Electron immunolocalization of type IV collagen, laminin and fibronectin synthesized by multilayered cells cultured from human oral epithelium.
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Biomedical subjects
Publications and source records attributed to M Faure.
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Cultured human epithelia obtained from epidermal cells in vitro were used to assay the activity of staphylococcal epidermolytic toxin and develop an in vitro experimental model for the staphylococcal scalded skin syndrome. Human epidermal cells were grown from single epidermal cell suspensions obtained through trypsinization of adult normal skin into multilayered epithelia (with a basal cell layer, several intermediate and one or two upper layers) on mouse 3T3 feeder cells. First passage cultures were incubated with exfoliative toxin A from phage Group II staphylococci at various concentrations in DMEM. They were examined at various time intervals by direct microscopic and histological examination of respectively the culture plates or the epidermal sheets after their detachment from the plates with dispase grad II. A total exfoliation could be obtained at 24 hour at concentrations of Img and 500 micrograms/ml, only local areas of epidermolysis noted at 100 micrograms/ml. The intraepithelial separation was noted to occur between the basal layer and the lowest intermediate layer. No exfoliation could be observed at lower concentrations. Up to 4-5 hours few changes were evident, but at this time small areas of epidermolysis developed. With exfoliatin 100 micrograms/ml, intraepidermal blisters were clearly visible, occurring either between the basal cells and the lowest intermediate layer or between the first two intermediate cell layers. At the ultrastructural level, desmosomes were sparse and altered, with enlargement of the intercellular spaces and condensation of tonofilaments. These data indicate that human epidermal cell cultures, although their differentiation in culture only mimics what occurs in vivo, can be used as an in vitro model of the staphylococcal TEN to further investigate the site of action of such a toxin and the cellular mechanism responsible for the syndrome.
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Extracellular matrices (ECM) have been reported to enhance epithelial cell attachment and proliferation as well as to induce differentiation in vitro. Since ECM components are physiological constituents of the dermoepidermal basement membrane, we studied the growth and differentiation of human keratinocytes on ECM in order to determine the benefits of culturing epidermal epithelial cells (keratinocytes) on reconstituted basement membranes. Disaggregated epidermal cells were grown in primary and subcultures in liquid medium; the attachment of the cells was greatly enhanced by ECM and noted within the first few hours after seeding; cells formed small islets that reached confluence within 2-12 days depending upon the plating density and the type of culture (primary or passages). Histological and ultrastructural cross-sections of the cultures clearly indicated that a multilayered epithelium can be obtained including a basal cell layer, several intermediate cell layers with cytoplasmic organelles, intermediate size filaments, desmosomes, and keratohyaline granules, and an upper layer of anucleated cells. Using immunofluorescence, both pemphigus and pemphigoid (basal membrane zone) antigens were expressed. The keratin pattern noted indicated that these epithelia differentiate and keratinize but do not express a complete program of keratinization, a finding usually noted when cells are grown submersed. These data show that ECM favor epidermal cell proliferation and differentiation and suggest that they may be used to obtain large amounts of epidermal equivalent suitable for grafting and/or in vitro studies.
In this work the ultrastructural features of cultured epithelial sheets (CES) used as skin allografts in humans are described, before and at various times after grafting. Prior to grafting, CES consisted of 4-5 layers of keratinocytes of a low to moderate degree of differentiation. However, after grafting, the CES developed progressively but rapidly features of a well-differentiated epidermis (including melanocytes and Langerhans' cells) and a dermal-epidermal junction. No evidence of rejection was observed. These results demonstrate the key role of normal dermis in the maturation of the surface epithelium and prove the suitability of CES as skin allografts.
Epidermal cells (EC) were cultured without stimulation and the effect of these EC culture supernatants (ECCS) on human in vitro B-cell response was determined. Supernatants obtained between Days 5 and 7 were able to replace monocytes in the antibody response to the particulate antigen trinitrophenyl-polyacrylamide (TNP-PAA). These results were obtained when highly monocyte-depleted cultures (less than 0.5% peroxidase-positive cells) were used and were reproduced with supernatants from several different EC cultures. ECCS could not substitute for T cells in the T-dependent response to TNP-PAA. They contained an interleukin 1 (IL-1) activity but no interleukin 2 or B-cell growth factor (BCGF) activities. We tested the effect of ECCS on the proliferative response of highly monocyte-depleted B cells cultured at low cell density costimulated with anti-u antibody and BCGF. ECCS had no BCGF-like activity of its own but did potentiate the effect of BCGF. Thus EC cultures produce IL-1-like factor(s) which act directly on the early stages of B-cell activation.
We have examined the biologic characteristics and immunologic properties of epidermal cell-derived lymphocyte differentiating factor (ELDIF), a lymphocyte differentiating factor produced by cultured human keratinocytes. The ELDIF was semipurified by a gel filtration procedure. This factor, which is distinct from prostaglandins, epidermal cell-derived thymocyte activating factor (ETAF), and the well-known thymic hormones (thymulin, thymopoietin, and thymosin alpha 1) did not exhibit any interleukin (IL)-1, IL-2, or IL-3 activity. It strongly inhibited in vitro lymphoproliferative responses of normal mouse spleen cells to phytohemagglutinin, concanavalin A, and lipopolysaccharide. This dose-dependent phenomenon was associated with a suppression of IL-2 production rather than any toxic effect. It can be concluded that ELDIF, a product of human epidermal cells, which displays in vitro T-cell differentiation and regulatory activities, could be of major importance in vivo in the control of cutaneous inflammatory reactions.
Human keratinocytes from small skin specimens were grown on mouse 3T3 cell feeder layers into epidermal sheets free from Langerhans cells and MHC class II antigen. These were found to be suitable for the permanent coverage of wounds when used as autografts or allografts. We report here the ultrastructural differentiation of this cultured epidermis after grafting onto autologous or allogeneic recipients. The cultured epidermis was a thin but multilayered Malpighian epithelium composed of keratinocytes at different stages of differentiation. The dermo-epidermal basement membrane was newly synthesized during the first few days following transplantation onto de-epidermized wounds. The analysis of keratins and examination of various keratinocyte membrane antigens by immunofluorescence indicated that full terminal epithelial differentiation was only achieved after in vivo transplantation of the cultured epidermis. Langerhans cells, absent in cultures, progressively colonized the grafts, while melanocytes, not detectable in sections of the cultures, were identified among the keratinocytes 2 weeks after grafting.
In vitro grown class II-MHC antigen free epidermal sheets were used as epidermal allografts (EAG) across a major histocompatibility barrier in 20 non-immunosuppressed recipients suffering from leg ulcers. Class I antigens were expressed on cell membranes of basal cell layer only on the epidermal sheets. After grafting, patchy areas of membrane fluorescence were observed among cells from the suprabasal layers on the epidermis from skin biopsies taken between days 5 and 28. All cells of the basal and the suprabasal layers expressed class I antigens on biopsies taken after day 28, as on normal human epidermis. This work demonstrates that class I antigens are expressed by epidermal cells in cultures used for grafting. The absence of rejection cannot be explained by the absence of class I-MHC antigens in EAG.
Human keratinocytes may be grown in vitro into living epithelia devoid of Langerhans cells and MHC class II antigens. These epithelia have been shown to be usable as epidermal allografts in patients with dermal wounds, without any apparent sign of rejection in the 12-month follow-up study. To evidence a progressive replacement by recipient cells of the grafted keratinocytes, we employed anti-MHC class I antigen monoclonal antibodies directed against tissue specificities expressed by either donors or recipients. At 2 and 4 weeks after grafting, some small epithelial cell islets from the recipient phenotype were clearly identified among cells from a donor origin by indirect immunofluorescence. At 6 months, all keratinocytes present at the grafted areas were labelled by antibodies directed toward recipient specificities only. This replacement may be related to the fact that, when placed on such superficial dermal wounds, the allografts are likely colonized by epithelial cells proliferating from residual recipient dermal appendages.
The expression of blood-group antigens was studied on human epidermal cultures used as allografts in 13 non-immunosuppressed patients treated for leg ulcers. The study was carried out using monoclonal antibodies to A and B antigens by an indirect immunofluorescence technique. Blood-group antigens are weakly expressed on the suprabasal layers of the cultured epidermal sheets. After grafting, the donor's blood-group antigens were detected on a few cells of the suprabasal layers. Furthermore, scattered keratinocytes as well as acrosyringia were found to express the recipient's blood-group antigens.
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Paranoid Reaction (or bouffée délirante) according to the french classification was the basic psychosis of primitive societies. It corresponds to a need of defense of restructuration or restitution according to a pattern of "misconduction" admitted by the cultural environment. Paranoid reaction, in this respect, offers some relative good pronostic as soon as the goal is reached even if the previous personality remains modified. Paranoid reaction may get closer to hysterical crepuscular states. Some symptoms may also evoke schizophrenias. Evolution toward a lasting psychosis may be considered only in case the process of deculturation/acculturation is already engaged in an irreversible way and the defensive mode presented in the new cultural system shows schizophrenic patterns. Quick transformations and strong cultural pressures, trough that primitive societies are going, might increase dramatically chronic evolution of paranoid reactions. A case has been related to illustrate those considerations.
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