Nestin+ progenitor cells isolated from adult human sweat gland stroma promote reepithelialisation and may stimulate angiogenesis in wounded human skin ex vivo.

The combination of an aging population and an increasing prevalence of diseases associated with impaired-wound healing, including obesity, peripheral vascular disease and diabetes, is likely to result in a dramatic increase in the incidence and prevalence of chronic skin wounds. Indeed, systemic reviews are now not only trying to establish both the prevalence and the often under-estimated socio-economic costs of chronic skin wounds, but most importantly are addressing the impact that chronic wounds have on quality of life. Given the clear need for novel approaches to the management of chronic skin ulceration, ideally developed and tested in the human system in a manner that can be rapidly translated into clinical practice, we examined the effects of multipotent primary human nestin+ progenitor cells on human wound healing in an ex vivo model. Human sweat gland-derived nestin+ cells demonstrated the capacity to significantly promote two key wound healing parameters, i.e., both reepithelialisation and angiogenesis in experimentally wounded, organ-cultured human skin. The current data further support the use of full-thickness human skin wound-healing models ex vivo to pre-clinically test wound healing-promoting candidate agents. Whilst larger studies are required to substantiate a firm "proof-of-concept," our preliminary studies encourage further efforts to systemically determine the potential of cell-based regenerative medicine strategies in general, and the use of skin appendage-associated human nestin+ cells in particular, as novel treatment strategies for chronic skin ulceration.

[The rationale for the application of laserophoresis of biologically active compounds].

Laserophoresis is a technique for the transcutaneous administration of biologically active compounds by means of low-intensity laser radiation (LFLR). It is currently regarded as a most promising method for the integrated application of a pharmaceutical substance and a physical factor. At present laserophoresis of various medicinal preparations is successfully used after preliminary experimental studies of their phoretic properties for the treatment of various inflammatory and dystrophic conditions as well as for the prevention of skin ageing. The most important route for the administration of the majority of drug preparations is through the shunts provided by perspiratory glands and hair follicles. Another essential factor determining the potential possibility of drug penetration through the skin is the characteristic of the substance chosen for the administration, such as its molecular weight, chemical structure, conformation, and hydrophilic properties. However, the most likely mechanism underlying the transport of the substance through the glandular cells of perspiratory glands and epithelial cells of hair follicles is pinocytosis, i.e. the process integrating exocytosis and endocytosis. To-day, the majority of the researchers lay emphasis on thermodynamic triggering of Ca2+-dependent processes as the primary mechanism behind the biological action of low-intensity laser radiation. Both exocytosis and endocytosis being the Ca2+-dependent processes, the liberation of Ca2+-ions under the influence of LFLR causes the activation of pinocytosis as a whole.

Effects of UTP on Na+, Cl- and K+ transport in primary cultures from human sweat gland coils.

Extracellular ATP and UTP can increase membrane permeability in the sweat gland, but the intracellular signalling regulating the response to these agonists is poorly understood. Stimulation of Cl- transport by nucleotides has been suggested as a pharmacological therapy to improve Cl- secretion in patients with cystic fibrosis. In the present study, regulation of Na+, Cl- and K+ transport in primary cultures of cells from the secretory coil of human sweat glands was investigated by electron probe X-ray microanalysis. Stimulation with 200 microM UTP for 2 min at room temperature caused a significant increase in intracellular Na but did not affect Cl and K. After 5 min, the Na concentration was still increased, but now also a significant decrease in Cl and K was observed, indicating an increase in Cl- and K+ permeability. The effect of UTP on Cl- secretion was enhanced in Mg2+-deficient buffer, indicating that the response is elicited by the extracellular fully ionized form of UTP (UTP4+), but not by MgUTP2+. The effects of UTP were abolished in Ca2+-deficient buffer supplemented with EGTA. Alloxan, an adenylate cyclase inhibitor, did not inhibit the response to UTP. These results indicate that the membrane Cl- and K+ permeability elicited by UTP in primary coil cell cultures is Ca2+-dependent. The response to UTP did not attenuate at 8 degrees C, suggesting that it could be activated, in part, via ligand-gated ion channels. The effect of UTP was not decreased in the presence of ouabain. Pre-treatment of the cells with pertussis toxin (24 h) had minor effects on Cl- secretion activated by UTP, indicating a role for G proteins in the UTP activation of Cl- secretion.

Cation transport by sweat ducts in primary culture. Ionic mechanism of cholinergically evoked current oscillations.

1. The coiled reabsorptive segment of human sweat ducts was cultured in vitro. Cells were then harvested and plated onto a dialysis membrane which was glued over a hole in a small disc. Cultures were maintained in a low serum, hormone-supplemented medium that allowed the cells to grow to confluency. The disc was then placed as a partition between two compartments of a miniature Ussing chamber. The chamber was mounted on the stage of an inverted microscope and intracellular potentials were recorded under transepithelial open-circuit or voltage clamp conditions. All values are given as means +/- S.E.M. and n refers to the number of preparations or duct cells. 2. Under control conditions, the cultured epithelia developed mucosa-negative transepithelial potentials (Vt) ranging from -2.5 to -38 mV (-13.5 +/- 1.5 mV, n = 36). The basolateral membrane potential (Vb) was -39.4 +/- 0.7 mV (n = 50 cells), and the apical membrane potential (Va) was linearly correlated with Vt:Va = 1.0 Vt -39.3 mV (r = -0.78, n = 50). 3. The epithelium generated inwardly directed short-circuit currents (Isc) of 12-95 microA cm-2 (45 +/- 4 microA cm-2, n = 36) with a steady-state intracellular potential. Vc = -31.1 +/- 0.6 mV and a fractional resistance of the apical membrane, fR = 0.59 +/- 0.01 (n = 115 cells). 4. The Na+ channel blocker amiloride (mucosal bath, 10 microM) abolished Isc -0.8 +/- 0.6 microA cm-2), the cells hyperpolarized to -61.0 +/- 1.2 mV, and fR increased to 0.85 +/- 0.01 (n = 44). These effects were fully reversible. 5. During initial stimulation with the cholinergic agonist, methacholine (serosa, 5 or 10 microM), the short-circuit current increased to 80 +/- 10 microA cm-2, the cells hyperpolarized to -55.8 +/- 1.2 mV, and fR increased to 0.82 +/- 0.01 (n = 35). 6. In short-circuited preparations stimulated with methacholine an increase in mucosal potassium concentration ([K+]m) from 5 to 25 mM had no significant effect, while a similar increase in the serosal K+ concentration ([K+]s) produced a change in Vc of 44 +/- 3 mV per log10[K+]s (n = 9). In non-stimulated preparations this change was only 16 +/- 2 mV per log10[K+]s (n = 13). After blocking the apical Na+ channels with amiloride the slope was 24 +/- 5 mV per log10[K+]s in unstimulated preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

Human sweat duct cells in primary culture. Basic bioelectric properties of cultures derived from normals and patients with cystic fibrosis.

Human sweat duct cells from the coiled reabsorptive segment have been cultured successfully, free from fibroblasts, in a low serum, hormone-supplemented medium, Ham's F12. The cultured cells exhibited a typical epithelial cobblestone pattern, and microvilli-covered luminal cells were seen joined together with typical junctional complexes. In cultures derived from normals and patients with cystic fibrosis (CF), growth and morphologic characteristics were indistinguishable. When grown on a membranous support, and mounted in an Ussing chamber, vectorial electroconductive ion-transport could be identified. The epithelial preparations produced active mucosa to serosa-directed sodium flux via amiloride-sensitive, apical sodium channels and ouabain-sensitive sodium pumps located in the basolateral membrane, which also contained a potassium shunt. These findings are consistent with a polarized epithelium with properties similar to the intact organ. High transepithelial resistance and increased amiloride sensitivity were typical for cells derived from CF, indicating that principal normal as well as pathologic properties of the sweat duct are preserved in culture.

Electrophysiological studies on isolated human eccrine sweat glands.

Human eccrine sweat glands were isolated by shearing and the potential differences across the basolateral membranes determined using bevelled micro-electrodes filled with 4 M potassium acetate. Stable resting potentials of up to -81 mV were recorded. Alterations in external potassium concentration from 1.2 to 100 mM caused the membrane potential to change over a 70 mV range in cells of high resting potential, indicating that the basolateral membrane is largely potassium permeable. Input impedance was determined by constant current injection and found to be in the range 4-80 M omega. On giving a bolus injection of acetylcholine to produce a final concentration of 10(-6)-10(-7) M, four types of response were observed: depolarization, in a proportion of cells with resting potentials of -66 to -80 mV (n = 19), hyperpolarization, in a group of cells with resting potentials of -47 to -70 mV (n = 22), no change, in some cells of -40 to -81 mV resting potential (n = 22) and micro-electrode dislodgement (n = 8). In cells depolarizing to acetylcholine, the depolarization was short-lived and in thirteen cases was followed by a 'rebound' hyperpolarization. Input impedance decreased during depolarization in one-third (n = 5) of the cells in which satisfactory measurement could be made and increased during the final phase of depolarization or during rebound hyperpolarization. In cells hyperpolarizing to acetylcholine, the hyperpolarization was usually accompanied by an increase in input impedance. In ten of the twenty-two cells which showed no change to a first dose of acetylcholine, the agonist was administered at least two more times. In two cells (resting potentials -62 mV, -64 mV) a hyperpolarization was observed whereas in three others (resting potentials -66 mV, -70 mV, -81 mV) depolarization occurred. The effects of acetylcholine, whether depolarizing or hyperpolarizing, were reversibly inhibited by atropine and irreversibly reduced by ouabain. Experiments performed on glands maintained for up to 30 h in supplemented RPMI 1640 tissue culture medium yielded essentially similar results to those performed on freshly isolated glands.

Sweating and its disorders.

Eccrine sweat is produced by millions of miniscule glands buried in the skin. Eccrine sweating from the general body surface is an extremely important function in human thermoregulation; disturbances either in the control of sweating activity or in the glands themselves can result in problems ranging from minor social embarrassment to fatal hyperpyrexia. A general review of the function and control of normal sweating precedes an overview of sweating abnormalities.