M. Ponec

Cell type heterogeneity of cytokeratin expression in complex epithelia and carcinomas as demonstrated by monoclonal antibodies specific for cytokeratins nos. 4 and 13

Three monoclonal antibodies, 1C7, 2D7 and 6B10, directed against cytokeratins of human esophagus were isolated and characterized by one- and two-dimensional gel electrophoresis and by immunohistochemical staining on sections of human epithelial tissues. In immunoblot experiments, antibodies of clones 1C7 (IgG2a) and 2D7 (IgG2b) react only with cytokeratin no. 13 of the acidic (type I) subfamily of cytokeratin polypeptides (Mr 54000; pI 5.1); antibodies of clone 6B10 (IgG1) detect only cytokeratin no. 4 (Mr 59000; pI 7.3) of the basic (type II) cytokeratin subfamily and allows the detection of this protein and possible degradation products at high sensitivity. In immunohistochemical staining all three antibodies stain non-cornifying squamous epithelium (e.g., tongue, esophagus, anus) and transitional epithelium of the bladder. Antibodies of clone 6B10 also stain cells in certain ciliated pseudostratified epithelia and ductal epithelia of various exocrine glands. These monoclonal antibodies are the first examples of antibodies specific for individual cytokeratin polypeptides characteristic of certain complex epithelia. They allow the identification of distinct minor populations of cells present in certain complex and glandular epithelia and in tumors derived therefrom which hitherto have not been distinguished. The possible reasons for the occurrence of cell type heterogeneity of cytokeratin expression in complex epithelia and in some carcinomas are discussed.

Effect of fibroblasts on epidermal regeneration.

Summary Backgroundu2003There is little information on specific interactions between dermal fibroblasts and epidermal keratinocytes. The use of engineered skin equivalents consisting of organotypic cocultures of keratinocytes and fibroblasts offers an attractive approach for such studies.

Crucial role of fibroblasts in regulating epidermal morphogenesis

Abstract. Epidermis reconstructed on de-epidermized dermis (DED) was used to investigate whether fibroblasts can substitute growth factors needed for generation of a fully differentiated epidermis. For this purpose, a centrifugal seeding method was developed to reproducibly incorporate different fibroblast numbers into DED. Using (immuno)histochemical techniques, we could demonstrate that in the absence of fibroblasts the formed epidermis consisted only of two to three viable cell layers with a very thin stratum corneum layer. However, in the presence of fibroblasts keratinocyte proliferation and migration was stimulated and epidermal morphology markedly improved. The stimulatory effect of fibroblasts showed a biphasic character: keratinocyte proliferation increased in the initial phase but decreased in later stages of cell culture. After 3xa0weeks culture at the air–liquid interface, the proliferation index decreased irrespective of the number of fibroblasts present within the dermal matrix to levels observed also in native epidermis. Keratinxa010 was localized in all viable suprabasal cell layers irrespective of the absence or presence of fibroblasts. Keratinxa06 was downregulated with increasing numbers of fibroblasts, and keratinsxa016 and 17 were absent in fibroblast-populated matrices. The expression of involucrin or transglutaminasexa01 showed a similar pattern as for the keratins. Irrespective of the number of fibroblasts incorporated into DED, the expression of α3, α6, β1, and β4 integrin subunits was upregulated. In fibroblast-free DED matrices normalization of epidermal differentiation was only achieved when the culture medium was supplemented by keratinocyte growth factor. The results of this study indicate that normalization of epidermal differentiation can be achieved using a non-contractile dermal matrix populated with fibroblasts.

The Lipid Organisation in the Skin Barrier

The main function of the skin is to protect the body against exogenous substances. The skin barrier is located in the outermost layer of the skin, the stratum corneum. This layer consists of keratin enriched cells embedded in lipid lamellae. These lamellae form the main barrier for diffusion of substances through the skin. In diseased skin the barrier function is often impaired. For a full understanding of the properties of the human skin barrier, insight in the stratum corneum lipid organisation is of great importance. In this paper a short description of the lipid organisation in normal human stratum corneum will be given, after which the role the main lipid classes play in the stratum corneum lipid organisation will be described. In addition the effect of cholesterol sulfate and calcium on the lipid organisation will be discussed. Finally a new model, the sandwich model, will be proposed that describe the localisation of the fluid phases in the stratum corneum.

Skin constructs for replacement of skin tissues for in vitro testing

Reconstructed human skin equivalents as an alternative to animal experimentation offer not only a way to concede to demands of regulatory authorities, animal welfare organizations, consumers and scientists, but also provide a means to improve and extend our knowledge on biological processes in the skin. Presently, various skin reconstructs are available composed either of the epidermal compartment only or of both the epidermal and dermal compartments. Within each compartment various types of cells can be incorporated, including keratinocytes, melanocytes and Langerhans cells in the epidermal, and fibroblasts and endothelial cells in the dermal compartment. The quality of the human skin equivalents has now reached a point that their suitability for skin toxicity testing will make great progress. Next to the field of toxicity and safety standards, skin equivalents offer a well-characterized model for studies of the basic skin biology, wound repair, regulation of melanogenesis, pathogenesis of skin diseases and skin cancer.

New Aspects of the Skin Barrier Organization

In the superficial layer of the skin, the stratum corneum (SC), the lipids form two crystalline lamellar phases with periodicities of 6.4 and 13.4 nm (long-periodicity phase). The main lipid classes in SC are ceramides, free fatty acids and cholesterol. Studies with mixtures prepared with isolated ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases, and the presence of ceramide 1 is crucial for the formation of the long-periodicity phase. This observation and the broad-narrow-broad sequence of lipid layers in the 13.4-nm phase led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains on both sides of a broad layer with a crystalline structure. This model is referred to as ‘the sandwich model’. While the presence of free fatty acids does not substantially affect the lipid lamellar organization, it is crucial for the formation of the orthorhombic sublattice, since the addition of free fatty acids to cholesterol/ceramide mixtures results in transition from a hexagonal to a crystalline lipid phase. Studies examining lipid organization in SC derived from dry or lamellar X-linked ichthyosis skin revealed that in native tissue the role of ceramide 1 and free fatty acids is similar to that observed with mixtures prepared with isolated SC lipids. From this we conclude that the results obtained with lipid mixtures can be used to predict the SC lipid organization in native tissue.

Epidermal growth factor and keratinocyte growth factor differentially regulate epidermal migration, growth, and differentiation.

Various growth factors such as epidermal growth factor and keratinocyte growth factor have been reported to promote wound closure and epidermal regeneration. In the present study epidermis reconstructed on de‐epidermized dermis was used to investigate the effects of epidermal growth factor and keratinocyte growth factor on keratinocyte proliferation, migration and differentiation. Our results show that epidermal growth factor supplemented cultures share many of the features which are observed during regeneration of wounded epidermis: a thickening of the entire epidermis, an enhanced rate of proliferation and migration, and an increase in keratin 6, keratin 16, skin‐derived antileukoproteinase, involucrin and transglutaminase 1 expression. The increase in transglutaminase 1 protein is accompanied by an increase in the amount of active transglutaminase 1 enzyme. Surprisingly no increase in keratin 17 is observed. Prolonging the culture period for more than two weeks results in rapid senescence and aging of the cultures. In contrast, keratinocyte growth factor supplemented cultures have a tissue architecture that is similar to healthy native epidermis and remains unchanged for at least 4 weeks of air‐exposure. The rate of proliferation and the expression of keratins 6, 16 and 17, skin‐derived antileukoproteinase, involucrin and transglutaminase 1 is similar to that found in healthy epidermis and furthermore keratinocyte migration does not occur. When the culture medium is supplemented with a combination of keratinocyte growth factor and a low concentration of epidermal growth factor, skin‐derived antileukoproteinase, involucrin and keratins 6, 16 and 17 expression is similar to that found in cultures supplemented with keratinocyte growth factor alone and in healthy epidermis. Only high transglutaminase 1 expression remains similar to that observed in cultures supplemented with epidermal growth factor alone. Our results show that the regulation of keratinocyte growth, migration and differentiation depends on the availability of these growth factors. Epidermal growth factor may play a dominant early role in wound healing by stimulating keratinocyte proliferation and migration while keratinocyte growth factor may play a role later in the repair process by stabilizing epidermal turnover and barrier function.

Barrier function parameters in various keratinization disorders: transepidermal water loss and vascular response to hexyl nicotinate

In this study, we characterized the stratum corneum barrier function in 39 patients with various keratinization disorders (autosomal dominant ichthyosis vulgaris [ADI] [n=7]. X‐linked recessive ichthyosis [XRI] [n=6], autosomal recessive congenital ichthyosis [C1] [N=10]. dyskeratosis follicularis [Dariers disease: DD] [n=8], erythrokeratoderma variabilis [EKV] [n=8]). and 21 healthy volunteers, using two non‐invasive methods: transepidermal water loss (TEWL) measuring outward transport of water through the skin by evaporimetry. and the vascular response to hexyl nicotinate (HN) penetration into the skin as determined by laser‐Doppler flowmetry.

Epidermal growth factor receptor expression related to differentiation capacity in normal and transformed keratinocytes

Epidermal growth factor (EGF) and Ca2+ have been indicated to play a major role in skin development. We have used normal keratinocytes, SV40-transformed keratinocytes (SVK14) and various squamous carcinoma cell (SCC) lines as in vitro model system to study the effect of the extracellular Ca2+ concentration of EGF-receptor expression in relation to the capability of cells to differentiate. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-12B2 greater than SCC-4, as judged from Ca2+-ionophore-induced cornified envelope formation. Under normal Ca2+ conditions, all cell lines (except for SCC-15) exhibited two classes of EGF-binding sites. The number of low-affinity binding sites increased considerably as cells were less able to differentiate, while the apparent dissociation constant (kd) was similar in all cell lines. In contrast, the properties of high-affinity EGF binding varied in the various cell lines without a clear relationship to the degree of differentiation capacity. Lowering the extracellular Ca2+ concentration to 0.06 mM resulted in a decrease of Ca2+ ionophore-induced cornified envelope formation, demonstrating the decreased ability to differentiate under these conditions. The decreased ability to differentiate was accompanied by a marked increase in the number of EGF-binding sites, but without a change of the kd. Furthermore, no high-affinity EGF-binding sites were detectable under these conditions. Finally, addition of Ca2+ to low Ca2+-cultured cells caused a rapid decrease of EGF binding in all cell lines, most prominently in normal keratinocytes and SCC-12F2 cells. The data presented demonstrate: The combination of normal keratinocytes, SVK14 and the various SCC lines provides an attractive model system to study differentiation in vitro; EGF-receptor expression is related to the state of differentiation, both phenomena being sensitive to the external Ca2+ concentration; and EGF-receptor expression is related to the capability of cells to differentiate.

Cell-seeding and in vitro biocompatibility evaluation of polymeric matrices of PEO/PBT copolymers and PLLA

A bilayered matrix has been evaluated in vitro as a carrier for autografts of cultured epidermal keratinocytes and dermal fibroblasts, to be used as a skin substitute in deep dermal skin defects. A poly-L-lactide (PLLA) and an elastomeric and biodegradable poly(ethyleneoxide)-poly(butyleneterephthalate)(PEO-PBT++ +)copolymer, called Polyactive, were chosen as the constituents of the matrix. The substrate properties of the bilayers for human and rat epidermal keratinocytes and dermal fibroblasts were assessed. Keratinocytes attached and expanded into confluent sheets on both the routine cell culture plastic (TCPS) and the experimental substrates. Morphology of the cells cultured on the biomaterials was found to be comparable with the morphology of those grown on TCPS. In contrast to dense films, porous PEO:PBT copolymer and PLLA appeared poor substrates for fibroblasts. Long-term (in vivo) degradation of the biomaterials was mimicked in vitro to screen the biomaterials for any release of toxic substances. Culturing keratinocytes and fibroblasts in media based on the artificially aged biomaterials did not result in any negative effects on proliferative activity or morphological appearance of the cells.