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Retinoids have been used for decades as efficacious topical agents to treat photoaged skin. The purpose of our present research is to evaluate whether the activity of the vitamin A ester retinyl propionate (RP) can be enhanced by niacinamide (Nam) and a flavonoid containing Ceratonia siliqua (CS) fruit extract in retinoid responsive in vitro models.Retinyl propionate was tested alone and in combination with Nam and CS in an RARα reporter cell line for promoter activation and compared to trans-retinoic acid (tRA) activation. These treatments were also tested in keratinocytes for gene expression profiling by qPCR using a panel of 40 retinoid responsive genes.tRA or RP elicited RARα reporter activation in a dose-dependent manner. The combination of 0.5 μM or 2 μM RP with 10 mM Nam had a 56% and 95% signal increase compared to RP, respectively. The addition of 1% CS to 0.5 μM or 2 μM RP with 10 mM Nam elicited a further increase of 114% and 156%, respectively, over RP and Nam combinations. All retinoids elicited an increase in expression of 40 retinoid sensitive genes over control levels. Of the 40 genes, 27 were enhanced by either 0.1 μM RP or 0.5 μM RP with 10 mM Nam and 1% CS. Nam or CS had very modest activity in both models.The combination of RP with Nam and CS showed a higher retinoid response than RP in two separate retinoid responsive in vitro models. We hypothesize Nam and CS enhances RP activity by modulating metabolism to tRA via increasing NADLes rétinoïdes sont utilisés depuis des décennies comme agents topiques efficaces pour traiter la peau photo-âgée. Le but de notre recherche actuelle est d’évaluer si l’activité du propionate rétinyl ester de vitamine A (RP) peut être augmentée par le niacinamide (Nam) et un flavonoïde contenant un extrait de fruit de Ceratonia Siliqua (CS) dans les modèles in vitro sensibles aux rétinoïdes. MÉTHODES: RP a été testé seul et en combinaison avec Nam et CS dans une ligne de cellule rapporteur de RARα pour l’activation du promoteur et par rapport à l’activation de l’acide transrétinoïque(tRA). Ces traitements ont également été testés dans les kératinocytes pour le profilage d’expression génique par qPCR à l’aide d’un panel de 40 gènes rétinoïdes sensibles. RÉSULTATS: tRA ou RP ont provoqué l’activation du promoteur RARα d’une manière dépendante de la dose. La combinaison de 0,5 µM ou 2 µM de RP avec 10 mM de Nam a permis une augmentation respectivement de 56% et 95% du signal par rapport à RP. L’ajout de 1 % de CS à 0,5 µM ou 2 µM de RP avec 10 mM de Nam a permis une nouvelle augmentation de 114 % et 156 %, respectivement, qu’avec la combinaison RP et Nam. Tous les rétinoïdes ont provoqué une augmentation de l’expression de 40 gènes sensibles aux rétinoïdes sur les niveaux de contrôle. Sur les 40 gènes, 27 ont été améliorés soit par 0,1 µM de RP ou 0.5 µM de RP avec 10 mM de Nam et 1% de CS. Nam ou CS avaient une activité très modeste dans les deux modèles.La combinaison de RP avec Nam et CS a montré une réponse rétinoïde plus élevée que RP dans deux modèles in vitro séparés sensibles aux rétinoïdes. Nous émettons l’hypothèse que Nam et CS améliorent l’activité RP en modulant le métabolisme de tRA par l’augmentation des groupement NAD

Ablative fractional laser treatment is considered the gold standard for skin rejuvenation. In order to understand how fractional laser works to rejuvenate skin, we performed microarray profiling on skin biopsies to identify temporal and dose-response changes in gene expression following fractional laser treatment. The backs of 14 women were treated with ablative fractional laser (Fraxel®) and 4 mm punch biopsies were collected from an untreated site and at the treated sites 1, 3, 7, 14, 21 and 28 days after the single treatment. In addition, in order to understand the effect that multiple fractional laser treatments have on skin rejuvenation, several sites were treated sequentially with either 1, 2, 3, or 4 treatments (with 28 days between treatments) followed by the collection of 4 mm punch biopsies. RNA was extracted from the biopsies, analyzed using Affymetrix U219 chips and gene expression was compared between untreated and treated sites. We observed dramatic changes in gene expression as early as 1 day after fractional laser treatment with changes remaining elevated even after 1 month. Analysis of individual genes demonstrated significant and time related changes in inflammatory, epidermal, and dermal genes, with dermal genes linked to extracellular matrix formation changing at later time points following fractional laser treatment. When comparing the age-related changes in skin gene expression to those induced by fractional laser, it was observed that fractional laser treatment reverses many of the changes in the aging gene expression. Finally, multiple fractional laser treatments resulted in continued changes in gene expression, with many genes either differentially regulated or continuously upregulated with increasing number of treatments, indicating that maximal skin rejuvenation requires multiple fractional laser treatments. In conclusion, fractional laser treatment of skin activates several biological processes involved in wound healing and tissue regeneration, all of which significantly contribute to the rejuvenating effect of fractional laser treatment on aged skin.

To explore synergistic effects related to skin regeneration, peptides with distinct biological mechanisms of action were evaluated in combination with different skin cell lines in the presence or absence of niacinamide (Nam). Furthermore, the synergistic responses of peptide combinations on global gene expression were compared with the changes that occur with fractional laser resurfacing treatment, a gold standard approach for skin rejuvenation, to further define optimal peptide combinations.Microarray profiling was used to characterize the biological responses of peptide combinations (+/- Nam) relative to the individual components in epidermal keratinocyte and dermal fibroblast cell lines. Cellular functional assays were utilized to confirm the synergistic effects of peptide combinations. Bioinformatics approaches were used to link the synergistic effects of peptide combinations on gene expression to the transcriptomics of the skin rejuvenation response from fractional laser treatment.Microarray analysis of skin cells treated with peptide combinations revealed synergistic changes in gene expression compared with individual peptide controls. Bioinformatic analysis of synergy genes in keratinocytes revealed the activation of NRF2-mediated oxidative stress responses by a combination of Ac-PPYL, Pal-KTTKS and Nam. Additional analysis revealed direct downstream transcriptional targets of NRF2/ARE exhibiting synergistic regulation by this combination of materials, which was corroborated by a cellular reporter assay. NRF2-mediated oxidative stress response pathways were also found to be activated in the transcriptomics of the early skin rejuvenation response to fractional laser treatment, suggesting the importance of this biology in the early stages of tissue repair. Additionally, the second combination of peptides (pal-KT and Ac-PPYL) was found to synergistically restore cellular ATP levels that had been depleted due to the presence of ROS, indicating an additional mechanism, whereby peptide synergies may accelerate skin repair.Through combinatorial synergy studies, we have identified additional in vitro skin repair mechanisms beyond the previously described functions of individual peptides and correlated these to the transcriptomics of the skin rejuvenation response of fractional laser treatment. These findings suggest that specific peptides can act together, via complementary and synergistic mechanisms, to holistically enhance the regenerative capacity of in vitro skin cells.Pour explorer les effets synergiques liés à la régénération cutanée, les peptides ayant des mécanismes d’action biologiques distincts ont été évalués en association dans différentes lignées cellulaires cutanées en présence ou en l’absence de niacinamide (Nam). De plus, les réponses synergiques des associations de peptides sur l’expression des gènes globale ont été comparées aux changements qui surviennent avec le traitement de resurfaçage au laser fractionné, une approche de référence pour le rajeunissement de la peau, afin de définir davantage les associations optimales de peptides. MÉTHODES: Le profilage de micro-réseau a été utilisé pour caractériser les réponses biologiques des combinaisons de peptides (+/-Nam) par rapport aux composants individuels dans les lignées cellulaires de kératinocytes épidermiques et de fibroblastes dermiques. Des tests fonctionnels cellulaires ont été réalisés pour confirmer les effets synergiques des associations de peptides. Des approches bio-informatiques ont été utilisées pour mettre en lien les effets synergiques des associations de peptides sur l’expression des gènes à la transcriptomique de la réponse de rajeunissement de la peau du traitement au laser fractionné. RÉSULTATS: L’analyse par micro-réseau des cellules cutanées traitées par des combinaisons de peptides a révélé des changements synergiques dans l’expression des gènes par rapport aux contrôles peptidiques individuels. L’analyse bio-informatique des gènes de synergie dans les kératinocytes a révélé une activation des réponses au stress oxydatif médiées par NRF2 par une association d’Ac-PPYL, de Pal-KTTKS et de Nam. Une analyse supplémentaire a révélé des cibles transcriptionnelles directes en aval de NRF2/ARE présentant une régulation synergique par cette combinaison de matériaux, qui a été corroborée par un test de gène rapporteur. Les voies de réponses au stress oxydatif médiées par NRF2 se sont également révélées activées dans la transcriptomique de la réponse précoce de rajeunissement cutané au traitement au laser fractionné, ce qui suggère l’importance de cette biologie dans les stades précoces de la réparation des tissus. De plus, une deuxième association de peptides (pal-KT et Ac-PPYL) s’est avérée restaurer de manière synergique les taux d’ATP cellulaire qui avaient été épuisés en raison de la présence de ROS, indiquant un mécanisme supplémentaire par lequel les synergies de peptides pourraient accélérer la réparation cutanée.Grâce à des études de synergie combinatoire, nous avons identifié des mécanismes de réparation cutanés in vitro supplémentaires au-delà des fonctions précédemment décrites des peptides individuels et les avons corrélés à la transcriptomique de la réponse de rajeunissement de la peau au traitement au laser fractionné. Ces résultats suggèrent que des peptides spécifiques peuvent agir ensemble, par le biais de mécanismes complémentaires et synergiques, pour améliorer de manière globale la capacité régénérative des cellules cutanées in vitro.

Ablative fractional laser treatment is considered the gold standard for skin rejuvenation. In order to understand how fractional laser works to rejuvenate skin, we performed microarray profiling on skin biopsies to identify temporal and dose-response changes in gene expression following fractional laser treatment. The backs of 14 women were treated with ablative fractional laser (Fraxel®) and 4 mm punch biopsies were collected from an untreated site and at the treated sites 1, 3, 7, 14, 21 and 28 days after the single treatment. In addition, in order to understand the effect that multiple fractional laser treatments have on skin rejuvenation, several sites were treated sequentially with either 1, 2, 3, or 4 treatments (with 28 days between treatments) followed by the collection of 4 mm punch biopsies. RNA was extracted from the biopsies, analyzed using Affymetrix U219 chips and gene expression was compared between untreated and treated sites. We observed dramatic changes in gene expression as early as 1 day after fractional laser treatment with changes remaining elevated even after 1 month. Analysis of individual genes demonstrated significant and time related changes in inflammatory, epidermal, and dermal genes, with dermal genes linked to extracellular matrix formation changing at later time points following fractional laser treatment. When comparing the age-related changes in skin gene expression to those induced by fractional laser, it was observed that fractional laser treatment reverses many of the changes in the aging gene expression. Finally, multiple fractional laser treatments, which cover different regions of a treatment area, resulted in a sustained or increased dermal remodeling response, with many genes either differentially regulated or continuously upregulated, supporting previous observations that maximal skin rejuvenation requires multiple fractional laser treatments. In conclusion, fractional laser treatment of human skin activates a number of biological processes involved in wound healing and tissue regeneration.

OBJECTIVESRetinoids have been used for decades as efficacious topical agents to treat photoaged skin. The purpose of our present research is to evaluate whether the activity of the Vitamin A ester retinyl propionate (RP) can be enhanced by niacinamide (Nam) and a flavonoid containingCeratonia siliqua(CS) fruit extract in retinoid responsivein vitromodels.METHODSRP was tested alone and in combination with Nam andCSin an RARα reporter cell line for promoter activation and compared totrans-retinoic acid (tRA) activation. These treatments were also tested in keratinocytes for gene expression profiling by qPCR using a panel of 40 retinoid responsive genes.RESULTStRA or RP elicited RARα reporter activation in a dose dependent manner. The combination of 0.5 μM or 2 μM RP with 10 mM Nam had a 56% and 95% signal increase compared to RP, respectively. The addition of 1%CSto 0.5 μM or 2 μM RP with 10 mM Nam elicited a further increase of 114% and 156%, respectively, over RP and Nam combinations. All retinoids elicited an increase in expression of 40 retinoid sensitive genes over control levels. Of the 40 genes 27 were enhanced by either 0.5 μM RP or 2 μM RP with 10 mM Nam and 1%CS. Nam orCShad very modest activity in both models.CONCLUSIONThe combination of RP with Nam andCSshowed a higher retinoid response than RP in two separate retinoid responsivein vitromodels. We hypothesize Nam andCSenhances RP activity by modulating metabolism to tRA via increasing NAD+pools and inhibiting reduction of retinal (RAL) back to retinol, respectively. The findings provide evidence that this combination may have enhanced efficacy for treating the appearance of photoaged skin.

Human skin is exposed daily to environmental stressors, which cause acute damage and inflammation. Over time this leads to morphological and visual appearance changes associated with premature aging. Topical vitamin A derivatives such as retinol (ROL), retinyl palmitate (RPalm), and retinyl propionate (RP) have been used to reverse these changes and improve the appearance of skin. This study investigated a stoichiometric comparison of these retinoids usingin vitroandex vivoskin models. Skin biopsies were treated topically to compare skin penetration and metabolism. Treated keratinocytes were evaluated for transcriptomics profiling and hyaluronic acid (HA) synthesis and treated 3D epidermal skin equivalents were stained for epidermal thickness, Ki67, and filaggrin. A retinoic acid receptor-alpha (RARα) reporter cell line was used to compare retinoid activation levels. Results fromex vivoskin found that RP and ROL have higher penetration levels compared to RPalm. RP is metabolized primarily into ROL in the viable epidermis and dermis whereas ROL is esterified into RPalm and metabolized into the inactive retinoid 14-hydroxy-4,14-retro-retinol (14-HRR). RP treatment yielded higher RARα activation and HA synthesis levels than ROL whereas RPalm had a null effect. In keratinocytes, RP and ROL stimulated similar gene expression patterns and pathway theme profiles. In conclusion, RP and ROL show a similar response directionality whereas RPalm response was inconsistent. Additionally, RP has a consistently higher magnitude of response compared with ROL or RPalm.

Alterations in metabolism in skin are accelerated by environmental stressors such as solar radiation, leading to premature aging. The impact of aging on mitochondria is of interest given their critical role for metabolic output and the finding that environmental stressors cause lowered energy output, particularly in fibroblasts where damage accumulates. To better understand these metabolic changes with aging, we performed an in‐depth profiling of the expression patterns of dermal genes in face, forearm, and buttock biopsies from females of 20–70 years of age that encode for all subunits comprising complexes I‐V of the mitochondrial electron transport chain. This complements previous preliminary analyses of these changes. “Oxidative phosphorylation” was the top canonical pathway associated with aging in the face, and genes encoding for numerous subunits had decreased expression patterns with age. Investigations on fibroblasts from older aged donors also showed decreased gene expression of numerous subunits from complexes I‐V, oxidative phosphorylation rates, spare respiratory capacity, and mitochondrial number and membrane potential compared to younger cells. Treatment of older fibroblasts with nicotinamide (Nam) restored these measures to younger cell levels. Nam increased complexes I, IV, and V activity and gene expression of representative subunits. Elevated mt‐Keima staining suggests a possible mechanism of action for these restorative effects via mitophagy. Nam also improved mitochondrial number and membrane potential in younger fibroblasts. These findings show there are significant changes in mitochondrial functionality with aging and that Nam treatment can restore bioenergetic efficiency and capacity in older fibroblasts with an amplifying effect in younger cells., A decline in mitochondrial quality was observed in primary human skin fibroblasts with age, with a decrease in mitochondrial complex gene expression, oxidative phosphorylation rates, spare respiratory capacity, mitochondrial number, membrane potential, and mitophagy. The addition of Nam (nicotinamide, vitamin B3) improved these age‐related changes in cells from older aged individuals to levels observed in younger individuals, with an amplifying effect seen in younger cells.

Objective To evaluate whether niacinamide (Nam) can mitigate production of inflammatory and senescence-related biomarkers induced by environmental stressors. Methods Human epidermal keratinocytes were exposed to UVB, urban dust, diesel exhaust and cigarette smoke extract and treated with Nam or vehicle control. Full thickness 3-D skin organotypic models were exposed to a combination of UVB and PM2.5 and treated with Nam or vehicle control. Quantitation of the SASP-related inflammatory mediators PGE2 , IL-6 and IL-8 was performed on cultured media. UVB-exposed keratinocytes treated with and without Nam were immunostained for the senescence biomarker Lamin B1 (LmnB1). Transcriptomics profiling of cigarette smoke extract effects on keratinocytes was performed. A double-blind, placebo-controlled clinical was conducted on 40 female panellists that were pretreated on back sites for two weeks with 5% Nam or vehicle and then exposed to 1.5 minimal erythemal dose (MED) solar-simulated radiation (SSR). Treated sites were compared with non-treated exposed sites for erythema and the skin surface IL-1αRA/IL-1α inflammatory biomarkers. Results Ultraviolet B induced synthesis of PGE2 , IL-8 and IL-6 and reduced LmnB1 levels in keratinocytes. Urban dust and diesel exhaust only stimulated synthesis of IL-8 whereas cigarette smoke extract only stimulated levels of PGE2 . In all exposures, treatment with Nam significantly mitigated synthesis of the inflammatory mediators and restored levels of UVB-reduced LmnB1. In the 3D skin equivalent model, Nam reduced IL-8 levels stimulated by a combination of topical PM2.5 and UV exposure. In a UV challenge clinical, pretreatment with 5% Nam reduced erythema and skin surface IL-1αRA/IL-1α inflammatory biomarkers that were induced by SSR. Conclusion Since it is known that Nam has anti-inflammatory properties, we tested whether Nam can inhibit environmental stress-induced inflammation and senescence-associated secretory phenotype (SASP) biomarkers. We show Nam can reduce PGE2 , IL-6 and IL-8 levels induced by environmental stressors. Additionally, in vivo pretreatment with Nam can reduce UV-induced erythema and skin surface inflammatory biomarkers. These findings add to the body of evidence that Nam can mitigate the skin's inflammatory response elicited by environmental stressors. This supports Nam can potentially inhibit senescence and premature ageing and thereby maintain skin's functionality and appearance.

Background Macromolecules in skin cells are damaged when exposed to environmental stressors, leading to disrupted cellular function and homeostasis. While epidermal turnover can eliminate some of this damage, autophagy can rapidly remove these defective components. Niacinamide (Nam) is known to induce autophagy and optimizing formulations to maximize this response could provide improved homeostasis in stressed skin. Objective To determine (i) whether Nam can induce autophagy related 5 (ATG5), an autophagy marker, in human keratinocytes and (ii) whether optimized low pH Nam formulations can enhance the response in 3D skin models. Methods Human keratinocytes treated with Nam were evaluated for autophagosome accumulation and induction of ATG5 by gene expression, immunoblotting and immune-fluorescence microscopy. 3D skin equivalents were topically treated with Nam formulations at pH 5.8 and 3.8. Gene expression profiling and immunoblot analysis of ATG5 were performed. Results Nam treatment of keratinocytes led to an accumulation of autophagosomes with a maximal signal at 48 h. Gene expression of ATG5 was induced by Nam, and immunoblots stained for ATG5 showed a significant increase after 6 h of treatment. Gene expression profiling of 3D epidermal skin equivalents treated with Nam at pH 3.8 showed stronger induction of autophagy-related genes, including ATG5, compared with pH 5.8 formulas. Enrichment for gene ontology terms on autophagy showed an increased linkage with Nam formulas at pH 3.8. Conclusions We found that Nam induces autophagosome accumulation and ATG5 levels in keratinocytes. We also discovered that a Nam formulation at pH 3.8 can further increase levels of ATG5 in 3D skin models when compared to Nam at pH 5.8. These data support that Nam can induce autophagy in keratinocytes and formulations at pH 3.8 can enhance the impact. We hypothesize that optimized formulations at pH 3.8 can improve skin ageing appearance via autophagy induction.

BackgroundFor circadian medicine to influence health, such as when to take a drug or undergo a procedure, a practical way to measure body time is needed. Recent machine learning algorithms show that gene expression data from blood and skin can provide reliable estimates of body time. However, for clinical viability, a biomarker must be easily measured and generalizable to a broad population. It is not clear that any circadian biomarker yet satisfies these criteria.ResultsWe analyzed 24 h molecular rhythms in human dermis and epidermis at three distinct body sites, leveraging both longitudinal and population data. Circadian clock function was strongest in the epidermis, regardless of body site. We identified a 12-gene biomarker set that reported circadian phase to within 3 hours from a single sample of epidermis—the skin’s most superficial layer. This set performed well across body sites, ages, sexes, and detection platforms.ConclusionsThis research shows that the clock in epidermis is more robust than dermis regardless of body site. To encourage ongoing validation of this biomarker in diverse populations, diseases, and experimental designs, we developed SkinPhaser—a user-friendly app to test biomarker performance in datasets (https://github.com/gangwug/SkinPhaser).

Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of the esophagus featuring increased esophageal interleukin-13 (IL-13) levels and impaired barrier function. Herein, we investigated leucine-rich repeat-containing protein 31 (LRRC31) in human EoE esophageal tissue and IL-13-treated esophageal epithelial cells. LRRC31 had basal mRNA expression in colonic and airway mucosal epithelium. Esophageal LRRC31 mRNA and protein increased in active EoE and strongly correlated with esophageal eosinophilia and IL13 and CCL26 (chemokine (C-C motif) ligand 26) mRNA expression. IL-13 treatment increased LRRC31 mRNA and protein in air-liquid interface-differentiated esophageal epithelial cells (EPC2s). At baseline, differentiated LRRC31-overexpressing EPC2s had increased barrier function (1.9-fold increase in transepithelial electrical resistance (P

Skin is the largest organ in the body and serves important barrier, regulatory, and sensory functions. The epidermal layer shows rhythmic physiological responses to daily environmental variation (e.g., DNA repair). We investigated the role of the circadian clock in the transcriptional regulation of epidermis using a hybrid experimental design, in which a limited set of human subjects ( n = 20) were sampled throughout the 24-h cycle and a larger population ( n = 219) were sampled once. We found a robust circadian oscillator in human epidermis at the population level using pairwise correlations of clock and clock-associated genes in 298 epidermis samples. We then used CYCLOPS to reconstruct the temporal order of all samples, and identified hundreds of rhythmically expressed genes at the population level in human epidermis. We compared these results with published time-series skin data from mice and found a strong concordance in circadian phase across species for both transcripts and pathways. Furthermore, like blood, epidermis is readily accessible and a potential source of biomarkers. Using ZeitZeiger, we identified a biomarker set for human epidermis that is capable of reporting circadian phase to within 3 hours from a single sample. In summary, we show rhythms in human epidermis that persist at the population scale and describe a path to develop robust single-sample circadian biomarkers.

Triggers of skin disease pathogenesis vary, but events associated with the elicitation of a lesion share many features in common. Our objective was to examine gene expression patterns in skin disease to develop a molecular signature of disruption of cutaneous homeostasis. Gene expression data from common inflammatory skin diseases (eg psoriasis, atopic dermatitis, seborrhoeic dermatitis and acne) and a novel statistical algorithm were used to define a unifying molecular signature referred to as the "unhealthy skin signature" (USS). Using a pattern-matching algorithm, analysis of public data repositories revealed that the USS is found in diverse epithelial diseases. Studies of milder disruptions of epidermal homeostasis have also shown that these conditions converge, to varying degrees, on the USS and that the degree of convergence is related directly to the severity of homeostatic disruption. The USS contains genes that had no prior published association with skin, but that play important roles in many different disease processes, supporting the importance of the USS to homeostasis. Finally, we show through pattern matching that the USS can be used to discover new potential dermatologic therapeutics. The USS provides a new means to further interrogate epithelial homeostasis and potentially develop novel therapeutics with efficacy across a spectrum of skin conditions.

Eosinophilic esophagitis (EoE) is an allergic inflammatory esophageal disorder with a complex underlying genetic etiology often associated with other comorbidities. Using whole-exome sequencing (WES) of 63 patients with EoE and 60 unaffected family members and family-based trio analysis, we sought to uncover rare coding variants. WES analysis identified 5 rare, damaging variants in dehydrogenase E1 and transketolase domain–containing 1 (DHTKD1). Rare variant burden analysis revealed an overabundance of putative, potentially damaging DHTKD1 mutations in EoE (P = 0.01). Interestingly, we also identified 7 variants in the DHTKD1 homolog oxoglutarate dehydrogenase-like (OGDHL). Using shRNA-transduced esophageal epithelial cells and/or patient fibroblasts, we further showed that disruption of normal DHTKD1 or OGDHL expression blunts mitochondrial function. Finally, we demonstrated that the loss of DHTKD1 expression increased ROS production and induced the expression of viperin, a gene previously shown to be involved in production of Th2 cytokines in T cells. Viperin had increased expression in esophageal biopsies of EoE patients compared with control individuals and was upregulated by IL-13 in esophageal epithelial cells. These data identify a series of rare genetic variants implicating DHTKD1 and OGDHL in the genetic etiology of EoE and underscore a potential pathogenic role for mitochondrial dysfunction in EoE.

Skin is the largest organ in the body and serves important barrier, regulatory, and sensory functions. Like other tissues, skin is subject to temporal fluctuations in physiological responses under both homeostatic and stressed states. To gain insight into these fluctuations, we investigated the role of the circadian clock in the transcriptional regulation of epidermis using a hybrid experimental design, where a limited set of human subjects (n=20) were sampled throughout the 24 h cycle and a larger population (n=219) were sampled once. By looking at pairwise correlations of core clock genes in 298 skin samples, we found a robust circadian oscillator in skin at the population level. Encouraged by this, we used CYCLOPS to reconstruct the temporal order of all samples and identified hundreds of rhythmically-expressed genes at the population level in human skin. We compared these results with published time-series skin data from mouse and show strong concordance in circadian phase across species for both transcripts and pathways. Further, like blood, skin is readily accessible and a potential source of biomarkers. Using ZeitZeiger, we identified a biomarker set for human skin that is capable of reporting circadian phase to within 3 h from a single sample. In summary, we show rhythms in human skin that persist at the population scale and a path to develop robust single-sample circadian biomarkers.One Sentence SummaryHuman epidermis shows strong circadian rhythms at the population scale and provides a better source for developing robust, single-sample circadian phase biomarkers than human blood.

Background Eosinophilic esophagitis (EoE) is increasingly common, but data on phenotypic aspects are still incomplete. Objectives To describe the clinical, endoscopic, and histopathologic features of a large number of children and adults with EoE across the United States. Methods This was a multisite single visit registry enrolling subjects aged 6 months to 65 years with EoE. Participants provided responses regarding their medical history, with verification of the diagnosis and history by the study teams. Results A total of 705 subjects were analyzed (median [interquartile range] age at enrollment 11.2 [6.7-17.7] years, 68.2% male, 87.9% whites). Of these, 67 subjects had concurrent gastrointestinal eosinophilia, with gastric mucosa most common. An age- and race-dependent time gap was present between symptom onset and time of diagnosis (adults and whites with longer gap). Food allergy and atopic dermatitis were associated with a decrease in this gap. Symptoms varied with age (more dysphagia and food impaction in adults) and with race (more vomiting in non-whites). Esophageal rings and strictures at diagnosis were more common in adults, although esophageal eosinophilia was comparable among age groups. Concomitant allergic disease (91%), infectious/immunologic disorders (44%), neurodevelopmental disorders (30%), and failure to thrive (21%) were common. Depression/anxiety increased with age. EoE was reported in 3% of parents and 4.5% of siblings. Conclusions Gastrointestinal eosinophilia is present in approximately 10% of patients with EoE; the symptom-diagnosis time gap is influenced by age, race, food allergy, and atopic dermatitis; symptoms vary with race; concurrent infectious/immunologic disorders and mental health disorders are common; and the level of esophageal eosinophils is comparable in patients with and without fibrostenotic features.

Background Eosinophilic esophagitis (EoE) is an inflammatory disorder of the esophagus defined by eosinophil infiltration and tissue remodeling with resulting symptoms of esophageal dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) promotes inflammation through upregulation of the E3 ubiquitin-ligase midline-1 (MID1), which binds to and deactivates the catalytic subunit of protein phosphatase 2Ac, resulting in increased nuclear factor κB activation. Objective We sought to elucidate the role of TRAIL in EoE. Methods We used Aspergillus fumigatus to induce EoE in TRAIL-sufficient (wild-type) and TRAIL-deficient (TRAIL −/− ) mice and targeted MID1 in the esophagus with small interfering RNA. We also treated mice with recombinant thymic stromal lymphopoietin (TSLP) and TRAIL. Results TRAIL deficiency and MID1 silencing with small interfering RNA reduced esophageal eosinophil and mast cell numbers and protected against esophageal circumference enlargement, muscularis externa thickening, and collagen deposition. MID1 expression and nuclear factor κB activation were reduced in TRAIL −/− mice, whereas protein phosphatase 2Ac levels were increased compared with those seen in wild-type control mice. This was associated with reduced expression of CCL24, CCL11, CCL20, IL-5, IL-13, IL-25, TGFB, and TSLP. Treatment with TSLP reconstituted hallmark features of EoE in TRAIL −/− mice and recombinant TRAIL induced esophageal TSLP expression in vivo in the absence of allergen. Post hoc analysis of gene array data demonstrated significant upregulation of TRAIL and MID1 in a cohort of children with EoE compared with that seen in controls. Conclusion TRAIL regulates MID1 and TSLP, inflammation, fibrosis, smooth muscle hypertrophy, and expression of inflammatory effector chemokines and cytokines in experimental EoE.

Cadherins (CDH) mediate diverse processes critical in inflammation, including cell adhesion, migration, and differentiation. Herein, we report that the uncharacterized cadherin 26 (CDH26) is highly expressed by epithelial cells in human allergic gastrointestinal tissue. In vitro, CDH26 promotes calcium-dependent cellular adhesion of cells lacking endogenous CDHs by a mechanism involving homotypic binding and interaction with catenin family members (alpha, beta, and p120), as assessed by biochemical assays. Additionally, CDH26 enhances cellular adhesion to recombinant integrin α4β7 in vitro; conversely, recombinant CDH26 binds αE and α4 integrins in biochemical and cellular functional assays, respectively. Interestingly, CDH26-Fc inhibits activation of human CD4(+) T cells in vitro including secretion of IL-2. Taken together, we have identified a novel functional CDH regulated during allergic responses with unique immunomodulatory properties, as it binds α4 and αE integrins and regulates leukocyte adhesion and activation, and may thus represent a novel checkpoint for immune regulation and therapy via CDH26-Fc.

Eosinophilic esophagitis (EoE) is a complex, polygenic disorder caused by genetic predisposition and environmental exposures. Because of the recent emergence of EoE as a bona fide global health concern, a paucity of available therapeutic and diagnostic options exists. However, rapid progress has been made in an effort to rectify this lack and to improve understanding of the factors that cause EoE. This article highlights key advances in elucidating the genetic (and epigenetic) components involved in EoE.

Eosinophilic esophagitis (EoE) is an allergic inflammatory disorder of the esophagus that is compounded by genetic predisposition and hypersensitivity to environmental antigens. Using high-density oligonucleotide expression chips, a disease-specific esophageal transcript signature was identified and was shown to be largely reversible with therapy. In an effort to expand the molecular signature of EoE, we performed RNA sequencing on esophageal biopsies from healthy controls and patients with active EoE and identified a total of 1607 significantly dysregulated transcripts (1096 upregulated, 511 downregulated). When clustered by raw expression levels, an abundance of immune cell-specific transcripts are highly induced in EoE but expressed at low (or undetectable) levels in healthy controls. Moreover, 66% of the gene signature identified by RNA sequencing was previously unrecognized in the EoE transcript signature by microarray-based expression profiling and included several long non-coding RNAs (lncRNA), an emerging class of transcriptional regulators. The lncRNA BRAF-activated non-protein coding RNA (BANCR) was upregulated in EoE and induced in interleukin-13 (IL-13)-treated primary esophageal epithelial cells. Repression of BANCR significantly altered the expression of IL-13-induced proinflammatory genes. Together, these data comprise new potential biomarkers of EoE and demonstrate a novel role for lncRNAs in EoE and IL-13-associated responses.

Eosinophilic esophagitis (EoE) is a complex genetic disorder characterized by eosinophilic inflammation within the esophagus. Multiple epidemiological studies estimate the prevalence of EoE is 4 in 10,000, with a higher disease prevalence in individuals of European ancestry and in males, highlighting a genetic etiology of the disease. EoE has often been noted to occur in multiple family members, particularly siblings, in a non-Mendelian pattern, indicating the heritable component of EoE is likely complex in nature. Although EoE is a newly diagnosed disorder involving a complex polygenic etiology, much progress has been made towards identifying the molecular pathways contributing to the disease pathogenesis and the genetic variants associated with disease susceptibility using a variety of approaches (genome-wide and candidate gene) as well as study designs (case-control and family-based cohorts). Here, we discuss the major scientific findings that have shaped the current molecular and genetic landscape of EoE as well as the major obstacles in the discovery of disease causal variants in complex disorders.

The desmosomal cadherin desmoglein-1 (DSG1) is an essential intercellular adhesion molecule that is altered in various human cutaneous disorders; however, its regulation and function in allergic disease remains unexplored. Herein, we demonstrate a specific reduction in DSG1 in esophageal biopsies from patients with eosinophilic esophagitis (EoE), an emerging allergic disorder characterized by chronic inflammation within the esophageal mucosa. Further, we show that DSG1 gene silencing weakens esophageal epithelial integrity, and induces cell separation and impaired barrier function (IBF) despite high levels of desmoglein-3 (DSG3). Moreover, DSG1 deficiency induces transcriptional changes that partially overlap with the transcriptome of inflamed esophageal mucosa; notably, periostin, a multipotent pro-inflammatory extracellular matrix molecule, is the top induced overlapping gene. We further demonstrate that IBF is a pathological feature in EoE, which can be partially induced through the downregulation of DSG1 by interleukin-13 (IL-13). Taken together, these data identify a functional role for DSG1 and its dysregulation by IL-13 in the pathophysiology of EoE and suggest that the loss of DSG1 may potentiate allergic inflammation through the induction of pro-inflammatory mediators such as periostin.

Background A key question in the allergy field is to understand how tissue-specific disease is manifested. Eosinophilic esophagitis (EoE) is an emerging tissue-specific allergic disease with an unclear pathogenesis. Objective Herein we tested the hypothesis that a defect in tissue-specific esophageal genes is an integral part of EoE pathogenesis. Methods We interrogated the pattern of expression of esophagus-specific signature genes derived from the Human Protein Atlas in the EoE transcriptome and in EPC2 esophageal epithelial cells. Western blotting and immunofluorescence were used for evaluating expression of esophageal proteins in biopsy specimens from control subjects and patients with active EoE. Whole-exome sequencing was performed to identify mutations in esophagus-specific genes. Results We found that approximately 39% of the esophagus-specific transcripts were altered in patients with EoE, with approximately 90% being downregulated. The majority of transcriptional changes observed in esophagus-specific genes were reproduced in vitro in esophageal epithelial cells differentiated in the presence of IL-13. Functional enrichment analysis revealed keratinization and differentiation as the most affected biological processes and identified IL-1 cytokines and serine peptidase inhibitors as the most dysregulated esophagus-specific protein families in patients with EoE. Accordingly, biopsy specimens from patients with EoE evidenced a profound loss of tissue differentiation, decreased expression of keratin 4 (KRT4) and cornulin (CRNN) , and increased expression of KRT5 and KRT14 . Whole-exome sequencing of 33 unrelated patients with EoE revealed 39 rare mutations in 18 esophagus-specific differentially expressed genes. Conclusions A tissue-centered analysis has revealed a profound loss of esophageal tissue differentiation (identity) as an integral and specific part of the pathophysiology of EoE and implicated protease- and IL-1–related activities as putative central pathways in disease pathogenesis.

The presence of numerous G protein-coupled receptor (GPCR) homologs within the herpesvirus genomes suggests an essential role for these genes in viral replication in the infected host. Such is the case for murine cytomegalovirus (MCMV), where deletion of the M33 GPCR or replacement of M33 with a signaling defective mutant has been shown to severely attenuate replication in vivo. In the present study we utilized a genetically altered version of M33 (termed R131A) in combination with pharmacological inhibitors to further characterize the mechanisms by which M33 activates downstream signaling pathways. This R131A mutant of M33 fails to support salivary gland replication in vivo and, as such, is an important tool that can be used to examine the signaling activities of M33. We show that M33 stimulates the transcription factor CREB via heterotrimeric G q/11 proteins and not through promiscuous coupling of M33 to the G s pathway. Using inhibitors of signaling molecules downstream of G q/11 , we demonstrate that M33 stimulates CREB transcriptional activity in a phospholipase C-β and protein kinase C (PKC)-dependent manner. Finally, utilizing wild-type and R131A versions of M33, we show that M33-mediated activation of other signaling nodes, including the mitogen-activated protein kinase family member p38α and transcription factor NF-κB, occurs in the absence of G q/11 and PKC signaling. The results from the present study indicate that M33 utilizes multiple mechanisms to modulate intracellular signaling cascades and suggest that signaling through PLC-β and PKC plays a central role in MCMV pathogenesis in vivo.

Epithelial differentiation is an essential physiological process that imparts mechanical strength and barrier function to squamous epithelia. Perturbation of this process can give rise to numerous human diseases, such as atopic dermatitis, in which antigenic stimuli can penetrate the weakened epithelial barrier to initiate the allergic inflammatory cascade. We recently described a simplified air-liquid interface (ALI) culture system that facilitates the study of differentiated squamous epithelia in vitro. Herein, we use RNA sequencing to define the genome-wide transcriptional changes that occur within the ALI system during epithelial differentiation and in response to allergic inflammation. We identified 2,191 and 781 genes that were significantly altered upon epithelial differentiation or dysregulated in the presence of interleukin 13 (IL-13), respectively. Notably, 286 genes that were modified by IL-13 in the ALI system overlapped with the gene signature present within the inflamed esophageal tissue from patients with eosinophilic esophagitis (EoE), an allergic inflammatory disorder of the esophagus that is characterized by elevated IL-13 levels, altered epithelial differentiation, and pro-inflammatory gene expression. Pathway analysis of these overlapping genes indicated enrichment in keratin genes; for example, the gene encoding keratin 78, an uncharacterized type II keratin, was upregulated during epithelial differentiation (45-fold) yet downregulated in response to IL-13 and in inflamed esophageal tissue from patients. Thus, our findings delineate an in vitro experimental system that models epithelial differentiation that is dynamically regulated by IL-13. Using this system and analyses of patient tissues, we identify an altered expression profile of novel keratin differentiation markers in response to IL-13 and disease activity, substantiating the potential of this combined approach to identify relevant molecular processes that contribute to human allergic inflammatory disease.

Although interleukin (IL)-13 and neurotrophins are functionally important for the pathogenesis of immune responses, the interaction of these pathways has not been explored. Herein, by interrogating IL-13-induced responses in human epithelial cells we show that neurotrophic tyrosine kinase receptor, type 1 (NTRK1), a cognate, high-affinity receptor for nerve growth factor (NGF), is an early transcriptional IL-13 target. Induction of NTRK1 was accompanied by accumulation of activating epigenetic marks in the promoter; transcriptional and epigenetic changes were signal transducer and activator of transcription 6 dependent. Using eosinophilic esophagitis as a model for human allergic inflammation, we found that NTRK1 was increased in inflamed tissue and dynamically expressed as a function of disease activity and that the downstream mediator of NTRK1 signaling early growth response 1 protein was elevated in allergic inflammatory tissue compared with control tissue. Unlike NTRK1, its ligand NGF was constitutively expressed in control and disease states, indicating that IL-13-stimulated NTRK1 induction is a limiting factor in pathway activation. In epithelial cells, NGF and IL-13 synergistically induced several target genes, including chemokine (C-C motif) ligand 26 (eotaxin-3). In summary, we have demonstrated that IL-13 confers epithelial cell responsiveness to NGF by regulating NTRK1 levels by a transcriptional and epigenetic mechanism and that this process likely contributes to allergic inflammation.

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder associated with allergic hypersensitivity to food. We interrogated1.5 million genetic variants in EoE cases of European ancestry and subsequently in a multi-site cohort with local and out-of-study control subjects. In addition to replicating association of the 5q22 locus (meta-analysis P=1.9×10(-16)), we identified an association at 2p23 spanning CAPN14 (P=2.5×10(-10)). CAPN14 was specifically expressed in the esophagus, was dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to interleukin (IL)-13, and was located in an epigenetic hotspot modified by IL-13. Genes neighboring the top 208 EoE-associated sequence variants were enriched for esophageal expression, and multiple loci for allergic sensitization were associated with EoE susceptibility (4.8×10(-2)P5.1×10(-11)). We propose a model to explain the tissue-specific nature of EoE that involves the interplay of allergic sensitization with an EoE-specific, IL-13-inducible esophageal response involving CAPN14.

Eosinophilic esophagitis (EoE) is a polygenic disorder characterized by the accumulation of eosinophils in the esophagus. We carried out a genome-wide association study on clinically and biopsy confirmed EoE patients to identify common variants associated with the disease risk. One hundred and eighty one EoE samples from Cincinnati Children’s Hospital (CCHMC) and 170 EoE samples and ~3100 controls from Children’s Hospital of Philadelphia (CHOP) were genotyped on the Illumina 550K BeadChip. All patients and controls were of European ancestry. Following standard quality control filtering of the genotype data we carried out Cochran-Armitage trend tests at each SNP using the CCHMC samples as a discovery cohort. We detected genome-wide association with variants on chr5q22 that mapped to a single LD block encompassing the TSLP and WDR36 genes. The most significantly associated SNP at that locus which maps upstream of the TSLP gene remained wide significant after Bonferroni correction (rs3806932, uncorrected P-value = 7.18×10−8, OR = 0.54). Eleven other SNPs in LD with rs3806932 were also significantly associated with EoE and mapped to the same LD block on 5q22. We subsequently replicated the association in the independent CHOP cohort (170 cases, 1130 controls) with rs3806932 P-value = 8×10−3 OR = 0.73; combined P-value for rs3806932 across CCHMC and CHOP cohorts = 3.19×10−9). In addition, TSLP was overexpressed in the esophagus of EoE patients compared with control individuals with no differences observed in the expression of WDR36. In conclusion, we have identified the first genetic association with EoE predisposition at 5q22 implicating TSLP and/or WDR36 as genes potentially involved in the pathogenesis of EoE.

In inflammatory bowel diseases (IBDs), particularly ulcerative colitis, intestinal macrophages (MΦs), eosinophils, and the eosinophil-selective chemokine CCL11, have been associated with disease pathogenesis. MΦs, a source of CCL11, have been reported to be of a mixed classical (NF-κB–mediated) and alternatively activated (STAT-6–mediated) phenotype. The importance of NF-κB and STAT-6 pathways to the intestinal MΦ/CCL11 response and eosinophilic inflammation in the histopathology of experimental colitis is not yet understood. Our gene array analyses demonstrated elevated STAT-6– and NF-κB–dependent genes in pediatric ulcerative colitis colonic biopsies. Dextran sodium sulfate (DSS) exposure induced STAT-6 and NF-κB activation in mouse intestinal F4/80+CD11b+Ly6Chi (inflammatory) MΦs. DSS-induced CCL11 expression, eosinophilic inflammation, and histopathology were attenuated in RelA/p65Δmye mice, but not in the absence of STAT-6. Deletion of p65 in myeloid cells did not affect inflammatory MΦ recruitment or alter apoptosis, but did attenuate LPS-induced cytokine production (IL-6) and Ccl11 expression in purified F4/80+CD11b+Ly6Chi inflammatory MΦs. Molecular and cellular analyses revealed a link between expression of calprotectin (S100a8/S100a9), Ccl11 expression, and eosinophil numbers in the DSS-treated colon. In vitro studies of bone marrow–derived MΦs showed calprotectin-induced CCL11 production via a p65-dependent mechanism. Our results indicate that myeloid cell–specific NF-κB–dependent pathways play an unexpected role in CCL11 expression and maintenance of eosinophilic inflammation in experimental colitis. These data indicate that targeting myeloid cells and NF-κB–dependent pathways may be of therapeutic benefit for the treatment of eosinophilic inflammation and histopathology in IBD.

We report the association of eosinophilic esophagitis (EoE) and hypertrophic cardiomyopathy (HCM) and provide genetic data indicating a linkage between EoE and HCM. The letter begins with an index patient diagnosed with both EoE and HCM and found to have a known genetic mutation for HCM. We then identify an odds ratio of nearly 8 for having both EoE and HCM following review of an electronic medical record with >1,000,000 individuals. Finally, via a candidate gene approach we identify significant association of HCM gene variants in a cohort of EoE patients versus control subjects. Collectively, we have identified a putative interaction between EoE and HCM with clinical and pathogenic implications.

The epidemiology of eosinophilic esophagitis (EoE), a chronic inflammatory condition of the esophagus, indicates a worldwide increase in disease prevalence over the last 10 years with a disease bias among certain demographic populations. Retrospective studies have shown the prevalence of EoE is almost three times higher in males and is primarily restricted to Caucasians. The disease risk among siblings of EoE patients is estimated to be 40 times higher than the risk of asthma, a more widely prevalent disease with a well-accepted genetic component. These factors suggest that EoE is a polygenic disorder with a heritable genetic basis. Snapshots of whole genome expression patterns from patient-derived biopsies and whole genome polymorphism mapping of patient DNA have provided great insight at the molecular level into the genetic influences contributing to EoE. In this chapter, we will highlight seminal studies that have identified critical gene regulatory networks that are operational in EoE and discuss genetic polymorphisms associated with increased disease susceptibility.

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder of the esophagus that is compounded by both genetic predisposition and aberrant responses to environmental antigens, particularly those that are food-derived. Data have indicated a unique transcriptional response in vivo that defines EoE and which is partially attributable to the Th2 cytokine interleukin 13 (IL-13). Moreover, a number of genetic risk variants in pro-inflammatory and epithelial cell genes associate with EoE susceptibility, demonstrating novel heritable mechanisms that contribute to disease risk. Here, we discuss recent advances in our understanding of the intrinsic (genetic) and extrinsic (environmental) components that illustrate the complex nature of EoE.

Members of the herpesvirus family, including human cytomegalovirus (HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), encode G protein-coupled receptor (GPCR) homologs, which strongly activate classical G protein signal transduction networks within the cell. In animal models of herpesvirus infection, the viral GPCRs appear to play physiologically important roles by enabling viral replication within tropic tissues and by promoting reactivation from latency. While a number of studies have defined intracellular signaling pathways activated by herpesviral GPCRs, it remains unclear if their physiological function is subjected to the process of desensitization as observed for cellular GPCRs. G protein-coupled receptor kinases (GRK) and arrestin proteins have been recently implicated in regulating viral GPCR signaling; however, the role that these desensitization proteins play in viral GPCR function in vivo remains unknown. Here, we review what is currently known regarding viral GPCR desensitization and discuss potential biological ramifications of viral GPCR regulation by the host cell desensitization machinery.

FEBRUARY 2015 AB40 Abstracts S A T U R D A Y 123 Adult Eosinophilic Oesophagitis: A UK Based Case Series Efrem Eren, MBBS, MRCP, FRCPath, PhD, Tak Chin, William Rae, BSc, BM, MRCP, Syed H. Arshad, DM, FRCP, Peter H. Howarth, MD, Anthony Williams, Elena Salagean, SpR, Bryan N. Fernandes, MD, Ramesh Kurukulaaratchy, Carina Venter, PhD, RD; Southampton General Hospital, UK, Southampton, United Kingdom, Southampton, University Hospital Southampton, Southampton, United Kingdom, The David Hide Asthma and Allergy Research Centre, United Kingdom, University of Southampton, United Kingdom, Southampton General Hospital, Southampton, United Kingdom, University of Southampton, University Hospital of NHS Foundation Trust, United Kingdom, University of Southampton, Southampton, United Kingdom, University of Portsmouth, United Kingdom. RATIONALE: Adult onset Eosinophilic Oesophagitis (EoE) presents frequently to the allergy clinics in the UK. However, no study to date has attempted to describe these patients. . This case series set out to clinically characterise adults patients with EoE seen over the past four years in a tertiary care center in the UK. METHODS: We screened Allergy/Immunology clinic lists and the edocument systems at Southampton General Hospital (SGH) for patients given a diagnosis of EoE since 2010. RESULTS: Thirty six patients were identified with a diagnostic label of EoE. They were predominantly male (77.8%; 28/36) with an age range of 19 – 71 years. The majority (61%, 22/36) suffered from other atopic conditions/or showed a positive total IgE level and suspected a wide range of potential foods triggers. Interestingly, patients often considered food that are difficult to swallow such as meat, chicken and starchy food as the cause of their problems. SPT and specific IgE tests were not helpful in identifying offending foods. As expected the majority (58.3%; 21/36) were sensitised to aero-allergens with 14 being sensitised to tree/birch pollen. Dietary interventions varied widely between these patients, ranging from elemental diets, 6 food elimination diets to test directed diets. CONCLUSIONS: Male sex and atopy were found to be primary characteristics, but age did not influence the diagnosis. A wide range of foods are suspected to trigger EoE pathology in adults, and dietarymeasure seems to vary greatly. Studies are needed to identify which foods are implicated in adult EoE in the UK.

S A T U R D A Y 247 Functional Analysis of Calpain-14 in Eosinophilic Esophagitis Benjamin P. Davis, MD, Leah Claire Kottyan, PhD, Emily Stucke, BA, Joseph D. Sherrill, PhD, Marc E. Rothenberg, MD, PhD; Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA, Center for Autoimmune Genomics and Etiology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, USA, Cincinnati, OH, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, Children’s Hospital Medical Center, Cincinnati, OH. RATIONALE: We recently identified a genome-wide association susceptibility locus for eosinophilic esophagitis (EoE) at 2q23 wherein CAPN14 (p52.5 3 10210) is located. CAPN14 encodes for a member of the calpain protease family. We have shown that CAPN14 was specifically expressed in the esophagus, dynamically regulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to IL-13. Herein, we aimed to determine the function of calpain-14. METHODS: We overexpressed calpain-14 in an esophageal progenitor cell line (EPC2) grown in an air-liquid interface (ALI) culture and analyzed barrier effects by transepithial electrical resistance (TEER), fluorescein isothiocyanate (FITC)-dextran flux, and hematoxylin and eosin stain. We also identified potential calpain-14 targets via western blot and immunofluorescence (IF) of the ALI culture. RESULTS: Calpain-14 over-expression induced a 2-3 fold (p50.0034) decrease in TEER and a 2-3 fold (P

The mouse cytomegalovirus M33 protein is highly homologous to mammalian G protein-coupled receptors (GPCRs) yet functions in an agonist-independent manner to activate a number of classical GPCR signal transduction pathways. M33 is functionally similar to the human cytomegalovirus-encoded US28 GPCR in its ability to induce inositol phosphate accumulation, activate NF-kappaB, and promote smooth muscle cell migration. This ability to promote cellular migration suggests a role for viral GPCRs like M33 in viral dissemination in vivo, and accordingly, M33 is required for efficient murine cytomegalovirus replication in the mouse. Although previous studies have identified several M33-induced signaling pathways, little is known regarding the membrane-proximal events involved in signaling and regulation of this receptor. In this study, we used recombinant retroviruses to express M33 in wild-type and Galpha(q/11)(-/-) mouse embryonic fibroblasts and show that M33 couples directly to the G(q/11) signaling pathway to induce high levels of total inositol phosphates in an agonist-independent manner. Our data also show that GRK2 is a potent regulator of M33-induced G(q/11) signaling through its ability to phosphorylate M33 and sequester Galpha(q/11) proteins. Taken together, the results from this study provide the first genetic evidence of a viral GPCR coupling to a specific G protein signaling pathway as well as identify the first viral GPCR to be regulated specifically by both the catalytic activity of the GRK2 kinase domain and the Galpha(q/11) binding activity of the GRK2 RH domain.

Background The role of microRNAs (miRNAs), a key class of regulators of mRNA expression and translation, in patients with eosinophilic esophagitis (EoE) has not been explored. Objective We aimed to identify miRNAs dysregulated in patients with EoE and assess the potential of these miRNAs as disease biomarkers. Methods Esophageal miRNA expression was profiled in patients with active EoE and those with glucocorticoid-induced disease remission. Expression profiles were compared with those of healthy control subjects and patients with chronic (noneosinophilic) esophagitis. Expression levels of the top differentially expressed miRNAs from the plasma of patients with active EoE and patients with EoE remission were compared with those of healthy control subjects. Results EoE was associated with 32 differentially regulated miRNAs and was distinguished from noneosinophilic forms of esophagitis. The expression levels of the most upregulated miRNAs (miR-21 and miR-223) and the most downregulated miRNA (miR-375) strongly correlated with esophageal eosinophil levels. Bioinformatic analysis predicted interplay of miR-21 and miR-223 with key roles in the polarization of adaptive immunity and regulation of eosinophilia, and indeed, these miRNAs correlated with key elements of the EoE transcriptome. The differentially expressed miRNAs were largely reversible in patients who responded to glucocorticoid treatment. EoE remission induced a single miRNA (miR-675) likely to be involved in DNA methylation. Plasma analysis of the most upregulated esophageal miRNAs identified miR-146a, miR-146b, and miR-223 as the most differentially expressed miRNAs in the plasma. Conclusions We have identified a marked dysregulated expression of a select group of miRNAs in patients with EoE and defined their reversibility with glucocorticoid treatment and their potential value as invasive and noninvasive biomarkers.