[Epidermolysis bullosa and dermo-epidermic junction. Clinico-pathologic correlation]. / Epidermólise bolhosa e zona de junção dermo-epidérmica. Correlação clínico-patológica.

Epidermolysis bullosa encompasses a group of hereditary diseases clinically and pathologically characteristic. In this review the grouping criteria commonly accepted for their classification are described. The cleavage phenomena observed in these diseases are analysed in accordance with the concept of a skin area including dermal and epidermal interrelated structures--the dermo-epidermal junction zone. Finally in the most expressive forms the clinical manifestations, diagnosis and treatment are reviewed.

Genetically determined coincidence of Kaposi sarcoma and psoriasis in an HIV-negative patient after prednisolone treatment. Spontaneous regression 8 months after discontinuing therapy.

We report the case of drug-induced, acrolocalized Kaposi sarcoma (KS), arising multicentrically in both palms and soles of a male patient who has had widespread psoriasis since 12 years of age. This 59-year-old man, of Mediterranean origin, was HIV antibody-negative and had received oral prednisolone treatment over 5 months for chronic obstructive lung disease (initial dose: 75 mg/d). Eight months after discontinuing oral treatment the KS nodules regressed spontaneously and finally disappeared completely without additional treatment. Light and electron microscopic investigations confirmed the diagnosis of KS, whereas laboratory tests excluded HIV infection and suggested mild immune dysfunction. The existence of HLA loci predisposing to KS and to psoriasis (A1, DR5, DR7, DR11) was characteristic for the simultaneous occurrence of these two diseases. This case report demonstrates the complex interrelationships between genetic predisposition, drugs leading to immune suppression, and the evolution of an unusual neoplasm.

Cultivation of human dermal microvascular endothelial cells in vitro: immunocytochemical and ultrastructural characterization and effect of treatment with three synthetic retinoids.

A new reliable and reproducible technique to culture endothelial cells from the small vessels and capillaries of human skin is introduced, and proliferation and differentiation of the growing cells are characterized. The endothelial origin of the culture cells was confirmed by light- and electron microscopy and by labelling with Ulex europaeus Agglutinin I and an antibody against Factor VIII-related antigen. Further immunocytochemical characterization showed that 92-100% of the cells were positive for beta 2-microglobulin and the entire cell population expressed vimentin, whereas cytokeratins, desmin, HLA-DR antigen, Leu 6 and S 100 protein, could not be detected. As vascular endothelium is a common site of inflammation and retinoids have been shown to be of good clinical efficacy in some chronic inflammatory skin diseases, we investigated the influence of etretinate, etretin and isotretinoin on proliferation and antigen expression of our culture cells. All retinoids applied inhibited proliferation of endothelial cells in a dose- and time-dependent manner whereas they induced neither HLA-DR nor intercellular adhesion molecule-1 (ICAM-1). Furthermore, none of the retinoids applied influenced the gamma-interferon-induced expression of these surface antigens on endothelial cells. Our results suggest that the action of retinoids in skin inflammation is not mediated by modulation of HLA-DR or ICAM-1. The cell culture technique described here is an interesting and reliable model for studying the influence of drugs on endothelial cell growth and differentiation in vitro.

[Skin aging. Physiopathology and clinical perspective]. / Envelhecimento cutâneo. Fisiopatologia e perspectiva clínica.

The main structural and functional alterations observed during skin ageing are reviewed, considering the intrinsic process of senescence and the damage induced by chronic exposure to actinic radiation. In photo-ageing the importance of skin type is stressed, concerning its capacity to tan in order to get protection against radiation. Finally some inflammatory, degenerative and neoplastic skin diseases associated with ageing and photo-ageing are briefly described.

Initial hyperproliferation and incomplete terminal differentiation of cultured human keratinocytes from lesional and uninvolved psoriatic skin.

Human epidermal keratinocytes (KCs) were isolated from lesional and from uninvolved skin of 8 patients with chronic plaque-like psoriasis and from the normal skin of 8 healthy volunteers. Primary KC cultures, grown on 3T3 cell feeder layers, were examined over a period of 4 weeks and their plating efficiency, colony growth area, DNA synthesis and ultrastructural cell differentiation were evaluated. Psoriatic KCs formed colonies one day earlier than non-psoriatic controls and proliferated faster during the first 2 weeks, as assessed by the mean colony growth area and 3H-thymidine incorporation. After 4 weeks, however, no significant differences were observed between the in vitro proliferation parameters of normal and psoriatic KCs. At the ultrastructural level, cultures of lesional psoriatic KCs consisted of more cell layers with adherent transitional cells and incomplete formation of cornified envelopes, even after 4 weeks, while KCs from uninvolved psoriatic skin were characterized by a transient delay of in vitro maturation. These results indicate that the characteristic hyperproliferation of psoriatic KCs may only be maintained over a short period of primary culture, whereas defective terminal differentiation of lesional psoriatic KCs was maintained throughout the culture period.

Isolation of human sebaceous glands and cultivation of sebaceous gland-derived cells as an in vitro model.

An experimental technique is presented as an in vitro model for the study of human sebaceous gland-derived cells. Intact sebaceous glands were isolated from full-thickness human skin after incubation in dispase (2.4 U/ml) and in deoxyribonuclease (0.02%) by using microsurgical instruments under microscopical observation of the epidermal underface. Subsequently, the ducts of the glands were removed, the isolated gland lobules were seeded on a 3T3-cell feeder layer in Dulbecco's modified Eagle's medium and Ham's F 12 medium (3:1) supplemented with fetal calf serum (10%), L-glutamine, antibiotics, epidermal growth factor (10 ng/ml), hydrocortisone (0.4 microgram/ml), and cholera toxin (10(-9) M), and were then cultivated in a CO2-incubator at 37 degrees C. After 2-3 wk cell outgrowths resulting from the periphery of the gland lobules were obtained and dispersed cells were passaged for three subcultures with or without 3T3-cell feeder layer. The cultured cells preserved in vitro morphologic characteristics and differentiation patterns comparable to those described for normal human sebocytes in vivo, with a high rate of viable cells. Their labeling pattern with MoAb showed close similarities to the pattern reported for sebocytes in vivo but differences to the pattern of keratinocytes in vivo and in vitro. In their cytoplasm oil red and nile red stained droplets were detected, and the observed density and distribution evidenced in vitro lipogenesis. The technique presented here may provide a promising model for further experimental studies on sebaceous gland cell development and function.

Effects of azelaic acid on proliferation and ultrastructure of mouse keratinocytes in vitro.

The effects of azelaic acid (C9-dicarboxylic acid, AZA) on the proliferation and ultrastructure of neonatal NMRI mouse keratinocyte cultures were studied to clarify the mechanisms of AZA action on normal and diseased epidermis. The dose- and time-dependency of the drug effects on DNA synthesis was examined by 3H-thymidine incorporation into DNA and by autoradiography. Electron microscopy was used to detect the target cell organelles of the drug. Azelaic acid decreased DNA synthesis in a dose- and time-dependent manner with a 50% inhibitory concentration of 20 mM. The inhibition of DNA synthesis was already observed after 1 h of treatment, reached its maximum after 4 h, and was stable for 24 h. A complete reversibility of the inhibitory effects was observed within 2 h after discontinuation of the treatment, and, interestingly, a rebound effect occurred with a temporary increase of DNA synthesis. Furthermore, treatment with AZA reduced the RNA and protein synthesis of the cells. Electron microscopic evaluation of treated cultures showed early marked damage of the mitochondria, followed by dilation of the rough endoplasmic reticulum (RER). These alterations were completely reversible after discontinuation of the treatment. Our findings show that AZA exerts a dose- and time-dependent, reversible antiproliferative effect on keratinocytes, acting primarily on mitochondria and RER. The antiproliferative action of AZA could explain its beneficial effect in some skin disorders characterized by alteration of keratinocytic differentiation.

Interferons in dermatology.

Interferons are a large family of proteins and glycoproteins, naturally occurring or artificially produced by recombinant biotechnology. Their antiviral, antiproliferative, antitumoral, and immunomodulatory activities are induced by alterations in cell metabolism after binding to specific membrane receptors. Interferons have been used for the treatment of viral papillomas (e.g., verruca vulgaris and condyloma acuminatum), human immunodeficiency virus (HIV)-associated Kaposi's sarcoma and cutaneous tumors (e.g., melanoma, cutaneous T cell lymphoma, and basal cell carcinoma), and inflammatory dermatoses (e.g., Behçet's syndrome and psoriatic arthropathy). Clinical trials have been performed worldwide with various regimens and have not always led to conclusive results. In our experience long-term therapy with high doses of subcutaneously injected, recombinant interferon-alpha-2a in patients with HIV-associated Kaposi's sarcoma induces a remission or stabilization of the disease. In malignant melanoma a low response rate is obtained in metastatic disease with the use of interferon as a single therapeutic agent. Combined with other antitumor agents, however, interferon seems to be a useful drug. Excellent control of Behçet's disease has been obtained, and the treatment of condylomata acuminata has been effective.

Effects of azelaic acid on sebaceous gland, sebum excretion rate and keratinization pattern in human skin. An in vivo and in vitro study.

The effects of azelaic acid (AZA) on the epidermis of 47 individuals (12 with normal skin, 15 with seborrheic skin and 20 suffering from acne) and on in vitro cultured keratinocytes are reported. Topical application of a 20% AZA cream significantly improved the lesions of acne patients, but failed to induce clinically detectable changes in normal or seborrheic epidermis. Complementary investigations clearly showed that AZA treatment failed to induce specific changes in sebum composition, excretion rate, or in the size of sebaceous glands, but modified epidermal keratinization. Keratohyalin granules and tonofilament bundles were reduced in size and number, mitochondria were swollen and the rough endoplasmic reticulum of malpighian keratinocytes enlarged. The infundibular epidermis of acne individuals showed marked reduction of the horny layer thickness, widening of the horny cell cytoplasm, transitional corneal cells, normalization of filaggrin distribution, and the comedo contained few bacteria and spores. In vitro, AZA exerted marked time- and dose-dependent antiproliferative cytostatic effects on cultured keratinocytes, with a 50% inhibitory dose of 20 mM, decreased some keratinocyte proteins (highly soluble fractions S2, keratohyalin macroaggregate R2, and non-cross-linked fibrous protein S4) and a 95 kD and a 35 kD protein of the cytosolic fraction. Mitochondria were frequently damaged and the rough endoplasmic reticulum enlarged. Our results indicate that AZA is an antikeratinizing agent, displaying antiproliferative cytostatic effects on keratinocytes and modulating the early and terminal phases of epidermal differentiation.

Monoclonal antibody labeling for cytokeratins and filaggrin in the human pilosebaceous unit of normal, seborrhoeic and acne skin.

The distribution of cytokeratins and filaggrin in human pilosebaceous unit was investigated in specimens obtained from normal (n = 15), seborrhoeic (n = 6), and acne skin (n = 6), using the monoclonal antibodies CK8.12, CK8.13, CK4.62, CK8.60, KL1, PKK2, RPN 1160, and an antibody for filaggrin. The type and amount of cytokeratin content was correlated with the stage of cell differentiation in these three skin types. In all specimens studied the sebocytes. The sebaceous duct cells, and the infundibular cells contained cytokeratins, no clear differences were found between normal, seborrhoeic, and acne skin. During sebocytic maturation the amount and type of cytokeratin content changed gradually and the labeling pattern was partly different compared to the interfollicular epidermal pattern. In the sebaceous duct and the infundibulum, the labeling pattern using KL1, CK8.12, and CK8.13 was similar to that seen in interfollicular epidermis, whereas labeling with CK8.60 and PKK2 was different. These findings indicate that sebaceous duct and infundibular cells express transitional patterns of differentiation between epidermal keratinocytes and sebocytes. Filaggrin was expressed only in some sebaceous duct cells and in infundibular cells. In seborrhoeic and in acne skin, however, the reactivity of antibody to filaggrin was more intense and was already observed in the lower parts of the sebaceous duct and the infundibulum. Although no filaggrin was found in the intermediate cells of the sebaceous duct and the infundibulum in normal skin, these cell types clearly contained filaggrin in seborrhoeic and acne skin.

Juvenile hyaline fibromatosis. A histologic and histochemical study.

Histochemical and routine light microscopic studies were performed in nodular skin lesions excised from one patient with juvenile hyaline fibromatosis. The lesions had different times of evolution. Recent lesions showed a high density of fibroblastlike cells embedded in an amorphous matrix of glycoproteins, hyaluronic acid, and small amounts of chondroitin sulfates A and C and of dermatan sulfate. The progressive enlargement of the lesions was due to an increase in the amount of intercellular matrix produced by the cells that progressively displayed a pattern of peripheral stratification. In the older lesions, the matrix was mainly composed by chondroitin sulfates A and C. We suggest that juvenile hyaline fibromatosis represents a disease of the connective tissue with progressive abnormal differentiation to chondroid tissue.

[Therapy of AIDS-induced Kaposi sarcoma and malignant melanoma with interferons]. / Therapie des AIDS-induzierten Kaposi-Sarkoms und des malignen Melanoms mit Interferonen.

Since 1980, disseminated Kaposi's sarcoma has been occurring in new epidemic proportions with a rapid clinical course in risk populations. Sixteen cases were under therapy and close surveillance from 1982 to 1986. Eight are still under therapy. In disseminated Kaposi's sarcoma with acquired immune deficiency syndrome (AIDS) our experience was encouraging. Following systemic, long-term treatment with recombinant alpha 2a interferon we observed complete remission of the lesions in 2 cases, partial remission and stabilization of the disease in 3 cases, at least temporary stabilization of the disease in 3 cases and progressive disease in 8 cases. Systemic rIFN-alpha 2a therapy was well tolerated; its long-term administration in patients with a relatively good immune status has an obviously beneficial effect on the course of Kaposi's sarcoma. In metastatic malignant melanoma Stage IV the results were only moderately encouraging. Regression of cutaneous metastases in 1 case and long-term stabilization of the disease in another patient point to antitumor activity of interferon in disseminated malignant melanoma. However, the administration of rIFN-alpha 2a in earlier stages appears more promising.

Disseminated Kaposi's sarcoma in AIDS: histogenesis-related populations and influence of long-term treatment with rIFN-alpha A.

Lesions (n = 19) of cutaneous Kaposi's sarcoma in different stages of development were obtained from 13 patients with acquired immunodeficiency syndrome (AIDS), and studied by light and electron microscopy. Six additional biopsies from 4 patients treated with recombinant alpha A interferon were obtained after treatment. Varying amounts of two proliferating cell populations were found: (1) Large cells showing cytologic and histochemical characteristics of endothelial cells. They were seen in close proximity to normal vessels, forming new vascular structures and large aggregates found in papular and nodular lesions. (2) Smaller spindle-shaped cells, probably of pericytic origin. They appeared in bundles and fascicles in the papillary dermis of the cutaneous Kaposi's sarcoma lesions and, in part, gave origin to thin-walled, bizarre-shaped vessels that show incomplete lumina proliferating from the upper to the deep dermis and are surrounded by extravasate erythrocytes and siderophages. After long-term systemic treatment with recombinant alpha A interferon, the endothelial type of tumor cell aggregates mostly disappeared, whereas most of the spindle-shaped pericytic-like cells were still present. Our findings lead us to suggest that some cellular product may, as a promoter factor, induce the proliferation and growth of endothelial cells. This factor may be blocked by alpha A interferon and cause regression of endothelial cell proliferation observed in AIDS patients undergoing long-term systemic therapy.

Growth of human hair follicle keratinocytes in vitro. Ultrastructural features of a new model.

A simple experimental technique was developed to provide an in vitro model for the study of human follicular keratinocytes. Anagen-phase human hairs were plucked from the scalp of healthy individuals; the follicles were separated, plated on coverslips coated with collagen G, and cultivated in McCoy 5A Medium in a CO2-incubator at 37 degrees C. Light and electron microscopy after 1, 2, 3, and 6 weeks showed selective and progressive cell growth with keratinocyte differentiation, producing multilayered cultures of cells joined with fully developed desmosomes. Three distinct patterns of differentiation, leading to the formation of an incomplete horny layer, were seen. The particular arrangement of tonofilaments, the considerable amounts of cytoplasmic glycogen, and the absence of malpighian differentiation were ultrastructural indicators of the follicular origin of the cultured cell population, which most likely grew from the outer root sheath of the hair. This technique may provide a promising model on which to base further studies of hair biologic processes and hair growth.

Kaposi's sarcoma in AIDS. Multicentric angioneoplasia in early skin lesions.

Light-microscopic, immunohistochemical, and ultrastructural studies were performed on biopsy material from 15 young homosexual men with AIDS-associated mucocutaneous Kaposi's sarcoma; 19 Kaposi's sarcoma lesions in different developmental stages were investigated. These lesions showed multicentrically arising and proliferating vascular endothelia forming thick-walled and thin-walled capillaries and larger vessels, as well as spindle-shaped cells forming fascicles and bundles around them. Different amounts and organization of these two major cellular components were found in all stages of evolution of Kaposi's sarcoma lesions. Immunohistochemical and electron-microscopic techniques suggested that the spindle-shaped cells were of pericyte origin in different stages of maturation or, more rarely, lymphatic endotheliocytes. The skin lesions of AIDS-associated Kaposi's sarcoma occurred as a result of multicentric angioneoplasia of rather slow progression, together with the proliferation of pericyte-like mesenchymal cells, possibly representing a stromal reaction to the vascular proliferation. Both blood and lymphatic vessels seemed involved in this process. In early stages, scattered lymphocytic infiltration was an additional feature. Mitotic figures and cytologic atypia were not seen more frequently in early AIDS-associated Kaposi's sarcoma than in proliferating granulation tissue.

Angioproliferative changes in clinically noninvolved, perilesional skin in AIDS-associated Kaposi's sarcoma.

In clinically noninvolved skin from the area surrounding early macular lesions of AIDS-associated disseminated Kaposi's sarcoma (KS), morphological evidence of vascular proliferation was seen in the upper papillary dermis. Poorly differentiated cells with cytoplasmic inclusions similar to Weibel-Palade bodies were found to form primitive vascular lumina. Around the vessels, dermal Langerhans' cells and lymphoid cells were detected. Spindle-shaped cell proliferation characteristic of fully developed KS lesions was not observed. The angioproliferative changes, undetectable clinically, were similar, though less evident, to those observed in early lesions of disseminated KS, but without stroma tissue reaction. These findings suggest that in AIDS-associated KS angioproliferation may not be restricted to the clinically detectable lesions and that some angioplastic factor(s) may cause widespread endothelial hyperproliferation.

Azelaic acid vs. placebo: effects on normal human keratinocytes and melanocytes. Electron microscopic evaluation after long-term application in vivo.

The effects of topically applied 20% azelaic acid (AA) on normal human epidermis were investigated vs. placebo in a double blind study by electron microscopy in 15 volunteers. After 3 months of local application twice daily, the pattern of epidermal keratinization was found altered in skin treated with AA. In particular, the number and thickness of tonofilament bundles and the number of keratohyaline granules seemed decreased; the remaining granules were smaller, occasionally showing irregular electron densities. The perinuclear endoplasmic reticulum and the cytoplasmic cisternae were enlarged and swollen mitochondria were regularly observed in most malpighian keratinocytes. Thorough quantitative evaluation of the number and distribution of melanocytes by a MOP-videoplan computer system showed no differences between verum and placebo sites, although, the mean number of melanocytes had increased in both, as compared to the untreated controls taken before onset of therapy. No significant qualitative changes of the normal melanocytes were found. These findings indicate that azelaic acid may influence the differentiation of normal human keratinocytes by reducing the synthesis of keratin precursors and may, therefore, act as a mild antikeratinizing agent, whereas, the pigmentary system in normal human epidermis does not show any specific change after 3 months of treatment with AA.