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Background Dupilumab is an IL-4 receptor α mAb inhibiting signaling of IL-4 and IL-13, key drivers of type 2–driven inflammation, as demonstrated by its efficacy in patients with atopic/allergic diseases. Objective This placebo-controlled, double-blind trial (NCT01979016) evaluated the efficacy, safety, and effects of dupilumab on molecular/cellular lesional and nonlesional skin phenotypes and systemic type 2 biomarkers of patients with moderate-to-severe atopic dermatitis (AD). Methods Skin biopsy specimens and blood were evaluated from 54 patients randomized 1:1 to weekly subcutaneous doses of 200 mg of dupilumab or placebo for 16 weeks. Results Dupilumab (vs placebo) significantly improved clinical signs and symptoms of AD, was well tolerated, and progressively shifted the lesional transcriptome toward a nonlesional phenotype (weeks 4–16). Mean improvements in a meta-analysis–derived AD transcriptome (genes differentially expressed between lesional and nonlesional skin) were 68.8% and 110.8% with dupilumab and −10.5% and 55.0% with placebo (weeks 4 and 16, respectively; P <.001). Dupilumab significantly reduced expression of genes involved in type 2 inflammation (IL13 , IL31 , CCL17 , CCL18 , and CCL26), epidermal hyperplasia (keratin 16 [K16] and MKi67), T cells, dendritic cells (ICOS , CD11c , and CTLA4), and T H 17/T H 22 activity (IL17A , IL-22 , and S100As) and concurrently increased expression of epidermal differentiation, barrier, and lipid metabolism genes (filaggrin [FLG] , loricrin [LOR] , claudins, and ELOVL3). Dupilumab reduced lesional epidermal thickness versus placebo (week 4, P =.001; week 16, P =.0002). Improvements in clinical and histologic measures correlated significantly with modulation of gene expression. Dupilumab also significantly suppressed type 2 serum biomarkers, including CCL17, CCL18, periostin, and total and allergen-specific IgEs. Conclusion Dupilumab-mediated inhibition of IL-4/IL-13 signaling through IL-4 receptor α blockade significantly and progressively improved disease activity, suppressed cellular/molecular cutaneous markers of inflammation and systemic measures of type 2 inflammation, and reversed AD-associated epidermal abnormalities. [ABSTRACT FROM AUTHOR]

Introduction: There is a controversy surrounding the existence of palmoplantar pustulosis (PPP) and palmoplantar pustular psoriasis (PPPP) as separate clinical entities or as variants of the same clinical entity. We used gene expression microarray to compare gene expression in PPP and PPPP. Methodology/Principal findings: Skin biopsies from subjects with PPP (3), PPPP (6), psoriasis vulgaris (10) and acral skin from normal subjects (7) were analyzed using gene expression microarray. Principal component analysis showed that PPP and PPPP were different from psoriasis vulgaris and normal acral skin. However gene expression of PPP and PPPP clustered together and could not be used to differentiate PPP from PPPP. Gene-wise comparison between PPP and PPPP found no gene to be differentially expressed at a false discovery rate lower than 0.05. Surprisingly we found a higher expression of several genes involved in neural pathways (e.g. GPRIN and ADAM23) in PPP/PPPP as compared to psoriasis vulgaris and normal acral skin. Immunohistochemistry confirmed those findings and showed a keratinocyte localization for those proteins. Conclusion significance: PPP and PPPP could not be differentiated using gene expression microarray suggesting that they are not distinct clinical entities. Increased expression of GPRIN1, and ADAM23 in keratinocytes suggests that these proteins could be new therapeutic targets for PPP/PPPP. [ABSTRACT FROM AUTHOR]

Alopecia areata ( AA) is a common inflammatory disease targeting the anagen-stage hair follicle. Different cytokines have been implicated in the disease profile, but their pathogenic role is not yet fully determined. We studied biopsies of pretreatment lesional and non-lesional ( NL) scalp and post-treatment (intra-lesional steroid injection) lesional scalp of 6 patchy patients with AA using immunohistochemistry and gene expression analysis. Immunohistochemistry showed increases in CD3+, CD8+ T cells, CD11c+ dendritic cells and CD1a+ Langerhans cells within and around hair follicles of pretreatment lesional scalp, which decreased upon treatment. qRT- PCR showed in pretreatment lesional scalp (compared to NL) significant increases ( P < 0.05) in expression of inflammatory markers ( IL-2, IL-2 RA, JAK3, IL-15), Th1 ( CXCL10 and CXCL9), Th2 ( IL-13, CCL17 and CCL18), IL-12/ IL-23p40 and IL-32. Among these, we observed significant downregulation with treatment in IL-12/ IL-23p40, CCL18 and IL-32. We also observed significant downregulation of several hair keratins in lesional scalp, with significant upregulation of KRT35, KRT75 and KRT86 in post-treatment lesional scalp. This study shows concurrent activation of Th1 and Th2 immune axes as well as IL-23 and IL-32 cytokine pathways in lesional AA scalp and defined a series of response biomarkers to corticosteroid injection. Clinical trials with selective antagonists coupled with cytokine-pathway biomarkers will be necessary to further dissect pathogenic immunity. [ABSTRACT FROM AUTHOR]

Scalp psoriasis shows a variable clinical spectrum and in many cases poses a great therapeutic challenge. However, it remains unknown whether the immune response of scalp psoriasis differs from understood pathomechanisms of psoriasis in other skin areas. We sought to determine the cellular and molecular phenotype of scalp psoriasis by performing a comparative analysis of scalp and skin using lesional and nonlesional samples from 20 Caucasian subjects with untreated moderate to severe psoriasis and significant scalp involvement and 10 control subjects without psoriasis. Our results suggest that even in the scalp, psoriasis is a disease of the inter-follicular skin. The immune mechanisms that mediate scalp psoriasis were found to be similar to those involved in skin psoriasis. However, the magnitude of dysregulation, number of differentially expressed genes, and enrichment of the psoriatic genomic fingerprint were more prominent in skin lesions. Furthermore, the scalp transcriptome showed increased modulation of several gene-sets, particularly those induced by interferon-gamma, compared with that of skin psoriasis, which was mainly associated with activation of TNFα/L-17/IL-22-induced keratinocyte response genes. We also detected differences in expression of gene-sets involving negative regulation, epigenetic regulation, epidermal differentiation, and dendritic cell or Th1/Th17/Th22-related T-cell processes. [ABSTRACT FROM AUTHOR]

The article discuss a study on in vitro alteration of gene expression of epidermal melanocytes by dendritic cells (DCs) of melanocytic nevus. It mentions the hematoxylin and eosin staining of nevus cells followed by immunohistiochemistry of several immune cell markers. It mentions the correlation of DCs and melanocytic cells and infers that the senescence-associated nevus cell phenotype supposedly affects the DCs within the nevus nest.

Background Severe atopic dermatitis (AD) has a high unmet need for effective and safe therapeutics. In early-phase trials, dupilumab, a fully human mAb targeting IL-4 receptor α, markedly improved disease activity, but the effect of IL-4/IL-13 blockade on AD at the molecular level has not been characterized. Objectives We sought to evaluate dupilumab modulation of the AD molecular signature. Methods We performed transcriptomic analyses of pretreatment and posttreatment skin biopsy specimens from patients with moderate-to-severe AD treated weekly with 150 or 300 mg of dupilumab or placebo. Results Exacerbation of the AD transcriptome was observed in placebo-treated patients. Dupilumab improved the AD signature in a dose-dependent manner. Expression of genes upregulated in AD lesions decreased in patients treated with dupilumab by 26% (95% CI, 21% to 32%) and 65% (95% CI, 60% to 71%) for treatment with 150 and 300 mg, respectively. Genes downregulated in AD lesions increased by 21% (95% CI, 16% to 27%) and 32% (95% CI, 26% to 37%) with dupilumab (150 and 300 mg, respectively). The molecular changes paralleled improvements in clinical scores. A dupilumab treatment signature of 821 probes (>2-fold change, P < .05) significantly modulated in the 300-mg dupilumab group at week 4 compared with baseline was identified in this sample set. Significant ( P < .05) decreases in mRNA expression of genes related to hyperplasia ( K16 and MKI67 ), T cells, and dendritic cells ( CD1b and CD1c ) and potent inhibition of T H 2-associated chemokines ( CCL17 , CCL18 , CCL22 , and CCL26 ) were noted without significant modulation of T H 1-associated genes (IFNG) . Conclusions This is the first report showing rapid improvement of the AD molecular signature with targeted anti–IL-4 receptor α therapy. These data suggest that IL-4 and IL-13 drive a complex, T H 2-centered inflammatory axis in patients with AD. [ABSTRACT FROM AUTHOR]

Mutations in the caspase recruitment domain, family member 14 (CARD14) gene have recently been described in psoriasis patients, and explain the psoriasis susceptibility locus 2 (PSORS2). CARD14 is a scaffolding protein that regulates NF-κB activation, and psoriasis-associated CARD14 mutations lead to enhanced NF-κB signaling. CARD14 is expressed mainly in epidermal keratinocytes, but also in unidentified dermal cells. In this manuscript, the identity of the dermal cell types expressing CARD14, as well the potential functional consequence of overactive CARD14 in these dermal cell types, was determined. Using two-color immunofluorescence, dermal CARD14 did not co-localize with T-cells, dendritic cells, or macrophages. However, dermal CARD14 did highly co-localize with CD31+ endothelial cells (ECs). CARD14 was also expressed non-dermal endothelial cells, such as aortic endothelial cells, which may indicate a role of CARD14+ECs in the systemic inflammation and cardiovascular comorbidities associated with psoriasis. Additionally, phosphorylated NF-κB was found in psoriatic CARD14+ CD31+ ECs, demonstrating this pathway is active in dermal ECs in psoriasis. Transfection of dermal ECs with psoriasis-associated CARD14 mutations resulted in increased expression of several chemokines, including CXCL10, IL-8, and CCL2. These results provide preliminary evidence that CARD14 expression in ECs may contribute to psoriasis through increased expression of chemokines and facilitating recruitment of immune cells into skin. [ABSTRACT FROM AUTHOR]

The precise mechanisms governing invasion at the leading edge of squamous cell carcinoma (SCC) and its subsequent metastasis are not fully understood. We aimed to define the cancer-related molecular changes that distinguish noninvasive tumor from invasive SCC. To this end, we combined laser capture microdissection with complementary DNA (cDNA) microarray analysis. We defined invasion-associated genes as those differentially regulated only in invasive SCC nests, but not in actinic keratosis or in situ SCC, compared with normal epidermis. There were 383 upregulated and 354 downregulated genes in the 'invasion set.' SCC invasion was characterized by aberrant expression of various proteolytic molecules. We noted increased expression of MMP7 and IL-24 in invasive SCC. IL-24 induced the expression of matrix metallopeptidase 7 (MMP7) in SCC cells in culture. In addition, blocking of MMP7 by a specific antibody significantly delayed the migration of SCC cells in culture. These results suggest a possible contribution of IL-24 to SCC invasion via enhancing focal expression of MMP7, although IL-24 has been suggested to have antitumor growth effects in other cancer types. Identification of regional molecular changes that regulate cancer invasion may facilitate the development of new targeted treatments for aggressive cancer. [ABSTRACT FROM AUTHOR]

Background: IL-17 is the defining cytokine of the Th17, Tc17, and γδ T cell populations that plays a critical role in mediating inflammation and autoimmunity. Psoriasis vulgaris is an inflammatory skin disease mediated by Th1 and Th17 cytokines with relevant contributions of IFN-γ, TNF-α, and IL-17. Despite the pivotal role IL-17 plays in psoriasis, and in contrast to the other key mediators involved in the psoriasis cytokine cascade that are capable of inducing broad effects on keratinocytes, IL-17 was demonstrated to regulate the expression of a limited number of genes in monolayer keratinocytes cultured in vitro. Methodology/Principal Findings: Given the clinical efficacy of anti-IL-17 agents is associated with an impressive reduction in a large set of inflammatory genes, we sought a full-thickness skin model that more closely resemble in vivo epidermal architecture. Using a reconstructed human epidermis (RHE), IL-17 was able to upregulate 419 gene probes and downregulate 216 gene probes. As possible explanation for the increased gene induction in the RHE model is that C/CAAT-enhancer-binding proteins (C/EBP) -β, the transcription factor regulating IL-17-responsive genes, is expressed preferentially in differentiated keratinocytes. Conclusions/Significance: The genes identified in IL-17-treated RHE are likely relevant to the IL-17 effects in psoriasis, since ixekizumab (anti-IL-17A agent) strongly suppressed the “RHE” genes in psoriasis patients treated in vivo with this IL-17 antagonist. [ABSTRACT FROM AUTHOR]

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 TH2 predominating in acute disease and a switch to TH1 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 TH22 and TH2 cytokines and smaller increases in IL-17 levels. A lesser induction of TH1-associated genes was detected in acute disease, although some were significantly upregulated in chronic disease. Further significant intensification of major TH22 and TH2 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 TH2 and TH22 cytokines. Our findings support a model of progressive activation of TH2 and TH22 immune axes from the acute to chronic phases, expanding the prevailing view of pathogenesis with important therapeutic implications. [Copyright &y& Elsevier]

The cause of psoriasis, a common chronic inflammatory skin disease, is not fully understood. Microarray experiments have been widely used in recent years to identify genes associated with psoriasis pathology, by comparing expression levels of lesional (LS) with adjacent non-lesional (NL) skin. It is commonly observed that the differentially expressed genes (DEGs) differ greatly across experiments, due to variations introduced in the microarray experiment pipeline. Therefore, a statistically based meta-analytic approach, which combines the results of individual studies, is warranted. In this study, a meta-analysis was conducted on 5 microarray data sets, including 193 LS and NL pairs. We termed this the Meta-Analysis Derived (MAD) transcriptome. In "MAD-5'' transcriptome, 677 genes were up-regulated and 443 were down-regulated in LS skin compared to NL skin. This represents a much larger set than the intersection of DEGs of these 5 studies, which consisted of 100 DEGs. We also analyzed 3 of the studies conducted on the Affymetrix hgu133plus2 chips and found a greater number of DEGs (1084 up- and 748 down-regulated). Top canonical pathways over-represented in the MAD transcriptome include Atherosclerosis Signaling and Fatty Acid Metabolism, while several ''new'' genes identified are involved in Cardiovascular Development and Lipid Metabolism. These findings highlight the relationship between psoriasis and systemic manifestations such as the metabolic syndrome and cardiovascular disease. Then, the Meta Threshold Gradient Descent Regularization (MTGDR) algorithm was used to select potential markers distinguishing LS and NL skin. The resulting set (20 genes) contained many genes that were part of the residual disease genomic profile (RDGP) or ''molecular scar''after successful treatment, and also genes subject to differential methylation in LS tissues. To conclude, this MAD transcriptome yielded a reference list of reliable psoriasis DEGs, and represents a robust pool of candidates for further discovery of pathogenesis and treatment evaluation. [ABSTRACT FROM AUTHOR]

Psoriasis is a complex inflammatory disease that usually heals without visible scarring. Histological evaluation often suggests complete resolution, but reversal of genomic disease-associated alterations has not yet been defined. Gene expression profiling was used to determine the extent to which the psoriasis genes were reversed after 3 months of etanercept treatment in patients who responded to treatment. We reviewed the histology, leukocyte counts, and PCR data for inflammatory genes, to compare recovery of these parameters and the genomic studies. Many cellular markers do return close to nonlesional levels, although five inflammatory genes did not improve by >75% (IL-12p35, MX1, IL-22, IL-17, and IFNγ). Psoriasis-related genes with <75% improvement were defined as comprising a 'residual disease genomic profile,' composed of 248 probe sets. Genes of interest in psoriasis tissue that did not return to baseline included LYVE-1, WNT5A, RAB31, and AQP9. It appears that even when the epidermal reaction in psoriasis is fully resolved, inflammation, as defined by expression of key cytokines and chemokines, is not completely resolved in treated lesions. We also found that structural cells of the skin continued to express molecular alterations, and that some subtle features of skin structure, for example, lymphatics, were not fully normalized with treatment. [ABSTRACT FROM AUTHOR]

Background: Alefacept treatment is highly effective in a select group patients with moderate-to-severe psoriasis, and is an ideal candidate to develop systems to predict who will respond to therapy. A clinical trial of 22 patients with moderate to severe psoriasis treated with alefacept was conducted in 2002-2003, as a mechanism of action study. Patients were classified as responders or non-responders to alefacept based on histological criteria. Results of the original mechanism of action study have been published. Peripheral blood was collected at the start of this clinical trial, and a prior analysis demonstrated that gene expression in PBMCs differed between responders and non-responders, however, the analysis performed could not be used to predict response. Methods: Microarray data from PBMCs of 16 of these patients was analyzed to generate a treatment response classifier. We used a discriminant analysis method that performs sample classification from gene expression data, via "nearest shrunken centroid method". Centroids are the average gene expression for each gene in each class divided by the within-class standard deviation for that gene. Results: A disease response classifier using 23 genes was created to accurately predict response to alefacept (12.3% error rate). While the genes in this classifier should be considered as a group, some of the individual genes are of great interest, for example, cAMP response element modulator (CREM), v-MAF avian musculoaponeurotic fibrosarcoma oncogene family (MAFF), chloride intracellular channel protein 1 (CLIC1, also called NCC27), NLR family, pyrin domain-containing 1 (NLRP1), and CCL5 (chemokine, cc motif, ligand 5, also called regulated upon activation, normally T expressed, and presumably secreted/RANTES). Conclusions: Although this study is small, and based on analysis of existing microarray data, we demonstrate that a treatment response classifier for alefacept can be created using gene expression of PBMCs in psoriasis. This preliminary study may provide a useful tool to predict response of psoriatic patients to alefacept. [ABSTRACT FROM AUTHOR]

Background: Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans show similar artefacts, which affect the analysis, particularly when one tries to detect subtle changes. However, most blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs). Results: We present a method that harnesses the statistical power provided by having several HDONAs available, which are obtained under similar conditions except for the experimental factor. This method "harshlights" blemishes and renders them evident. We find empirically that about 25% of our chips are blemished, and we analyze the impact of masking them on screening for differentially expressed genes. Conclusion: Experiments attempting to assess subtle expression changes should be carefully screened for blemishes on the chips. The proposed method provides investigators with a novel robust approach to improve the sensitivity of microarray analyses. By utilizing topological information to identify and mask blemishes prior to model based analyses, the method prevents artefacts from confounding the process of background correction, normalization, and summarization. [ABSTRACT FROM AUTHOR]

Background: Atopic dermatitis (AD) is a common inflammatory skin disease with a TH2 and “T22” immune polarity. Despite recent data showing a genetic predisposition to epidermal barrier defects in some patients, a fundamental debate still exists regarding the role of barrier abnormalities versus immune responses in initiating the disease. An extensive study of nonlesional AD (ANL) skin is necessary to explore whether there is an intrinsic predisposition to barrier abnormalities, background immune activation, or both in patients with AD. Objective: We sought to characterize ANL skin by determining whether epidermal differentiation and immune abnormalities that characterize lesional AD (AL) skin are also reflected in ANL skin. Methods: We performed genomic and histologic profiling of both ANL and AL skin lesions (n = 12 each) compared with normal human skin (n = 10). Results: We found that ANL skin is clearly distinct from normal skin with respect to terminal differentiation and some immune abnormalities and that it has a cutaneous expansion of T cells. We also showed that ANL skin has a variable immune phenotype, which is largely determined by disease extent and severity. Whereas broad terminal differentiation abnormalities were largely similar between involved and uninvolved AD skin, perhaps accounting for the “background skin phenotype,” increased expression of immune-related genes was among the most obvious differences between AL and ANL skin, potentially reflecting the “clinical disease phenotype.” Conclusion: Our study implies that systemic immune activation might play a role in alteration of the normal epidermal phenotype, as suggested by the high correlation in expression of immune genes in ANL skin with the disease severity index. [ABSTRACT FROM AUTHOR]

The article offers information on the difference in gene expression of the outermost layer of skin keratinocyte which is obtained by laser capture microdissection (LCM) and cell separation methods. It informs that the LCM assess differentiation-related transcription factors in basal and superbasal human epidermis but limited number of genes were analyzed by polymerase chain reaction (PCR) on LCM.

Cutaneous T-cell lymphomas (CTCLs) form a heterogeneous group of non-Hodgkin's lymphomas characterized by only poor prognosis in advanced stage. Despite significant progress made in the identification of novel genes and pathways involved in the pathogenesis of cutaneous lymphoma, the therapeutic value of these findings has still to be proven. Here, we demonstrate by gene expression arrays that Aurora kinase A is one of the highly overexpressed genes of the serine/threonine kinase in CTCL. The finding was confirmed by quantitative reverse transcriptase-PCR, western blotting, and immunohistochemistry in CTCL cell lines and primary patient samples. Moreover, treatment with a specific Aurora kinase A inhibitor blocks cell proliferation by inducing cell cycle arrest in G2 phase, as well as apoptosis in CTCL cell lines. These data provide a promising rationale for using Aurora kinase A inhibition as a therapeutic modality of CTCL. [ABSTRACT FROM AUTHOR]

In the early stages of wound healing, keratinocytes (KCs) become 'activated' and release inflammatory molecules such as IL-1 and IL-8, which are linked to innate immune responses and neutrophil recruitment. It is unclear, however, whether KCs release molecules linked to adaptive immune responses, e.g., CCL20, in their early state of activation without signals from infiltrating T cells. This study aims to isolate the immediate alterations in protective and inflammatory gene expression that occur in epidermal KCs, with a particular focus on molecules associated with cell-mediated immunity. We used dispase-separated epidermis, followed by intercellular disassociation by trypsinization, as a model for epidermal injury. We obtained a pure population of KCs using flow cytometry. As a control for uninjured epidermis, we performed laser capture microdissection on normal human skin. Sorted KCs had an early burst of upregulated gene expression, which included CCL20, IL-15, IL-23A, IFN-κ, and several antimicrobial peptides. Our results provide insight into the potential role of KCs as contributors to cell-mediated inflammation, and expand knowledge about gene modulation that occurs during early wound healing. Our findings may be relevant to cutaneous diseases such as psoriasis, where micro-injury can trigger the formation of psoriatic plaques at the site of trauma. [ABSTRACT FROM AUTHOR]

Metastases from primary cutaneous squamous cell carcinoma (SCC) account for the majority of the ∼10,000 non-melanoma skin cancer deaths in the United States annually. We studied lymphangiogenesis in human SCC because of the potential link to metastasis. SCC samples were stained for lymphatic endothelial vessel marker LYVE-1 and positive cells were counted and compared with cells in normal skin. Gene set enrichment analysis and reverse transcription (RT)-PCR were performed on SCC, on adjacent non-tumor-bearing skin, and on normal skin to determine the differential expression of lymphangiogenesis-associated genes. Laser capture microdissection (LCM) was performed to isolate tumor cells and tumor-associated inflammatory cells for further gene expression analysis. Immunofluorescence was performed to determine the source of vascular endothelial growth factor-C (VEGF-C) in the tumor microenvironment. We found increased lymphatic density and reorganized lymphatic endothelial vessels in the dermis immediately adjacent to SCC nests. RT-PCR confirmed the presence of VEGF-C in skin immediately adjacent to SCC. LCM confirmed the increased expression of VEGF-C, the SCC inflammatory infiltrate. The presence of CD163+/CD68+/VEGFC+ cells and absence of VEGF-C expression by CD3+ or CD11C+ cells suggested that VEGF-C is derived from tumor-associated macrophages. Clarification of mechanisms governing SCC-mediated lymphangiogenesis may identify potential targets for therapeutic intervention against aggressive or inoperable disease. [ABSTRACT FROM AUTHOR]