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  • 1
    Keywords: EXPRESSION ; CELL ; evaluation ; Germany ; MICROSCOPY ; DISEASE ; DISTINCT ; PROTEIN ; COMPONENTS ; DIFFERENTIATION ; MARKER ; IMPACT ; SKIN ; ALPHA ; MEMBRANE ; MUTATION ; REPAIR ; COMPONENT ; ABERRATIONS ; EXTRACELLULAR-MATRIX ; MUTATIONS ; BETA ; ADHESION ; CELL-ADHESION ; HUMAN KERATINOCYTES ; INTEGRIN ; epidermis ; ultrastructure ; MATRIX ; assembly ; basement membrane ; BASEMENT-MEMBRANE ; extracellular matrix ; ARRAY ; DEFECTS ; INTEGRINS ; analysis ; methods ; JAPANESE ; DEFECT ; immunoelectron microscopy ; EXTRACELLULAR-MATRIX PROTEIN-1 ; lipoid proteinosis ; microvasculature ; aberration ; DERMO-EPIDERMAL JUNCTION ; ECM1 ; HEMIDESMOSOMES ; LAMININ ISOFORMS ; LICHEN-SCLEROSUS ; skin and oral mucosa ; VII COLLAGEN
    Abstract: Background: Excessive basement membrane (BM) deposition in skin and mucosa is characteristic for lipoid proteinosis (LP; hyalinosis cutis et mucosae), an inherited disease caused by extracellular matrix protein 1 (ECM1) mutations. According to ultrastructure there are striking differences between junctional. and microvascular BM. Objective: Distinct analysis of the junctional zone in epidermis and oral mucosa, contrasting concentric BM arrays in the microvasculature; evaluation of impact on epithelial. histogenesis and differentiation, and specifically on adhesion structures to BM (hemidesmosomes). Methods: LP-epithelia were analyzed for alterations in differentiation, BM composition and texture, and hemidesmosomal components by indirect immunofluorescence (IIF), electron microscopy (EM), and immunoelectron microscopy (ImEM). Results: Most striking was the irregular deposition of collagen IV and VII, BM-laminin, and laminin-5 at the junctional. zone, accompanied by lamellate or punctuated structures below BM (IIF), whereas integrin alpha 6 beta 4 and bullous pemphigoid antigen-1 and -2 (BPAG-1/-2) were regularly aligned. Also integrins alpha 2 beta 1 and alpha 3 beta 1 remained restricted to the epidermal basal layer, while the tissue-specific differentiation markers keratin K1/10 (mucosa, additionally K4/13) appeared delayed indicating mild hyperplasia, further confirmed by focal K6/16 expression. Ultrastructure (EM) disclosed abundance of extended basal cell protrusions and junctional aberrations like exfoliating excessive BM material. Hemidesmosomes were complete, but ImEM indicated weakened interactions between their components (BRAG-1, -2, and HD1). Confirming IIF, collagen IV and VII, and laminin-5 appeared extensively scattered, the latter two probably remaining associated. Conclusions: Subtle defects in anchorage assembly, spanning the entire BM zone, apparently compromise epithelial-matrix adhesion, which may provoke (mechanical stress-induced) erroneous BM repair. (c) 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17175139
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  • 2
    Keywords: GENES ; GENOME ; ASSOCIATION ; HEALTH ; PHENOTYPE ; ARCHITECTURE ; THORACIC AORTIC-ANEURYSMS ; CONNECTIVE-TISSUE ABNORMALITIES
    Abstract: Cervical artery dissection (CeAD) occurs in healthy young individuals and often entails ischemic stroke. Skin biopsies from most CeAD-patients show minor connective tissue alterations. We search for rare genetic deletions and duplication that may predispose to CeAD. Forty-nine non-traumatic CeAD-patients with electron microscopic (EM) alterations of their dermal connective tissue (EM+ patients) and 21 patients with normal connective tissue in skin biopsies (EM- patients) were analyzed. Affymetrix 6.0 microarrays (Affymetrix) from all patients were screened for copy number variants (CNVs). CNVs absent from 403 control subjects and from 2402 published disease-free individuals were considered as CeAD-associated. The genetic content of undentified CNVs was analyzed by means of the Gene Ontology (GO) Term Mapper to detect associations with biological processes. In 49 EM+ patients we identified 13 CeAD-associated CNVs harboring 83 protein-coding genes. In 21 EM- patients we found five CeAD-associated CNVs containing only nine genes (comparison of CNV gene density between the groups: Mann-Whitney P=0.039). Patients' CNVs were enriched for genes involved in extracellular matrix organization (COL5A2, COL3A1, SNTA1, P=0.035), collagen fibril organization COL5A2, COL3A1, (P=0.0001) and possibly for genes involved in transforming growth factor beta (TGF)-beta receptor signaling pathway (COL3A1, DUPS22, P=0.068). We conclude that rare genetic variants may contribute to the pathogenesis of CeAD, in particular in patients with a microscopic connective tissue phenotype.European Journal of Human Genetics advance online publication, 23 May 2012; doi:10.1038/ejhg.2012.82.
    Type of Publication: Journal article published
    PubMed ID: 22617347
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  • 3
    Keywords: EXPRESSION ; GENE ; ACQUISITION ; MUTATION ; CHROMOSOMAL LOCALIZATION ; ALOX12B ; GENOMIC STRUCTURE ; RECESSIVE CONGENITAL ICHTHYOSIS ; ADIPOCYTE DIFFERENTIATION ; 12R-LIPOXYGENASE
    Abstract: Loss-of-function mutations in the lipoxygenase (LOX) genes ALOX12B and ALOXE3 are the second most common cause of autosomal recessive congenital ichthyosis. The encoded proteins, 12R-LOX and epidermal LOX-3 (eLOX-3), act in sequence to convert fatty acid substrates via R-hydroperoxides to specific epoxyalcohol derivatives and have been proposed to operate in the same metabolic pathway during epidermal barrier formation. Here, we show that eLOX-3 deficiency in mice results in early postnatal death, associated with similar but somewhat less severe barrier defects and morphological changes than reported earlier for the 12R-LOX-knockout mice. Skin lipid analysis demonstrated that the severity of barrier failure is related to the loss of covalently bound ceramides in both 12R-LOX- and eLOX-3-null mice, confirming a proposed functional linkage of the LOX pathway to ceramide processing and formation of the corneocyte lipid envelope. Furthermore, analysis of free oxygenated fatty acid metabolites revealed strongly reduced levels of hepoxilin metabolites in eLOX-3-deficient epidermis, indicating an additional function of eLOX-3 in mammalian skin as a hepoxilin synthase linked to the 12S-LOX pathway.
    Type of Publication: Journal article published
    PubMed ID: 22832496
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  • 4
    Keywords: SYSTEM ; TOOL ; validation ; INDEX ; score ; CONSENSUS ; DERMATITIS ; AREA ; ATOPIC ECZEMA SEVERITY ; OUTCOME MEASURES
    Abstract: Background Autosomal recessive congenital ichthyoses (ARCIs) are keratinization disorders caused by impaired skin barrier function. Mutations in the genes encoding the lipoxygenases 12R-LOX and eLOX-3 are the second most common cause of ARCIs. In recent years, human skin equivalents recapitulating the ARCI phenotype have been established. Objectives To develop a murine organotypic tissue culture model for ARCI. Methods Epidermal keratinocytes were isolated from newborn 12R-LOX-deficient mice and cocultivated with mouse dermal fibroblasts embedded in a scaffold of native collagen type I. Results With this experimental set-up the keratinocytes formed a well-organized multilayered stratified epithelium resembling skin architecture in vivo. All epidermal layers were present and the keratinocytes within showed the characteristic morphological features. Markers for differentiation and maturation indicated regular epidermal morphogenesis. The major components of epidermal structures were expressed, and were obviously processed and assembled properly. In contrast to their wild-type counterparts, 12R-LOX-deficient skin equivalents showed abnormal vesicular structures in the upper epidermal layers correlating with altered lipid composition and increased transepidermal water loss, comparable with 12R-LOX-deficient mice. Conclusions The mouse skin equivalents faithfully recapitulate the 12R-LOX-deficient phenotype observed in vivo, classifying them as appropriate in vitro models to study molecular mechanisms involved in the development of ARCI and to evaluate novel therapeutic agents. In contrast to existing human three-dimensional skin models, the generation of these murine models is not constrained by a limited supply of material and does not depend on in vitro expansion and/or genetic manipulations that could result in inadvertent genotypic and phenotypic alterations.
    Type of Publication: Journal article published
    PubMed ID: 25078898
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  • 5
    Abstract: The genetic basis of epidermolysis bullosa, a group of genetic disorders characterized by the mechanically induced formation of skin blisters, is largely known, but a number of cases still remain genetically unsolved. Here, we used whole-exome and targeted sequencing to identify monoallelic mutations, c.1A〉G and c.2T〉C, in the translation initiation codon of the gene encoding kelch-like protein 24 (KLHL24) in 14 individuals with a distinct skin-fragility phenotype and skin cleavage within basal keratinocytes. Remarkably, mutation c.1A〉G occurred de novo and was recurrent in families originating from different countries. The striking similarities of the clinical features of the affected individuals point to a unique and very specific pathomechanism. We showed that mutations in the translation initiation codon of KLHL24 lead to the usage of a downstream translation initiation site with the same reading frame and formation of a truncated polypeptide. The pathobiology was examined in keratinocytes and fibroblasts of the affected individuals and via expression of mutant KLHL24, and we found mutant KLHL24 to be associated with abnormalities of intermediate filaments in keratinocytes and fibroblasts. In particular, KLHL24 mutations were associated with irregular and fragmented keratin 14. Recombinant overexpression of normal KLHL24 promoted keratin 14 degradation, whereas mutant KLHL24 showed less activity than the normal molecule. These findings identify KLHL24 mutations as a cause of skin fragility and identify a role for KLHL24 in maintaining the balance between intermediate filament stability and degradation required for skin integrity.
    Type of Publication: Journal article published
    PubMed ID: 27889062
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  • 6
    Abstract: BACKGROUND: Colorectal cancers (CRCs) that lack DNA mismatch repair function exhibit the microsatellite unstable (MSI) phenotype and are characterized by the accumulation of frameshift mutations at short repetitive DNA sequences (microsatellites). These tumors recurrently show inactivating frameshift mutations in the tumor suppressor Transforming Growth Factor Beta Receptor Type 2 (TGFBR2) thereby abrogating downstream signaling. How altered TGFBR2 signaling affects exosome-mediated communication between MSI tumor cells and their environment has not been resolved. Here, we report on molecular alterations of exosomes shed by MSI cells and the biological response evoked in recipient cells. METHODS: Exosomes were isolated and characterized by electron microscopy, nanoparticle tracking, and western blot analysis. TGFBR2-dependent effects on the cargo and functions of exosomes were studied in a MSI CRC model cell line enabling reconstituted and inducible TGFBR2 expression and signaling. Microsatellite frameshift mutations in exosomal and cellular DNA were examined by PCR-based DNA fragment analysis and exosomal protein profiles were identified by mass spectrometry. Uptake of fluorescent-labeled exosomes by hepatoma recipient cells was monitored by confocal microscopy. TGFBR2-dependent exosomal effects on secreted cytokine levels of recipient cells were analyzed by Luminex technology and ELISA. RESULTS: Frameshift mutation patterns in microsatellite stretches of TGFBR2 and other MSI target genes were found to be reflected in the cargo of MSI CRC-derived exosomes. At the proteome level, reconstituted TGFBR2 expression and signaling uncovered two protein subsets exclusively occurring in exosomes derived from TGFBR2-deficient (14 proteins) or TGFBR2-proficient (five proteins) MSI donor cells. Uptake of these exosomes by recipient cells caused increased secretion (2-6 fold) of specific cytokines (Interleukin-4, Stem Cell Factor, Platelet-derived Growth Factor-B), depending on the TGFBR2 expression status of the tumor cell. CONCLUSION: Our results indicate that the coding MSI phenotype of DNA mismatch repair-deficient CRC cells is maintained in their exosomal DNA. Moreover, we uncovered that a recurrent MSI tumor driver mutation like TGFBR2 can reprogram the protein content of MSI cell-derived exosomes and in turn modulate the cytokine secretion profile of recipient cells. Apart from its diagnostic potential, these TGFBR2-dependent exosomal molecular and proteomic signatures might help to understand the signaling routes used by MSI tumors. Fricke et al. uncovered coding microsatellite instability-associated mutations of colorectal tumor driver genes like TGFBR2 in MSI tumor cellderived exosomes. Depending on the TGFBR2 expression status of their donor cells, shed exosomes show distinct proteomic signatures and promote altered cytokine secretion profiles in recipient cells.
    Type of Publication: Journal article published
    PubMed ID: 28376875
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  • 7
    Keywords: EXPRESSION ; BLOOD ; Germany ; PROTEIN ; DIFFERENTIATION ; MARKER ; EXTRACELLULAR-MATRIX ; MUTATIONS ; HUMAN KERATINOCYTES ; MORPHOLOGY ; INTEGRIN ; INVOLVEMENT ; TUMOR ANGIOGENESIS ; ultrastructure ; MATRIX ; VESICLES ; extracellular matrix ; laminin ; HISTOLOGY ; function ; LOSSES ; JAPANESE ; POLARITY ; immunoelectron microscopy ; HUMAN-SKIN ; microvascular density ; basement membrane components ; EXTRACELLULAR-MATRIX PROTEIN-1 ; GENE ECM1 ; lipoid proteinosis ; microvasculature ; PERLECAN
    Abstract: Background: In lipoid proteinosis (LP) vascular anomalies represent severe functional defects caused by excessive deposition of basement membrane (BM)-like matrix, particularly around small subepithelial blood vessels. Objective: Correlation of microvascular anomalies in morphology and ultrastructure with extracellular matrix composition and cell interactions for elucidating vascular involvement in LP-pathophysiology. Methods: Biopsies from non-related LP-patients were analyzed by indirect immunofluorescence (IIF), electron microscopy (EM), and immune-EM (ImEM). Results: In LP-skin and mucosa the thickened vessel walls stained strongly for the BM-components type IV collagen, laminin, perlecan, and nidogen (IIF). Integrin (alpha 6 beta 4 was regularly collocated with endothelial surface markers such as PECAM (CD31). Ultrastructure (EM) revealed highly ordered matrix deposits around microvessels, with frequently collapsed lumina, functionally compensated by increased vascular density (histology, IIF). Pericytes were trapped between these concentric BM-layers at varying distances towards the periphery (EM), contrasting their regularly close endothelial apposition. Periodic type IV collagen patterns (ImEM) corroborated the multiple BM-leaflet structure and the lack of a common 'fused' endothelial-pericyte BM, seen normally. Presumptive secretory vesicles, abundant in both cell types, implied an equal contribution to BM-synthesis, but also indicated partial loss of endothelial polarity. Conclusions: In LP thickened vessel walls, composed of multiple BM, profoundly alter microvascular properties, also by interference with endothelial-pericyte interactions. The increased microvascular density reflects compensatory restoration for disabled function. Most remarkable was the exaggerated secretory activity (also at luminal surfaces) underlining the regulatory key role of extracellular matrix protein 1 (ECM1; mutated in LP) in export or turnover of all major BM-components. (c) 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16497486
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  • 8
    Abstract: We want to report and discuss the indication for open surgery for an asymptomatic penetrating aortic ulcer (PAU) in the era of thoracic endovascular aortic repair (TEVAR). A 31-year-old female presented with the diagnosis of an aneurysm in the distal aortic arch. With respect to the patients young age, the controversial status of connective tissue disorders and in the absence of concomitant disease, open repair was indicated. There was no proof of a mycotic plaque or connective tissue disease in the microbiological-, pathological analysis and at electron-microscopy. The patient was discharged on the thirteenth postoperative day. In spite of good preliminary results of TEVAR in PAU, in selective cases there is still an indication for open surgery.
    Type of Publication: Journal article published
    PubMed ID: 20464674
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  • 9
    Abstract: The multicomponent type VI secretion system (T6SS) mediates the transport of effector proteins by puncturing target membranes. T6SSs are suggested to form a contractile nanomachine, functioning similar to the cell-puncturing device of tailed bacteriophages. The T6SS members VipA/VipB form tubular complexes and are predicted to function in analogy to viral tail sheath proteins by providing the energy for secretion via contraction. The ATPase ClpV disassembles VipA/VipB tubules in vitro, but the physiological relevance of tubule disintegration remained unclear. Here, we show that VipA/VipB tubules localize near-perpendicular to the inner membrane of Vibrio cholerae cells and exhibit repetitive cycles of elongation, contraction and disassembly. VipA/VipB tubules are decorated by ClpV in vivo and become static in DeltaclpV cells, indicating that ClpV is required for tubule removal. VipA/VipB tubules mislocalize in DeltaclpV cells and exhibit a reduced frequency of tubule elongation, indicating that ClpV also suppresses the spontaneous formation of contracted, non-productive VipA/VipB tubules. ClpV activity is restricted to the contracted state of VipA/VipB, allowing formation of functional elongated tubules at a T6SS assembly. Targeting of an unrelated ATPase to VipA/VipB is sufficient to replace ClpV function in vivo, suggesting that ClpV activity is autonomously regulated by VipA/VipB conformation.
    Type of Publication: Journal article published
    PubMed ID: 23289512
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  • 10
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