Quantitative and qualitative study in keratinocytes from foreskin in children: Perspective application in paediatric burns.

BACKGROUND: We performed a quantitative and qualitative evaluation of keratinocytes from foreskin in children. MATERIALS AND METHODS: We harvested 18 foreskins after circumcision. The mean average age of the operated children was 4 years. The keratinocytes were isolated after double-enzymatic digestion. After filtration and centrifugation we put the keratinocytes in culture. Then, the keratinocytes were cultivated on collagen lattices. The keratinocytes were cultured in submerged condition for 2 days and then in an air-liquid interface condition for further differentiation. After cultures, the cells were counted and a histological examination was done. An immunohistologic analysis enabled us to highlight the markers characteristic of neo-epidermis differentiation. RESULTS: After enzymatic digestion, we obtained 11.4 million cells per foreskin. After 10 days of culture and from 2 million cells, we obtained 24 million cells. In contact with the collagen lattices, we obtained a neo-epidermis and we described the markers of keratinocytes differentiation as well as the markers of the dermo-epidermal junction. CONCLUSION: Keratinocytes from foreskin have a high capacity for division. These cells can divide for long periods before differentiation. These observations allow us to propose foreskin keratinocytes as a potential source of cells to provide coverage in burns.

Non-organ-specific autoantibodies in chronic hepatitis C patients: association with histological activity and fibrosis.

BACKGROUND: Non-organ-specific autoantibodies (NOSAs) are frequently found in the sera of patients with Hepatitis C Virus (HCV) infection. However, no conclusive answers have been produced concerning the clinical relevance of these antibodies. AIM: To determine whether a relationship might exist between the presence of NOSA and the severity of liver disease in chronic hepatitis C. METHODS: 186 treatment-naïve chronic hepatitis C patients were studied consecutively for autoantibodies. Liver biopsies were analyzed according to the Metavir score. RESULTS: NOSAs were present in 75 patients (40%). Anti-nuclear antibodies were found in 32% of patients (speckled pattern), anti-smooth muscle in 15% without F-actin specificity, anti-mitochondria in 0.5%, and anti-LKM1 in 0.5%, respectively. No liver-cytosol1 or soluble liver antigen antibodies were detected. There was a highly significant correlation between the positivity of NOSA and the degree of inflammation and hepatocellular injury (p = 0.001) and also with the degree of fibrosis (p < 0.0001). The presence of NOSA was associated with higher aspartate aminotransferase, gamma-glutamyl-transpeptidase, gamma-globulin and immunoglobulin G levels. By contrast, no differences were observed regarding age, gender, route of infection, duration of disease, HCV genotypes or viral load. CONCLUSION: NOSAs were associated with the most severe forms of chronic HCV infections.

[Cultured keratinocyte cells from foreskin and future application for burns in children]. / Modèles de cultures cellulaires kératinocytaires de prépuce. Possibilité d'application chez les enfants brûlés.

PURPOSE OF THE STUDY: We tested in vitro the keratinocytes capacity for division and differentiation. The donor site was the human foreskin. PATIENTS AND METHODS: For 12 months, we harvested 18 foreskins after circumcision. The middle age of the operated children was four years. The keratinocytes were isolated after double enzymatic digestion (thermolysin and trypsin, respectively). After filtration and centrifugation we put the keratinocytes in culture. In parallel, the keratinocytes were cultivated on the surface of collagen lattices. The keratinocytes were cultured in submerged condition for two days and then in an air-liquid interface condition for further differentiation. After nine days of culture, a histological examination and immunostain were used. An immunohistologic analysis made it possible to highlight the markers characteristic of epidermal skin differentiation. RESULTS: We obtained an average of 8.8 10(6) cells per foreskin. After seven days of culture, we obtained on average 23.7 10(6) cells by culture. In contact with the collagen lattices, we obtained an epidermal skin and we highlighted the markers of keratinocytes differentiation as well as the markers of the dermoepidermic junction. CONCLUSION: The keratinocytes resulting from foreskin have a high capacity of division. These cells can divide a long time before differentiation. The observations enable us to propose with our patients the keratinocytes from foreskin for wound healing especially for burns in children.

Stimulation of the proliferation of human dermal fibroblasts in vitro by a lipidocolloid dressing.

OBJECTIVE: The effect of Urgotul on normal human dermal fibroblast proliferation was studied in vitro and compared with that of two other dressings: Mepitel and Tulle Gras. METHOD: Proliferation was measured by the extent of thymidine incorporation into the replicating DNA of proliferative fibroblasts in contact with the complete dressing.Additional cell viability and metabolism were evaluated using MTT assay. Morphology and ultrastructure analysis were based on immunolabelling and confocal laser microscopy. RESULTS: Only Urgotul significantly stimulated thymidine incorporation, generally with a maximal proliferative effect at a contact time of 48 hours. This was confirmed by the observation of a greater number of dividing cells (mitotic cells) than in the control cultures. No cytotoxicity was observed following treatment with this dressing. Cells exhibited normal structural and ultrastructural features. CONCLUSION: Fibroblasts play a key role in dermal wound repair. The ability of Urgotul to promote fibroblast proliferation could explain its efficiency in the healing process of acute and chronic wounds. DECLARATION OF INTEREST: This study was supported by Urgo Laboratories.

Development of a highly sensitive in vitro phototoxicity assay using the SkinEthic reconstructed human epidermis.

The reconstituted human epidermis model SkinEthic was used to evaluate the phototoxicity of topically applied chemicals. For comparison with published data, we first tested a library of 13 nonphototoxic (NPT) and phototoxic (PT) compounds, applied onto SkinEthic reconstituted human epidermal tissues, in a protocol as close as possible to the one described by Liebsch using another skin tissue model. The results showed that, under these nonoptimized conditions, the SkinEthic model was already able to fully discriminate between known NPT and PT compounds. Furthermore, these epidermal tissues being highly resistant to UVA irradiation, it was possible to increase irradiation by (at least) 3-fold without decrease in tissue viability. In such conditions, the phototoxicity assay is much more sensitive, so that the model is expected to be of great interest for the detection not only of strong but also of weak phototoxic compounds.

Expression of type 1 5alpha-reductase and metabolism of testosterone in reconstructed human epidermis (SkinEthic(R)): a new model for screening skin-targeted androgen modulators.

The steroid 5alpha-reductase isoenzymes (5alphaR) transform testosterone into 17beta-hydroxy-5alpha-androstan-3-one (5-dihydrotestosterone, DHT), which exerts a much stronger biological activity than does testosterone. Briefly, the two 5alphaR isoenzymes are differentially expressed in the two major target organs of steroid action, the prostate (isoenzyme 2, 5alphaR2) and the skin (isoenzyme 1, 5alphaR1). We analysed the potential of a human epidermal tissue reconstituted by cell culture (RHE, provided by SkinEthic Laboratories, Nice, France) as a model for assessing 5alphaR activity. The epidermal model was found to express the type-1 (skin) isoform of 5alphaR and thus could be used as an enzyme source for the screening of 5alphaR modulators for dermatological/cosmetic purposes. A reproducible and convenient assay method was developed, allowing both the evaluation of testosterone transformation into DHT (5alphaR activity) and an outlook on the general metabolism process of testosterone. This could be important for the detection of any compound that could act mainly on another target enzyme than 5alphaR. The assay gave evidence of the inhibitory activity of finasteride against type-1 5alphaR, which is now established both in vitro and in clinical studies. In addition to enzyme inhibitors, this in situ cellular assay can detect transcriptional modulators of 5alphaR gene expression, or any compound that could modulate enzyme processing or post-translational activation. RT-PCR analysis of RNA samples from RHE failed to show any notable effect of finasteride, testosterone, or DHT treatment on the expression of 5alphaR1 at the transcriptional level.

Purification and characterisation of a metallopeptidase of Candida albicans.

A novel aminopeptidase was purified by high performance liquid chromatography from a cytosoluble 100,000 g extract of Candida albicans on the basis of its ability to cleave L-arginine 7-amino-4-methylcoumarin. The purification factor was 36 and the yield was 20%. The native enzyme had a mol. wt of 52 kDa as demonstrated by SDS-PAGE in the presence or absence of reducing conditions and exhibited an iso-electric point of 4.3. The aminopeptidase showed optimum activity at pH 7.2, a Michaelis constant of c. 50 microM and a Vmax at 19 mM AMC released/min/mg of protein for L-Arg-AMC. This enzyme was shown to cleave at low affinity L-leucine-7-amino-4-methylcoumarin as demonstrated by the spectrofluorimetric method. The enzyme was strongly inhibited by specific metallo-enzyme inhibitors-EDTA and o-phenanthroline. Furthermore, there is evidence that a similar or identical enzyme occurs in other C. albicans clinical isolates and other Candida spp.

Plasmodium falciparum proteinases and red blood cell invasion.

Malarial proteinases of the erythrocytic life-cycle are used to design new inhibitors capable of blocking the parasite's development. The Merozoite Proteinase for Erythrocytic Invasion (MPEI) of Plasmodium falciparum, a neutral proteinase, and the acidic Pf37 proteinase acting on spectrin as substrate, are good candidates for this kind of strategy.

Peptide derivatives specific for a Plasmodium falciparum proteinase inhibit the human erythrocyte invasion by merozoites.

A specific proteinase of P. falciparum merozoites has been detected by using hydrosoluble fluorogenic peptidic substrates synthesized by classical peptide chemistry; their N-terminal end was acylated by a gluconoyl group that protects them from aminopeptidase degradation and increases their hydrosolubility, and their carboxylic end was substituted by a 3-amino-9-ethylcarbazole group. The sequence Val-Leu-Gly-Lys was found to be the most specific substrate. On this basis, reversible peptidic inhibitors were synthesized by substituting the C-terminal lysyl residue, at the proteolytic site, by different alkylamines and amino alcohols. The activity of these compounds, studied on the P. falciparum proteinase and in in vitro cultures, strongly suggests a specific effect of this peptidic sequence on the reinvasion process. The peptidic inhibitors do not impair the release of merozoites from schizonts, but selectively inhibit the invasion step leading to the formation of rings. Although the natural target of this enzyme is not yet known, these specific peptide inhibitors could lead to a new antimalarial approach.