Publications by authors named "Cristina de Guzman Strong"

22 Publications

  • Page 1 of 1

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq).

J Vis Exp 2018 10 5(140). Epub 2018 Oct 5.

Division of Dermatology, Center for Pharmacogenomics, Center for the Study of Itch, Department of Medicine, Washington University School of Medicine;

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.
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http://dx.doi.org/10.3791/58030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235426PMC
October 2018

Tiled array-based sequencing identifies enrichment of loss-of-function variants in the highly homologous filaggrin gene in African-American children with severe atopic dermatitis.

Exp Dermatol 2018 09 28;27(9):989-992. Epub 2018 Jun 28.

Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA.

Filaggrin (FLG) loss-of-function (LOF) variants are a major risk factor for the common inflammatory skin disease, atopic dermatitis (AD) and are often population-specific. African-American (AA) children are disproportionately affected with AD, often later developing asthma and/or allergic rhinitis and comprise an atopy health disparity group for which the role of FLG LOF is not well known. Discovery of FLG LOF using exome sequencing is challenging given the known difficulties for accurate short-read alignment to FLG's high homology repeat variation. Here, we employed an array-based sequencing approach to tile across each FLG repeat and discover FLG LOF in a well-characterized cohort of AA children with moderate-to-severe AD. Five FLG LOF were identified in 23% of our cohort. Two novel FLG LOF singletons, c.488delG and p.S3101*, were discovered as well as p.R501*, p.R826* and p.S3316* previously reported for AD. p.S3316* (rs149484917) is likely an African ancestral FLG LOF, reported in African individuals in ExAC (Exome Aggregation Consortium), Exome Variant Server (ESP), and 4 African 1000G population databases (ESN, MSL, ASW, and ACB). The proportion of FLG LOF (11.5%) among the total FLG alleles in our cohort was significantly higher in comparisons with FLG LOF reported for African individuals in ExAC (2.5%; P = 4.3 × 10 ) and ESP (1.7%; P = 3.5 × 10 ) suggesting a disease-enrichment effect for FLG LOF. Our results demonstrate the utility of array-based sequencing in discovering FLG LOF, including novel and population-specific, which are of higher prevalence in our AA severe AD group than previously reported.
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http://dx.doi.org/10.1111/exd.13691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440476PMC
September 2018

Recent evolution of the human skin barrier.

Exp Dermatol 2018 08 28;27(8):859-866. Epub 2018 Jun 28.

Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA.

The skin is the first line of defense against the environment, with the epidermis as the outermost tissue providing much of the barrier function. Given its direct exposure to and encounters with the environment, the epidermis must evolve to provide an optimal barrier for the survival of an organism. Recent advances in genomics have identified a number of genes for the human skin barrier that have undergone evolutionary changes since humans diverged from chimpanzees. Here, we highlight a selection of key and innovative genetic findings for skin barrier evolution in our divergence from our primate ancestors and among modern human populations.
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http://dx.doi.org/10.1111/exd.13689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392702PMC
August 2018

Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.

Nature 2018 04 18;556(7702):501-504. Epub 2018 Apr 18.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines , including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-IκBζ regulatory axis could be an important new strategy for the treatment of IL-17-IκBζ-mediated autoimmune diseases.
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http://dx.doi.org/10.1038/s41586-018-0052-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6037913PMC
April 2018

Novel oxytocin receptor variants in laboring women requiring high doses of oxytocin.

Am J Obstet Gynecol 2017 08 26;217(2):214.e1-214.e8. Epub 2017 Apr 26.

Department of Obstetrics and Gynecology, Center for Reproductive Health Sciences, Washington University in St. Louis, School of Medicine, St. Louis, MO. Electronic address:

Background: Although oxytocin commonly is used to augment or induce labor, it is difficult to predict its effectiveness because oxytocin dose requirements vary significantly among women. One possibility is that women requiring high or low doses of oxytocin have variations in the oxytocin receptor gene.

Objectives: To identify oxytocin receptor gene variants in laboring women with low and high oxytocin dosage requirements.

Study Design: Term, nulliparous women requiring oxytocin doses of ≤4 mU/min (low-dose-requiring, n = 83) or ≥20 mU/min (high-dose-requiring, n = 104) for labor augmentation or induction provided consent to a postpartum blood draw as a source of genomic DNA. Targeted-amplicon sequencing (coverage >30×) with MiSeq (Illumina) was performed to discover variants in the coding exons of the oxytocin receptor gene. Baseline relevant clinical history, outcomes, demographics, and oxytocin receptor gene sequence variants and their allele frequencies were compared between low-dose-requiring and high-dose-requiring women. The Scale-Invariant Feature Transform algorithm was used to predict the effect of variants on oxytocin receptor function. The Fisher exact or χ tests were used for categorical variables, and Student t tests or Wilcoxon rank sum tests were used for continuous variables. A P value < .05 was considered statistically significant.

Results: The high-dose-requiring women had greater rates of obesity and diabetes and were more likely to have undergone labor induction and required prostaglandins. High-dose-requiring women were more likely to undergo cesarean delivery for first-stage arrest and less likely to undergo cesarean delivery for nonreassuring fetal status. Targeted sequencing of the oxytocin receptor gene in the total cohort (n = 187) revealed 30 distinct coding variants: 17 nonsynonymous, 11 synonymous, and 2 small structural variants. One novel variant (A243T) was found in both the low- and high-dose-requiring groups. Three novel variants (Y106H, A240_A249del, and P197delfs*206) resulting in an amino acid substitution, loss of 9 amino acids, and a frameshift stop mutation, respectively, were identified only in low-dose-requiring women. Nine nonsynonymous variants were unique to the high-dose-requiring group. These included 3 known variants (R151C, G221S, and W228C) and 6 novel variants (M133V, R150L, H173R, A248V, G253R, and I266V). Of these, R150L, R151C, and H173R were predicted by Scale-Invariant Feature Transform algorithm to damage oxytocin receptor function. There was no statistically significant association between the numbers of synonymous and nonsynonymous substitutions in the patient groups.

Conclusion: Obesity, diabetes, and labor induction were associated with the requirement for high doses of oxytocin. We did not identify significant differences in the prevalence of oxytocin receptor variants between low-dose-requiring and high-dose-requiring women, but novel oxytocin receptor variants were enriched in the high-dose-requiring women. We also found 3 oxytocin receptor variants (2 novel, 1 known) that were predicted to damage oxytocin receptor function and would likely increase an individual's risk for requiring a high oxytocin dose. Further investigation of oxytocin receptor variants and their effects on protein function will inform precision medicine in pregnant women.
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http://dx.doi.org/10.1016/j.ajog.2017.04.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5644384PMC
August 2017

The Molecular Revolution in Cutaneous Biology: EDC and Locus Control.

J Invest Dermatol 2017 05;137(5):e101-e104

Department of Internal Medicine, Division of Dermatology, Center for Pharmacogenomics, Center for the Study of Itch, Washington University School of Medicine, St. Louis, Missouri, USA. Electronic address:

The epidermal differentiation complex (EDC) locus consists of a cluster of genes important for the terminal differentiation of the epidermis. While early studies identified the functional importance of individual EDC genes, the recognition of the EDC genes as a cluster with its shared biology, homology, and physical linkage was pivotal to later studies that investigated the transcriptional regulation of the locus. Evolutionary conservation of the EDC and the transcriptional activation during epidermal differentiation suggested a cis-regulatory mechanism via conserved noncoding elements or enhancers. This line of pursuit led to the identification of CNE 923, an epidermal-specific enhancer that was found to mediate chromatin remodeling of the EDC in an AP-1 dependent manner. These genomic studies, as well as the advent of high-throughput sequencing and genome engineering techniques, have paved the way for future investigation into enhancer-mediated regulatory networks in cutaneous biology.
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http://dx.doi.org/10.1016/j.jid.2016.03.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400479PMC
May 2017

Recent Positive Selection in Genes of the Mammalian Epidermal Differentiation Complex Locus.

Front Genet 2016 10;7:227. Epub 2017 Jan 10.

Division of Dermatology, Department of Internal Medicine, Center for Pharmacogenomics and Center for the Study of Itch, Washington University School of Medicine, St. Louis MO, USA.

The epidermal differentiation complex (EDC) is the most rapidly evolving locus in the human genome compared to that of the chimpanzee. Yet the EDC genes that are undergoing positive selection across mammals and in humans are not known. We sought to identify the positively selected genetic variants and determine the evolutionary events of the EDC using mammalian-wide and clade-specific branch- and branch-site likelihood ratio tests and a genetic algorithm (GA) branch test. Significant non-synonymous substitutions were found in , and genes across 14 mammals. By contrast, we identified recent positive selection in in primates. Additionally, the GA branch test discovered lineage-specific evolution for distinct EDC genes occurring in each of the nodes in the 14-mammal phylogenetic tree. Multiple instances of positive selection for , and were noted among the primate branch nodes. Branch-site likelihood ratio tests further revealed positive selection in specific sites in , and across 14 mammals. However, in addition to continuous evolution of , site-specific positive selection was also found in , and in primates and , and in great apes. Very recent human positive selection was identified in the L41 site that was present in Neanderthal. Together, our results identifying recent positive selection in distinct EDC genes reveal an underappreciated evolution of epidermal skin barrier function in primates and humans.
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http://dx.doi.org/10.3389/fgene.2016.00227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5222828PMC
January 2017

Effect of Counting Genetic Variants on Precision Treatment for Pediatric Atopic Dermatitis: Do Count Your Chickens Before They Hatch.

JAMA Dermatol 2017 03;153(3):269-270

Division of Dermatology, Department of Internal Medicine, Washington University School of Medicine, St Louis, Missouri2Center for Pharmacogenomics, Washington University School of Medicine, St Louis, Missouri3Center for the Study of Itch, Washington University School of Medicine, St Louis, Missouri.

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http://dx.doi.org/10.1001/jamadermatol.2016.4468DOI Listing
March 2017

The Asian atopic dermatitis phenotype combines features of atopic dermatitis and psoriasis with increased TH17 polarization.

J Allergy Clin Immunol 2015 Nov 1;136(5):1254-64. Epub 2015 Oct 1.

Laboratory for Investigative Dermatology, The Rockefeller University, New York, NY; Department of Dermatology, Icahn School of Medicine at Mount Sinai, New York, NY; Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY. Electronic address:

Background: Atopic dermatitis (AD) shows very high prevalence in Asia, with a large unmet need for effective therapeutics. Direct comparisons between European American (EA) and Asian patients with AD are unavailable, but earlier blood studies detected increased IL-17(+)-producing cell counts in Asian patients with AD.

Objective: We sought to characterize the Asian AD skin phenotype and compare it with the EA AD skin phenotype.

Methods: We performed genomic profiling (real-time PCR) and immunohistochemistry on lesional and nonlesional biopsy specimens from 52 patients with AD (25 EAs and 27 Asians), 10 patients with psoriasis (all EAs), and 27 healthy subjects (12 EAs and 15 Asians).

Results: Although disease severity/SCORAD scores were similar between the AD groups (58.0 vs 56.7, P = .77), greater acanthosis, higher Ki67 counts, and frequent parakeratosis were characteristics of lesional epidermis from Asian patients with AD (P < .05). Most (24/27) Asian patients had high IgE levels. A principal component analysis using real-time PCR data clustered the Asian AD phenotype between the EA AD and psoriasis phenotypes. TH2 skewing characterized both Asian and EA patients with AD but not patients with psoriasis. Significantly higher TH17 and TH22 (IL17A, IL19, and S100A12 in lesional and IL-22 in nonlesional skin; P < .05) and lower TH1/interferon (CXCL9, CXCL10, MX1, and IFNG in nonlesional skin; P < .05) gene induction typified AD skin in Asian patients.

Conclusion: The Asian AD phenotype presents (even in the presence of increased IgE levels) a blended phenotype between that of EA patients with AD and those with psoriasis, including increased hyperplasia, parakeratosis, higher TH17 activation, and a strong TH2 component. The relative pathogenic contributions of the TH17 and TH2 axes in creating the Asian AD phenotype need to be tested in future clinical trials with appropriate targeted therapeutics.
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http://dx.doi.org/10.1016/j.jaci.2015.08.015DOI Listing
November 2015

Low filaggrin monomer repeats in African American pediatric patients with moderate to severe atopic dermatitis.

JAMA Dermatol 2015 May;151(5):557-9

Center for Pharmacogenomics, Center for the Study of Itch, Division of Dermatology, Department of Internal Medicine, Washington University School of Medicine, St Louis, Missouri.

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http://dx.doi.org/10.1001/jamadermatol.2014.4916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659403PMC
May 2015

Highlighting blondes: a tissue-specific KITLG enhancer shows us how.

Pigment Cell Melanoma Res 2014 Nov 17;27(6):1007-8. Epub 2014 Sep 17.

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http://dx.doi.org/10.1111/pcmr.12311DOI Listing
November 2014

Epigenetic coordination of embryonic heart transcription by dynamically regulated long noncoding RNAs.

Proc Natl Acad Sci U S A 2014 Aug 28;111(33):12264-9. Epub 2014 Jul 28.

Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110

The vast majority of mammalian DNA does not encode for proteins but instead is transcribed into noncoding (nc)RNAs having diverse regulatory functions. The poorly characterized subclass of long ncRNAs (lncRNAs) can epigenetically regulate protein-coding genes by interacting locally in cis or distally in trans. A few reports have implicated specific lncRNAs in cardiac development or failure, but precise details of lncRNAs expressed in hearts and how their expression may be altered during embryonic heart development or by adult heart disease is unknown. Using comprehensive quantitative RNA sequencing data from mouse hearts, livers, and skin cells, we identified 321 lncRNAs present in the heart, 117 of which exhibit a cardiac-enriched pattern of expression. By comparing lncRNA profiles of normal embryonic (∼E14), normal adult, and hypertrophied adult hearts, we defined a distinct fetal lncRNA abundance signature that includes 157 lncRNAs differentially expressed compared with adults (fold-change ≥ 50%, false discovery rate = 0.02) and that was only poorly recapitulated in hypertrophied hearts (17 differentially expressed lncRNAs; 13 of these observed in embryonic hearts). Analysis of protein-coding mRNAs from the same samples identified 22 concordantly and 11 reciprocally regulated mRNAs within 10 kb of dynamically expressed lncRNAs, and reciprocal relationships of lncRNA and mRNA levels were validated for the Mccc1 and Relb genes using in vitro lncRNA knockdown in C2C12 cells. Network analysis suggested a central role for lncRNAs in modulating NFκB- and CREB1-regulated genes during embryonic heart growth and identified multiple mRNAs within these pathways that are also regulated, but independently of lncRNAs.
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http://dx.doi.org/10.1073/pnas.1410622111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143054PMC
August 2014

Cyclosporine in patients with atopic dermatitis modulates activated inflammatory pathways and reverses epidermal pathology.

J Allergy Clin Immunol 2014 Jun 29;133(6):1626-34. Epub 2014 Apr 29.

Laboratory for Investigative Dermatology, Rockefeller University, New York, NY; Department of Dermatology, Icahn School of Medicine at Mount Sinai, New York, NY. Electronic address:

Background: Atopic dermatitis (AD) is the most common inflammatory disease. Evolving disease models link changes in epidermal growth and differentiation to T(H)2/T(H)22 cytokine activation. However, these models have not been tested by in vivo suppression of T-cell cytokines. Cyclosporine (CsA) is an immunosuppressant that is highly effective for severe disease, but its mechanism in AD skin lesions has not been studied.

Objective: We sought to establish the ability of a systemic immunosuppressant to modulate immune and epidermal alterations that form the pathogenic disease phenotype and to correlate changes with clinical improvement.

Methods: CsA's effects on AD skin pathology were evaluated by using gene expression and immunohistochemistry studies in baseline, week 2, and week 12 lesional and nonlesional biopsy specimens from 19 patients treated with 5 mg/kg/d CsA for 12 weeks.

Results: After 2 and 12 weeks of treatment, we observed significant reductions of 51% and 72%, respectively, in SCORAD scores. Clinical improvements were associated with significant gene expression changes in lesional but also nonlesional skin, particularly reductions in levels of T(H)2-, T(H)22-, and some T(H)17-related molecules (ie, IL-13, IL-22, CCL17, S100As, and elafin/peptidase inhibitor 3), and modulation of epidermal hyperplasia and differentiation measures.

Conclusions: This is the first study that establishes a relationship between cytokine activation and molecular epidermal alterations, as well as correlations between disease biomarkers in the skin and clinical improvement. The reversal of the molecular phenotype with CsA and the associated biomarkers can serve as a reference for the successful modulation of tissue inflammation with specific immune antagonists in future studies, contributing to the understanding of the specific cytokines involved in epidermal pathology.
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http://dx.doi.org/10.1016/j.jaci.2014.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4122665PMC
June 2014

Regulation of the dynamic chromatin architecture of the epidermal differentiation complex is mediated by a c-Jun/AP-1-modulated enhancer.

J Invest Dermatol 2014 Sep 27;134(9):2371-2380. Epub 2014 Jan 27.

Division of Dermatology, Department of Internal Medicine, Center for Pharmacogenomics, Center for the Study of Itch, Washington University School of Medicine, St Louis, Missouri, USA. Electronic address:

The epidermal differentiation complex (EDC) locus comprises a syntenic and linear cluster of genes whose concomitant expression is a hallmark feature of differentiation in the developing skin epidermis. Many of the EDC proteins are cross-linked together to form the cornified envelope, an essential and discrete unit of the mammalian skin barrier. The mechanism underlying coordinate transcriptional activation of the EDC is unknown. Within the human EDC, we identified an epidermal-specific regulatory enhancer, 923, which responded to the developmental and spatiotemporal cues at the onset of epidermal differentiation in the mouse embryo. Comparative chromosomal conformation capture assays in proliferating and differentiated primary mouse keratinocytes revealed multiple physiologically sensitive chromatin interactions between the 923 enhancer and EDC gene promoters, thus depicting the dynamic chromatin topology of the EDC. We elucidate a mechanistic link between c-Jun/AP-1 and 923, whereby AP-1- and 923-mediated EDC chromatin remodeling are required for functional EDC gene activation. Thus, we identify a critical enhancer/transcription factor axis governing the dynamic regulation of the EDC chromatin architecture and gene expression and provide a framework for future studies toward understanding gene regulation in cutaneous diseases.
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http://dx.doi.org/10.1038/jid.2014.44DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112170PMC
September 2014

Intrinsic atopic dermatitis shows similar TH2 and higher TH17 immune activation compared with extrinsic atopic dermatitis.

J Allergy Clin Immunol 2013 Aug 15;132(2):361-70. Epub 2013 Jun 15.

Laboratory for Investigative Dermatology, Rockefeller University, New York, NY, USA.

Background: Atopic dermatitis (AD) is classified as extrinsic and intrinsic, representing approximately 80% and 20% of patients with the disease, respectively. Although sharing a similar clinical phenotype, only extrinsic AD is characterized by high serum IgE levels. Because most patients with AD exhibit high IgE levels, an "allergic"/IgE-mediated disease pathogenesis was hypothesized. However, current models associate AD with T-cell activation, particularly TH2/TH22 polarization, and epidermal barrier defects.

Objective: We sought to define whether both variants share a common pathogenesis.

Methods: We stratified 51 patients with severe AD into extrinsic AD (n = 42) and intrinsic AD (n = 9) groups (with similar mean disease activity/SCORAD scores) and analyzed the molecular and cellular skin pathology of lesional and nonlesional intrinsic AD and extrinsic AD by using gene expression (real-time PCR) and immunohistochemistry.

Results: A significant correlation between IgE levels and SCORAD scores (r = 0.76, P < 10(-5)) was found only in patients with extrinsic AD. Marked infiltrates of T cells and dendritic cells and corresponding epidermal alterations (keratin 16, Mki67, and S100A7/A8/A9) defined lesional skin of patients with both variants. However, higher activation of all inflammatory axes (including TH2) was detected in patients with intrinsic AD, particularly TH17 and TH22 cytokines. Positive correlations between TH17-related molecules and SCORAD scores were only found in patients with intrinsic AD, whereas only patients with extrinsic AD showed positive correlations between SCORAD scores and TH2 cytokine (IL-4 and IL-5) levels and negative correlations with differentiation products (loricrin and periplakin).

Conclusions: Although differences in TH17 and TH22 activation exist between patients with intrinsic AD and those with extrinsic AD, we identified common disease-defining features of T-cell activation, production of polarized cytokines, and keratinocyte responses to immune products. Our data indicate that a TH2 bias is not the sole cause of high IgE levels in patients with extrinsic AD, with important implications for similar therapeutic interventions.
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http://dx.doi.org/10.1016/j.jaci.2013.04.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3991240PMC
August 2013

Translational profiling of hypocretin neurons identifies candidate molecules for sleep regulation.

Genes Dev 2013 Mar 21;27(5):565-78. Epub 2013 Feb 21.

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA.

Hypocretin (orexin; Hcrt)-containing neurons of the hypothalamus are essential for the normal regulation of sleep and wake behaviors and have been implicated in feeding, anxiety, depression, and reward. The absence of these neurons causes narcolepsy in humans and model organisms. However, little is known about the molecular phenotype of these cells; previous attempts at comprehensive profiling had only limited sensitivity or were inaccurate. We generated a Hcrt translating ribosome affinity purification (bacTRAP) line for comprehensive translational profiling of all ribosome-bound transcripts in these neurons in vivo. From this profile, we identified >6000 transcripts detectably expressed above background and 188 transcripts that are highly enriched in these neurons, including all known markers of the cells. Blinded analysis of in situ hybridization databases suggests that ~60% of these are expressed in a Hcrt marker-like pattern. Fifteen of these were confirmed with double labeling and microscopy, including the transcription factor Lhx9. Ablation of this gene results in a >30% loss specifically of Hcrt neurons, without a general disruption of hypothalamic development. Polysomnography and activity monitoring revealed a profound hypersomnolence in these mice. These data provide an in-depth and accurate profile of Hcrt neuron gene expression and suggest that Lhx9 may be important for specification or survival of a subset of these cells.
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http://dx.doi.org/10.1101/gad.207654.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605469PMC
March 2013

Progressive activation of T(H)2/T(H)22 cytokines and selective epidermal proteins characterizes acute and chronic atopic dermatitis.

J Allergy Clin Immunol 2012 Dec 27;130(6):1344-54. Epub 2012 Aug 27.

Albert Einstein College of Medicine, Bronx, NY, USA.

Background: Atopic dermatitis (AD) is a common disease with an increasing prevalence. The primary pathogenesis of the disease is still elusive, resulting in the lack of specific treatments. AD is currently considered a biphasic disease, with T(H)2 predominating in acute disease and a switch to T(H)1 characterizing chronic disease. Elucidation of the molecular factors that participate in the onset of new lesions and maintenance of chronic disease is critical for the development of targeted therapeutics.

Objectives: We sought to characterize the mechanisms underlying the onset and maintenance of AD.

Methods: We investigated intrapersonal sets of transcriptomes from nonlesional skin and acute and chronic lesions of 10 patients with AD through genomic, molecular, and cellular profiling.

Results: Our study associated the onset of acute lesions with a striking increase in a subset of terminal differentiation proteins, specifically the cytokine-modulated S100A7, S100A8, and S100A9. Acute disease was also associated with significant increases in gene expression levels of major T(H)22 and T(H)2 cytokines and smaller increases in IL-17 levels. A lesser induction of T(H)1-associated genes was detected in acute disease, although some were significantly upregulated in chronic disease. Further significant intensification of major T(H)22 and T(H)2 cytokines was observed between acute and chronic lesions.

Conclusions: Our data identified increased S100A7, S100A8, and S100A9 gene expression with AD initiation and concomitant activation of T(H)2 and T(H)22 cytokines. Our findings support a model of progressive activation of T(H)2 and T(H)22 immune axes from the acute to chronic phases, expanding the prevailing view of pathogenesis with important therapeutic implications.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3991245PMC
http://dx.doi.org/10.1016/j.jaci.2012.07.012DOI Listing
December 2012

Preparing the next generation in academic medicine: recruiting and retaining the best.

J Invest Dermatol 2012 Mar;132(3 Pt 2):1018-25

Department of Medicine, Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63130, USA.

In many ways, we are living and working in an unprecedented time in academic medicine. New technologies, scientific discovery, unparalleled availability of medical information and knowledge are currently paired with increasing (albeit slow) gender, cultural, and now generational diversity of the faculty. To prepare the next generation, we must simultaneously be the student and the teacher. As the student, our charge is to understand the current medical and academic environs and recognize the attributes, experiences, and expectations that each generational cohort brings to medicine. As the teacher, we must identify, extract, and communicate the tenets that remain constants for success in academic medicine today and reject those that are no longer relevant. Throughout the years, the basic motivation that drives success has remained constant while the individuals (the players), the environment, and the definition of success in academics have become more varied.
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http://dx.doi.org/10.1038/jid.2011.407DOI Listing
March 2012

A milieu of regulatory elements in the epidermal differentiation complex syntenic block: implications for atopic dermatitis and psoriasis.

Hum Mol Genet 2010 Apr 20;19(8):1453-60. Epub 2010 Jan 20.

Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA.

Two common inflammatory skin disorders with impaired barrier, atopic dermatitis (AD) and psoriasis, share distinct genetic linkage to the Epidermal Differentiation Complex (EDC) locus on 1q21. The EDC is comprised of tandemly arrayed gene families encoding proteins involved in skin cell differentiation. Discovery of semi-dominant mutations in filaggrin (FLG) associated with AD and a copy number variation within the LCE genes associated with psoriasis provide compelling evidence for the role of EDC genes in the pathogenesis of these diseases. To date, little is known about the potentially complex regulatory landscape within the EDC. Here, we report a computational approach to identify conserved non-coding elements (CNEs) in the EDC queried for regulatory function. Coordinate expression of EDC genes during mouse embryonic skin development and a striking degree of synteny and linearity in the EDC locus across a wide range of mammalian (placental and marsupial) genomes suggests an evolutionary conserved regulatory milieu in the EDC. CNEs identified by comparative genomics exhibit dynamic regulatory activity (enhancer or repressor) in differentiating or proliferating conditions. We further demonstrate epidermal-specific, developmental in vivo enhancer activities (DNaseI and transgenic mouse assays) in CNEs, including one within the psoriasis-associated deletion, LCE3C_LCE3B-del. Together, our multidisciplinary study features a network of regulatory elements coordinating developmental EDC gene expression as an unexplored resource for genetic variants in skin diseases.
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http://dx.doi.org/10.1093/hmg/ddq019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846160PMC
April 2010

Navigating the genome.

J Cell Sci 2008 Apr;121(Pt 7):921-3

National Human Genome Research Institute, National Institutes of Health, 49 Convent Drive, Bethesda, MD 20892, USA.

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http://dx.doi.org/10.1242/jcs.022400DOI Listing
April 2008

Lipid defect underlies selective skin barrier impairment of an epidermal-specific deletion of Gata-3.

J Cell Biol 2006 Nov;175(4):661-70

National Human Genome Research Institute and 2National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Skin lies at the interface between the complex physiology of the body and the external environment. This essential epidermal barrier, composed of cornified proteins encased in lipids, prevents both water loss and entry of infectious or toxic substances. We uncover that the transcription factor GATA-3 is required to establish the epidermal barrier and survive in the ex utero environment. Analysis of Gata-3 mutant transcriptional profiles at three critical developmental stages identifies a specific defect in lipid biosynthesis and a delay in differentiation. Genomic analysis identifies highly conserved GATA-3 binding sites bound in vivo by GATA-3 in the first intron of the lipid acyltransferase gene AGPAT5. Skin from both Gata-3-/- and previously characterized barrier-deficient Kruppel-like factor 4-/- newborns up-regulate antimicrobial peptides, effectors of innate immunity. Comparison of these animal models illustrates how impairment of the skin barrier by two genetically distinct mechanisms leads to innate immune responses, as observed in the common human skin disorders psoriasis and atopic dermatitis.
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http://dx.doi.org/10.1083/jcb.200605057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064601PMC
November 2006