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A quantitative histochemical study was carried out on the distribution of protein thiol and disulphide groups in normal human plantar epidermal tissue. Histochemical demonstration of reactive groups was achieved by addition of N-(4-aminophenyl) maleimide, subsequent diazotization and final coupling with a Nitro Red or chromotropic acid label as first described by Sippel. The quantitative reliability of the method was tested by absorption cytophotometry, and evaluated on the basis of the internal consistency of the results reported. Our histological observations and histophotometric data support accepted views on epidermal keratinization. A limited, though reproducible, amount of disulphide bonds was observed near the basement membrane. The free thiol concentration in basal and prickle cells was low and almost constant, but was higher in the granular cells, where deposition of sulphur-containing proteins on cell membranes is initiated. In Malpighian layers, disulphide cross-links only occurred just beneath the transition zone in thickened cell membranes. The staining pattern of the inner stratum corneum resembled a mosaic and was characterized by a Sharp rise of the disulphide content, which exceeded the decrease in free thiol groups. The free thiol concentration decreased further throughout the cornified layers whilst the disulphide content remained fairly constant. Staining of thiol and disulphide groups together corresponded, within the limits of the standard error, to the sum of the thiol and disulphide concentrations when they were assayed separately in living ahd horny cells. These results confirm that living cells are the main site of free thiol groups, while horny cells are the most prominent of site of disulphide cross-links.

Cytokeratin expression was studied in human middle ear cholesteatoma lesions, using a variety of immunohistological techniques and a wide range of polyclonal antisera and monoclonal antibodies against cytokeratin (CK) subgroups or individual CK polypeptides. The expression of the other cytoskeletal proteins, vimentin and desmin, was also investigated. Middle ear mucosa and epidermal tissues were used as reference tissues. Our investigations also included epithelial structures present in the cholesteatoma perimatrix and in dermal tissues. The results indicate that, compared with epidermal tissues, the expression profile of CKs in cholesteatoma matrix is representative of a hyperproliferative disease. Evaluating the presence of a marker of terminal keratinization - the 56.5 kD acidic CK n degrees 10 - we found supportive evidence of a pronounced retardation of its expression, which did not parallel histological differentiation. In epidermal tissues, the first prickle cell layers are CK10 positive whereas in many cholesteatomas this finding was observed near the stratum granulosum only. Probing the early stages of keratinization - the 58 kD basic CK n degrees 5 and the 50 kD acidic CK n degrees 14 - we regularly observed an extended staining area in the cholesteatoma matrix. In epidermal reference tissues, only the basal and nearest suprabasal layers were convincingly labeled. As a rule, non-epidermal CKs did not belong to the cholesteatoma CK set. However, exceptions to that rule were noticed as a focal or more extended expression of one or more non-epidermal CKs in about half of the cases. Together with the extended CK5 topography, this is further evidence that CK expression is seriously affected by the diseased state. CK expression in the perimatrix is limited to mucous glands, either normal, atrophic or hyperplastic. CKs n degrees 4, 5, 7, 14, 18 and 19, also displayed by middle ear mucosa, were consistently observed. Where ductal arrangements were present, CK10 was also detected, in analogy with the CK10 registration in ductal portions of mucous glands in the external ear canal skin. The absence of CK8 in mucous glands of the perimatrix, however, strongly differentiates these structures from the mucous gland acini and ducti in the external ear canal, where CK8 is systematically expressed. Vimentin staining was restricted to dendritic cells of the matrix (Langerhans cells) and to perimatrix fibroblasts, blood cells and vascular endothelium. Coexpression of CK and vimentin was not observed.

For the extraction of soft keratins from bovine snout or human epidermis the best results were obtained with a detergent (sodium dodecyl sulphate, SDS) or hydrogen bond breaking agent (urea) in a reducing medium (2-mercaptoethanol, 2-ME). N-acetylcysteine was a little less effective. Keratins from both sources gave typical sets of protein bands with molecular weights between 40.000 and 70.000 daltons. Upon electrofocusing special precautions had to be taken to avoid reoxidation and reaggregation of protein subunits. Keratins from aural cholesteatomas were obtained by extraction with SDA and 2-ME, with N-acetylcysteine alone and to a lesser extent with urea and 2-ME. The pattern of these keratins on SDS-gel is less complicated than that obtained from human skin or bovine snout. Histophotometric results argue for a clear analogy between the nuclear DNA metabolism in normal skin epidermis and cholesteatoma matrix. The only differences of potential relevance are the much wider range of the nuclear DNA amount in cholesteatoma stratum basale cells compared with basal cells in normal epidermis, and the higher persistence of nuclear DNA in cholesteatoma stratum granulosum, indicating postponement of keratinocyte terminal differentiation.

Quantitative DNA cytophotometric investigations were performed to clarify some aspects of the differentiation and fate of nuclei in bovine snout and human epidermis representing various sites and different degrees of keratinization. We elaborated optimal conditions for hydrolysis and Feulgen staining. Diverse cytophotometric techniques, including computerized scanning cytophotometry and image analysis were applied. This approach provided the first quantitative data concerning changes of nucleotype during soft keratinization. Cytophotometric DNA measurements provide evidence for a continuous decline of nuclear DNA content from immediately beyond the basal layer to the transition zone. The overall loss of DNA is an orderly process that intensifies gradually and culminates in the stratum granulosum. Gradual nuclear degeneration, however, is not a general phenomenon, and a significant number of nuclei retains a DNA content within the diploid limits throughout the entire stratum spinosum and part of the stratum granulosum. At any level of differentiation or decay, residual nucleoprotein complexes remain intact, as judged from their resistance to acid hydrolysis. Karyological features change completely during keratinization. Basal cell nuclei are rather compact, ellipsoid and heterochromatic. Beyond the basal layer, nuclei enlarge, round up and obviously evolve to an extremely euchromatic state, with preferential localization of the dispersed heterochromatic clumps at the more peripheral sites. In the upper stratum spinosum, nuclei undergo even more drastic changes: nuclear area and volume shrink, nuclei partially regain the ellipsoid shape and revert to heterochromasia. Nevertheless, euchromatin remains the major constituent of decaying nuclei. Terminal differentiation stages, except in human sole, are marked by heterochromatin clumping. In human sole, persistence or even progression of heterochromatin dispersion is observed. Heterochromatic dots are situated along the nuclear membrane in human terminal keratinocytes, but are almost randomly distributed in bovine stratum granulosum nuclei. Finally, nuclear contrast analysis partially reveals statistically significant changes throughout keratinization.

Quantitative DNA cytophotometric techniques were applied to judge the alteration (differentiation) and ultimate fate of nuclei during keratinization in human middle ear cholesteatoma. Compared with a healthy epidermis, a tendency towards postponed nuclear degradation was noticed. Two patterns governing the loss of DNA are recognized. In one group, the mean nuclear DNA content declines continuously, starting in the nearest suprabasal layers and continuing throughout the prickle and granular cell stages, where the ultimate degeneration of nuclei takes place. This pathway corresponds to that observed in epidermis, but evolves more slowly. In another group of samples, the onset of the DNA decline is delayed to the upper prickle cells, exceptionally to more terminal stages of keratinization. During matrix keratinization, a profound nuclear remodelling takes place, similar to that in epidermal tissues, as far as eu- and heterchromatin DNA and area data are concerned. However, euchromatinization of nuclei in matrix prickle cells is more pronounced than in epidermal tissues. The topography of residual heterochromatic clumps does not reflect a persistent margination as in epidermal nuclei, but is the result of more individualized rearrangements. The changes in karyotype are less elaborate when the complete decline of the nuclear DNA content only occurs during terminal keratinization.