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Background: There is currently no insight into biomarkers that can predict the onset of pediatric atopic dermatitis (AD). Methods: Nested in a prospective birth cohort study that examined the occurrence of physician‐diagnosed AD in 300 children, 44 random children with onset of AD in the first year of life were matched on sex and season of birth with 44 children who did not develop AD. Natural moisturizing factor (NMF), corneocyte surface protrusions, cytokines, free sphingoid bases (SBs) of different chain lengths and their ceramides were analyzed from tape strips collected at 2 months of age before onset of AD using liquid chromatography, atomic force microscopy, multiplex immunoassay, and liquid chromatography mass spectrometry, respectively. Results: Significant alterations were observed for four lipid markers, with phytosphingosine ([P]) levels being significantly lower in children who developed AD compared with children who did not (median 240 pmol/mg vs. 540 pmol/mg, p < 0.001). The two groups of children differed in the relative amounts of SB of different chain lengths (C17, C18 and C20). Thymus‐ and activation‐regulated chemokine (TARC/CCL17) was slightly higher in children who developed AD, whereas NMF and corneocyte surface texture were similar. AD severity assessed by the eczema area and severity index (EASI) at disease onset was 4.2 (2.0;7.2). [P] had the highest prediction accuracy among the biomarkers (75.6%), whereas the combination of 5 lipid ratios gave an accuracy of 89.4%. Conclusion: This study showed that levels and SB chain length were altered in infants who later developed AD, and that TARC/CCL17 levels were higher. [ABSTRACT FROM AUTHOR]

Triple‐negative breast cancer (TNBC) is defined by aggressive behavior, limited response to chemotherapy and lower overall survival rates. The increased metastatic potential of TNBC is a combined result of extensive extracellular matrix (ECM) remodeling that leads to cytoskeleton rearrangement and activation of epithelial‐to‐mesenchymal transition (EMT). The overexpression of epidermal growth factor receptor (EGFR) in TNBC tumors has been linked to induced expression of EMT‐related molecules. EMT activation has often been associated with increased metastasis and stemness. Recently, we described the crucial role of EGFR/estrogen receptor beta (ERβ) interplay in the regulation of invasion and cell–matrix interactions. In this study, we report on the EGFR‐ERβ functional relationship in connection to the aggressiveness and cancer stem cell (CSC)‐like characteristics of TNBC cells. ERβ‐suppressed and MDA‐MB‐231 cells were subjected to downstream EGFR inhibition and/or estradiol stimulation to assess alterations in functional parameters as well as in morphological characteristics, studied by scanning electron, atomic force, and immunofluorescence microscopies. Moreover, the expression and localization of key EMT and CSC‐related markers were also evaluated by real‐time qPCR, immunofluorescence microscopy, and flow cytometry. EGFR inhibition resulted in an overall suppression of aggressive functional characteristics, which occurred in an ERβ‐mediated manner. These changes could be attributed to a reduction, at the molecular level, of EMT and stemness‐linked markers, most notably reduced expression of Notch signaling constituents and the cell surface proteoglycan, syndecan‐1. Collectively, our study highlights the importance of EGFR signaling as a key effector of aggressiveness, EMT, and stemness in an ERβ‐dependent way in TNBC. [ABSTRACT FROM AUTHOR]

Irritant contact dermatitis is a wide spread occupational skin condition. In addition to generalized protection and teaching schemes, a knowledge about individual risk might add to an improved awareness for hazards. Here, we report on a novel candidate biomarker, which might stage individual susceptibility to irritant skin damage. Subclinical sensitivity was proven in recent studies. As a nano-anatomical measure, it works non-invasively on corneocytes from tape strips. Here, we report on a 7-day course after exposure to sodium lauryl sulphate and compare the novel cell texture index with the classical markers water loss (transepidermal water loss) and natural moisturizing factor. All parameters show a high degree of correlation. [ABSTRACT FROM AUTHOR]

Stabilization of the blood-brain barrier during and after stroke can lead to less adverse outcome. For elucidation of underlying mechanisms and development of novel therapeutic strategies validated in vitro disease models of the blood-brain barrier could be very helpful. To mimic in vitro stroke conditions we have established a blood-brain barrier in vitro model based on mouse cell line cerebEND and applied oxygen/glucose deprivation (OGD). The role of astrocytes in this disease model was investigated by using cell line C6. Transwell studies pointed out that addition of astrocytes during OGD increased the barrier damage significantly in comparison to the endothelial monoculture shown by changes of transendothelial electrical resistance as well as fluorescein permeability data. Analysis on mRNA and protein levels by qPCR, western blotting and immunofluorescence microscopy of tight junction molecules claudin-3,-5,-12, occludin and ZO-1 revealed that their regulation and localisation is associated with the functional barrier breakdown. Furthermore, soluble factors of astrocytes, OGD and their combination were able to induce changes of functionality and expression of ABC-transporters Abcb1a (P-gp), Abcg2 (bcrp), and Abcc4 (mrp4). Moreover, the expression of proteases (matrixmetalloproteinases MMP-2, MMP-3, MMP-9, and t-PA) as well as of their endogenous inhibitors (TIMP-1, TIMP-3, PAI-1) was altered by astrocyte factors and OGD which resulted in significant changes of total MMP and t-PA activity. Morphological rearrangements induced by OGD and treatment with astrocyte factors were confirmed at a nanometer scale using atomic force microscopy. In conclusion, astrocytes play a major role in blood-brain barrier breakdown during OGD in vitro. [ABSTRACT FROM AUTHOR]

AVP and atrial natriuretic peptide (ANP) have opposite effects in the kidney. AVP induces antidiuresis by insertion of aquaporin-2 (AQP2) water channels into the plasma membrane of collecting duct principal cells. ANP acts as a diuretic factor. An ANPand nitric oxide (NO)/soluble guanylate cyclase (sGC)-induced insertion of AQP2 into the plasma membrane is reported from different models. However, functional data on the insertion of AQP2 is missing. We used primary cultured inner medullary collecting duct (IMCD) cells and digital holographic microscopy, calcein-quenching measurements, and immunofluorescence and Western blotting to analyze the effects of ANP and NO donors on AQP2 phosphorylation, membrane expression, and water permeability. While AVP led to acceleration in osmotically induced swelling, ANP had no effect. However, in AVP-pretreated cells ANP significantly decreased the kinetics of cell swelling. This effect was mimicked by 8-bromo-cGMP and blunted by PKG inhibition. Stimulation of the NO/sOC pathway or direct activation of sGC with BAY 58-2667 had similar effects to ANP. In cells treated with AVP, AQP2 was predominantly localized in the plasma membrane, and after additional incubation with ANP AQP2 was mostly localized in the cytosol, indicating an increased retrieval of AQP2 from the plasma membrane by ANP. Western blot analysis showed that ANP was able to reduce AVP-induced phosphorylation of AQP2 at position S256. In conclusion, we show that the diuretic action of ANP or NO in the IMCD involves a decreased localization of AQP2 in the plasma membrane which is mediated by cGMP and PKG. [ABSTRACT FROM AUTHOR]

Generally, a formation of paracellular gaps is considered to be the main pathway for fluid passage across endothelia. A model substance for studies in vitro is the vasodilatory peptide bradykinin, which has important functions in inflammation and vascular fluid balance. The mechanisms by which it increases endothelial permeability are not as yet clearly defined. Paracellular gap formation was approached using atomic force microscopy (AFM) on human umbilical vein endothelial cells grown on permeable filter supports. To further distinguish between para- vs transcellular fluid passage, a standard permeability assay was modified by a rapid cooling protocol to specifically inhibit vesicular transport pathways. Cell layers stimulated with bradykinin (1 μM) did not show significant alterations at the cellular junctions. However, gap formation was easily detectable by AFM after addition of the Ca2+-ionophore ionomycin (1 μM), which was taken as a positive control for cellular contraction. At 37°C, bradykinin enhanced fluorescein isothiocyanate-dextran permeability by 48 ± 11%. This was blocked by rapid cooling of the sample, indicating a vesicular mechanism of fluid transport. Contrastingly, ionomycin-induced permeability (259 ± 43%) persisted after cooling (230 ± 44%), thereby confirming paracellular gap formation. Accordingly, endocytotic vesicle formation, as detected by fluorescence microscopy, was upregulated by 68 ± 15% through bradykinin action, while ionomycin did not show a significant effect (7 ± 26%). The combined results of both permeability and morphometric studies lead to the conclusion that bradykinin promotes transcellular fluid passage rather than increasing paracellular diffusion. [ABSTRACT FROM AUTHOR]