Organic acids and anaerobic microorganisms in the contents of the cholesteatoma sac.

Organic acids in the contents of the cholesteatoma sac from 28 cases were studied by gas chromatographic technique. Five volatile fatty acids (acetate, propionate, isobutyrate, butyrate and isovalerate) and lactate were detected in large amounts, which may lower the pH of the cholesteatoma content. These acids were considered to be derived from products of anaerobic microorganisms. Therefore, the contents from 12 cases were cultured anaerobically in a glove box. Obligate microorganisms were identified in 92% of the cases and Peptococcus, Bacteroides, and Clostridium species were frequently isolated. In vitro, such obligate anaerobes produced various organic acids from the cholesteatoma content. Facultatives such as Staphylococcus aureus and Proteus mirabilis produced acetate in the content under aerobic and anaerobic conditions, whereas no organic acid was produced by Pseudomonas aeruginosa. Organic acids in the cholesteatoma content could be fermentative products made by the microorganisms, anaerobes and facultatives, which use the content as a substrate for acid production.

[Estrogen receptors in patients with brain tumors].

Estrogen receptor (ER) of brain tumors was assayed in 45 patients. In this series there were 39 cases contained ER, ranged from 0.7 to 188 fmol/mg protein of tumors (mean: 16.1 fmol/mg). The mean level of ER was 10.3 fmol/mg in 20 cases with meningiomas, 8.4 fmol/mg in 11 cases with gliomas, 51.1 fmol/mg in 5 cases with neurilemmomas, 10.5 fmol/mg in 3 cases with medulloblastomas, 16 fmol/mg in 2 organized hematomas due to ruptured AVM, 5.7 fmol/mg in a chordoma, 3.9 fmol/mg in a metastatic carcinoma from thyroid gland, and 0.8 fmol/mg in a cholesteatoma. The presence of ER in the latter several kinds of brain tumors have not been reported in previous literatures. The ER level of the tumors correlated with histological type of tumors, as well as the sex and age of the patients.

A comparative immunohistochemical study of cytokeratin and vimentin expression in middle ear mucosa and cholesteatoma, and in epidermis.

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.

Structural and topological studies of cholesteatoma proteins, in relation to the keratinization process.

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.