Development of chicken epidermis cultured with embryo extract

The epidermis from 11-day-old chick embryo shank skin was cultured with 11-day-old chick embryo extract. The growth and the differentiation of the epidermis in culture were studied histologically, electron microscopically and with polyacrylamide gel electrophoresis of keratin proteins. The epidermis cultured with the chick embryo extract proliferated and stratum structures developed simultaneously with the increase in epidermal cell layers. Finally, a keratinized layer was observed after 10 days in culture. Electron microscopic observations revealed that tonofilaments were produced after 2 days in culture and increased thereafter with culture time, becoming condensed with desmosomes. Keratohyaline granules were observed in 7-day cultures. These keratinization characteristics occurring during culture showed the general characteristics of the alpha stratum observed in the skin of in ovo embryos during the early stages of development. However, the development of peridermal and subperidermal granules was poor and the stratum granulosum, which develops at the late stages between the stratum intermedium and the stratum corneum, was not observed. Polyacrylamide gel electrophoresis of S-carboxymethylated keratin proteins showed that the keratin protein band patterns of the culture differed from those of in ovo skin epidermis.     

白边油蝠表皮角质化细胞末端分化的超微结构

蝙蝠是一种唯一能够飞行的哺乳动物,其皮肤的超微结构尚未见报道。在电镜下观察了白边油蝠(Pipistrellus kuhlii)背部和翼膜皮肤的超微结构。表皮的厚度较低(10~12μm),角质层下有1~2层的刺细胞,该刺细胞由相似于鸟类无羽表皮的纤细角化细胞形成。颗粒层不连续且仅有少量小型透明角质颗粒(<0.3μm)。在翼膜的若干区域,表皮简化为一层与角质层相连的基底层。过渡期的角化细胞几乎不存在,提示其角质化过程非常迅速。基底膜上的无数半桥粒在真皮下面形成密集的附着点。大量胶原纤维直接维系在半桥粒和基底膜的致密层上,稀疏的弹性纤维使得蝙蝠表皮在飞行时易于伸展、在飞行后易于迅速折叠而不会受到损伤。与鸟类的表皮相似,蝙蝠角化细胞富有大量的脂质。由于脂质有助于蝙蝠皮肤在飞行中与冷空气流的传热绝缘,大量脂质的存在可能是为补偿蝙蝠翼膜的真皮缺乏厚的脂肪层。研究还表明,毛发较薄(4~7μm),并具有与皮层相似的突状物组成的精细表皮,其表皮细胞形成钩状抓握点使毛发紧紧粘结在一起,通过这种方式毛皮保持紧凑以恒定体温。     

CD44 standard and variant isoform expression in normal human skin appendages and epidermis

 CD44 isoforms have been implicated in tumor progression and metastasis formation. This study presents a thorough immunohistochemical analysis of CD44 standard and isoform expression in normal human skin appendages and epidermis applying monoclonal antibodies against CD44s, CD44v3, -v4, -v5, -v6, and -v9. An improved immunohistochemical protocol with microwave-based antigen retrieval in paraffin sections and heavy metal amplification of the diaminobenzidine reaction product provided enhanced resolution and sensitivity as compared to studies on frozen sections. The hair follicle, the seborrheic and eccrine sweat glands were strongly positive for all CD44 isoforms studied. In the latter, the clear cells but not the dark (intercalated) cells were positive. The sudoriferous ducts adjacent to the glands were weakly positive for all CD44 isoforms and strongly positive near the skin surface. In the apocrine glands, the basal cells showed only a moderate positivity. The myoepithelial cells expressed only CD44s. In the epidermis, all CD44 isoforms were detectable, with strongest CD44 immunostaining in the lower third of the stratum spinosum and weaker staining in the stratum basale and the upper two-thirds of the stratum granulosum. The stratum granulosum and corneum were unreactive. Thus, a regional and cell type-specific CD44 expression was revealed. Accepted: 10 May 1996     

SD大鼠皮肤组织病理学观察

目的观察不同日龄SD大鼠皮肤组织学结构。方法10％甲醛固定,行石蜡切片,HE染色。结果新生大鼠皮肤较薄,透明层缺乏,皮脂腺发育良好。6月龄时表皮、真皮和皮下组织明显增厚,毛囊增粗,生长旺盛,毛囊深入皮下脂肪层。24月龄时,大鼠皮脂腺及汗腺萎缩,表皮变薄,真皮成纤维细胞、血管数量减少,弹力纤维变细。结论不同日龄SD大鼠皮肤组织学结构有差异。     

Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis

Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains omega-hydroxy very long chain fatty acids (C30-C36). These omega-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular omega-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free omega-hydroxy ceramides increased 4-fold and omega-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier.     

Human tissue kallikreins as promiscuous modulators of homeostatic skin barrier functions

Human tissue kallikreins (KLKs) are the largest family of secreted serine protease endopeptidases encoded by 15 genes clustered on chromosome 19q13.4. Multiple KLK enzymes are co-localized in the upper stratum granulosum and stratum corneum of human epidermis, and in associated appendages such as hair follicle epithelia and sweat glands. Until recently, kallikrein proteolytic activity in the skin was exclusively attributed to KLK5 and KLK7. However, wider cutaneous roles of kallikreins became evident in recent years as the proposal of KLK proteolytic activation cascades emerged. We postulate that these proteolytic enzymes may serve as promiscuous mediators of different skin barrier functions, since they are capable of proteolysing different substrates that govern skin desquamation, antimicrobial defense, and lipid permeability. Growing evidence now attests to potential kallikrein involvement in skin inflammation, pigmentation, and tumor suppression via their ability to target proteinase-activated receptor signaling pathways. Current knowledge on kallikrein roles in skin physiology and pathobiology is described in this review.     

Characterization of metamorphic changes in anuran larval epidermis using lectins as probes

The skin of an adult frog of Xenopus laevis was characterized by the reactivity of 20 lectins. The lectins were classified into six groups in their binding to the epidermal cells: Lycopersicon esculentum lectin (LEL)-type which was positive for all epidermal cells; Pisum sativum agglutinin (PSA)-type for stratum germinativum; succinylated wheat germ agglutinin (sWGA)-type for strata spinosum, granulosum and corneum; Dolichos biflorus agglutinin (DBA)-type for strata germinativum and spinosum; peanut agglutinin (PNA)-type for stratum spinosum; and Ulex europaeus agglutinin (UEA-I)-type for strata granulosum and corneum. PSA and sWGA were utilized as markers of mitotically active germinative cells and the differentiated cells of the epidermis, respectively, to describe the metamorphic conversion of larval epidermal cells to adult type. PSA stained all epidermal cells of tadpoles before metamorphic climax. At the end of metamorphosis, PSA-positive cells were restricted to cells in the basal layer of body epidermis while all the tail epidermis remained PSA-positive. The other cell marker, sWGA, only stained apical cells in tadpole epidermis. During the metamorphic climax, sWGA-positive cells appeared in the cells beneath the stratum corneum of the body region, but not in the tail region. The present study demonstrates that PSA and sWGA are useful to investigate metamorphic changes in tadpole epidermal cells.     

In vitro indentation to determine the mechanical properties of epidermis

The lack of understanding of the mechanical behavior of the human skin layers makes the development of drug delivery using microneedles or microjets a challenging task. In particular, the key mechanical properties of the epidermis composed of stratum corneum and viable epidermis should be better understood. Micro-indentation experiments were applied, using a spherical tip with a large diameter to the sample thickness ratio. The Young's moduli were derived via an analytical and a numerical method. The tests showed that the analytical method was not appropriate to assess the Young's moduli. That is why a numerical model was used to obtain the correct stiffness. When loaded perpendicularly, the stiffness of both the epidermis and stratum corneum vary between 1 and 2MPa. No significant differences in stiffness between the stratum corneum and viable epidermis were observed.     

Effects of the sheep-chewing louse (Damalinia ovis) on the epidermis of the Australian merino

Frozen longitudinal skin sections taken from the dorsal thoraco-lumbar region of adult Merino sheep that were infested with the sheep-chewing louse were examined by light microscopy. The epidermis of infested sheep exhibited acanthosis due to hyperplasia of the stratum spinosum, and orthokeratosis. The thickness of the uncornified epidermis, the stratum corneum, and the sudanophilic region were significantly greater (P less than 0.005) than equivalent regions in louse-free Merinos and the results suggest that a positive correlation exists between the thickness of each region and the level of louse infestation. The results indicate that the variance in region thickness was greater in lousy than in louse-free sheep (P less than 0.005).     

Biological and physical factors influencing the successful engraftment of a cultured human skin substitute

Skin tissue may be engineered in a variety of ways. Our cultured skin substitute (Graftskin, living skin equivalent or G-LSE), Apligraftrade mark, is an organotypic culture of skin, containing both a "dermis" and "epidermis." The epidermis is an important functional component of skin, responsible for biologic wound closure. The epidermis possesses a stratum corneum which develops with time in culture. The stratum corneum provides barrier function properties and gives the LSE improved strength and handling characteristics. Clinical experience indicated that the stratum corneum might play an important role in improving the clinical utility of the LSE. Handling and physical characteristics improved with time in culture. We examined the LSE at different stages of epidermal maturation for barrier function and ability to persist as a graft. LSE grafted onto athymic mice before significant development of barrier function did not withstand bandage removal at 7 days postgraft. LSE grafted after barrier function had been established in vitro were able to withstand bandage removal at day 7. Corneum lipid composition and structure are critical components for barrier function. Media modifications were used in an attempt to improve the fatty acid composition of the stratum corneum. The barrier developed more rapidly and was improved in a serum-free, lipid-supplemented condition. Lipid lamellar structure was improved with 10% of the stratum corneum exhibiting broad-narrow-broad lipid lamellar arrangements similar to human skin. Fatty acid metabolism was not appreciably altered. Barrier function in vitro was 4- to 10-fold more permeable than human skin. Epidermal differentiation does not compromise engraftment or the wound healing ability of the epidermis. The stratum corneum provides features beneficial for engraftment and clinical use. (c) 1996 John Wiley & Sons, Inc.     

A Spontaneous Fatp4/Scl27a4 Splice Site Mutation in a New Murine Model for Congenital Ichthyosis

Congenital ichthyoses are life-threatening conditions in humans. We describe here the identification and molecular characterization of a novel recessive mutation in mice that results in newborn lethality with severe congenital lamellar ichthyosis. Mutant newborns have a taut, shiny, non-expandable epidermis that resembles cornified manifestations of autosomal-recessive congenital ichthyosis in humans. The skin is stretched so tightly that the newborn mice are immobilized. The genetic defect was mapped to a region near the proximal end of chromosome 2 by SNP analysis, suggesting Fatp4/Slc27a4 as a candidate gene. FATP4 mutations in humans cause ichthyosis prematurity syndrome (IPS), and mutations of Fatp4 in mice have previously been found to cause a phenotype that resembles human congenital ichthyoses. Characterization of the Fatp4 cDNA revealed a fusion of exon 8 to exon 10, with deletion of exon 9. Genomic sequencing identified an A to T mutation in the splice donor sequence at the 3′-end of exon 9. Loss of exon 9 results in a frame shift mutation upstream from the conserved very long-chain acyl-CoA synthase (VLACS) domain. Histological studies revealed that the mutant mice have defects in keratinocyte differentiation, along with hyperproliferation of the stratum basale of the epidermis, a hyperkeratotic stratum corneum, and reduced numbers of secondary hair follicles. Since Fatp4 protein is present primarily at the stratum granulosum and the stratum spinosum, the hyperproliferation and the alterations in hair follicle induction suggest that very long chain fatty acids, in addition to being required for normal cornification, may influence signals from the stratum corneum to the basal cells that help to orchestrate normal skin differentiation.     

Functional organization of the bovine rumen epithelium

The functional organization of the bovine rumen epithelium has been examined by electron and light microscopy combined with immunocytochemistry to define a transport model for this epithelium. Expression of connexin 43, an integral component of gap junctions, the tight-junction molecules claudin-1 and zonula occludens 1 (ZO-1), and the catalytic alpha-subunit of Na(+)-K(+)-ATPase was demonstrated by SDS-PAGE and Western blotting. From the lumen surface, four cell layers can be distinguished: the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale. Both claudin-1 and ZO-1 immunostaining showed plasma membrane staining, which was present at the stratum granulosum with decreasing intensity through the stratum spinosum to the stratum basale. The stratum corneum was negative for claudin-1 immunostaining. Transmission electron microscopy confirmed that occluding tight junctions were present at the stratum granulosum. Plasma membrane connexin 43 immunostaining was most intense at the stratum granulosum and decreased in intensity through stratum spinosum and stratum basale. There was intense immunostaining of the stratum basale for Na(+)-K(+)-ATPase, with weak staining of the stratum spinosum. Both the stratum granulosum and the stratum corneum were essentially negative. Stratum basale cells also displayed a high mitochondrial density relative to more apical cell layers. We conclude that epithelial barrier function may be attributed to the stratum granulosum and that cell-cell gap junctions allow diffusion to interconnect the barrier cell layer with the stratum basale where Na(+)-K(+)-ATPase is concentrated.     

Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient

Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (< or =50 microm x 50 microm) are acquired every 1.7 microm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 microm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 microm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the stratum corneum increases with depth because of a decrease in the ratio of acidic to neutral regions within the stratum corneum. The data definitively show that the stratum corneum acid mantle results from the presence of aqueous acidic pockets within the lipid-rich extracellular matrix.     

Factors controlling the expressed activity of histidine ammonia-lyase in the epidermis and the resulting accumulation of urocanic acid.

The synthesis of urocanic acid by histidine ammonia-lyase in guinea-pig epidermis was investigated in various ways. 1. In epidermal homogenates the enzyme obeys Michaelis-Menten kinetics and shows marked dependence of its activity of pH, such that below pH 6 it is inactive. 2. Part-thickness skin samples cultured with radioactive histidine do not accumulate detectable radioactive urocanic acid during 3 days in culture. 3. Very little histidine ammonia-lyase activity can be detected in the living cells of the epidermis. The enzyme is almost completely restricted to the dead cells of the stratum corneum. 4. Radioactive histidine injected into living animals does not result immediately in the accumulation of radioactive urocanic acid. By 3 days after the injection, however, both radioactive urocanic acid and histidine appear, apparently at the expense of radioactive proteins, 5. In isolated stratum corneum, the residual histidine can be converted into urocanic acid by the histidine ammonia-lyase in the tissue only if the natural acidity of the tissue is neutralized. It is concluded from these observations that the biosynthesis of urocanic acid occurs naturally only in the stratum corneum, which contains only dead cells. The amount of urocanic acid accumulated is limited by the availability of free histidine produced by proteolysis of residual protein in these cells and by the penetration into the stratum corneum of the 'acid mantle' of the skin.     

Ontogenetic variation in the stratum granulosum of the epidermis of Chaetophractus vellerosus (Xenarthra,Dasypodidae) in relation to the development of cornified scales

The epidermis of mammals is characterized by having a stratum granulosum that produces an orthokeratotic stratum corneum, different from the typical reptilian parakeratotic stratum. Nonetheless, some mammals show distinct degrees of parakeratosis in epidermal regions with few or no pilose follicles (e.g., areas subjacent to cornified scales). With respect to the epidermis and the development of cornified scales in the Dasypodidae, previous studies have supported the presence of a continuous stratum granulosum without any variations during ontogeny. This condition, in which the cornified scales develop without a loss of the stratum granulosum, was interpreted as primitive for eutherians. The present contribution expands the knowledge on the epidermis of Chaetophractus vellerosus in distinct ontogenetic stages in order to determine whether the cornified scales show the same developmental pattern as in other eutherians. The presence of a stratum granulosum in C. vellerosus neonates and its reduction in more advanced ontogenetic stages, in direct relationship with cornified scale development, supports the hypothesis that the partial parakeratosis in the xenarthran integument is secondary, as in other eutherians, and can be interpreted as a derived character state.     

Effect of a sake concentrate on the epidermis of aged mice and confirmation of ethyl alpha-D-glucoside as its active component

Generations of Japanese have appreciated the positive effects that sake can have on skin conditions, and studies have shown that concentrated sake suppressed the epidermal barrier disruption caused by ultraviolet B (UVB) irradiation. We investigated the effect of a topical application of a sake concentrate on the murine epidermis and found that the intercellular lipid content in an aged epidermis was significantly increased. Furthermore, the topical application of ethyl alpha-D-glucoside (alpha-EG), a component of sake, brought about a similar improvement in the levels of intercellular lipids. Following on from this, we confirmed that alpha-EG also significantly increased the content of loricrin protein, an indicator of successful corneocyte differentiation, while reducing the number of corneocyte layers in the aged stratum corneum. These results confirmed alpha-EG as the primary active component of the sake concentrate that had a positive effect on the epidermis. alpha-EG increased the intercellular lipid content, accelerated the differentiation of corneocytes, and reduced the thickness, thus improving the functions of the stratum corneum.     

Human epidermal lipids: characterization and modulations during differentiation

Using thin-layer chromatography and glass capillary gas-liquid chromatography, we have quantitated the lipids in the germinative, differentiating, and fully cornified layers in human epidermis. As previously noted in nonhuman species, we found progressive depletion of phospholipids coupled with repletion of sterols and sphingolipids during differentiation. The sphingolipids, present only in small quantities in the lower epidermis, accounted for about 20% of the lipid in the stratum corneum, and were the major repository for the long-chain fatty acids that predominate in the outer epidermis. Although the absolute quantities of sphingolipids increased in the outer epidermis, the glycolipid:ceramide ratio diminished in the stratum corneum, and glycolipids virtually disappeared in the outer stratum corneum. Squalene and n-alkanes were distributed evenly in all epidermal layers, suggesting that these hydrocarbons are not simply of environmental or pilosebaceous origin. Cholesterol sulfate, previously considered only a trace metabolite in epidermis, was found in significant quantities, with peak levels immediately beneath the stratum corneum in the stratum granulosum. These studies: 1) provide new quantitative data about human epidermal lipids; 2) implicate certain classes of lipids for specific functions of the stratum corneum; and, 3) shed light on possible product-precursor relationships of these lipids.     

Gap junctions in the epidermis of fetal rats studied by transmission electron microscopy

In the ventral epidermis of fetal rats the size and distribution of intercellular gap junctions changed during differentiation. In the young fetus, between 13 and 17 days, large gap junctions sometimes exceeding 3 micron in profile length were found predominantly in basal cells. As the epidermis increased in thickness the mean profile length diminished until only small gap junctions were present mainly in more superficial layers even persisting into the stratum corneum. Endocytosis of the intercellular gap junctions gave rise to intracytoplasmic annular gap junctions (AGJs) which occurred after 17 days predominantly in the superficial three layers of the epidermis. The AGJs diminished in mean diameter with the age of the fetuses possibly as a consequence of the decreasing size of the intercellular gap junctions from which they had formed. Rarely sequestration of AGJs by cytoplasmic membranes occurred but many recognizable AGJs persisted into the stratum corneum. As in other developing systems, the function of gap junctions in epidermis is unknown but the extensive junctions of younger epidermis might be related to the maintenance of a greater level of uniformity both of mitotic activity and of differentiation.     

Effects of intradermally injected and topically applied mouse epidermal growth factor on wool growth, skin and wool follicles of merino sheep

Twice daily intradermal (ID) injections of mouse epidermal growth factor (mEGF) in sterile saline for 1-4 days into delineated areas of skin of Merino sheep produced dose-dependent changes in wool follicles and fibres, ranging from slight reduction in follicle bulb size and transient disturbance of cuticle formation on some fibres to the induction of catagen of follicles and shedding of fibres with distorted, tapered ends. Regeneration of follicles commenced by day 7. By contrast, ID injections of saline did not affect follicle activity. The epidermis became thicker and more parakeratotic after multiple injections of mEGF than after injection of saline, but was almost normal again by day 14. Persistent small increases in sebaceous gland size, additional to those induced by ID injections of saline, and delayed small increases in sweat gland size also occurred after multiple injections of mEGF. Daily topical applications of mEGF in 50% (v/v) aqueous propylene glycol 5 days each week for 4 weeks did not affect wool growth or the follicles and other skin components. The only effect observed, due to application of the aqueous propylene glycol, was an increase in the number of layers of cornified cells in the stratum corneum of the epidermis, with the cells arranged in clearly discernible stacks. The effects produced by ID injections of mEGF indicate that mEGF acts directly on the pilosebaceous and epidermal components of skin.     

Epidermal differentiation: the role of proteases and their inhibitors

Dermatological diseases range from minor cosmetic problems to life-threatening conditions, as seen in some severe disorders of keratinization and cornification. These disorders are commonly due to abnormal epidermal differentiation processes, which result in disturbed barrier function of human skin. Elucidation of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis is therefore of great importance for the understanding and therapy of these disorders. Much of the barrier function of human epidermis against the environment is provided by the cornified cell envelope (CE), which is assembled by transglutaminase (TGase)-mediated cross-linking of several structural proteins and lipids during the terminal stages of normal keratinocyte differentiation. The major constituents of the stratum corneum and the current knowledge on the formation of the stratum corneum will be briefly reviewed here. The discovery of mutations that underlie several human diseases caused by genetic defects in the protein or lipid components of the CE, and recent analyses of mouse mutants with defects in the structural components of the CE, catalyzing enzymes, and lipid processing, have highlighted their essential function in establishing the epidermal barrier. In addition, recent findings have provided evidence that a disturbed protease-antiprotease balance could cause faulty differentiation processes in the epidermis and hair follicle. The importance of regulated proteolysis in epithelia is well demonstrated by the recent identification of the SPINK5 serine proteinase inhibitor as the defective gene in Netherton syndrome, cathepsin C mutations in Papillon-Lefevre syndrome, cathepsin L deficiency infurless mice, targeted ablation of the serine protease Matriptase/MTSP1, targeted ablation of the aspartate protease cathepsin D, and the phenotype of targeted epidermal overexpression of stratum corneum chymotryptic enzyme in mice. Notably, our recent findings on the role of cystatin M/E and legumain as a functional dyad in skin and hair follicle cornification, a paradigm example of the regulatory functions exerted by epidermal proteases, will be discussed.