Bo L. Chawes

Reduced diversity of the intestinal microbiota during infancy is associated with increased risk of allergic disease at school age

BACKGROUND Changes in the human microbiome have been suggested as a risk factor for a number of lifestyle-related disorders, such as atopic diseases, possibly through a modifying influence on immune maturation in infancy. OBJECTIVES We aimed to explore the association between neonatal fecal flora and the development of atopic disorders until age 6 years, hypothesizing that the diversity of the intestinal microbiota influences disease development. METHODS We studied the intestinal microbiota in infants in the Copenhagen Prospective Study on Asthma in Childhood, a clinical study of a birth cohort of 411 high-risk children followed for 6 years by clinical assessments at 6-month intervals, as well as at acute symptom exacerbations. Bacterial flora was analyzed at 1 and 12 months of age by using molecular techniques based on 16S rRNA PCR combined with denaturing gradient gel electrophoresis, as well as conventional culturing. The main outcome measures were the development of allergic sensitization (skin test and specific serum IgE), allergic rhinitis, peripheral blood eosinophil counts, asthma, and atopic dermatitis during the first 6 years of life. RESULTS We found that bacterial diversity in the early intestinal flora 1 and 12 months after birth was inversely associated with the risk of allergic sensitization (serum specific IgE P = .003; skin prick test P = .017), peripheral blood eosinophils (P = .034), and allergic rhinitis (P = .007). There was no association with the development of asthma or atopic dermatitis. CONCLUSIONS Reduced bacterial diversity of the infants intestinal flora was associated with increased risk of allergic sensitization, allergic rhinitis, and peripheral blood eosinophilia, but not asthma or atopic dermatitis, in the first 6 years of life. These results support the general hypothesis that an imbalance in the intestinal microbiome is influencing the development of lifestyle-related disorders, such as allergic disease.

Chromosome 17q21 Gene Variants Are Associated with Asthma and Exacerbations but Not Atopy in Early Childhood

RATIONALE An asthma predisposition locus on chromosome 17q12-q21 has recently been replicated in different ethnic groups. OBJECTIVES To characterize the asthma and atopy phenotypes in early childhood that associate with the 17q12-21 locus. METHODS The single nucleotide polymorphism (SNP), rs7216389, was genotyped in 376 of 411 children from the Copenhagen Prospective Study on Asthma in Childhood (COPSAC) birth cohort born to mothers with asthma together with 305 mothers and 224 fathers. Nineteen additional SNPs in the region were genotyped in the children. Investigator-diagnosed clinical endpoints were based on diary cards and clinic visits every 6 months and at acute symptoms from birth. Lung function, bronchial responsiveness, and sensitization were tested longitudinally from early infancy. MEASUREMENTS AND MAIN RESULTS rs7216389 was significantly associated with the development of wheeze (hazard ratio 1.64 [1.05-2.59], P value = 0.03), asthma (hazard ratio, 1.88 [1.15-3.07], P = 0.01), and acute severe exacerbations (hazard ratio 2.66 [1.58-4.48], P value = 0.0002). The effect on wheeze and asthma was observed for early onset but not late onset of disease. The increased risk of exacerbations persisted from 1 to 6 years of age (incidence ratio 2.48 [1.42-4.32], P value = 0.001), and increased bronchial responsiveness was present in infancy and at 4 years of age, but not at 6 years. In contrast, rs7216389 conferred no risk of eczema, rhinitis, or allergic sensitization. CONCLUSIONS Variation at the chromosome 17q12-q21 locus was associated with approximately twofold increased risk of recurrent wheeze, asthma, asthma exacerbations, and bronchial hyperresponsiveness from early infancy to school age but without conferring risk of eczema, rhinitis, or allergic sensitization. These longitudinal clinical data show this locus to be an important genetic determinant of nonatopic asthma in children.

Effect of Vitamin D3 Supplementation During Pregnancy on Risk of Persistent Wheeze in the Offspring: A Randomized Clinical Trial.

IMPORTANCE Observational studies have suggested that increased dietary vitamin D intake during pregnancy may protect against wheezing in the offspring, but the preventive effect of vitamin D supplementation to pregnant women is unknown. OBJECTIVE To determine whether supplementation of vitamin D3 during the third trimester of pregnancy reduces the risk of persistent wheeze in the offspring. DESIGN, SETTING, AND PARTICIPANTS A double-blind, single-center, randomized clinical trial conducted within the Copenhagen Prospective Studies on Asthma in Childhood 2010 cohort. Enrollment began March 2009 with a goal of 708 participants, but due to delayed ethical approval, only 623 women were recruited at 24 weeks of pregnancy. Follow-up of the children (N = 581) was completed when the youngest child reached age 3 years in March 2014. INTERVENTIONS Vitamin D3 (2400 IU/d; n = 315) or matching placebo tablets (n = 308) from pregnancy week 24 to 1 week postpartum. All women received 400 IU/d of vitamin D3 as part of usual pregnancy care. MAIN OUTCOMES AND MEASURES Age at onset of persistent wheeze in the first 3 years of life. Secondary outcomes included number of episodes of troublesome lung symptoms, asthma, respiratory tract infections, and neonatal airway immunology. Adverse events were assessed. RESULTS Of the 581 children, persistent wheeze was diagnosed during the first 3 years of life in 47 children (16%) in the vitamin D3 group and 57 children (20%) in the control group. Vitamin D3 supplementation was not associated with the risk of persistent wheeze, but the number of episodes of troublesome lung symptoms was reduced, and the airway immune profile was up-regulated (principal component analysis, P = .04). There was no effect on additional end points. Intrauterine death was observed in 1 fetus (<1%) in the vitamin D3 group vs 3 fetuses (1%) in the control group and congenital malformations in 17 neonates (5%) in the vitamin D3 group vs 23 neonates (8%) in the control group. [table: see text]. CONCLUSIONS AND RELEVANCE The use of 2800 IU/d of vitamin D3 during the third trimester of pregnancy compared with 400 IU/d did not result in a statistically significant reduced risk of persistent wheeze in the offspring through age 3 years. However, interpretation of the study is limited by a wide CI that includes a clinically important protective effect. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00856947.

Children with allergic and nonallergic rhinitis have a similar risk of asthma

BACKGROUND Both allergic and nonallergic rhinitis have been associated with increased prevalence of asthma. OBJECTIVE To characterize asthma and intermediary asthma endpoints in young children with allergic and nonallergic rhinitis. METHODS Thirty-eight 7-year-old children with allergic rhinitis, 67 with nonallergic rhinitis, and 185 without rhinitis from the Copenhagen Prospective Study on Asthma in Childhood birth cohort were compared for prevalence of asthma, eczema, food sensitization, filaggrin null-mutations, total IgE, blood eosinophil count, fractional exhaled nitric oxide (FeNO), lung function, and bronchial responsiveness. RESULTS Children with allergic rhinitis compared with asymptomatic controls had increased prevalence of asthma (21% vs 5%; P = .002), food sensitization (47% vs 13%; P < .001), and eczema (66% vs 43%; P = .01) and increased total IgE (155 kU/L vs 41 kU/L; P < .001), blood eosinophil count (0.46 x 10(9)/L vs 0.30 x 10(9)/L; P = .01), FeNO (15.9 ppb vs 6.6 ppb; P < .001), and bronchial hyperresponsiveness (23% vs 9%; P = .008). Filaggrin null-mutations were associated with allergic rhinitis (odds ratio, 3.3; 95% CI, 1.3-8.3) but did not modify these associations. Children with nonallergic rhinitis also had increased asthma prevalence (20% vs 5%; P = .001) but showed no association with filaggrin null-mutations, eczema, food sensitization, total IgE, blood eosinophil count, FeNO, or bronchial responsiveness. CONCLUSION Asthma is similarly associated with allergic and nonallergic rhinitis, suggesting a link between upper and lower airways beyond allergy associated inflammation. Only children with allergic rhinitis had increased bronchial responsiveness and elevated FeNO, suggesting different endotypes of asthma symptoms in young children with allergic and nonallergic rhinitis.

Fish Oil–Derived Fatty Acids in Pregnancy and Wheeze and Asthma in Offspring

BACKGROUND Reduced intake of n-3 long-chain polyunsaturated fatty acids (LCPUFAs) may be a contributing factor to the increasing prevalence of wheezing disorders. We assessed the effect of supplementation with n-3 LCPUFAs in pregnant women on the risk of persistent wheeze and asthma in their offspring. METHODS We randomly assigned 736 pregnant women at 24 weeks of gestation to receive 2.4 g of n-3 LCPUFA (fish oil) or placebo (olive oil) per day. Their children formed the Copenhagen Prospective Studies on Asthma in Childhood2010 (COPSAC2010) cohort and were followed prospectively with extensive clinical phenotyping. Neither the investigators nor the participants were aware of group assignments during follow-up for the first 3 years of the childrens lives, after which there was a 2-year follow-up period during which only the investigators were unaware of group assignments. The primary end point was persistent wheeze or asthma, and the secondary end points included lower respiratory tract infections, asthma exacerbations, eczema, and allergic sensitization. RESULTS A total of 695 children were included in the trial, and 95.5% completed the 3-year, double-blind follow-up period. The risk of persistent wheeze or asthma in the treatment group was 16.9%, versus 23.7% in the control group (hazard ratio, 0.69; 95% confidence interval [CI], 0.49 to 0.97; P=0.035), corresponding to a relative reduction of 30.7%. Prespecified subgroup analyses suggested that the effect was strongest in the children of women whose blood levels of eicosapentaenoic acid and docosahexaenoic acid were in the lowest third of the trial population at randomization: 17.5% versus 34.1% (hazard ratio, 0.46; 95% CI, 0.25 to 0.83; P=0.011). Analyses of secondary end points showed that supplementation with n-3 LCPUFA was associated with a reduced risk of infections of the lower respiratory tract (31.7% vs. 39.1%; hazard ratio, 0.75; 95% CI, 0.58 to 0.98; P=0.033), but there was no statistically significant association between supplementation and asthma exacerbations, eczema, or allergic sensitization. CONCLUSIONS Supplementation with n-3 LCPUFA in the third trimester of pregnancy reduced the absolute risk of persistent wheeze or asthma and infections of the lower respiratory tract in offspring by approximately 7 percentage points, or one third. (Funded by the Lundbeck Foundation and others; ClinicalTrials.gov number, NCT00798226 .).

Increased Risk of Pneumonia and Bronchiolitis after Bacterial Colonization of the Airways as Neonates

RATIONALE The frequency of pneumonia and bronchiolitis exhibits considerable variation in otherwise healthy children, and suspected risk factors explain only a minor proportion of the variation. We hypothesized that alterations in the airway microbiome in early life may be associated with susceptibility to pneumonia and bronchiolitis in young children. OBJECTIVES To investigate the relation between neonatal airway colonization and pneumonia and bronchiolitis during the first 3 years of life. METHODS Participants comprised children of the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort, a prospective birth cohort study of 411 children born to mothers with asthma. Aspirates from the hypopharynx at age 4 weeks were cultured for Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus. Clinical information on pneumonia and bronchiolitis within the first 3 years of life was prospectively collected by the research physicians at the center. Analyses were adjusted for covariates associated with pneumonia and bronchiolitis and bacterial airway colonization. MEASUREMENTS AND MAIN RESULTS Hypopharyngeal aspirates and full clinical follow-up until 3 years of age were available for 265 children. Of these, 56 (21%) neonates were colonized with S. pneumoniae, H. influenzae, and/or M. catarrhalis at 4 weeks of age. Colonization with at least one of these microorganisms (but not S. aureus) was significantly associated with increased incidence of pneumonia and bronchiolitis (adjusted incidence rate ratio, 1.79 [1.29-2.48]; P < 0.005) independently of concurrent or later asthma. CONCLUSIONS Neonatal airway colonization with S. pneumoniae, H. influenzae, or M. catarrhalis is associated with increased risk of pneumonia and bronchiolitis in early life independently of asthma. This suggests a role of pathogenic bacterial colonization of the airways in neonates for subsequent susceptibly to pneumonia and bronchiolitis.

Pathogenic Bacteria Colonizing the Airways in Asymptomatic Neonates Stimulates Topical Inflammatory Mediator Release

RATIONALE Bacterial colonization of neonatal airways with the pathogenic bacterial species, Moraxella catarrhalis, Streptococcus pneumoniae, and Haemophilus influenzae, is associated with later development of childhood asthma. OBJECTIVES To study a possible association between colonization with pathogenic bacterial strains and the immune signature of the upper airways in healthy neonates. METHODS A total of 20 cytokines and chemokines were quantified in vivo in the airway mucosal lining fluid of 662 neonates from the Copenhagen Prospective Study of Asthma in Childhood 2010 birth cohort. Colonization of the hypopharynx with M. catarrhalis, S. pneumoniae, H. influenzae, and Staphylococcus aureus was assessed simultaneously. The association between immune signatures and bacterial colonization or noncolonized controls was analyzed using conventional statistical methods supplemented by a multivariate approach for pattern identification. MEASUREMENTS AND MAIN RESULTS Colonization with M. catarrhalis and H. influenzae induced a mixed T helper cell (Th) type 1/Th2/Th17 response with high levels of IL-1β (M. catarrhalis, P = 2.2 × 10(-12); H. influenzae, P = 7.1 × 10(-10)), TNF-α (M. catarrhalis, P = 1.5 × 10(-9); H. influenzae, P = 5.9 × 10(-7)), and macrophage inflammatory protein-1β (M. catarrhalis, P = 1.6 × 10(-11); H. influenzae, P = 2.7 × 10(-7)). S. aureus colonization demonstrated a Th17-promoting profile with elevated IL-17 levels (P = 1.6 × 10(-24)). S. pneumoniae colonization was not significantly associated with any of the mediators. CONCLUSIONS M. catarrhalis and H. influenzae colonization of the airways of asymptomatic neonates is associated with an inflammatory immune response of the airway mucosa, which may result in chronic inflammation.

Cord blood 25(OH)-vitamin D deficiency and childhood asthma, allergy and eczema: the COPSAC2000 birth cohort study.

Background Epidemiological studies have suggested an association between maternal vitamin D dietary intake during pregnancy and risk of asthma and allergy in the offspring. However, prospective clinical studies on vitamin D measured in cord blood and development of clinical end-points are sparse. Objective To investigate the interdependence of cord blood 25-hydroxyvitamin D (25(OH)-Vitamin D) level and investigator-diagnosed asthma- and allergy-related conditions during preschool-age. Methods Cord blood 25(OH)-Vitamin D level was measured in 257 children from the Copenhagen Prospective Studies on Asthma in Childhood (COPSAC2000) at-risk mother-child cohort. Troublesome lung symptoms (TROLS), asthma, respiratory infections, allergic rhinitis, and eczema, at age 0–7 yrs were diagnosed exclusively by the COPSAC pediatricians strictly adhering to predefined algorithms. Objective assessments of lung function and sensitization were performed repeatedly from birth. Results After adjusting for season of birth, deficient cord blood 25(OH)-Vitamin D level (<50 nmol/L) was associated with a 2.7-fold increased risk of recurrent TROLS (HR = 2.65; 95% CI = 1.02–6.86), but showed no association with respiratory infections or asthma. We saw no association between cord blood 25(OH)-Vitamin D level and lung function, sensitization, rhinitis or eczema. The effects were unaffected from adjusting for multiple lifestyle factors. Conclusion Cord blood 25(OH)-Vitamin D deficiency associated with increased risk of recurrent TROLS till age 7 years. Randomized controlled trials of vitamin D supplementation during pregnancy are needed to prove causality.

Azithromycin for episodes with asthma-like symptoms in young children aged 1–3 years: a randomised, double-blind, placebo-controlled trial

Summary Background Bacteria and viruses are equally associated with the risk of acute episodes of asthma-like symptoms in young children, suggesting antibiotics as a potential treatment for such episodes. We aimed to assess the effect of azithromycin on the duration of respiratory episodes in young children with recurrent asthma-like symptoms, hypothesising that it reduces the duration of the symptomatic period. Methods In this randomised, double-blind, placebo-controlled trial, we recruited children aged 1–3 years, who were diagnosed with recurrent asthma-like symptoms from the Copenhagen Prospective Studies on Asthma in Childhood 2010 cohort; a birth cohort consisting of the general Danish population of Zealand, including Copenhagen. Exclusion criteria were macrolide allergy, heart, liver, neurological, and kidney disease, and, before each treatment, one or more clinical signs of pneumonia (respiratory frequency of ≥50 breaths per min; fever of ≥39°C; C-reactive protein concentration of ≥476·20 nmol/L [≥50 mg/L]). Each episode of asthma-like symptoms lasting at least 3 days was randomly allocated to a 3-day course of azithromycin oral solution of 10 mg/kg per day or placebo after thorough examination by a study physician at the Copenhagen Prospective Studies on Asthma research unit. Each episode was randomly allocated independently of previous treatment from a computer-generated list of random numbers in blocks of ten (generated at the Pharmacy of Glostrup). Investigators and children were masked until the youngest child turned 3 years of age and throughout the data validation and analysis phases. The primary outcome was duration of the respiratory episode after treatment, verified by prospective daily diaries and analysed with Poisson regression. Analyses were per protocol (excluding those without a primary outcome measure or who did not receive treatment). This trial is registered with ClinicalTrials.gov, number NCT01233297. Findings Between Nov 17, 2010, and Jan 28, 2014, we randomly allocated 158 asthma-like episodes in 72 children (79 [50%] to azithromycin and 79 [50%] to placebo). The mean duration of the episode after treatment was 3·4 days for children receiving azithromycin compared with 7·7 days for children receiving placebo. Azithromycin caused a significant shortening of the episode of 63·3% (95% CI 56·0–69·3; p<0·0001). The effect size increased with early initiation of treatment, showing a reduction in episode duration of 83% if treatment was initiated before day 6 of the episode compared with 36% if initiated on or after day 6 (p<0·0001). We noted no differences in clinical adverse events between the azithromycin (18 [23%] of 78 episodes included in final analysis) and placebo (24 [30%] of 79) groups (p=0·30), but we did not investigate bacterial resistance patterns after treatment. Interpretation Azithromycin reduced the duration of episodes of asthma-like symptoms in young children, suggesting that this drug could have a role in acute management of exacerbations. Further research is needed to disentangle the inflammatory versus antimicrobial aspects of this relation. Funding Lundbeck Foundation, Danish Ministry of Health, Danish Council for Strategic Research, Capital Region Research Foundation.

Elevated Exhaled Nitric Oxide in High-Risk Neonates Precedes Transient Early but Not Persistent Wheeze

RATIONALE Elevated fractional exhaled nitric oxide (Fe(NO)) concentration has been suggested to predict early childhood wheeze and sensitization. OBJECTIVES To investigate the association between Fe(NO) in asymptomatic neonates and the development of wheeze patterns and atopic intermediary phenotypes in the first 6 years of life. METHODS We measured Fe(NO) in 253 healthy 1-month-old neonates from the Copenhagen Prospective Study on Asthma in Childhood birth cohort and monitored prospectively wheezy episodes by daily diary cards during the first 6 years of life. Total IgE, specific IgE, and blood eosinophil count were assessed at age 6 months, 4 years, and 6 years. Associations were studied by Cox regression, logistic regression, and generalized linear models. MEASUREMENTS AND MAIN RESULTS Increased neonatal Fe(NO) level was significantly associated with the development of recurrent wheeze in the first year of life (hazard ratio, 2.63; 95% confidence interval, 1.1 to 6.2; P = 0.026) but not thereafter. The association was unaffected by environmental tobacco smoke exposure. Fe(NO) was not associated with elevated levels of total IgE, specific IgE, or blood eosinophil count at any age point and was unrelated to neonatal lung function. CONCLUSIONS An elevated Fe(NO) level in asymptomatic neonates born to mothers with asthma preceded the development of transient early wheezing, but not persistent wheezing during preschool age, and was unrelated to atopy. This suggests an early disease process other than small airway caliber contributing to the transient wheezing phenotype.