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M Haftek

Publications and source records attributed to M Haftek.

At least 55 records · Page 3Linked to original sources
PubMed1991

Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.

Cross-linked cornified envelopes are cell structures specifically synthesized by terminally differentiating keratinocytes. They are composed of proteins deposited at the cell periphery under the plasma membrane, and can be purified from epidermis by physicochemical extractions. The resulting keratinocyte "shells" are highly insoluble structures devoid of cytoplasmic components. The rigidity of the stratum corneum cell envelope seems to be one of the essential factors contributing to the physical resistance of this most superficial epidermal layer. We studied the purified cell envelopes from human plantar horny layer to determine their antigenic composition and protein distribution. The extraction protocol consisted of four 10-min cycles of boiling in 10 mM Tris-HCl buffer containing 2% SDS and 1% beta-mercaptoethanol. The absence of any extractable proteins persisting in the purified pellets was checked with SDS-PAGE of the sample electroeluates. Indirect immunofluorescence as well as pre- and post-embedding immunogold labeling for electron microscopy revealed the persistence of several keratinocyte antigenic determinants on the purified substrates. The antibodies directed against involucrin, keratin 10, desmoplakin I + II, desmoglein (intracellular epitope), intercellular corneodesmosome proteins, and filaggrin (a considerably weaker reactivity) labeled the cell envelopes according to the ultrastructural localization pattern characteristic for a given antigen. We conclude that the cytoskeletal and desmosomal components become "embedded" in the highly cross-linked cornified envelope structures during the process of keratinocyte terminal differentiation. This underlines the central role of cornified envelopes in the physical resistance of superficial epidermal layers and indicates a possible importance of junctional proteins in this function.

Cross Reactions↗
PubMed1990

Histological and ultrastructural effects of cyclosporin A on normal human skin xenografted on to nude mice.

Cyclosporin A (CsA) is a potent immunosuppressant with a selective activity on T-helper lymphocytes. However, CsA also exerts biological effects on non-lymphoid cells (fibroblasts, endothelial and epithelial cells). CsA can inhibit in vivo and in vitro DNA synthesis of epidermal keratinocytes (EK) and induces in vivo morphological alterations of kidney epithelial cells. In the present study we investigated the in vivo effects of a short-term CsA treatment (50 mg/kg per day) on DNA synthesis (evaluated through 5-bromo-2'-deoxyuridine incorporation) and on the histological features of normal human skin xenografted (NHSX) on to congenitally athymic nude mice. When compared with control NHSX, CsA induced a statistically significant inhibition of DNA synthesis of NHSX EK. At the light- and electron-microscopic level, apart from a decrease in the thickness of the viable epidermis of NHSX (statistically non-significant), no noticeable differences between treated and control NHSX could be detected. EK, Langerhans cells and melanocytes appeared morphologically unaffected by CsA and no signs of acute toxicity (giant mitochondria, vacuolization, microcalcifications) were seen. These results suggest that CsA exerts a subtle effect on human EK; indeed, despite an unequivocal antiproliferative activity, no significant histological changes related to the acute CsA toxicity seem to be induced on the various epidermal cell types.

Animals↗
PubMed1990

The effect of cyclosporin A on proliferation and differentiation-associated antigens of normal human skin xenografted onto nude mice.

Cyclosporin A (CsA) has been shown to inhibit, in vitro, the proliferation of cultured normal and neoplastic keratinocytes and to exert also in vivo an antiproliferative effect on keratinocytes of normal human skin xenografted onto nude mice. To gain further insight into the effects of CsA on human skin we investigated the immunohistochemical expression of several epidermal proliferation- and differentiation-associated antigens in the same model: six-week-old nude mice received a xenograft of full-thickness normal human skin; six animals subsequently received a daily subcutaneous injection of 50 mg/kg of CsA diluted in olive-oil while the others received an equivalent volume of olive-oil. The rate of epidermal proliferation was evaluated through a BrdU pulse-labelling technique, and was found to be decreased by 56% in the CsA-treated epidermal xenografts as compared to the controls. The xenografts were further examined for the expression of the following antigens: Epidermal Growth Factor- and Transferrin-receptors, Ki-67, 56.5 kD keratin polypeptide, Filaggrin, Involucrin, beta 2-microglobulin, Ulex Europaeus I- and Peanut-Agglutinin-binding sites. Most of these antigens were unchanged on CsA-treated human xenografts. However, the 56.5 kD keratin polypeptide which was consistently expressed by both basal and suprabasal epidermal keratinocytes in control xenografts showed a normal expression pattern (i.e. suprabasal keratinocytes only) in three out of the six CsA-treated xenografts. These results raise the possibility that, concurrently with a cytostatic effect, CsA may also affect keratinocyte differentiation and that this effect, possibly contributes in the beneficial effect of CsA in diseases of abnormal keratinization.

Animals↗
PubMed1990

Effects of cyclosporin A on cultured human epidermal keratinocytes. Growth and 5-bromo-2'-deoxyuridine incorporation.

We have studied the in vitro effect of CsA at various doses (0.5, 1, 5, 10 micrograms/ml) on the growth and 5-bromo-2'-deoxyuridine (BrdU) incorporation by cultured normal human epidermal keratinocytes (EK). CsA was found to reduce the growth of EK at all doses used after 24, 48 and 72 h in culture, but the difference with appropriate controls became statistically significant for the dose of 10 micrograms/ml after a 48- and 72-h culture. On the other hand, CsA-treated EK comprised a reduced fraction of BrdU + (S-phase) cells, the difference being statistically significant for the dose of 10 micrograms/ml at 24, 48 and 72 h. Ultrastructural examination of CsA-treated EK, despite the presence of cytoplasmic vacuoles also observed in control EK did not show signs of severe cytoplasmic or nuclear damage. These results confirm the antiproliferative effect of CsA on cultured EK and suggest that at the concentration used, CsA acts through a cytostatic rather than a cytotoxic mechanism, most likely by blocking EK in an early phase (G0/G1) of the cell cycle.

Cell Count↗
PubMed1989

Re-expression of disease-characteristic features of non-bullous congenital ichthyosiform erythroderma (CIE) after grafting of the pathological keratinocyte cultures to athymic mice.

Epidermal keratinocytes separated from skin lesions of non-bullous congenital ichthyosiform erythroderma were investigated in an attempt at experimental reproduction of this keratinization disorder. In vitro studies on growth and differentiation of pathological keratinocytes isolated from the influence of the host's dermal and humoral components were performed using the immersed epidermal cell culture technique. Ten to 25-day-old confluent and stratified cultures were examined by means of photonic and electron microscopy, and stained with various differentiation markers for indirect immunofluorescence studies. The cultured epidermis showed low-grade differentiation and no clear-cut evenly distributed signs of the original disease. Grafting on congenitally athymic nude mice allowed further differentiation of the epidermal sheets and re-expression of the histologic and ultrastructural features of non-bullous congenital ichthyosiform erythroderma. Thus, the purely epidermal origin of this particular form of autosomal recessive ichthyosis could be confirmed. Large amounts of pathological keratinocytes generated from small skin biopsies may be used for experimental purposes after grafting on several athymic animals.

Animals↗
PubMed1989

Nonimmunosuppressive ciclosporin H inhibits the growth and DNA synthesis of cultured normal human epidermal keratinocytes.

Ciclosporin H (CsH) is an epimer (11-D-N-methyl-L-valine) of ciclosporin A (CsA) devoid of immunosuppressive properties. The known antiproliferative effect of CsA on epidermal keratinocytes (EK) grown in vitro prompted us to study the effect of CsH (at the doses of 0.5, 1, 3.5 and 5 micrograms/ml) on the growth and DNA synthesis (5-bromo-2'-deoxyuridine incorporation). CsH was observed to reduce both the number and DNA synthesis of EK in a statistically significant manner at the dosage of 5 micrograms/ml after 24, 48 and 72 h in culture. These results show that the immunosuppressive properties of the ciclosporins (Cs) are dissociated from the antiproliferative ones on EK and highlight the interest of further in vivo studies that may lead to therapeutic applications of Cs in hyperproliferative epidermal diseases requiring no immunosuppression.

Bromodeoxyuridine↗
PubMed1989

The hapten in contact hypersensitivity to dinitrochlorobenzene: immunoelectron microscopic and immunofluorescent studies.

Six hours after challenge with dinitrochlorobenzene (DNCB) in sensitized volunteers, infrequent cells reacting with both OKT6 (CD 1a) and with an antibody to DNCB were demonstrated in the upper dermis in 3 out of 4 subjects, using a double fluorescence technique. Single marker studies using polyclonal and monoclonal antibodies to DNCB showed cytoplasmic reactivity with peripheral accentuation in keratinocytes throughout the epidermis, most intense in the granular and basal layers, and occasional positive cells in the papillary dermis. Immunoperoxidase electron microscopy using polyclonal and monoclonal antibodies to DNCB demonstrated reactivity on keratinocyte and Langerhans cell membranes, with intracellular deposition of reaction product on mitochondria. Reaction product was present on the external leaflets of Birbeck granules, suggesting that the penetration of DNCB was passive rather than due to an active endocytic process. The immunofluorescent studies suggest that the double-fluorescing upper dermal cells were Langerhans cells but this could not be confirmed by electron microscopy.

Aged↗
PubMed1989

Quantitative studies of keratin expression with the post-embedding immunogold labeling method.

Immunoreactivity of the 56.5 KD acidic (type I) keratin was localized ultrastructurally and quantified in normal human epidermis using the specific monoclonal antibody KL1 and post-embedding immunogold labeling. The protein was detected in keratin intermediate filament bundles of all suprabasal keratinocytes. Keratohyalin granules and desmosomal plaques were labeled only on the periphery, in regions where keratin filaments penetrate these structures. The 56.5 KD keratin immunoreactivity increased from the first suprabasal layer onwards and reached its maximum in the outmost spinous layer. A subsequent abrupt decrease of the specific immunogold labeling was observed in the granular layer. This low reactivity, which persisted also in the horny layer, may be partially explained by either protein degradation or masking of the antigenic sites by a filament-aggregating material occurring at these stages of keratinocyte terminal differentiation. Statistical comparison of the quantitative results obtained in various cell and tissue compartments revealed no significant differences between the background labeling levels observed in the basal layer of epidermis with KL1, a control monoclonal antibody, or the immunogold conjugate alone. Our results confirm the specificity of 56.5 KD keratin for terminally differentiating suprabasal keratinocytes and demonstrate the importance of appropriate control studies when a post-embedding immunogold labeling method is employed.

Adult↗
PubMed1989

Cyclosporin A does not modify Langerhans' cell number and distribution in normal human skin.

We used the model of human skin graft on nude mouse to investigate a possible influence of systemically administered cyclosporin A (CsA) on epidermal Langerhans' cells (LC). This experimental model has the advantage of being independent of the systemic humoral and cellular influences of a human host. No change in the human LC distribution or number could be observed after 3 weeks of CsA therapy as revealed by anti-CD1 and anti-HLA-DR immunohistochemical stainings and the cell counting, despite the evidence of the drug effect on the keratinocyte proliferation. However, our findings do not rule out the possibility that CsA influences the LC functional capacities responsible for the local cell-mediated immunity responses.

Animals↗
PubMed1988

Structure, function, and immunogenicity of human insulinoma cells.

Dissociated human insulinoma cells were plated onto plastic multiwell dishes. Cells were maintained for 1 mo on plastic with three passages. Cultures consisted of small colonies with some areas of stratification and few intercellular spaces. Ultrastructural studies indicated that cultured cells had epithelial features with desmosomes at cell-to-cell contacts and intermediate filaments in addition to secretory granules in the cytoplasm. Insulin and C-peptide were released in equimolar amounts in culture media. When challenged for 30 min with 16.7 mM glucose, 1 mM 3-isobutyl-1-methylxanthine, 4 mM tolbutamide, or 10(-6) M glucagon, insulinoma cells responded by a 1.5-, 1.5-, 2-, or 3-fold increase, respectively, in insulin release above baseline levels. A 15-min challenge with 10(-5) M isoproterenol increased insulin secretion by 1.85-fold. By indirect immunofluorescence, an anti-insulin antibody reacted positively with cell cytoplasm, whereas anti-somatostatin and anti-glucagon antibodies did not. Insulinoma cell surface expressed class I MHC molecules but not class II molecules. Immediately after isolation, crude insulinoma cells were contaminated by 2% of DR+ cells from nonislet components that disappeared after several weeks in culture. The ability of insulinoma cells to stimulate allogenic T-lymphocyte proliferation was assessed by [3H]thymidine incorporation in mixed culture combinations. Crude insulinoma cells elicited a strong lymphoproliferative response with a stimulation index ranging between 3.5 and 7, whereas no stimulation was found after 1 mo in culture. It is postulated that absence of class II-positive cells in the stimulatory cell preparation conditioned this immune tolerance across the major histocompatibility barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1988

Sequence of changes in psoriatic epidermis. Immunocompetent cell redistribution precedes altered expression of keratinocyte differentiation markers.

Recent studies suggest that the immune system is involved in the pathogenesis of psoriasis. We studied the expression and distribution of immunocompetent cells and of some chosen differentiation antigens of keratinocytes at various stages of lesion development, using indirect and amplified immunofluorescence, and avidin-biotin-peroxidase method. Serial cryostat sections were collected so as to allow comparative studies of adjacent parts of each biopsy sample with various immunocytochemical markers. Our results indicate that focal intra-epidermal infiltration of otherwise unaltered epidermis with lymphocytes, mostly of the T-helper phenotype, was the first perceptible change occurring in patients with eruptive psoriasis. Modification of the Langerhans' cell staining was observed in these initial subclinical lesions. A significant reduction of the cell frequency was noted in psoriatic papules and plaques. Changes in the epidermal antigen expression could be observed in the developed lesions only. The simultaneous appearance of histologic signs of psoriasis and the modification of keratinocyte antigens indicates that both events are related to the epidermis hyperproliferation, possibly induced by focal inflammatory reaction.

Adolescent↗
PubMed1987

Langerhans cells in epidermodysplasia verruciformis.

T cell defect and energy to contact sensitizers are characteristic of the depressed cell-mediated immunity in epidermodysplasia verruciformis (EV). In this disease the generalized infection with human papilloma viruses is associated with a high risk of skin cancers. Langerhans cell density was studied quantitatively in lesions and noninvolved epidermis of EV using OKT6 and anti-HLA-DR monoclonal antibodies with indirect immunofluorescence technique. No significant changes were found in apparently normal skin suggesting no primary defect of Langerhans cells in EV. A marked decrease in Langerhans cell number per unit of epidermal volume could be observed in EV lesions. This additional reduction of immunological surveillance at sites of potentially oncogenic human papilloma virus infection may contribute to the increased risk of malignant transformation in EV lesions.

Antigens, Surface↗
PubMed1987

Effects of PUVA and mechlorethamine treatment of psoriatic patients on epidermal Langerhans' cells.

Using OKT6 monoclonal antibody, we investigated the number of epidermal Langerhans' cells (LCs) in involved skin from patients with psoriasis, before and after mechlorethamine (HN2) or PUVA treatment. The number of LCs remained at about pretreatment number during three weeks of HN2 treatment alone, though they were reduced after 10 systemic PUVA treatments. Therefore, in contrast to PUVA which influences LCs, HN2 seems to have little effect on LCs. LCs in psoriatic plaques were, in number, 3-4 times less numerous than those in uninvolved, nontreated epidermis.

Antibodies, Monoclonal↗