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The pH gradient over the stratum corneum differs in X-linked recessive and autosomal dominant ichthyosis: a clue to the molecular origin of the "acid skin mantle"?

In a search for pathogenetic mechanisms underlying retention hyperkeratosis, we examined the pH gradient over the stratum corneum in 13 male patients suffering from either x-linked recessive (XRI) or autosomal dominant ichthyosis vulgaris. For recording pH values, a flat glass electrode was repeatedly applied to the skin during tape stripping of mildly involved forearm skin. Before stripping, surface pH was higher in ichthyosis vulgaris (5.3 +/- 0.7; n = 7) than in XRI (4.6 +/- 0.4; n = 6; p < 0.05) and healthy control men (4.5 +/- 0.2; n = 7; p < 0.01). Removal of stratum corneum, which required 100-240 strippings in ichthyotic skin and 80-120 strippings in healthy control skin, disclosed markedly different pH variations in the two types of ichthyosis. The major abnormality in ichthyosis vulgaris skin was that a neutral pH was attained already halfway through the horny layer, possibly reflecting a congenital lack of acidic breakdown products from keratohyaline. By contrast, stripping of XRI skin revealed a shallow pH gradient that plateaued at 6.2-6.6, instead of about 7 as in normal and ichthyosis vulgaris skin. A likely explanation is the XRI-associated accumulation of cholesterol sulfate in lower stratum corneum. Our results suggest that the "acid mantle" of normal skin, which penetrates deep into the stratum corneum, is the combined result of cornification-associated organic acids and back-diffusion of acid material from the surface. Because corneocyte desquamation involves many pH-dependent enzymes, abnormalities in the transcorneal pH gradient might play a role in the pathogenesis of ichthyosis.

Epidermis↗

Water disrupts stratum corneum lipid lamellae: damage is similar to surfactants.

Using electron microscopy, we investigated the effect of (i) a dilute surfactant and of water alone on the ultrastructure of stratum corneum lipids in pig skin exposed in vitro at 46 degrees C, and (ii) of water alone on human skin exposed in vivo at ambient temperature. For pig skin, the surfactant sodium dodecyl sulfate disrupts stratum corneum intercellular lamellar bilayers, leading to bilayer delamination and "roll-up" in a water milieu after 1 h, extensive bilayer disruption after 6 h, and nearly complete dissociation of corneocytes after 24 h. Corneodesmosomes show progressive degradation with exposure time. Water alone also disrupts the stratum corneum, but with a slower onset. Alterations in intercellular lamellar bilayers, but not intercellular lamellar bilayer roll-up, are detected after 2 h. Intercellular lamellar bilayer roll-up occurs after 6 h. Extensive dissociation of corneocytes occurs after 24 h of water exposure. Unlike sodium dodecyl sulfate, water exposure results in the formation of amorphous intercellular lipid. Corneodesmosome degradation parallels intercellular lamellar bilayer disruption; calcium appears to offer some protection. Similar disruption of intercellular lamellar bilayers occurs in human skin in vivo at ambient temperature. Our studies show that water can directly disrupt the barrier lipids and are consistent with surfactant-induced intercellular lamellar bilayer disruption being due at least in part to the deleterious action of water. Intercellular lamellar bilayer disruption by water would be expected to enhance permeability and susceptibility to irritants; accordingly, increased attention should be given to the potential dangers of prolonged water contact. For common in vitro procedures, such as skin permeation studies or isolation of stratum corneum sheets, exposure to water should also be minimized.

Animals↗

Defective stratum corneum and early neonatal death in mice lacking the gene for transglutaminase 1 (keratinocyte transglutaminase).

The stratum corneum of the skin serves as an effective barrier for maintenance of the internal milieu against the external environment. At the cell periphery of the stratum corneum is the cell envelope, a highly insoluble membranous structure composed of precursor proteins cross-linked by epsilon-(gamma-glutamyl)lysine bonds. Transglutaminase 1 (TGase 1; keratinocyte TGase), a membrane-bound isozyme of the TGase family, has been proposed to catalyze this process of assembly. Deficient cross-linking of the cell envelope in some patients with the autosomal recessive skin disorder lamellar ichthyosis (LI) and several mutations of the TGase 1 gene that have been identified in families with LI suggest the importance of this gene in production of the cell envelope. In this study, we generated mice lacking the TGase 1 gene, and we report that they have erythrodermic skin with abnormal keratinization. In their stratum corneum, degradation of nuclei and keratohyalin F-granules was incomplete and cell envelope assembly was defective. The skin barrier function of TGase 1-null mice was markedly impaired, and these mice died within 4-5 h after birth. These results clearly demonstrate that the TGase 1 gene is essential to the development and maturation of the stratum corneum and to adaptation to the environment after birth. Thus, these TGase 1 knockout mice may be a useful model for severe cases of LI.

Absorption↗

Very intense pulse in the groundwater flow in fissurized-porous stratum.

An asymptotic solution is obtained corresponding to a very intense pulse: a sudden strong increase and fast subsequent decrease of the water level at the boundary of semi-infinite fissurized-porous stratum. This flow is of practical interest: it gives a model of a groundwater flow after a high water period or after a failure of a dam around a collector of liquid waste. It is demonstrated that the fissures have a dramatic influence on the groundwater flow, increasing the penetration depth and speed of fluid penetration into the stratum. A characteristic property of the flow in fissurized-porous stratum is the rapid breakthrough of the fluid at the first stage deeply into the stratum via a system of cracks, feeding of porous blocks by the fluid in cracks, and at a later stage feeding of advancing fluid flow in fissures by the fluid, accumulated in porous blocks.

Journal Article↗

Antileukoprotease inhibits stratum corneum chymotryptic enzyme. Evidence for a regulative function in desquamation.

The stratum corneum chymotryptic enzyme (SCCE) has been previously purified from human stratum corneum and resembles a chymotryptic serine endopeptidase involved in physiological detachment of corneocytes from human stratum corneum. From human stratum corneum two inhibitory activities of SCCE could be extracted. These were due to serine protease inhibitors already known to be present in human epidermis, antileukoprotease (secretory leukocyte protease inhibitor) and elafin (skin-derived antileukoprotease). The Inhibition of SCCE by antileukoprotease shows a hyperbolic, mixed type inhibition with an equilibrium dissociation constant of 63 n. Antileukoprotease also inhibits detachment of corneocytes from human plantar callus in vitro almost completely (>96%). In addition, elafin was shown to be a weak inhibitor for SCCE activity, and elafin significantly reduces the shedding of corneocytes. Thus, antileukoprotease, which is known to be produced by human keratinocytes, is likely to be the major physiological inhibitor of SCCE in the epidermis. It seems to be involved in the regulation of desquamation under physiological and pathophysiological conditions.

Aprotinin↗

The effect on the stratum-specific odds ratios of nondifferential misclassification of a dichotomous covariate.

Precise measurement of confounding or effect modifying variables is seldom possible. Nondifferential misclassification of such a variable can introduce bias in the odds ratios within the strata of the confounding variable. For two strata, the effect on the odds ratios is dependent on separate measures of the degree of nondifferential misclassification of the confounding variable. The authors investigate the bias in the stratum-specific odds ratios from nondifferential misclassification of a confounder in a case-control study, and show how estimates of the true stratum-specific odds ratios can be derived. For one stratum, the degree of bias introduced in the odds ratio is dependent only on the sensitivity of the measure of the confounding variable while only the specificity of this measure determines the bias introduced in the other stratum-specific odds ratio. The results are applied to a case-control study of fenoterol prescription and asthma death.

Bias↗

Lipid thermotropic transitions in human stratum corneum.

The techniques of differential scanning calorimetry (DSC) and thermal perturbation infrared (IR) spectrometry were used to investigate thermal transitions in intact, fractionated, and lipid-extracted human stratum corneum. The DSC results show 3 major and one minor thermal transition in the range of 30-120 degrees C. Of particular interest to this study are 2 transitions seen near 65 degrees and 75 degrees C in intact stratum corneum and a stratum corneum membrane preparation, but absent from lipid-extracted samples. Results of IR spectrometry show that thermally induced spectral changes related to enhanced motion of the lipid acyl chains also occur in the region of about 60-80 degrees C. The combined DSC and IR results show that the thermal transitions near 65 degrees and 75 degrees C depend on water concentration in a manner identical to that seen for a variety of lipid-water systems. Taken together, these results suggest that thermal transitions occurring near 65 degrees and 85 degrees C involve increased acyl chain motion of stratum corneum lipids.

Calorimetry, Differential Scanning↗

Preparation of liposomes from stratum corneum lipids.

Mammalian stratum corneum contains multiple intercellular lipid bilayers that constitute the epidermal water barrier. Unlike all other biologic membranes, the epidermal lamellae do not contain phospholipids, as a result of which the ability of the stratum corneum lipid mixture to form bilayers has been questioned. In the present study, a lipid mixture containing only epidermal ceramides (40%), cholesterol (25%), palmitic acid (25%), and cholesteryl sulfate (10%), approximating the composition of stratum corneum lipids, formed stable, unilamellar liposomes when sonicated at 80 degrees C in buffer containing 100 mM NaCl, 5 mM Tris, and 1 mM EDTA at pH 7.5. The size and form of the liposomes were studied by both freeze fracture and negative staining electron microscopy. Lipid mixtures from which either the palmitic acid or the cholesteryl sulfate were omitted were still capable of forming similar liposomes, but a mixture of ceramides and cholesterol, or ceramides alone, were incapable of forming liposomes. The results indicate that lipid mixtures similar to those found in stratum corneum are capable of forming bilayers at physiologic pH.

Animals↗

Preparation and characterization of the nonionic detergent-soluble proteins of human stratum corneum.

Nonionic detergent-soluble (NIDS) proteins from human stratum corneum have been prepared by the combined action of detergent and mechanical stimulation of normal human skin. SDS-polyacrylamide gel electrophoresis studies revealed approximately 15 components falling into the molecular weight ranges of 10,000-15,000, 24,000-37,000, and 44,000-68,000. Immunization of rabbits with this material gave antisera which demonstrated 3 or 4 antigenic components using 2-dimensional immunoelectrophoresis. Studies with 125I-labeled NIDS protein indicated that the main precipitate on 2-dimensional immunoelectrophoresis was associated with SDS-polyacrylamide gel components of molecular weight 15,000 and 30,000. Indirect immunofluorescence studies on human skin sections revealed localization of NIDS protein antigens throughout the suprabasal epidermis but concentrated in the stratum granulosum and stratum corneum. Localization studies using strips of stratum corneum obtained by the skin surface biopsy technique revealed a pericellular type of distribution of the NIDS protein antigens.

Detergents↗

Immunologic cross-reaction of stratum corneum basic protein and a keratohyalin granule protein.

Antiserum to the stratum corneum basic protein (SCBP) of newborn rat epidermis was used to test for cross-reactive proteins in extracts of skin and in tissue sections. The antibody reacts strongly with epidermal extracts but very poor with dermal extracts. Buffer extracts of epidermis give a reaction of partial identity or identity with the antigen, SCBP. Urea extracts of isolated stratum corneum give a reaction of identity with SCBP. When the proteins of these extracts are separated by SDS polyacrylamide gel electrophoresis, the immunoreactive peak in the 4 M urea extract corresponds to the 49,000 MW SCBP. The immunoreactive peak in the 1 M potassium phosphate extract corresponds to a 52,000 MW protein. This protein is rapidly and transiently labeled after injection of 3H-histidine into newborn rats, in contrast to the SCBP which is labeled after a 5-hr lag. The 52,000 MW protein appears to be an immunologically related precursor of the SCBP. Immunoreactive proteins were localized in tissue sections by the indirect immunoperoxidase method. A strong positive reaction was seen in keratohyalin granules and in the stratum corneum. The reaction of keratohyalin granules corroborates the extraction of a cross-reactive protein by 1 M potassium phosphate, a method for extraction of keratohyalin granules from epidermis. These results are consistent with the hypothesis that a protein of 52,000 MW is present in keratohyalin and is converted to the SCBP (49,000 MW) concomitantly with the conconversion of a granular cell to a stratum corneum cell.

Animals↗

The influence of water content, chemical treatment and temperature on the rheological properties of stratum corneum.

The elastic modulus is determined as a function of water content for untreated stratum corneum, and stratum corneum treated with urea and LiBr. The modulus is also determined for stratum corneum at temperatures from 2 degrees C to 45 degrees C and on samples with the water solubles extracted. The modulus is independent of temperature, but may be modified by binding of small molecules to the protein in stratum corneum. The modulus is found to be a function of water content, not water activity.

Body Water↗

Composition of free long-chain (sphingoid) bases in stratum corneum of normal and pathologic human skin conditions.

The composition of molecular species of free long-chain bases (FLCB) isolated from stratum corneum of various human skin conditions was analyzed by the high-performance liquid chromatographic and fast atom bombardment mass spectrometric methods. FLCB with carbon length ranging from 16 to 20 comprised about 0.3% of total lipids in stratum corneum of normal and pathologic skin conditions. Major FLCB included sphinganines and sphingenines with 18 to 20 carbons and some phytosphingosines such as t17:1, t18:1, t18:0, t20:1, and t20:0. Compared with stratum corneum of normal lower legs, molar percentages of FLCB having 18 carbons and those with 20 carbons were slightly higher and lower, respectively, in normal plantar epidermis, showing site-related differences in normal skin. Psoriatic scales and hyperkeratotic stratum corneum from clavus and plantar keratoderma contained increased levels of FLCB with 18 carbons and decreased levels of FLCB with 20 carbons. These findings may reflect abnormal keratinization in hyperkeratotic skin conditions.

Adolescent↗

Covalently bound lipids of human stratum corneum.

In the present study, we demonstrate that human stratum corneum contains covalently bound lipids accounting for 1.4% of the dry weight of the tissue. The major component (53.3% of the total by weight) is a ceramide (CER-A) consisting of 30 through 34-carbon omega-hydroxyacids amide-linked to sphingosine. The other bound lipids in human stratum corneum include fatty acids (12.7%), omega-hydroxy acids (9.4%), and a second, more polar, omega-hydroxyacid-containing ceramide (CER-B, 24.8%). The predominant omega-hydroxyacids in both ceramides, as well as the free hydroxyacid fraction, are the 30-carbon saturated and 32- and 34-carbon monoenoic species. The bound fatty acids consist largely of 14 through 22-carbon saturated species, but significant proportions of monoenoic species and linoleic acid are also present. Psoriatic scale contains a similar total concentration of the same covalently bound lipids, but the proportions of the individual bound lipids are different from those found in normal stratum corneum. It is suggested that the principal function of the covalently bound lipids in human stratum corneum is the formation of a lipid envelope on the outer surface of the keratinized cells.

Animals↗

Effects of glycolic acid on desquamation-regulating proteinases in human stratum corneum.

In order to investigate the mechanism of glycolic acid (GA) function in human stratum corneum, we monitored changes in cathepsin D-like (CD) and chymotrypsin-like (SCCE) proteinases for 3 weeks following topical GA application (50% w/v, pH 0.9) for 30 min to human skin. In the early phase, weakened stratum corneum cohesion in the lower layers was observed on day 2 and the amount of active CD in the upper layer of the stratum corneum was significantly decreased from 30 min until day 2, whereas that in the lower layer remained normal. In contrast, the amount of active SCCE showed no change during the experimental period. The surface pH of the stratum corneum drastically decreased to pH 2 at 30 min and slightly recovered to around pH 3 until 1 day after treatment. From 9 to 19 days, a decrease in corneocyte cell area and a remarkable long-term increase in the amount of active CD in the upper layer were observed. In an in vitro study, the activities of desquamation-regulating proteinases were shown to have remarkably increased at around pH 3, due to activation of CD at its optimal pH. These results suggest that GA functions via at least two different mechanisms, acute activation of CD in the lower layer by acidification around pH 3, along with inactivation of CD in the upper layer, and long-term enhancement of de novo CD production in the few weeks following GA treatment.

Administration, Cutaneous↗

The demonstration of bacteria on and within the stratum corneum using scanning electron microscopy.

Using scanning electron microscopy it has been possible to demonstrate the location of bacteria on and within the stratum corneum of the human foot. Biopsies taken either by sectioning or by removing stratum corneum with cyanoacrylate ester adhesive were examined using a Jeol JSM-T20 scanning electron microscope. Bacteria could be seen easily on specimens from skin which had been occluded to increase the number of bacteria present. On the surface, bacteria were scattered widely in small colonies (usually containing less than ten bacteria). Although bacteria could be seen around the orifice of sweat ducts they did not preferentially favour these sites. Within the stratum corneum, bacteria could be found as relatively large colonies but these were usually associated with sweat ducts or the underside of the furrows in the skin surface. This study suggests that, in normal skin, bacteria are able to colonize both the surface and the depths of the stratum corneum.

Bacteria↗

The effect of washing on the thickness of the stratum corneum in normal and atopic individuals.

The thickness of, and number of cell layers in, the stratum corneum and the living epidermis were determined on frozen sections of washed and unwashed skin from normal and atopic individuals of both sexes. The stratum corneum of atopic patients was thinner and had fewer layers of dead cells and intercellular lipid than normal, although the living epidermis was thicker. Regular washing with soap and water had no appreciable effect on the dimensions of the living epidermis of either group but caused a reduction in the number of cell layers and the amount of sudanophilic material in the stratum corneum of both. In atopic patients little surface lipid remained, suggesting that washing with soap and water may be detrimental to the barrier function of the stratum corneum in such patients.

Administration, Topical↗

The Azerbaijani scrub: a simple method to harvest large amounts of stratum corneum.

BACKGROUND: The study of skin barrier constituents may require collection of much stratum corneum. Existing methods are inadequate and/or difficult. METHODS: A simple and safe method to harvest stratum corneum from glabrous human skin derives from the bathing practices of people in Azerbaijan. The method requires water immersion of the subject for 30 min immediately followed by vigorous scrubbing of skin with a moist rough crepe mitten. RESULTS: This scrubbing method causes the separation of large amounts of stratum corneum which is easily harvested. CONCLUSION: The method facilitates study of stratum corneum components, including intercellular lipids.

Adult↗

Quantitative screening of human stratum corneum lipids by sequential one-dimensional thin layer chromatography.

There is a need in many studies on the epidermal permeability barrier for practical and reliable separation of all major fractions of human stratum corneum lipids. Various methods have been described including thin layer chromatography, high performance thin layer chromatography and iatroscan. However, none of these methods seems to be applicable for an inexpensive, rapid and reliable analysis of a large number of samples. Here, such a method for the separation and quantification of all major stratum corneum lipid fractions is presented. This method employs the one-dimensional separation of stratum corneum lipids using thin layer chromatography. The systems used herein are based upon various modifications of the solvents, solvent ratio and developing distance of each system. For quantification of chromatographed and charred lipids a Desaga densitometer is used. The system described here had been successfully applied to 550 samples of human stratum corneum lipids.

Chromatography, Thin Layer↗