Christopher Schuster

Topical Treatment of Basal Cell Carcinomas in Nevoid Basal Cell Carcinoma Syndrome with a Smoothened Inhibitor

Basal cell carcinoma (BCC) is a distinctive manifestation in nevoid basal cell carcinoma syndrome (NBCCS) patients. Both inherited and acquired mutations of patched 1 (PTCH1), a tumor-suppressor gene controlling the activity of Smoothened (SMO), are the primary cause of the constitutive activation of the Hedgehog (HH) pathway, leading to the emergence of BCCs in NBCCS. LDE225, a distinct, selective antagonist of SMO, showed potent inhibition of basaloid tumor nest formation and mediated regression of preformed basaloid tumors in organ cultures of skin derived from Ptch1 heterozygous knockout mice. In a double-blind, randomized, vehicle-controlled, intraindividual study, a total of 8 NBCCS patients presenting 27 BCCs were treated twice daily with 0.75% LDE225 cream or vehicle for 4 weeks. Application of 0.75% LDE225 cream was well tolerated and showed no skin irritation. Of 13 LDE225-treated BCCs, 3 showed a complete, 9 a partial, and only 1 no clinical response. Except for one partial response, the vehicle produced no clinical response in any of the 14 treated BCCs. Treatment with 0.75% LDE225 cream in NBCCS patients was very well tolerated and caused BCC regression, thus potentially offering an attractive therapeutic alternative to currently available therapies for this indication.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.

Human Dermis Harbors Distinct Mesenchymal Stromal Cell Subsets

Multipotent mesenchymal stromal cells (MSCs) are found in a variety of adult tissues including human dermis. These MSCs are morphologically similar to bone marrow–derived MSCs, but are of unclear phenotype. To shed light on the characteristics of human dermal MSCs, this study was designed to identify and isolate dermal MSCs by a specific marker expression profile, and subsequently rate their mesenchymal differentiation potential. Immunohistochemical staining showed that MSC markers CD73/CD90/CD105, as well as CD271 and SSEA-4, are expressed on dermal cells in situ. Flow cytometric analysis revealed a phenotype similar to bone marrow–derived MSCs. Human dermal cells isolated by plastic adherence had a lower differentiation capacity as compared with bone marrow–derived MSCs. To distinguish dermal MSCs from differentiated fibroblasts, we immunoselected CD271+ and SSEA-4+ cells from adherent dermal cells and investigated their mesenchymal differentiation capacity. This revealed that cells with increased adipogenic, osteogenic, and chondrogenic potential were enriched in the dermal CD271+ population. The differentiation potential of dermal SSEA-4+ cells, in contrast, appeared to be limited to adipogenesis. These results indicate that specific cell populations with variable mesenchymal differentiation potential can be isolated from human dermis. Moreover, we identified three different subsets of dermal mesenchymal progenitor cells.

Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation

Bone morphogenetic protein 7 (BMP7) promotes the differentiation of Langerhans cells in the epidermis during prenatal development.

Phenotypic Characterization of Leukocytes in Prenatal Human Dermis

The adult human skin harbors a variety of leukocytes providing immune surveillance and host defense, but knowledge about their ontogeny is scarce. In this study we investigated the number and phenotype of leukocytes in prenatal human skin (dermal dendritic cells (DDCs), macrophages, T cells (including FoxP3+ regulatory T cells), and mast cells) to unravel their derivation and to get a clue as to their putative function in utero. By flow cytometry and immunofluorescence, we found a distinction between CD206+CD1c+CD11c+ DDCs and CD206+CD209+CD1c− skin macrophages by 9 weeks estimated gestational age (EGA). T cells appear at the end of the first trimester, expressing CD3 intracytoplasmatically. During midgestation, CD3+FoxP3− and CD3+FoxP3+ cells can exclusively be found in the dermis. Similarly, other leukocytes such as CD117+ (c-kit) mast cells were not identified before 12–14 weeks EGA and only slowly acquire a mature phenotype during gestation. Our data show at which time point during gestation antigen-presenting cells, T cells, and mast cells populate the human dermis and provide a step forward to a better understanding of the development of the human skin immune system.

14-3-3ζ escorts CCTα for calcium-activated nuclear import in lung epithelia

Integrity of animal biomembranes is critical to preserve normal cellular functions and viability. Phosphatidylcholine, an indispensible membrane component, requires the enzyme CCTα for its biosynthesis. Nuclear expression of CCTα is needed for expansion of the nuclear membrane network, but mechanisms for CCTα nuclear import are unknown. Herein, we show that in epithelia, extracellular Ca2+ triggers CCTα cytoplasmic‐nuclear translocation. CCTα nuclear import was associated with binding to 14‐3‐3ζ, a key regulator of protein trafficking. 14‐3‐3ζ was both sufficient and required for CCTα nuclear import. Helix G within the 14‐3‐3ζ binding groove interacts with a putative molecular signature within the CCTα carboxyl‐terminal phosphoserine motif (residues 328‐343). 14‐3‐3ζ was critically involved in preserving phosphatidylcholine synthesis and cell viability in a model of Pseudomonas aeruginosa infection where Ca2+ concentrations increase within epithelia. Thus, 14‐3‐3ζ controls CCTα nuclear import in response to calcium signals, thereby regulating mammalian phospholipid synthesis. Agassandian, M., Chen, B. B., Schuster, C. C., Houtman, J. C. D., Mallampalli, R. K. 14‐3‐3ζ escorts CCTα for calcium‐activated nuclear import in lung epithelia. FASEB J. 24, 1271–1283 (2010). www.fasebj.org

Human embryonic epidermis contains a diverse Langerhans cell precursor pool

Despite intense efforts, the exact phenotype of the epidermal Langerhans cell (LC) precursors during human ontogeny has not been determined yet. These elusive precursors are believed to migrate into the embryonic skin and to express primitive surface markers, including CD36, but not typical LC markers such as CD1a, CD1c and CD207. The aim of this study was to further characterize the phenotype of LC precursors in human embryonic epidermis and to compare it with that of LCs in healthy adult skin. We found that epidermal leukocytes in first trimester human skin are negative for CD34 and heterogeneous with regard to the expression of CD1c, CD14 and CD36, thus contrasting the phenotypic uniformity of epidermal LCs in adult skin. These data indicate that LC precursors colonize the developing epidermis in an undifferentiated state, where they acquire the definitive LC marker profile with time. Using a human three-dimensional full-thickness skin model to mimic in vivo LC development, we found that FACS-sorted, CD207- cord blood-derived haematopoietic precursor cells resembling foetal LC precursors but not CD14+CD16- blood monocytes integrate into skin equivalents, and without additional exogenous cytokines give rise to cells that morphologically and phenotypically resemble LCs. Overall, it appears that CD14- haematopoietic precursors possess a much higher differentiation potential than CD14+ precursor cells.

Development of the prenatal cutaneous antigen-presenting cell network

The skin, and in particular the epidermis, is a physical barrier that protects the body from external threats and is critically involved in immune reactivity. Professional antigen‐presenting cells, such as epidermal Langerhans cells and dermal dendritic cells, are gaining prominence as principal players orchestrating the decision between immunity and tolerance. A focus of research interest in recent years has been the investigation of these cells in mammalian prenatal skin. In this review, we will compare the recent progress in dissecting the phenotype and functional role of antigen‐presenting cells in the developing human and mouse skin before birth and perinatally, and will discuss how this knowledge improves our understanding of the level of immunocompetence of the skin in utero.

A Comparative Proteomic Study of Human Skin Suction Blister Fluid from Healthy Individuals Using Immunodepletion and iTRAQ Labeling

Aberrations in skin morphology and functionality can cause acute and chronic skin-related diseases that are the focus of dermatological research. Mechanically induced skin suction blister fluid may serve as a potential, alternative human body fluid for quantitative mass spectrometry (MS)-based proteomics in order to assist in the understanding of the mechanisms and causes underlying skin-related diseases. The combination of abundant-protein removal with iTRAQ technology and multidimensional fractionation techniques improved the number of identified protein groups. A relative comparison of a cohort of 8 healthy volunteers was thus recruited in order to assess the net variability encountered in a healthy scenario. The technology enabled the identification, to date, of the highest number of reported protein groups (739) with concomitant relative quantitative data for over 90% of all proteins with high reproducibility and accuracy. The use of iTRAQ 8-plex resulted in a 66% decrease in protein identifications but, despite this, provided valuable insight into interindividual differences of the healthy control samples. The geometric mean ratio was close to 1 with 95% of all ratios ranging between 0.45 and 2.05 and a calculated mean coefficient of variation of 15.8%, indicating a lower biological variance than that reported for plasma or urine. By applying a multistep sample processing, the obtained sensitivity and accuracy of quantitative MS analysis demonstrates the prospective value of the approach in future research into skin diseases.

Human skin dendritic cell fate is differentially regulated by the monocyte identity factor Kruppel-like factor 4 during steady state and inflammation.

Background Langerhans cell (LC) networks play key roles in immunity and tolerance at body surfaces. LCs are established prenatally and can be replenished from blood monocytes. Unlike skin‐resident dermal DCs (dDCs)/interstitial‐type DCs and inflammatory dendritic epidermal cells appearing in dermatitis/eczema lesions, LCs lack key monocyte‐affiliated markers. Inversely, LCs express various epithelial genes critical for their long‐term peripheral tissue residency. Objective Dendritic cells (DCs) are functionally involved in inflammatory diseases; however, the mechanisms remained poorly understood. Methods In vitro differentiation models of human DCs, gene profiling, gene transduction, and immunohistology were used to identify molecules involved in DC subset specification. Results Here we identified the monocyte/macrophage lineage identity transcription factor Kruppel‐like factor 4 (KLF4) to be inhibited during LC differentiation from human blood monocytes. Conversely, KLF4 is maintained or induced during dermal DC and monocyte‐derived dendritic cell/inflammatory dendritic epidermal cell differentiation. We showed that in monocytic cells KLF4 has to be repressed to allow their differentiation into LCs. Moreover, respective KLF4 levels in DC subsets positively correlate with proinflammatory characteristics. We identified epithelial Notch signaling to repress KLF4 in monocytes undergoing LC commitment. Loss of KLF4 in monocytes transcriptionally derepresses Runt‐related transcription factor 3 in response to TGF‐&bgr;1, thereby allowing LC differentiation marked by a low cytokine expression profile. Conclusion Monocyte differentiation into LCs depends on activation of Notch signaling and the concomitant loss of KLF4. Graphical abstract Figure. No caption available.

Prenatal human skin expresses the antimicrobial peptide RNase 7

Antimicrobial peptides and proteins (AMPs) play important roles in skin immune defense due to their capacity to inhibit growth of microbes. During intrauterine life, the skin immune system has to acquire the prerequisites to protect the newborn from infection in the hostile environment after birth, which includes the production of skin AMPs. The aim of this study was to analyze the expression of RNase 7, HBD-2/3 and psoriasin during human skin development, thus, providing a deeper insight about the maturity of a fundamental component of the innate immune system. We found low RNase 7 expression levels in the periderm but no expression of HBD-2/3 and psoriasin in first trimester human skin using immunohistochemistry. At the end of the second trimester, RNase 7 is expressed weakly in all epidermal layers with a marked signal in the stratum corneum. HBD-3 and psoriasin are focally expressed while HBD-2 is not detectable. Analysis of supernatants from cultured prenatal skin cells showed that in contrast to adult control, RNase 7 and psoriasin are not found in prenatal skin, suggesting that AMPs are detectable but are not secreted. This study shows the differential expression of AMPs in developing, non-perturbed human prenatal skin. It is conceivable that the combined expression of RNase 7, HBD-3 and psoriasin in fetal skin constitutes a developmental program to exert a broad spectrum of antimicrobial activity to maintain sterility in the amniotic cavity.