Establishment and Characterization of Two Merkel Cell Tumor Cultures

Two Merkel cell tumor cultures (MC-MA1, MC-MA2) have been established from metastases of typical Merkel cell tumors. The mestastases in vivo were characterized by co-expression of cytokeratins 8, 18, 19, 20 and neurofilaments, presence of intermediate filament whirls, expression of synaptophysin, neuron-specific enolase, and chromogranin A, rare and weak immunostaining for plakoglobin but absence of cadherins and desmoplakins. Both cultures grow, using supplemented RPMI medium on human irradiated fibroblast feeder layers, as loosely arranged floating small aggregates. Their karyotypes are mostly hyperdiploid. The mean doubling times were about 84 h in the first 8 months and later increased.…

Comparative cytokeratin analysis of sweat gland ducts and eccrine poromas.

Human eccrine sweat gland ducts and benign and malignant eccrine poromas were studied for the expression of various cytokeratins (CK) and vimentin by applying immunoperoxidase and immunofluorescence microscopy to frozen or paraffin-embedded sections, and using two-dimensional gel electrophoresis and immunoblotting. In acrosyringia and dermal eccrine ducts, the luminal cells exhibited intense staining for CKs 1/10/11 and 19. The periluminal cell layers of acrosyringia contained CKs 1/10/11, while CK 5 was absent. In contrast, the basal cell layer of dermal ducts was only positive with the antibody against CK 5, i. e. a pattern resembling that seen in epidermal basal cells. CK 9 was detected …

Cytokeratin 20 Is a General Marker of Cutaneous Merkel Cells While Certain Neuronal Proteins Are Absent

Merkel cells are difficult to identify in tissue sections. Previous studies have used cytokeratins (CK) 8, 18, and 19 as histologic markers of Merkel cells. However, these CKs are also expressed in some outer root sheath keratinocytes and some early fetal epidermal cells and thus are not truly specific of Merkel cells in general. Using selective antibodies against a newly described CK, number 20--originally found in intestinal epithelium and Merkel cell carcinomas--in comparison to a key protein of neuroendocrine cells, chromogranin A, we established CK 20 as a specific Merkel cell marker in skin of humans, pigs, and mice. CK 20 seems to be an even more general and sensitive Merkel cell mar…

Early development of human Merkel cells.

Human fetal Merkel cells are now generally considered to be epidermal derivatives. Previous studies using antibodies against the simple epithelial cytokeratins (CKs), 8 and 18, have demonstrated the presence of these cells in the epidermis at as early as fetal week 10 to 12. Using antibodies against CK 20 whose expression within the skin is restricted to Merkel cells, we applied immunofluorescence and immunoperoxidase microscopy to analyze earlier embryonic and fetal human skin (wk 7 to 9). We were able to demonstrate the first Merkel cells at as early as fetal wk 8, i.e., at the same time as the epidermis starts to develop an intermediate, third layer, characterized by the expression of CK…

Distribution of a special subset of keratinocytes characterized by the expression of cytokeratin 9 in adult and fetal human epidermis of various body sites.

Biochemical analyses have previously shown that palmar and plantar epidermis, unlike the epidermis of other body sites, contain cytokeratin 9 (Mr 64,000), an unusually large acidic (type I) cytokeratin. Guinea-pig antibodies that specifically and selectively react with bovine and human cytokeratin 9 were used for the immunocytochemical identification of cytokeratin 9 in adult and fetal human epidermis from various body sites. In the epidermis of palms and soles, antibodies against cytokeratin 9 stained a high proportion of the keratinocytes in suprabasal locations. These suprabasal cytokeratin-9-positive keratinocytes were often arranged in vertical columns and concentrated around intraepid…

Cells of extramammary Paget's disease express cytokeratins different from those of epidermal cells.

The patterns of expression of cytokeratin polypeptides which are closely correlated to routes of differentiation of epithelial cells were studied in extramammary Paget's disease. Cytokeratins of uninvolved and involved epidermis were analyzed by two-dimensional gel electrophoresis of microdissected tissue preparations as well as by immunofluorescence microscopy using cytokeratin antibodies with different specificities. In uninvolved epidermis, cytokeratins Nos. 1, 5, 6, 10, 11, 14, and 16, characteristic of keratinocytes, were found. Epidermis infiltrated by Paget's cells contained the same components and, in addition, cytokeratins Nos. 7, 8, 18, and 19, the latter being characteristic of s…

Diagnostics of autoimmune bullous diseases in German dermatology departments

Summary Background: No consistent data are available on the currently employed diagnostic tools for autoimmune bullous diseases in Germany. The aim of this survey was to describe currently performed diagnostic methods for bullous autoimmune diseases in German dermatology departments. Methods: A standardized questionnaire evaluated the available diagnostic methods i. e. direct immunofluorescence microscopy (IFM), indirect IFM, commercial ELISA systems, and non-commercial serological tests as well as the number of samples per year in all 34 university and 39 non-university dermatology departments. Results: The overall return rate was 89 %, 100 % and 79 % for the university and non-university …

Identification of Merkel cells in human skin by specific cytokeratin antibodies:

Merkel cells are special neurosecretory cells which, in adult human skin, are usually very scarce. By immunofluorescence microscopy using antibodies to human cytokeratin polypeptide no. 18, we localized distinct non-keratinocyte cells in the glandular ridges of human fetal and adult plantar epidermis. Using electron and immunofluorescence microscopy, these cells were identified as Merkel cells containing typical neurosecretory granules as well as bundles of intermediate-sized filaments and desmosomes. Two-dimensional gel electrophoresis of the cytoskeletal fractions of microdissected epidermal preparations highly enriched in Merkel cells indicated the presence of cytokeratin polypeptides no…

Cytokeratin Analysis of Pilomatrixoma: Changes in Cytokeratin-Type Expression During Differentiation

The various structural components of pilomatrixoma (calcifying epithelioma of Malherbe) were studied for the expression of hair-specific (trichocytic) cytokeratins as well as epithelial cytokeratins, using immunoperoxidase as well as epithelial cytokeratins, using immunoperoxidase and immunofluorescence microscopy of frozen sections as well as two-dimensional gel electrophoresis and immunoblotting. Trichocyte-type cytokeratins were detected in only a minor subpopulation of basophilic cells but more prominently in most “transitional” cells as well as in “shadow” cells. in contrast, antibodies against certain epithelial cytokeratins (including antibody KA1 against cytokeratins of stratified s…

Merkel cells in ontogenesis of human nails

Digital skin of human fetuses is known to contain a particularly high concentration of Merkel cells. Using antibodies against the simple epithelial cytokeratins (CK) 18 and 20, which are sensitive and specific Merkel cell markers, we studied immunohistochemically the main adnexal structure of digital skin, the nail anlage, in human fetuses (9-22 weeks of gestation) for the presence of Merkel cells. As early as week 9 some clustered Merkel cells were detected in the early matrix primordium. In specimens of week 12-15, abundant Merkel cells were found in the nail anlagen, particularly in the epithelium of the proximal nail-fold and the dorsal and ventral side of the apex region. In contrast, …

Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues

Abstract Cells forming hair and nail material are characterized by the synthesis of members of a particular group of α-keratin polypeptides (trichocytic cytokeratins, “T cytokeratins”) different from epithelial cytokeratins (“E cytokeratins”). As the precursor cells to trichocytes are derived from fetal epidermal keratinocytes expressing only E cytokeratins, we have studied the patterns of expression of both T and E cytokeratins in developing human hair-and nailforming tissues of different fetal stages, by immunocyto-chemistry using antibodies specific for certain T or E cytokeratins and by two-dimensional gel electrophoresis and immunoblotting. In developing hair follicles up to the early …

First International Merkel Cell Symposium, Heidelberg, Germany

Changes of Expression of Intermediate Filament Proteins During Ontogenesis of Eccrine Sweat Glands

The intermediate filament expression in fetal and adult human eccrine sweat glands was studied by immunoperoxidase microscopy performed on cryostat sections using monoclonal antibodies against various cytokeratins (CK), vimentin, and actin. In palmar skin of 14-week-old fetuses, the early dermal cords showed a primitive CK pattern similar to that of epidermal basal cells. From week 15 on (distal finger skin), inner cells of the proximal (ductal) portion of the glandular anlagen expressed CK 1/10/11 and 19 (markers of adult eccrine ductal luminal cells). In addition, CK 4 was expressed in ductal luminal cells mainly in the fetal period. In the distal portion of the sweat gland anlagen the in…

Formation of Epidermal and Dermal Merkel Cells During Human Fetal Skin Development

The origin of Merkel cells is still a matter of debate, specifically the question of whether they are derived from epithelial cells of the epidermis or from immigrated neural crest cells. As an argument for the latter hypothesis the occurrence of dermal, nerve-associated Merkel cells in human fetal skin has often been mentioned. Therefore, we analyzed the distribution of Merkel cells in epidermis and dermis of plantar skin of human embryos and fetuses, ranging in gestational age between 7 and 17 weeks. Merkel cells were identified by immunocytochemistry on frozen sections using antibodies against simple epithelium-type cytokeratins and by electron microscopy. In the 17-week-old fetus, 17% o…

Intraepidermal formation of Merkel cells in xenografts of human fetal skin.

An experimental transplantation model using human fetal skin was applied to approach the question of the embryologic origin of human Merkel cells. Palmar and plantar skin from five fetuses, between 8 and 11 weeks of estimated gestational age (EGA), was xenografted to subcutaneous beds of nude mice. After 4 or 8 weeks of growth, biopsies were taken from these xenografts and examined for the presence of Merkel cells, using immunocytochemistry with antibodies specific for simple epithelial-type cytokeratins and neuron-specific enolase (NSE) as well as using electron microscopy. Skin from the same fetuses at the time of transplantation was screened in the same way. In all fetuses, no (or very s…

Diagnostik blasenbildender Autoimmundermatosen an deutschen Hautkliniken

Induction of bone morphogenetic protein-6 in skin wounds. Delayed reepitheliazation and scar formation in BMP-6 overexpressing transgenic mice.

Growth factors of the transforming growth factor-beta superfamily are involved in cutaneous wound healing. In this study we analyze the expression of the bone morphogenetic protein-6 (BMP-6) gene, a transforming growth factor-beta related gene, in skin wounds. In normal mouse skin high levels of BMP-6 mRNA and protein are expressed by postmitotic keratinocytes of stratified epidermis until day 6 after birth. BMP-6 expression is strongly reduced in adult epidermis with diminished mitotic activity. After skin injury we found large induction of BMP-6-specific RNA and protein in keratinocytes at the wound edge and keratinocytes of the newly formed epithelium as well as in fibroblast shaped cell…

Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging : quantifying neuronal dysfunction in neuroinflammation

Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Forster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lif…

Special Program of Differentiation Expressed in Keratinocytes of Human Haarscheiben: An Analysis of Individual Cytokeratin Polypeptides

Human haarscheiben, epidermal Merkel cell-rich sensory organs of hairy skin, were studied for the expression of various cytokeratin (CK) polypeptides and other epithelial and neuronal differentiation markers by applying immunoperoxidase and immunofluorescence microscopy to frozen sections and by two-dimensional gel electrophoresis. The basal clusters of Merkel cells were specifically detected by antibodies against CK 20. Haarscheiben keratinocytes were unique mainly by the prominent expression of CK 17 in the lower and middle layers. Further differences as compared to keratinocytes of usual epidermis included the enlargement of the basal compartment, characterized by the expression of CK 5 …