Barrier functions of human skin: a holistic view.

The fascinating topic of skin barrier continues to engage researchers from diverse disciplines both in academia and industry. Much of the information on the basic biology of barrier formation, its ontogeny as well as repair and homeostasis comes from studies on animal models. A smaller number of human studies have validated the usefulness of animal models, while highlighting some essential differences. We submit that the human skin barrier is unique in several ways, as much due to our adaptive ability as our control over the environment (macro and micro) that none of the other species have exerted. The human skin is not only exposed to the greatest variations of environment due to our phenomenal mobility but also to the largest number of xenobiotics, both chemical and microbial, resulting from human activity. In this overview, we attempt to evaluate the interdependent relation of skin barriers to environmental stressors hoping to raise interest in some of the lesser known or neglected aspects of human skin barriers as they relate to skin health and dysfunctions.

Glucocorticoids accelerate fetal maturation of the epidermal permeability barrier in the rat.

The cutaneous permeability barrier to systemic water loss is mediated by hydrophobic lipids forming membrane bilayers within the intercellular domains of the stratum corneum (SC). The barrier emerges during day 20 of gestation in the fetal rat and is correlated with increasing SC thickness and increasing SC lipid content, the appearance of well-formed lamellar bodies in the epidermis, and the presence of lamellar unit structures throughout the SC. Because glucocorticoids accelerate lung lamellar body and surfactant maturation in man and experimental animals, these studies were undertaken to determine whether maternal glucocorticoid treatment accelerates maturation of the epidermal lamellar body secretory system. Maternal rats were injected with betamethasone or saline (control) on days 16-18, and pups were delivered prematurely on day 19. Whereas control pups exhibited immature barriers to transepidermal water loss (8.16 +/- 0.52 mg/cm2 per h), glucocorticoid-treated pups exhibited competent barriers (0.74 +/- 0.14 mg/cm2 per h; P < 0.001). Glucocorticoid treatment also: (a) accelerated maturation of lamellar body and SC membrane ultrastructure; (b) increased SC total lipid content twofold; and (c) increased cholesterol and polar ceramide content three- to sixfold. Thus, glucocorticoids accelerate the functional, morphological, and lipid biochemical maturation of the permeability barrier in the fetal rat.

Sphingolipids are required for mammalian epidermal barrier function. Inhibition of sphingolipid synthesis delays barrier recovery after acute perturbation.

Stratum corneum lipids comprise an approximately equimolar mixture of sphingolipids, cholesterol, and free fatty acids, arranged as intercellular membrane bilayers that are presumed to mediate the epidermal permeability barrier. Prior studies have shown that alterations in epidermal barrier function lead to a rapid increase in cholesterol and fatty acid synthesis which parallels the early stages of the repair process. Despite an abundance of indirect evidence for their role in the barrier, the importance of sphingolipids has yet to be demonstrated directly. Whereas sphingolipid synthesis also increases during barrier repair, this response is delayed in comparison to cholesterol and fatty acid synthesis (Holleran, W.M., et al. 1991. J. Lipid Res. 32:1151-1158). To further delineate the role of sphingolipids in barrier homeostasis, we assessed the impact of inhibition of sphingolipid synthesis on epidermal barrier recovery. A single topical application of beta-chloro-L-alanine (beta-CA), an irreversible inhibitor of serine-palmitoyl transferase (SPT), applied to acetone-treated skin of hairless mice resulted in: (a) greater than 75% inhibition of SPT activity at 30 min (P less than 0.001); (b) a global decrease in sphingolipid synthesis between 1 and 3 h (P less than 0.02); (c) reduction of epidermal sphingolipid content at 18 h (P less than 0.01); (d) delayed reaccumulation of histochemical staining for sphingolipids in the stratum corneum; and (e) reduced numbers and contents of lamellar bodies in the stratum granulosum. Finally, despite its immediate, marked diminution of sphingolipid synthesis, beta-CA slowed barrier recovery only at late time points (greater than 6 h) after acetone treatment. This inhibition was overridden by coapplications of ceramides (the distal SPT product), indicating that the delay in repair was not due to non-specific toxicity. These studies demonstrate a distinctive role for epidermal sphingolipids in permeability barrier homeostasis.

Processing of epidermal glucosylceramides is required for optimal mammalian cutaneous permeability barrier function.

The interstices of the mammalian stratum corneum contain lipids in a system of continuous membrane bilayers critical for the epidermal permeability barrier. During the transition from inner to outer stratum corneum, the content of polar lipids including glucosylceramides, decreases while ceramide content increases. We investigated whether inhibition of glucosylceramide hydrolysis would alter epidermal permeability barrier function. Daily topical applications of bromoconduritol B epoxide (BrCBE) to intact murine skin selectively inhibited beta-glucocerebrosidase, increased glucosylceramide content of stratum corneum with ceramide content remaining largely unchanged, and caused a progressive, reversible decrease in barrier function. Histochemistry of inhibitor-treated epidermis revealed persistence of periodic acid-Schiff-positive staining in stratum corneum cell membranes, consistent with retention of hexose moieties. Electron microscopy of inhibitor-treated samples revealed no evidence of toxicity or changes in the epidermal lipid delivery system. However, immature membrane structures persisted in the intercellular spaces throughout the stratum corneum, with reappearance of mature membrane structures progressing outward from the lower stratum corneum upon termination of BrCBE. Finally, the induced barrier abnormality was not reversed by coapplications of ceramide. These data demonstrate that glucosylceramide hydrolysis is important in the formation of the epidermal permeability barrier, and suggest that accumulation of glucosylceramides in stratum corneum intercellular membrane domains leads to abnormal barrier function.

Cholesterol synthesis is required for cutaneous barrier function in mice.

Previous studies have shown that topical acetone treatment results in the removal of stratum corneum lipids and disruption of the permeability barrier. This disruption stimulates epidermal lipid synthesis which is associated with the rapid restoration of stratum corneum lipids and barrier function. The aim of this study was to determine the role of cutaneous cholesterol synthesis in the barrier recovery. Here we show that topical lovastatin, a competitive inhibitor of HMG CoA reductase, inhibits cholesterol synthesis. After acetone disruption of the barrier, the normal rapid return of cholesterol to the stratum corneum and recovery of barrier function is impaired in animals treated topically with lovastatin. When lovastatin animals are simultaneously treated topically with either mevalonate, the immediate product of HMG CoA reductase, or cholesterol, the final end product of the pathway, the recovery of the barrier is normalized. Lovastatin resulted in the delayed secretion and abnormal appearance of lamellar bodies. These results provide the first evidence demonstrating that cholesterol synthesis is required for the maintenance of barrier structure and function and suggests a crucial role for cholesterol synthesis in allowing for terrestrial existence.

Calcium and potassium are important regulators of barrier homeostasis in murine epidermis.

Topical solvent treatment removes lipids from the stratum corneum leading to a marked increase in transepidermal water loss (TEWL). This disturbance stimulates a variety of metabolic changes in the epidermis leading to rapid repair of the barrier defect. Using an immersion system we explored the nature of the signal leading to barrier repair in intact mice. Initial experiments using hypotonic to hypertonic solutions showed that water transit per se was not the crucial signal. However, addition of calcium at concentrations as low as 0.01 mM inhibited barrier repair. Moreover, both verapamil and nifedipine, which block calcium transport into cells, prevented the calcium-induced inhibition of TEWL recovery. Additionally, trifluoroperazine or N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide, which inhibit calmodulin, prevented the calcium-induced inhibition of TEWL recovery. Although these results suggest an important role for calcium in barrier homeostasis, calcium alone was only modestly effective in inhibiting TEWL recovery. Potassium alone (10 mM) and phosphate alone (5 mM) also produced a modest inhibition of barrier repair. Together, however, calcium and potassium produced a synergistic inhibition of barrier repair (control 50% recovery vs. calcium + potassium 0-11% recovery in 2.5 h). Furthermore, in addition to inhibiting TEWL recovery, calcium and potassium also prevented the characteristic increase in 3-hydroxy-3-glutaryl CoA reductase activity that occurs after barrier disruption. Finally, the return of lipids to the stratum corneum was also blocked by calcium and potassium. These results demonstrate that the repair of the epidermal permeability barrier after solvent disruption can be prevented by calcium, potassium, and phosphate. The repair process may be signalled by a decrease in the concentrations of these ions in the upper epidermis resulting from increased water flux leading to passive loss of these ions.

Consequences of beta-glucocerebrosidase deficiency in epidermis. Ultrastructure and permeability barrier alterations in Gaucher disease.

Hydrolysis of glucosylceramide by beta-glucocerebrosidase results in ceramide, a critical component of the intercellular lamellae that mediate the epidermal permeability barrier. A subset of type 2 Gaucher patients displays ichthyosiform skin abnormalities, as do transgenic Gaucher mice homozygous for a null allele. To investigate the relationship between glucocerebrosidase deficiency and epidermal permeability barrier function, we compared the stratum corneum (SC) ultrastructure, lipid content, and barrier function of Gaucher mice to carrier and normal mice, and to hairless mice treated topically with bromoconduritol B epoxide (BrCBE), an irreversible inhibitor of glucocerebrosidase. Both Gaucher mice and BrCBE-treated mice revealed abnormal, incompletely processed, lamellar body-derived sheets throughout the SC interstices, while transgenic carrier mice displayed normal bilayers. The SC of a severely affected type 2 Gaucher's disease infant revealed similarly abnormal ultrastructure. Furthermore, the Gaucher mice demonstrated markedly elevated transepidermal water loss (4.2 +/- 0.6 vs < 0.10 g/m2 per h). The electron-dense tracer, colloidal lanthanum, percolated between the incompletely processed lamellar body-derived sheets in the SC interstices of Gaucher mice only, demonstrating altered permeability barrier function. Gaucher and BrCBE-treated mice showed < 1% and < 5% of normal epidermal glucocerebrosidase activity, respectively, and the epidermis/SC of Gaucher mice demonstrated elevated glucosylceramide (5- to 10-fold), with diminished ceramide content. Thus, the skin changes observed in Gaucher mice and infants may result from the formation of incompetent intercellular lamellar bilayers due to a decreased hydrolysis of glucosylceramide to ceramide. Glucocerebrosidase therefore appears necessary for the generation of membranes of sufficient functional competence for epidermal barrier function.

The effect of vitamin D status on cutaneous sterologenesis in vivo and in vitro.

Recent studies have shown that cutaneous sterologenesis is autonomous from the influence of circulating sterols, and that the epidermis is an important site of sterologenesis which is regulated by permeability barrier requirements. In addition to barrier function, an additional, important function of the epidermis is to synthesize sterol precursors of vitamin D3. The present study was designed, first, to determine whether vitamin D status and/or circulating levels of 1,25-dihydroxyvitamin D3 might play a role in regulating cutaneous sterol synthesis in vivo and, second, whether 1,25-dihydroxyvitamin D3 modulates sterologenesis in cultured human keratinocytes. Hairless mice were maintained on a vitamin D-deficient diet in the dark and supplemented with various doses of vitamin D3/day. Despite demonstrating serum 25-hydroxyvitamin D3 levels ranging from less than 10 to 343 ng/ml, the incorporation of tritiated water into cholesterol and total nonsaponifiable lipids in both the epidermis and dermis was similar in the four groups of animals. Likewise, administration of various doses of 1,25-dihydroxyvitamin D3 to vitamin D-deficient mice resulted in serum levels of 1,25-dihydroxyvitamin D3 ranging from less than 10 to 85 pg/ml; yet, cholesterol and total nonsaponifiable lipid synthesis was similar in both the dermis and epidermis in all groups of animals. Moreover, administration of 0.6 micrograms/kg per day of 1,25-dihydroxyvitamin D3 to 'normal' vitamin D-replete mice also had no effect on cutaneous sterol synthesis. Furthermore, conversion of 7-dehydrocholesterol to cholesterol in vitamin D-deficient vs. supplemented animals did not differ. Finally, addition of 1,25-dihydroxyvitamin D3 to cultured keratinocytes over a concentration range of 10(-12)-10(-7) M did not affect sterologenesis, except at supraphysiologic doses (10(-7) M). Together, these results suggest that vitamin D status does not influence sterol synthesis in the skin.

Cytochemical and biochemical localization of lipase and sphingomyelinase activity in mammalian epidermis.

Despite a wealth of new information on epidermal lipids and their role in permeability barrier function and desquamation, little is known about the location of the enzymes that regulate their catabolism. In this study we have localized lipase (triacylglycerol hydrolase) and sphingomyelinase in the outer epidermis simultaneously by cytochemical and cell fractionation techniques. Aldehyde-fixed tissues (100-microns slices) incubated in either Tween 85 or triolein plus taurocholate/calcium chloride-containing buffer, pH 7.2 or 4.5, were then exposed to lead to form insoluble soaps, and processed for electron microscopy. Simultaneously, cell homogenates and isolated lamellar body fractions were incubated with methylumbelliferyl oleate under similar conditions, with released, free methylumbelliferone serving as an index of lipase activity. On electron microscopy and cell fractionation, both lipase and sphingomyelinase were localized primarily to intercellular domains in the stratum corneum. In the stratum granulosum lipases were found, both ultrastructurally and biochemically, in lamellar bodies and ultrastructurally in both the perinuclear cistern and mitochondria. In summary, these studies: by demonstrating lipid-catabolic enzymes in the intercellular domains of the stratum corneum, lend further support to the 2-compartment model of the stratum corneum; provide new information about the location of lipid-catabolic enzymes in differentiating epidermis; and provide insights about how lipids are processed during permeability barrier formation and desquamation.

Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-capture cytochemistry.

Although calcium ions have been shown to regulate the differentiation of keratinocytes in vitro, the role of divalent cations in vivo is not known. Prior attempts to localize divalent cations in epithelial tissues have been impeded by a lack of specificity of ultrastructural techniques, as well as translocation of precipitates within tissues. The availability of an improved cytochemical method (oxalate-pyroantimonate technique) has facilitated more precise, reliable localization of calcium. When this technique (+/- 10 mM EGTA) was applied to neonatal mouse epidermis, Ca++-containing precipitates localized primarily within the cytosol, mitochondria, and nuclear chromatin of some basal and spinous cells, suggesting a possible relationship of Ca++ with the cell cycle. In the lower granular layer, progressively more Ca++ precipitates appeared intercellularly, with the only intracellular Ca++ localized within mitochondria and lamellar bodies (limiting membranes and discs). The most apical granular cells always demonstrated dense extracellular deposits, and high intracellular Ca++, free in the cytosol. The extruded contents of lamellar bodies, at the granular-cornified layer interface, also demonstrated significant amounts of Ca++-containing precipitates between the lamellar discs. Although some corneocytes in the lower stratum corneum demonstrated intracellular precipitates, most were deviod of Ca++. The striking intercellular Ca++ accumulation in the mid granular layer, coupled with Ca++ influx in the upper granular layer, supports the view that changes in intracellular Ca++ may regulate epidermal differentiation. Finally, the association of Ca++ with lamellar body disc membranes and contents suggests that divalent cations may contribute to both lamellar body secretion and to the formation of intercorneocyte membrane bilayers.

Membrane structural alterations in murine stratum corneum: relationship to the localization of polar lipids and phospholipases.

During the formation of the mammalian epidermal permeability barrier, lipids are sequestered in the stratum corneum intercellular spaces, transforming from a relatively polar lipid mixture to predominantly nonpolar species. Certain lipid catabolic enzymes, which co-localize with these lipids, may regulate this process. In order to localize the sites within the outer epidermis where polar lipids are catabolized, and their relationship to the alterations in membrane structure that occur in these layers, we compared the biochemical localization of polar lipids, the ultrastructure, and freeze-fracture morphology, as well as the localization of phospholipases within the outer epidermis. Both histochemical staining of frozen sections and biochemical studies of protease- and tape-stripped whole stratum corneum demonstrated small amounts of polar lipids in the stratum compactum, while in contrast, the stratum disjunctum was devoid of both phospholipids and glycosphingolipids. Phospholipase activity was present within lamellar bodies, among secreted lamellar body disks at the granular-cornified layer interface, and within the intercellular spaces of the stratum compactum. Both the depletion of polar lipids from the stratum compactum and deletion of these substances from the stratum disjunctum correlated with sequential changes in membrane structure observed by transmission electron microscopy and freeze-fracture. Thus, a phospholipase-mediated attack on phospholipids (with a parallel assault by other lipid catabolic enzymes on other polar species), may induce both the initial fusion and elongation of lamellar body disks and the subsequent formation of the hydrophobic membrane bilayers found in the mid-to-outer stratum corneum. These studies also may require modification of traditional views of the stratum corneum as a metabolically inert tissue, revealing its intercellular lipid domains to be partially in an active state of flux.

Lamellar body secretory response to barrier disruption.

Abundant evidence points to an important role for epidermal lamellar body secretion in permeability-barrier maintenance. However, the response of the lamellar body secretory system to barrier disruption has not been examined. Hence, we examined the lamellar body secretory response at various points after acetone-induced barrier abrogation in hairless mice in air-exposed animals and those occluded with impermeable versus vapor-permeable membranes. Tape-stripped animals served as a control for chemical toxicity. Barrier perturbation with either acetone or tape stripping was followed by rapid secretion of lamellar body contents from the uppermost granular cell layer, leaving the cytosol largely devoid of lamellar bodies. The newly secreted lamellar body contents comprised pleated sheets (not "discs," as previously thought), which unfurled in the intercellular spaces at the granular-cornified cell interface. At this time (15-30 min), the basic unit structure of the lamellar bilayers in the mid-to-upper stratum corneum appeared disorganized and interspersed with large lacunae, reflecting solvent extraction. Nascent lamellar bodies began to reappear in the granular cell cytosol by 30 min and by 360 min the cells displayed a full complement of normal-appearing lamellar bodies. Between 60 and 360 min, the density of lamellar body sheets at the granular-cornified cell interface increased, whereas the membrane bilayers of the outer stratum corneum remained disorganized. New lamellar bilayer units first appeared in the lower stratum corneum between 60 and 180 min, as a result of the transformation of secreted lamellar body sheets and over time these lamellae appeared at more apical locations. Occlusion with a water vapor-impermeable but not a vapor-permeable membrane resulted in a) decreased quantities of lamellar bodies and lamellar body-derived intercellular products; b) formation of lamellar bodies with abnormal internal contents; c) inhibition of lamellar body secretion; and d) inhibition of transformation of lamellar body-derived sheets into lamellar bilayer units. These results demonstrate the central role of the lamellar body-secretory system in barrier repair and homeostasis.

Selective obliteration of the epidermal calcium gradient leads to enhanced lamellar body secretion.

The epidermal permeability barrier is formed by lipids delivered to the intercellular spaces through the secretion of lamellar bodies. Prior studies have shown that the rate of lamellar body secretion appears to be regulated by the extracellular calcium content of the upper epidermis, which is altered following permeability barrier disruption. To determine directly whether changes in extracellular calcium content in the upper epidermis versus disruption of the barrier regulate lamellar body secretion, we experimentally manipulated the Ca++ content of the upper epidermis by sonophoresis of aqueous solutions containing physiologic Ca++ (and K+) versus ion-free solutions across hairless mouse stratum corneum. Sonophoresis at 15 MHz did not alter barrier function, but in the absence of Ca++ the extracellular calcium content of the outer epidermis, as revealed by ion capture cytochemistry, was displaced downward toward the basal layer and dermis. In contrast, following sonophoresis of Ca(++)-containing solutions, the extracellular Ca++ gradient became obscured by excess Ca++ in the cytosol at all levels of the epidermis. These changes in the extracellular calcium content lead, in turn, to accelerated lamellar body secretion (with low Ca++), or basal rates of lamellar body secretion (with normal Ca++). These results demonstrate that the epidermal extracellular calcium content in the upper epidermis can be manipulated by sonophoresis without prior barrier disruption, and that changes in the Ca++ gradient induce lamellar body secretion, independent of barrier disruption.

Structural basis for the barrier abnormality following inhibition of HMG CoA reductase in murine epidermis.

Recent studies have shown that increased epidermal 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase activity is crucial for the barrier recovery response that follows solvent-induced barrier perturbation. Upregulation of this enzyme leads to increased cholesterologenesis, formation and secretion of cholesterol-enriched lamellar bodies, and barrier repair. Topical lovastatin-induced inhibition of HMG CoA reductase activity both delays the acute barrier-repair response, as well as leading to a chronic barrier abnormality when applied repeatedly to intact skin. Presently, we assessed the effects of repeated topical applications of two different specific inhibitors of HMG CoA reductase on barrier function, the lamellar body-secretory system, and stratum corneum intercellular domains, with functional and morphologic parameters. Once-daily applications of lovastatin or fluindostatin (XU62-320; Sandoz) for 4-8 d to intact hairless mouse epidermis produced a progressive abnormality in barrier function (transepidermal water loss greater than 2.0-5.0 in treated versus less than 0.25 mg/cm2/h for weakly active analogues or vehicle controls). The barrier defect was preceded by alterations in lamellar body internal structure and a partial failure of lamellar body secretion into the stratum corneum interstices, further confirmed by enzyme cytochemistry. Moreover, the deposition of abnormal lamellar body contents resulted in the formation of clefts in the intercellular spaces at the stratum granulosum-stratum corneum interface, resulting in increased permeability through these domains shown by lanthanum perfusion. Applications of irritants, even when producing a barrier abnormality, did not alter the lamellar body secretory system. Co-applications of cholesterol with the inhibitors reversed both the barrier abnormality and the abnormalities in the lamellar body secretory system that occur with the inhibitor alone. Finally, membrane bilayer structures in the mid-to-outer stratum corneum of inhibitor-treated specimens appeared normal, but the intercellular domains displayed enormously expanded lacunae. However, because similar dilatations also occurred in vehicle-treated samples, they can be attributed to the vehicle alone. These studies provide further evidence that the inhibitor-induced defect in barrier function a) is initiated by inhibition of HMG CoA reductase; b) can be attributed to defects in both lamellar body structure and deposition with resultant abnormalities in intercellular membrane domains in the lower stratum corneum; and c) is further enhanced by permissive effects of the vehicle on the permeability of the outer stratum corneum.

The lovastatin-treated rodent: a new model of barrier disruption and epidermal hyperplasia.

Recent studies have linked epidermal cholesterol synthesis with maintenance of the permeability barrier. To assess directly the importance of cholesterol synthesis, we applied lovastatin, a potent inhibitor of cholesterol synthesis, to hairless mouse skin. Transepidermal water loss (TEWL) began to increase after four to six daily applications. Co-application of cholesterol blocked the expected increase in TEWL, demonstrating the importance of cholesterol for development of the lesion. The histology of lovastatin-treated skin revealed epidermal hyperplasia, accompanied by accelerated DNA synthesis. Whereas cholesterol synthesis initially was reduced in lovastatin-treated epidermis, with further treatment cholesterol synthesis normalized, while fatty acid synthesis accelerated greatly. Although the total free sterol content of lovastatin-treated epidermis remained normal, the fatty acid content increased coincident with barrier disruption. Finally, morphologic abnormalities of both lamellar body structure and their deposited, intercellular contents occurred coincident with the emerging biochemical abnormalities. Thus, the abnormal barrier function in this model can be ascribed to an initial inhibition of epidermal sterol synthesis followed by an alteration in cholesterol and fatty acid synthesis, leading to an imbalance in stratum corneum lipid composition and abnormal membrane bilayer structure.

Membrane structures in normal and essential fatty acid-deficient stratum corneum: characterization by ruthenium tetroxide staining and x-ray diffraction.

Despite the importance of intercellular lamellar bilayers for stratum corneum (SC) barrier function, knowledge about the structure of these bilayers is limited due to their poor visualization and/or retention. Whereas substitution of ruthenium tetroxide (RuO4) for osmium tetroxide fixation provides clear images of these bilayers, the usefulness of RuO4 has been limited by its slow penetration and cytotoxicity. Utilizing a new fixation protocol for RuO4, we obtained clear images of lamellar domains at all levels of murine SC. Computer-aided image reconstructions demonstrated a lamellar spacing of 129 +/- 2 A, which agreed with x-ray diffraction data from parallel, unfixed samples (131 +/- 2 A), a spacing not affected by hydration. Furthermore, novel structures were seen in the intercellular spaces of normal SC. Finally, in murine essential fatty acid deficiency (EFAD), the overall lamellar spacing is comparable to normal [127 +/- 7 A by computer transform vs. 131.9 +/- 2 A (hydrated) and 129.6 +/- 2.2 A (dry) by x-ray diffraction]. Yet, these domains are structurally abnormal, displaying regions with either an excess or absence of lamellae. The new RuO4 protocol provides quantitative information about SC lamellar dimensions and morphologic abnormalities in bilayer distribution and substructure in EFAD stratum corneum that are not detected by either x-ray diffraction or computer-aided image reconstruction. Thus, the barrier abnormality in EFAD stratum corneum can be ascribed either to focal depletion of lamellae or abnormalities in lamellar substructure.

Pattern recognition. Classification of therapeutic agents according to pharmacophores.

One of the preprocessing methods used in pattern recognition-factor analysis-is shown to be well suited to the derivation of structure-activity relationships. Applications of the procedure developed are illustrated using sets of compounds which are of accepted therapeutic utility.

Ultrastructural localization of calcium in psoriatic and normal human epidermis.

With ion capture cytochemistry, we previously demonstrated the distribution of calcium ions in murine epidermis, a pattern consistent with a role for this ion in the regulation of epidermal differentiation. Because of the known proliferation and differentiation defects in psoriasis, we compared the calcium distribution of involved vs uninvolved psoriatic lesions and normal human epidermis. Whereas normal human and uninvolved psoriatic epidermis revealed increased calcium-containing precipitates in the uppermost stratum granulosum, in contrast the basal layer of psoriatic lesions contained less extracellular calcium, a condition that favored enhanced proliferation. Moreover, all psoriatic suprabasal cell layers displayed heavier than normal concentrations of calcium, indicating loss of the normal calcium gradient that programs terminal differentiation. This abnormal profile may account for the differentiation defects (eg, parakeratosis) that occur in psoriasis. Finally, psoriatic lesions displayed retained ionic Ca in intercellular domains of the upper stratum granulosum with absence of normal intercellular bilayers, findings that may underlie the abnormal desquamation and permeability barrier in psoriasis.

Mode of action of glycolic acid on human stratum corneum: ultrastructural and functional evaluation of the epidermal barrier.

Alpha-hydroxy acids (AHA) such as glycolic acid have recently been used extensively in cosmetic and dermatological formulas. In low concentration (2-5%) glycolic acid is believed to facilitate progressive weakening of cohesion of the intercellular material of the stratum corneum (SC), resulting in uniform exfoliation of its outermost layers (the stratum disjunctum). Since thinning of the SC as well as changes of intercellular lipids could theoretically compromise the barrier functions of the skin, we investigated the mode of AHA action on the SC to determine whether enhanced desquamation compromises the barrier structures of the SC and changes transepidermal water loss (TEWL) values. Electron microscopy of the epidermis biopsied from the volar forearm of human volunteers after 3 weeks of treatment with a 4% glycolic acid formulation twice daily was employed to evaluate 1) epidermal morphology and thickness of the SC, (2) the lamellar body and SC lipid bilayer organization, and (3) desquamative events based on degradation of desmosomes. TEWL values and SC hydration were recorded prior to and at the end of the study. Electron microscopy revealed no ultrastructural changes in the nucleated layers of the epidermis. The lamellar body (LB) secretory system in the stratum granulosum (SG), and intercellular lipid lamellae in the SC in both vehicle- and glycolic acid-treated samples were comparable to normal human SC. Within the SC, enhanced desmosomal breakdown, promoting loss of cohesion and desquamation, was restricted to the stratum disjunctum while desmosomes of the stratum compactum were unaffected. Treated areas displayed histologically, a more compact appearing SC. TEWL values remained unchanged in glycolic acid- and vehicle-treated skin. Our findings indicate that the barrier structures of the SC are not disrupted by glycolic acid formulations at the concentration used. One of the mechanism of action of AHA on the SC seemed to be a "targeted" desmosomal (corneosomal) action without compromising the barrier structures of the skin.

Ultrastructural organization of avian stratum corneum lipids as the basis for facultative cutaneous waterproofing.

The ultrastructure of naked neck epidermis from the ostrich (Struthio camelus) and ventral apterium from watered, and water-deprived, Zebra finches (Taeniopygia [Poephila] guttata castanotis) is presented. The form and distribution of the fully differentiated products of the lipid-enriched multigranular bodies are compared in biopsies post-fixed with osmium tetroxide or ruthenium tetroxide. The fine structure of ostrich epidermis suggests it is a relatively poor barrier to cutaneous water loss (CWL). The fine structure from watered, and 16-hr water-deprived Zebra finches, considered in conjunction with measurements of CWL, confirms previous reports of "facultative waterproofing," and emphasizes the rapidity of tissue response to dehydration. The seemingly counterintuitive facts that one xerophilic avian species, the ostrich, lacks a "good barrier" to CWL, whereas another, the Zebra finch, is capable of forming a good barrier, but does not always express this capability, are discussed. An explanation of these data in comparison to mammals centers on the dual roles of the integument of homeotherms in thermoregulation and conserving body water. It is concluded that birds, whose homeothermic control depends so much on CWL, cannot possess a permanent "good barrier," as such would compromise the heat loss mechanism. Facultative waterproofing (also documented in lizards) protects the organism against sudden reductions in water availability. In birds, and probably in snakes and lizards, facultative waterproofing involves qualitative changes in epidermal cell differentiation. Possible control mechanisms are discussed.