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Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.

Autophagy is the central cellular mechanism for delivering organelles and cytoplasm to lysosomes for degradation and recycling of their molecular components. To determine the contribution of autophagy to melanocyte (MC) biology, we inactivated the essential autophagy gene Atg7 specifically in MCs using the Cre-loxP system. This gene deletion efficiently suppressed a key step in autophagy, lipidation of microtubule-associated protein 1 light chain 3 beta (LC3), in MCs and induced slight hypopigmentation of the epidermis in mice. The melanin content of hair was decreased by 10–15% in mice with autophagy-deficient MC as compared with control animals. When cultured in vitro, MCs from mutant and control mice produced equal amounts of melanin per cell. However, Atg7-deficient MCs entered into premature growth arrest and accumulated reactive oxygen species (ROS) damage, ubiquitinated proteins, and the multi-functional adapter protein SQSTM1/p62. Moreover, nuclear factor erythroid 2–related factor 2 (Nrf2)–dependent expression of NAD(P)H dehydrogenase, quinone 1, and glutathione S-transferase Mu 1 was increased, indicating a contribution of autophagy to redox homeostasis in MCs. In summary, the results of our study suggest that Atg7-dependent autophagy is dispensable for melanogenesis but necessary for achieving the full proliferative capacity of MCs.

Urocanic acid (UCA) is produced by the enzyme histidase and accumulates in the stratum corneum of the epidermis. In this study, we investigated the photoprotective role of endogenous UCA in the murine skin using histidinemic mice, in which the gene encoding histidase is mutated. Histidase was detected by immunohistochemistry in the stratum granulosum and stratum corneum of the normal murine skin but not in the histidinemic skin. The UCA content of the stratum corneum and the UVB absorption capacity of aqueous extracts from the stratum corneum were significantly reduced in histidinemic mice as compared with wild-type mice. When the shaved back skin of adult mice was irradiated with 250mJcm-2 UVB, histidinemic mice accumulated significantly more DNA damage in the form of cyclobutane pyrimidine dimers than did wild-type mice. Furthermore, UVB irradiation induced significantly higher levels of markers of apoptosis in the epidermis of histidinemic mice. Topical application of UCA reversed the UVB-photosensitive phenotype of histidinemic mice and increased UVB photoprotection of wild-type mice. Taken together, these results provide strong evidence for an important contribution of endogenous UCA to the protection of the epidermis against the damaging effects of UVB radiation.

Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SEC(PBMC)). Six mm punch biopsy wounds were set on the backs of C57BL/6J-mice and SEC(PBMC) containing emulsion or controls were applied daily for three days. Morphology and neo-angiogenesis were analyzed by H&E-staining and CD31 immuno-staining, respectively. In vitro effects on diverse skin cells were investigated by migration assays, cell cycle analysis, and tube formation assay. Signaling pathways were analyzed by Western blot analysis. Application of SEC(PBMC) on 6 mm punch biopsy wounds significantly enhanced wound closure. H&E staining of the wounds after 6 days revealed that wound healing was more advanced after application of SEC(PBMC) containing emulsion. Furthermore, there was a massive increase in CD31 positive cells, indicating enhanced neo-angiogenesis. In primary human fibroblasts (FB) and keratinocytes (KC) migration but not proliferation was induced. In endothelial cells (EC) SEC(PBMC) induced proliferation and tube-formation in a matrigel-assay. In addition, SEC(PBMC) treatment of skin cells led to the induction of multiple signaling pathways involved in cell migration, proliferation and survival. In summary, we could show that emulsions containing the secretome of PBMC derived from healthy individuals accelerates wound healing in a mouse model and induce wound healing associated mechanisms in human primary skin cells. The formulation and use of such emulsions might therefore represent a possible novel option for the treatment of non-healing skin ulcers.

Autophagy is a ubiquitous degradation mechanism, which plays a critical role in cellular homeostasis. To test whether autophagy suppresses or supports the growth of tumors in the epidermis of the skin, we inactivated the essential autophagy gene Atg7 specifically in the epidermal keratinocytes of mice (Atg7∆ep) and subjected such mutant mice and fully autophagy-competent mice to tumorigenesis. The lack of epithelial Atg7 did not prevent tumor formation in response to 7, 12-dimethylbenz(a)anthracene (DMBA) as the initiator and 12-O tetradecanoylphorbol-13-acetate (TPA) as the promoter of tumor growth. However, the number of tumors per mouse was reduced in mice with epithelial Atg7 deficiency. In the K5-SOS EGFRwa2/wa2 mouse model, epithelial tumors were initiated by Son of sevenless (SOS) in response to wounding. Within 12 weeks after tumor initiation, 60% of the autophagy-competent K5-SOS EGFRwa2/wa2 mice had tumors of 1 cm diameter and had to be sacrificed, whereas none of the Atg7∆ep K5-SOS EGFRwa2/wa2 mice formed tumors of this size. In summary, the deletion of Atg7 reduced the growth of epithelial tumors in these two mouse models of skin cancer. Thus, our data show that the inhibition of autophagy limits the growth of epithelial skin tumors.

Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SECPBMC). Six mm punch biopsy wounds were set on the backs of C57BL/6J-mice and SECPBMC containing emulsion or controls were applied daily for three days. Morphology and neo-angiogenesis were analyzed by H&E-staining and CD31 immuno-staining, respectively. In vitro effects on diverse skin cells were investigated by migration assays, cell cycle analysis, and tube formation assay. Signaling pathways were analyzed by Western blot analysis. Application of SECPBMC on 6 mm punch biopsy wounds significantly enhanced wound closure. H&E staining of the wounds after 6 days revealed that wound healing was more advanced after application of SECPBMC containing emulsion. Furthermore, there was a massive increase in CD31 positive cells, indicating enhanced neo-angiogenesis. In primary human fibroblasts (FB) and keratinocytes (KC) migration but not proliferation was induced. In endothelial cells (EC) SECPBMC induced proliferation and tube-formation in a matrigel-assay. In addition, SECPBMC treatment of skin cells led to the induction of multiple signaling pathways involved in cell migration, proliferation and survival. In summary, we could show that emulsions containing the secretome of PBMC derived from healthy individuals accelerates wound healing in a mouse model and induce wound healing associated mechanisms in human primary skin cells. The formulation and use of such emulsions might therefore represent a possible novel option for the treatment of non-healing skin ulcers.

Background The Deepwater Horizon disaster was the largest marine oil spill in history, and total vertical exposure of oil to the water column suggests it could impact an enormous diversity of ecosystems. The most vulnerable organisms are those encountering these pollutants during their early life stages. Water-soluble components of crude oil and specific polycyclic aromatic hydrocarbons have been shown to cause defects in cardiovascular and craniofacial development in a variety of teleost species, but the developmental origins of these defects have yet to be determined. We have adopted zebrafish, Danio rerio , as a model to test whether water accumulated fractions (WAF) of the Deepwater Horizon oil could impact specific embryonic developmental processes. While not a native species to the Gulf waters, the developmental biology of zebrafish has been well characterized and makes it a powerful model system to reveal the cellular and molecular mechanisms behind Macondo crude toxicity. Results WAF of Macondo crude oil sampled during the oil spill was used to treat zebrafish throughout embryonic and larval development. Our results indicate that the Macondo crude oil causes a variety of significant defects in zebrafish embryogenesis, but these defects have specific developmental origins. WAF treatments caused defects in craniofacial development and circulatory function similar to previous reports, but we extend these results to show they are likely derived from an earlier defect in neural crest cell development. Moreover, we demonstrate that exposure to WAFs causes a variety of novel deformations in specific developmental processes, including programmed cell death, locomotor behavior, sensory and motor axon pathfinding, somitogenesis and muscle patterning. Interestingly, the severity of cell death and muscle phenotypes decreased over several months of repeated analysis, which was correlated with a rapid drop-off in the aromatic and alkane hydrocarbon components of the oil. Conclusions Whether these teratogenic effects are unique to the oil from the Deepwater Horizon oil spill or generalizable for most crude oil types remains to be determined. This work establishes a model for further investigation into the molecular mechanisms behind crude oil mediated deformations. In addition, due to the high conservation of genetic and cellular processes between zebrafish and other vertebrates, our work also provides a platform for more focused assessment of the impact that the Deepwater Horizon oil spill has had on the early life stages of native fish species in the Gulf of Mexico and the Atlantic Ocean.

Background It is crucial to identify in large population samples the most important determinants of excessive fetal growth. The aim of the study was to evaluate the independent role of pre-pregnancy body mass index (BMI), gestational weight gain and gestational diabetes on the risk of macrosomia. Methods A prospective study collected data on mode of delivery and maternal/neonatal outcomes in eleven Hospitals in Italy. Multiple pregnancies and preterm deliveries were excluded. The sample included 14109 women with complete records. Associations between exposure variables and newborn macrosomia were analyzed using Pearson’s chi squared test. Multiple logistic regression models were built to assess the independent association between potential predictors and macrosomia. Results Maternal obesity (adjusted OR 1.7, 95% CI 1.4-2.2), excessive gestational weight gain (adjusted OR 1.9, 95% CI 1.6-2.2) and diabetes (adjusted OR 2.1, 95% CI 1.5-3.0 for gestational; adjusted OR 3.0, 95% CI 1.2-7.6 for pre-gestational) resulted to be independent predictors of macrosomia, when adjusted for other recognized risk factors. Since no significant interaction was found between pre-gestational BMI and gestational weight gain, excessive weight gain should be considered an independent risk factor for macrosomia. In the sub-group of women affected by gestational or pre-gestational diabetes, pre-gestational BMI was not significantly associated to macrosomia, while excessive pregnancy weight gain, maternal height and gestational age at delivery were significantly associated. In this sub-population, pregnancy weight gain less than recommended was not significantly associated to a reduction in macrosomia. Conclusions Our findings indicate that maternal obesity, gestational weight gain excess and diabetes should be considered as independent risk factors for newborn macrosomia. To adequately evaluate the clinical evolution of pregnancy all three variables need to be carefully assessed and monitored.

High-grade glioma with pleomorphic and pseudopapillary features (HPAP) is a recently identified methylation cluster comprised of relatively circumscribed gliomas enriched for variants in TP53 , RB1 , NF1 , NF2 , BRAF and with a more favorable clinical outcome than IDH-wildtype glioblastoma. Here, we present two cases occurring in young adults, one of which occurred in the background of NF2-related schwannomatosis. Both cases demonstrated characteristic histologic features including ependymoma-like areas (Case #1) and an astroblastoma-like phenotype (Case #2), as well as archetypal pseudopapillary structures and pleomorphic tumor cells. High-grade features were present and pathogenic variants in RB1 and TP53 were detected. Cytogenetic analysis revealed aneuploidy involving multiple whole chromosomes, including copy neutral LOH in chromosome 13 (Case #1). Both cases were classified as “no match” using the Heidelberg Brain Tumor Classifier (v12.5 and 12.8). Results from a preliminary classification model (“Bethesda Classifier”) were consistent with HPAP. Confirmatory dimensionality reduction (t-SNE) showed clustering within (Case #2) or near (Case #1) the HPAP group. Patient #1 is currently receiving maintenance temozolomide following concomitant chemo-radiotherapy, 10 months post-surgery. Patient #2, treated with temozolomide, remains disease-free at 42 months. Our study highlights additional clinical and pathologic insights into this proposed tumor type and may suggest an association with NF2-related schwannomatosis and evolution from low-grade precursors. These observations support the consideration of HPAP as a distinct clinicopathological entity.

We report on a case of EWSR1‐PATZ1 rearranged brain tumor occurring in a 17 month‐old child, originally interpreted as an infantile glioblastoma. Our case shows important analogies with the 2 previously reported cases, including the intraventricular location, the histologic appearance (pushing borders, oligodendrocyte‐like morphology, rich vascular network) and the glioneural immunophenotype, supporting the role of these features as relevant clues to the diagnosis. On the other hand, our case displays unique characteristics, i.e. the onset in an infant, the presence of a focal high‐grade component and the leptomeningeal dissemination, pointing to the importance of considering this entity in the differential diagnosis of an infantile glial/glioneural tumor.