Expression and ultrastructural localization of plasmin(ogen) in the terminally differentiated layers of normal human epidermis.

OBJECTIVE: Plasmin, a relatively unspecific trypsin-like serine protease, is involved in many physiological and pathological conditions, particularly in dermatoses with barrier impairment. It is secreted as the inactive zymogen plasminogen and is activated to plasmin by plasminogen activators, such as urokinase. There still exists a paucity of data on the precise localization of epidermal plasmin(ogen) within the epidermis and the stratum corneum. The aim of the present study was to get information about its origin and ultrastructural localization within normal human epidermis. METHOD: We performed immunoelectron transmission electron microscopy immunogold labelling in normal abdominal human skin. RESULT: Plasmin was only observed in the terminally differentiated cell layers of the epidermis and was largely associated with the corneocyte envelopes and to some extent with the intercellular lipid matrix in the stratum corneum. CONCLUSION: Our results indicate that in normal human skin, plasmin(ogen) is synthesized by differentiated epidermal keratinocytes of the stratum granulosum and is not serum-born.

OBJECTIF: Plasmine, une relativement peu spécifique ' trypsin-like' protéase sérine, participe aux plusieurs processus physiologiques et pathologiques et, plus particulièrement, à la physiopathologie des dermatoses caractérisées par l'altération de la barrière de perméabilité. Elle est sécrétée sous forme d'un zymogene inactif, plasminogène, et devient activée par les activateurs du plasminogène, telle urokinase. A l'heure actuelle, on manque de précision quant à la localisation de plasmine (ou son précurseur) dans l'épiderme et le stratum corneum. Le but du présent travail a été de d'apporter l'information sur la provenance et la localisation ultrastructurale de plasmine/plasminogène présents dans l'épiderme humain. MÉTHODE: L'étude ultrastructurale de l'épiderme humain normal (plastie abdominale) a fait appel à l'immunomarquage à l'or colloïdal sur coupes ultrafines des tissus inclus à froid dans des résines acryliques. RÉSULTAT: L'anticorps monoclonal anti -plasmine/plasminogène a détecté l'antigène situé exclusivement dans la partie la plus différenciée de l'épiderme et persistant dans la couche cornée. Il n'y a pas eu de réactivité dans les couches épineuse et basale. Le marquage a été prédominant sur les enveloppes cornifiées des kératinocytes granuleux et cornéocytes. Des foyers du marquage ont été également présents dans le cytoplasme et les espaces intercellulaires de la couche granuleuse, ainsi que dans la matrice lipidique de la couche cornée profonde. CONCLUSION: Nos résultats indiquent la production de novo de plasmine/plasminogène dans les kératinocytes le plus différenciés et ne suggèrent pas l'origine sérique de cette enzyme dans l'épiderme.

[The epidermal barrier]. / Barrière épidermique.

The skin acts as an interface between the body and its surrounding environment. The epidermis, the surface layer of the skin, is chiefly responsible for this interactive protective function. The epidermal barrier may be subdivided into three defensive systems: the photoprotective barrier, the immune barrier, and the physical and chemical barrier of the stratum corneum or horny layer. To protect against harmful ultraviolet radiation, the epidermis has absorption factors such as melanin, produced by melanocytes, and urocanic acid, which is a degradation product of filaggrin. The epidermal immune defence system comprises an innate component, which is rapid but non-specific, together with adaptive response, which is systemic and antigen-specific, initiated by Langerhans cells. The stratum corneum, derived from terminal differentiation of epidermal keratinocytes, plays a key role as a physical and chemical permeability barrier. This horny layer is made up of corneocytes, covered with horny envelopes and linked to one another by corneodesmosomes and by extracellular matrix sheets. The epidermal barrier, which is constantly being renewed, is characterised by its extremely great capacity of adaptation to changing conditions in the environment.

Fragility of epidermis in newborns, children and adolescents.

Within their first days of life, newborns' skin undergoes various adaptation processes needed to accommodate the transition from the wet uterine environment to the dry atmosphere. The skin of newborns and infants is considered as a physiological fragile skin, a skin with lower resistance to aggressions. Fragile skin is divided into four categories up to its origin: physiological fragile skin (age, location), pathological fragile skin (acute and chronic), circumstantial fragile skin (due to environmental extrinsic factors or intrinsic factors such as stress) and iatrogenic fragile skin. Extensive research of the past 10 years have proven evidence that at birth albeit showing a nearly perfect appearance, newborn skin is structurally and functionally immature compared to adult skin undergoing a physiological maturation process after birth at least throughout the first year of life. This article is an overview of all known data about fragility of epidermis in 'fragile populations': newborns, children and adolescents. It includes the recent pathological, pathophysiological and clinical data about fragility of epidermis in various dermatological diseases, such as atopic dermatitis, acne, rosacea, contact dermatitis, irritative dermatitis and focus on UV protection.

'Memory' of the stratum corneum: exploration of the epidermis' past.

The stratum corneum (SC) is the final product of the process of epidermal differentiation. Besides its crucial protective role as a physical permeability barrier, this composite structure made of cornified keratinocytes embedded in a layered lipid matrix is also, by nature, a tissue that keeps track of past events occurring in the outermost living layers. In normal human epidermis, formation of the SC is very rapid, and during this cornification process several structures expressed by the last granular layer of keratinocytes become entrapped and immobilized at the cells' periphery. Cell-cell junctions are obvious targets of transglutaminases that cross-link junctions' components within the corneocyte envelopes. Thus, desmosomes and tight junctions (TJs) in living cells become fixed at the corneocyte periphery and cannot be recycled anymore. We have quantified the TJ-like structures residing in the SC of human skin explants subjected to environmental stress and compared these results with fresh skin controls. Significant overexpression of TJ-like cell-cell envelope fusions has been observed in the stressed epidermis and in two different hereditary skin diseases characterized by increased SC cohesion. Quantitation of TJ-like structures has contributed to the interpretation of the diseases' physiopathology. Other examples of information retrieved from the SC concern fluctuating lipid expression in the course of atopic dermatitis and patterns of corneodesmosome breakdown influencing SC desquamation. It is, therefore, possible to analyse and quantify the traces left in the SC and to draw conclusions on the dynamics of living tissue over the past several days.

Retention of corneodesmosomes and increased expression of protease inhibitors in dandruff.

BACKGROUND: Dandruff is a common, relapsing and uncomfortable scalp condition affecting a large proportion of the global population. The appearance of flakes on the scalp and in the hair line, and associated itch are thought to be consequences of a damaged skin barrier, altered corneocyte cohesion and abnormal desquamation in dandruff. The balance between skin proteases and protease inhibitors is essential for driving the key events, including corneodesmosome degradation, in the desquamation process and to maintain stratum corneum (SC) barrier integrity. OBJECTIVES: To investigate the distribution of corneodesmosomes, the key component of the SC cohesivity and barrier function, and the protease inhibitors lympho-epithelial Kazal-type-related inhibitor (LEKTI-1) and squamous cell carcinoma antigen (SCCA1) in the scalp of dandruff-affected participants. METHODS: The methods utilized were immunohistochemistry, scanning immunoelectron microscopy, phase-contrast microscopy, Western blotting and serine protease activity assay on tape-stripped SC or scalp skin biopsies. RESULTS: In SC samples from healthy subjects, corneodesmosomes were peripherally located in the corneocytes. In samples of dandruff lesions, corneodesmosomes were located both peripherally and on the entire surface area of the corneocytes. LEKTI-1 and SCCA1 protein levels and parakeratosis were found to be highly elevated in the lesional samples. CONCLUSIONS: The persistence of nonperipheral corneodesmosomes is a characteristic feature of the perturbed desquamation seen in dandruff. The increased expression levels of LEKTI-1 and SCCA1 are consistent with the view that the dandruff condition is characterized by an imbalance in protease-protease inhibitor interaction in the SC.

Development and organization of human stratum corneum after birth: electron microscopy isotropy score and immunocytochemical corneocyte labelling as epidermal maturation's markers in infancy.

BACKGROUND: There is growing evidence for the ongoing structural and functional adaptation of the skin after birth. OBJECTIVES: The aim of this study was the definition of scanning electron microscopy markers of skin maturation in different age groups (birth to adulthood). We propose a semiquantitative score to analyse the maturation of the skin surface and a complementary evaluation of the distribution of corneodesmosin and corneodesmosomes. MATERIAL AND METHODS: An electron microscopy isotropy (E.M.I.) score was performed in six age-groups to include fullterm neonates, babies, children and adults. The distribution of corneodesmosome remnants was analysed by corneodesmosin distribution with immunocytochemical corneocyte labelling. RESULTS: The E.M.I. score showed the highest anisotropy in neonates. The youngest groups displayed irregular and thick cell clusters composed of poorly individualized cells. In the older groups, the distribution of superficial corneocytes was more regular. The cells evenly covered the surface and displayed easily visualized single cell outlines. The distribution of immune-labelled corneodesmosome remnants and the corneocyte projected area showed a correlation between age and structural maturation. The observed evolution indicated a poorly controlled process of corneocyte desquamation in infants and confirmed the relative immaturity of the epidermal barrier up to 1-2 years after birth under basal conditions. CONCLUSION: Our study is the first attempt at semiquantitative evaluation of the micromorphology maturation of the epidermal surface at the ultrastructural level. The E.M.I. score and the associated pattern of corneodesmosome breakdown may be used as markers of the stratum corneum maturation.

Fragility of epidermis and its consequence in dermatology.

The skin is the largest organ of the body, providing a protective barrier against bacteria, chemicals and physical insults while maintaining homeostasis in the internal environment. Such a barrier function the skin ensures protection against excessive water loss. The skin's immune defence consists of several facets, including immediate, non-specific mechanisms (innate immunity) and delayed, stimulus-specific responses (adaptive immunity), which contribute to fending off a wide range of potentially invasive microorganisms. This article is an overview of all known data about 'fragile skin'. Fragile skin is defined as skin with lower resistance to aggressions. Fragile skin can be classified into four categories up to its origin: physiological fragile skin (age, location), pathological fragile skin (acute and chronic), circumstantial fragile skin (due to environmental extrinsic factors or intrinsic factors such as stress) and iatrogenic fragile skin. This article includes the epidemiologic data, pathologic description of fragile skin with pathophysiological bases (mechanical and immunological role of skin barrier) and clinical description of fragile skin in atopic dermatitis, in acne, in rosacea, in psoriasis, in contact dermatitis and other dermatologic pathologies. This article includes also clinical cases and differential diagnosis of fragile skin (reactive skin) in face in adult population. In conclusion, fragile skin is very frequent worldwide and its prevalence varies between 25% and 52% in Caucasian, African and Asian population.

Aqueous dispersions of organogel nanoparticles - potential systems for cosmetic and dermo-cosmetic applications.

OBJECTIVE: The preparation and physicochemical characterization of organogel nanoparticles dispersed in water have been developed. These systems could be employed as nanocarrier for cosmetic applications or as hydrophobic reservoirs for drug delivery. METHODS: Gelled particles of organic liquid and 12-hydroxystearic acid (organogelator) were obtained by hot emulsification (T>Tgel), with a surfactant (acetylated glycol stearate) and polymers (sodium hyaluronate and polyvinyl alcohol) as stabilizing agents, and cooling at room temperature (T

The lipid alterations in the stratum corneum of dogs with atopic dermatitis are alleviated by topical application of a sphingolipid-containing emulsion.

BACKGROUND: Atopic dermatitis (AD) results from an altered skin barrier associated with defects in the lipid composition of the skin. Dogs with AD present similar clinical symptoms to humans, and may be a useful model for investigations into AD. AIM: To analyse the changes occurring in the lipids of the stratum corneum (SC) of dogs with AE after 3 weeks of topical treatment with an emulsion containing ceramides, free fatty acids (FFAs) and cholesterol (skin lipid complex; SLC). METHODS: Nonlesional SC was collected by tape stripping from control and treated areas. Free and protein-bound lipids were purified, and the various classes were isolated by column chromatography, analysed by thin-layer chromatography and assayed. RESULTS: Ceramides, FFA and cholesterol were all found to be lower in the skin of untreated dogs with AD than in normal dogs, and the topical treatment resulted in significantly increased values for ceramides. Conversely, only trace amounts of glucosylceramides were present in normal SC, but a high concentration (27 µg per mg protein) was detected in canine atopic SC, which disappeared after treatment with SLC. There was a heterogeneous distribution of all of the lipids in the different layers of canine atopic SC, which was more pronounced for protein-bound than for free lipids. Following topical treatment, the protein-bound lipid content normalized. CONCLUSIONS: Topical treatment with SLC resulted in a significant improvement of the lipid biosynthesis of keratinocytes in atopic dogs, thereby potentially enabling the formation of a tighter epidermal barrier.

Circumscribed palmo-plantar hypokeratosis: a disease of desquamation? Immunohistological study of five cases and literature review.

BACKGROUND: Circumscribed palmoplantar hypokeratosis (CPH) is a recently recognized, rarely reported dermatosis that shows characteristic clinicopathological features; however, its pathogenesis remains unknown. OBJECTIVE: The aim of this study was to get further insight into the pathogenesis of CPH. METHODS: An immunohistological study was performed on five cases of CPH to investigate the expression of several epidermal proliferation and differentiation proteins, with emphasis on those involved in corneocyte desquamation [including corneodesmosin, kallikrein 5 and lympho-epithelial Kazal type inhibitor (LEKTI)]. RESULTS: In three of five cases, a decreased expression of LEKTI, corneodesmosin and filaggrin was found, along with an increased expression of kallikrein 5 and keratin 6. The expression of several other antigens (including involucrin, Ki67, p63, CD138/syndecan I, EGF-R) did not present a consistently different pattern as compared with the unaffected epidermis. CONCLUSION: The immunohistopathologic features of CPH suggest that an altered (accelerated) corneocyte desquamation process could be the main pathological mechanism underlying the development of lesions.

Responsiveness of human monocyte-derived dendritic cells to thimerosal and mercury derivatives.

Several cases of skin sensitization have been reported following the application of thimerosal, which is composed of ethyl mercury and thiosalicylic acid (TSA). However, few in vitro studies have been carried out on human dendritic cells (DCs) which play an essential role in the initiation of allergic contact dermatitis. The aim of the present study was to identify the effect of thimerosal and other mercury compounds on human DCs. To address this purpose, DCs derived from monocytes (mono-DCs) were used. Data show that thimerosal and mercury derivatives induced DC activation, as monitored by CD86 and HLA-DR overexpression associated with the secretion of tumor necrosis factor alpha and interleukin 8, similarly to lipopolysaccharide and the sensitizers, 1-chloro-2,4-dinitrobenzene (DNCB) and nickel sulfate, which were used as positive controls. In contrast, TSA, the non-mercury part of thimerosal, as well as dichloronitrobenzene, a DNCB negative control, and the irritant, sodium dodecyl sulfate, had no effect. Moreover, oxidative stress, monitored by ROS induction and depolarization of the mitochondrial membrane potential, was induced by thimerosal and mercury compounds, as well as DNCB, in comparison with hydrogen peroxide, used as a positive control. The role of thiol oxidation in the initiation of mono-DC activation was confirmed by a pre-treatment with N-acetyl-l-cysteine which strongly decreased chemical-induced CD86 overexpression. These data are in agreement with several clinical observations of the high relevance of thimerosal in patch-test reactions and prove that human mono-DCs are useful in vitro tools for determining the allergenic potency of chemicals.

A multitechnique evaluation of topical corticosteroid treatment.

BACKGROUND/PURPOSE: Corticosteroids are widely prescribed for systemic or local treatment of inflammatory autoimmune disorders. Long-term therapy is associated with side effects and causes cutaneous atrophy of the epidermis and the dermis. The present study aims to evaluate with several noninvasive techniques, the skin modifications observed during corticosteroids treatment. The potential of skin mechanical measurement and ultrasound radio frequency (RF) signal analysis are proposed as new measures more closely related to the functional impairments. METHODS: Thirteen young healthy women volunteers had two applications per day on one arm of topical Clobetasol propionate 0.05% for 28 days, and they were followed for 28 days more. Skin modifications were studied by high-frequency ultrasound imaging, ultrasound RF signal analysis, optical coherence tomography and by the suction test. RESULTS: For all the techniques, a statistically significant change is observed with treatment. Large variations, around 30%, are observed for all techniques, but less for ultrasound imaging (10%). Dermis and epidermis thickness presented stable measurements on the nontreated zone. At the end of the study, measures returned to normal. The dynamic is mainly observed within the first 14 days of treatment and within the first 14 days after its cessation. CONCLUSION: Similar dynamics of skin modification during corticosteroid treatment was observed with very different techniques. Moreover, the potential of RF ultrasound analysis and mechanical skin measurement for characterizing skin structural and functional impairments has been evaluated.

Simultaneous characterization of oxygen transport into and through porcine skin exposed to oxygen-saturated water.

Oxygen delivery to the skin is a promising approach for treatment of dermatological diseases (e.g. ischemic wound healing). However, characterization of oxygen transport into and through the skin exposed to oxygen carrier formulations has not been reported. In the present study, we developed an original lab-made static diffusion cell mounted with porcine skin enabling the assessment of oxygen uptake into the skin (i.e., oxygen penetration) and passage through the skin (i.e., oxygen permeation). Oxygen penetration and permeation were recorded by using an optical probe implanted into the skin tissue and a Clark-type electrode plunged into the receptor solution of the diffusion cells. Permeability parameters (i.e., maximal and steady-state flux; permeability coefficient) of oxygen were determined after a 2-hour application of oxygen-saturated water to either the skin surface (exogenous delivery) or the dermis (endogenous delivery). Similar experiments were performed by using intact or stripped skin in order to appreciate the role of the stratum corneum as oxygen barrier. Exogenous delivery of oxygen to skin tissue was found more effective than endogenous delivery through intact and stripped skin. However, exogenous oxygen permeation was found smaller than that determined from endogenous delivery. The upper layers of the skin would constitute a potential oxygen reservoir created by the high solubility of oxygen in epidermal lipids. Therefore, oxygen carrier formulations might significantly improve the oxygen status in the skin for further biological effects.

Effects of a topically applied preparation of epidermal lipids on the stratum corneum barrier of atopic dogs.

Canine atopic dermatitis (AD) is characterized ultrastructurally by disorganization of the lamellar lipids (LLs) in the stratum corneum (SC), similar to that seen in the human disease. This study, based on the examination of biopsy samples, was designed to investigate the expression of canine epidermal lipids and to evaluate quantitatively, by means of electron microscopy and ruthenium tetroxide post-fixation, the effect of a new topical skin lipid complex (SLC) on the structural deficit in the skin of five dogs with AD. The non-lesional skin of atopic dogs differed from the skin of healthy dogs in that the LLs were reduced in number and highly disorganized. After repeated applications of SLC to the non-lesional skin of dogs with AD, numerous LLs were observed in the deepest part of the SC, occupying 74% of the inter-corneocyte space, while they accounted for only 31.8% of the inter-corneocyte space in comparable biopsy samples from untreated (control) skin of the same dogs. In contrast, the LLs filled 89.5% of the deepest inter-corneocyte spaces in the SC of healthy dogs. Many keratinosomes were observed at the interface between living epidermis and SC after treatment of non-lesional AD skin. Stacks of short LL discs represented 57.6% of the total LLs found in the newly formed SC compactum in the treated atopic dogs. It is suggested that the treatment with SLC stimulated the production and secretion of endogenous SC lipids, contributing to the formation of an improved epidermal barrier.

Percutaneous absorption of benzophenone-3 loaded lipid nanoparticles and polymeric nanocapsules: A comparative study.

For the last years, the increase of the number of skin cancer cases led to a growing awareness of the need of skin protection against ultraviolet (UV) radiations. Chemical UV filters are widely used into sunscreen formulations as benzophenone-3 (BP-3), a usually used broad spectrum chemical UV filter that has been shown to exercise undesirable effects after topical application. Innovative sunscreen formulations are thus necessary to provide more safety to users. Lipid carriers seem to be a good alternative to formulate chemical UV filters reducing their skin penetration while maintaining good photo-protective abilities. The aim of this work was to compare percutaneous absorption and cutaneous bioavailability of BP-3 loaded into solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), nanostructured polymeric lipid carriers (NPLC) and nanocapsules (NC). Particle size, zeta potential and in vitro sun protection factor (SPF) of nanoparticle suspensions were also investigated. Results showed that polymeric lipid carriers, comprising NPLC and NC, significantly reduced BP-3 skin permeation while exhibiting the highest SPF. This study confirms the interesting potential of NPLC and NC to formulate chemical UV filters.

Structure, function, and immunogenicity of human insulinoma cells.

Dissociated human insulinoma cells were plated onto plastic multiwell dishes. Cells were maintained for 1 mo on plastic with three passages. Cultures consisted of small colonies with some areas of stratification and few intercellular spaces. Ultrastructural studies indicated that cultured cells had epithelial features with desmosomes at cell-to-cell contacts and intermediate filaments in addition to secretory granules in the cytoplasm. Insulin and C-peptide were released in equimolar amounts in culture media. When challenged for 30 min with 16.7 mM glucose, 1 mM 3-isobutyl-1-methylxanthine, 4 mM tolbutamide, or 10(-6) M glucagon, insulinoma cells responded by a 1.5-, 1.5-, 2-, or 3-fold increase, respectively, in insulin release above baseline levels. A 15-min challenge with 10(-5) M isoproterenol increased insulin secretion by 1.85-fold. By indirect immunofluorescence, an anti-insulin antibody reacted positively with cell cytoplasm, whereas anti-somatostatin and anti-glucagon antibodies did not. Insulinoma cell surface expressed class I MHC molecules but not class II molecules. Immediately after isolation, crude insulinoma cells were contaminated by 2% of DR+ cells from nonislet components that disappeared after several weeks in culture. The ability of insulinoma cells to stimulate allogenic T-lymphocyte proliferation was assessed by [3H]thymidine incorporation in mixed culture combinations. Crude insulinoma cells elicited a strong lymphoproliferative response with a stimulation index ranging between 3.5 and 7, whereas no stimulation was found after 1 mo in culture. It is postulated that absence of class II-positive cells in the stimulatory cell preparation conditioned this immune tolerance across the major histocompatibility barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

[Netherton syndrome: a type of infantile erythroderma with failure to thrive, immune deficiency, rickets. Report of 3 cases]. / Le syndrome de Netherton: une ectodermatose du petit nourrisson avec retard de croissance, déficit immunitaire et rachitisme. A propos de 3 cas.

We report the cases of 2 boys and 1 girl suffering from Netherton syndrome. Both boys presented with a non-bullous congenital erythroderma and were diagnosed early as Netherton syndrome with hair biopsies. Both had severe failure to thrive, signs of atopy, several episodes of bacterial infection, and rickets (with a high blood level of vitamin D in the first boy, and vitamin D deficiency in the second). In the third case, the pilar abnormality appeared at the age of 3 years. The girl had ichtyosis linearis circumflexa, failure to thrive and severe constipation. Netherton syndrome is a rare disorder characterized by severe ichtyosis, signs of atopy, immune deficiency and failure to thrive. The disease is severe and comprises many complications in early infancy. It is due to a genetic disorder of recessive autosomal transmission, and the gene, SPINK5, is located in the chromosome 5. Prenatal diagnosis is possible. Two of our patients had rickets, which has never been described in such patients population.

Measurement, analysis and prediction of topical UV filter bioavailability.

The aim of the present study was to objectively quantify and predict bioavailability of three sunscreen agents (i.e., benzophenone-3, 2-ethylhexylsalicylate, and 2 ethylhexyl-4-methoxycinnamate) in epidermis treated by petrolatum and emulsion-based formulations for 7 and 30min on four human volunteers. Profiles of sunscreen agents through stratum corneum (SC), derived from the assessment of chemical amounts in SC layers collected by successive adhesive tape-stripping, were successfully fitted to Fick's second law of diffusion. Therefore, permeability coefficients of sunscreen agents were found lower with petrolatum than with emulsion based formulations confirming the crucial role of vehicle in topical delivery. Furthermore, the robustness of that methodology was confirmed by the linear relationship between the chemical absorption measured after 30min and that predicted from the 7-min exposure experiment. Interestingly, in this dermatopharmacokinetic method, the deconvolution of permeability coefficients in their respective partition coefficients and absorption constants allowed a better understanding of vehicle effects upon topical bioavailability mechanisms and bioequivalence of skin products.

Flow cytometry for separation of keratinocyte subpopulations from the viable epidermis.

Human epidermal cell suspensions were analyzed and sorted with flow cytometry. The desmosome and differentiation-related KM48 monoclonal antibody was used for indirect immunofluorescence and permitted staining of keratinocytes at various stages of the cell maturation. Intensity of the staining correlated with the degree of differentiation. Three sorting gates were chosen to obtain subpopulations which varied distinctly in KM48 expression. The flow cytometry-sorted cells were characterized by their ultrastructural appearance and by the bullous pemphigoid antigen expression. According to the ultrastructure criteria, about 50% of the cells obtained from the "IF negative" gate were basal layer keratinocytes (45.5% expressed bullous pemphigoid antigen); 90% of the "intermediate" gate cells were spinal layer keratinocytes, and over 80% of the cells sorted through the "strongly IF positive" gate were of the granular layer type. The method of keratinocyte separation proposed allows samples to be obtained for further biochemical and functional studies on keratinocyte subpopulations in normal and pathologic skin.