Non identity of non ionic detergent soluble (NIDS) protein and stratum corneum autoantigens

The relationship between naturally occurring circulating stratum corneum autoantibodies and experimentally produced antibodies to a non ionic detergent soluble (NIDS) protein fraction from human stratum corneum has been investigated. Using indirect immunofluorescence and indirect haemagglutination techniques, anti NIDS protein serum was shown not to inhibit the autoantibody-antigen reaction. Furthermore no direct interaction could be demonstrated between sheep red blood cells coated with stratum corneum autoantigen and anti NIDS protein serum. The NIDS protein antigens therefore represent a separate and distinct class of stratum corneum antigens.     

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.     

Flow cytometric investigations of corneocytes from psoriatic scales and the effects of Ingram therapy

Flow cytometry has been used to study the stratum corneum. Following mechanical disruption and propidium iodide staining for DNA, analysis of the fluorescence intensity profile permits discrimination between nucleated and anucleate corneocytes. It was established that the nucleated corneocyte from parakeratotic stratum corneum contains double-stranded DNA in the normal diploid amount. A model investigation is described following the normalization of the psoriatic stratum corneum during Ingram therapy. It is shown that the initially high percentage of nucleated corneocytes (50%) falls exponentially during the first weeks of therapy. The rate of normalization seems to offer a quantitative and objective approach for evaluation and optimization of therapy.     

Three-dimensional analyses of individual corneocytes with atomic force microscope: morphological changes related to age, location and to the pathologic skin conditions

Background/aims: The past morphological studies on individual corneocytes have so far mainly focused on their two-dimensional characteristics, particularly on their projected area, which have been widely employed for the estimation of the turnover rate of the stratum corneum because of the practical use. However, sometimes a poor correlation has been reported between the projected area of corneocytes and actually measured turnover time of the stratum corneum. The objective of the present study is to perform detailed three-dimensional measurements of individual corneocytes with atomic force microscope. Through analyses of the obtained data, we tried to find morphological parameters that reflect more closely the differentiation process of the corneocytes in the stratum corneum than the frequently used two-dimensional projected area. Methods: We measured such morphological parameters as the volume, average thickness and real surface area of individual corneocytes isolated from the covered skin (the flexor surface of the upper arm) and the exposed skin (the cheek) of 12 healthy individuals belonging to different age brackets, in addition to their projected area. We further introduced a new parameter, a flatness index calculated by dividing the projected area of corneocytes by their thickness. Similarly, we measured corneocytes obtained from eight patients with atopic dermatitis (AD) and psoriatic patients. Results: Obtained results showed that most of these morphological parameters varied greatly depending upon the anatomical location and age of the subjects. Needless to say great differences were found between healthy skin and lesional skin of atopic dermatitis or psoriasis. However, the volume and thickness of corneocytes collected from the same location of normal skin of the covered area (upper arm) with tape-strippings were noted to decrease as they differentiated in the stratum corneum, showing an increase in their surface area and projected area with a resultant increase in the flatness index. Moreover, the corneocyte collected from the lesional skin of AD or psoriasis showed a great decrease in flatness index, reflecting their poor differentiation in the stratum corneum due to its enhanced turnover rate. Most of all, we found a poor correlation between the projected area and the various three-dimensional morphologic parameters of the corneocytes, indicating that the projected area does not reflect the volume or thickness of corneocytes that are also greatly influenced by the differentiation process of corneocytes in the stratum corneum. Conclusions: To estimate the differentiation speed of the corneocytes, we suggest using their flatness index rather than the two-dimensional cell surface area, because the former is a concept that takes into account the three-dimensional characteristics of corneocytes.     

Nanoscale infrared (IR) spectroscopy and imaging of structural lipids in human stratum corneum using an atomic force microscope to directly detect absorbed light from a tunable IR laser source

An atomic force microscope (AFM) and a tunable infrared (IR) laser source have been combined in a single instrument (AFM‐IR) capable of producing ~200‐nm spatial resolution IR spectra and absorption images. This new capability enables IR spectroscopic characterization of human stratum corneum at unprecendented levels. Samples of normal and delipidized stratum corneum were embedded, cross‐sectioned and mounted on ZnSe prisms. A pulsed tunable IR laser source produces thermomechanical expansion upon absorption, which is detected through excitation of contact resonance modes in the AFM cantilever. In addition to reducing the total lipid content, the delipidization process damages the stratum corneum morphological structure. The delipidized stratum corneum shows substantially less long‐chain CH₂‐stretching IR absorption band intensity than normal skin. AFM‐IR images that compare absorbances at 2930/cm (lipid) and 3290/cm (keratin) suggest that regions of higher lipid concentration are located at the perimeter of corneocytes in the normal stratum corneum.     

Water distribution and related morphology in human stratum corneum at different hydration levels

This study focused on the water distribution in human stratum corneum and on the swelling of the corneocytes. For this purpose stratum corneum was hydrated to various levels and used either for Fourier transform infrared spectroscopy or for cryo-scanning electron microscopy. The images were analyzed with respect to water localization and cell shape. The Fourier transform infrared spectra were measured to study the water-lipid interactions. The results show that water only slightly changes the lipid transitions in the stratum corneum even at a hydration level of 300% wt/wt compared to stratum corneum and that water is inhomogeneously distributed in the stratum corneum. No gradual increase in water level was observed in depth. At 57%-87% wt/wt water content the hydration level in the central part of stratum corneum is higher than in the superficial and deeper cell layers. Water domains are mainly present within the corneocytes and not in the intercellular regions. At a very high hydration level (300% wt/wt), the corneocytes are strongly swollen except for the deepest cell layers adjacent to the viable epidermis. The corneocytes in these layers are not swollen. At 300% wt/wt hydration level water domains are also present in intercellular regions. Between 17% wt/wt and 300% wt/wt the cell thickness increases linearly with the hydration level suggesting that swelling of cells mainly occurs in the direction perpendicular to the skin surface. At an increased hydration level, the corneocyte envelope more efficiently surrounds the cell content compensating for the increased cell volume. The changes in stratum corneum morphology with increasing water level have also been observed in dermatomed skin.     

Molecular models of the intercellular lipid lamellae in mammalian stratum corneum

Intercellular lipid lamellae in the stratum corneum constitute the barrier to water diffusion and may also play a role in cohesion between corneocytes. The lamellae arise from stacks of lamellar disks that are extruded from the granular cells and then fuse edge-to-edge to form sheets. It has been proposed that each lamellar disk is formed from a flattened vesicle, and therefore consists of two lipid bilayers in close apposition. In the present study, electron microscopic examination of ruthenium-tetroxide-fixed stratum corneum from mouse, pig, and human skin revealed that the double bilayer pattern persists in the intercellular lamellae. In addition, distinctive patterning of the intercellular lamellae has led us to propose novel molecular arrangements of the intercellular lipids. These include interlamellar sharing of lipid chains to produce lipid monolayers between pairs of bilayers. The pattern reflects the provenance of the intercellular lamellae from lamellar granule disks and the nonrandom orientation of the lamellar lipids.     

Interaction of liposomes with human epidermis reconstructed in vitro

Summary Upon topical application of liposomes of the large unilamellar vesicle type to human epidermis reconstructed in vitro , there is a dose-dependent alteration of the morphology of both the stratum corneum and the living part of the epidermis. In particular. shrunken lipid droplets are found between corneocytes and keratinocytes, Sometimes, corneocytes show inclusions reminiscent of 'cholesterol crystals', Corneocytes, moreover, show a decreased density. Both corneocytes of the various layers of the stratum corneum and keratinocytes belonging to the uppermost layer of the living epidermis show particularly osmophilic membranes. indicating lipid transfer. Intact liposomes or their remnants can sometimes be seen between corneocytes of the upper strata, The presence of liposomal lipid within the stratum corneum is supported by the presence of gold particles used as a marker. There is, however, no evidence for the uptake of intact liposomes by the living epidermis, or their passage through this compartment of the skin.     

Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature.

The outermost layer of mammalian skin, the stratum corneum, provides the body with a barrier against transepidermal water loss and penetration of agents from outside. The lipid-rich extracellular matrix surrounding the corneocytes in the stratum corneum is mainly responsible for this barrier function. In this study (cryo-) electron diffraction was applied to obtain information about the local lateral lipid organization in the extracellular matrix in relation to depth in human stratum corneum. For this purpose, stratum corneum grid-strips were prepared from native skin in vivo and ex vivo. It was found that the lipid packing in samples prepared at room temperature is predominantly orthorhombic. In samples prepared at 32 degrees C the presence of a hexagonal packing is more pronounced in the outer layers of the stratum corneum. Gradually increasing the specimen temperature from 30 to 40 degrees C induced a further transition from an orthorhombic to a hexagonal sublattice. At 90 degrees C all lipids were present in a fluid phase. These results are in good agreement with previously reported wide angle X-ray diffraction and Fourier transformed infrared spectroscopy studies. We conclude that the lipids in human stratum corneum are highly ordered throughout the stratum corneum and that electron diffraction allows monitoring of the local lipid organization, which contributes to the understanding of stratum corneum barrier function.     

The role of the corneocyte lipid envelopes in cohesion of the stratum corneum

Treatment of isolated stratum corneum with certain detergents results in complete disaggregation of the corneocytes within hours at 45 degrees C without agitation. This is prevented by prior heating of the tissue to 80 degrees C or by solvent extraction of the intercellular lipids. In the present study, electron microscopy revealed that the heated or solvent-extracted tissue was characterized by cell-to-cell contacts that appeared to involve the chemically bound hydroxyceramides which constitute the corneocyte lipid envelope. It is proposed that the irreversible bonding between corneocytes that results from heating or lipid extraction results from interdigitation of the sphingosine chains belonging to those hydroxyceramides that are bound to the corneocyte protein envelope by the omega-hydroxyl function of the 30- and 32-carbon hydroxyacid moieties. Similar interdigitation of adjacent envelopes might be involved in natural stratum corneum cohesion, limited mostly to the periphery of corneocytes where the absence of intercellular lamellae allows the appropriate cell-to-cell contact.     

Are corneocytes elastic?

By using a microhandling system, we have recorded the force necessary to elongate isolated corneocytes. According to certain hypothesis, it appears that the elastic modulus of corneocyte is far higher than that of stratum corneum itself. Aggregates of corneocytes are disassembled by forces with a wide range of intensity. In the light of these experiments, stratum corneum appears as a very supple material where the deformations occur more by a plastification of the intercellular medium and an unfolding of the microrelief lines than an elongation of the corneocytes themselves. Moreover, desmosomes remnants could play a certain role in the corneocytes cohesiveness.     

Barrier function of the skin: "la raison d'être" of the epidermis

The primary function of the epidermis is to produce the protective, semi-permeable stratum corneum that permits terrestrial life. The barrier function of the stratum corneum is provided by patterned lipid lamellae localized to the extracellular spaces between corneocytes. Anucleate corneocytes contain keratin filaments bound to a peripheral cornified envelope composed of cross-linked proteins. The many layers of these specialized cells in the stratum corneum provide a tough and resilient framework for the intercellular lipid lamellae. The lamellae are derived from disk-like lipid membranes extruded from lamellar granules into the intercellular spaces of the upper granular layer. Lysosomal and other enzymes present in the extracellular compartment are responsible for the lipid remodeling required to generate the barrier lamellae as well as for the reactions that result in desquamation. Lamellar granules likely originate from the Golgi apparatus and are currently thought to be elements of the tubulo-vesicular trans-Golgi network. The regulation of barrier lipid synthesis has been studied in a variety of models, with induction of several enzymes demonstrated during fetal development and keratinocyte differentiation, but an understanding of this process at the molecular genetic level awaits further study. Certain genetic defects in lipid metabolism or in the protein components of the stratum corneum produce scaly or ichthyotic skin with abnormal barrier lipid structure and function. The inflammatory skin diseases psoriasis and atopic dermatitis also show decreased barrier function, but the underlying mechanisms remain under investigation. Topically applied "moisturizers" work by acting as humectants or by providing an artificial barrier to trans-epidermal water loss; current work has focused on developing a more physiologic mix of lipids for topical application to skin. Recent studies in genetically engineered mice have suggested an unexpected role for tight junctions in epidermal barrier function and further developments in this area are expected. Ultimately, more sophisticated understanding of epidermal barrier function will lead to more rational therapy of a host of skin conditions in which the barrier is impaired.     

Characterization of vernix caseosa: water content, morphology, and elemental analysis

Recent studies have prompted interest in the use of epidermal barrier creams as protective biofilms for very low birthweight preterm infants. The key to understanding the role of epidermal barrier films is an elucidation of their interaction with water and a basic knowledge of their composition. In this study, we investigated the morphologic properties and elemental composition of the naturally occurring biofilm, vernix caseosa. This biofilm is typically lacking in preterm infants and its production coincides in utero with terminal differentiation of the epidermis and formation of the stratum corneum. Significantly, vernix (80.5+/-1.0% H2O) had a much higher water content than other barrier creams (Eucerin: 17.1+/-0.6%, Aquaphor: 0.33+/-0.03%, Ilex: 0.19+/-0.02%, petrolatum: 0.03+/-0.01%; all p<0.05). Phase contrast microscopy of vernix showed multiple cellular elements with nucleic "ghosts" embedded in a putative lipid matrix. Transmission electron microscopy revealed flattened structures approximately 1-2 microm in thickness with distinct cellular envelopes indicative of differentiated corneocytes. Compared with mature corneocytes in adult stratum corneum, vernix corneocytes appeared swollen, the density of the keratin filaments was less, and there was a relative lack of tonofilament orientation. Cryofractured specimens were examined by cryoscanning electron microscopy with subsequent elemental localization by X-ray beam analysis. The findings indicate the high water content of vernix is largely compartmentalized within fetal corneocytes. These results are consistent with the novel view of vernix as a "fluid phase" stratum corneum consisting of a hydrophobic lipid matrix with embedded fetal corneocytes possessing unique biomechanical and water-binding properties.     

Hydration disrupts human stratum corneum ultrastructure

Using transmission and cryo-scanning electron microscopy, we confirm that extended water exposure leads to extensive disruption of stratum corneum intercellular lipid lamellae. We define the in vivo swelling behavior of the stratum corneum: exposure to water for 4 or 24 h results in a 3- or 4-fold expansion of the stratum corneum thickness, respectively. Corneocytes swell uniformly with the exception of the outermost and inner two to four corneocyte layers, which swell less. We show that hydration induces large pools of water in the intercellular space, pools that can exceed the size of water-swollen corneocytes. By 4 h of water exposure there are numerous small and large intercellular pools of water ("cisternae") present throughout the stratum corneum, and at 24 h these cisternae substantially increase in size. Within cisternae the lipid structure is disrupted by lamellar delamination ("roll-up"). Cisternae appear to be disk-shaped structures that do not obviously communicate. Cisternae appear to contain considerable lipidic and other material and to contain a substantial fluid volume that can rival the volume of the dry stratum corneum. Similar results are obtained following urine exposure. With urine exposure, cisternae communicate with salts in the external solution. This study illustrates the disruptive effect of overhydration on the stratum corneum intercellular space, identifies large and numerous unanticipated intercellular cisternal structures, defines the magnitude of stratum corneum swelling, and identifies stratum corneum cell layers that swell less. The study suggests the stratum corneum is a more chaotic structure than previously envisioned, and provides a framework for better understanding desquamation, irritancy, and percutaneous transport.     

Lipids,proteins and corneocyte adhesion

Summary Three factors were examined for their relative contribution to corneocyte cohesion in normal adult pig ear: (1) extracellular lipids derived from membrane-coating granules (MCG); (2) corneosomes (modified stratum corneum desmosomes); and (3) corneocyte covalently bound lipid envelopes. Cohesion strength of the outer stratum corneum was measured directly by cohesometry, then altered by removing MCG lipids with solvents of varying potency. Cohesion changes were related to degree of lipid removal and ultrastructural alterations. Trypsin was also used to see if proteolysis of corneosomes promoted squame shedding. Potent solvents increased cohesion in relation to the amount of MCG lipid extracted. Tighter cohesion was due to fusion of the outer leaflets from covalently bound lipid envelopes on adjacent corneocytes. However, lipid envelopes are unlikely to mediate normal stratum corneum cohesion since MCG lipids play a significant anti-cohesive role preventing their apposition. Mild solvents partially removed MCG lipids causing a slight decrease in cohesion compared with untreated samples. This suggests a minor cohesive role for MCG lipids, consistent with maintaining their barrier function. We believe that corneosomes are the major determinant of stratum corneum cohesiveness because, in untreated skin, both cohesion and the number of corneosomes increased from the surface towards the granular layer. Furthermore, corneosome digestion with trypsin induced superficial squame shedding.     

Lipid composition of cohesive and desquamated corneocytes from mouse ear skin

An organ culture system has been used to examine differences in the lipid compositions of materials derived from cohesive and desquamated mouse ear stratum corneum. Within this culture system, skin explants display rates of cell replication and differentiation comparable to those observed in vivo for up to 2 weeks and, during this period, loosened or dishesive material accumulates at the surface. Lipid compositions were determined for both intact and loosened stratum corneum derived from cultured skin and also for freshly prepared stratum corneum. In all 3 cases, the profiles of the nonpolar lipids and the ceramides were essentially the same; some of the nonpolar lipids appeared to be of sebaceous origin. The only changes detected upon desquamation were reductions of cholesteryl sulfate and a second unidentified lipid of similar polarity. Cholesteryl sulfate constitutes 4-5% of the polar lipid in fresh stratum corneum or stratum corneum from organ culture. This is reduced to 0.4% in the desquamated material which accumulates in the culture system. The unidentified lipid decreases from 1-2% of the polar lipid in intact fresh or cultured stratum corneum to 0.1% in the desquamated material. The possible function of cholesteryl sulfate in corneocyte cohesion is discussed.     

Eigenwirkungen von Emulsionen auf die Hornschichtbarriere und -hydratation

The appearance of the skin depends greatly on the hydration of the stratum corneum which is regulated by water binding substances of the corneocytes and also by the quality of the stratum corneum lipids. Furthermore these lipids are responsible for the barrier function. In patients with atopic dermatitis, the water binding capacity and the barrier function of the stratum corneum are reduced even in clinically healthy skin areas. Emollients can damage the stratum corneum and lead to desiccation and a disturbance of the barrier. This effect is a result of an increased permeability of the barrier lipids and direct damage to the keratinocytes and corneocytes. The degree of damage of the barrier caused by emollients in dermatological vehicles has not been sufficiently investigated. As suggested by hypothetical considerations, such an effect is not expected and cannot be demonstrated in water-in-oil-emulsions. Oil-in-water-emulsions without glycerol as well as lipophilic and hydrophilic microemulsions do damage the barrier function. Both types of microemulsions additionally lead to a dehydration of the stratum corneum. The damaging effect of oil-in-water-emulsions can be reduced by the addition of glycerol and urea.     

Oriented structure in human stratum corneum revealed by X-ray diffraction

Various types of human stratum corneum (sheets or callus) were exposed, in parallel and perpendicular geometry, to the high flux of X rays produced by a synchrotron radiation source. Under these conditions, very clear and rich diffraction patterns, corresponding to the supramolecular organization of stratum corneum proteins and lipids, were obtained. The comparative study of normal or delipidized stratum corneum sheets and membrane couplets allows one to attribute certain diffraction features to lipids. Our results in the 3-7-nm range show two different distances for lipid bilayers. Concerning the protein nature of normal stratum corneum, the results show that keratin would occur in the beta form, whereas for callus it is in the alpha form. Indeed, normal stratum corneum sheets never display the 0.514-nm characteristic of alpha keratin. This result means that the supramolecular organization of keratin could depend on the keratinization process. Finally, our studies also confirm the presence of a still-unknown protein component existing in the beta form that would be located either inside the corneocytes or in some dilatated zones of the intercellular spaces.     

Structural modifications in the stratum corneum by effect of different solubilizing agents: a study based on high-resolution low-temperature scanning electron microscopy

The action of different solubilizing agents (chloroform-methanol mixtures and the nonionic surfactant octyl glucoside, OG) on the structural organization of the stratum corneum (SC) was investigated by means of the double-layer high-resolution low-temperature scanning electron microscopy technique. Chloroform-methanol mixtures were able to remove mainly the lipid without a significant loss of cohesion in the SC tissue. However, OG treatment caused a partial disaggregation of the corneocytes and their envelopes and, at a macroscopic level, a loss of cohesion in the whole SC tissue. As for lipid domains, after OG treatment the formation of rough structures was detected, probably associated with the disorder in the lipid lamellae. The formation of these new structures could be attributable to the interaction of the lipids with the proteins liberated from the corneocytes. Hence, a direct correlation may be established between the preservation of the structure of the corneocytes and the corneocyte envelope and the cohesion of the whole SC tissue.     

Microfabrication of individual 200 microm diameter transdermal microconduits using high voltage pulsing in salicylic acid and benzoic acid

We describe an extension of semiconductor fabrication methods that creates individual approximately 200 microm diameter aqueous pathways through human stratum corneum at predetermined sites. Our hypothesis is that spatially localized electroporation of the multilamellar lipid bilayer membranes provides rapid delivery of salicylic acid to the keratin within corneocytes, leading to localized keratin disruption and then to a microconduit. A microconduit penetrating the isolated stratum corneum supports a volumetric flow of order 0.01 ml per s with a pressure difference of only 0.01 atm (about 10(2) Pa). This study provides a method for rapidly microengineering a pathway in the skin to interface future devices for transdermal drug delivery and sampling of biologically relevant fluids.