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1.  SERPINB3/B4 contributes to early inflammation and barrier dysfunction in an experimental murine model of atopic dermatitis 
Serine proteases are critical for epidermal barrier homeostasis and their aberrant expression and/or activity is associated with chronic skin diseases. Elevated levels of the serine protease inhibitors SERPINB3 and SERPINB4 are seen in patients with atopic dermatitis and psoriasis. However their mechanistic role in the skin is unknown. To evaluate the contribution of Serpinb3a (mouse homolog of SERPINB3 and SERPINB4) in atopic dermatitis, we examined the effect of topical Aspergillus fumigatus extract exposure in wild-type and Serpinb3a null mice on transepidermal water loss (TEWL), sensitization and inflammation. Allergen exposure induced Serpinb3a expression in the skin, along with increased TEWL, epidermal thickness, and skin inflammation, all of which were attenuated in the absence of Serpinb3a. Attenuated TEWL correlated with decreased expression of the pro-inflammatory marker S100A8. Silencing of SERPINB3/B4 in human keratinocytes decreased S100A8 expression supporting a role for SERPINB3/B4 in initiation of the acute inflammatory response. RNA-Seq analysis following allergen exposure identified a network of pro-inflammatory genes induced in the wild type mice that was absent in the Serpinb3a null mice. In conclusion, Serpinb3a deficiency attenuates barrier dysfunction and the early inflammatory response following cutaneous allergen exposure, supporting a role for Serpinb3a (mice) and SERPINB3/B4 (humans) early in atopic dermatitis.
PMCID: PMC4268075  PMID: 25111616
Atopic dermatitis; SERPINB3/B4; Serpinb3a; TEWL; S100A8; inflammation
2.  IL-13 Receptor Alpha 2 Contributes to Development of Experimental Allergic Asthma 
The Journal of allergy and clinical immunology  2013;132(4):10.1016/j.jaci.2013.04.016.
IL-13 receptor alpha2 (IL-13Rα2) binds IL-13 with high affinity and modulates IL-13 responses. There are soluble and membrane forms of IL-13Rα2 generated by alternative splicing in mice but humans express only the membrane form (memIL-13Rα2).
We determined the role of memIL-13Rα2 in development of allergic inflammation in mouse models of asthma.
IL-13Rα2-deficient and memIL-13Rα2 lung epithelium-specific transgenic mice were challenged with house dust mite (HDM). Airway hyperresponsiveness (AHR) and inflammation were assessed by airway pressure time index, bronchoalveolar lavage (BAL) cell counts and lung histology. The mucus production was determined by periodic acid-Schiff (PAS) staining of lung sections, western blot analysis of chloride channel calcium activated 3 (CLCA3) expression in lung homogenates, and ELISA of Muc5ac in BAL fluid (BALF). The expression of cytokines and chemokines was determined by RT-quantitative PCR.
In IL-13Rα2-deficient mice, AHR and airway inflammation were attenuated compared to wild type mice following HDM challenge. Lung epithelium overexpression of memIL-13Rα2 in the IL-13Rα2-deficient mice reconstituted AHR and inflammation to levels similar to those observed in HDM-challenged wild type mice. Mucus production was attenuated in lungs from HDM-treated IL-13Rα2-deficient mice while lung epithelium overexpression of memIL-13Rα2 increased mucus production. Lung epithelium overexpression of memIL-13Rα2 had no effect on the levels of sIL-13Rα2 in the serum or BALF and did not affect IL-13-dependent STAT6 activation in the lungs.
These data collectively support a distinct role for memIL-13Rα2 in lung, and suggest that memIL-13Rα2 may contribute to allergic inflammation.
PMCID: PMC3836839  PMID: 23763980
IL-13; IL-13 receptor; lung; airway hyperresponsiveness; airway inflammation
3.  The Greater Cincinnati Pediatric Clinic Repository: A Novel Framework for Childhood Asthma and Allergy Research 
Allergic disorders, including asthma, allergic rhinitis, atopic dermatitis, eosinophilic esophagitis, and food allergy, are a major global health burden. The study and management of allergic disorders is complicated by the considerable heterogeneity in both the presentation and natural history of these disorders. Biorepositories serve as an excellent source of data and biospecimens for delineating subphenotypes of allergic disorders, but such resources are lacking.
In order to define subphenotypes of allergic disease accurately, we established an infrastructure to link and efficiently utilize clinical and epidemiologic data with biospecimens into a single biorepository called the Greater Cincinnati Pediatric Clinic Repository (GCPCR). Children with allergic disorders as well as healthy controls are followed longitudinally at hospital clinic, emergency department, and inpatient visits. Subjects' asthma, allergy, and skin symptoms; past medical, family, social, diet, and environmental histories; physical activity; medication adherence; perceived quality of life; and demographics are ascertained. DNA is collected from all participants, and other biospecimens such as blood, hair, and nasal epithelial cells are collected on a subset.
To date, the GCPCR has 6,317 predominantly Caucasian and African American participants, and 93% have banked DNA. This large sample size supports adequately powered genetic, epidemiologic, environmental, and health disparities studies of childhood allergic diseases.
The GCPCR is a unique biorepository that is continuously evaluated and refined to achieve and maintain rigorous clinical phenotype and biological data. Development of similar disease-specific repositories using common data elements is necessary to enable studies across multiple populations of comprehensively phenotyped patients.
PMCID: PMC3377950  PMID: 22768387
4.  Genetic variation in small proline rich protein 2B as a predictor for asthma among children with eczema 
Small proline rich protein 2B (SPRR2B) is a skin and lung epithelial protein associated with allergic inflammation in mice that has not been evaluated in human atopic diseases.
To determine whether single-nucleotide polymorphisms (SNPs) in SPRR2B are associated with childhood eczema and with the phenotype of childhood eczema combined with asthma.
Genotyping for SPRR2B and filaggrin (FLG) was performed in 2 independent populations: the Cincinnati Childhood Allergy & Air Pollution Study (CCAAPS; N = 762; birth-age, 4 years) and the Greater Cincinnati Pediatric Clinical Repository (GCPCR;N = 1152; ages 5–10 years). Eczema and eczema plus asthma were clinical outcomes based on parental report and clinician’s diagnosis. Genetic analyses were restricted to whites and adjusted for sex in both cohorts and adjusted for environmental covariates in CCAAPS.
Variants in SPRR2B were not significantly associated with eczema in either cohort after Bonferroni adjustment. Children from both cohorts with the CC genotype of the SPRR2B rs6693927 SNP were at 4 times the risk for eczema plus asthma (adjusted odds ratio, 4.1; 95% confidence interval, 1.5– 10.9; P = .005 in CCAAPS; and adjusted odds ratio, 4.0; 95% confidence interval, 1.8 –9.1; P <.001 in the GCPCR), however. SNPs in SPRR2B were not in strong linkage disequilibrium with the R501X and del2282 FLG mutations, and these findings were independent of FLG.
An SNP in SPRR2B was predictive of asthma among white children with eczema from 2 independent populations. SPRR2B polymorphisms may serve as important predictive markers for the combined eczema plus asthma phenotype.
PMCID: PMC3759990  PMID: 22374195
5.  Down-regulation of Glutathione S-transferase Pi in Asthma Contributes to Enhanced Oxidative Stress 
Glutathione S-transferase Pi (GSTPi) is the predominant redox regulator in the lung. While evidence implicates an important role for GSTPi in asthma, the mechanism for this has remained elusive.
To determine how GSTPi is regulated in asthma and to elucidate its role in maintaining redox homeostasis.
We elucidated the regulation of GSTPi in children with asthma and utilized murine models of asthma to determine the role of GSTPi in redox homeostasis.
Measurements and Main Results
Our findings demonstrate that GSTPi transcript levels are markedly down-regulated in allergen and IL-13 treated mouse models of asthma via STAT6 dependent and independent pathways. Nuclear factor-erythroid 2 related factor 2 (Nrf2) was also down-regulated in these models. The decrease in GSTPi expression was associated with decreased total GST activity in the lungs of mice. Examination of cystine intermediates uncovered a functional role for GSTPi in regulating Cys oxidation, whereby GSTPi-deficient mice exhibited increased oxidative stress (increase in % cystine) compared with wild-type mice following allergen challenge. GSTPi expression was similarly down-regulated in children with asthma.
These data collectively suggest that down-regulation of GSTPi following allergen challenge may contribute to the asthma phenotype due to disruption of redox homeostasis and increased oxidative stress. Furthermore, GSTPi may be an important therapeutic target for asthma, and evaluation of GSTPi expression may prove beneficial in identifying individuals who would benefit from therapy targeting this pathway.
PMCID: PMC3164907  PMID: 21570714
GSTPi; asthma; oxidative stress; redox homeostasis; gene
6.  Functional Variant in the Autophagy-Related 5 Gene Promotor is Associated with Childhood Asthma 
PLoS ONE  2012;7(4):e33454.
Rationale and Objective
Autophagy is a cellular process directed at eliminating or recycling cellular proteins. Recently, the autophagy pathway has been implicated in immune dysfunction, the pathogenesis of inflammatory disorders, and response to viral infection. Associations between two genes in the autophagy pathway, ATG5 and ATG7, with childhood asthma were investigated.
Using genetic and experimental approaches, we examined the association of 13 HapMap-derived tagging SNPs in ATG5 and ATG7 with childhood asthma in 312 asthmatic and 246 non-allergic control children. We confirmed our findings by using independent cohorts and imputation analysis. Finally, we evaluated the functional relevance of a disease associated SNP.
Measurements and Main Results
We demonstrated that ATG5 single nucleotide polymorphisms rs12201458 and rs510432 were associated with asthma (p = 0.00085 and 0.0025, respectively). In three independent cohorts, additional variants in ATG5 in the same LD block were associated with asthma (p<0.05). We found that rs510432 was functionally relevant and conferred significantly increased promotor activity. Furthermore, Atg5 expression was increased in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic controls.
Genetic variants in ATG5, including a functional promotor variant, are associated with childhood asthma. These results provide novel evidence for a role for ATG5 in childhood asthma.
PMCID: PMC3335039  PMID: 22536318
7.  A non-redundant role for mouse Serpinb3a in the induction of mucus production in asthma 
Asthma is a major public health burden worldwide. Studies from our group and others have demonstrated that SERPINB3 and B4 are induced in asthmatics; however their mechanistic role in asthma has yet to be determined.
To evaluate the role of Serpin3a, the murine homolog of human SERPINB3 and B4, in asthma.
We studied wild type Balb/c and Serpinb3a null mice in house dust mite or IL-13 induced asthma models and evaluated airway hyperresponsiveness, inflammation, and goblet cell hyperplasia.
Airway hyperresponsiveness and goblet cell hyperplasia were markedly attenuated in the Serpinb3a null mice compared to the wild type mice following allergen challenge, with minimal effects on inflammation. Expression of SPDEF, a transcription factor that mediates goblet cell hyperplasia, was decreased in the absence of Serpinb3a. IL-13 treated Serpinb3a null mice showed attenuated AHR, inflammation, and mucus production.
Excessive mucus production and mucus plugging are key pathologic features of asthma, yet the mechanisms responsible for mucus production are not well understood. Our data reveal a novel non-redundant role for Serpinb3a in mediating mucus production through regulation of SPDEF expression. This pathway may be used to effectively target mucus hypersecretion.
PMCID: PMC3058372  PMID: 21126757
goblet cells; SPDEF; IL-13; hyperplasia
8.  Identification of KIF3A as a Novel Candidate Gene for Childhood Asthma Using RNA Expression and Population Allelic Frequencies Differences 
PLoS ONE  2011;6(8):e23714.
Asthma is a chronic inflammatory disease with a strong genetic predisposition. A major challenge for candidate gene association studies in asthma is the selection of biologically relevant genes.
Methodology/Principal Findings
Using epithelial RNA expression arrays, HapMap allele frequency variation, and the literature, we identified six possible candidate susceptibility genes for childhood asthma including ADCY2, DNAH5, KIF3A, PDE4B, PLAU, SPRR2B. To evaluate these genes, we compared the genotypes of 194 predominantly tagging SNPs in 790 asthmatic, allergic and non-allergic children. We found that SNPs in all six genes were nominally associated with asthma (p<0.05) in our discovery cohort and in three independent cohorts at either the SNP or gene level (p<0.05). Further, we determined that our selection approach was superior to random selection of genes either differentially expressed in asthmatics compared to controls (p = 0.0049) or selected based on the literature alone (p = 0.0049), substantiating the validity of our gene selection approach. Importantly, we observed that 7 of 9 SNPs in the KIF3A gene more than doubled the odds of asthma (OR = 2.3, p<0.0001) and increased the odds of allergic disease (OR = 1.8, p<0.008). Our data indicate that KIF3A rs7737031 (T-allele) has an asthma population attributable risk of 18.5%. The association between KIF3A rs7737031 and asthma was validated in 3 independent populations, further substantiating the validity of our gene selection approach.
Our study demonstrates that KIF3A, a member of the kinesin superfamily of microtubule associated motors that are important in the transport of protein complexes within cilia, is a novel candidate gene for childhood asthma. Polymorphisms in KIF3A may in part be responsible for poor mucus and/or allergen clearance from the airways. Furthermore, our study provides a promising framework for the identification and evaluation of novel candidate susceptibility genes.
PMCID: PMC3166061  PMID: 21912604
9.  Differences in Candidate Gene Association between European Ancestry and African American Asthmatic Children 
PLoS ONE  2011;6(2):e16522.
Candidate gene case-control studies have identified several single nucleotide polymorphisms (SNPs) that are associated with asthma susceptibility. Most of these studies have been restricted to evaluations of specific SNPs within a single gene and within populations from European ancestry. Recently, there is increasing interest in understanding racial differences in genetic risk associated with childhood asthma. Our aim was to compare association patterns of asthma candidate genes between children of European and African ancestry.
Methodology/Principal Findings
Using a custom-designed Illumina SNP array, we genotyped 1,485 children within the Greater Cincinnati Pediatric Clinic Repository and Cincinnati Genomic Control Cohort for 259 SNPs in 28 genes and evaluated their associations with asthma. We identified 14 SNPs located in 6 genes that were significantly associated (p-values <0.05) with childhood asthma in African Americans. Among Caucasians, 13 SNPs in 5 genes were associated with childhood asthma. Two SNPs in IL4 were associated with asthma in both races (p-values <0.05). Gene-gene interaction studies identified race specific sets of genes that best discriminate between asthmatic children and non-allergic controls.
We identified IL4 as having a role in asthma susceptibility in both African American and Caucasian children. However, while IL4 SNPs were associated with asthma in asthmatic children with European and African ancestry, the relative contributions of the most replicated asthma-associated SNPs varied by ancestry. These data provides valuable insights into the pathways that may predispose to asthma in individuals with European vs. African ancestry.
PMCID: PMC3046166  PMID: 21387019

Results 1-9 (9)