[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.

A role for T cell-derived interleukin 22 in psoriatic skin inflammation.

Interleukin (IL)-22 is a T cell-derived cytokine that has been reported recently to induce cutaneous inflammation in an experimental murine model of psoriasis, and to induce in vitro an inflammatory-like phenotype. In the present study, we assessed the presence of IL-22 and the IL-22 receptor 1 (IL-22R1) in skin lesions, skin-derived T cells, as well as IL-22 levels in sera from patients with psoriasis. IL-22R1 and IL-10R2 transcripts are expressed at a similar level in psoriatic and healthy skin. In contrast, IL-22 mRNA expression was up-regulated in psoriatic skin lesions compared to normal skin, whereas IL-22 mRNA levels in peripheral blood mononuclear cells from psoriatic patients and normal subjects were similar. Circulating IL-22 levels were significantly higher in psoriatic patients than in normal subjects. T cells isolated from psoriatic skin produced higher levels of IL-22 in comparison to peripheral T cells isolated from the same patients. IL-10 was expressed at similar levels in skin biopsies and peripheral blood mononuclear cells of psoriatic patients and normal subjects. Finally, we show here that supernatants of lesional psoriatic skin-infiltrating T cells induce an inflammatory response by normal human epidermal keratinocytes, resembling that observed in psoriatic lesions. Taken together, the results reported in this study indicate that IL-22 is a cytokine produced by skin-infiltrating lymphocytes that is potentially involved in initiation and/or maintenance of the pathogenesis of psoriasis.

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.

Isotypic analysis, antigen specificity, and inhibitory function of maternally transmitted Plasmodium falciparum-specific antibodies in Gabonese newborns.

An analysis of Plasmodium falciparum-specific antibodies was performed in pairs of maternal and cord sera from Gabon, a region endemic for malaria. All paired sera (n = 59) had P. falciparum-specific antibodies. Immunofluorescence assays detected parasite-specific IgG1, IgG2, and IgG3 in 100% of the tested pairs (n = 26) and IgG4 in 42% of them. The titers of specific IgG2 and IgG3 were significantly lower in cord than in maternal sera. All maternal sera had specific IgM. Of the seven P. falciparum-IgM positive cord sera, six were associated with malaria-related histological placental changes (MRHPC). In addition, higher titers of specific IgG1 in maternal and cord sera and of specific IgG3 in cord sera were associated with MRHPC. Similar P. falciparum antigens were recognized by cord and corresponding maternal sera in radioimmunoprecipitation and Western blot assays (n = 40). Sixteen of 20 cord sera and 15 of 20 paired maternal sera significantly inhibited in vitro parasite growth. The extent of inhibition did not correlate with the titer of specific antibodies. These data confirm the very effective placental transfer of anti-malarial antibodies. The presence of IgM in some cord sera raise the question of intrauterine sensitization to malaria antigens.

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.

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.

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.

Glycosylated flavones as selective inhibitors of topoisomerase IV.

Three flavonoids which promoted Escherichia coli topoisomerase IV-dependent DNA cleavage were isolated from cottonseed flour and identified as quercetin 3-O-beta-D-glucose-[1,6]-O-alpha-L-rhamnose (rutin), quercetin 3-O-beta-D-galactose-[1,6]-O-alpha-L-rhamnose, and quercetin 3-O-beta-D-glucose (isoquercitrin). The most active one (rutin) also inhibited topoisomerase IV-dependent decatenation activity (50% inhibitory concentration, 64 microg/ml) and induced the SOS response of a permeable E. coli strain. Derivatives of quercetin glycosylated at position C-3 were shown to induce two site-specific DNA cleavages of pBR322 DNA, which were mapped by DNA sequence analysis to the gene encoding resistance to tetracycline. Cleavage at these sites was hardly detectable in cleavage reactions with quercetin or fluoroquinolones. None of the three flavonoids isolated from cottonseeds had any stimulatory activity on E. coli DNA gyrase-dependent or calf thymus topoisomerase II-dependent DNA cleavage, and they were therefore specific to topoisomerase IV. These results show that selective inhibitors of topoisomerase IV can be derived from the flavone structure. This is the first report on a DNA topoisomerase inhibitor specific for topoisomerase IV.

Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases.

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.