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Biomedical subjects

M Ponec

Publications and source records attributed to M Ponec.

At least 19 recordsLinked to original sources
PubMed1992

LDL receptors in keratinocytes.

The presence of sufficient amounts of cholesterol in the epidermis is necessary for proper functioning of plasma membranes in the viable epidermal cell layers and also for the barrier quality of lipid intercellular bilayers of the stratum corneum. Cholesterol can be generated by local epidermal synthesis, or imported from the circulation as low-density lipoprotein (LDL), which is internalized by the cells by receptor-mediated endocytosis. Because of the complex structure of the skin, a model consisting of cultured human keratinocytes has been used to study in detail the regulation of epidermal sterologenesis in relation to keratinocyte differentiation. Experimental modulation of the differentiation of normal human keratinocytes has been achieved by varying extracellular calcium concentration or by comparison of a number of human squamous carcinoma cell lines and normal keratinocytes. These studies have clearly demonstrated a reciprocal correlation between the ability of cells to differentiate and LDL receptor activity. Regulation of LDL receptor expression has been found to occur at the DNA, mRNA, and protein levels, depending on the cell line studied. In normal but not malignant keratinocytes, the induction of keratinocyte differentiation was associated not only with a decrease of functional LDL receptors but also with changes in their cellular distribution. This conclusion is drawn from the observations that only in normal human keratinocytes, cultured at physiologic calcium concentrations, high levels of intracellular, cytoskeleton-associated receptors were found. Differentiation-related modulations of the LDL-receptor expression and of the cellular LDL-receptor distribution found in cultured keratinocytes were in agreement with observations made in the epidermis in situ.

Carcinoma, Squamous Cell↗
PubMed1992

Regulation of parathyroid hormonelike protein production in cultured normal and malignant keratinocytes.

We have recently demonstrated that parathyroid hormone-like protein (PLP) production by cultured human squamous carcinoma cells (SCC) can be modulated by co-culture with fibroblasts. The interaction of SCC with fibroblasts, possibly occurring during the invasive phase of SCC, may be the stimulus for enhanced PLP production, thus contributing to the genesis of humoral hypercalcemia of malignancy in this type of cancer (Cancer Res 50:3589-3594, 1990). In the present study we show that the fibroblast-induced increase in PLP level in the medium of SCC-4 cells is paralleled by an increase in PLP messenger ribonucleic acid (mRNA) expression in these cells. We also found that the inhibition of secretion of PLP by monensin for 2 h resulted in a marked increase in immunodetectable PLP intracellularly, suggesting that secretion of PLP was a fast process. The modulation of the production of PLP by calcium and hydrocortisone was further examined in SCC-4 cells and was compared to that in normal keratinocytes and in SCC-9 cells. PLP levels in conditioned media were highest in poorly differentiating SCC-4 cells, intermediate in moderately differentiating SCC-9 cells, and lowest in normal keratinocytes showing high differentiating capacity. Furthermore, in each of the cell types used, PLP production was highest in cultures grown under low calcium conditions; at both calcium concentrations used, the presence of hydrocortisone reduced the PLP release into the medium. This reduction was probably due to a direct effect of hydrocortisone on PLP synthesis because the expression of PLP mRNA was also reduced in the presence of hydrocortisone when tested in SCC-4 cells. In conclusion, our findings indicate that the induction of differentiation in both normal and malignant keratinocytes is associated with the inhibition of PLP production.

Carcinoma, Squamous Cell↗
PubMed1992

Safety aspects of non-ionic surfactant vesicles: a toxicity study related to the physicochemical characteristics of non-ionic surfactants.

Two different toxicity models were used to assess the relationship between the physicochemical properties of non-ionic surfactant vesicles (NSVs), and the safety of these vesicles for topical drug administration. The vesicles used in this study consisted of polyoxyethylene alkyl ethers (CnEOm) in which the number of C atoms (n) varied between 12 and 18 and the number of oxyethylene units (m) between 3 and 7. The physicochemical properties of the vesicles are described in terms of hydrophilic-lipophilic balance (HLB) values, and critical micelle concentrations (CMC), and the rigidity of the bilayers as determined by the gel-liquid transition temperatures and the cholesterol content of the bilayers. The first toxicity model, comprising the measurement of the ciliary beat frequency, is a tool to assess the safety of intranasally applied formulations. Studies using this ciliotoxicity model revealed that by increasing the length of the alkyl chain of the surfactant, a decrease in toxicity was observed. The opposite correlation was found if the length of the polyoxyethylene headgroup was increased. Furthermore, it was observed that gel-state vesicles produce less of an effect on the ciliary beat frequency than liquid state vesicles. The second toxicity model, comprising the determination of cell proliferation of human keratinocytes, is a method to assess skin irritancy. In contrast to the ciliotoxicity model the length of the polyoxyethylene headgroup and of the alkyl chains did not seem to have an effect on the safety of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Intranasal↗
PubMed1991

Regulation of epidermal growth factor receptor expression in normal and transformed keratinocytes.

Transformed keratinocytes (SCC-4, SCC-15, SCC-12F2, SVK14) or normal keratinocytes which differ in their differentiation programme were used to study the regulation of EGF-receptor expression. The capacity of the cells to differentiate was modulated by changing the extracellular calcium concentration. We were able to demonstrate that EGF-receptor expression in normal and transformed keratinocytes depends upon the cell type and one or more levels of regulatory control. At the DNA level, EGF-receptor gene amplification occurred in poorly differentiating cells. At the mRNA level, cells showing EGF-receptor gene amplification expressed elevated mRNA and protein levels when cultured under low Ca2+ conditions. Cells not exhibiting EGF-receptor gene amplification showed equal mRNA expression, regardless the Ca2+ concentration in the culture medium. At the protein level, EGF-receptor protein was decreased in cells exhibiting EGF-receptor gene amplification when extracellular Ca2+ was increased (to 1.6 mM) to stimulate differentiation, the decrease in protein being comparable to mRNA expression. Cells not exhibiting EGF-receptor gene amplification showed equal protein expression, regardless of the Ca2+ concentration in the culture medium. Under the same conditions, SV40 transformed keratinocytes showed equal mRNA but elevated protein expression in cells grown under low Ca2+ conditions. At the membrane level, normal keratinocytes and SCC-17F2 cells showed elevated numbers of cell surface exposed EGF-receptors in cells grown under low Ca2+ conditions, but equal mRNA and protein expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Western↗
PubMed1991

Acylceramides and lanosterol-lipid markers of terminal differentiation in cultured human keratinocytes: modulating effect of retinoic acid.

Epidermal differentiation is accompanied by profound changes in the synthesis of a variety of intracellular proteins and intercellular lipids. In conventional, submerged culture keratinocytes have been shown to lose the ability to synthesize the protein markers of differentiation. They re-express them, however, when they are cultured in medium supplemented with delipidized [retinoic acid (RA)-depleted] serum or in air-exposed cultures using de-epidermized dermis (DED) as a substrate. Recent studies have revealed that acylceramides (AC) and lanosterol (LAN), which are present only in trace amounts in cultures of keratinocytes grown under submerged conditions on DED in medium supplemented with normal serum, become expressed in significant amounts when the culture is lifted to the air-liquid interface. Inasmuch as culture conditions may markedly affect the extent of keratinocyte differentiation, the present study aimed to investigate the effect of normal (RA-containing) or delipidized (RA-depleted) serum and of RA administration on lipid composition (especially of the AC and LAN contents) in cells cultured under submerged and air-exposed conditions. To test a possible effect of dermal substrate (used in the air-exposed model), the lipid composition of keratinocytes grown under submerged conditions on a plastic and on a dermal substrate (de-epidermized dermis, DED) has also been compared. The results revealed that under all culture conditions, RA deprivation of fetal bovine serum resulted in a marked increase of total ceramide content. Even under submerged conditions, the presence of both AC and LAN could be detected. In air-exposed culture, the content of these lipids was markedly increased. Addition of RA at 1 microM concentration to cultures grown in RA-depleted medium induced marked changes in lipid composition under all culture conditions tested. In cells grown under submerged conditions (both on plastic and on DED) AC and LAN were no longer present in detectable amounts. Also in air-exposed culture, a marked decrease in the content of these lipids was observed. These results suggest that liposoluble serum components, like RA, control the synthesis of lipids that are present in later stages of epidermal differentiation.

Biomarkers↗
PubMed1991

Distribution of low-density lipoprotein receptors and apolipoprotein B on normal and on reconstructed human epidermis.

Immunoelectronmicroscopy was employed to investigate the ultrastructural localization of the low density lipoprotein (LDL) receptor and the protein moiety of LDL (apo B) on normal human epidermis in situ and reconstructed human epidermis in vitro. For this purpose ultrathin sections of Lowicryl K4M embedded material were incubated with monoclonal antibodies against the LDL receptor and apo B followed by a second antibody conjugated to 15 nm colloidal gold. Examination of the sections revealed a similar distribution of the LDL receptor and apo B in normal human epidermis and in reconstructed epidermis. In both systems the amount of LDL receptors decreases during keratinocyte differentiation. In contrast, apo B molecules are more abundantly expressed in the upper layers of the stratum spinosum and the stratum granulosum. The great similarities in the distribution of the LDL receptor and apo B between the in vitro reconstructed epidermis and its in vivo counterpart provide additional proof that reconstructed epidermis is an excellent tool to investigate proliferation and differentiation processes of keratinocytes in vitro.

Apolipoproteins B↗
PubMed1991

Regulation of low-density lipoprotein receptor expression during keratinocyte differentiation.

Transformed keratinocytes (i.e., SCC-4, SCC-15, SCC-12F2, SVK14) or normal keratinocytes which differ in their differentiation program, were used to study the regulation of low-density lipoprotein (LDL)-receptor expression. The capacity of the cells to differentiate was modulated by changing the extracellular calcium concentration. We now demonstrate that LDL-receptor expression in normal and transformed keratinocytes depends on the cell type and one or more levels of regulatory control. Cells express elevated mRNA levels when cultured under low Ca++ (proliferating) conditions. In contrast, SV40-transformed keratinocytes express decreased message under similar condition. In addition, LDL-receptor protein is decreased in transformed cells when extracellular Ca++ is increased (1.6 mM) to stimulate differentiation; the decrease in protein is comparable to the decrease in mRNA expression. Under the same conditions, normal keratinocytes show markedly decreased LDL-receptor protein relative to the decrease in mRNA. Incubation with LDL-cholesterol decreases the number of cell surface-exposed LDL-receptors. The LDL-receptor in fibroblasts is regulated differently from SCC-4 cells. The addition of LDL-cholesterol to fibroblasts causes decreased LDL-receptor mRNA and protein expression whereas SCC-4 cells show the opposite effect. The addition of cholesterol in non-lipoprotein form causes decreased LDL-receptor mRNA and protein expression in both cell types. These results suggest another, yet unidentified, regulatory mechanism that affects LDL-receptor expression in these two cell types.

Cell Differentiation↗
PubMed1990

Modulation of the production of a parathyroid hormone-like protein in human squamous carcinoma cell lines by interaction with fibroblasts.

Normal human keratinocytes as well as human squamous cell carcinomas produce a parathyroid hormone-like protein (PLP). However, PLP production by these cells is not a constant phenomenon. Since nothing is known about factors which regulate the production of PLP, in vitro studies were performed with normal keratinocytes and squamous carcinoma cell lines in order to establish conditions under which PLP production may vary. PLP was measured as cyclic AMP production in parathyroid hormone target cells (osteoblasts) which could be inhibited by a parathyroid hormone antagonist. The presence of PLP was confirmed using a radioimmunoassay specific for PLP. Results from the bioassay correlated very well with the data obtained by radioimmunoassay for PLP. The results confirm that human squamous carcinoma cells and normal keratinocytes produce PLP. PLP production appeared to be very sensitive to modulation of coculture of squamous carcinoma cells with fibroblasts. The effect of fibroblasts was not mediated by an effect on squamous carcinoma cell viability. Murine transformed fibroblasts (3T3 cells) as well as human normal foreskin fibroblasts were equally effective in inducing PLP production in these cells. The fibroblastic factor was apparently present in a soluble form in the coculture system which prevented direct cell-cell contact but allowed communication through the medium. Nevertheless, conditioned medium from 3T3 cells failed to induce PLP production by squamous carcinoma cells. This suggests a more complicated interaction between the two cell types than a one way message from fibroblasts to keratinocytes. Production of PLP by a number of squamous carcinoma cell lines was variable and not evidently correlated with the ability of these carcinoma cells to differentiate. Production of parathyroid hormone-like protein not only is the expression of a disturbed metabolism of a specific cell type but also reflects the cell-cell interaction in tumor tissue.

Animals↗
PubMed1990

Retinol and retinal metabolism. Relationship to the state of differentiation of cultured human keratinocytes.

Cultured keratinocytes offer an attractive model for studying the metabolism of retinol in relation to cell differentiation, since the extent of keratinocyte differentiation can be modulated experimentally. The metabolism of retinol and retinal was studied in cytosol fractions prepared from two distinct keratinocyte populations, differentiating and non-differentiated. The enzymic activities were analysed using physiological concentrations of [3H]retinol and [3H]retinal in the presence of cofactors. The products formed were quantified by h.p.l.c. In the population of differentiating keratinocytes, the formation of retinoic acid from retinol occurred at a rate of 4.49 +/- 0.17 pmol/h per mg of protein, but no such conversion was observed in the population of non-differentiated cells. However, when retinal was used as substrate, retinoic acid was formed in both cell populations, at rates of 14.4 pmol/h per mg of protein in non-differentiated and 51.6 pmol/h per mg of protein in differentiating keratinocytes. Using PAGE/radiobinding assay, we demonstrated that retinoic acid formed from retinol was bound in differentiating keratinocytes to endogenous cellular retinoic acid-binding protein (CRABP). Furthermore, retinal was reduced to retinol in the presence of NADH in both differentiating and non-differentiated keratinocytes at a similar rate (8 pmol/h per mg of protein). Although retinal could not be detected under physiological conditions, it was found in significant amounts at pH 8.5-9, which is optimal for enzymic activity. This indicates that in keratinocytes retinal is an intermediate metabolite in retinoic acid formation from retinol. The enzymes catalysing the conversion of retinol into retinoic acid were found to differ from other alcohol and aldehyde dehydrogenases, since the formation of retinoic acid was not significantly affected by specific inhibitors of alcohol metabolism, such as 4-methylpyrazole and disulfiram. Moreover, the cytosol of non-differentiated keratinocytes did not generate retinoic acid from retinol despite showing alcohol dehydrogenase activity. The results suggest that: (1) retinol metabolism in human keratinocytes is different from that of other alcohols, (2) retinal is an intermediate metabolite in the conversion of retinol into retinoic acid, and (3) differentiating keratinocytes rich in CRABP are probably target cells for retinoic acid action.

Adolescent↗
PubMed1990

A new sensitive assay for measurement of cell-mediated cytotoxicity to intact layers of cultured human keratinocytes.

A cytotoxicity assay for sensitive measurement of cell-mediated lympholysis (CML) of human cultured keratinocytes (cK) is described. The usage of 51Cr-labeled keratinocytes in intact layers as target cells in this assay allows objective and accurate determination of lysis of keratinocytes which have not undergone trypsin- and suspension-induced membrane changes. Furthermore, the problem of high spontaneous 51Cr release values encountered with suspended keratinocytes is overcome. The assay was applied to study antigen-specific CML of cK by cloned cytotoxic T cells (CTL) and to determine the effect of IFN-gamma on the susceptibility of cK to lysis. The results showed that HLA-A2 specific CTLs could reproducibly lyse cK of HLA-A2 positive healthy skin donors both with and without incubation of cK with IFN-gamma. Applications of this keratinocyte cytotoxicity assay lie in determining the antigenic expression of human cK, in analysis of effector cell/keratinocyte interactions in CML and of the modulatory effects of cytokines on these mechanisms. The assay thus may provide a helpful tool in gaining insight into the role of CML of keratinocytes in the destruction of inflamed skin.

Cell Adhesion↗
PubMed1990

Use of human keratinocyte and fibroblast cultures for toxicity studies of topically applied compounds.

Penetration enhancers are often used as additives in pharmaceutical and dermatological preparations. It should be expected that in many cases penetration enhancers not only enter the stratum corneum but also reach the viable cells of the epidermis and exert a toxic effect. This study focused on a series of well-known compounds that are often used as skin penetration enhancers; namely, ethanol, propylene glycol, dimethylsulfoxide, dimethylformamide, and Brij 96. In order to obtain more insight in the potential skin toxicity of these agents, they were administrated to cultured human keratinocytes and fibroblasts and the following cytotoxicity assays were performed: inhibition of the proliferation of fibroblasts and keratinocytes; inhibition of collagen contraction by fibroblasts; and cell morphology changes in confluent cultures of fibroblasts and keratinocytes. In all assays performed, the same trend was observed: ethanol was the least toxic, propylene glycol, dimethylsulfoxide, and dimethylformamide were moderately potent, and Brij 96 was the most toxic agent. An obvious advantage of the in vitro model presented here is its immediate availability and reproducibility, which allows for the comparison of a large series of topical agents (e.g., penetration enhancers) with respect to their cell toxicity under standardized conditions. However, this single-cell model lacks some of the properties found in intact skin, such as the stratum corneum barrier, and interactions between keratinocytes and other cells, such as Langerhans cells. Hence, extrapolation of these data to in vivo should be done with caution.

Adjuvants, Pharmaceutic↗
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↗