Cultured human epidermal allografts are not rejected for a long period.
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Aged↗
Biomedical subjects
M Faure
Publications and source records attributed to M Faure.
Effects of temperature, ouabain and diuretics on the cell sodium and potassium contents of isolated rat kidney tubules.
Cell Na+ and K+ contents were measured by flame photometry in single pieces of rat medullary thick ascending limb (MAL) and medullary collecting tubules (MCT) after an overnight incubation at various temperatures. Below 8 degrees C, MCT samples were no more able to sustain high-K+, low-Na+ cell concentrations, and sodium progressively replaced cell potassium as the temperature decreased. The loss of potassium and the accompanying accumulation of sodium by MCT cells occurred at lower or higher temperature when amiloride (20 mumol/l) or ouabain (1 mmol/l) was present respectively in the incubation medium compared to that observed on control non-treated MCT. In contrast, MAL samples maintained normal cation gradients across their membrane at all temperatures, including 0 degree C, even in the presence of ouabain. However, MAL cells lost nearly all their potassium which was replaced by sodium when they were incubated in K+-free solution. These Na+-loaded, K+-depleted MAL cells restored high-K+ and low-Na+ contents similar to those of control samples when they were further incubated for 1 h at 0 degree C in presence of 5 mmol/l external potassium. Even in the presence of 1 mmol/l ouabain and at 0 degree C, a restoration of almost normal cation gradients occurred provided that Na+-loaded MAL were incubated for 24 h after potassium addition to the external medium. The results are discussed in relation to the respective effects of low temperatures on the passive and active components of the cation balance in the cells of the two nephron segments.
Amiloride↗
Effects of external potassium concentrations on the cell sodium and potassium contents of isolated rat kidney tubules.
The effects of 20 mumol/l amiloride, 10 mumol/l furosemide and 1 mmol/l ouabain on cell Na and K concentrations were investigated by flame microphotometry in isolated rat medullary collecting tubules and medullary thick ascending limbs (MCT and MAL) as a function of the external potassium concentration [Ke]. The results are expressed as Na and K concentrations per liter cell volume ([Nac] and [Kc], mmol/l) and relative sodium content, [Nac]/([Nac] + [Kc]). From the experimental curves, [Ke]1/2 is defined as the [Ke] value corresponding to half maximal exchange of K against Na in cells. When [Ke] was 5 mmol/l, the relative Na content was less than 15% in control and amiloride-treated MCT as well as in control and furosemide-treated MAL, and about 24% in ouabain-treated MCT and MAL. In MCT, relative cell Na content increased up to 90% or more when [Ke] was reduced from 2.5 to 0.25 mmol/l. [Ke]1/2 was 0.55, 0.45 and 1.25 mmol/l for control, amiloride-treated and ouabain-treated MCT respectively. In MAL, similar increases in relative Na content were observed when [Ke] was reduced from 0.5 to 0.05 mmol/l. [Ke]1/2 was 0.25, 0.10 and 1.75 mmol/l for control, furosemide-treated and ouabain-treated MAL respectively. When [Ke] was reduced from 5 to 1 mmol/l, [Nac] dropped from 16.4 to 8.4 mmol/l (P less than 0.01) in control MAL. When [Ke] was 5 mmol/l, [Nac] was lower in furosemide-treated MAL (7.8 mmol/l) than control MAL (P less than 0.01). At 1 mmol/l [Ke], [Nac] was similar in both groups. These results are discussed in terms of the balance between the active and passive components of Na and K fluxes across apical and basolateral cell membranes. They indicate that a K-dependent passive Na entry process exists in the membranes of MAL cells but not of MCT cells. This process was proportionally more inhibited than the active Na pump when [Ke] was reduced from 5 to 1 mmol/l. In addition, it was found sensitive to furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Loss of allogeneic T-cell activating ability and Langerhans cell markers in human epidermal cell cultures.
Human Langerhans cells are the only epidermal cells that express the T6 and HLA-DR antigens and are responsible for the in vitro allogeneic T-cell proliferative responses in the mixed skin cell lymphocyte reaction (MSLR). To investigate the presence of Langerhans cells in normal human epidermal cell cultures, epidermal cell suspensions obtained from normal human skin specimens and from the subsequent epidermal cell cultures were analyzed by indirect immunofluorescence for the presence of T6 and HLA-DR determinants. In parallel, MSLRs were conducted with suspensions of cultured epidermal cells as stimulatory cells. These studies present evidence that when human epidermal cells are grown in culture, they loose both the ability to stimulate the proliferation of allogeneic T lymphocytes in vitro and their expression of HLA-DR and T6 antigens. The T6 antigens were lost during the first 2 weeks of culture, while HLA-DR determinants were still expressed by a small number of cells and were progressively lost through duration of cultures. The loss of HLA-DR antigens closely paralleled the progressive inability of human epidermal cells in culture to stimulate allogenic T cells in MSLR.
Antigens, Differentiation, T-Lymphocyte↗
Long-term survival and immunological tolerance of human epidermal allografts produced in culture.
Human epidermal cells from a small skin specimen can be grown in culture into multilayered sheets suitable for the permanent coverage of large burn wounds when used as epidermal autografts. We report here on the long-term survival of such cultured epidermal sheets used as epidermal allografts (EAG) across a major histocompatibility barrier in three nonimmunosuppressed adult patients, suffering from large chronic grafted leg ulcers, where the EAG have been placed to cover the conventional split-thickness skin autograft donor site. The absence of rejection was based upon clinical, histological, and immunopathological observation of the allografted sites at various intervals after grafting of the EAG. The identity of the epidermal cells on the grafted area with cultured cells from allogeneic donor was then established after blood substance typing by indirect immunofluorescence. Furthermore, epidermal cells from cultured sheets, but not control human cells from freshly excised normal epidermis, failed to stimulate the recipient peripheral blood cells in the mixed epidermal cell lymphocyte culture reaction, a finding that is related to the complete absence of class-II-antigen-bearing cells in cultured epidermis. This absence of T cell stimulation was noted not only on the day of grafting but throughout the follow-up. Altogether, these findings show that Langerhans cell and other class-II-antigen-bearing cell-depleted cultured epidermal allografts, are tolerated in unrelated recipients. EAG may serve as a skin substitute in patients with large wounds or burns. Since EAG may be grown continuously, the coverage of burns may not then be limited by the availability of the donor site, or by the time necessary to produce epidermal tissue in cultures.
Adult↗
Ultrastructure of multilayered cultures of human gingival epithelial cells: attachment to enamel surfaces in vitro.
Human gingival cells were collected using an enzymatic procedure and seeded on a feeder layer of irradiated mouse 3T3 fibroblasts. Epithelial cells generate stratified colonies ultimately forming an epithelium which was studied using electron microscopy. When this epithelium-like structure was dispased and transferred to enamel surfaces, the relationship between basal cells and enamel corresponded to half-desmosomes and a discontinuous extracellular matrix.
Adult↗
[Rapid production of large surface epidermal autografts].
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Burns↗
[Human epidermis grown in in vitro culture is not rejected and may be used as a skin allograft].
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Culture Techniques↗
Production of a lymphocyte differentiating factor (ELDIF) by cultured human epidermal cells.
T lymphocyte maturation activity of supernatants from cultured human epidermal cells was investigated using a biological rosette assay based upon Thy 1 induction on T cell precursors. Between days 5 and 18 of keratinocyte cultures, supernatants exhibited Thy 1-inductive properties. Optimal activity was found between days 10 and 14 of culture, which correspond to the beginning of epidermal cell stratification. This activity was independent of the presence of prostaglandin E. Absorption experiments using monoclonal and polyclonal antibodies showed that ELDIF was different from other recognized thymic hormones (thymulin, thymopoietin, thymosin alpha 1), produced by thymic epithelial cells and known to induce T cell markers.
Antigens, Differentiation, T-Lymphocyte↗
Immunogold technique applied to simultaneous identification of T6 and HLA-DR antigens on Langerhans cells by electron microscopy.
A double-labeling immunogold technique in electron microscopy and specific monoclonal antibodies to surface antigens of Langerhans cells (OKT6 and BL2) were applied to assess directly the coexpression of two cell surface antigens (T6 and HLA-DR antigens) in a heterogeneous epidermal cell suspension. Electron microscopic examination of double-labeled cells revealed that all Birbeck granule-containing Langerhans cells bound OKT6 and BL2. The preparation of markers with colloidal gold particles and the procedure for double labeling are described. Several problems related to the steric hindrance and current artifacts are illustrated by micrographs and also discussed.
Antibodies, Monoclonal↗
Recent advances of ultrastructural immunocytochemistry of epidermal Langerhans cells.
Using electron microscopy, the immunological visualization of the membrane antigens of Langerhans cells (LC) can be performed by immunoperoxidase and immunogold techniques. The immunoperoxidase labelling permits the identification of only one antigen and the observation of qualitative variations of surface antigens. The immunogold method allows the identification of one antigen or simultaneously two or three surface antigens using gold particles of various sizes. This technique can be used to quantify the surface density of antigens on the cell membrane. The simultaneous identification of different surface antigens can be correlated with the ultrastructural characteristics of the cells. Using this technique we have recently demonstrated the existence of LC subsets in normal epidermis, and the presence of circulating T6-positive cells in normal subjects. In addition, a very low density of T4 antigenic sites on the LC membrane surface was observed. Several problems of a double-labelling immunogold technique related to steric hindrance and current artifacts are discussed.
Antigens, Differentiation, T-Lymphocyte↗
Langerhans cell induced cytotoxic T-cell responses against normal human epidermal cell targets: in vitro studies.
We investigated the role of class II MHC (HLA-DR Ia-like) antigen-bearing Langerhans cells in the in vitro generation of human alloreactive cytotoxic T lymphocytes (CTL) directed against epidermal cells (EC). Normal EC in suspensions (from reconstructive surgery specimens) and anti-DR monoclonal antibody plus complement (C')-treated EC were used to stimulate peripheral blood (PB) T cells (T cells allogeneic or autologous to EC) in a classical 6-day human mixed skin cell-lymphocyte culture reaction (MSLR). CTL responses were then tested in 51Cr release assays against cultured EC (EC grown on collagen-coated plates in parallel to MSLR). CTL responses to EC were observed only after allogeneic MSLR and when targets and EC used in MSLR were from the same donor. They were comparable in magnitude to those seen in parallel studies using PBL from the same donor as the stimulating EC as target cells. They were abolished when EC used in MSLR were depleted in class II MHC Langerhans cells (preincubation of EC suspensions with anti-DR + C'). These data show: (a) alloreactive CTL to human normal EC may be generated in MSLR, as previously shown in animals; (b) the MSLR is an in vitro model of primary immunization against EC; (c) Langerhans cells are necessary for the generation of cell-mediated cytotoxicity reactions occurring against epidermal cells.
Cytotoxicity, Immunologic↗
Human epidermal cell-induced generation of alloreactive cytotoxic T-lymphocyte responses against epidermal cells. Requirement for DR-positive Langerhans cells.
Human epidermal cells act as stimulators in the mixed-skin cell lymphocyte culture reaction (MSLR). To analyse the generation in MSLR of alloreactive cytotoxic T lymphocytes (CTL) in cell-mediated cytolysis of human epidermal cells. a phenomenon suggested by various observations of skin inflammatory processes in vivo, 18-h 51Cr-release assays against epidermal cells cultivated on collagen-coated plates (epidermal cells autologous to the stimulator cells in MSLR) were conducted after allogenic human MSLR. To analyse the role of human Langerhans cells and related epidermal dendritic cells, which are the only cells expressing the DR-Ia-like (class II) antigens in normal epidermis and in suspensions of normal epidermal cells, MSLR and CTL assays were conducted with, as stimulator cells, suspensions of normal human epidermal cells as controls, and, in parallel, suspensions of epidermal cells after preincubation with anti-class II monoclonal antibody and complement. The generation of alloreactive CTL to epidermal cells occurred only after allogenic MSLR and when targets and stimulator cells were from the same donor; it was abolished when epidermal cell suspensions used in MSLR were depleted in HLA-DR-expressing cells. These findings demonstrate that an epidermal cell-induced generation of cell-mediated cytotoxicity to human epidermal cells may occur in vitro. Langerhans cells and other class II antigen-expressing epidermal cells (dendritic indeterminate cells) are necessary for an optimal in vitro sensitization in MSLR and the subsequent generation of alloreactive CTL towards epidermal cells in man.
Cytotoxicity, Immunologic↗
[Paraneoplastic erythroderma: apropos of a case].
A 63-year-old male patient had severe exfoliative dermatitis (erythroderma) which led to the discovery of squamous cell carcinoma of the larynx. The possibility of paraneoplastic erythroderma is discussed. Although such an aetiology is rare, 5 cases with a similar paraneoplastic syndrome have been previously reported, which cannot be considered as a merely fortuitous association of exfoliative dermatitis and visceral cancer.
Carcinoma, Squamous Cell↗
[Pruritus vulvae].
Vulvogenital pruritus is due to infectious, dystrophic or dermatological causes. Diagnoses of "senile" or "psychogenic" pruritus do not obviate the need for regular surveillance. Treatment is easy if the cause is duly recognised. It is more difficult in essential forms and the authors describe the principles and methods of symptomatic treatment.
Female↗
Ultrastructural immunogold labelling of human Langerhans cells enriched epidermal cell suspension.
Colloidal gold particles are well suited as markers in electron microscopy. Indirect immunogold staining was used to identify cell membrane antigens defined by monoclonal antibodies OKT6 and BL6 on human Langerhans cells (LC) in suspensions. Isolated epidermal cells were obtained by skin trypsinization and enriched or depleted in OKT6 positive on BL6 positive LC using the panning method: incubation of OKT6 or BL6 preincubated cells on immunoglobulin coated dishes. Indirect immunogold staining was then performed after prefixation in 2% paraformaldehyde. In LC enriched suspensions, only LC exhibited a specific membrane labelling with OKT6 or BL6 recognized by the presence of small evently distributed gold granules. Neither Birbeck granules, nor other cytoplasmic organelles, were labelled. No other epidermal cells were found positive. In LC depleted suspensions, no labelling was observed. Immunogold labelling on LC enriched suspensions after panning is now in progress for the qualitative evaluation and the quantitative analysis of cell surface constituents and antigens expressed by human dendritic epidermal cells.
Antibodies, Monoclonal↗