PubMed Health⌕ Search

Biomedical subjects

M Ponec

Publications and source records attributed to M Ponec.

At least 109 records · Page 6Linked to original sources
PubMed1990

Association of EGF and LDL receptors with the cytoskeleton of cultured keratinocytes.

In this paper we demonstrate that isolated cytoskeletons of normal keratinocytes cultured under differentiation inducing conditions exhibit a high level of epidermal growth factor (EGF) binding. This binding is approximately 300% higher than the binding of intact cells. In contrast, various squamous carcinoma cell lines or normal keratinocytes cultured under differentiation retarding conditions exhibit EGF binding to isolated cytoskeletons which is around 10-20% of the binding to intact cells. Incubation of normal keratinocytes in the presence of arotinoid ethyl sulfone resulted in a marked decrease of the ability of the cells to differentiate, and a decrease of EGF binding to isolated cytoskeletons. These results suggest a close relationship between the differentiation capacity of the cells and the presence of cytoskeleton-associated EGF receptors. Similar results were obtained for low density lipoprotein (LDL) binding.

Calcium↗
PubMed1990

The use of cultured autologous epidermis in the treatment of extensive burn wounds.

Seventeen patients with deep second- and third-degree burn wounds have been grafted with cultured autologous epidermis. These epidermal cell sheets were cultivated according to the feeder layer technique as described by Rheinwald and Green. After dispase treatment and detachment from the culture vessel, the cell sheets, mounted on a polyamide mesh, were ready for grafting. Patients with wounds excised at an early stage, prepared with human cadaver allografts or synthetic dressings, showed a significantly better graft take than nonexcised, chronic granulating wounds which were grafted at a later stage (47% versus 15%; p less than 0.002). Sandwich treatment of expanded mesh autografts and cultured autograft overlay did not improve the graft take, although in some cases wound healing was accelerated. The graft take was inversely correlated with the age of the patient (p = 0.01), and showed a weak inverse correlation with the day of first (and subsequent) culture grafting (p = 0.07). Wound infection was the main cause of graft failure. Up to 4 years after grafting, the grafted areas showed continued stability and the regenerated skin became supple, smooth, and pliable. Hypertrophic scar formation was less than observed in comparable areas treated with meshed grafts. Wound contraction occurred approximately to the same extent as in split-thickness skin grafts. We emphasize that by a better control of wound infection the graft take, also in secondary-stage procedures, can significantly improve.

Adolescent↗
PubMed1990

Freeze-fracture electron microscopy of in vitro reconstructed human epidermis.

Epidermis has been reconstructed in vitro by seeding human keratinocytes on a human dermal substrate in an air-exposed culture. The end product has been examined by freeze-fracture electron microscopy, transmission electron microscopy (TEM) of thin sections, light microscopy, and lipid analysis using thin-layer chromatography. Light microscopic observation of hematoxylin-eosin stained, paraffin embedded cross-sections of the cell culture revealed a strong resemblance to its intact human counterpart, especially with respect to the morphologic organization in basal, spinous, granular, and horny layers. Freeze-fracture electron microscopy and TEM of thin sections generally confirmed the observed resemblances and additionally suggested the presence of lamellar bodies in the stratum granulosum, and of lamellar (lipid) structures between the corneocytes. However, some imperfections were also observed, including some anomalous lipid structures in the intercellular space. Lipid analyses in conjunction with essential fatty acid enrichment studies suggested that the structural anomalies observed in the cultured system may be caused by a lack of linoleyl-ceramides resulting from "immobilization" of linoleyl moieties in the form of triglycerides and phospholipids. In its present form, the air-exposed cell culture already looks very promising as a model for studies of, e.g., skin differentiation disorders such as psoriasis or ichthyosis, studies of the percutaneous penetration and intra(epi)dermal biotransformation of drugs, and skin toxicity screenings. It is furthermore expected that the aforementioned imperfections in the air-exposed cell culture should be avoidable by changing culture conditions such as the relative humidity and the pH, the composition of the medium, or both.

Cells, Cultured↗
PubMed1990

Fresh versus cryopreserved cultured allografts for the treatment of chronic skin ulcers.

Both fresh and cryopreserved cultured epithelial allografts were used to treat 20 patients with a total of 30 chronic ulcers. A profound stimulation of host epithelialization from the wound edges and epidermal appendages was observed. Our data suggest that allografts, prepared using a simple cryopreservation technique, retain their morphological and functional characteristics, and are as effective as fresh allografts in healing chronic ulceration.

Adult↗
PubMed1990

Nitroglycerin and sucrose permeability as quality markers for reconstructed human epidermis.

In order to evaluate the epidermal permeability barrier of in vitro reconstructed epidermis, the penetration of nitroglycerin (NG) and sucrose were measured across human keratinocytes cultured at the air-liquid interface, using de-epidermized dermis (DED) as a substrate. In the presence of reconstructed epidermis on top of DED the penetration rate of sucrose is about 100 times and that of NG 2 times lower, as compared to DED only, indicating that the stratum corneum of the cultured epidermis exhibits considerable barrier capacity. The permeability of reconstructed epidermis was for both solutes higher (3- to 10-fold) than that of freshly excised human skin. Based on the impaired barrier function and distribution of various differentiation markers it can be concluded that the reconstructed human epidermis used in the present study shows a high extent of similarity with hyperproliferating epidermis.

Air↗
PubMed1989

Susceptibility of human male keratinocytes to MHC-restricted H-Y-specific lysis.

We studied the expression of the male-specific mH antigen H-Y on cultured human skin cells by investigating susceptibility to H-Y-specific cytolysis using conventional class I-restricted CTL clones in a modified cell-mediated cytotoxicity assay. In contrast to what was found in the rodent system, we observed H-Y-specific lysis of human male keratinocytes. Susceptibility for H-Y-specific lysis was efficiently enhanced by exposure of the keratinocytes to IFN-gamma. Our results demonstrate that human skin cells are equally sensitive for the activity of H-Y-specific CTLs as target cells of lymphoid origin. Finally, the cellular recognition of the H-Y mH antigen in the skin further supports its possible target function in the local graft versus host attack.

Cytotoxicity, Immunologic↗
PubMed1989

Toxicity screening of N-alkylazacycloheptan-2-one derivatives in cultured human skin cells: structure-toxicity relationships.

A number of N-alkylazacycloheptan-2-one derivatives, with the hydrocarbon chain lengths systematically varied from C2 to C16, were tested for their possible skin toxic effects. For this purpose, three in vitro cytotoxicity assays were used: (1) inhibition of proliferation of cultured human fibroblasts and keratinocytes; (2) inhibition of collagen contraction by human fibroblasts; and (3) cell morphology changes in confluent cultures of human fibroblasts and keratinocytes. With all assays used, the toxicity of N-alkylazacycloheptan-2-one derivatives increased from C2 to C8, remained constant at a hydrocarbon chain length between C8 and C14, and subsequently decreased with increasing alkyl chain length. A similar trend has been observed for flux enhancement of nitroglycerine in the presence of these N-alkylazacycloheptan-2-one derivatives, suggesting that with these compounds a parallelism exists between skin cell toxicity and penetration enhancing capacity. Since for practical use it is preferable to find a balance between skin toxicity and the penetration enhancement effect of a particular enhancer, it would be advisable to do QSAR studies of this kind with a number of congeners of a particular compound in order to optimize the choice. In this particular case, further modification of the N-alkylazacycloheptan-2-one structure might lead to an even better choice than the often propagated dodecyl derivative.

Cell Division↗
PubMed1989

Expression of EGF receptor, involucrin, and cytokeratins in basal cell carcinomas and squamous cell carcinomas of the skin.

The distribution of several markers of keratinocyte differentiation was studied in normal epidermis, basal cell carcinomas (BCCs), and squamous cell carcinomas (SCCs) using the immunoperoxidase technique on frozen sections of punch biopsy specimens. As markers a panel of chain-specific monoclonal antibodies (MoAbs) directed against cytokeratin (CK) 4, 8, 10, 13, 18 and 19, a polyclonal antiserum against involucrin, as well as a MoAb against the epidermal growth factor (EGF) receptor were used. In 15 out of 19 BCCs tested, expression of CK 8 was seen. Only a few individual cells in a limited number of BCCs showed positive staining for CK 4, 18, or 19. No expression of CK 10 was seen except for some foci of cell keratinization. Involucrin was not found in BCCs except for some squamous horn cysts. In all BCC cells expression of EGF receptor was found. In the suprabasal layers of normal epidermis from SCC patients, positive staining for CK 10 was seen. A few individual cells in a limited number of SCCs showed positive staining for CK 4, 8, or 18. Involucrin was expressed in the center of SCCs and in the upper layers of normal epidermis. Expression of EGF receptor was found in all SCC cells. These results demonstrate differences in cellular origin and differentiation between BCC and SCC.

Basal Cell Carcinoma↗
PubMed1989

Differentiation of cultured human keratinocytes: effect of culture conditions on lipid composition of normal vs. malignant cells.

Differentiation in keratinocytes can be experimentally modulated by changing the culture conditions. When cultured under conventional, submerged conditions, the extent of cellular differentiation is reduced in the presence of low calcium medium and is enhanced in medium containing physiologic calcium concentrations. Moreover, cultures grown at the air-medium interface or on a dermal substrate, or both, differentiate even further. Herein we report the effect of culture conditions on lipid composition in normal human keratinocytes and three squamous carcinoma cell (SCC) lines that vary in their capacity to differentiate as assessed by cornified envelope formation. Under submerged conditions, the total phospholipid content was lower, triglyceride content higher, and phospholipid:neutral lipid ratio lower in direct correlation to the degree of differentiation in these cultures. When grown at the air-medium interface on de-epidermized dermis, evidence of further morphologic differentiation was found only for well-differentiated SCC cells and normal keratinocytes. Similarly, the phospholipid content remained high in poorly differentiated SCC cells and it decreased modestly in well-differentiated SCC cells and markedly in normal keratinocytes. In all cell lines the triglyceride content was increased and cholesterol content decreased when compared to parallel submerged cultures, but these differences were most pronounced in well-differentiated cell lines. Acylceramides and acylglucosylceramides were found only in normal keratinocytes and only under the most differentiation-enhancing conditions. These studies demonstrate differentiation-related changes in the lipid content of both normal and neoplastic keratinocytes.

Cell Differentiation↗
PubMed1989

Contraction of collagen by human fibroblasts and keratinocytes.

In the process of wound healing keratinocytes and fibroblasts play an important role, keratinocytes in the re-epithelization process and fibroblasts in the process of wound contraction. We have studied the role of human keratinocytes and fibroblasts in the rearrangement of collagen in a collagen lattice model system. Our results revealed that keratinocytes as well as fibroblasts rearrange the collagen lattice; this occurs in a cell number and collagen concentration dependent manner. The optimal gel contraction is obtained in the presence of keratinocytes on the top of and of fibroblasts in the collagen lattice, the situation most closely approaching the in vivo situation. Between the two types of cells, differences in morphologic behavior were observed: when incorporated into the gel the keratinocytes retained their spherical shape throughout the whole culture period, but fibroblasts became elongated and formed extensions. Our data suggest that not only fibroblasts but also keratinocytes may be involved in the process of wound contraction.

Animals↗
PubMed1989

Proliferating, transformed keratinocytes cultured under low Ca2+ conditions exhibit high-affinity epidermal growth factor receptors.

In a previously published report (Exp. Cell. Res. 161:421 (1985] we have demonstrated that cultured normal and transformed keratinocytes exhibit two classes of EGF binding sites after growth under normal Ca2+ conditions but only low-affinity binding sites after growth under low Ca2+ conditions. Here we demonstrate the presence of high-affinity binding sites in transformed keratinocytes grown under low Ca2+ conditions, using a specific monoclonal anti EGF-receptor antibody.

Antibodies, Monoclonal↗
PubMed1989

The competence of transformed keratinocytes to differentiate is accompanied by amplification of the LDL- and EGF-receptor genes but not of the insulin receptor gene.

The possible relationship between cell surface receptor numbers, receptor gene expression for low density lipoprotein (LDL), insulin and epidermal growth factor (EGF), and differentiation capacity has been studied in normal and SV40 transformed (SVK14) keratinocytes, various squamous carcinoma cell (SCC) lines and A431 cells. Our recent studies demonstrated that an inverse relationship exists between LDL- and EGF-receptor binding and the ability to differentiate of both normal and transformed keratinocytes. In the present study cloned LDL- and EGF-receptor complementary DNAs were used as probes to identify both LDL and EGF receptor gene fragments on genomic DNA blots. The extent of hybridisation was found to be increased to the highest extent in A431 cells and decreased in other cells in the following order SCC-4 greater than SCC-15. In SCC-12F2, SVK14 and normal keratinocytes no increase has been observed. The increased hybridisation of LDL- and EGF-receptors in A431, SCC-4 and SCC-15 cells was found to be due to gene amplification and not to aneuploidy. In contrast to the LDL- and EGF-receptor binding, no correlation has been found between insulin receptor binding and ability of cells to differentiate. Furthermore, no amplification of insulin receptor gene has been observed in any of the cells under study.

Cell Differentiation↗
PubMed1989

Transdermal peptide delivery.

The transdermal delivery of peptide drugs, though ill-favoured by their hydrophilicity and high molecular mass, would seem very attractive from the pharmacotherapeutical and patient compliance point of view. In some cases, effective transdermal dosing has been achieved in vivo, especially with the aid of iontophoresis. This paper deals with a dodecapeptide, des-enkephalin-gamma-endorphin, of which the transepidermal permeation and the intra(epi-)dermal biotransformation were both studied in vitro. Small, though measurable, fluxes through human stratum corneum were obtained in vitro, which could be enhanced by using a skin lipid fluidizer. The half-life of the peptide, both in the epidermis and in the dermis, was surprisingly long as compared with that in human plasma. Hence, improvement of the transdermal bioavailability of the peptide will most likely be obtained chiefly by enhancing its flux (possibly through iontophoresis), intra(epi-)dermal degradation being a problem of only minor importance.

Administration, Cutaneous↗
PubMed1988

Lateral mobility of plasma membrane lipids in normal and transformed keratinocytes.

In this study we have examined possible differentiation-dependent modulations in plasma membrane lipid properties in normal keratinocytes, SV-40 transformed keratinocytes (SVK14) and a number of squamous carcinoma (SCC) cells. In normal keratinocytes the lateral diffusion coefficient of plasma membrane lipids (D) differs significantly for cells cultured permanently under low and normal Ca2+-conditions (5.16 x 10(-9) and 3.27 x 10(-9) cm2/s, respectively). When differentiation is induced by exposing low Ca2+-cultured cells to normal Ca2+ concentrations D increases to 7.07 x 10(-9) cm2/s during the initial hours of differentiation followed by a gradual sustained decrease to values also observed in cells cultured permanently under normal Ca2+-conditions. In SCC and SVK14 cells a similar initial transient increase in lateral lipid mobility is observed upon initiation of differentiation, but, in contrast to normal keratinocytes, no sustained decrease in D is seen upon prolonged culturing under normal Ca2+ conditions. The results indicate that the deficiency of the transformed cells to respond to Ca2+-induced differentiation might involve transformation-dependent alterations in membrane structure and function.

Calcium↗
PubMed1988

Proliferation and differentiation of human squamous carcinoma cell lines and normal keratinocytes: effects of epidermal growth factor, retinoids, and hydrocortisone.

Exposure of squamous carcinoma cell (SCC) lines, exhibiting high levels of epidermal growth factor (EGF) receptors, to EGF for 6 d caused a dose-dependent inhibition of cell proliferation. This EGF-induced inhibition of cell proliferation occurred under both low (0.06 mM) and normal (1.6 mM) Ca2+ concentrations. Furthermore, the extent of EGF-induced inhibition of cell proliferation seemed to be independent of the number of EGF-receptors. This conclusion is based on the notion that the various SCC lines exhibited an increasing number of EGF receptors accompanied by a decreasing ability to differentiate, whereas no relationship was observed with the EGF-induced inhibition of cell proliferation in these cell lines. Retinoids caused also a dose-dependent inhibition of cell proliferation. The effects of EGF and retinoids were additive, indicating that different regulatory mechanisms are involved. On the other hand, hydrocortisone caused a stimulation of SCC-proliferation, also independent of EGF. In contrast to SCC cells, EGF did not affect significantly the rate of proliferation of normal keratinocytes. However, the simultaneous addition of EGF and hydrocortisone resulted in a significant increase in the rate of keratinocyte proliferation only in cells grown under normal calcium conditions. Differentiation capacity of normal keratinocytes and SCC lines was not affected by EGF. Furthermore, the retinoid-induced decrease and hydrocortisone-induced increase of competence of cells to form cornified envelopes was not affected by EGF. These observations suggest that the action of retinoids and hydrocortisone on both cell proliferation and cell differentiation occurs independently of EGF receptors.

Calcium↗
PubMed1988

Induction of proteins and mRNAs after uv irradiation of human epidermal keratinocytes.

uv sensitivity of cultured human epidermal keratinocytes was analyzed at different growth conditions and compared with the sensitivity of dermal fibroblasts derived from the same skin specimen. No significant differences in survival curves were found between these two cell types, although keratinocytes grown under standard conditions were slightly more resistant to uv irradiation than fibroblasts. The extracellular concentration of calcium appeared to be critical not only in the regulation of keratinocyte proliferation and differentiation, but also in the uv sensitivity of these cells: keratinocytes grown under conditions which favor cell proliferation (low calcium concentration) are more resistant to uv irradiation than those grown under conditions favoring differentiation (high calcium concentration). Two-dimensional protein gel electrophoresis was used to detect a possible effect of uv irradiation on the accumulation of specific mRNAs in the cytoplasm and/or on the synthesis of specific proteins. Proteins were pulse labeled in vivo with [35S]methionine or synthesized in vitro in rabbit reticulocyte lysates on mRNA isolated from keratinocytes that were irradiated with different uv doses at different periods of time prior to isolation. Alterations in expression were demonstrated for several proteins in both in vivo and in vitro experiments.

Calcium↗
PubMed1988

Terminal differentiation in cultured human keratinocytes is associated with increased levels of cellular retinoic acid-binding protein.

The control of normal epithelial cell growth and differentiation by retinoids (vitamin A and its analogs) may involve, at least in part, cellular retinoid-binding proteins. In the present study the levels of cellular retinoic acid (CRABP)- and retinol (CRBP)-binding proteins were measured in cultured normal human epidermal keratinocytes. To this end cells were cultured under either low calcium (nondifferentiating) or normal calcium (differentiating) conditions; in the latter culture the cells were also separated into nondifferentiated, attached cells, and differentiating, shed, cell populations. Two different techniques [gel filtration and polyacrylamide gel electrophoresis (PAGE)] were used, for both the qualitative and the quantitative determinations of retinoid-binding proteins. Gel filtration analysis on Sephadex G75 columns showed the presence of high-molecular-weight binding sites for retinol and retinoic acid in both nondifferentiated and the differentiating keratinocytes; the nature of these binding proteins is unclear. Free CRABP was found in the cytosol of differentiating cells, but was undetectable in nondifferentiated cells. Using a more sensitive PAGE technique, very low levels of CRABP and CRBP could be detected in nondifferentiated keratinocytes. By this technique it was possible to demonstrate very high levels of CRABP only in differentiating keratinocytes; the CRBP levels were found to be very low in differentiating cells and comparable to the amount found in nondifferentiated cells. On the basis of molecular weight determinations by gel exclusion and by electrophoretic mobility, the CRABP and CRBP of cultured keratinocytes were found to be identical to CRBP and CRABP from human epidermis.

Carrier Proteins↗