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Bad Münster am Stein-Ebernburg, Germany

Riethmuller C.,Serend ip GmbH | McAleer M.A.,Our Ladys Childrens Hospital | McAleer M.A.,National Childrens Research Center | Koppes S.A.,Coronel Institute of Occupational Health | And 11 more authors.
Journal of Allergy and Clinical Immunology

Background Loss-of-function (LOF) mutations in the filaggrin gene (FLG) are a well-replicated risk factor for atopic dermatitis (AD) and are known to cause an epidermal barrier defect. The nature of this barrier defect is not fully understood. Patients with AD with FLG LOF mutations are known to have more persistent disease, more severe disease, and greater risk of food allergies and eczema herpeticum. Abnormalities in corneocyte morphology have been observed in patients with AD, including prominent villus-like projections (VP); however, these ultrastructural features have not been systematically studied in patients with AD in relation to FLG genotype and acute and convalescent status. Objective We sought to quantitatively explore the relationship between FLG genotype, filaggrin breakdown products (natural moisturizing factor [NMF]), and corneocyte morphology in patients with AD. Methods We studied 15 children at first presentation of AD and after 6 weeks of standard therapy. We applied atomic force microscopy to study corneocyte conformation in patients with AD stratified by FLG status and NMF level. By using a new quantitative methodology, the number of VPs per investigated corneocyte area was assessed and expressed as the Dermal Texture Index score. Corneocytes were also labeled with an anti-corneodesmosin antibody and visualized with scanning electron microscopy. Results We found a strong correlation between NMF levels and Dermal Texture Index scores in both acute and convalescent states (respective r = -0.80 and -0.75, P <.001 and P =.002). Most, but not all, VPs showed the presence of corneodesmosin abundantly all over the cell surface in homozygous/compound heterozygous FLG patients and, to a lesser extent, in heterozygous and wild-type patients. Conclusions NMF levels are highly correlated with corneocyte morphology in patients with AD. These corneocyte conformational changes shed further insight into the filaggrin-deficient phenotype and help explain the barrier defect in patients with AD with FLG LOF mutations. © 2015 The Authors. Source

Isac L.,University of Munster | Isac L.,University of Bucharest | Thoelking G.,University of Munster | Schwab A.,University of Munster | And 3 more authors.
Analytical and Bioanalytical Chemistry

A demanding task of medicine is to understand and control the immune system. Central players in the cellular immune response are the leukocytes that leave the blood stream for host defense. Endothelial cells limit the emigration rate of leukocytes. Being located between blood and tissues, they permit or deny the passage. The exact mechanism of this process called diapedesis is not solved yet. Leukocytes can principally traverse either between cells (paracellularly) or directly through an individual endothelial cell (transcellularly). The transcellular way has recently gained experimental support, but it is not clear how the endothelial cytoskeleton manages to open and close a transmigratory channel. Atomic force microscopy was used to investigate the endothelial cytoskeleton. In order to directly access the leukocyte-endothelial interaction site, we applied a special protocol ("nanosurgery"). As a result, the endothelial cell turned out to become softer in a confined region strictly underneath the leukocyte. Fluorescence microscopy confirmed a depolymerization of the f-actin strands at the invasion site. Leukocytes dramatically rearrange the endothelial cytoskeleton to form transmigratory channels. © 2010 Springer-Verlag. Source

Franz J.,University of Munster | Brinkmann B.F.,Institute Assoc Research Group Cell Adhesion And Cell Polarity | Konig M.,University of Munster | Huve J.,Hannover Medical School | And 3 more authors.

Endothelial barriers have a central role in inflammation as they allow or deny the passage of leukocytes from the vasculature into the tissue. To bind leukocytes, endothelial cells form adhesive clusters containing tetraspanins and ICAM-1, so-called endothelial adhesive platforms (EAPs). Upon leukocyte binding, EAPs evolve into docking structures that emanate from the endothelial surface while engulfing the leukocyte. Here, we show that TNF-is sufficient to induce apical protrusions in the absence of leukocytes. Using advanced quantitation of atomic force microscopy (AFM) recordings, we found these structures to protrude by 160 80 nm above endothelial surface level. Confocal immunofluorescence microscopy proved them positive for ICAM-1, JAM-A, tetraspanin CD9 and f-Actin. Microvilli formation was inhibited in the absence of CD9. Our findings indicate that stimulation with TNF-induces nanoscale changes in endothelial surface architecture and that- via a tetraspanin CD9 depending mechanism-the EAPs rise above the surface to facilitate leukocyte capture. © 2016 Franz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source

Thoelking G.,University of Munster | Reiss B.,University of Munster | Reiss B.,University of Regensburg | Wegener J.,University of Regensburg | And 4 more authors.

Inflammation and cellular fibrosis often imply an involvement of the cytokine TGF-β1. TGF-β1 induces epithelial-to-mesenchymal transdifferentiation (EMT), a term describing the loss of epithelium-specific function. Indicative for this process are an elongated cell shape parallel to stress fibre formation. Many signalling pathways of TGF-β1 have been discovered, but mechanical aspects have not yet been investigated. In this study, atomic force microscopy (AFM) was used to analyse surface topography and mechanical properties of EMT in proximal kidney tubule epithelium (NRK52E). Elongated cells, an increase of stress fibre formation and a loss of microvillus compatible structures were observed as characteristic signs of EMT. Furthermore, AFM could identify an increase in stiffness by 71% after six days of stimulation with TGF-β1. As a novel topographical phenomenon, nodular protrusions emerged at the cell-cell junctions. They occurred preferentially at sites where stress fibres cross the border. Since these nodular protrusions were sensitive to inhibitors of force generation, they can indicate intracellular tension. The results demonstrate a manifest impact of elevated tension on the cellular topography. © 2010 IOP Publishing Ltd. Source

Schwickert A.,University of Munster | Weghake E.,University of Munster | Bruggemann K.,University of Munster | Engbers A.,University of Munster | And 9 more authors.

MicroRNAs (miRNAs, micro ribonucleic acids) are pivotal post-transcriptional regulators of gene expression. These endogenous small non-coding RNAs play significant roles in tumorigenesis and tumor progression. miR-142-3p expression is dysregulated in several breast cancer subtypes. We aimed at investigating the role of miR-142-3p in breast cancer cell invasiveness. Supported by transcriptomic Affymetrix array analysis and confirmatory investigations at the mRNA and protein level, we demonstrate that overexpression of miR- 142-3p in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells leads to downregulation of WASL (Wiskott-Aldrich syndrome-like, protein: N-WASP), Integrin-αV, RAC1, and CFL2, molecules implicated in cytoskeletal regulation and cell motility. ROCK2, IL6ST, KLF4, PGRMC2 and ADCY9 were identified as additional targets in a subset of cell lines. Decreased Matrigel invasiveness was associated with the miR-142-3p-induced expression changes. Confocal immunofluorescence microscopy, nanoscale atomic force microscopy and digital holographic microscopy revealed a change in cell morphology as well as a reduced cell volume and size. A more cortical actin distribution and a loss of membrane protrusions were observed in cells overexpressing miR-142-3p. Luciferase activation assays confirmed direct miR-142-3p-dependent regulation of the 3'-untranslated region of ITGAV and WASL. siRNA-mediated depletion of ITGAV and WASL resulted in a significant reduction of cellular invasiveness, highlighting the contribution of these factors to the miRNAdependent invasion phenotype. While knockdown of WASL significantly reduced the number of membrane protrusions compared to controls, knockdown of ITGAV resulted in a decreased cell volume, indicating differential contributions of these factors to the miR-142-3p-induced phenotype. Our data identify WASL, ITGAV and several additional cytoskeleton- associated molecules as novel invasion-promoting targets of miR-142-3p in breast cancer. © 2015 Schwickert et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source

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