RATIONALE:Early childhood represents a critical window for asthma susceptibility, marked by developmental and molecular changes, yet their longitudinal pattern remains unclear. OBJECTIVES:To identify differences in longitudinal whole-blood gene expression during early childhood in future asthmatics compared to healthy children. METHODS:We conducted a longitudinal whole-blood transcriptomic analysis at 4 timepoints (1, 4.5, 6, 10.5 years) in a sample of the birth cohort Protection against Allergy Study in Rural Environments (PASTURE) (n = 378), comparing children who developed asthma between ages 6 and 10.5 years with nonasthmatic controls (83/295). Analyses included longitudinal differential gene expression, weighted gene co-expression network analysis, and cis-expression quantitative trait loci analysis. MEASUREMENTS AND MAIN RESULTS:At age 1 year, 42 genes, mostly upregulated in future asthmatics, were associated with neutrophilic inflammation and NLRP3 inflammasome-markers. By 4.5 years, this shifted to a novel eosinophil-related signature (40 genes), remaining increased in asthmatics until 10.5 years. Co-expression analysis confirmed a neutrophilic module at 1 year and eosinophilic modules at 4.5, 6, and 10.5 years, all associated with asthma. Fractional exhaled nitric oxide was associated with the eosinophilic module at age 6 years (P = .003). A total of 86 SNPs were identified modulating the expression of 10 eosinophil-associated genes and GSDMB from this eosinophilic signature. A variant-based genetic risk score was associated with asthma diagnosis (adjusted odds ratio [aOR], 1.47; 95% CI, 1.13-1.93). CONCLUSIONS:We identified a shift from a neutrophil-driven gene signature at age 1 year to a persistent eosinophilic signature at 4.5-10.5 years in asthmatic children, highlighting the 1- to 4.5-year period as the most vulnerable period. Genetic variants strongly influenced the persistent eosinophilic gene signature, comprising potential novel therapeutic targets.

Abstract Background The relevance of high-sensitivity C-reactive protein (hsCRP), a marker of low-grade systemic inflammation, remains unclear with regard to its association with severity and clinical outcomes in wheeze/asthma. We aimed to assess the role of hsCRP across different phenotypes and severity levels. Methods We studied children with preschool wheeze (≥ 2 episodes), and patients with GINA-defined asthma (school-age/adult) compared with healthy controls (HCs) in the well-characterized ALLIANCE (All Age Asthma Cohort) study. HsCRP was measured (AU5800®-CRP-Latex test) in 944 study participants (pediatric: n = 728; adult: n = 216) at baseline. Age-stratified analyses (age groups 0–5, 6–18, ≥ 18 years) of standardized log 10 -transformed hsCRP concentrations (age, sex, BMI, site) were performed using univariable tests and regression models. The validated ASSESS score and its dimensions (exacerbations, lung function, inhaled corticosteroids, symptom control) were primary outcomes. Results Adult patients with asthma showed higher hsCRP than HCs (OR 2.22, 95% CI 1.56–3.24). Across all ages, hsCRP increased with clinical severity of wheeze/asthma. The ASSESS score correlated positively with hsCRP in patients aged ≥ 6 years ( R = 0.19, p = 0.007 ). hsCRP was increased in school-age asthmatics with prior exacerbations (OR 1.37, 95% CI 1.01–1.87), and in adult asthmatics with impaired lung function ( R = 0.2, p = 0.013). Inhaled corticosteroid use was associated with lower hsCRP in preschool wheezers (OR 0.66, 95% CI 0.50–0.85) but higher levels in adults (OR 1.99, 95% CI 1.02–4.09). Conclusions HsCRP was increased in adult asthmatics compared to HCs and was associated with several severity-related clinical characteristics. ICS use was associated with higher hsCRP levels in adults, potentially reflecting greater disease severity, whereas ICS use in preschool wheezers was associated with lower hsCRP levels. These age-dependent effects may mirror varying disease courses across the lifespan and progression of asthma. The association of hsCRP with asthma severity in child- and adulthood may indicate its potential relevance for the course of disease and monitoring clinical outcomes. Future longitudinal studies are needed to assess, whether hsCRP may support therapy monitoring. Trial registration ClinicalTrials.gov; Pediatric arm: NCT02496468, Registration date: 03 July 2015; Adult arm: NCT02419274, Registration date: 14 April 2015.

Introduction: There is growing evidence that small airway disease indicated by ventilation heterogeneity plays an important role in subgrouip of children with bronchial asthma. Yet clinical implications of conducting N2 Multiple Breath Washout (N2MBW) measurements remain unclear. This study aims to evaluate the role of Lung clearance index (LCI) in the diagnosis of childhood asthma and in identifying children at risk for exacerbations and a potentially more severe course of disease. Materials and Methods: Children with preschool wheeze (6 mo.- 5 ys, min. 2 episodes of wheeze within past 12 months), or asthma (age 6-17 ys, doctor‘s diagnosis of asthma according to current guideline), and healthy age matched controls were included from the multicentre longitudinal All Age Asthma Cohort (ALLIANCE). Exacerbations were defined as increased intake of short-acting-beta-agonists (SABA) in the last twelve month. N2MBW was performed according to standard operating procedures using Spiroware and LungSim software. The upper limit of normal for LCI2,5% was set to 7.1. N2MBW, clinical data and spirometry were obtained during follow-up over up to 8 years. Results: We included 91 children with wheeze (6,6 years median age, 3.2-10.7I), 171 with asthma (13 years median age, 6.5-21.8) and 53 controls (13 years median age, 4.7-20.3). 51/364 (14 %) asthmatic patients, 44/141 (31.2%) wheezers and 2/53 (3.8%) healthy controls had abnormal LCI results. LCI2.5% baseline measurements had a high specificity (96.2%) to discriminate between asthmatic and healthy children, but low sensitivity (20.3%). The majority of asthmatic children with abnormal LCI2.5% 69% (24/35) had a normal FEV1 z-score. Children with evidence of more frequent exacerbations preceding baseline measurements presented with significantly higher LCI2.5% (p = 0.014, 6.68 – 6.86 LCI2.5%), and comparable FEV1 z-score (p=0.3). Mixed linear models identified eosinophil counts (beta= 0.15; 0.05-0.24CI; p=0.0043) to predict baseline LCI2.5% after adjusting for confounders, like BMI, FEV1, age, gender and center specific effects. Discussion: Our data shows that N2MBW might help to confirm asthma in symptomatic children with normal spirometry. Furthermore, it could help to identify a special phenotype with an increased T2 inflammation, increased exacerbation frequency and potentially more severe course of the disease.

BACKGROUND:Atopic eczema often develops in the first year of life, when the composition of the gut microbiota is most plastic as illustrated by the decrease in bifidobacteria after weaning. This may provide the opportunity for microbial stimuli and their environmental determinants to alter the disease course. OBJECTIVES:To determine the role of the genus Bifidobacterium for atopic eczema in early childhood. METHODS:We analysed the bacterial composition in fecal samples of 618 children of the PASTURE ("Protection against Allergy-Study in Rural Environments") birth cohort using 16S rRNA amplicon sequencing of fecal samples collected at 2 and 12 months of age. Atopic eczema was defined as a parent-reported doctor's diagnosis until 2 years, and patterns of rash symptoms were classified by latent class analysis. We applied mediation models to assess direct and microbiota-mediated effects of environmental determinants on atopic eczema. RESULTS:The Bifidobacterium composition observed at 2 months was inversely related to atopic eczema (OR = 0.68 [0.53-0.87], p = .002) and persistent rash. This association was not seen at 12 months, when the composition of Bifidobacterium amplicon sequence variants (ASVs) was altered. The effect of beneficial ASVs at 2 months (OR = 0.72 [0.57-0.91]) was lost at 12 months (OR = 0.97 [0.76-1.24]), when distinct bifidobacteria tended to be positively related to late-onset rash. CONCLUSIONS:The subgenus composition of Bifidobacterium undergoes substantial changes in the first year of life. The protective effect of Bifidobacterium depends on the ASV composition at the respective age of the infant, highlighting the importance of timing in prevention strategies targeting infant-microbe interactions.

This report is a summary of the presentations given at the European Respiratory Society's Research Seminar on Asthma Prevention. The seminar reviewed both epidemiological and mechanistic studies and concluded that; (i) reducing exposure of pregnant women and children to air pollution will reduce incident asthma, (ii) there are promising data that both fish oil and a component of raw cow's milk prevent asthma, and (iii) modulating trained immunity by either mimicking helminth infection or oral and sublingual bacterial products is a promising area of research.

Introduction: The course of asthma and wheezing episodes differs depending on age. During the pandemic, the number of respiratory infections was markedly reduced during lockdowns and turned to higher rates once hygiene measures were eased. Objective: To investigate the course of wheeze and asthma across all ages in the setting of a natural experiment with high and low exposure to infections. Methods: Within the ALL Age Asthma Cohort (ALLIANCE), a clinical multicenter longitudinal study, we assessed asthma course and infection burden using questionnaires during and after lockdown periods (Lockdown (L): Feb-Aug 2020, Oct–Apr 2020/21 n= 412, Post-Lockdown (PL) Jun-Oct 2021: n= 603). We investigated asthma features such as atopy and genetic background and conducted multivariate statistical models to identify factors affecting asthma course in children and adults. Results: During lockdown, the number of children with controlled asthma was higher than during post-lockdown (0-5y: L= 52% vs PL= 30%; 6-17y L= 68% vs PL = 60%). Change in asthma control was fully explained by a reduction in infectious burden in atopic children carrying a polymorphism in the IFNL2 gene. In contrast, the effect was much less pronounced in adults (18-59y L=32% vs PL= 28%; >60y L= 36% vs PL= 36%) and the genetic background did not play a significant role. IFNL2 gene expression in nasal mucosa showed higher expression levels in preschool children as compared to older children. Conclusion: Children with a polymorphism in the IFNL2 gene and co-occurrence of atopy are particularly prone to loss of wheeze or asthma control when exposed to viral infections. This effect is age-dependent, with higher susceptibility in young children and diminished effect in adults.

Background Transepidermal water loss (TEWL) has been used to measure skin barrier function and has been associated with atopic dermatitis and allergic diseases in infancy. However, few studies have assessed the association between TEWL and allergic diseases in adolescents. Objective To investigate the association between TEWL and allergic sensitizations in 16-year-old adolescents. Materials and methods The study was conducted in 78 adolescents of the PASTURE study. Different types of sensitization (seasonal, perennial, inhalant, food and any) were defined using serum specific immunoglobulin E (IgE) and skin prick test. TEWL was measured on the crook of either right or the left arm using a TEWAMETER® TM 300 (Courage + Khazaka electronic, Cologne, Germany) at mean temperature and humidity of 24.1 °C and 36.1%, respectively. The mean TEWL and interquartile range (IQR) were 11.9 ± 4.4 and 4.8 g/m 2 /h respectively. Results In our study, TEWL was positively associated with any sensitization (adjusted odds ratio [OR] per-IQR of the probability of increased TEWL [95% confidence interval]: OR 2.64; [1.12–6.19]; p = 0.03) and allergic rhinoconjunctivitis (ARC; OR 1.92; [1.04–3.54]; p = 0.04), but not with asthma or atopic dermatitis. When separating any sensitization into perennial and seasonal, only perennial sensitization (OR 3.30; [1.42–7.43]; p = 0.005) was associated with TEWL. Conclusion In the present study, we show the association of defective skin barrier function measured as TEWL with perennial sensitization and ARC suggesting its possible role in the pathogenesis of ARC through sensitization.

BACKGROUND:Consumption of raw cow's milk has repeatedly been shown to protect from asthma, allergies, and respiratory infections. As raw milk bears potential health hazards, it cannot be recommended for prevention. Therefore, we performed an intervention study with microbially safe but otherwise minimally processed cow's milk. Here we describe feasibility and safety of the trial. METHODS:The MARTHA trial (DRKS00014781) was set up as a double-blind randomized intervention in a population residing in Bavaria. Infants from 6 to 36 months of age consumed minimally processed cow's milk (intervention arm) or ultra-heat-treated (UHT) semi-skimmed milk (comparator arm). RESULTS:At the age of 6 to 12 months, 260 infants were enrolled, with 72% having a family history of atopy. The extensive screening system for milk consumption and symptoms suggestive of adverse events was well accepted with 22,988 completed weekly surveys and an average completion of 82% surveys sent out. The children consumed the study milk on average on 457 days (61% of intervention days). The intervention proved to be safe without any case of milk allergy or milk intolerance under the intervention in both arms. All 6 cases of serious adverse events were unrelated to milk. The most common reason was unscheduled hospitalization of more than 3 days. CONCLUSIONS:The intervention with minimally processed milk and the study instruments proved feasible. During the age of 6 to 36 months, there was no increased risk of milk allergy in a population with a substantial proportion of family history of atopy.

Patients with hypomorphic mutations in the RAG1 or RAG2 gene present with either Omenn syndrome or atypical combined immunodeficiency with a wide phenotypic range. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but data are scarce. We report on a worldwide cohort of 60 patients with hypomorphic RAG variants who underwent HSCT, 78% of whom experienced infections (29% active at HSCT), 72% had autoimmunity, and 18% had granulomas pretransplant. These complications are frequently associated with organ damage. Eight individuals (13%) were diagnosed by newborn screening or family history. HSCT was performed at a median of 3.4 years (range 0.3-42.9 years) from matched unrelated donors, matched sibling or matched family donors, or mismatched donors in 48%, 22%, and

PURPOSE:Numerous genes have been associated with allergic diseases (asthma, allergic rhinitis, and eczema), but they explain only part of their heritability. This is partly because most previous studies ignored complex mechanisms such as gene-environment (G-E) interactions and complex phenotypes such as co-morbidity. However, it was recently evidenced that the co-morbidity of asthma-plus-eczema appears as a sub-entity depending on specific genetic factors. Besides, evidence also suggest that gene-by-early life environmental tobacco smoke (ETS) exposure interactions play a role in asthma, but were never investigated for asthma-plus-eczema. To identify genetic variants interacting with ETS exposure that influence asthma-plus-eczema susceptibility. METHODS:To conduct a genome-wide interaction study (GWIS) of asthma-plus-eczema according to ETS exposure, we applied a 2-stage strategy with a first selection of single nucleotide polymorphisms (SNPs) from genome-wide association meta-analysis to be tested at a second stage by interaction meta-analysis. All meta-analyses were conducted across 4 studies including a total of 5,516 European-ancestry individuals, of whom 1,164 had both asthma and eczema. RESULTS:Two SNPs showed significant interactions with ETS exposure. They were located in 2 genes, NRXN1 (2p16) and TNS1 (2q35), never reported associated and/or interacting with ETS exposure for asthma, eczema or more generally for allergic diseases. TNS1 is a promising candidate gene because of its link to lung and skin diseases with possible interactive effect with tobacco smoke exposure. CONCLUSIONS:This first GWIS of asthma-plus-eczema with ETS exposure underlines the importance of studying sub-phenotypes such as co-morbidities as well as G-E interactions to detect new susceptibility genes.

In order to summarize recent research on the prevention of allergies-particularly asthma-and stimulate new activities for future initiatives, a virtual workshop sponsored by the EAACI Clemens von Pirquet foundation and EUFOREA was held in October 2021. The determinants of the "allergic march" as well as the key messages from intervention studies were reviewed by an international faculty of experts. Several unmet needs were identified, and a number of priorities for future studies were proposed.

BACKGROUND:Comprehensive studies investigated the role of T-cells in asthma which led to personalised treatment options targeting severe eosinophilic asthma. However, little is known about the contribution of B-cells to this chronic inflammatory disease. In this study we investigated the contribution of various B-cell populations to specific clinical features in asthma. METHODS:In the All Age Asthma Cohort (ALLIANCE), a subgroup of 154 adult asthma patients and 28 healthy controls were included for B-cell characterisation by flow cytometry. Questionnaires, lung function measurements, blood differential counts and allergy testing of participants were analysed together with comprehensive data on B-cells using association studies and multivariate linear models. RESULTS:Patients with severe asthma showed decreased immature B-cell populations while memory B-cells were significantly increased compared with both mild-moderate asthma patients and healthy controls. Furthermore, increased frequencies of IgA+ memory B-cells were associated with impaired lung function and specifically with parameters indicative for augmented resistance in the peripheral airways. Accordingly, asthma patients with small airway dysfunction (SAD) defined by impulse oscillometry showed increased frequencies of IgA+ memory B-cells, particularly in patients with mild-moderate asthma. Additionally, IgA+ memory B-cells significantly correlated with clinical features of SAD such as exacerbations. CONCLUSIONS:With this study we demonstrate for the first time a significant association of increased IgA+ memory B-cells with asthma and SAD, pointing towards future options for B-cell-directed strategies in preventing and treating asthma.

Here we report our results of a multi-center, open cohort study ("COVID-Kids-Bavaria") investigating the distribution of SARS-CoV-2 among children and staff in 99 daycare facilities and 48 elementary schools in Bavaria, Germany. Overall, 2568 children (1337 school children, 1231 preschool children) and 1288 adults (466 teachers, 822 daycare staff) consented to participate in the study and were randomly tested in three consecutive phases (September/October 2020, November/December 2020, March 2021). In total, 7062 throat swabs were analyzed for SARS-CoV-2 by RT-PCR. In phase I, only one daycare worker tested positive. In phase II, SARS-CoV-2 was detected in three daycare workers, two preschool children, and seven school children. In phase III, no sample tested positive. This corresponds to a positive test rate of 0.05% in phase I, 0.4% in phase II and 0% in phase III. After phase III, antibody testing was offered to 713 study participants in elementary schools. A seroprevalence rate of 7.7% (students) and 4.5% (teachers) was determined. We conclude that during the initial waves of the SARS-CoV-2 pandemic, the risk of a positive SARS-CoV-2 result correlated positively with the local 7-day incidence. Thus, an increased risk of SARS-CoV-2 transmission in the setting of daycare and elementary schooling was unlikely.

Background: The asthma syndrome comprises various phenotypes, which may manifest with similar symptoms in early childhood but follow different trajectories ranging from complete remission to gradual impairment of lung function. Objectives: To define longitudinal data-driven phenotypes in the pediatric arm of the ALLIANCE cohort. Methods: Individuals in the pediatric arm (N=607) were classified by a latent class analysis (LCA) based on clinical features including frequency of asthma symptoms, GINA asthma control items, hospitalization, atopic and eczematic symptoms. Data were collected at recruitment and three annual follow-up visits. The latent classes (LCs) were compared for age, heredity, biological measurements, medication, and genetics. Results: LCA yielded an 8-class solution, which assigned n=335 children to 4 less, and n=272 to 4 more severe phenotypes. Among the two most severe LCs, LC5 (n=59; mean age=3.8 yrs.) was characterized by absence of atopy in the beginning but a substantial increase in total specific IgE and eosinophils and an impaired lung function. LC8 (n=28; mean age=7.7 yrs.) was characterized by an early increase in IgE (p<0.01), FeNO (p=0.046), decrease in FEV1/FV-z-score (p<0.01) and an association with the risk allele encoded on chromosome 17q21 (OR=3.55 (95%CI: 1.37-9.17) for rs7216389). In both classes, lung function impairment was mainly determined by preceding hospitalisation. Conclusions: The current analysis revealed a substantial heterogeneity of disease entities subsumed as asthma or wheeze cases in the pediatric arm of ALLIANCE. The high resolution of this classification might be useful for assessing corresponding endotypes and for prediction of exacerbation and resolution of symptoms.

BACKGROUND:An important window of opportunity for early-life exposures has been proposed for the development of atopic eczema and asthma. OBJECTIVE:However, it is unknown whether hay fever with a peak incidence around late school age to adolescence is similarly determined very early in life. METHODS:In the Protection against Allergy-Study in Rural Environments (PASTURE) birth cohort potentially relevant exposures such as farm milk consumption and exposure to animal sheds were assessed at multiple time points from infancy to age 10.5 years and classified by repeated measure latent class analyses (n = 769). Fecal samples at ages 2 and 12 months were sequenced by 16S rRNA. Hay fever was defined by parent-reported symptoms and/or physician's diagnosis of hay fever in the last 12 months using questionnaires at 10.5 years. RESULTS:Farm children had half the risk of hay fever at 10.5 years (adjusted odds ratio [aOR] 0.50; 95% CI 0.31-0.79) than that of nonfarm children. Whereas early life events such as gut microbiome richness at 12 months (aOR 0.66; 95% CI 0.46-0.96) and exposure to animal sheds in the first 3 years of life (aOR 0.26; 95% CI 0.06-1.15) were determinants of hay fever, the continuous consumption of farm milk from infancy up to school age was necessary to exert the protective effect (aOR 0.35; 95% CI 0.17-0.72). CONCLUSIONS:While early life events determine the risk of subsequent hay fever, continuous exposure is necessary to achieve protection. These findings argue against the notion that only early life exposures set long-lasting trajectories.

In order to summarize recent research on the prevention of allergies-particularly asthma-and stimulate new activities for future initiatives, a virtual workshop sponsored by the EAACI Clemens von Pirquet foundation and EUFOREA was held in October 2021. The determinants of the "allergic march" as well as the key messages from intervention studies were reviewed by an international faculty of experts. Several unmet needs were identified, and a number of priorities for future studies were proposed.

Collective flow observables are known to be a sensitive tool to gain insights on the equation of state of nuclear matter from heavy-ion collision observations. Towards more quantitative constraints one has to carefully assess other influences on the collective behaviour. In this work a hadronic transport approach SMASH (Simulating Many Accelerated Strongly-interacting Hadrons) is applied to study the first four anisotropic flow coefficients in Au+Au collisions at Elab = 1.23A GeV in the context of the recently measured data by the HADES collaboration. In particular, the formation of light nuclei is important in this energy regime. Two different approaches are contrasted to each other: A clustering algorithm inspired by coalescence as well as microscopic formation of deuterons via explicit cross-sections. The sensitivity of directed and elliptic flow observables to the strength of the Skyrme mean field is explored. In addition, it is demonstrated that the rapidity-odd v3 coefficient is practically zero in this energy regime and the ratio of v4/v 2 2 is close to the value of 0.5 expected from hydrodynamic behaviour. This study establishes the current understanding of collective behaviour within the SMASH approach and lays the ground for future more quantitative constraints on the equation of state of nuclear matter within improved mean field calculations.

IntroductionHere we report our results of a multi-center, open cohort study (“COVID-Kids-Bavaria”) investigating the distribution of acute SARS-CoV-2 infections among children and staff in 99 daycare facilities and 48 elementary schools in Bavaria, Germany.Materials and MethodsOverall, 2,568 children (1,337 school children, 1,231 preschool children) and 1,288 adults (466 teachers, 822 daycare staff) consented to participate in the study and were randomly tested in three consecutive phases (September/October 2020, November/December 2020, March 2021). In total, 7,062 throat swabs were analyzed for SARS-CoV-2 by commercial RT-PCR kits.ResultsIn phase I, only one daycare worker tested positive. In phase II, SARS-CoV-2 was detected in three daycare workers, two preschool children, and seven school children. In phase III, no sample tested positive. This corresponds to a positive test rate of 0.05% in phase I, 0.4% in phase II and 0% in phase III. Correlation of a positive PCR test result with the local-7-day incidence values showed a strong association of a 7-day-incidence of more than 100/100,000 as compared to <100/100,000 (OR = 10.3 [1.5–438], p < 0.005). After phase III, antibody testing was offered to 713 study participants in elementary schools. A seroprevalence rate of 7.7% (students) and 4.5% (teachers) was determined.DiscussionDuring the initial waves of the SARS-CoV-2 pandemic, the risk of a positive SARS-CoV-2 result correlated positively with the local 7-day incidence. Hence, the occurrence of SARS-CoV-2 infections were reflected in schools and daycare facilities. An increased risk of SARS-CoV-2 transmission in the setting of daycare and elementary schooling was unlikely.