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The adult human skin contains a vast number of T cells that are essential for skin homeostasis and pathogen defense. T cells are first observed in the skin at the early stages of gestation; however, our understanding of their contribution to early immunity has been limited by their low abundance and lack of comprehensive methodologies for their assessment. Here, we describe a new workflow for isolating and expanding significant amounts of T cells from fetal human skin. Using multiparametric flow cytometry and in situ immunofluorescence, we found a large population with a naive phenotype and small populations with a memory and regulatory phenotype. Their molecular state was characterized using single-cell transcriptomics and TCR repertoire profiling. Importantly, culture of total fetal skin biopsies facilitated T cell expansion without a substantial impact on their phenotype, a major prerequisite for subsequent functional assays. Collectively, our experimental approaches and data advance the understanding of fetal skin immunity and potential use in future therapeutic interventions., Summary: This study provides versatile tools for the isolation, phenotyping and expansion of human fetal skin T cells, enabling the study of their complexity and heterogeneity allowing new research in skin development and immunity.

Various autoantibodies are detected more frequently in HIV-infected individuals than in HIV-negative controls; however, limited data exist regarding autoimmune blistering skin diseases. Using enzyme-linked immunoassay (ELISA) and indirect immunofluore-scence, no difference in the frequency and magnitude of autoantibodies against BP180, BP230, desmoglein 1 and 3 was found between 594 HIV-infected patients and 248 uninfected controls in this cross-sectional study (16.0% vs. 11.7%, respectively, for at least one positive ELISA, p = 0.11). Interestingly, reactive syphilis serology in both HIV-infected individuals and uninfected controls was associated with positive anti-BP180 ELISA results (adjusted odds ratio (OR) 2.14, 95% confidence interval (CI) 1.07-4.29, p = 0.03 and OR 4.70, CI 1.3-16.86; p = 0.0180). Our study shows a comparably low prevalence of cutaneous autoantibodies in both HIV-infected patients and uninfected controls lacking signs of autoimmune blistering skin disease. Positive BP180 ELISA in the absence of clinical signs of bullous pemphigoid should prompt further evaluation for syphilis antibodies.

By characterizing the poorly defined T cell composition in developing human fetal skin, Reitermaier et al. identify a unique population of TCR αβγδ+ T cells. These cells may contribute to early skin development and immune defense against invading pathogens in utero., T cells in human skin play an important role in the immune defense against pathogens and tumors. T cells are present already in fetal skin, where little is known about their cellular phenotype and biological function. Using single-cell analyses, we identified a naive T cell population expressing αβ and γδ T cell receptors (TCRs) that was enriched in fetal skin and intestine but not detected in other fetal organs and peripheral blood. TCR sequencing data revealed that double-positive (DP) αβγδ T cells displayed little overlap of CDR3 sequences with single-positive αβ T cells. Gene signatures, cytokine profiles and in silico receptor–ligand interaction studies indicate their contribution to early skin development. DP αβγδ T cells were phosphoantigen responsive, suggesting their participation in the protection of the fetus against pathogens in intrauterine infections. Together, our analyses unveil a unique cutaneous T cell type within the native skin microenvironment and point to fundamental differences in the immune surveillance between fetal and adult human skin., Graphical Abstract

RTN1 is an endoplasmic reticulum-associated protein that was initially identified in neuronal tissues. Here we show that the main isoform RTN1A is a marker for dendritic cells. In the skin, HLA-DR+CD1ahighCD207+CD11cweak Langerhans cells were the only cells in the epidermis, and HLA-DR+CD11c+ dendritic cells were the main cells in the dermis, expressing this protein. RTN1A+ dendritic cells were also found in gingiva, trachea, tonsil, thymus, and peripheral blood. During differentiation of MUTZ-3 cells into Langerhans cells, expression of RTN1A mRNA and protein preceded established Langerhans cell markers CD1a and CD207, and RTN1A protein partially co-localized with the endoplasmic reticulum marker protein disulfide isomerase. In line with this observation, we found that RTN1A was expressed by around 80% of Langerhans cell precursors in human embryonic skin. Our findings show that RTN1A is a marker for cells of the dendritic lineage, including Langerhans cells and dermal dendritic cells. This unexpected finding will serve as a starting point for the elucidation of the, until now, elusive functional roles of RTN1A in both the immune and the nervous system.

Blepharoplasty is the third most common plastic surgical procedure in the USA. Due to the emergence of multiresistant bacteria, optimising the antiseptic procedure is crucial. Choice of antiseptics plays an important role as they may cause skin irritation and colouring of disinfected areas. In this study, the use of the aqueous antiseptic octenisept® (octenidine) was evaluated in the outcome of blepharoplasties: incidence of wound dehiscence; haematoma; and infection in correlation with gender, medication, smoking habits and time of year. This retrospective surveillance study included 352 patients (median age 58·3 years). Skin disinfection was performed thrice prior to blepharoplasty. Sutures were removed on day 6. None of the patients suffered from wound infection. The total rate of wound dehiscence was 6·3%, with a higher ratio among male patients. Smokers and patients on anticoagulant medication showed a significantly higher incidence of wound dehiscence. Throughout the year, rates of wound dehiscence were highest in summer. Aseptic surgical preparation for blepharoplasty via full-face scrub with octenisept® without oral antibiotic prophylaxis is well tolerated, with no report of wound infection, which may improve antibiotic stewardship as well as patient comfort. Elective upper eyelid blepharoplasty may ideally be performed in winter.

// Abigail J. Deloria 1 , Doris Hoflmayer 2, 6 , Philip Kienzl 1 , Justyna Łopatecka 1 , Sandra Sampl 1 , Martin Klimpfinger 2 , Tamara Braunschmid 3 , Fabienne Bastian 3 , Lingeng Lu 4 , Brigitte Marian 1 , Stefan Stattner 3, 5 , Klaus Holzmann 1 1 Division of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University Vienna, Austria 2 Department of Pathology and Bacteriology, Social Medical Center South, Kaiser Franz Josef Hospital, Vienna, Austria 3 Department of Surgery, Social Medical Center South, Kaiser Franz Josef Hospital, Vienna, Austria 4 Department of Chronic Disease Epidemiology, Yale School of Public Health, School of Medicine, Yale Cancer Center, Yale University, New Haven, USA 5 Department of Visceral, Transplantation and Thoracic Surgery, Innsbruck, Austria 6 Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Correspondence to: Klaus Holzmann, email: klaus.holzmann@meduniwien.ac.at Keywords: colorectal cancer, epithelial splicing regulatory protein, microsatellite instability, overall survival, prognostic marker Received: April 25, 2016 Accepted: September 02, 2016 Published: September 16, 2016 ABSTRACT ESRPs are master splice regulators implicated in alternative mRNA splicing programs important for epithelial-mesenchymal transition (EMT) and tumor progression. ESRP1 was identified in some tumors as good or worse predictor of outcome, but in colorectal cancer (CRC) the prognostic value of ESRPs and relation with mesenchymal splice variants is not clear. Here, we studied 68 CRC cases, compared tissue expression of ESRPs with clinical data and with EMT gene splice patterns of conditional CRC cells with deficient ESRP1 expression. Around 72% of patients showed global decreased transcript expression of both ESRPs in tumor as compared to matched non-neoplastic colorectal epithelium. Reduction of ESRP1 in tumor cells was evaluated by immunohistochemistry, associated with microsatellite stability and switch to mesenchymal splice signatures of FGFRs, CD44, ENAH and CTNND1(p120-catenin). Expression of ESRPs was significantly associated with favorable overall survival (log-rank test, P =0.0186 and 0.0408), better than prognostic stratification by tumor staging; and for ESRP1 confirmed with second TCGA cohort (log-rank test, P =0.0435). Prognostic value is independent of the pathological stage and microsatellite instability (ESRP1: HR=0.36, 95%CI 0.15–0.91, P =0.032; ESRP2: HR=0.23, 95%CI 0.08–0.65, P =0.006). Our study supports the role of ESRP1 as tumor suppressor and strongly suggests that ESRPs are candidate markers for early detection, diagnosis, and prognosis of CRC.

Hypertrophic scar formation because of surgical procedures is associated with higher levels of pain, a lower quality of life, and poor cosmetic outcome and requires more resources in follow-up management. An octenidine-based hydrogel has been shown to modulate immunological function in an in vitro wound model, suggesting an improved scar formation. In this prospective, randomised, observer-blinded, and intra-patient-controlled study, 45 patients who underwent abdominoplasty or mastectomy with transverse rectus abdominis muscle (TRAM) flap reconstruction were given both a standard postoperative wound dressing on one wound side and an octenidine-based hydrogel with transparent film dressing, covered with standard postoperative dressing on the other side. Four instances of hypertrophia were reported in the gel side versus 12 in the standard dressing side. Visual Analogue Scale (VAS) pain scores taken during postoperative dressing changes showed reduced scores on the gel side at all time points. Vancouver Scar Scale (VSS) scores showed improvement in the gel side at 3, 6, and 12 months postoperatively. Skin distensibility measured using a cutometer showed significantly improved measures in gel-treated wounds, similar to measures of healthy skin. Trans-epidermal water loss (TEWL), measured using a tewameter, showed improved values on the gel side soon after surgery, with both the control and the gel side normalising after approximately 6 months. The octenidine-based wound dressing demonstrates improved wound healing associated with a lower incidence of hypertrophic scar formation.

Together with keratinocytes (KCs) and the dense network of Langerhans cells (LCs), the epidermis is an ideal portal for vaccine delivery. Pattern recognition receptor agonists, in particular polyinosinic–polycytidylic acid [p(I:C)], are promising adjuvant candidates for therapeutic vaccination to generate protective T-cell immunity. Here we established an ex vivo skin explant model to study the expression and activation of double-stranded RNA (dsRNA)-sensing pattern recognition receptors in LCs and KCs in human skin. Whereas KCs expressed all known dsRNA sensing receptors at a constitutive and inducible level, LCs exclusively expressed melanoma differentiation–associated protein 5 (MDA5) in untreated skin and freshly isolated cells. Comparative assessments of downstream signaling pathways induced by p(I:C) revealed distinct mitochondrial antiviral-signaling protein, IFN-regulatory factor 3, and NF-κB activation in LCs and KCs. Consequently, p(I:C) treatment of LCs significantly induced IFN-α and IFN-β mRNA expression, while in KCs an up-regulation of IFN-β and TNF-α mRNA was detectable. Stimulation of LCs with specific ligands revealed that not the TLR3- but only the MDA5-specific ligand induced IFN-α2, IFN-β, and TNF-α cytokines, but no IL-6 and -8. In KCs, both ligands induced production of high IL-6 and IL-8 levels, and low IFN-α2 and IFN-β levels, indicating that different dsRNA-sensing receptors and/or downstream signaling pathways are activated in both cell types. Our data suggest that MDA5 may be an attractive adjuvant target for epicutaneous delivery of therapeutic vaccines with the goal to target LCs.—Tajpara, P., Schuster, C., Schön, E., Kienzl, P., Vierhapper, M., Mildner, M., Elbe-Bürger, A. Epicutaneous administration of the pattern recognition receptor agonist polyinosinic–polycytidylic acid activates the MDA5/MAVS pathway in Langerhans cells.

Background: In highrisk populations, allergenspecific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of viruslike nanoparticles (VNP) expressing surfaceexposed or shielded allergens. Methods: Fulllength major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. Results: Viruslike nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surfaceexposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergenspecific T cells in vitro. Upon intranasal application in mice, VNP expressing surfaceexposed but not shielded allergen induced allergenspecific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergenprotected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Tregdominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. Conclusion: Viruslike nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization. DKW1248 SFB F4605 SFB F4609 (VLID)3132477

A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer.

LINE-1 elements (L1s, long interspersed nuclear elements-1) are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Here we show the involvement of L1 in telomere maintenance in cells using the less characterized alternative lengthening of telomeres (ALT) mechanism. Interestingly, ALT cells show significantly higher expression levels of L1 when compared to TA cells. Knock-down (KD) of L1 in ALT cell lines was associated with reduced length of telomeres, an increase in telomere dysfunction foci, a reduced expression of ALT characteristics such as c-circles and ALT associated PML bodies, and a decreased growth. On the other hand, overexpression of L1 in ALT cell lines lead to a higher rate of c-circles and increased length of telomeres. LINE-1 not only bound to the C-strand of telomeric DNA but also to telomere-repeat-containing RNA (TERRA), a multifunctional component of human telomeres that is highly expressed in ALT cells and has been described to be involved in telomere stabilization. Whereas L1-KD decreased overall TERRA, L1 overexpression increased this RNA. Moreover, L1 KD abrogated the nuclear retention of TERRA. Moreover, the L1-ribonucleoprotein can use the polyadenylated form of TERRA as a template to generate telomere-specific DNA. Thus, L1 appears to contribute to telomere maintenance by a mechanism involving TERRA, either by ensuring its nuclear localization or by using this RNA as a template for the generation of telomere-specific DNA. Our findings now render L1 proteins a promising target in cancer therapy by interfering with telomere lengthening in ALT cells. Citation Format: Thomas Aschacher, Brigitte Wolf, Philip Kienzl, Florian Enzmann, Barbara Messner, Klaus Holzmann, Michael M. Bergmann. A role of long interspersed nuclear element-1 (LINE-1) for telomere maintenance in cells with alternative lengthening of telomeres. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-084. doi:10.1158/1538-7445.AM2015-LB-084

Tumors can overcome cellular senescence, one of the main barriers to tumor development and immortality by telomere maintenance mechanism (TMM). 90% of colorectal carcinomas (CRC) use telomerase activity (TA) as TMM. Mechanisms of remaining CRC tumors are not well analyzed yet and may utilize alternative lengthening of telomeres (ALT) or a not defined telomere maintenance mechanism (NDTMM). Both TA and expression of telomeric repeat containing RNA (TERRA) were recently identified to correlate with tumor grade. This led us to evaluate possible correlations of TMM and TERRA levels to clinical data. Tissues from 49 CRC patients (35 grade II, 14 grade III, 2 polyps) and cell lines (N = 7) were assessed. Tumor tissues were compared to matched adjacent non-tumor tissues. Mean telomere length (TL) was measured by real-time PCR and telomere restriction fragment (TRF) length analyses. TMM were evaluated by measuring TA with telomere amplification protocol (TRAP) and by detection of ALT with c-circles. Real-time RT-PCR assays for relative quantity (RQ) of transcript levels for TERRA, telomerase genes hTERT and hTERC were determined in relation to levels of house-keeping genes and normal tissues. RQ values were analyzed for significant differences between groups by Mann-Whitney test and were assessed after log-transformation with the Pearson's correlation calculation. TL of grade II and grade III tumors were in the range of 5.5 ± 1.3kbp and 5.3 ± 0.5kbp, respectively (mean ± SD), and were comparable with TL of normal tissue and cell lines. TA was detected with mean of 27 TPG units in 90% (34 of 38) and with mean of 4 units in 24% (9 of 38) of tumor and normal tissues, respectively. All cell lines analyzed demonstrate TA with mean of 51 units. A restricted set of tumors consisting of 2 with and 4 without TA was analyzed for ALT. One of the analyzed tumors without detectable TA showed significantly elevated levels of c-circles, but did not reach levels of sarcomas and cell models with ALT, indicating the existence of NDTMM in CRC. Within the transcripts analyzed, TERRA levels showed a moderate negative correlation with TA (Pearson r=−0.45, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4597. doi:1538-7445.AM2012-4597