Stratum Corneum Of Epidermis Research Articles

Role of the FAD-dependent polyamine oxidase in the selective formation of N1,N8-bis(γ- glutamyl)spermidine protein cross-links

Protein-bound gamma-glutamylpolyamines have highlighted a new pathway in polyamine metabolism. Human foreskin keratinocytes offer a suitable model for this study. Indeed, they develop polymerized envelopes, as they differentiate, rich in epsilon-(gamma-glutamyl)lysine and N(1),N(8)-bis(gamma-glutamyl)spermidine cross-links. We have found that the selective oxidation of N(1)-(gamma-glutamyl)spermidine and N-(gamma-glutamyl)spermine by FAD-dependent polyamine oxidase (PAO) may be one of the cellular mechanisms regulating the preferential formation of a sterically defined bis(gamma-glutamyl)spermidine cross-link. The significance of this finding is unknown, but it suggests that the target of this PAO-modulation is to achieve the biochemical prerequisite for production of a normal epidermal stratum corneum.

Pathogenicity of theChrysosporiumAnamorph ofNannizziopsis vriesiifor veiled chameleons (Chamaeleo calyptratus)

Veiled chameleons (Chamaeleo calyptratus) were experimentally challenged with the fungus Chrysosporium anamorph of Nannizziopsis vriesii (CANV). Chameleons were exposed to conidia in their captive environment, or were inoculated by direct application of a conidial suspension inoculum on intact and on abraded skin. The CANV induced lesions in all experimental groups and was recovered from infected animals, fulfilling Koch's postulates and confirming that it may act as a primary fungal pathogen in this species of reptile. A breach in cutaneous integrity, as simulated by mild scarification, increased the risk of infection but was not required for the CANV to express pathogenicity. Initial hyphae proliferation occurred in the outer epidermal stratum corneum, with subsequent invasion of the deeper epidermal strata and dermis. A spectrum of lesions was observed ranging from liquefactive necrosis of the epidermis to granulomatous inflammation in the dermis. CANV dermatomycosis appears to be contagious and can readily spread within a reptile collection, either directly through contact with infective arthroconidia or indirectly via fomites. Dense tufts of arthroconidiating hyphae were demonstrated histologically on the skin surface of many animals that developed dermatomycosis, and these arthroconidia may act as infective propagules involved in the transfer of disease between reptiles.

Genetic and Environmental Influences on Skin Pattern Deterioration

Sun exposure has been known to cause histological changes in the dermal layer of the skin. Using deterioration in the fine reticular patterning of the epidermal stratum corneum (skin pattern, as measured on the Beagley-Gibson scale) as a proxy measure of histological changes in the dermal layer, previous studies have typically assumed that degradation of skin pattern is largely caused by sun exposure. A twin study comprising 332 monozygotic twin pairs and 488 dizygotic twin pairs at ages 12, 14, and 16 was used to investigate the etiology of variation in skin pattern, particularly in relation to measured sun exposure and skin color. Our results indicate that although self-reported sun exposure is a significant contributor to variation in skin pattern, its effect is small, explaining only 3.4% of variation in skin pattern at age 14. Additive genetic effects explain 86% of variation in skin pattern at age 12 but these effects reduce with age so that 75% of variation is due to additive genetic effects at age 14 and 72% at age 16. This trend of diminishing genetic influences continues into adulthood, with 62% of variation due to non-additive genetic factors in a smaller adult sample (aged 32-86). Skin color explains 10.4% of variation in skin pattern at age 12, which is due to additive genetic influences common to both. Melanin content appears to provide a protective effect against skin pattern deterioration, perhaps because of the structural differences in melanosomes between different skin types or the free radical scavenging properties of melanin.

Skin damage and mitochondrial dysfunction after acute ultraviolet B irradiation: relationship with nitric oxide production

Ultraviolet (UV) radiation is the main environmental carcinogen. It is able to induce injury in the keratinocytes, which triggers mechanisms in order to protect the skin against molecular alterations that may lead to the development of skin cancer. UVB is capable of producing genotoxic damage, directly or indirectly through reactive oxygen species, inducing DNA alterations and mutations. UVB radiation has also been associated with the generation of nitric oxide (NO), which is able to induce many physiological and physiopathological processes. The aim of the current study was to investigate the effect of UVB irradiation in hairless mice skin. We evaluated the effect of an acute dose (200 mJ/cm(2)) of UVB irradiation correlating with histological alterations, nitric oxide synthase expression and activity, mitochondrial respiratory function, superoxide anion production and lipid peroxidation, 0, 6, 17 and 24 h post-irradiation treatment. Morphological analysis showed disruption of the epidermal stratum corneum and basale after UVB irradiation. The results indicated that skin UVB irradiation was associated with an increased cytosolic inducible nitric oxide synthase (iNOS) expression, inversely related to lipid peroxidation processes. An increase in mitochondrial superoxide anion (O(2) (*-)) and NO production 17 h post-irradiation was correlated with a mitochondrial dysfunction, all of them integrating the skin response to acute UVB irradiation. UVB irradiation of the skin produces morphological alterations as a consequence of the induction of molecular mechanisms associated with mitochondrial respiratory dysfunction and O(2) (*-) production, probably mediated by the increased mitochondrial NO production. On the other hand lipid peroxidation decrease inversely correlates with cytosolic iNOS expression, suggesting a protective role for the inflammatory response.

Cubosome Dispersions as Delivery Systems for Percutaneous Administration of Indomethacin

The present study concerns the production and characterization of monooleine (MO) dispersions as drug delivery systems for indomethacin, taken as model anti-inflammatory drug. Dispersions were produced by emulsification and homogenization of MO and poloxamer in water. Morphology and dimensional distribution of the disperse phase have been characterized by cryo-transmission electron microscopy and photon correlation spectroscopy, respectively. X-ray diffraction has been performed to determine the structural organization of the disperse phase. Sedimentation field flow fractionation (SdFFF) has been performed to investigate drug distribution in the dispersion. An in vitro diffusion study was conducted by Franz cell associated to stratum corneum epidermis membrane on cubosome dispersions viscosized by carbomer. In vivo studies based on skin reflectance spectrophotometry and tape stripping were performed to better investigate the performance of cubosome as indomethacin delivery system. Microscopy studies showed the coexistence of vesicles and cubosomes. X-ray diffraction revealed the presence of a bicontinuous cubic phase of spatial symmetry Im3m (Q229). SdFFF demonstrated that no free drug was present in the dispersion. Indomethacin incorporated in viscosized MO dispersions exhibited a lower flux with respect to the analogous formulation containing the free drug in the aqueous phase and to the control formulation based on carbomer gel. Reflectance spectroscopy demonstrated that indomethacin incorporated into MO dispersions can be released in a prolonged fashion. Tape-stripping experiments corroborated this finding. MO dispersions can be proposed as nanoparticulate systems able to control the percutaneous absorption of indomethacin.

Synthesis of Sphingosine Relatives. Part 26. Synthesis of Ceramide 1 {(2S,3R,4E)‐N‐[30′‐(Linoleoyloxy)triacontanoyl]‐4‐sphingenine}, a Unique Constituent of Human Skin.

Ceramide 1 ((2S,3R,4E)-N-[30'-(linoleoyloxy)triacontanoyl]-4-sphingenine, 1}, a unique ceramide of human epidermal stratum corneum, was synthesized from (S)-Garner's aldehyde (2), pentadecan-15-olide (5) and linoleic acid.

Evaluation of the anti-Trichophyton activity of a prodigiosin analogue produced by γ-proteobacterium, using stratum corneum epidermis of the Yucatan micropig

Prodigiosins (PGs) are known to be a family of natural red pigments, characterized by a common pyrrolydipyrrolylmethane skeleton structure with a C-4 methoxy group, and some of these pigments have been isolated from some microorganisms. Members of the PG family have been reported to show several biological activities, such as immunosuppressive and cytotoxic activities. Recently, we discovered a bacterial strain (MS-02-063), from our microbial library, that produces large amounts of a PG analogue (PG-L-1). In this study, we examined the anti-Trichophyton activity of PG-L-1 (produced by strain MS-02-063) against clinically isolated Trichophyton spp., by a method using stratum corneum epidermis (SCE) of the Yucatan micropig, which is suitable for estimating the antifungal activity of drugs in vitro. In the National Committee for Clinical Laboratory Standards (NCCLS) method, PG-L-1 showed potent antifungal activity against nine clinically isolated strains of Trichophyton spp., although the minimum inhibitory concentration (MIC) values were slightly higher than those of bifonazole. In spite of the lower efficiency of PG-L-1 transfer into SCE from medium than that of bifonazole, PG-L-1 transferred into SCE showed more potent antifungal activity than bifonazole, at lower concentrations.

Molecular models of the intercellular lipid lamellae from epidermal stratum corneum

The purpose of the present study was to test the hypothesis that the 13 nm trilamellar repeat units within the intercellular spaces of epidermal stratum corneum are composed of lamellae with alternating 5–3–5 nm dimensions as presented in previous models [J. Invest. Dermatol. 92 (1989) 251, P.W. Wertz, Integral lipids in hair and stratum corneum, in: P. Jolles, H. Zahn, H. Hocker (Eds.), Hair: Biology And Structure, Birkhauser Verlag, Basel, 1996, pp. 227–238, Acta Derm.-Venereol., Suppl. 208 (2000) 23]. Electron density profiles were measured from transmission electron micrographs of porcine stratum corneum prepared using ruthenium tetroxide [J. Invest. Dermatol. 92 (1989) 251]. Center-to-center distances of adjacent electron-dense bands as well as adjacent lucent bands were measured. Dense band center-to-center measurements were consistent with a 5–3–5 nm arrangement. However, lucent band center-to-center measurements revealed uniform lamellar thickness. It is suggested that linoleate chains in the central lamella reduce more ruthenium than the predominantly saturated chains in the outer lamellae and that this additional reduced ruthenium accumulates under the polar head group regions. A similar phenomenon involving the sphingosine moieties of the covalently bound ω-hydroxyceramide molecules accounts for the three-band pattern seen between the ends of adjacent corneocytes. It is concluded that the component lamellae of the several types of 13 nm trilamellar units of the stratum corneum are all of equal thickness.

Terahertz pulse imaging in reflection geometry of human skin cancer and skin tissue

We demonstrate the application of terahertz pulse imaging (TPI) in reflection geometry for the study of skin tissue and related cancers both in vitro and in vivo. The sensitivity of terahertz radiation to polar molecules, such as water, makes TPI suitable for studying the hydration levels in the skin and the determination of the lateral spread of skin cancer pre-operatively. By studying the terahertz pulse shape in the time domain we have been able to differentiate between diseased and normal tissue for the study of basal cell carcinoma (BCC). Basal cell carcinoma has shown a positive terahertz contrast, and inflammation and scar tissue a negative terahertz contrast compared to normal tissue. In vivo measurements on the stratum corneum have enabled visualization of the stratum corneum–epidermis interface and the study of skin hydration levels. These results demonstrate the potential of terahertz pulse imaging for the study of skin tissue and its related disorders, both in vitro and in vivo.

Desmoglein isoform distribution affects stratum corneum structure and function.

Desmogleins are desmosomal cadherins that mediate cell-cell adhesion. In stratified squamous epithelia there are two major isoforms of desmoglein, 1 and 3, with different distributions in epidermis and mucous membrane. Since either desmoglein isoform alone can mediate adhesion, the reason for their differential distribution is not known. To address this issue, we engineered transgenic mice with desmoglein 3 under the control of the involucrin promoter. These mice expressed desmoglein 3 with the same distribution in epidermis as found in normal oral mucous membranes, while expression of other major differentiation molecules was unchanged. Although the nucleated epidermis appeared normal, the epidermal stratum corneum was abnormal with gross scaling, and a lamellar histology resembling that of normal mucous membrane. The mice died shortly after birth with severe dehydration, suggesting excessive transepidermal water loss, which was confirmed by in vitro and in vivo measurement. Ultrastructure of the stratum corneum showed premature loss of cohesion of corneocytes. This dysadhesion of corneocytes and its contribution to increased transepidermal water loss was confirmed by tape stripping. These data demonstrate that differential expression of desmoglein isoforms affects the major function of epidermis, the permeability barrier, by altering the structure of the stratum corneum.

Does epidermal turnover reduce percutaneous penetration?

After its removal from the skin surface, chemical remaining within the skin can become systemically available. The fraction of chemical in the skin that eventually enters the body depends on the relative rates of percutaneous transport and epidermal turnover (i.e., stratum corneum desquamation). Indeed, some investigators have claimed that desquamation is an efficient mechanism for eliminating dermally absorbed chemical from the skin. The fate of chemical within the skin following chemical contact was examined using a mathematical model representing turnover of and absorption into the stratum corneum and viable epidermis. The effects of turnover rate, exposure duration, penetrant lipophilicity, and lag time for chemical diffusion were explored. These calculations show that significant amounts of chemical can be removed from skin by desquamation if epidermal turnover is fast relative to chemical diffusion through the stratum corneum. However, except for highly lipophilic and/or high molecular weight (>350 Da) chemicals, the normal epidermal turnover rate is not fast enough and most of the chemical in the skin at the end of an exposure will enter the body. Epidermal turnover can significantly reduce subsequent chemical absorption into the systemic circulation only for highly lipophilic or high molecular weight chemicals.

Modulation of T-lymphocyte proliferation by exogenous natural ceramides and sphingosylphosphorylcholine.

Sphingolipids such as ceramide and sphingosine are abundantly present in the stratum corneum of epidermis. In atopic stratum corneum, sphingosylphosphorylcholine (SPC) is present in association with a reduction in the amount of ceramides. We have previously shown that the cellular kinetics of T cells are affected by exogenous addition of sphingosine and synthetic ceramides, raising the possibility that sphingolipids diffusing from the stratum corneum modulate skin-infiltrating T cells. By using two natural ceramides and murine T cells, this study further clarified the conditions under which exogenous ceramides enhance the proliferation of T cells. KLH-specific T cell clones 28-4 and 24-2 proliferated in response to natural ceramides when cultured for 44-48 h in the presence of concanavalin A at 1 microg per ml. Elongation of culture periods adversely inhibited the T cell proliferation, suggesting the existence of an optimal exposure time. Augmentation of DNA synthesis by natural ceramides was more pronounced in tumor necrosis factor alpha (TNFalpha)-sensitive 28-4 cells than in less sensitive 24-2 cells, and TNFalpha-induced proliferation of 28-4 cells was suppressed by the concomitant addition of natural ceramides. Similar to ceramides, SPC augmented the proliferation of resting spleen cells. Our study suggests that ceramide modulation of T cell proliferation depends on the TNFalpha sensitivity and activation level of T cells and that SPC also has a mitogenic potential for T cells.

Low Frequency Dielectric Dispersion of Microporous Membranes in Electrolyte Solution

The electrical properties of microporous membranes in 1 mM KCl solution have been in vestigated in the frequency range 1 mHz to 1 kHz, using a four-electrode measuring cell. An alpha-dispersion centred around 0.1 Hz was detected and this was assumed to be caused by counterion relaxation effects in the pores of the membrane. Schwarz theory was used to provide a rough estimate on the results to be expected from the measurements. This study is part of a project of which the objective is to reveal the contribution from the electrical properties of the sweat ducts to the total electrical properties of the epidermal stratum corneum. Although the phase angles measured on the membranes were small (<2.9°), more data are needed before any conclusions regarding human skin can be drawn.

Isolation of a rat histidase cDNA sequence and expression in Escherichia coli--evidence of extrahepatic/epidermal distribution.

Histidase (histidine ammonia-lyase) is a cytosolic enzyme responsible for catalyzing the non-oxidative deamination of histidine to urocanic acid. Full-length cDNAs encoding rat histidase have been isolated from a lambdaZAP liver cDNA library using a partial cDNA fragment obtained by PCR. Whereas the initial description of the rat histidase 3' untranslated sequence contained a rare polyadenylation signal sequence, the data presented encompass a more distant 28-bp region, possessing a nucleotide stretch (AATATAAA), identical to that in the mouse histidase cDNA. Dideoxynucleotide chain-termination sequencing of two clones obtained by in vivo excision yielded an additional 376 bp and 105 bp of 5' and 3' untranslated sequences, respectively. A selected rat histidase cDNA clone was introduced into the pET-16b prokaryotic vector and expressed in BL21(DE3)pLysS Escherichia coli. After purification by nickel-chelation chromatography, recombinant histidine-tagged protein was employed to raise anti-(rat histidase) immunoglobulin in a Japanese white rabbit. The polyclonal rabbit antibody recognized and formed immune complexes with rat and recombinant human histidase proteins. Immunoblots of crude rat organ extracts detected a spectrum of histidase expression extending beyond that observed in liver and skin. Among other histidase-positive cells were those of the renal cortex tubular epithelium, fundic mucosal glands of stomach, gastric intramuscular (Auerbach's) plexus, and adrenal cortex. Immunohistochemical studies of histidase in rat liver produced discrete staining of hepatocytes in association with portal triads (Rappaport zone I). Furthermore, in contrast with previous reports of activity confined to epidermal stratum corneum, our findings demonstrate immunoreactive protein within and limited to the adjacent stratum granulosum.

Determination and correlation of in vitro viability for hairless mouse and human neonatal whole skin and stratum corneum/epidermis.

Viable tissue is essential to assess the rate and extent of biotransformation during percutaneous absorption in vitro. We assessed the viability of hairless mouse whole skin (WS) and stratum corneum/epidermis (SCE) and human neonatal SCE following separation from the dermis by EDTA phosphate-buffered saline (EDTA-PBS) incubation or by heat treatment by measuring the conversion of dextrose to lactate. Lactate concentrations in receptor fluid samples were determined using a Sigma diagnostic lactate determination kit. A standard curve was prepared and samples assayed spectrophotometrically at 340 nm using a lambda 2 beta spectrophotometer. Standard curves were prepared for each experiment and correlation coefficient values (r) were calculated. Our results showed that hairless mouse SCE was associated with glucose conversion to lactic acid at an increased rate if incubated in EDTA-PBS for 4 h and used immediately. Lactate production was greater with the dermis present (EDTA-PBS WS). The rate of glucose to lactate conversion in hairless mouse SCE was 20-25% of that found in WS. Compared with Dulbecco's modified PBS (DMPBS)-treated WS controls, the rate of lactate production in EDTA-PBS-treated WS was nearly a 50% less. Heat treatment in water at 60 degrees C to separate SCE from hairless mouse WS appeared to eliminate viability. Viability of hairless mouse SCE, as measured by glucose conversion to lactate, was comparable to human neonatal SCE. These results suggest that the dermis is a significant contributor to glucose metabolism and that incubation in EDTA-PBS is a contributing factor to the overall decrease in metabolic capacity of the tissue. As a result of these findings, hairless mouse SCE appears to be useful as a model for human neonatal SCE in percutaneous absorption studies.

Integral lipids of hair and stratum corneum.

Within the past 10 years, covalently bound lipids have been identified both in wool and hair and in epidermal stratum corneum. In hair, fatty acids, the most abundant of which is 18-methyleicosanoic acid, are attached via thioester linkages to protein at the outer surface of cuticle cells. This lipid layer, the fatty layer or F-layer, is thought to be of major significance in determining the surface properties of hair and wool. In epidermal stratum corneum, the covalently attached lipid has been identified as an omega-hydroxyceramide consisting of 30-through 34-carbon omega-hydroxyacids amide-linked to sphingosine bases. The hydroxyceramide molecules are attached to the outer surface of the cornified envelope through ester linkages, and provide a monolayer covering the outer surface of all corneocytes. This covalently bound lipid layer, or lipid envelope, interacts with free lipids and has a major influence on the organization of lipid lamellar structures in the intercellular space.

Photoprotection in vitiligo and normal skin. A quantitative assessment of the role of stratum corneum, viable epidermis and pigmentation.

Pigmentation, stratum corneum and viable epidermis are considered to be the main factors protecting against ultraviolet radiation. We quantitatively investigated the degree of photoprotection provided by these structures in vitiligo and adjacent normally pigmented skin. In 14 patients 61 MED tests were performed in vitiligo and adjacent normally pigmented skin using a solar simulator. The thickness of stratum corneum and viable epidermis was determined from frozen skin sections, and pigmentation was calculated by measuring skin reflectance at 555 nm and 660 nm. To analyse photoprotection, the UV dose necessary to evoke erythema was regressed against the thickness of stratum corneum and viable epidermis, pigmentation and the erythema grade in the MED test. By analysing regression coefficients we found that stratum corneum was the main photoprotective factor not only in vitiligo but also in normally pigmented skin. The effect of pigmentation in normal skin was slightly less prominent. Stratum corneum was thicker in vitiligo than in normally pigmented skin. However, the photoprotection due to stratum corneum was similar in both groups because significantly less photoprotection was achieved per thickness unit of stratum corneum in vitiligo than in normal skin. Neither in vitiligo nor in normally pigmented skin did the photoprotection depend on viable epidermis. Our data quantitatively document the importance of stratum corneum and pigmentation. Hyperkeratosis in vitiligo offers just as efficient photoprotection as does the normal stratum corneum in pigmented skin.

An Alpha Hydroxy Acid Derivative Suitable for Sensitive Skin

Individuals with sensitive skin may not tolerate some products formulated with alpha hydroxy acids (AHAs) due to unacceptable levels of stinging and irritation. To develop a neutral molecule that is well tolerated by individuals with sensitive skin without stinging or irritation, yet retains the beneficial effects of AHAs. A neutral amide derivative, methoxypropylgluconamide (MPG), was invented. Formulations containing MPG (15%) were tested by cummulative irritation, repeat insult patch testing, and lactic acid stinging assay. Bilateral comparison clinical and histological studies of xerosis and photoaging assessments were performed. MPG was nonirritating and nonsensitizing. Clinically significant improvements in xerosis and photoaging including uneven pigmentation were seen. Improvement in the morphology of the stratum corneum epidermis and in the amount of glycosaminoglycans in the epidermis and dermis were seen histologically. A molecule was invented (MPG) that could be formulated in a neutral vehicle to avoid skin irritation and produced improvements in xerosis and photoaging.

Regional variation in content, composition and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and transmission electron microscopy

Epidermis and oral epithelia provide permeability barriers that limit penetration of potentially harmful agents. Barrier function is determined by lipids in the superficial epithelial layers and varies regionally by more than 10-fold. The purpose of this study was to determine whether differences in lipid content, composition or organization could account for this variation in barrier function. Stratum corneum from skin, gingiva and palate and superficial layers from buccal regions and the floor of the mouth were isolated, and lipids were extracted and analysed by thin-layer chromatography. Tissue from each region was examined by electron microscopy. There was an inverse correlation between permeability and ceramide content and a direct correlation with triglyceride content. Electron microscopy revealed that the intercellular space in epidermal stratum corneum contained multiple lipid lamellae displaying an alternating broad-narrow-broad spacing. In palatal and gingival stratum corneum, uniformly spaced lamellae were present at the periphery of dilations of the intercellular space, but the interiors of the dilations contained disorganized lamellae and electron-dense material. In the non-keratinized barriers, there was a single, broad lamella at the cell periphery and occasional short stacks of lamellae traversing the intercellular space. These intercellular lamellae may be derived from a population of membrane-coating granules that contain internal lamellae. The results suggest that ceramides may be important barrier components, even in non-keratinizing epithelia where they are very minor components. Regional differences in the physical organization of barrier lipids may also contribute to differences in barrier function.

Organization of the intercellular spaces of porcine epidermal and palatal stratum corneum: a quantitative study employing ruthenium tetroxide

Previous studies have demonstrated that the intercellular spaces of the stratum corneum contain multilamellar lipid sheets with variable ultrastructure in addition to desmosomes or desmosomal remnants. The intercellular lamellae are thought to provide a permeability barrier whereas the desmosomes are responsible for cell-cell cohesion. In this study, transmission electron microscopy of RuO4-fixed tissue was used to compare the proportions of the intercellular spaces in epidermal and palatal stratum corneum occupied by desmosomes and by different patterns of lamellae. Desmosomes are more abundant in palatal than in epidermal stratum corneum (46.9 vs 15.0% length of intercellular space). In epidermis the most frequent lamellar arrangements involve 3 (23.5%) or 6 (24.2%) lucent bands with an alternating broad-narrow-broad pattern, whereas the most frequent lamellar arrangements in palatal tissue are 2 (17.2%) or 4 (10.5%) lucent bands of uniform width. Most of the nondesmosomal portion of the intercellular space in palatal stratum corneum was dilated and had elongated lamellae at the periphery and short disorganized lamellae and amorphous electron-dense material in the interior. It is concluded that the multilamellar lipid sheets are less extensive in palatal than in epidermal stratum corneum, which could explain the greater permeability of the palate.