D. Broekaert

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 no 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 no 5 and the 50 kD acidic CK no 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 no 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.

Immunohistochemical Analysis of the Cytokeratin Expression in Middle Ear Cholesteatoma and Related Epithelial Tissues

Immunohistochemical investigations were carried out to determine the pattern of cytokeratin (CK) expression in middle ear cholesteatoma and related epithelia. Using monoclonal antibodies specific for CK chains and the indirect immunoperoxidase technique, we examined 10 CK polypeptides for expression. The external stratified squamous epithelium of the tympanic membrane generally expressed CKs 5, 10, and 14. In addition, basal keratinocytes in the annular region of the pars tensa expressed CK 19 (a simple epithelium marker), while suprabasally the hyperproliferative marker CK 16 was expressed. These data reflect the unusual proliferative nature of this region. The unexpected appearance of CK 16 (known to have a limited distribution in healthy epidermis) clearly relates to its expression in the neighboring deep meatus. The medial simple epithelium of the eardrum revealed mucosal CKs 7, 8, 14, 18, and 19. Acquired cholesteatoma lesions, besides CKs 5, 10, and 14, consistently expressed CK 16 in suprabasal layers. These results constitute the first direct molecular evidence for the hyperproliferative nature of the cholesteatoma matrix. Overall, our CK data suggest that aural cholesteatoma lesions and epidermal tissue in this area are related. However, they do not explain the mechanism(s) by which the eardrum or meatal epithelia might invade the middle ear cavity. Congenital cholesteatomas expressed CKs 5, 10, 14, and 16 equally. These CK data do not support the idea of a metaplastic origin from middle ear mucosa; instead, they suggest activation of an ectodermal rest in the middle ear cavity.

An investigation of cytokeratin expression in skin epithelial cysts and some uncommon types of cystic tumours using chain-specific antibodies

SummaryThe differentiation state of skin epithelial cysts and some uncommon types of epithelial skin tumours was investigated by immunohistochemical staining, mainly using cytokeratin (CK) polypeptide-specific monoclonal antibodies. Samples of interfollicular epidermis, hair follicles and eccrine sweat glands were included as reference tissues. The CK reactivity in epidermoid cysts and milia is not restricted to CKs involved in epidermal-type differentiation, i.e. CK1, 5, 10 and 14, but in addition CK16, a hyperproliferative keratinocyte marker is suprabasally expressed. CK1 and 10 are other prominent suprabasal markers, while CK14 seems to be preferentially expressed in the basal cell layer. Of the non-epidermal CKs, only CK4 was focally or more extensively detected in about 50% of the cases. In terms of CK reactivity, keratinization of trichilemmal cysts corresponds to the keratinization of the anagen-phase hair follicle in the isthmus. The CK reactivity is again restricted to CK1, 5, 10, 14 and 16. However, the CK1 as well as CK10 reactivity is subject to serious limitations, since both CKs were only convincingly observed in foci of terminal differentiation. Eccrine hydrocystoma obligatorily expresses a complex CK set, including CK7, 8, 14, 18 and 19. This CK set perfectly corresponds to the CK composition observed in acini of eccrine sweat glands. In addition, a discontinuous CK4 and 16 reactivity was seen in about 50% of the sites, while CK1 and 10 displayed a strictly focal appearance. On the other hand, syringoma produces in its distinct structures, a CK pattern reminiscent of the one observed in eccrine sweat gland ducts and includes CK1, 5, 10, 14, 16 and 19. Finally, the CK expression pattern of pilomatricoma includes CK1, 8, 10, 14 and 19, and is reminiscent of the CK staining of hair bulb matrix cells differentiating in the keratogenous zone in the direction of hair cortex. The reactivity of CK1 and 10 was mainly restricted to foci of squamoid differentiation and also to transitional cells bordering on shadow cells, as far as it concerns CK10. Occasionally, CK7 and 16 were observed in individual cells or small cell groups. In our view these CK reactivity patterns are useful to judge the differentiation state reached in pathological conditions, but so far do not allow us unequivocally to determine the site of origin of these lesions.

A quantitative histochemical study of sulphydryl and disulphide content during normal epidermal keratinization.

SummaryA 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 ofN-(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 and horny cells. These results confirm that living cells are the main site of free thiol groups, while horny cells are the most prominent site of disulphide cross-links.

An immunohistochemical and histochemical study of cytokeratin, involucrin and transglutaminase in seborrhoeic keratosis

The mode of differentiation of seborrhoeic keratoses was investigated by immunohistochemical staining using cytokeratin (CK) polypeptide-specific monoclonal antibodies and an antibody specific for the particulate form of epidermal transglutaminase (ETgase), and by applying an anti-human involucrin serum. The role played by (E)Tgase was further evaluated using an activity assay based on the covalent attachment of monodansylcadaverine. Samples of uninvolved epidermis served as reference tissue. CK reactivities suggested that seborrhoeic keratoses is a hyperproliferative disease with an epidermal CK composition. CK5 and CK14 were prominent markers of basal and basaloid keratinocytes, whereas a decrease in staining occurred in advanced maturation stages and areas of terminal keratinization. In contrast, CK1 and CK10 were prominent markers of suprabasaloid differentiation stages and produced complementary stainings to those of CK5 and 14. Generally, CK10 staining was more impressive than CK1 staining and seemed to start before CK1 staining. In contrast to CK10 staining, cornified areas lost CK1 reactivity. These staining patterns were similar to those observed in uninvolved reference tissues. The epidermal CK subset was further supplemented with the ‘hyperproliferative’ CK6 and 16 which occur sequentially. Positive staining for CK6 was noted from basal and proximal basaloid cells onwards, whereas distal basaloid cells additionally showed CK16 staining. The presence of other non-epidermal CK polypeptides could not be shown. The competence for other differentiation markers belonging to the group of (E)Tgase and cornifying cell membranes also evolved with a typical epidermal pattern. (E)Tgase activity was restricted to advanced and terminal stages of keratinization and was dual in nature, i.e. a diffuse cytoplasmic staining occurred together with a prominent staining of cornifying cell membranes. Similarly, involucrin first detected in the cytosol of distal basaloid cells, was soon translocated to the cornifying cell membrane, reflecting its function as an ETgase substrate and precursor of the marginal band. Finally, the immunolocalization of the particulate form of ETgase was strikingly similar to the location of the first two markers. Taken together, the results allow us to conclude that seborrhoeic keratoses exhibits a hyperproliferative variant of the epidermal keratinization process. Maturation of basal keratinocytes is greatly retarded leading to an accumulation of basaloid cells which retain the molecular markers of basal cells in proximal areas, but progressively gain the molecular markers of advancing maturation in distal areas.

Nuclear differentiation and ultimate fate during epidermal keratinization

SummaryQuantitative 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.

The origin of wound-induced satellite DNA in pea seedlings

DNA synthesis in wounded pea seedlings was studied by incorporation of [6-3H]-thymidine. It was shown that the satellite DNA, synthesized within the first two days after wounding, is not of plant origin, but produced by contaminating bacteria, although a strong sterilizing procedure was used. Main-band DNA synthesized after wounding seems to be the only plant DNA synthesized during the stress-period.

Characterization of the DNA Synthesized in Pea Seedlings after Wounding

Summary The characteristics of the newly synthesized main-band DNA in wounded and control pea seedlings were studied by analytical and preparative CsCl density gradient centrifugation in neutral and alkaline conditions, by Cs2SO4 gradient analysis in the absence or presence of Ag+ and Hg2+ in different concentrations, by examination of thermal denaturation, using the spectrophotometric technique or column chromatography on hydroxylapatite and by polyamino acid-kieselguhr column chromatography. The base composition calculated from the melting point is 43.2% GC and differs from the composition deduced from the density (35.7% GC). This discrepancy is caused by the presence of 4.9 to 5.3% 5-methylcytosine. All experiments show the similarity of the newly synthesized DNA (radioactivity profile) and the total DNA isolated from wounded and unwounded tissues (UV absorbancy) and point to the conclusion of a random base distribution in the pea genome. The presence of an AT or GC rich satellite DNA could not be demonstrated, even using selective Ag+ or Hg2+ complex formation and Cs2SO4 gradient centrifugation in conditions which reveal such DNA in other plant species.

Histophotometric DNA measurements onUmbelliferae, Solanaceae andCompositae, before and after crown gall tumour induction

SummaryHistophotometric measurements of the relative amount of DNA per nucleus in control cells (meristematic cells of the plumula) and in differentiated cells (procambium and parenchyma cells of the first or second internode or leaf mesophyll cells) reveal that the investigated species, belonging to theUmbelliferae, Solanaceae andCompositae, differentiate within the diploid conditions. After crown gall transformation, the nuclear DNA amount remains within the diploid limits. However, a limited number of cells, grouped or spread in the tumour goes through an endomitotic cell cycle and becomes octaploid or even 16-ploid. The conclusion seems justified that a strong relationship exists between the nuclear condition in the host plant and in the tumour tissues. The importance of “primary” (releated to the mitotic stimulus and dedifferentiation of existing cells at the onset of tumour growth) and “secondary” phenomena (resulting from the specific physiology in the tumours) is discussed.

Quantitative determination of free thiol and di8ulphide groups by a fluorescent maleimide procedure

To determine the quantitative reliability of thiol histofluorometry, the distribution of protein thiol and disulphide groups was reinvestigated in normal human epidermis, labelled with DACM-3 after cryocutting. Microscopical observations roughly confirmed, that living keratinocytes and inner stratum corneum are the main sites of free thiol groups, while disulphide crosslinks are almost exclusively found in cornified cells. Histofluorometric quantitation led to free--SH, --S--S-- or combined --SH and --S--S-profiles through the different strata, that were not compatible with previous absorption-histophotometric studies and lacked internal consistency. The difficulties reside mainly at the methodological level and may partially be resolved--at least in keratinocytes--by extraction of nonstructural SH-groups in a prerinsing step. Permanent mounting further contribute to the realization of normal fluorescence behaviour, as visualized by the decay curves. DACM-saturation of structural protein reactive groups was only reached after prolonged staining.