Fine autoradiographical study on scale morphogenesis in the regenerating tail of lizards

Regenerating scales in lizards originate as pockets in the epidermis instead of epidermal elevations as during embryo development. The morphogenesis of scales in the regenerating tail of the lizards. Anolis and Lamphropholis was studied after peritoneal injection of 3H-thymidine. The tracer was localized in the forming epidermis after progressive post-injection times, by means of autoradiography on plastic sections. After 4-5 hours post-injection of 3H-thymidine, the radioactivity was localized in the basal layer. After 2 to 4 days post-injection labelled cells were seen in the basal and intermediate spinosus layers but not in the uppermost keratinizing layers. Labelled cells were seen in the differentiating cornifying layers (pre-beta and pre-alpha) 6-8 days post-injection. At 12-14 days post-injection almost no radioactivity was seen in the basal layer or in the living part of the epidermis. A few labelled cells were present in the dense keratinizing layers of the sloughing wound and interscale lacunar layers. This study shows that scale formation and morphogenesis in the regenerating tail is brought about by a localized cell proliferation along the regenerating epidermis. In the forming scales the percentage of labelled cells in the distal side (future dorsal part of the new scale) is much higher than in the proximal side (the future ventral side of the scale), so that overlapped scales are generated.     

Effects of food on plasma catecholamine and gastrin levels in patients with duodenal ulcer and normal volunteers

The expression of sugar residues on human epidermal cells was investigated by means of lectin binding, as a way of determining membrane structural changes occurring during the differentiation of the epidermis. Fourteen lectins of different sugar specificity were conjugated with fluorescein isothiocyanate (FITC-lectins) and tested in fluorescence microscopy on frozen sections of normal human epidermis. In parallel, FITC-lectins were tested on psoriatic-involved epidermis to visualize differences in the expression of sugar residues that might occur during abnormal epidermal differentiation. No labelling could be obtained with lectins from Bandeira simplicifolia I, Dolichos biflorus, Limulus poyphemus, Tetragonolobus purpureas, Ulex europeus I, and Triticum vulgaris (group 1 lectins). A "pemphigus-like" intercellular labelling of the whole epidermis, except the stratum corneum, was obtained with lectins from Canavalia ensiformis. Maclura pomifera, Phaseolus vulgaris, and Ricinus communis I (group 2 lectins). A selective intercellular labelling of the stratum spinosum and the stratum granulosum was seen in normal epidermis with lectins from Arachis hypogaea, Glycine max, Helix pomatia, and Sophora japonica (group 3 lectins). In psoriatic epidermis, not only the basal cell layer, but also cells from the adjacent lower stratum spinosum were found to be negative, using FITC-lectins of group 3. These data indicate that the expression of lectin binding sites in normal epidermis differs according to the maturation of the cell from the basal cell to the more mature keratinocyte in the stratum granulosum. They suggest that lectins may be used as markers of epidermal cells in various stages of normal and abnormal differentiation.     

Transformation of skin from larval to adult types in normally metamorphosing and metamorphosis-arrested salamander, Hynobius retardatus

Transformation of skin from larval to adult types in a salamander, Hynobius retardatus, which had been reported to show neotenic reproduction in a specific environment, was examined morphologically in normally metamorphosing, precociously metamorphosing and metamorphosis-arrested larvae. Typical larval skin was composed of an epidermis constituted by three types of cells such as apical, Leydig, and basal cells. The Leydig cells were larval specific, and thus disappeared and were replaced by adult epidermal cells during the metamorphosis. Disappearance of the Leydig cells was accomplished by apoptosis as confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end-labeling method and electron microscopy during the normal metamorphosis and precocious metamorphosis induced by exogenously applied triiodothyronine. Typical adult skin was composed of epidermis constituted by stratified squamous cells and of dermis mainly occupied with two types of dermal glands, mucous and serous glands. When the metamorphosis was arrested by different procedures (thyroidectomy, hypophysectomy, goitrogen treatment, and rearing at low temperature), the larval-specific Leydig cells fully remained in the epidermis, suggesting that the disappearance of these depended on the thyroid activity. Contrary to this, dermal glands behaved differently from the Leydig cells, though they developed and differentiated from epidermal basal cells and constituted the same skin. Those in the metamorphosis-arrested (thyroidectomized, hypophysectomized, or goitrogen-treated) larvae, except in the larvae reared at 4 degrees C, appeared a little later than in the controls. Thus, the aged, metamorphosis-arrested larvae had skin which consisted of larval type epidermis (Leydig cells) and adult type dermis (mucous and serous glands).     

Selection and extended growth of murine epidermal stem cells in culture

Continuously renewing epithelia contain small undifferentiated stem cells capable of self-renewal and maintenance of the differentiating cell population. In murine epidermis stem cells have been identified as label-retaining cells (LRCs) by long-term retention of tritiated thymidine or BrdU. It has been suggested that epidermal stem cells adhere to basement membranes through differential expression of specific integrins. To determine whether we could use a specific integrin to enrich for murine epidermal stem cells, we tested adherence of LRCs to several substrates. Regardless of the substrate used, approximately 10% of total basal cells and 100% of LRCs adhered in 10 min. In our medium specifically formulated for murine keratinocytes, rapidly adherent stem cells formed large colonies and could be used to form a structurally complete epidermis in organotypic culture. They showed a fivefold greater transient transfection efficiency than total basal cells, and when individual adherent cells were transduced with a retroviral vector, they formed large clones. Although these stem cells grew more slowly than the total basal cell population, they could be subcultured more times. Our results indicate that murine epidermal stem cells can be selected by rapid attachment to a substrate, but not by one specific integrin, and that they can be expanded in culture if the appropriate conditions are maintained.     

Incorporation of bromodeoxyuridine in regenerating fin tissue of the goldfish Carassius auratus

We have investigated the pattern of incorporation of 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU) by proliferating cells during regeneration of the tail fin of Carassius auratus. Fifteen days after amputation, intraperitoneal injection of a single dose of 0.25 mg/g wet weight of BrdU and subsequent immunocytochemical detection on sections revealed groups of replicating cells in the blastema and epidermis at different proximodistal levels. Proliferating blastemal cells were confined to a crowded, compact distal area that lost its replicative capacity laterally, causing the differentiation of scleroblasts, which synthesize the lepidotrichia hemisegments. Proximally, but centrally located, the blastemal cells did not incorporate BrdU and they differentiated giving rise to the mature intraray connective tissue. An independent cell-proliferation process was noted in the epidermis. The distal cap did not proliferate; the lateral faces of the epidermis showed high rates of cell replication in the central layer at every level of the regenerate rays; quiescent cells remained in the superficial layers. The basal epidermal cells did not incorporate BrdU when actinotrichia were present. The possible role of basal epidermal cells in the synthesis of actinotrichia, the contribution of these collagen macrofibrils to the morphogenetic process, and the different pathways of cell differentiation during fin regeneration are discussed.     

TC findings in a case of Castleman''s disease with subclavian localization

An 85-year-old Japanese woman had noticed erythema on her vulvar region 3 years before. The erythema gradually increased in size and followed erosion and ulceration with pigmentation on the edge of the erythema. A skin biopsy from the pigmented area showed large round cells with ample cytoplasm, which formed nests or glandular structures. In the dermis there was mass formation of basophilic cells and peripheral cells in a palisade arrangement. The tumor cells in the epidermis showed positive immunoreactivity for carcinoembryonic antigen; on the other hand, the dermal tumor was negative. We diagnosed that the tumor in the epidermis was vulvar Paget's disease, and the dermal tumor was a solid type of basal cell carcinoma. We speculate that the vulvar Paget's disease preceded and then the basal cell carcinoma developed in the area of Paget's disease. This is the first report in which basal cell carcinoma in the area of Paget's disease was documented.     

Successful ultraviolet B treatment of psoriasis is accompanied by a reversal of keratinocyte pathology and by selective depletion of intraepidermal T cells

Skin irradiation with ultraviolet B (UVB) is a common and often durable treatment for psoriasis and other inflammatory skin disorders. We studied the effects of UVB on keratinocytes and leukocytes in psoriatic tissue and in culture. In nine patients treated repetitively, most of the cellular and molecular changes that typify the psoriatic epidermis reverted to normal. Keratinocyte hyperplasia, assessed by expression of the Ki-67 cell cycle antigen, decreased by 70%, and residual cell proliferation was appropriately confined to the basal layer. Epidermal thickening was reduced by 60%, and a granular layer reformed. Expression of keratin 16, as well as suprabasal integrin alpha 3 and insulin-like growth factor-1 receptors, was eliminated, whereas filagrin increased markedly. UVB also depleted > 90% of the CD3+, CD8+, and CD25+ T cells from the psoriatic epidermis, whereas dermal T cells were only minimally depressed. The latter finding parallels the known inability of these doses of UVB to penetrate the dermis. In tissue culture, UVB was antiproliferative and cytotoxic toward T cells and keratinocytes, but the T cells were 10-fold more sensitive. Furthermore, low doses of UVB induced apoptosis in lymphocytes but not keratinocytes, as detected by the TUNEL (TdT-mediated dUTP-biotin nick end labeling) technique. The selective effects of UVB on intraepidermal T cells in situ and in culture support the hypothesis that epidermal alterations in psoriasis can be normalized by a depletion of activated intraepidermal T cells.     

Refeeding varying fatty acid and cholesterol diets alters phospholipids in rat intestinal brush border membrane

The epidermis is an attractive site for therapeutic gene delivery because it is accessible and capable of delivering polypeptides to the systemic circulation. A number of difficulties, however, have emerged in attempts at cutaneous gene delivery, and central among these is an inability to sustain therapeutic gene production. We have examined two major potential contributing factors, viral vector stamina and involvement of long-lived epidermal progenitor cells. Human keratinocytes were either untreated or transduced with a retroviral vector for beta-galactosidase (beta-Gal) at > 99% efficiency and then grafted onto immunodeficient mice to regenerate human epidermis. Human epidermis was monitored in vivo after grafting for clinical and histologic appearance as well as for gene expression. Although integrated vector sequences persisted unchanged in engineered epidermis at 10 weeks post-grafting, retroviral long terminal repeat (LTR)-driven beta-Gal expression ceased in vivo after approximately 4 weeks. Endogenous cellular promoters, however, maintained consistently normal gene expression levels without evidence of time-dependent decline, as determined by immunostaining with species-specific antibodies for human involucrin, filaggrin, keratinocyte transglutaminase, keratin 10, type VII collagen, and Laminin 5 proteins out to week 14 post-grafting. Transduced human keratinocytes generated multilayer epidermis sustained through multiple epidermal turnover cycles; this epidermis demonstrated retention of a spatially appropriate pattern of basal and suprabasal epidermal marker gene expression. These results confirm previous findings suggesting that viral promoter-driven gene expression is not durable and demonstrate that keratinocytes passaged in vitro can regenerate and sustain normal epidermis for prolonged periods.     

Temporal variation in cellular proliferation during recornification of mouse tail skin

The influence of the time of injury on subsequent epidermal regeneration is unknown. Epidermal cell proliferation of tail skin in C57BL/6J mice in response to tape stripping was followed for 7 days by radiolabelled thymidine incorporation and autoradiography. The homeostatic labelling index (LI) of the basal epidermis of unmanipulated, unwounded (control) animals was 7.6% and did not vary depending on the time of day. Tape stripping increased the LI of epidermal basal cells 110% above control values 24 h after injury. Labelling indexes of epidermal basal cells in the skin adjacent to the wounded area were 7.0%. Basal cell DNA synthesis stimulated by wounding exhibited a distinct temporal variation at 24 h postinjury, with tail skin wounded at 12.00 h found to be 275% greater than control values and elevated 78% from LIs recorded at any other time point. This temporal spike was due to the time of day at which wounding occurred rather than the time point when the LI was determined. Mice wounded at 12.00 h and terminated 27 h later (15.00 h) had LIs that were 52% greater than wounds created at 09.00 h and examined at 12.00 h the following day. Higher levels of DNA synthesis in tail skin injured at 12.00 h compared to wounding at 09.00 h was detected 12-48 h after injury. Furthermore, DNA synthesis in wounds created at 12.00 h returned to baseline levels 1-2 days earlier than tail skin wounded at 09.00 h. Investigation of other strains of mice detected differences in radiolabelling of epidermal basal cells 24 h after tape stripping at 12.00 h or 09.00 h in CD-1 and BALB/cJ mice, but not in the C3H/HeJ strain. These results indicate: (a) there is no diurnal variation in the LI of mouse tail skin under normal homeostatic conditions (b) tape stripping is a potent stimulator of basal cell turnover in the epidermis (c) the time of wounding determines the magnitude of the increase in the LI of basal cells following injury, and (d) the proliferative response to wounding of the tail is dependent on the strain of mouse.     

Fine needle aspiration of basal cell adenocarcinoma of the parotid gland. Report of a case with assessment of DNA ploidy in aspirates and tissue sections by image analysis

BACKGROUND: Basal cell adenocarcinoma of the parotid gland is a low grade malignant neoplasm. It has cytologic features of basal cell adenoma and a histologically infiltrative growth pattern of malignant tumors with perineural and vascular invasion. CASE: Fine needle aspiration biopsy findings of basal cell adenocarcinoma of the parotid gland in a 77-year-old male were supplemented by DNA ploidy analysis. CONCLUSION: No single cytologic feature was found to unequivocally distinguish this lesion from basal cell adenoma and/or solid variant of adenoid cystic carcinoma. Therefore, for diagnostic purposes, we grouped all three lesions under the term basal cell tumor. Evaluation of DNA content of tumor cells revealed diploid histograms in both cytologic material and paraffin-embedded tissue. Infiltrative tumor nests, the histologic basis for differentiating basal cell adenocarcinoma from adenoma, showed the same diploid pattern. Though DNA quantitation may not discriminate basal cell adenoma from basal cell adenocarcinoma, it may prove useful in separating them from adenoid cystic carcinoma, which is considered to be a tumor with high malignant potential.     

Demand feeding and locomotor circadian rhythms in the goldfish, Carassius auratus: dual and independent phasing

Freshly isolated human lymphocytes from normal epidermis were characterized with respect to distribution of subsets. The major T cell receptor-alpha beta + compartment was enriched for CD4+, for CD8 alpha alpha +, and for CD4-CD8-T lymphocytes compared with peripheral blood lymphocytes. Furthermore, the majority of epidermal T lymphocytes expressed a CD45RA- CD45ROhigh Fas+ memory/effector phenotype; many also expressed early-intermediate activation markers, suggesting antigenic exposure in vivo. The cutaneous lymphocyte-associated antigen was expressed by almost all epidermal T lymphocytes. A large portion also expressed the mucosal-associated alpha e beta 7-integrin, which may mediate retention to epithelium. These data show that T lymphocytes present in normal human epidermis constitute a distinct T cells compartment with characteristics similar to that of other epithelial-associated T cell compartments.     

Induction of basal cell carcinoma features in transgenic human skin expressing Sonic Hedgehog

Hedgehog (HH) signaling proteins mediate inductive events during animal development. Mutation of the only known HH receptor gene, Patched (PTC), has recently been implicated in inherited and sporadic forms of the most common human cancer, basal cell carcinoma (BCC). In Drosophila, HH acts by inactivating PTC function, raising the possibility that overexpression of Sonic Hedgehog (SHH) in human epidermis might have a tumorigenic effect equivalent to loss of PTC function. We used retroviral transduction of normal human keratinocytes to constitutively express SHH. SHH-expressing cells demonstrated increased expression of both the known HH target, BMP-2B, as well as bcl-2, a protein prominently expressed by keratinocytes in BCCs. These keratinocytes were then used to regenerate human skin transgenic for long terminal repeat-driven SHH (LTR-SHH) on immune-deficient mice. LTR-SHH human skin consistently displays the abnormal specific histologic features seen in BCCs, including downgrowth of epithelial buds into the dermis, basal cell palisading and separation of epidermis from the underlying dermis. In addition, LTR-SHH skin displays the gene expression abnormalities previously described for human BCCs, including decreased BP180/BPAG2 and laminin 5 adhesion proteins and expression of basal epidermal keratins. These data indicate that expression of SHH in human skin recapitulates features of human BCC in vivo, suggest that activation of this conserved signaling pathway contributes to the development of epithelial neoplasia and describe a new transgenic human tissue model of neoplasia.     

Low humidity stimulates epidermal DNA synthesis and amplifies the hyperproliferative response to barrier disruption: implication for seasonal exacerbations of inflammatory dermatoses

Although seasonal changes in humidity are thought to exacerbate various skin diseases, whether these flares can be attributed to prolonged exposure to extremes in environmental humidities has not been studied systematically. We recently showed that prolonged exposure to high versus low humidities induced profound changes in epidermal structure and permeability barrier homeostasis. Therefore, we asked here whether comparable extremes in humidity could initiate not only homeostatic, but also potentially pathophysiologic alterations. We showed first that exposure to low humidity increases epidermal DNA synthesis in normal murine epidermis. Moreover, exposure to a low humidity for 48 h further amplifies the DNA synthetic response to barrier disruption, resulting in marked epidermal hyperplasia. Additionally, exposure to a dry environment for 48 h prior to barrier disruption results in dermal mast cell hypertrophy, degranulation, as well as histologic evidence of inflammation. To demonstrate the role of changes in external moisture on these phenomena, we applied either an occlusive, water-impermeable plastic membrane, Petrolatum, or a nonocclusive humectant, both to nonperturbated and to perturbed skin. All three forms of treatment prevented the epidermal hyperplasia and dermal mast cell hypertrophy and degranulation induced by exposure to low humidity. These studies indicate that (i) exposure to changes in environmental humidity alone induces increased keratinocyte proliferation and markers of inflammation, and (ii) that these changes are attributable to changes in stratum corneum moisture content. Finally, these studies provide evidence that changes in environmental humidity contribute to the seasonal exacerbations/amelioration of cutaneous disorders, such as atopic dermatitis and psoriasis, diseases which are characterized by a defective barrier, epidermal hyperplasia, and inflammation.     

Intraepidermal mass of M. leprae in a case of seborrheic keratosis due to Hansen's disease (LLs)

A 67-year-old patient has had exanthema in the lower right limb since 51 years ago (16 years old at onset), which underwent repeated remission and recurrence. At present, he has bilateral symmetrical widespread infiltrating exanthema and asymmetrical marked neuralhypertrophy, and has been diagnosed typical LLs (His father had the same disease). The exanthema recurred several years ago, and the patient is being treated for Hansen's disease. He had a dark brown flat elevation with a rough surface and the size of a small finger tip in his right abdominal skin for approximately 20 years. A biopsy was performed, and the specimen was fixed in 10% formalin and paraffin sections were prepared for histopathologic examination. A part of the specimen was processed forscanning electron microscopic examination. Seborrheic keratosis was diagnosed by H & E staining. Acid-fast (FITE) staining, immunohistochemical staining (keratin, S-100 protein, anti-PGL antibody and anti-BCG antibody) and scanning electron microscopy revealed the presence of bacteria (M. leprae) in the dermal foam cells, the matrix with a banded structure and the squamous epithelial cells which normally lack phagocytosis function. Compared to the basal cells of normal epidermis, the basal cells located adjacent to the dermis affected with seborrheic keratosis showed increased proliferation and more marked characteristics of a germinative cell. The degree of differentiation of the basal cells appeared regressed, and they probably possessed augmented phagocytic activity. The phagocytosed bacteria were probably carried by the epidermal cell cycle toward the surface layer. However, bacteria could not be found in the stratum corneum, probably due to an association with the lysosome.     

Apoptosis, necrosis, and proliferation: possible implications in the etiology of keloids

Keloids are collagenous lesions acquired as a result of abnormal wound heating. In this study we have assessed the potential role of proliferation, apoptosis, and necrosis in keloids. Samples were immunolabeled for proliferating cell nuclear antigen or DNA strand breaks or stained with acridine orange. Proliferating cells were observed in the basal layer of the epidermis and fibroblasts in the dermis, the numbers of the latter being increased in comparison with normal skin. No proliferating cells were observed in the central region of the keloid. In normal skin, apoptotic cells were restricted to the basal layer of the epidermis. In keloid samples, numerous apoptotic cells were observed in the epidermis and dermis; the number and distribution of positive cells decreased more distal to the keloid lesion. Apoptotic endothelial cells of a small proportion of blood vessels in the dermis were also observed. Evidence of necrosis was also seen in the dermis. These results suggest that, with maturity, progressive cell degeneration primarily by apoptosis results in clearance of certain cellular populations resulting in the typical keloid lesion. However, the persistence of fibroblast proliferation at the dermal/keloid interface propagates the fibrosis.     

IL-7 overexpression in transgenic mouse keratinocytes causes a lymphoproliferative skin disease dominated by intermediate TCR cells: evidence for a hierarchy in IL-7 responsiveness among cutaneous T cells

IL-7 is a keratinocyte-derived lymphocyte growth factor critical for the development of gammadelta T cells including murine dendritic epidermal T cells (DETC). We derived transgenic mice that overexpress IL-7 in basal keratinocytes under the control of the human K14 promoter. These K14/IL-7 mice develop dermal and epidermal T cell infiltrates associated with alopecia. This lymphoproliferative skin disease is substantially more severe in mice homozygous for the K14/IL-7 transgene. Conventional DETC expressing a Vgamma5 Vdelta1 TCR are rare or absent among the cutaneous T cells in these mice. The T cells in the skin infiltrates of young K14/IL-7 mice are predominantly gammadelta T cells that express intermediate levels of TCR, are negative for E-cadherin, often lack expression of CD2, and include cells that coexpress NK1.1. T cells expressing intermediate levels of a TCR-alphabeta are also present in transgenic skin, and progressively increase in number as the mice age. Phenotypically similar intermediate gammadelta and alphabeta T cell subsets also constitute the major lymphocyte populations recovered from organ culture of normal mouse skin in the presence of IL-7, suggesting that the T cells that accumulate in the epidermis of K14/IL-7 mice are derived from precursors normally resident in skin. We conclude that intermediate TCR cells, some of which coexpress NK1.1, can be selectively expanded in skin under the influence of IL-7 produced locally. Our results also suggest that features of the epidermal microenvironment besides keratinocyte-derived IL-7 account for the normal predominance of Vgamma5 Vdelta1 DETC in mouse epidermis.     

Neuropathological diagnosis and CAG repeat expansion in Huntington''s disease

Vitamin D3 analogs interfere with various aspects of epidermal growth, inflammation, and cellular differentiation. Most data are derived from in vitro studies. In the present review, the in vivo effects of vitamin D3 analogues on the psoriatic plaque are discussed. Calcipotriol, tacalcitol, and calcitriol in ointment modulate aspects of epidermal growth, differentiation, and inflammation. Immunohistochemical studies suggest that the inflammatory changes might be more expressed after treatment with calcitriol and tacalcitol. Flow cytometric quantification of the percentage of cells in SG2M phase and of keratin 10-positive cells revealed that calcipotriol reduced both indices significantly during treatment of psoriatic plaques. Flow cytometric analysis of epidermal cell suspensions using triple labeling for epidermal proliferation, expression of keratin 10, and vimentin permits a quantitative assessment of DNA synthesis selectively in the basal cells of the epidermis, an estimation of the distribution of the basal and suprabasal compartments, and a quantification of the distribution of mesenchymal and nonmesenchymal cells. Using this approach, the interference of tacalcitol with growth control of basal cells was demonstrated. Remarkably, recompartmentalization of basal and suprabasal cells and mesenchymal and nonmesenchymal cells proved to be inconspicuous during this treatment.