Effects of antiinflammatory triterpenes isolated from Leptadenia hastata latex on keratinocyte proliferation.

Several triterpenes isolated from Leptadenia hastata latex were tested for their anti-inflammatory activity. Lupeol (1), its acetate (2) and palmitate (3) esters were found to be the main antiinflammatory constituents in the croton oil-induced ear oedema test. Furthermore, lupeol hemisuccinate (4), synthesized from lupeol, exhibited a higher activity than lupeol in the test. These results prove that the triterpenes play a pivotal role in the topical antiinflammatory effect of this latex. In addition, an in vitro model of human skin keratinocytes (epidermal explants) cultured at an air-liquid interface on a de-epidermized human dermis (DED) was used to investigate the effects of lupeol esters on skin repair in vitro. Compared with the control, compounds 2 and 3 improved keratinocyte proliferation at a concentration of 5 microM in the culture medium; however, they remained less active than compounds 1 and 4. In contrast to compound 1, all the lupeol esters (2-4), and particularly compound 4, induced a good differentiation of keratinocytes with a well-formed stratum corneum without parakeratosis. These results substantiate the topical use of Leptadenia hastata latex in traditional medicine and showed that both antiinflammatory activity and the effect on keratinocyte proliferation of compound 1 could be improved by its hemisuccinylation; on the contrary, esterification by acetylation or palmitoylation decreased these activities.

Apoptosis in established and healing psoriasis.

BACKGROUND: Previous studies have described apoptosis in the stratum granulosum and in the stratum corneum, but not in the germinative compartment in normal skin. In psoriasis, an increased epidermal apoptosis has been observed in the differentiated compartment, suggesting that apoptosis has a key role in the pathogenesis of psoriasis, as a counteracting factor to the overproduction of cells. Little is known on apoptosis in the germinative compartment. METHODS: Apoptosis was studied on biopsies of normal skin, established lesions of psoriasis and PUVA-treated psoriasis using the transferase-mediated uridine nick end labelling method, which detects fragmented DNA, and electron microscopy. Counting of apoptotic cells was restricted to the germinative compartment as defined by Mib1 staining to evaluate the impact of cell loss on cell production and tissue architecture. RESULTS: The apoptotic index was 0.12% in normal epidermis, 0.035% in established psoriasis and 0.31% in regressive psoriasis. CONCLUSION: These results have three implications: (1) they show the physiological presence of apoptosis in the germinative compartment in normal epidermis; (2) they suggest that induction of apoptosis is involved in the regression of psoriatic hyperplasia after PUVA therapy; (3) the decrease of physiological apoptosis in the psoriatic lesion suggests that this phenomenon could play a role in the induction of psoriatic hyperplasia.

Methotrexate induces apoptotic cell death in human keratinocytes.

The mechanism by which low doses of methotrexate act in psoriasis to restore a clinically normal skin is poorly understood. Apoptosis is a programmed cell death activated when cell removal is needed. The purpose of the present work was to examine using an organotypical model of keratinocyte culture, the possibility that low doses of methotrexate can induce apoptosis of keratinocytes. Epidermal explants were cultivated on dead deepidermized dermis under air-exposed conditions. After 10 days, methotrexate (10(-7) M) was added. After a further 5 days, one part of each culture was fixed and submitted to routine histology, DNA nick end labelling (TUNEL) to detect DNA fragmentation (a molecular marker of apoptotic cell death) and immunohistochemical detection of p53 (a protein involved in apoptosis induced by DNA-damaging agents). The other part of each culture was processed for electron microscopy. A significant proportion of keratinocytes (1%) were damaged and exhibited the morphological features of apoptotic cell death. Immunohistochemical overexpression of p53 was detected in the basal layer of the cultures treated with methotrexate. Low doses of methotrexate induce apoptosis. This mode of action could explain the reduction in epidermal hyperplasia during treatment of psoriasis with methotrexate.

Effects of low frequency pulsed electrical current on keratinocytes in vitro.

The effects of low frequency pulsed electrical current on epidermal repair in vitro were examined. Charge-balance current stimuli proposed for chronic wound treatment were tested on skin keratinocytes cultured at an air-liquid interface on dead human dermis. Results imply that the balance between proliferation and differentiation in electrically treated samples is significantly modified in favor of differentiation. More advanced differentiation, shown through epidermal histology, was obtained in cultures exposed to electrical current, whereas the culture growth, the result of keratinocyte migration and proliferation, was greater in control samples.

Renewal and differentiation of keratinocytes cultured on dead de-epidermalized dermis.

Human keratinocytes grown at an air-liquid interface on dead de-epidermalized dermis exhibit a pattern of organization similar to that seen in vivo. Cell renewal is limited to the basal layer. The cell cycle time determined after 7 days of culture, using a percentage labelled mitoses (PLM) technique, was about 15 h. This result is comparable with published data for cultivated keratinocytes but is shorter than the parameter proposed for epidermis in vivo. Appearance of labelled cells in the granular layer was observed 4 days after pulse labelling. Despite this high cell renewal, a normal cell differentiation with expression of various keratinization markers was maintained.

In vitro acantholysis induced by D-penicillamine, captopril, and piroxicam on dead de-epidermized dermis.

Drug-induced pemphigus has been recognized for 20 years, but the mechanisms leading to acantholysis are still unclear. It has recently been demonstrated that penicillamine, captopril, and thiopronin may produce acantholytic lesions, either by direct toxic or biochemical effect, in human skin explants. Our work confirms that penicillamine and captopril may induce acantholysis on the model of keratinocyte culture on dead, de-epidermized dermis. Moreover, it demonstrates that piroxicam, a new non-steroidal anti-inflammatory drug, of which one side effect is a pemphigus vulgaris-like eruption, is also able to produce in vitro acantholysis.

Kinetics of the calcium induced stratification of human keratinocytes in vitro.

In a low concentration of calcium (0.1 mM), keratinocytes form a monolayer with about 30% of cells synthesizing involucrin. After addition of calcium to the culture medium to a concentration of 1.2 mM, the monolayer stratifies within 24 h, with a preferential migration of involucrin positive keratinocytes. In the present study, we tried to determine if keratinocytes control the decision to migrate at a distinct cell cycle point. A percentage labelled mitosis (PLM) curve was constructed for keratinocytes grown in low calcium medium and values for the length of the cell cycle (47 h), S phase duration (11 h) and G2+M period (6 h), were obtained. Monolayer cultures at 80% confluence were switched to high calcium concentration at various times (from 0 to 48 h), after pulse labelling with [3H]-thymidine. Based on the PLM data, the behaviour of cells known to be in S, G1 and G2 at the time of the migration stimulus were followed. No significant difference in the percentage of labelled suprabasal cells was found for any point of the cell cycle. For cells submitting to stratification, in S phase involucrin staining showed that about 60% of the [3H]-thymidine labelled cells were also involucrin negative. These results indicate that upward migration of keratinocytes in cultured epithelium can be triggered at all points in the cell cycle with equal probability and is not restricted to those cells that already contained involucrin.

Reproduction of the characteristic morphologic changes of familial benign chronic pemphigus in cultures of lesional keratinocytes onto dead deepidermized dermis.

We report the in vitro reproduction of the classic histologic and ultrastructural features of familial benign chronic pemphigus (FBCP) by seeding suspensions of lesional keratinocytes onto healthy heterologous dead deepidermized dermis (DED). With the use of normal keratinocytes, control cultures showed a well-differentiated epidermis, with keratohyaline granules and lamellar bodies. To the best of our knowledge this is the first successful culture of FBCP with the use of dispersed lesional keratinocytes. The results suggest that in FBCP the epidermis is the site of the defect leading to acantholysis, without any dermal contribution.