Project description:Epigenetics presents a dynamic approach to assess complex individual variation in obesity susceptibility. However, few studies have examined epigenetic patterns in preschool-age children, despite the relevance of this developmental stage to trajectories of weight gain, because of difficulties obtaining blood tissue samples. This proof of principle study examined DNA methylation in 92 saliva samples, comparing Latino preschool children of normal weight mothers (Body Mass Index [BMI] <27 kg/m2 and WC <90 cm) to children of obese mothers (BMI >30 kg/m2 and WC >100 cm). We hypothesized that salivary DNA methylation patterns in Latino preschool age children born of normal weight vs obese weight mothers would be: 1) associated with maternal BMI phenotype in continuous linear regression analysis; 2) saliva could demonstrate epigenetic variation across individuals; and 3) preschool child saliva would be differentially methylated when comparing those children with obese versus normal weight mothers. One hundred and nineteen CpG sites were significantly (p-value <1.56 X 10-5, p-value adjusted <.05) associated with maternal BMI in linear regression models controlling for childâs age, gender, and BMI. Of these 119 CpG sites, 41 were found within the transcription start site, 5â UTR, 3â UTR, or another regulatory region outside of the gene body. Saliva, a practical human tissue to obtain in naturalistic settings and in pediatric populations, was confirmed to be a viable medium for genome-wide epigenetic testing with maternal weight. Although not identical to results yielded from other human tissue types (i.e., cord blood samples), saliva findings indicate potential epigenetic differences in Latino preschool children at risk for pediatric obesity. This proof of principle study examined DNA methylation in 92 saliva samples, comparing Latino preschool children of normal weight mothers (Body Mass Index [BMI] <27 kg/m2 and WC <90 cm) to children of obese mothers (BMI >30 kg/m2 and WC >100 cm). Antropometry was measured objectively according to a standardized protocol.Saliva from preschool Latino children at risk for obesity (BMI>50% < 95% participating in WIC/SNAP programs) was collected using the Oragene DNA saliva kit following a strict data collection protocol. DNA extraction was performed as per DNA Genotek's recommendations using the PrepIT L2P reagent. Extracted DNA was stored in individually barcoded cryovials at â80 degrees Fahrenheit. For children, saliva was obtained using the âbaby brushâ approach, in which small sponges attached to plastic handles are inserted between cheek and gumline to absorb saliva .Arrays were processed using standard protocol [34], with 3 samples randomly selected to serve as duplicates and 1 sample run with HapMap DNA to test functionality of reagents. Duplicates were measured for high technique consistency with Pearson correlation coefficient (>.99). Methylation data were quality controlled using Illumina GenomeStudio (V2011.1), Methylation module (V1.9.0). Samples with lower than 98% call rate (i.e. <485,000 probes) were excluded. Any non-specific cross-reacting probes, probes carrying common SNPs (MAF >1%), or any probes with p-values greater than 0.05 for more than 20% of the sample were sequentially excluded. Validation via pyrosequencing was conducted.

Project description:Preschool children with recurrent wheezing are heterogeneous, with differing responses to respiratory viral infections. Although neutrophils are crucial for host defense, their function has not been studied in this population. We performed functional immunophenotyping on isolated blood neutrophils from 52 preschool children with recurrent wheezing (aeroallergen sensitization, n=16; no sensitization, n=36). Blood neutrophils were purified and cultured overnight with polyinosinic:polycytidylic acid (poly(I:C)) as a viral analog stimulus. Neutrophils underwent next-generation sequencing with Reactome pathway analysis and were analyzed for cytokine secretion, apoptosis, myeloperoxidase and extracellular DNA release. CD14+ monocytes were also exposed to neutrophil culture supernatant and analyzed for markers of M1 and M2 activation. 495 genes, related largely to the innate immune system and neutrophil degranulation, were differently expressed in children with versus without aeroallergen sensitization. Functional experiments identified more neutrophil degranulation and extracellular trap formation (i.e., more myeloperoxidase and extracellular DNA) and less neutrophil pro-inflammatory cytokine secretion in children with aeroallergen sensitization. Neutrophils also shifted CD14+ monocytes to a more anti-inflammatory (i.e., M2) phenotype in sensitized children and a more pro-inflammatory (i.e., M1) phenotype in non-sensitized children. Although both groups experienced viral exacerbations, annualized exacerbation rates prompting unscheduled healthcare were also higher in children without aeroallergen sensitization after enrollment. Systemic neutrophil responses to viral infection differ by allergic phenotype and may be less effective in preschool children without allergic inflammation. Further studies of neutrophil function are needed in this population, which often has less favorable therapeutic responses to inhaled corticosteroids and other therapies directed at T2-high inflammation.

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