Michel Prunieras

A syndrome associating partial albinism and immunodeficiency

Two unrelated patients with partial albinism, frequent pyogenic infections and acute episodes of fever, neutropenia and thrombocytopenia are described. Their pigmentary dilution was characterized by large clumps of pigments in the hair shafts and an accumulation of melanosomes in melanocytes. Melanocytes had few short dendritic expansions, and keratinocytes were hypopigmented. No or few Langerhans cells were detected in skin by electron microscopy and ATP-ase reactions. This pigmentary dilution, different from all other human albinisms, resembles the unique defect of the mutant dilute (d-d) mouse. Despite the presence of an adequate number of T and B lymphocytes, the patients were hypogammaglobulinemic, deficient in antibody production and incapable of manifesting delayed skin hypersensitivity or of rejecting skin grafts. Their leukocytes did not stimulate normal lymphocytes and could not generate cytotoxic cells during mixed leukocyte reaction. T lymphocytes of one patient were unable to exert a helper effect on the maturation of B lymphocytes into immunoglobulin-containing cells following in vitro stimulation with pokeweed mitogen. This suggests that the humoral deficiency might be secondary to a defect of helper T lymphocytes. Granulocytes did not show any morphologic abnormality, and their bactericidal activity was only moderately reduced. An increased number of polymorphonuclear leukocytes with polar distribution of Concanavaline A (Con A) receptors (capping) was found in one patient and her parents. The family histories suggest that this syndrome is transmitted as an autosomal recessive character.

Cutaneous wound healing: A model for cell-matrix interactions

Molecules of the extracellular matrix may influence the biologic behavior of basal cell keratinocytes. In the unwounded situation, basal cells are in contact with the basement membrane of the dermo-epidermal junction and eventually detach from this structure, move vertically, and differentiate. During wound healing these cells are in contact with an entirely different set of matrix molecules (including elastin, fibronectin, and interstitial collagen). The cells migrate laterally to cover the wound and remain in a proliferative, undifferentiated state. When 25-day-old epidermal cultures were compared, adult human keratinocyte migration across an interstitial collagen matrix was found to be approximately double that across a basement membrane matrix. This finding suggests that adult human keratinocyte migration is influenced by the matrix in contact with the cells. This type of cell-matrix interaction may be important to the understanding of cutaneous wound healing.

Expression of high molecular weight (67K) keratin in human keratinocytes cultured on dead de-epidermized dermis

The influence of living dermal tissue upon epidermal differentiation during embryonic development as well as in vitro culture has been documented. Living dermal tissue contains both cellular and matricial elements. In the present study, third-passage subcultured adult human keratinocytes were either seeded on plastic dishes or recombined with dead de-epidermized dermis and further cultured for 3 weeks. After this time, keratins were extracted and analysed by one- and two-dimensional gel electrophoresis. The 67K keratin subunit, which is thought to be involved in the process of in vivo type skin differentiation, was absent in ordinary cultures; however, it was expressed in air-exposed cultures on dead de-epidermized dermis. Quantitatively, however, it did not reach the in vivo level. This suggests that in principle, the induction of the expression of this protein does not require the presence of living dermal cells.

PIGMENT DILUTION AND IMMUNODEFICIENCY: A NEW SYNDROME

Pigment changes and hematologic disorders are not uncommon. Just as the association of oculocutaneous albinism with bleeding and platelet abnormalities is familiar to dermatologists, so are the pigmented macules found in Fanconi’s pancytopenia. The Chediak-Higashi syndrome (CHS) combines pigment dilution and hematologic changes.’ Pigment dilution is related to structural abnormalities of melanosomes. These abnormalities include an unusully large size together with irregular shape and disorganized internal arrangement. Such changes are likely due to aggregation of several melanosomes through fusion of melanosome membranes. The dilution of pigmentation can be explained on several grounds including decrease in melanosome number, impaired transfer to keratinocytes and, possibly dysfunction of tyrosinase.

Recent advances in epidermal cell cultures

SummaryAmong the many skin culture systems, three have been selected in this short review because of their specific potentials in Dermatological research. H. Green cultures newborn human forsekin keratinocytes on a mouse 3T3 feeder layer. Keratinocytes grow and keratinize. The feeder cells release factor (s) which allows serial propagation of keratinocytes to be achieved. The cell yield is further increased by adding epidermal growth factor. This system has already proved to be a potent tool for the study of keratinization at the molecular level. A. Freeman has described a system in which explants of adult human skin are cultured on the dermal aspect of dead split-thickness pig skin. Keratinocytes can be passaged several times. Their differentiation is remarkable: it includes the production of keratohyaline, membrane coating granules, pemphigus as well as pemphigoid antigens. This system is interesting in the study of epidermal morphogenesis and may be applicable to the treatment of burns.The culture of epidermal cells from adult guinea pig ear in comparison with that of dermal fibroblasts is being used to study the specificity of action of pharmacological compounds on growth and keratinization of epidermal cells. Furthermore, the isolation (and culture) of pure populations of basal cells appears as a promising approach to the study of the mechanisms which moderate epidermal cell proliferation.ZusammenfassungAus den vielen Hautkultursystemen, haben wir, für diese kurze Übersicht, drei ausgewählt, ihrer spezifischen Wichtigkeit, im Gebiet der dermatologischen Forschung, wegen. H. Green kultiviert Keratinocyten der Vorhaut menschlicher Neugeborenen auf Maus-3T3-Nährlage. Die Keratinocyten wachsen und keratinisieren. Die Nährzellen geben einen Faktor (s) ab, der die seriele Weiterkultur der Keratinocyten ermöglicht. Der Zellertrag ist noch größer wenn man Epidermiswachstumsfaktor (epidermal growth factor) dazu gibt. Dieses System hat sich bereits als ein wichtiges Werkzeug für das Studium der Keratinisation auf dem molekularen Gebiet, bewährt. A. Freeman hat ein System beschrieben in dem Hautexplante erwachsener Menschen auf der dermalen Seite dünnschichtiger toter Schweinshaut kultiviert werden. Die Keratinocyten können mehrere Male versetzt werden. Ihre Differentiation ist bemerkenswert: Sie umfaßt Keratohyalin, membran coating granules (Keratinosome), und Pemphigus sowie Pemphigoidantigen, Produktion. Dieses System ist interessant im Bereich des Epidermismorphogenese Studium, und kann für die Behandlung der Brandwunden angewendet werden.Die Kultur von Epidermiszellen der Ohren erwachsener Meerschweinchen, im Vergleich zu der aus dermalen Fibroblasten, wird zum Studium der Spezifizität der Wirkung pharmakologischer Mittel auf das Wachstum und die Keratinisation epidermaler Zellen, verwendet. Überdies erscheint die Absonderung (und Kultur) reiner basaler Zellen, als eine vielversprechende Methode zur Erforschung der Mechanismen welche die Epidermiszellproliferation mäßigen.

A keratin of fetal skin is reexpressed in human keratinocytes transformed by SV40 virus or treated with the tumor promoter TPA

SV40-transformation as well as treatment with tumor promoters produce alterations in morphology, differentiation and keratinization of human keratinocytes. Two cell lines of SV40-transformed keratinocytes and primary cultures of keratinocytes treated with the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) were found to contain an additional protein of 52.5 kD molecular weight (MW). This protein was identified by its reactivity with the monoclonal antibody TROMA-I as being keratin no. 8, a keratin normally present only in simple epithelia. Since this keratin is present in fetal epidermis but disappears gradually when fetal skin becomes multilayered after week 13 of development (Moll et al., Differentiation 23 (1982) 170. [23]), it suggests that SV40 virus and TPA are able to induce in human keratinocytes the reexpression of fetal characters.

CULTURE OF THE SKIN: HOW AND WHY

The skin is made up of two compartments: the cell population and the intercellular matrix. The cell population has three major classes of cells. The first, the ectodermal, is formed of cells which synthesize keratin: the keratinocytes. Keratinocytes are found in the epidermis and in dermal appendages. The second, the neuroectodermal, is composed of cells which synthesize melanin: the melanocytes. These are located in the basal layer of the epidermis, and in the dermis as well. The third is mesenchymal in origin. It includes all cells responsible for the synthesis of intercellular matrix macromolecules, namely collagen, elastin, glycosaminoglycans (acid mucopolysaccharides) and structural glycoproteins. Traditionally, these cells are called fibroblasts. Additional classes of cells are present in the skin, such as blood and nerve cells; however, they are considered of little importance in culture because they do not survive in vitro conditions more than a few days. ln this paper we shall refer to cell cultures when skin cells are cultured

Antimelanoma hybridoma antibodies against partially purified melanoma antigen

We have immunised BALB/c mice with a melanoma antigen obtained after papain solubilisation of the membranes of a metastatic melanoma tumour and fused the immune spleen cells to the mouse myeloma line P3-NS1/1-Ag4.1. The produced hybridoma antibodies (Mel-PV antibodies) recognised the initial melanoma antigen in haemagglutination, but did not react with any of the HLA phenotypes tested by cytotoxicity on a panel of B lymphocytes with known HLA-A and B phenotypes. We rosetted red blood cells coated with protein A with dispersed cells from fresh melanoma tumours, and a high degree of specificity for human malignant melanocytes was observed. Purified Mel-PV antibodies were also tested by indirect immunofluorescence and found to be oriented towards cytoplasmic components of malignant melanoma cells. These results indicate that the use of melanoma antigens for preparing monoclonal antibodies maintained a satisfactory degree of specificity and may be an adequate starting point for defining common and specific antigenic determinants on human melanoma.

On the Use of Cell Cultures in Skin Toxico-Pharmacology

The development of cell culture models for skin pharmacology and toxicology is justified on several grounds. A large number of laboratory animals are killed each year for drug testing, in particular in cosmetic industries. It is legitimate to determine to what extent laboratory animals can be replaced by in vitro cultures. Ethical considerations also act as incentive to replace drug testing in human volunteers by in vitro systems. This is particularly important when chemicals with sensitization potential are applied to human skin, since this application may result in life-long sensitivity ioward a class of chemicals of as yet unknown therapeutic value but that may be vital in the future for the individuals in question. In addition, drug testing may turn out to be less expensive and trrne-consumjng in culture systems than in the animal in vivo. The use of cell cultures in skin pharmacology and toxicology appears to be a particular case of a more general problem; the use of in vitro systems in pharmaco-toxicology at large. In this latter field, a number of recent pubficatfons stem for the most part from research on anti-cancer drugs and evaluation of mutagenic-carcinogenic potentials of environmental agents. Cellular systems for toxicity testing have been reviewed recently. In the more restricted fields of toxicology in dermatology and cosmetology, two important advances have been made recently. First, it has been shown that cultured epidermal cells of mouse, hamster, and

Hyperplasia and Tumor Promotion

When repeated doses of chemicai carcinogens, such as dimethyibenzanthracene (DMBA), are appiied to the si<in of aduit mice, skin carcinomas deveiop. If a single application of DMBA is made, the dose can be so adjusted that carcinomas do not appear. In this iatter case the effect of DMBA is not clinlcaliy visibie even though the carcinogen in question interacted with epidermal cells at the molecular level. Such a moiecular action is calied initiation. When repeated doses of croton oii are applied to initiated mouse skin, carcinomas develop, whereas croton oil alone induces few, if any at all. The action of croton oil on the initiated skin is cailed promotion, The promoting action is not exactly carcinogenic, but favors the development of malignant growth and renders initiation clinically apprehendable, Croton oii is an irritant which induces chronic epidermai hyperpiasia in both initiated and normai skin, Croton oil-induced hyperplasia is linked to promotion. Indeed, if the applications of croton oii are interrupted before the deveiopment of tumors, the epidermis regains its normal thickness and the promoting action is arrested, In psoriasis, there aiso exists chronic hyperpiasia; however, psoriatic hyperpiasia never transforms into carcinomas, even in patients who repeatedly applied to their skin for years or decades such medications as coaltar and tar derivatives which contain various amounts of