Significant decreases in the intensity of staining for proteins and protein thiols in basal-cell epitheliomas (basaliomas) as compared to normal skin

Microphotometric measurements of fast reacting protein thiols (PSHr) and proteins were performed on freshly frozen sections of samples from normal skin (26 cases as controls) and from 45 basal cell epitheliomas (basalioma; BCE). The intensity of the staining (E/micron2) for both proteins and PSHr was significantly higher in normal epidermis than in the adjacent dermis. The values of QE (quotient of values observed in the epidermis divided by those observed in the dermis) were calculated to be 3.48 for proteins (QE, Prot) and 4.62 for PSHr (QE, PSHr). In cases of BCE, significantly lower QE values were found: QE, Prot = 2.16 and QE, PSHr = 1.72. The decrease of QE, PSHr was due to a decrease in the staining intensity observed in the BCEs, whereas practically no changes occurred in the adjacent dermis. The decrease of QE, Prot was mainly caused by a decrease in the staining intensity in the BCE (by 68%) as well as in the adjacent dermis (by 36%). By dividing the mean extinction value (E/micron2) for PSHr by the E/micron2 for proteins, a new quotient, PSHr/Prot, is obtained which can serve as a quantitative measure of the content of the tissue proteins of PSHr. The proteins of normal epidermis contained more PSHr than dermal proteins. The proteins of BCEs also contained more PSHr than those of the adjacent dermis, but the PSHr/Prot values of both tissues were 1.5 to 1.6 times greater than the corresponding values for normal epidermis and dermis, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melanin and dopa-positive cells in the skin of tropical cattle.

Various histochemical and histological techniques were used to study the melanin and dopa-positive cell distribution in the skin of some tropical and temperate breeds of cattle in Nigeria. Melanin pigments were concentrated in the basal and lower spinous layers of the epidermis and in the hair cortex, follicle sheaths and papillae of the various breeds. In the White Fulani and N'Dama breeds, melanin pigments were however found in all layers of the epidermis. Dopa-positive cells (melanocytes) were observed in the epidermis, dermis and hair follicles; the distribution pattern varied among breeds, being copiously disposed in the basal epidermis and papillary dermis in the White Fulani and Muturu and, except in areas of thick epidermal ridges, scanty in the epidermis and dermis of the Friesian and N'Dama. Mast cell distribution pattern in the various breeds was similar to that of the dopa-positive cells. Peroxidase-positive cells were present in the basal epidermis and upper dermis of the Muturu, widespread in the subepidermal layer of the N'Dama and very scanty in the dermis of the White Fulani and Friesian. Acid phosphatase activity was intense in the granular layer of the Muturu and N'Dama breeds and also in the papillary dermis and hair follicles, whereas alkaline phosphatase-positive dendritic cells, and 'clear' cells were also observed in the basal and upper epidermis.     

Analysis of morphogenesis and keratinization in transfilter recombinants of feather-forming skin

The relationships between feather morphogenesis, histogenesis, and biochemical differentiation were examined by recombining backskin epidermis and dermis, from chick embryos (Hamburger-Hamilton stages 27-31), with an intervening Nucleopore filter (pore size of 0.4 micron). The filter inhibited normal feather morphogenesis and histogenesis of barb ridges, yet feather-like filaments, which were free of dermal cells, formed from the epidermal cells. Using indirect immunofluorescence, with antiserum against alpha- and beta-keratins, the biochemical differentiation of the feather-like filaments was compared to normal feathers. In the feather-like filaments resulting from tissues of stages 27-29, cells containing beta keratins were occasionally seen at the periphery of the filaments, yet cells containing alpha-keratins were inappropriately located throughout the filaments. In a few feather-like filaments on recombinants resulting from tissues of stages 29.5-31, cells positive for beta-keratins were found in the center of the filament, but again alpha-keratins were also found. Surrounding these cells there were several layers of cells, arranged circumferentially, resembling sheath cells. Some sheath-like cells contained beta-keratins. We conclude that although feather epidermal cells, which are separated from their dermis by a Nuclepore filter, can undergo limited morphogenesis and the production of alpha- and beta-keratins, normal feather morphogenesis, histogenesis, and biochemical differentiation require the intimate associations of epidermis and dermis.     

A survey of polar and nonpolar lipids from epidermis and epidermal appendages of the chicken (Gallus domesticus)

Total lipid was extracted from chicken (Gallus domesticus) epidermis, leg scale, claws, feathers and preen glands and analyzed by quantitative thin-layer chromatography. All of the tissue lipids contained large proportions of wax diesters, triglycerides, and free sterols and variable proportions of phospholipids, steryl esters and free fatty acids. All of the keratinized tissues, but not the preen gland, contained ceramides, acylceramides and cholesteryl sulfate. Acylglucosylceramides were found only in full thickness epidermis. Glucosylsterols and acylglucosylsterols were found in the keratinized tissues, and may be of significance in the evolutionary history of the epidermal water barrier.     

Fibronectin involvement in granulation tissue and wound healing in rabbits

This study describes the distribution of fibronectin and its association with reticulin fibers (type III collagen) and hyaluronic acid in shallow rabbit wounds. Linear incisions were made dorsally with a surgical blade. Animals were sacrificed and 1,2,3,4,5, and 8 day wounds were examined using peroxidase-antiperoxidase to localize affinity-purified antibodies to fibronectin. Tissue samples were also stained with hematoxylin and eosin in addition to silver stains for reticulin, and Alcian blue for hyaluronic acid. After wounding, the incision filled with a fibrin clot that stained positively for fibronectin. The underlying dermis and adjacent, unwounded dermis also contained fibronectin. Epidermal cells that migrate from the wound margin between the clot and the dermis were in direct association with fibronectin in these wound components. By 72 hr, epidermal continuity was reestablished. Early granulation tissue formation was apparent just below the epidermis 5 day wounds. Fibronectin was observed in the matrix surrounding individual fibroblasts and codistributed with reticulin fibers and hyaluronic acid in both 5 and 8 day wounds. Granulation tissue of 8 day wounds stained intensely for fibronectin and extended to a greater depth in the reticular dermis. Dense fibrillar networks of fibronectin and fibroblasts were aligned parallel to the epidermis, giving the granulation tissue a highly structured and organized appearance. Fibroblasts contained fibronectin and were surrounded by less fibronectin at the wound periphery than within the granulation tissue. These findings suggest that fibronectin may be important in the reconstruction of tissues during repair by functioning as an extracellular scaffold for migrating cells.     

Differentiation of embryonic chick feather-forming and scale-forming tissues in transfilter cultures

The dermal-epidermal tissue interaction in the chick embryo, leading to the formation of feathers and scales, provides a good experimental system to study the transfer between tissues of signals which specify cell type. At certain times in development, the dermis controls whether the epidermis forms feathers or scales, each of which are characterized by the synthesis of specific beta-keratins. In our culture system, a dermal effect on epidermal differentiation can still be observed, even when the tissues are separated by a Nuclepore filter, although development is abnormal. Epidermal morphological and histological differentiation in transfilter cultures are distinct and recognizable, more closely resembling feather or scale development, depending on the regional origin of the dermis. Differentiation is more advanced when epidermis is cultured transfilter from scale dermis than from feather dermis, as assessed by morphology and histology, as well as the expression of the tissue-specific gene products, the beta-keratins. Two-dimensional polyacrylamide gel analysis of the beta-keratins reveals that scale dermis cultured transfilter from either presumptive scale or feather epidermis induces the production of 7 of the 9 scale-specific beta-keratins that we have identified. Feather dermis, although less effective in activating the feather gene program when cultured transfilter from either presumptive feather or scale epidermis, is able to turn on the synthesis of 3 to 6 of the 18 feather-specific beta-keratins that we have identified. However, scale epidermis in transfilter recombinants with feather dermis also continues to synthesize many of the scale-specific beta-keratins. Using transmission and scanning electron microscopy, we detect no cell contact between tissues separated by a 0.2-micron pore diameter Nuclepore filter, while 0.4-micron filters readily permit cell processes to traverse the filter. We find that epidermal differentiation is the same with either pore size filter. Furthermore, we do not detect a basement membrane in transfilter cultures, implying that neither direct cell contact between dermis and epidermis, nor a basement membrane between the tissues is required for the extent of epidermal differentiation that we observe.     

Purification and characterization of beta-galactoside-binding lectin from chick embryonic skin

Lectin activity was found in tarsometatarsal skin of chick embryo. It was specific for beta-linked galactosyl residues and required a thiol-reducing agent for hemagglutination activity. The lectin was extracted from dermis and epidermis (skin) with lactose and purified to apparent homogeneity by affinity chromatography on asialofetuin-Sepharose. Examination of their biochemical properties showed that although dermis and epidermis develop from different origins, they contain the same lectin. The apparent subunit Mr of lectin was 14000 and its isoelectric point was 7.0. Under non-dissociating conditions, the lectin exists mainly as a dimer. Radioimmunoassay showed that this skin-type lectin is present in many tissues including skin, muscle, bone, eye, heart, liver and brain at various developmental stages. A wide distribution and a marked change in its content during development strongly suggest that the lectin might have a fundamental role in cellular function, embryonic development and tissue differentiation.     

Embryonic dermal condensation and adult dermal papilla induce hair follicles in adult glabrous epidermis through different mechanisms

Hair induction in the adult glabrous epidermis by the embryonic dermis was compared with that by the adult dermis. Recombinant skin, composed of the adult sole epidermis and the embryonic dermis containing dermal condensations (DC), was transplanted onto the back of nude mice. The epidermis of transplants formed hairs. Histology on the induction process demonstrated the formation of placode-like tissues, indicating that the transplant produces hair follicles through a mechanism similar to that underlying hair follicle development in the embryonic skin. An isolated adult rat sole skin piece, inserted with either an aggregate of cultured dermal papilla (DP) cells or an intact DP between its epidermis and dermis, was similarly transplanted. The transplant produced hair follicles. Histology showed that the epidermis in both cases surrounded the aggregates of DP cells. The epidermis never formed placode-like tissues. Thus, it was concluded that the adult epidermal cells recapitulate the embryonic process of hair follicle development when exposed to DC, whereas they get directly into the anagen of the hair cycle when exposed to DP. The expression pattern of Edar and Shh genes, and P-cadherin protein during the hair follicle development in the two types of transplants supported the above conclusion.     

Depilated (dep), a Mutant Gene That Affects the Coat of the Mouse and Acts in the Epidermis

Depilated is a recessive mutation on Chromosome 4 in the position b^-1.93±0.51^- dep^-3.45±0.68.^-Pt. It causes severe abnormalities of hair structure. The site of action of dep was investigated by the method of dermal-epidermal recombination. Skins from 14-day mutant and normal mouse embryos were separated into dermal and epidermal components, recombined, and grown in histocompatible mouse testes for 20 days. The recombinations made were +/+ epidermis with +/+ dermis, +/+ epidermis with dep/dep dermis, dep/dep epidermis with +/+ dermis and dep/dep epidermis with dep/dep dermis. Grafts that contained mutant epidermis as one of the components produced hairs that were similar to those found in depilated mice. There was no observable effect of the dermis on hair types produced in this experiment.     

Region-specific deposition of dermal proteins between dermis and epidermis during induction of chick feather and scale rudiments

To begin to study the role of particular proteins in inductive tissue interactions, we have used density labelling techniques to determine whether any dermal proteins are found between embryonic chick dermis and epidermis at a stage when the dermis plays an important inductive role in epidermal differentiation. Epidermis will form feathers or scales depending on whether it interacts with dorsal or foot dermis, respectively, and the dermis can still influence epidermal differentiation when direct cell contact between the tissues is blocked by a membrane filter during culturing (Peterson & Grainger, 1985). In transfilter experiments, we detect a subset of dermal proteins within the filter between the tissues. Several of these dermal proteins are deposited in a region-specific manner, that is, they are only found associated with filters from either dorsal or foot dermis. We have previously shown that the expression of some of these proteins is specific to particular regions of dermis and is also associated with the inductive potential of the dermis (Peterson & Grainger, 1986). We detect only 17 dermal proteins which are transferred across the filter in these cultures and found in direct association with epidermis; of these 14 are common to both dorsal and foot dermis, and 3 are deposited in a region-specific manner. Our results lead us to hypothesize a significant function for certain dermal proteins in this inductive interaction either as part of the extracellular matrix or in direct association with epidermis.     

Structural and functional evaluation of modifications in the composite skin graft: cryopreserved dermis and cultured keratinocytes.

Structural and functional aspects of modifications in the composite skin graft consisting of cultured keratinocytes and cryopreserved dermis were determined. Cryopreserved human cadaveric dermis separated from skin by short and mild trypsinization was compared with dermis obtained by prolonged incubation in medium and with fresh dermis obtained by the same methods. All types of dermis were shown to retain normal ultrastructure and topographic organization, as detected by scanning and transmission electron microscope and immunofluorescence analysis. However, in fresh skin, the layers were more firmly attached, mechanical separation was more difficult, and residual epidermis often remained attached to the dermis. Keratinocytes attached better, began replication earlier, and generally reached higher cell numbers when cultured on trypsinized dermis than on medium-treated dermis. The performance of several modifications in the reconstitution and grafting procedures of the composite skin graft after transplantation to athymic mice was examined. Cultured epidermis combined onto trypsinized or medium-treated whole and meshed dermis, dermis pregrafted and allowed to take before transplanting epidermis on top, and keratinocytes grown into multiple epithelia on top of trypsinized meshed or whole dermis prior to grafting. The best grafting results were obtained with an "instant" reconstituted skin model: multiple epithelia grown in vitro combined immediately before grafting onto meshed trypsinized dermis. The transplantation results of this modification were significantly better than those of all the other modifications, including initial growth of keratinocytes into multiple epithelia on top of trypsinized dermis prior to grafting.     

An Ex Vivo Study of Congenital Pigmented Nevi in Epidermal Reconstructs

In order to study morphologic and functional characteristics of pigment cells in congenital pigmented nevi, autologous or heterologous reconstructs have been made using normal keratinocytes and nevus cells from the dermal-epidermal junction or from the dermis. All these cells, keratinocytes and nevus cells, were used as cell suspensions immediately after dissociation from the tissues or after subsequent brief cultivation in a serum-free medium. Reconstructed epidermis were cultured for 15 days at the air-liquid interface with or without ultraviolet (UV) B exposure. The reconstructs were examined macroscopically (formation of hyperpigmented macules), histologically (pigment cell nesting) and ultrastructurally (pigment structure and transfer). Typical nesting of nevus cells was observed in the dermal-epidermal junction or in the superficial dermis associated with macroscopically detectable small pigmented macules. UVB exposure induced an upward migration of nevus cells in the suprabasal layers of the epidermis. This tissue model can be considered as an excellent system for the ex vivo reproduction of pigmented nevi and as an assay of the sensitivity of nevus cells towards UVB irradiation.     

An ex vivo study of congenital pigmented nevi in epidermal reconstructs.

In order to study morphologic and functional characteristics of pigment cells in congenital pigmented nevi, autologous or heterologous reconstructs have been made using normal keratinocytes and nevus cells from the dermal-epidermal junction or from the dermis. All these cells, keratinocytes and nevus cells, were used as cell suspensions immediately after dissociation from the tissues or after subsequent brief cultivation in a serum-free medium. Reconstructed epidermis were cultured for 15 days at the air-liquid interface with or without ultraviolet (UV) B exposure. The reconstructs were examined macroscopically (formation of hyperpigmented macules), histologically (pigment cell nesting) and ultrastructurally (pigment structure and transfer). Typical nesting of nevus cells was observed in the dermal-epidermal junction or in the superficial dermis associated with macroscopically detectable small pigmented macules. UVB exposure induced an upward migration of nevus cells in the suprabasal layers of the epidermis. This tissue model can be considered as an excellent system for the ex vivo reproduction of pigmented nevi and as an assay of the sensitivity of nevus cells towards UVB irradiation.     

The keratin polypeptide patterns in heterotypically recombined epithelia of skin and mucosa of adult mouse

Epithelial-mesenchymal interactions were investigated considering both morphologic criteria and keratin polypeptide expression in homotypic and heterotypic recombinants of adult mouse skin and oral mucosa. Two series of cross-recombinants of epithelia with different morphology and keratin patterns were chosen: (a) footpad epidermis/ear dermis and ear epidermis/footpad dermis; (b) palate epithelium/cheek connective tissue and cheek epithelium/palate connective tissue. Homotypic and heterotypic recombinants were prepared after EDTA-separation of the original tissues and then grown on syngeneic mice in subcutaneously prepared protected graft chambers. EDTA-separation is especially suited to completely separate the epidermal-dermal union, and the transplantation procedure used strictly prevents contamination with host epithelium. Five weeks after implantation keratins were analyzed by one and two-dimensional gel electrophoresis and peptide mapping. In both series, homotypic recombination of the tissues did not alter the original morphology and keratin polypeptide composition of the individual epithelial components. Ear epidermis displayed no significant changes in structure or keratin pattern in heterotypic recombinants. Recombined with ear dermis, footpad epidermis showed acquisition of some morphologic features typical for ear epidermis and slight changes in keratin composition which were, however, difficult to interpret due to the normal similarities of footpad keratin with that of ear. In contrast, the heterorecombinants of the palate/cheek series exhibited considerable alterations in their keratin patterns. Either epithelium showed suppression of distinct keratin subunits and de novo expression of subunits characteristic of the epithelium normally associated with the connective tissue component. The keratin patterns of both matches closely resembled each other and represented patterns intermediate between the normal patterns. This partial, however, significant modulation in the expression of differentiation markers was paralleled by similarly directed changes in the architecture of the heterotransplanted tissues, thus indicating that both morphogenesis and cytodifferentiation of certain adult epithelia can be influenced by extrinsic mesenchymal factors.     

Avian scale development. XIII. Epidermal germinative cells are committed to appendage-specific differentiation and respond to patterned cues in the dermis.

The ability of the germinative cell population of scutate scale epidermis to continue to generate cells that undergo their appendage-specific differentiation (beta stratum formation), when associated with foreign dermis, was examined. Tissue recombination experiments were carried out which placed anterior metatarsal epidermis (scutate scale forming region) from normal 15-day chick embryos with either the anterior metatarsal dermis from 15-day scaleless (sc/sc) embryos or the dermis from the metatarsal footpad (reticulate scale forming region) of 15-day normal embryos. Neither of these dermal tissues are able to induce beta stratum formation in the simple ectodermal epithelium of the chorion, however, the footpad dermis develops an appendage-specific pattern during morphogenesis of the reticulate scales, while the sc/sc dermis does not. Morphological and immunohistological criteria were used to assess appendage-specific epidermal differentiation in these recombinants. The results show that the germinative cell population of the 15-day scutate scale epidermis is committed to generating suprabasal cells that follow their appendage-specific pathways of histogenesis and terminal differentiation. Of significance is the observation that the expression of this determined state occurred only when the epidermis differentiated in association with the footpad dermis, not when it was associated with the sc/sc dermis. The consistent positioning of the newly generated beta strata to the apical regions of individual reticulate-like appendages demonstrates that the dermal cues necessary for terminal epidermal differentiation are present in a reticulate scale pattern. The observation that beta stratum formation is completely missing in the determined scutate scale epidermis when associated with the sc/sc dermis adds to our understanding of the sc/sc defect. The present data support the conclusion of earlier studies that the anterior metatarsal dermis from 15-day sc/sc embryos lacks the ability to induce beta stratum formation in a foreign epithelium. In addition, these observations evoke the hypothesis that the sc/sc dermis either lacks the cues (generated during scutate and reticulate scale morphogenesis) necessary for terminal differentiation of the determined scutate scale epidermis or inhibits the generation of a beta stratum.     

Sensory reinnervation and sensibility in skin transplants

Reinnervation and sensibility were investigated in patients previously subjected to split thickness skin transplantation after excision of skin and subcutaneous fat for treatment of malignant melanoma. All patients except one showed poor sensibility and no neurofilament positive sensory nerve fibres were detected. Most patients had heat and pain sensibility but with a higher threshold than on the control side. It is suggested that this heat pain sensibility is mediated from nerve fibres in the underlying tissue. In one patient, however, sensibility was encountered and in this patient neurofilament positive sensory nerve fibres were observed in the dermis and epidermis.     

Significant decreases in the intensity of staining for proteins and protein thiols in basal-cell epitheliomas (basaliomas) as compared to normal skin

Summary Microphotometric measurements of fast reacting protein thiols (PSHr) and proteins were performed on freshly frozen sections of samples from normal skin (26 cases as controls) and from 45 basal cell epitheliomas (basalioma; BCE).The intensity of the staining (/m²) for both proteins and PSHr was significantly higher in normal epidermis than in the adjacent dermis. The values of QE (quotient of values observed in the epidermis divided by those observed in the dermis) were calculated to be 3.48 for proteins (QE, Prot) and 4.62 for PSHr (QE, PSHr). In cases of BCE, significantly lower QE values were found: QE, Prot=2.16 and QE, PSHr=1.72. The decrease of QE, PSHr was due to a decrease in the staining intensity observed in the BCEs, whereas practically no changes occured in the adjacent dermis. The decrease of QE, Prot was mainly caused by a decrease in the staining intensity in the BCE (by 68%) as well as in the adjacent dermis (by 36%). By dividing the mean extinction value (/m²) for PSHr by the (/m²) for proteins, a new quotient, PSHr/Prot, is obtained which can serve as a quantitative measure of the content of the tissue proteins of PSHr. The proteins of normal epidermis contained more PSHr than dermal proteins. The proteins of BCEs also contained more PSHr than those of the adjacent dermis, but the PSHr/Prot values of both tissues were 1.5 to 1.6 times greater than the corresponding values for normal epidermis and dermis, respectively. As essentially similar results have previously been obtained for carcinoma of the uterine cervix, it is assumed that the decrease in PSHr in malignant epithelium (or epidermis) might possibly be a more general expression of malignancy.     

Purification and characterization of β-galactoside-binding lectin from chick embryonic skin

Lectin activity was found in tarsometatarsal skin of chick embryo. It was specific for β-linked galactosyl residues and required a thiol-reducing agent for hemagglutination activity. The lectin was extracted from dermis and epidermis (skin) with lactose and purified to apparent homogeneity by affinity chromatography on asialofetuin-Sepharose. Examination of their biochemical properties showed that although dermis and epidermis develop from different origins, they contain the same lectin. The apparent subunit M_r of lectin was 14 000 and its isoelectric point was 7.0. Under non-dissociating conditions, the lectin exists mainly as a dimer. Radioimmunoassay showed that this skin-type lectin is present in many tissues including skin, muscle, bone, eye, heart, liver and brain at various developmental stages. A wide distribution and a marked change in its content during development strongly suggest that the lectin might have a fundamental role in cellular function, embryonic development and tissue differentiation.     

Effect of diethylstilbestrol on skin sterols of the male rat

Diethylstilbestrol (DES) was injected in doses ranging from 600 micro g to 0.4 micrograms/kg body weight into mature male rats over a 3 wk period. Profound effects on skin morphology and on sterol content of skin were noted. The sebaceous glands atrophied and the epidermis lost granularity. The concentrations of all skin sterols, with the exception of cholesterol, were reduced. At a dose level of DES of 4 micrograms/kg there was still a perceptible reduction in the concentration of Delta(7)-cholestenol. Incubation of skin fragments with acetate-2-(14)C for 2 hr demonstrated a reduced uptake of (14)C into the nonsaponifiable fraction of skin lipids at all dose levels studied. Preliminary thin-layer chromatography of the nonsaponifiable fraction revealed that the uptake of (14)C into cholesterol was only slightly decreased; uptake into cholesterol precursors was decreased somewhat more. The epidermis and dermis were separated by incubation of skin with elastase and hyaluronidase. The epidermis contained at least three times as much sterol per mg dry weight as did the dermis. Unesterified cholesterol was the major sterol present in both layers; the other sterols were present mainly as esters. DES injection resulted in no change in the free sterol content but markedly reduced the ester content of the epidermis and dermis.