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Synopsis Glutathione (GSH) plays an important role in cellular protection during ageing. The hair plucking technique is a non-invasive method for the direct biochemical study of keratinocytes. Hair was taken from the suboccipital area of 63 volunteers (men and women whose ages ranged from 13 to 103 years). The results show a diminution in the glutathione content as a function of age. We compare two groups of population: group A (less than 80) and group B (more than 80). A remarkable fact is observed: group B displays a weak dispersion of the values as compared to A. The glutathione content (nmol 10(-3) mg DNA) is 5.42 +/- 0.60 for A and 1.86 +/- 0.35 for B. A reduction of 88% was observed in glutathione reductase activity and of 78% in the activity of glutathione-S-transferase from group A to group B. The glutathione peroxidase activity remains relatively constant. The decrease in the GSH concentration and the constancy of the glutathione peroxidase suggest that the capacity of the cell to protect itself from peroxides remains unchanged but that the GSH concentration may become the limiting factor.

In this investigation, glutathione peroxidase (GSH-PX), glutathione reductase (GSSG-RD), glutathione- S -transferase (GSH-S-T), γ-glutamyl transpeptidase (γ-GT) and glucose-6-phosphate deshydrogenase (G6PDH) were measured in human hair follicle obtained by plucking as source of keratinized cells. This non-invasive method was used on 27 men and women volunteers ranging from 19 to 102 years. Our results show that GSSG-RD, GSH-S-T, γ-GT and G6PDH activities decrease as a function of age, whereas GSH-PX activity does not vary. We discriminated 2 groups: a first one from 19 to 60 years with a large dispersion of the enzymatic activities and a second one corresponding to elderly people (up to 70) with a smaller dispersion of the values. This study suggests the keratinocytes possess an age-correlated enzymatic detoxification response potential.

We have addressed the question whether the level of UV-B induced DNA damage can be accurately assessed by the measurement of the rate of unscheduled DNA synthesis (UDS). Cultured human fibroblasts were irradiated with UV radiation at 290, 313 or 365 nm. The LD50 was 85 J/m2 at 290 nm, 4500 J/m2 at 313 nm, and 70 kJ/m2 at 365 nm. The analysis of UDS measurements indicate complete arrest of repair processes within 24 h after irradiation, irrespective of the dose (in the range 10-60 J/m2 at 290 nm, and 250-1000 J/m2 at 313 nm). Irradiation at 365 nm failed to yield detectable evidence of UDS. Incubation of irradiated cells with an antiserum directed against both 6-4 type and cyclobutane-type pyrimidine dimers shows a clear parallelism between the disappearance of the antibody-binding determinants and the variation of the rate of UDS vs time after the end of the irradiation. Thus it is concluded that in UV-B irradiated normal cultured human fibroblasts, the lack of UDS reflects the absence of immunodetectable pyrimidine dimers.

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.

Isolated adult human keratinocytes were grown either on plastic coverslips or a nonviable basement membrane surface containing intact laminin, type IV and V collagens, and heparan sulfate proteoglycan and examined by indirect immunofluorescence for the expression of bullous pemphigoid, pemphigus and Prlh antigens. Initial cell suspensions had a mean of 23% and 30%, respectively, of bullous pemphigoid and PrIh positive staining cells, while those stained with pemphigus serum were usually negative (19 of 22 series). Pemphigus antigen was expressed as intercellular staining between keratinocytes within 24hr in both cultures on plastic and basement membrane. Likewise, Prlh antigen was expressed within 24hr as a homogeneous cytoplasmic fluorescence leaving the basement membrane zone unstained. In contrast, pemphigoid antigen was expressed as a linear fluorescent band at the basement membrane zone between days 3 and 4 of culture. Systematic cell counts of builous pemphigoid antigen positive cells from trypsin disrupted primary cultures made on plastic over time showed a nadir (8%) of positive cells in early cultures after which the percentage rapidly rose to a peak of 58% between days 14 and 21 of culture. In subcultures repeatedly disrupted at short intervals, the percentage of bullous pemphigoid positive cells remained low when compared to those interrupted and passaged over longer intervals. The percentage of bullous pemphigoid antigen bearing cells in culture over time is similar, but not identical, to the percentage of basal cells and is related to the age and known growth kinetics of the culture system. Bullous pemphigoid, pemphigus and Prlh antigens are synthesized by the epidermal cell whether cultured on basement membrane or plastic.

Epidermal cells are natural targets for physical and chemical environmental agents, some of which are potential mutagens. Therefore, cytogenetic studies including chromosome morphology and sister chromatid exchanges (SCE) are desirable. Because there is usually poor growth on glass substrates (which are commonly needed for good chromosome spreading) cytogenetic studies of adult human keratinocytes have remained scarce. A retinal extract (RE) has been found to contain a cellular growth factor (eye-derived growth factor or EDGF) having a definite mitogenic effect on lens epithelial cells. In the present study EDGF was used as a mitogen to expand primary cultures of adult human keratinocytes and to prepare metaphase chromosome spreads from small biopsy specimens of the skin. EDGF was obtained as previously described [1] and 20 μl (equivalent to 100 μg protein) was added to the culture medium of freshly trypsinized skin biopsies of about 2 cm 2 . We used 0.25% trypsin in phosphate-buffered saline at 4°C overnight to separate the epidermis from the dermis, and then keratinocyte suspensions were made. Keratinocyte cultures on plastic substrate responded to EDGF by expansion (doubling the surface area covered in a given time) and twofold increase in mitotic activity. The same cultures made on glass coverslips allowed the preparation of about 100 well-spread metaphases. The average number of SCE per chromosome was 0.065 in keratinocyte cultures treated with EDGF. Control cultures of dermal fibroblasts (untreated) yielded similar values (0.08 per chromosome), which indicates that EDGF does not increase SCE per se. The epidermal versus fibroblastic nature of the cultured cells was assessed directly by the leucine aminopeptidase reaction (which was negative in keratinocyte cultures and positive in control dermal fibroblasts) and indirectly by showing that EDGF had no growth-promoting action on human fibroblasts in vitro. The exact composition of EDGF is yet unknown, but biochemical, immunological and biological studies indicate that it is different from both FGF (fibroblast growth factor) and EGF (epidermal growth factor).

Human keratinocytes were grown on a dermal equivalent (or lattice) at the liquid-air interface in an attempt to reconstitute a functional epidermis in vitro. Although the multilayered epithelium thus obtained is well differentiated, as shown by the presence of keratohyaline granules and horny layer, several differences from its in vivo counterpart were also observed: (1) In the reconstructed epidermis, basal keratinocytes do not have the cuboidal shape found in vivo; they synthesize bullous pemphigoid antigen and laminin, but the distribution of these antigens is not linear as in vivo; they contain the plasma-membrane antigens restricted to the basal layer in vivo (VM 1 , BC 1 ), but these antigens are not polarized; lack of polarization is also evidenced by the distribution of actin. (2) Differentiation markers appear but with a topography slightly different from that of epidermis in vivo; the 67-kD keratin does not appear in the first suprabasal layer as in vivo but above; involucrin, which appears in the granular layers in vivo appears as soon as the cells leave the basal layer. (3) ψ 3 antigen and fibronectin found in vivo only in hyperproliferative epidermis (wound healing, psoriasis) are detected. Hyperproliferation would also explain the unexpected staining of basal cells by KL1 monoclonal antibody. Because of the potential clinical or pharmacologic use of artificial epidermis, the question of whether the epidermis obtained in vitro can be considered as "normal" is discussed.

Two months after transplantation of human skin onto the nude mouse, excisional wounds were made through the entire thickness of the skin, at the center of the graft, using a 2-mm punch. At various time intervals thereafter, ranging from 2 days to 9 weeks, the graft sites were harvested and processed for an immunohistological study. With a monoclonal antibody directed against HLA-ABC antigens, it was demonstrated that the healing epidermis is of human origin. Moreover, with three different monoclonal antibodies directed against human keratins, named respectively AE1, AE3, and KL1 and with an anti-involucrin antiserum, it is reported that the keratinization and involucrin distribution patterns observed in normal human epidermis are reconstituted, 2 months after transplantation, in the major part of the grafted epidermis, undergo changes during the reepithelialization process, and are restored in the healed epidermis 9 weeks after injury. This study indicates that the nude mouse/human skin model could be a valuable tool to study a major aspect of regeneration such as the reepidermization of human skin without recourse to human volunteers.

In vivo, keratinocyte cytoplasmic antigens linked to keratinization have been traced with 2 distinct types of antibodies found in human sera. One type of antibody is specific for the basal cell compartment of keratinocytes while the other one is specific for upper keratinocytes. Using these markers, we followed a well-defined, in vitro human keratinocyte culture system that produces a keratinizing epithelial cell outgrowth juxtaposed to dead dermal substrate for the in vitro expression of keratinocyte cytoplasmic antigens. These antigens were found to be expressed in vitro independently from dermal influences. Their chronology, localization and topography in culture matched the in vivo situation.

The author gives a summary of the recent researches concerning Sézary’s syndrome.

The reconstruction of human epidermis during healing of human skin wounded after grafting onto the nude mouse was described in a previous paper (M. Demarchez, P. Sengel, and M. Prunieras, 1986, Dev. Biol. 113, 90-96). The regeneration of the epidermal basement membrane zone (BMZ) and the reorganization of the connective tissue are the subjects of the present study. They were investigated by two complementary methods: electron microscopy to analyze the BMZ reorganization, and indirect immunofluorescence with species-specific and cross-reacting antibodies directed against laminin, bullous pemphigoid antigen, mouse or human collagens of types I or IV, human elastic fibers, fibronectin, fibrin, actin, and human vimentin, to examine the species origin and distribution of BMZ and connective tissue components during the regeneration process. It is reported that grafted human skin preserves its own immunological markers not only in the epidermis but also in the BMZ and dermis as well, and that, after injury, its regeneration proceeds according to the following sequence of overlapping events: production of a mouse granulation tissue; reepidermization by human cells; reconstruction of a BMZ with human characteristics; formation of a human neodermis. It is concluded that human skin grafted onto the nude mouse is able to regenerate its three structural compartments, namely, the epidermis, BMZ, and dermis. Interestingly, it appeared, also, that the connective tissue regeneration would be a two-step mechanism including the sequential formation of two tissues of distinct sources, namely, a granulation tissue and a neodermis.

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.

Pigmented melanocytes isolated in culture from the epidermis of guinea pig ears were used to study the unscheduled DNA synthesis (UDS) induced by ultraviolet (UV) irradiation at 254 nm. After irradiation, the cells were labeled for 6 hr with 3H-thymidine (3HTdR), fixed, dehydrated, and flat embedded in Epon. Radioautographs were made on serial 0.5-1 micrometer thick sections of the cultures in order to distinguish silver grains from pigment granules. Cells irradiated with 5, 10, or 50 joules (J) m-2 showed a 3HTdR uptake due to UDS, whereas their proliferative ability, measured by the 3HTdR uptake due to scheduled DNA synthesis, was very low. For the same UV doses, UDS was lower in melanocytes than in guinea pig fibroblasts and keratinocytes.

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.

Since the papers by Steedman (1), Lison (2) and Mo wry (3), the use of Alcian Blue stain has undergone considerable change.

Cultured living human epidermal cells migrating on the surface of irradiated non-viable pig dermis, were found to produce bullous pemphigoid antigen at the epidermis-pig dermal junction between days 10 and 50 in culture. In certain series, the antigen was detected within the cytoplasm of the basal cells as well. These findings suggest that the bullous pemphigoid antigen is synthesized by epidermal cells.

Human Langerhans cells (LC) are located in the epidermal tissue which is naturally accessible to UV irradiation. They may be the first immunocompetent cells exposed to its effect. In the present study, the epidermal tissue was dissociated with trypsin, and epidermal cell (EC) suspensions, which contain keratinocytes, melanocytes, and LC were irradiated with UVB (10 or 20 mJ/ cm[sup2]). After irradiation LC retained their surface determinants: T-6 and HLA-Dr. In addition, their number did not decrease during 3 days of culture following UVB exposure as compared with nonirradiated EC cultured in parallel. On the contrary, UV irradiation of EC resulted in decreased lymphocyte-stimulating ability in a mixed skin cell-lymphocyte culture reaction (MSLR). EC used directly after irradiation in MSLR induced about half the lymphocyte response compared to nonirradiated EC. After 24-h culture, the irradiated EC did not produce any lymphocyte response, whereas the 48- h cultures showed a slight lymphocyte stimulation. At 72 h the cultures from irradiated and nonirradiated EC showed similar responses in MSLR. The doses of UV radiation which decreased MSLR responses did not affect EC viability and did not significantly reduce their DNA content. It is suggested that under the experimental conditions used in this study the defect induced by IJV irradiation was essentially functional and was the result of the transient inhibition of the antigen processing function of LC rather than of an alteration in membrane antigen expression (T-6 and HLA-Dr).

In early studies, the bullous pemphigoid antigen (BPA) has been localized extracellularly in the lamina lucida in the basement membrane zone. However, trypsin-dissociated basal cells can be tagged with bullous pemphigoid sera (BPS). By immunofluorescence, BPA appears located at the dermal pole of basal cells (BC). This may indicate that when BC are separated from the underlying matrix molecules, chunks of BPA remain attached to them. In the present study, fresh crude initial suspensions (CIS) of epidermal cells were prepared by trypsin-EDTA dissociation. The cells were smeared and air-dried. Polar fluorescent cells (i.e., BC) amounted to 42% +/- 7%. CIS were then passed through a fluorescence-activated cell sorter (FACS). In the fluorescent-positive fractions selected by FACS, 34% +/- 7% only of the BC were present. FACS-negative cell fractions were smeared on glass slides, air-dried, and restained with BPS + fluorescein isothiocyanate; 66% +/- 10% of BC were present in these fractions. This is evidence that trypsin-isolated BC comprise two subpopulations: one with BPA directly accessible, the other not. Viability tests and tissue culture studies indicated that the FACS-positive cell fractions were not viable. BPA was extracted from CIS, FACS-positive, and FACS-negative fractions and immunoblotted against BPS. Identical blots were found. FACS-negative cell fractions were treated with heparitinase, nitrous acid, methanol-chloroform, or EDTA without modifying the number of reacting cells. When BC were treated with Triton X-100 or permeabilized by successive freezings and thawings, the number of positive cells became comparable to those obtained by air-drying smears. Finally, BPA was localized on the intracellular part of hemidesmosomes of BC by immunoelectron microscopy. To see whether BPA was also present extracellularly, suction blisters were raised in minipigs and BPS injected into the blister cavity. BPA was found attached to all cells of the cellular roof but not to the dermal base of the blisters. When pieces of skin kept overnight in cold trypsin were reacted with BPS, BPA was found on both sides (epidermal and dermal) of the split. It is concluded that BPA has two localizations: one extracellular, essentially labile which accumulates at the dermal-epidermal junction; the other essentially stable which remains on the intracellular part of basal cell hemidesmosomes and which can be detected after permeabilization of the cells.

In ordinary cultures, cells are grown on artificial substrates and immersed in culture medium. In vivo, interfollicular epidermal cells grow on the basement membrane and are exposed to air. In a first effort to render the culture of these cells more physiological it seems legitimate to raise the cultured cells to the air-medium interface. Epidermal cells can be raised by the use of collagen gels maintained on a rigid support. They can also be grown on nitrocellulose filters coated with collagen or coated with a basement-membrane equivalent (BME) previously deposited by bovine corneal endothelial cells. By raising the cultures to the air-medium interface there is some evidence of a more complete differentiation, as evaluated by morphologic criteria. However, biochemically, the raising of the cultures does not seem to induce the synthesis of those keratin polypeptides which are not expressed in immersed cultures. Epidermal cells can also be raised by culturing them on dermal substrates or dermal equivalents. When they were cultured on inverted dead pig skin, epidermal cells synthesized membrane-coating granules (MCG). MCG were not found in immersed controls. By culturing epidermal-cell suspensions on dead deepidermized dermis (DED), all morphologic markers of differentiation were seen except the keratin pattern. In addition, partial reexpression of high-molecular-weight keratin polypeptides occurred. However, the complete expression of keratins by cultured cells depends on the filtering action of the dermal substrate (the cultures are fed from underneath) more than on exposure to the air-liquid interface. In summary, several methods are available to culture epidermal cells at the air-liquid interface that are of interest in an investigation of the response of these cells to epigenetic influences.

On histologic vertical sections of skin, the epidermis is separated from the dermis by an amorphous thin membrane, the basal lamina. Ultrastructurally, the basal lamina is composed of four areas, including the basal-cell plasma membrane and hemidesmosomes, the lamina incida, the lamina densa, and the sub-lamina densa fibrillar region. In culture, epidermal keratinocytes are able to produce hemidesmosomes, lamina lucida, and lamina densa. There is no evidence that cultured keratinocytes can produce sub-lamina densa fibrils. Biochemically, the lamina lucida contains two major glycoproteins. One, the bullous pemphigoid antigen, is synthesized by epidermal keratinocytes in vitro. These cells also synthesize laminin, the other glycoprotein of lamina lucida. At the interface between lamina lucida and lamina densa there is probably a heparan sulfate proteoglycan. Whether this proteoglycan is produced by keratinocytes in culture is not known, but the possibility can be considered. Lamina densa contains collagen IV, and this collagen is synthesized by keratinocytes in culture. However, cultured keratinocytes may also synthesize collagen types I, III, and V. Type V is associated with the basal lamina, but its exact location is unknown. Types I and III (if they are produced in vivo) would be situated in the sub-basal lamina region. The problem of fibronectin remains unsolved. There is "some" fibronectin in the lamina lucida, but its origin is not clear.

The limited number of Langerhans cells (LC) in human epidermis and the resultant technical difficulties have left open the question of LC kinetics. In the present study using flow cytometry (FCM) we have applied 3 methods to estimate LC-DNA distribution: (1) FCM-DNA measurement on highly enriched LC suspensions, (2) FCM-correlated analysis of DNA and OKT-6(+) cells in total epidermal cell suspensions, (3) LC-enriched suspensions (70–90%) were FACS (fluorescence-activated cell sorter) sorted on microscopic slides, and stained with the Feulgen technique, and DNA was measured densitometrically. In the latter method, contaminating keratinocytes were counterlabeled with antikeratin serum to eliminate them from LC-DNA estimation. All 3 in vitro analyses clearly showed that human LC are a cycling cell population in the epidermis. The number of LC in S (1.3–3.3%) and G 2 /M (1.0–2.5%) phase compares with those found for keratinocytes. Assuming that this percentage of keratinocytes in S and G 2 /M phases is sufficient to maintain the structural integrity of the epidermis, it was suggested that LC may represent a stable, self-reproducing cell population in normal epidermis.

The epidermis is composed of closely packed cells with very little intercellular matrix. Most of epidermal cells are keratinocytes which synthesize keratin and are eventually lost by desquamation. This loss is compensated by permanent proliferation of basal cells so that the biology of living epidermis is dominated by keratinization and regulation of growth. Mainly 2 mechanisms have been proposed for this regulation. The first is based on the fact that the epidermis is always in contact with the underlying dermis. The idea is that growth as well as keratinization are controlled by connective tissue factors passing through the basement membrane. According to the second, signals are emitted by maturing, keratinizing cells, which control the activity of basal cells by blocking them at various stages along the cell cycle. One major point in using epidermal cell cultures as models for living epidermis is to isolate epidermal cells to eventually study the various factors influencing their growth at the molecular level. Besides keratinocytes there are other epidermal cells such as melanocytes and Langerhans cells. But in contrast to keratinocytes which accomplish their biological function (keratinization) independently of other cell types, the functions of melanocytes and Langerhans cells are more or less dependent upon their interactions with adjacent keratinocytes with which they form some sort of epidermal “symbionts.” For example skin pigmentation is largely influenced by interactions between melanocytes and keratinocytes. Also, contacts between these latter cells and Langerhans cells are important in epidermal immunology. Thus, the epidermis is a complex tissue, the biology of which rests not only on that of the keratinocyte taken as a separate element, but also on the interactions that it (the keratinocyte) has with other epidermal cells. Consequently, when epidermal cell cultures are considered as models for living epidermis at least 2 types of culture Systems must be envisaged: one for the study of the biology of keratinocytes, proper, and one for that of keratinocytic-nonkeratinocytic cell interactions. In addition, living epidermis is morphologically organized. The proliferative compartment is composed of basal cells which appear like a palisade on vertical bistology sections whereas the maturing compartment is made of flattened cells which pile up into adjacent columns. This raises the question as to whether epidermal morphogenesis can be approached through tissue culture methods. Finally, living epidermis can regenerate after wounding and, here again the question is to see if regeneration can be studied in culture.

Eye-derived growth factor (EDGF) has been found in several ocular tissues and shown to be able to stimulate the in vitro proliferation of cells from various tissues and organisms. It had already been shown that EDGF differs biochemically and biologically from other growth factors such as epidermal growth factor (EGF) and fibroblast growth factor in that it is the only one that can stimulate the in vitro growth of human adult keratinocytes. Moreover EDGF stimulates reepithelialization and neovascularization. In this paper we report data concerning the effect on the rate of epidermal wound healing in guinea pigs of different extracts obtained from adult bovine retina. Our results show that EDGF can significantly increase the rate of reepithelialization when epidermis is detached from dermis and removed after induction of a blister. The doses used were comparable to the ones used to obtain maximal increase of cell proliferation in vitro. However no attempt was made to further investigate the mechanism accounting for the observed wound healing. At 24 h, control wounds maintained under occlusive dressing had only about 50% of their surface covered with cells as opposed to EDGF-treated wounds which were covered up to about 80% (p = 0.05). On the other hand, EGF does not increase the rate of wound healing in this model even at 1000-fold higher doses than those used in in vitro bioassays. Although EDGF is still not purified to homogeneity and another 10- to 100-fold purification might be necessary to achieve homogeneity, our results suggest that EDGF may find therapeutic applications as a potent in vivo epidermal wound healing agent.

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.

Stratified epithelia such as epidermis are classically considered to comprise 2 cell compartments, one consisting of undifferentiated proliferative cells occupying the basal layer, and the other consisting of differentiated postmitotic cells occupying the suprabasal layers. It is also generally assumed that the 58K basic-50K acidic couple of keratins is expressed in basal cells, while the 67K basic-56K acidic couple appears in suprabasal cells. In the present work we demonstrate that the population of basal keratinocytes is heterogeneous, since 8% of them are found to express the 67-56K "suprabasal" set of keratins. The morphology of these transitional cells suggests that they are in the process of detaching from the basement membrane to move upward to the epidermis. Cytoflowfluorometric studies showed that the fraction of cells in S plus G 2 / M phases is 4 times higher in transitional keratinocytes than in basal or suprabasal keratinocytes. Altogether, these results suggest that the onset of terminal differentiation occurs in human epidermis in a subpopulation of keratinocytes which are still located in the basal layer, and that a transient increase in proliferation occurs when the cells engage in terminal differentiation and are ready to move toward the suprabasal layers.

The major and vital function of skin is protection: protection against environmental elements that can be hazardous if they penetrate into the blood stream; protection against body fluid depletion. This latter point is examplified by the case of large burn patients who do not survive if their ion plasmatic concentrations are not adjusted and if their lost skin is not replaced.

Two main criticisms can be leveled against the standard methods of skin culture: they are poorly quantifiable and the cultured cell populations are heterogeneous. A new technique based mainly on enzymatic dissociation allows specific cell types to be extracted from the skin before cultivation. In this way, separate cultures of epidermal keratinocytes and dermal fibroblasts can be obtained from the same piece of skin. These purified systems have been used to study the kinetics of epidermal cell growth and to quantify the effect of various chemically defined substances on the growth and differentiation of keratinocytes. With further refinements in technique, purified populations of melanocytes can be extracted. The co-culture of pigmented melanocytes with albino keratinocytes has been proposed as a model to study pigment donation in vitro. The usual organ culture technique, including the use of large explants of skin immersed in the culture fluid, has been modified to show that adult human skin partially regenerates in vitro and that mitotic activity goes on for months in the regenerated epidermis. The use of nucleic acid hybridization techniques, combined with skin cell cultures from human tumors, opens new avenues of research on human cancer.

Among 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.

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 keratlnocytes were hypoplgmented. 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.

Human adult keratinocytes migrating on a nonviable dermal substrate in cultures without fibroblasts induce thinning and degradation of the collagen substrate beneath the migrating epithelium. Further, unconcentrated conditioned medium from the cultures exhibit collagenolytic activity against both type I and type IV collagen which is inhibited by EDTA but not by phenylmethylsulfonyl fluoride or N-ethylmaleimide. Since the migrating epithelium and dermal substrate do not contain fibroblasts, this study shows that migratory keratinocytes in contact with interstitial collagen are capable of producing collagenases against type I and type IV collagen. Moreover, migratory keratinocytes appear to be similar to highly metastatic cells in their ability to degrade basement membrane collagen.

The skin of three cases of generalized morphea in the active stage was studied by electron microscopy. The following cells were identified within areas of panniculitis: plasma cells, macrophages, and lymphocytes. Fibroblasts revealed a well developed rough endoplasmic reticulum filled with granular material. Phagocytosis of fat by macrophages was a prominent feature. Fat trabeculae were increased in thickness due to the deposition of immature collagen fibrils, ranging in diameter from 200 to 400 Angstroms. Collagen fibrils alternating in their orientation at a 90° angle were present in the lower dermis and in the subcutaneous tissue. A bimodal distribution of collagen fibrils was noted and consisted of fibrils of 350 and 950 A in diameter.