Search

Because outer root sheath (ORS) cells are valuable substitutes for interfollicular epidermal keratinocytes, we wanted to determine whether epidermal equivalents generated from ORS cells and containing cultured melanocytes can serve as an in vitro model for skin pigmentation. In such epidermal equivalents prepared with ORS cells and melanocytes from donors of phototypes II, III and VI, a stratified epithelium resembling normal epidermis developed within 14 days, as documented by histological, ultrastructural (e.g. basement membrane-like structure, keratohyalin granules, keratinosomes) and immunohistochemical (e.g. keratins, integrins, gp80, involucrin, filaggrin) criteria. The melanocytes were localized in the basal layer and accounted for 10% of the total cell number. Heavily pigmented melanocytes from black donors contained regular melanosomes in all stages of maturation, whereas melanocytes derived from white donors contained predominantly melanosomes of stages I and II. Melanosome-laden dendrites were readily detected extending from the heavily pigmented melanocytes, while they were less conspicuous in melanocytes from white donors. The extent of melanosome transfer was independent of the racial origin of the ORS cells. Melanosomes could also be transferred "through racial barriers". Melanosomes, mainly of stages III and IV, were detected in the ORS cells, being distributed either as single or compound melanosomes, again irrespective of the racial origin of the ORS cells. In conclusion, pigmented epidermal equivalents generated from ORS cells offer practical advantages over other in vitro pigmentation models: (1) the ORS cells are easily and repeatedly available from any donor regardless of age; (2) primary cultures of ORS cells are free of contaminating melanocytes, a bias if using interfollicular epidermal keratinocytes; (3) a high degree of epidermal differentiation is maintained for 3 weeks in fully defined medium, enabling labelling and stimulation experiments to be performed and compounds interfering with melanin pigmentation to be tested.

Depending on environmental influences, follicular outer root sheath (ORS) cells in vivo can differentiate either towards interfollicular keratinocytes or, as demonstrated in the rat vibrissa, hair matrix cells. Crucial regulators of both their proliferation and differentiation are the mesenchymal cells of the respective tissues. The interactions of human ORS cells with human hair papilla cells (HPC) or human dermal fibroblasts (HDF) were studied using a two-chamber model separating the two cell types either by a microporous membrane or additionally by a medium layer. The results of 3H-thymidine incorporation studies indicated that ORS cell growth was markedly enhanced in co-culture with either HPC or HDF, the highest stimulatory effect resulting when ORS cells were in close association with the mesenchymal cells. No correlation was found between ORS cell proliferation and IL-6 production in the co-culture system, thus pointing to the secretion by HPC and HDF of growth-promoting soluble factors that are different form IL-6 as well as from EGF, bFGF and insulin present in the culture medium.

By means of microsurgery a peripheral subcutaneous lymph vessel draining a defined skin area was isolated and cannulated on the lower leg of six healthy volunteers. Lymph was collected over a period of 8 days. During the first 2 days baseline values for lymph flow and output of cells were established. A contact dermatitis was then induced in the drained skin area by the application of 10% sodium lauryl sulphate. All six probands developed a mild to moderate irritant contact dermatitis. Lymph flow as well as output of cells increased with the intensity of the skin reaction. Subsequent local treatment with clobetasol propionate decreased the cell output, but the lymph flow increased further. Neither lymph flow nor output of cells returned to the initial baseline values at the end of the study, when the clinical signs of contact dermatitis had completely disappeared. During the experiment significant individual variations were found, with means ranging from 0.10 to 0.48 ml/h for lymph flow and from 8700 to 174000/h for cells, which probably depended mainly on the different topographies and calibres of the cannulated lymph vessels.

In humans and in animals, biotin deficiency causes pathological changes in the skin and its appendages. High doses of biotin may also have beneficial effects on skin, hair and fingernails in humans and animals with normal biotin status. Therefore, we investigated the effects of low and high concentrations of biotin on proliferation and differentiation of cultured outer root sheath cells from human hair follicles as an in vitro model for skin. The activities of biotin-dependent carboxylases were measured to evaluate the biotin status of the cells. In monolayer cultures of outer root sheath cells, proliferation and expression of the differentiation-specific keratins K1 and K10 were not influenced by extremely low concentrations of biotin (

Using microsurgery a superficial peripheral lymph vessel draining the skin of the upper and medial part of the foot was cannulated on the lower leg of two healthy human volunteers. An irritant contact dermatitis was induced 2 days later by the application of 10% sodium lauryl sulphate to the drained skin area. After a further 3 days the spontaneously regressing skin reaction was treated with clobetasol propionate. Lymph was continuously collected in two aliquots per day for 7 days. The levels of total protein, of albumin and globulins, and of complement components of the classical, the alternative and the lytic pathway as well as the C4A and C4B gene products and the regulatory proteins FB, C1INH, C4BP, FH and FI were determined by ELISA and radial immunodiffusion techniques. Postoperatively, the levels of complement proteins and globulins in the lymph were 5-10 times lower than those in normal human serum, but increased during the course of the skin reaction, while the irritant contact dermatitis did not induce a change in their plasma concentration. In comparison to the baseline, the mean values for C1q, C1r, C2, C5, C6, C7, C8, C9, FB, C1INH, C4BP, FH and FI exhibited a 3-5-fold increase, C3, total C4, albumin and the alpha 1-globulin fraction a 6-9-fold increase, and C1s, C4A, C4B, FB and alpha 2-, beta- and gamma-globulins a 10-20-fold increase.(ABSTRACT TRUNCATED AT 250 WORDS)

In the human hair follicle, outer root sheath (ORS) cells constitutively express the hyperproliferation-associated keratins 6, 16 and 17 instead of keratins 1 and 10 found in interfollicular epidermis. In organotypic cultures. ORS cells form a stratified epithelium which in many respects resembles psoriatic skin: it has a hyperplastic tissue architecture and a poorly developed granular layer, and expresses hyperproliferation-associated keratins. Therefore, we studied the effects of the antipsoriatic compounds 1 alpha,25-dihydroxy-vitamin D3 (1 alpha,25-(OH)2-D3) and its synthetic derivative calcipotriol on cultured ORS cells. In monolayer cultures, 10(-6) M 1 alpha,25-(OH)2-D3 or calcipotriol completely blocked ORS cell proliferation. This inhibitory effect was substantially reduced at 10(-8) M. Incubation of organotypic ORS cultures with both vitamin D analogues resulted in a marked thinning of the living cell compartment concomitant with a thickening of the horny layer. A reduced expression of differentiation markers such as keratins 10, 16 and 17, involucrin and filaggrin paralleled the thinning of the stratum Malpighi. As determined by quantification of BrdU-positive cells, ORS cell proliferation was apparently not affected by the vitamin D analogues, indicating that these compounds mainly operate by accelerating the differentiation pathway within the suprabasal living cell compartment. No alteration in the expression of the alpha 6- and beta 1-integrin chains was found.

Addition of human plasminogen to three different pemphigus plasma samples showed a synergistic effect on acantholysis in the skin organ culture model. Human plasmin itself, without addition of pemphigus plasma, induced typical acantholytic changes in the skin explants, causing different types of acantholysis in a dose- and time-dependent manner: in the presence of 3 CU plasmin per ml culture medium, focal suprabasilar acantholysis of pemphigus vulgaris type could be detected after 72 h incubation, whereas 15 CU/ml caused extended acantholysis of pemphigus foliaceus type in the upper epidermal layers after 24 h, and extended acantholysis of benign chronic pemphigus (Hailey-Hailey disease) type comprising all layers of the epidermis after 48 h incubation. Plasminogen activator levels (Mr 55,000 urokinase type) in tissue extracts of skin explants and in culture media were reduced after 24 and 48 h incubation with pemphigus IgG as compared to control experiments with normal human IgG; this probably resulted from urokinase inactivation by reaction with inhibitors. These results lend support to the hypothesis proposed by Hashimoto et al. in 1983 that the plasminogen activator-plasmin system could play an essential role in the protease mechanisms of pemphigus acantholysis.

A patient suffering from severe pemphigus vulgaris was treated using large-volume plasma exchange in combination with an immunosuppressive regimen. As some recent reports have shown evidence that polyclonal, polyspecific human IgG in high doses through the i.v. route (IGIV) protect target platelets in idiopathic thrombocytopenic purpura from attack by antiplatelet autoantibodies and/or immune complexes, we also administered IGIV to this pemphigus-vulgaris patient. In order to test the hypothesis that IGIV might protect in vitro-cultured human skin from acantholysis induced by pemphigus antibodies, studies with skin organ cultures were carried out using plasma from another pemphigus-vulgaris patient who had undergone plasma exchange. The preincubation of either the skin explants or the pemphigus plasma with various concentrations of IGIV (ranging from 0.15 to 15 mg/ml in the culture medium) did not prevent acantholysis induced by the pemphigus plasma nor did it inhibit the binding of the specific antibodies visualized by direct immunofluorescence. Thus, the assumption that IGIV may coat the pemphigus antigens on epidermal cells making them inaccessible to pathogenic autoantibodies was not substantiated by our tests in vitro; likewise, the hypothesis of functionally blocking autoantibody activity by means of anti-idiotype effects of IGIV cannot be supported.