Search

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.

In a series of cholesteatoma patients the benefit of local application of acetylcysteine was demonstrated. Chemical studies and fractional analyses were used to reveal the breakdown of keratin. Histochemically the influence of acetylcysteine on keratin is confirmed.

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.