limitation to synchrotron small-angle X-ray scattering analysis of biomacromolecules. Flowing the sample during exposure helps to reduce the problem, but its effectiveness in the laminar-flow regime is limited by slow flow velocity at the walls of sample cells. To overcome this limitation, the coflow method was developed, where the sample flows through the centre of its cell surrounded by a flow of matched buffer. The method permits an order-of-magnitude increase of X-ray incident flux before sample damage, improves measurement statistics and maintains low sample concentration limits. The method also efficiently handles sample volumes of a few microlitres, can increase sample throughput, is intrinsically resistant to capillary fouling by sample and is suited to static samples and size-exclusion chromatography applications. The method unlocks further potential of third-generation synchrotron beamlines to facilitate new and challenging applications in solution scattering.

The nasopharynx (NP) is a reservoir for microbes associated with acute respiratory infections (ARIs). Lung inflammation resulting from ARIs during infancy is linked to asthma development. We examined the NP microbiome during the critical first year of life in a prospective cohort of 234 children, capturing both the viral and bacterial communities and documenting all incidents of ARIs. Most infants were initially colonized with Staphylococcus or Corynebacterium before stable colonization with Alloiococcus or Moraxella. Transient incursions of Streptococcus, Moraxella, or Haemophilus marked virus-associated ARIs. Our data identify the NP microbiome as a determinant for infection spread to the lower airways, severity of accompanying inflammatory symptoms, and risk for future asthma development. Early asymptomatic colonization with Streptococcus was a strong asthma predictor, and antibiotic usage disrupted asymptomatic colonization patterns. In the absence of effective anti-viral therapies, targeting pathogenic bacteria within the NP microbiome could represent a prophylactic approach to asthma.

Rationale: Vitamin D deficiency is associated with chronic lung disease. We have previously shown in an in vivo mouse model that maternal vitamin D deficiency is associated with alterations in early life lung structure and function. However, there are limited data to support a relationship between maternal vitamin D deficiency during the early stages of lung development and postnatal lung function in human populations. Objectives: To assess the association between maternal vitamin D deficiency, postnatal lung function, and asthmatic status in a longitudinal birth cohort. Methods: Serum was collected at 16 to 20 weeks' gestation at the time of recruitment in a community-based prospective birth cohort for measurement of vitamin D (25[OH]D). Lung function was assessed by spirometry according to American Thoracic Society guidelines in children at 6 and 14 years of age. Demographic and clinical history data were collected by questionnaire at recruitment and at the follow-up visits. Measurements and Main Results: FVC Z-scores in both sexes (ß, 0.007 [95% confidence interval (CI), 0.001-0.013]; P = 0.02) and FEV1 Z-scores in girls (ß, 0.007 [95% CI, 0.001-0.013]; P = 0.02) were positively associated with maternal serum 25(OH)D at 6 years of age. These associations were mostly absent at 14 years of age. Maternal vitamin D deficiency was positively associated with asthma at 6 years of age but only in boys (odds ratio, 3.03 [95% CI, 1.02-9.02]; P = 0.04). Conclusions: This study supports the notion that vitamin D deficiency during lung development may impact on postnatal lung growth and increase the risk of developing lung disease.

Background: The impact of breast milk feeding on susceptibility to asthma in childhood is highly controversial, due in part to failure of the majority of studies in the area to adequately account for key confounders exemplified by respiratory infection history, plus the effects of recall bias. Methods: As part of a prospective cohort study on the role of respiratory infections in asthma development in high-risk children, we measured the concentration of a panel of anti-infective proteins in maternal milk samples and analyzed associations between these and subsequent atopy-, infection-, and asthma-related outcomes prospectively to age 10 years. Results: We observed significant but transient inverse associations between the concentration of milk proteins and susceptibility to upper respiratory infections in year 1 only, and parallel but positive transient associations with early lower respiratory infections and atopy. No associations were seen with asthma-related outcomes. Conclusions: Breast milk feeding may influence the expression of inflammatory symptoms associated with respiratory infections and atopy in early life, but these effects appear to be inconsistent and transient. The heterogeneous nature of breast-feeding effects suggests it may influence systemic immunoinflammatory function at several different levels.

Background: The forced oscillation technique (FOT) can be used in children as young as 2 years of age and in those unable to perform routine spirometry. There is limited information on changes in FOT outcomes in healthy children beyond the preschool years and the level of bronchodilator responsiveness (BDR) in healthy children. We aimed to create reference ranges for respiratory impedance outcomes collated from multiple centers. Outcomes included respiratory system resistance (Rrs) and reactance (Xrs), resonant frequency (Fres), frequency dependence of Rrs (Fdep), and the area under the reactance curve (AX). We also aimed to define the physiological effects of bronchodilators in a large population of healthy children using the FOT. Methods: Respiratory impedance was measured in 760 healthy children, aged 2–13 years, from Australia and Italy. Stepwise linear regression identified anthropometric predictors of transformed Rrs and Xrs at 6, 8, and 10 Hz, Fres, Fdep, and AX. Bronchodilator response (BDR) was assessed in 508 children after 200 µg of inhaled salbutamol. Results: Regression analysis showed that Rrs, Xrs, and AX outcomes were dependent on height and sex. The BDR cut-offs by absolute change in Rrs8, Xrs8, and AX were −2.74 hPa s L−1, 1.93 hPa s L−1, and −33 hPa s L−1, respectively. These corresponded to relative and Z-score changes of −32%; −1.85 for Rrs8, 65%; 1.95 for Xrs8, and −82%; −2.04 for AX. Conclusions: We have established generalizable reference ranges for respiratory impedance and defined cut-offs for a positive bronchodilator response using the FOT in healthy children.

Background: Infants who develop house dust mite (HDM) allergy and HDM-sensitised children with severe persistent asthma have low antibody responses to the P6 antigen of Haemophilus influenzae. Objective: To measure the development of antibody to two ubiquitous bacteria of the respiratory mucosa in a prospective birth cohort at high risk of allergic disease and to assess which responses are associated with asthma and atopy. Methods: IgG1 and IgG4 antibody to H influenzae (P4 and P6) and Streptoccocus pneumoniae (PspA and PspC) surface antigens was measured in yearly blood samples of children aged 1-5 years. IgE to the P6 antigen was examined for the 5-year group. The children were stratified based on HDM sensitisation and asthma at 5 years of age. Results: HDM-sensitised children had lower IgG1 antibody titres to the bacterial antigens, and early responses (<3 years and before the development of HDM sensitisation and asthma) corrected for multiple antigens were significantly reduced for P4, P6 and PspC (p=0.008, p=0.004 and p=0.028, respectively). Similar associations with asthma were also found (p=0.008, p=0.004 and p=0.032 for P4, P6 and PspC, respectively). The IgG4 antibody titre and prevalence were similar in both HDM-sensitised and non-sensitised groups, but sensitised children had a slower downregulation of the IgG4 response. Children with asthma (27/145 at 5 years) had lower anti-P6 IgE responses (p<0.05). Conclusions: HDM-sensitised children have early defective antibody responses to bacteria that are associated with asthma. Surprisingly, antibacterial IgE was associated with a reduced risk for asthma.

Background: Exhaled breath temperature (EBT) has been proposed for the non-invasive assessment of airway inflammation. Previous studies have not examined the influence of room temperature or lung size on the EBT. Objective: This study aimed to address these issues in healthy children. Methods: We assessed the effects of room temperature and lung volume in 60 healthy children aged 9–11 years (mean age 10.3 years, 33 male). Static lung volumes were assessed using multiple breath nitrogen washout. Questionnaire and skin prick tests were also used to establish respiratory health in the children. We obtained the EBT parameters of slope, end plateau temperature (PLET) and normalized plateau temperature (nPLET; plateau temperature minus inspired air temperature), and ascertained physiological factors influencing EBT. Results: End plateau temperature was shown to be proportionally affected by room temperature (r = 0.532, P < 0.001) whereas slope and nPLET decreased with increasing room temperature (r = −0.392 P < 0.02 and r = −0.507 P = 0.002). After adjusting for room temperature, height and age, the total lung capacity (r2 = 0.435, P = 0.006) and slow vital capacity (SVC; r2 = 0.44, P = 0.005) were found to be the strongest predictors of end PLET in healthy children. When all factors were included in a multiple regression model, SVC and room temperature were the only predictors of plateau and nPLET. Slope was only influenced by room temperature. Conclusions: Exhaled breath temperature measurements are highly feasible in children with a 95% success rate in this healthy population. Room temperature and SVC significantly influence EBT variables in healthy children. Further studies are required to investigate the ability of EBT to assess airway inflammation in children with respiratory disease.

Background: Atopy and asthma are commonly initiated during early life, and there is increasing interest in the development of preventive treatments for at-risk children. However, effective methods for assessing the level of risk in individual children are lacking. Objective: We sought to identify clinical and laboratory biomarkers in 2-year-olds that are predictive of the risk for persistent atopy and wheeze at age 5 years. Methods: We prospectively studied 198 atopic family history-positive children to age 5 years. Clinical and laboratory assessments related to asthma history and atopy status were undertaken annually; episodes of acute respiratory illness were assessed and classified throughout and graded by severity. Results: Aeroallergen-specific IgE titers cycled continuously within the low range in nonatopic subjects. Atopic subjects displayed similar cycling in infancy but eventually locked into a stable pattern of upwardly trending antibody production and TH2-polarized cellular immunity. The latter was associated with stable expression of IL-4 receptor in allergen-specific TH2 memory responses, which was absent from responses during infancy. Risk for persistent wheeze was strongly linked to early sensitization and in turn to early infection. Integration of these data by means of logistic regression revealed that attaining mite-specific IgE titers of greater than 0.20 kU/L by age 2 years was associated with a 12.7% risk of persistent wheeze, increasing progressively to an 87.2% risk with increasing numbers of severe lower respiratory tract illnesses experienced. Conclusion: The risk for development of persistent wheeze in children can be quantified by means of integration of measures related to early sensitization and early infections. Follow-up studies along similar lines in larger unselected populations to refine this approach are warranted. © 2010 American Academy of Allergy, Asthma & Immunology.

Rationale: Respiratory infections in early life are associated with risk for wheezing bronchiolitis, especially in children at high risk of atopy. The underlying mechanisms are unknown, but are suspected to involve imbalance(s) in host defense responses against pathogens stemming from functional immaturity of the immune system in this age group. Objectives: To assess the contribution of eosinophil-trophic IL-5, and the potent antiinflammatory cytokine IL-10, to risk for infection in early life. Measurements and Main Results: We prospectively monitored a cohort of 198 high-risk children to age 5 years, recording every acute respiratory infection episode and classifying them by severity. We measured cord blood T-cell capacity to produce IL-10 and IL-5, and related these functions to subsequent infection history. IL-10 and IL-5 were associated, respectively, with resistance versus susceptibility to infections. The greatest contrasting effects of these two cytokines were seen when they were considered in combination by generating IL-10/IL-5 response ratios for each subject. The low IL-10/high IL-5 T-cell response phenotype was strongly associated with susceptibility to all grades of acute respiratory infection, relative to the more resistant high IL-10/low IL-5 phenotype. Conclusions: Excessive production of IL-5 by T cells at birth is associated with heightened risk for subsequent severe respiratory infections, and this risk is attenuated by concomitant IL-10 production. The underlying mechanisms may involve IL-10-mediated feedback inhibition of IL-5-dependent eosinophil-induced inflammation, which is a common feature of host antiviral responses in early life.

Background: It has been proposed that immune dysfunction during early childhood plays an important role in asthma pathogenesis. However, it is not known specifically whether changes in dendritic cells (DCs) during infancy antedate the development of respiratory tract infections, asthma, and related clinical phenotypes. Objectives: We sought to assess the association between the level of blood DCs during the first year and the subsequent development of respiratory tract infections, wheezing, and allergic sensitization. Methods: A community-based cohort of children with a family history of atopy was followed to age 5 years. Children were monitored intensively for respiratory tract infections. History of wheeze and asthma was collected annually, atopy was documented at 5 years, and flow cytometry was used to identify DC subsets in blood samples collected when children were well. Results: Levels of plasmacytoid DCs (pDCs) during infancy were inversely correlated with symptoms of lower respiratory tract infections, parent-reported wheezing, and the cumulative rate of physician-diagnosed asthma up to age 5 years. These relationships were independent of atopy, as determined by allergy skin test results and total and specific IgE levels. In contrast, levels of myeloid DCs were not associated with respiratory tract infections, asthma, or wheezing but were associated with total IgE levels at age 5 years. Conclusion: In children with a family history of atopy, relative deficiency of circulating pDCs during infancy appears to be a risk factor for more frequent and more severe respiratory tract infections, wheezing, and a diagnosis of asthma. Infants with higher numbers of pDCs are protected against these outcomes. © 2009 American Academy of Allergy, Asthma & Immunology.

Atopy is characterized by eosinophilic inflammation associated with recruitment of eosinophil/basophil (Eo/B) progenitors. We have previously shown that Eo/B progenitor phenotypes are altered in cord blood (CB) in infants at high risk of atopy/asthma, and respond to maternal dietary intervention during pregnancy. As respiratory tract viral infections have been shown to induce wheeze in infancy, we investigated the relationship between CB progenitor function and phenotype and acute respiratory illness (ARI), specifically wheeze and fever. CB from 39 high-risk infants was studied by flow cytometry for CD34(+) progenitor phenotype and by ex vivo Eo/B-colony forming unit (CFU) responses to cytokine stimulation in relation to ARI in the first year of life. A consistent relationship was observed between increased numbers of granulocyte/macrophage (GM)-colony-stimulating factor (CSF)- and IL-3-responsive Eo/B-CFU in CB and the frequency/characteristics of ARI during infancy. Comparable associations were found between ARI and CB IL-3R(+) and GM-CSFR(+)CD34(+) cell numbers. Conversely, a reciprocal decrease in the proportion of CB IL-5R(+) cells was found in relation to the clinical outcomes. The elevation of IL-3/GM-CSF-responsive Eo/B progenitors in high-risk infants in relation to ARI outcomes suggests a mechanism for the increased severity of inflammatory responses in these subjects following viral infection.

The aim of this study was to investigate the relationship between air pollution and respiratory symptoms in young children. A total of 263 children at high risk of developing asthma or atopy were recruited antenatally and all respiratory symptoms experienced by the children were recorded by their parents for five years. Daily pollutant concentrations and meteorological data (ambient temperature and humidity) were collected from network monitoring sites. Logistic regression models investigating relationships between individual air pollutants and respiratory symptoms showed significant associations between Ozone (O3) (1 h and 8 h) concentrations and raised body temperature (lag 0); Carbon monoxide (CO) (8 h) and wheeze/rattle and runny/blocked nose (lag 5 and additive exposure over 5 days); Nitrogen dioxide (NO2) (24 h) concentrations and cough (lag 0 and additive exposure over 5 days) and PM2.5 and visibility (BSP) (1 h) with cough (lag 0). These associations were observed even though air pollutant concentrations were below national standards throughout the study period.

Interferon- (IFN) gene expression is tightly regulated in early life, and exaggerated negative control of IFN production in CD4+ T cells has been associated with risk for subsequent development of atopy. Recent studies have demonstrated hypermethylation of CpG sites in the IFN promoter in neonates, a mechanism which in mice leads to strong suppression of IFN gene transcription. In the present study, the methylation status of six CpG sites in the proximal promoter of the human IFN gene was determined by bisulphite sequencing. Cell populations studied were Th1 or Th2 polarized cell lines derived from neonatal and adult CD4+/CD45RA+ T cells, CD4+ and CD8+ naive T cells from cord blood of children followed to outcome age 2 for assessment of atopy status, and CD4+ and CD8+ naive T cells from 6 yr old and adult atopics and controls. We demonstrate that in vitro differentiation of CD4+ T cells down the Th1 pathway (but not the Th2 pathway) is accompanied by progressive demethylation of CpG sites in the IFN promoter, which is most marked in neonatal cells. Atopy development by age 2 was not associated with variations in methylation patterns in cord blood T cells. However, IFN promoter methylation was reduced in CD8+ T cells from atopic children in the age range in which hyperproduction of IFN as recently been identified as a common feature of the atopic phenotype. The findings demonstrate the potency of IFN promoter methylation as a mechanism for control of human IFN gene expression, particularly during early life. Differential regulation of IFN promoter methylation in T cells may be an important contributory factor in atopy development in childhood, and this possibility warrants further detailed investigation.