Organotypic cultures of autologous hair follicle keratinocytes for the treatment of recurrent leg ulcers.

Our purpose was to evaluate, in an open study, the efficacy of epidermal equivalents (EEs), a tissue-engineered epidermis prepared from autologous hair follicle keratinocytes, for the treatment of recurrent leg ulcers (n = 50). To generate EEs, keratinocytes expanded from the outer root sheaths of plucked anagen hair follicles were seeded on cell culture inserts at air-liquid interface. The total culture time was 5 to 6 weeks. Three days after the procedure, 95% of EEs adhered to the wound bed. After 8 weeks, 70% of the total wound surface was re-epithelialized and 32% of the ulcers were healed. After applying the EEs, a major relief of wound pain was noticed by the patients. EEs were applied in ambulatory patients without surgical facilities. Because 92% of the cases included in this study presented a recurrence of their ulcers after a split-thickness skin graft, we consider these ulcers as difficult to treat and propose the EEs as an alternative effective treatment of recurrent leg ulcers.

Intracellular localization of keratinocyte Fas ligand explains lack of cytolytic activity under physiological conditions.

Acquired Fas ligand (FasL)-mediated cytolytic activity of human keratinocytes causes the massive keratinocyte cell death that occurs during toxic epidermal necrolysis, a deadly adverse drug eruption. Under normal conditions keratinocyte apoptosis is a rare event in the epidermis although keratinocytes express the death receptor Fas and its ligand. Here we have investigated why this is so. We show that Fas, FasL, Fas-associated death domain, and caspase-8 mRNA are detectable in the epidermis, primary keratinocyte cultures, and keratinocyte cell line and that Fas protein is expressed in keratinocytes of all subcorneal layers of the epidermis, whereas FasL is only expressed in the basal and first suprabasal layers. Coexpression of Fas and FasL therefore occurs in basal and suprabasal keratinocytes. In vitro, keratinocytes are killed by recombinant FasL in a dose-dependent manner, but they are unable to kill Fas-sensitive target cells despite FasL expression. Analysis of keratinocyte culture supernatants and treatment of keratinocytes with metalloproteinase inhibitors excluded cell surface expression of FasL and rapid metalloproteinase-mediated cleavage of cell surface FasL. Fluorescence-activated cell sorter, confocal microscopical, and electron microscopical analysis revealed that keratinocyte FasL is localized intracellularly predominantly associated to intermediate filaments. These data suggest that the observed inability of keratinocyte FasL to induce apoptosis under physiological conditions is due to its cellular localization and also indicate that intermediate filaments may be involved in regulating the subcellular localization of FasL.

Acute generalized exanthematous pustulosis: role of cytotoxic T cells in pustule formation.

Extensive formation of nonfollicular sterile pustules on erythematous background combined with fever and peripheral blood leukocytosis are the characteristics of acute generalized exanthematous pustulosis. This uncommon eruption most often is an allergic reaction because of drugs such as aminopenicillins and sulfonamides inter alia. We recently demonstrated the important role of drug-specific T cells in the pathogenesis of this disease, showing that they produce high amounts of the neutrophil-attracting chemokine interleukin-8 and therefore stand out as a special subgroup of T cells, differing from the usual Th1 and Th2 subsets. In this study we use immunohistochemistry as well as cytotoxicity assays (4- and 18-hour assays) and fluorescence-activated cell-sorting analysis of drug-specific circulating T cells and of cells eluted from the skin of five patients with acute generalized exanthematous pustulosis, to analyze whether cytotoxic T-cell functions are important in the pathogenesis of this disease, in particular for the formation of vesicles. The data reveal that drug-specific CD4(+) as well as CD8(+) T cells both are activated and cytotoxic; perforin/granzyme B and to a variable degree the Fas/FasL-killing mechanism is involved in tissue destruction. These features allow the formation of vesicles. Additional secretion of interleukin-8 by T cells and keratinocytes attracts neutrophils that fill the vesicles and transform them into pustules.

Use of epidermal equivalents generated from follicular outer root sheath cells in vitro and for autologous grafting of chronic wounds.

During wound healing, outer root sheath (ORS) cells of hair follicles can substitute for interfollicular epidermal keratinocytes and thus act as precursor cells for interfollicular epidermal keratinocytes. Owing to improved culture techniques, ORS cells can be induced to develop highly differentiated epidermal equivalents, which are close to the normal human epidermis in terms of histological, ultrastructural, biochemical and immunohistological criteria. Such epidermal equivalents provide a versatile system for various applications in vitro, e.g. the study of epidermal homeostasis, cell interactions, pigmentation as well as toxicity testing and metabolism of xenobiotics. The easy and repeated availability of ORS cells, their successful multiplication in culture irrespective of the age of the hair follicle donor as well as the extended tissue normalization of epidermal equivalents prepared with ORS cells prompted us to test the usefulness of autologous epidermal equivalents for the treatment of recalcitrant chronic wounds. Autologous grafting of such epidermal equivalents in more than 50 recalcitrant leg ulcers of a mainly vascular origin resulted in an initial take rate of around 90%, with subsequent complete closure of the ulcers in about 45% and a significant size reduction in another 40% within 8 weeks. These positive results are probably due to the large compartment of proliferative cells as well as to the well-developed horny layer, which prevents rapid disintegration of the grafts. Practical advantages of this technology are its noninvasiveness and thus repeated availability, the fact that surgical facilities are not necessary and the short immobilization period after grafting, allowing a strategy of sequential application in an outpatient setting as an alternative to surgical autografting.

Biotin-dependent carboxylase activities in different CNS and skin-derived cells, and their sensitivity to biotin-depletion.

The validity of various transformed and untransformed CNS and skin-derived cell cultures as a model for studying effects of biotin deficiency was tested. In biotin-sufficient conditions (0.1-10 mumol/L) all cell types showed considerable activities of the four biotin-dependent carboxylases. Notably, pyruvate carboxylase activity was also present in the different neuronal cells. One passage in low-biotin medium (6-130 pmol/L) lowered mitochondrial carboxylase activities in all cell types, but to varying degrees. Sensitivity to biotin depletion was greatest in three neuronal cell types, Roc-1 oligodendroglia, and three keratinocyte cell types (carboxylase activities decreased to 2-11% of maximal); intermediate in primary astrocytes and C6 glioma (decreased to 12-28%), and least in SAOS2 sarcoma and skin fibroblasts (decreased to 32-85%). Transformed and untransformed cell lines of the same cell type showed similar sensitivity. We conclude that cultures of different transformed CNS and keratinocyte cell types allow the study of effects of biotin deprivation. Carboxylase activities of neurons, oligodendroglia, and keratinocytes were much more sensitive to biotin depletion than fibroblasts. This may be an important factor in the pathogenesis of neurological and cutaneous abnormalities in congenital biotinidase deficiency where recycling of biotin is deficient.