Psoriasis vulgaris: immunologlobulin binding to human epidermis after limited proteolysis]

Immune complexes isolated from sera of psoriatic patients and healthy controls show an identical pattern in sodiumdodecylsulfate polyacrylamide gel electrophoresis. And so it seems to be unlikely that the increased provable serum immune complexes by psoriatics contain specific protein antigens.--Following to limited proteolysis of normal human epidermis, antibodies which solely are provable in sera of psoriatic patients could bind to proteolytic altered structures. For achieving a similar process in vivo, the following conditions are to be discussed: (1) an elevated concentration of certain autoantibodies in the sera of psoriatic patients and a temporary increased vessels permeability for antibodies, (2) the availability of structures in the epidermis of psoriatics with increased affintiy to certain antibodies. These structures may be produced by limited proteolysis or as a result of the disturbed differentiation of epidermal cells.     

Precursor keratin protein from human epidermis

Prekeratin has been isolated from human epidermis using the method of Matoltsy for bovine nose epidermis. Analysis by sedimentation equilibrium indicates that human prekeratin is heterogeneous in solution. The molecular weight range is from about 200 X 10(3) to at least 2 X 10(6). Polyacrylamide gel electrophoresis shows three polypeptide chains of molecular weights 65,000 60,000 and 58,000. A lower than average ratio of polar/apolar amino acid residues suggests that hydrophobic interactions are important for stability.     

Characterization of pemphigus foliaceus antigen from human epidermis

Pemphigus foliaceus (PF) and its endemic form, Fogo Selvagem (FS), are characterized by subcorneal vesicles and pathogenic IgG autoantibodies directed against keratinocyte surface antigens. A major pool of FS antigen(s) remains bound to the insoluble epidermal envelope fraction. In this paper we demonstrate that this antigen(s) can be released from the envelope fraction by sonication. By immune precipitation four components can be detected, having molecular weights (MW) of 260, 80, 62, and 45 kD. The 260-kD component is lost by boiling or extraction with glycine HCl at pH 2.8. The major components appear to be the 80- and 62-kD poly-peptides. They chromatograph as a unit by gel filtration in 0.1% SDS, in the MW range of 115-120 kD. The FS antigen(s) appears to be cationic, forming insoluble complexes at low pH with SDS, and is labile to ammonium sulfate and freezing and thawing. It is unaffected by positive pressure concentration, 50% acetone precipitation, and reduction/alkylation. The FS antigen(s) is precipitated by all FS and nonendemic PF sera except those in complete clinical and serologic remission. The FS antigen(s) is also precipitated by 50% of pemphigus vulgaris but none of the bullous pemphigoid sera tested. All FS antigenic components are immunoprecipitated by IgG4 autoantibodies, but the IgG1 subclass from the same patients appear to immunoprecipitate only the 62-kD polypeptide. The FS antigen(s) is able to adsorb human autoantibodies against human desmoglein 1 (DG1), but not rabbit antisera against bovine DG1 or 2. This paper shows that physical stress, i.e., sonication, may be able to solubilize sufficient FS antigen(s) from the epidermal envelope fractions for further chemical characterization. The relationship of these FS antigen(s) to other reported FS antigens is presently unknown.     

Age incidence of antibodies to normal human epidermis

Antibodies reactive with various layers of normal human epidermis can be detected in a high percentage of sera from normal adults. Stimulation to produce these antibodies occurs at a very early age, with some suggestion that in utero stimulation may occur in a small number of cases.     

Binding of nickel to human epidermis in vitro

Human epidermis was homogenized, dried and incubated with nickel chloride solutions in the concentration range 0.008-3.4 mM/50 mg epidermis. Nickel uptake was found to occur according to the Freundlich adsorption isotherm, giving a slope of 0.55, indicating that nickel is associatively bound to constituents of the epidermis. The binding characteristics of cobalt were very similar to those of nickel, but the two metals were not found to compete significantly for binding sites in the epidermis. The metal-chelating agents, ethylenediamine tetra-acetic acid disodium, (Na-EDTA) L-histidine and D-penicillamine were tested for ability to remove nickel from its binding sites. Na-EDTA was found to be the most efficient, removing 70-90% of the nickel bound to the tissue. The possible clinical significance of these in vitro observations are discussed in relation to percutaneous absorption and allergic contact dermatitis.     

Identification and partial purification of chalone from human epidermis

Crude human skin extracts having tissue-specific mitotic inhibitory activity (chalone), were studied by ethanol precipitation and disc gel electrophoresis. Precipitates obtained at various ethanol concentrations were dialyzed, lyophilized and tested for their mitotic inhibitory activity. It was found that the greatest inhibitory activity was concentrated in the precipitate formed in 81% ethanol, the active principle being concentrated about 200 times. Proteins in crude extracts were separated by disc gel electrophoresis, and at least eight bands were identified. Groups of bands were extracted from the electrophoretic gels and tested for their mitotic inhibitory activity. Fraction II, containing two bands, had the highest activity. Disc gel electrophoretic analysis of the 81% ethane precipitate revealed only two major bands, one of which could also be found in fraction II of the crude extract. This protein band, which is present in both of the mitotic inhibitory samples, may represent the human epidermal chalone.     

Protein phosphatase activity in human keratinocytes cultured from normal epidermis and epidermis from patients with harlequin ichthyosis

We investigated serine/threonine protein phosphatase (PP) activity and the expression of PP2A during growth and differentiation of epidermal keratinocytes in culture. Keratinocyte PP activity was strongly inhibited by calyculin A and okadaic acid. indicating that the activity was mainly due to PP2A and PP1. The phosphatase activity decreased to about 20% of the initial (day 1) level by the time of confluence and to about 10% at day 7 postconfluence. In contrast to activity. the level of expression of the PP2A catalytic subunit protein and the mRNA for the two isoforms increased slightly over the period of growth. Keratinocyte differentiation was shown by a significant increase in profilaggrin expression after confluence. Keratinocytes were also cultured from individuals affected with harlequin ichthyosis. This severe hyperkeratotic skin disorder has abnormal lipid structures and is blocked in the PP2A-dependent conversion of phosphorylated profilaggrin to the non-phosphorylated filaggrin. The PP activity in harlequin cultures was lower than in normal cultures (about 20% of the subconfluent normal control value) and decreased even further in confluent cultures. In contrast.the level of expression of the PP2A catalytic subunit protein and mRNA for the two isoforms was similar to that of normal keratinocytes and increased with confluence. These results suggest that PP activity in keratinocytes is regulated in a post-translational manner: they also support the possibility of impaired or reduced function of PPs in harlequin ichthyosis.     

Selective cultivation of human melanocytes from newborn and adult epidermis

Development of adequate culture systems for the human epidermal melanocyte is critical to further advances in pigment cell biology. We now report selective growth and long-term maintenance of melanocytes derived from both newborn and adult skin specimens. Disaggregated epidermal cell suspensions were plated in a hormone-supplemented medium containing cholera toxin and a hypothalamic extract treated to remove keratinocyte growth-promoting activity. After 3-4 weeks, pure melanocyte populations could be harvested and serially passaged up to 6 times over several months for a total of 10 or more cumulative population doublings in vitro. Electron microscopic studies revealed metabolically active cells with abundant melanosomes in various stages of melanization throughout the culture lifespan. Differences in size and number of melanosomes attributable to race of the tissue donor were readily apparent, and pigment content of melanocytes from both black and Caucasian donors appeared to increase with time in culture. Newborn melanocytes proliferated more rapidly and survived longer than did adult melanocytes, but there were no consistent morphologic differences as a function of donor age. Comparison of growth potential for the 3 major skin-derived cell types in this hormone-supplemented medium revealed striking specificity for melanocytes, with total elimination of keratinocytes over 1-2 weeks, and no fibroblast proliferation whatever in the absence of serum supplementation. This system promises to facilitate in vitro investigation of epidermal melanocytes in normal and diseased human skin.