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In view of the increasing need to identify non-animal tests able to predict acute skin irritation of chemicals, the European Centre for the Validation of Alternative Methods (ECVAM) focused on the evaluation of appropriate in vitro models. In vitro tests should be capable of discriminating between irritant (I) chemicals (EU risk: R38) and non-irritant (NI) chemicals (EU risk: “no classification”). Since major in vivo skin irritation assays rely on visual scoring, it is still a challenge to correlate in vivo clinical signs with in vitro biochemical measurements. Being particularly suited to test raw materials or chemicals with a wide variety of physical properties, in vitro skin models resembling in vivo human skin were involved in prevalidation processes. Among many other factors, cytotoxicity is known to trigger irritation processes, and can therefore be a first common event for irritants. A refined protocol (protocol15min–18hours) for the EPISKIN model had been proposed for inclusion in the ECVAM formal validation study. A further improvement on this protocol, mainly based on a post-treatment incubation period of 42 hours (protocol15min–42hours), the optimised protocol, was applied to a set of 48 chemicals. The sensitivity, specificity and accuracy with the MTT assay-based prediction model (PM) were 85%, 78.6% and 81.3% respectively, with a low rate of false negatives (12%). The improved performance of this optimised protocol was confirmed by a higher robustness (homogeneity of individual responses) and a better discrimination between the I and NI classes. To improve the MTT viability-based PM, the release of a membrane damage marker, adenylate kinase (AK), and of cytokines IL-1α and IL-8 were also investigated. Combining these endpoints, a simple two-tiered strategy (TTS) was developed, with the MTT assay as the first, sort-out, stage. This resulted in a clear increase in sensitivity to 95%, and a fall in the false-positive rate (to 4.3%), thus demonstrating its usefulness as a “decision-making” tool. The optimised protocol proved, both by its higher performances and by its robustness, to be a good candidate for the validation process, as well as a potential alternative method for assessing acute skin irritation.

Several tight junction (TJ) proteins were detected in the living layers of adult human epidermis, and TJ-like membrane ridges were observed at the top of the stratum granulosum (SG) in freeze-fracture studies. We applied standard and immunoelectron microscopy to look for TJ-derived structures in the stratum corneum (SC) of human adult epidermis and in cornified envelopes purified from the plantar SC. Besides confirming claudin-1 labelling in the proximity of SG desmosomes, we also observed immunolocalization near corneodesmosomes in the lower SC. In addition, TJ proteins were consistently detected in the purified cornified envelopes. Lateral but not horizontal walls of the corneocytes showed frequent points of molecular fusion between lipid envelopes. These structural associations were very frequently localized at the top of the lateral corneocyte membranes, thus sealing the extremities of lateral intercorneocyte spaces. We propose that TJ-like structures persist in the SC and contribute to the reinforcement of lateral contacts and to the formation of membrane interdigitations between corneocytes. Their presence could contribute to subdivision of the extracellular spaces of SC into consecutive individualized compartments. Intercellular lipids, enzymes and other (glyco)protein content could thus evolve in the keratinized epidermal layer at different paces, as preprogrammed in the underlying living cells and influenced by the environment, e.g. humidity. Such situation might explain differences in the degradation rates between the 'peripheral' and the 'non-peripheral' corneodesmosomes observed during physiological desquamation, as previously suggested by us and others.Key words: cornified envelopes -desquamation -epidermal cohesionstratum corneum -tight junctions Accepted for publication 26 April 2011Human stratum corneum (SC) is the final product of epidermal differentiation that provides barrier function to the skin. Composed of cornified epithelial cells, called corneocytes, and sealed with the lipid-rich extracellular matrix, this thin epidermal layer is relatively impermeable to water and water-soluble substances. Like the entire epidermis, SC is constantly recycled in a highly regulated and interactive process in which superficial cell losses by desquamation at the skin surface are readily compensated by the conversion of the uppermost living keratinocytes into corneocytes (1,2). Such a programmed cell death is associated with several important changes in the cell shape and structure leading to the accumulation of flattened, laterally interdigitated, cornified keratinocytes forming the lower part of the SC, the SC compactum. During cornification, phospholipids of the keratinocyte plasma membrane are replaced by a single layer of ceramides. The latter are covalently bound by transglutaminase 1 to the protein envelope that is also cross-linked at the cell periphery by the same enzyme (3,4). The proper functioning of the SC barrier depends largely on its cohesion. Therefore, it is important to note tha...

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Human reconstituted epidermis Episkin can be useful to study in vitro the onset of cutaneous phototoxic reactions and particularly to identify weak phototoxic compounds.