BackgroundObesity is frequently complicated by comorbid conditions, yet how excess adipose contributes is poorly understood. Although adipocytes in obese individuals induce systemic inflammation via secreted cytokines, another potential mediator has recently been identified (i.e. adipocyte-derived exosomes). We hypothesized that adipocyte-derived exosomes contain mediators capable of activating end-organ inflammatory and fibrotic signaling pathways.MethodsWe developed techniques to quantify and characterize exosomes shed by adipocytes from 7 obese (age: 12–17.5 years, BMI: 33–50 kg/m2) and 5 lean (age: 11–19 years, BMI: 22–25 kg/m2) subjects.ResultsAbundant exosomal miRNAs, but no mRNAs, were detected. Comparison of obese vs. lean visceral adipose donors detected 55 differentially-expressed miRNAs (p<0.05; fold change≥|1.2|). qRT-PCR confirmed downregulation of miR-148b (ratio = 0.2 [95% confidence interval = 0.1, 0.6]) and miR-4269 (0.3 [0.1, 0.8]), and upregulation of miR-23b (6.2 [2.2, 17.8]) and miR-4429 (3.8 [1.1 to 13.4]). Pathways analysis identified TGF-β signaling and Wnt/ β-catenin signaling among the top canonical pathways expected to be altered with visceral adiposity based on projected mRNA targets for the 55 differentially expressed miRNAs. A select mRNA target was validated in vitro.ConclusionThese data show that visceral adipocytes shed exosomal-mediators predicted to regulate key end-organ inflammatory and fibrotic signaling pathways.

Background Given strong environmental influence on both epigenetic marks and allergic asthma in children, the epigenetic alterations in respiratory epithelia may provide insight into allergic asthma. Objective To identify DNA methylation and gene expression changes associated with childhood allergic persistent asthma. Methods We compared genomic DNA methylation patterns and gene expression in African American children with persistent atopic asthma[N=36] versus healthy controls[N=36]. Results were validated in an independent population of asthmatic children[N=30] using a shared healthy control population[N=36] and in independent population of Caucasian adult atopic asthmatics[N=12] and controls[N=12]. Results We identified 186 genes with significant methylation changes, differentially methylated regions(DMRs) or differentially methylated probes(DMPs), after adjustment for age, gender, race/ethnicity, batch effects, inflation, and multiple comparisons. Genes differentially methylated included those with established roles in asthma and atopy, genes related to extracellular matrix, immunity, cell adhesion, epigenetic regulation, and airflow obstruction. The methylation changes were substantial (median 9.5%, range:2.6–29.5%). Hypo- and hyper-methylated genes were associated with increased and decreased gene expression respectively (P<2.8x10−6 for DMRs and P<7.8x10−10 for DMPs). Quantitative analysis in 53 differentially expressed genes demonstrated that 32(60%) have significant methylation-expression relationships within 5kb of the gene. 10 loci selected based on the relevance to asthma, magnitude of methylation change, and methylation-expression relationships were validated in an independent cohort of children with atopic asthma. 67/186 genes also have significant asthma-associated methylation changes in nasal epithelia of adult Caucasian asthmatics. Conclusions Epigenetic marks in respiratory epithelia are associated with allergic asthma and gene expression changes in inner-city children.

Objective Low vitamin D levels have been implicated in the development of and increased morbidity from asthma. The prevalence of asthma among urban African American (AA) youth is high. The goal of this study was to examine the prevalence of vitamin D insufficiency and deficiency among urban AA youth with asthma compared with non-asthmatic controls. Study Design A cross-sectional case-control study was conducted at an urban pediatric medical center. Total 25-hydroxyvitamin D insufficiency (< 30 ng/mL) and deficiency (< 20 ng/mL) were assessed in urban self-reported AA patients, aged 6 to 20 years, with (n = 92) and without (n = 21) physician-diagnosed asthma. Results Blood samples were available for 85 (92%) cases. After adjusting for age, gender, body mass index percentile, and season of sampling, the median vitamin D level of cases [18.5 (interquartile range (IQR): 11.3, 25.1)] was significantly lower than that of controls [40.4 (IQR: 34.6, 49.5), P = 0.002]. The prevalences of vitamin D insufficiency and deficiency were significantly greater among cases than controls [73/85 (86%) vs. 4/21 (19%), adjusted odds ratio (OR) = 41.7 (95% confidence interval (95%CI): 4.4 to 398.5) for insufficiency and 46/85 (54%) vs. 1/21 (5%), adjusted OR = 19.5 (95%CI: 1.4 to 272.0) for deficiency]. Conclusions A majority of this sample of urban AA youth with persistent asthma were vitamin D deficient and/or insufficient. Given the emerging associations between low vitamin D levels and asthma, strong consideration should be given to routine vitamin D testing in urban AA youth, particularly those with asthma. Clinical trials of vitamin D supplementation among urban AA youth with asthma are warranted.

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Asthma is an inflammatory condition for which anti-inflammatory glucocorticoids are the standard of care. However, similar efficacy has not been shown for agents targeting inflammatory cells and pathways. This suggests a noninflammatory cell contributor (e.g., epithelium) to asthmatic inflammation. Herein, we sought to define the intrinsic and glucocorticoid-affected properties of asthmatic airway epithelium compared with normal epithelium. Human primary differentiated normal and asthmatic airway epithelia were cultured in glucocorticoid-free medium beginning at 248 hours. They were pulsed with dexamethasone (20 nM) or vehicle for 2 hours at 226, 22, 122, and 146 hours. Cultures were mechanically scrapewounded at 0 hours and exposed continuously to bromodeoxyuridine (BrdU). Cytokine secretions were analyzed using cytometric bead assays. Wound regeneration/mitosis was analyzed by microscopy and flow cytometry. Quiescent normal (n 5 3) and asthmatic (n 5 6) epithelia showed similar minimal inflammatory cytokine secretion and mitotic indices. After wounding, asthmatic epithelia secreted more basolateral TGF-b1, IL-10, IL-13, and IL-1b (P , 0.05) and regenerated less efficiently than normal epithelia (148 h wound area reduction 5 [mean 6 SEM] 50.2 6 7.5% versus 78.6 6 7.7%; P 5 0.02). Asthmatic epithelia showed 40% fewer BrdU 1 cells at 148 hours (0.32 6 0.05% versus 0.56 6 0.07% of total cells; P 5 0.03), and those cells were more dyssynchronously distributed along the cell cycle (52 6 10, 25 6 4, 23 6 7% for G1/G0, S, and G2/M, respectively) than normal epithelia (71 6 1, 12 6 2, and 17 6 2% for G1/G0, S, and G2/M, respectively). Dexamethasone pulses improved asthmatic epithelial inflammation and regeneration/mitosis. In summary, we show that inflammatory/fibrogenic cytokine secretions are correlated with dyssynchronous mitosis upon injury. Intermittent glucocorticoids simultaneously decreased epithelial cytokine secretions and resynchronized mitosis. These data, generated in an airway model lacking inflammatory cells, support the concept that epithelium contributes to asthmatic inflammation.

Background Pathway analyses can be used to determine how host and environmental factors contribute to asthma severity. Objective Investigate pathways explaining asthma severity in inner-city children. Methods Based on medical evidence in the published literature, we developed a conceptual model to describe how eight risk-factor domains (allergen sensitization, allergic inflammation, pulmonary physiology, stress, obesity, vitamin D, environmental tobacco smoke (ETS) exposure and rhinitis severity) are linked to asthma severity. To estimate the relative magnitude and significance of hypothesized relationships among these domains and asthma severity, we applied a causal network analysis to test our model in an Inner-City Asthma Consortium study. Participants comprised 6–17 year old children (n=561) with asthma and rhinitis from 9 U.S. inner-cities who were evaluated every two months for one year. Asthma severity was measured by a longitudinal composite assessment of day and night symptoms, exacerbations, and controller usage. Results Our conceptual model explained 53.4% of the variance in asthma severity. An allergy pathway (linking allergen sensitization, allergic inflammation, pulmonary physiology, and rhinitis severity domains to asthma severity) and ETS exposure pathway (linking ETS exposure and pulmonary physiology domains to asthma severity) exerted significant effects on asthma severity. Among the domains, pulmonary physiology and rhinitis severity had the largest significant standardized total effects on asthma severity (−0.51 and 0.48 respectively), followed by ETS exposure (0.30) and allergic inflammation (0.22). While vitamin D had modest but significant indirect effects on asthma severity, its total effect was insignificant (0.01). Conclusions The standardized effect sizes generated by a causal network analysis quantify the relative contributions of different domains and can be used to prioritize interventions to address asthma severity.

Introduction Low vitamin D levels have been associated with asthma severity in children. Young, urban African Americans (AA) have high rates of hypovitaminosis D and asthma. Our objective was to determine associations between variants in vitamin D metabolism genes and asthma characteristics in a pilot study of young urban AAs. Materials and Methods Two urban AA cohorts of subjects aged 6 to 20 years (139 subjects with asthma and 74 subjects without asthma) were genotyped for 12 single nucleotide polymorphisms (SNPs) in 3 vitamin D metabolism genes: VDR (vitamin D receptor), CYP24A1 (cytochrome P450 vitamin D 24-hydroxylase), and CYP2R1 (cytochrome P450 vitamin D 25-hydroxylase). In a case-control analysis, SNPs were studied for associations with an asthma diagnosis. Within the asthmatic cohort, SNPs were analyzed for associations with quantitative asthma characteristics. All analyses were adjusted for age, gender, and BMI percentile. Results Only the CYP2R1 SNP rs10766197 homozygous minor genotype was associated with asthma (P=0.044). CYP24A1 SNP rs2248137 was associated with lower vitamin D levels (P=0.006). Within the asthma cohort, multiple significant associations between SNPs and asthma characteristics were identified; VDR SNP rs2228570 was associated with the higher nighttime asthma morbidity scores (P=0.04), lower baseline spirometric measures (P<0.05), ≥1 positive aeroallergen skin test (P=0.003), and increased IgE levels (P<0.001). Discussion This pilot study demonstrates that variants in vitamin D metabolism genes are associated with quantitative asthma characteristics in young, urban AAs. The collection of these associations provides evidence for the need for a large population-based study of vitamin D relevant SNPs in this cohort.

Proteomic analysis of human body fluids is highly challenging, therefore many researchers are redirecting efforts towards secretome profiling. The goal is to define potential biomarkers and therapeutic targets in the secretome that can be traced back in accessible human body fluids. However, currently there is a lack of secretome profiles of normal human primary cells making it difficult to assess the biological meaning of current findings. In this study we sought to establish secretome profiles of human primary cells obtained from healthy donors with the goal of building a human secretome atlas. Such an atlas can be used as a reference for discovery of potential disease associated biomarkers and eventually novel therapeutic targets. As a preliminary study, secretome profiles were established for six different types of human primary cell cultures and checked for overlaps with the three major human body fluids including plasma, cerebrospinal fluid and urine. About 67% of the 1054 identified proteins in the secretome of these primary cells occurred in at least one body fluid. Furthermore, comparison of the secretome profiles of two human glioblastoma cell lines to this new human secretome atlas enabled unambiguous identification of potential brain tumor biomarkers. These biomarkers can be easily monitored in different body fluids using stable isotope labeled standard proteins. The long term goal of this study is to establish a comprehensive online human secretome atlas for future use as a reference for any disease related secretome study.