Monoclonal antibodies specific for subsets of epidermal keratins: biochemical and immunocytochemical characterization--applications in pathology and cell culture

Keratin composition has been widely used as a biochemical marker of differentiation in normal epithelia, cell culture systems and tumours of epithelial tissues. We have been developing a model system for the study of human squamous epithelial cell differentiation, and among a panel of monoclonal antibodies we have generated for analysing this system are two antibodies recognizing subsets of epidermal keratins. The two antibodies, designated LICR-LON-16a and LICR-LON-29b, were raised to the human squamous carcinoma cell line LICR-LON-HN-5, and we describe here their biochemical and immunocytochemical characterization. Antibody 16a reacts with only epidermal basal cells in normal human skin and shows specificity for the 45 and 46 kdalton keratins. Antibody 29b stains all living layers of the epidermis, and reacts with a broad range of ketain polypeptides, (45-56 kdaltons) in immunoblotting analyses. We have investigated the alterations of cellular staining that occur in chronic hyperproliferative skin diseases and carcinomas and compared this with the staining of multilayered cultures of normal keratinocytes and the HN-5 cell line. We show that in squamous cell carcinomas and in HN-5 cell xenografts 16a and 29b stain only the well-differentiated cell types. Furthermore we found that the basal cell specificity of 16a was lost in all of the hyperproliferative skin lesions examined including psoriasis and eczema. This transition to suprabasal staining pattern was also seen in the cultures of normal keratinocytes and HN-5 cells. We conclude that aberrant keratin synthesis or abnormal post-translational processing of keratins associated with an increased rate of cell turnover could account for the altered expression of the epitope recognized by antibody 16a.     

Keratin expression in cutaneous lichen planus

The characteristic expression of keratins by keratinocytes is well documented. A typical 'hyperproliferative' profile of epidermal keratin expression occurs in psoriasis, wound healing and warts. This study analyses keratin expression in cutaneous lichen planus to determine abnormalities of differentiation occuring in this inflammatory disorder. Using a panel of monoclonal antibodies 28 samples (20 patients) were studied. The results showed that squamous differentiation was unaffected, with keratins K1 and K10 being expressed normally for the site sampled. The main abnormalities included extension of reactivity of the basal cell marker, LH8, into the suprabasal compartment. Keratin K17, usually restricted to adnexal structures, was variably expressed in the basal and suprabasal layers of the interfollicular epithelium of affected epidermis. Keratins K6 and K16, found suprabasally in hyperproliferative states, were detected both basally and suprabasally in all diseased samples. The keratin profile in lichen planus is analogous to the wound healing response. Suprabasal keratin K17 is found in psoriasis, wound healing and viral warts so the changes in keratin K17 may reflect hyperproliferative changes. It is likely that the changes in epidermal keratin expression are due to up-regulation of specific keratin genes by the production of cytokines and inflammatory mediators from the lymphocytic infiltrate typical of lichen planus.     

Keratin subsets in papillary and follicular thyroid lesions

 Previous studies indicate that keratins 7, 8 and 18 are present in all thyroid papillary and follicular lesions, but the distribution of other keratins has been incompletely characterized. The profile of individual keratin (K) polypeptides was evaluated immunohistochemically in over 200 non-neoplastic and neoplastic thyroid papillary and follicular lesions. Monoclonal antibodies to K19, K17, K16, K5/6 and K10 were applied in paraffin sections of formaldehyde-fixed tissue. K19 was present variably, often only focally in goitres, and was present only sporadically in papillary hyperplasia. However, K19 was strongly and uniformly expressed in virtually all papillary carcinomas, indicating differential diagnostic usefulness in differentiating papillary hyperplasia and papillary carcinoma. About half of the follicular carcinomas (defined as tumours strictly excluding the follicular variant of papillary carcinoma) were also strongly K19-positive, suggesting that K19 patterns are not reliable in differentiating papillary and follicular carcinoma. K17 and K5/6 were present in cysts and squamous metaplasia of goitres, and focally in papillary but only exceptionally in follicular carcinoma in areas of squamous differentiation and tumour cells in desmoplastic stroma. K16 in turn was present only focally in well-developed squamous metaplasia in goitres but was not found in differentiated thyroid carcinomas. K10, a high-molecular-weight keratin typical of epidermal differentiation, was identified neither in non-neoplastic nor in neoplastic differentiated thyroid lesions, including squamous metaplasia. These results indicate that papillary carcinomas differ from other differentiated thyroid tumours in their varying, usually focal, expression of stratified epithelial keratins that are partly but not exclusively related to squamous differentiation in such lesions. However, papillary carcinomas do not express truly epidermally restricted keratins; their previously described reactivity with polyclonal ”epidermal keratin” antibodies most probably results from the reactivity of such antibodies with K19. Received: 14 April 1997 / Accepted: 28 May 1997     

Antibodies to intermediate filament proteins as molecular markers in clinical tumor pathology. Differentiation of carcinomas by their reaction with different cytokeratin antibodies

Antibodies to human and bovine epidermal prekeratin and antibodies to mouse liver cytokeratin component D (Mr 49 000) have been applied in indirect immunofluorescence microscopy on sections of human tumors of mammary gland and liver. In non-neoplastic mammary gland all epithelial cells were stained with these antibodies. In pre-invasive and invasive ductal and lobular carcinomas a cell population was observed which was not significantly stained with antibodies to epidermal prekeratin but did strongly react with antibodies to liver cytokeratin D. In the liver, the antibodies to epidermal prekeratin as well as those directed against liver cytokeratin D strongly decorated bile duct epithelia. In contrast, significant staining of the hepatocytes was only achieved with antibodies to liver cytokeratin D. This different staining reaction was maintained in liver tumors of hepatocellular and cholangiocellular origin. Antibodies to vimentin stained mesenchymal cells and tumors of mesenchymal derivation but reacted not significantly with any of the epithelial and carcinoma cells examined. The difference is of practical importance for the discrimination between anaplastic carcinomas and sarcomas of unknown origin. Cytokeratin could also be detected by antibody staining using the peroxidase-antiperoxidase (PAP) technique in formaldehyde-fixed and paraffin-embedded material of skin, gastrointestinal, respiratory, urinary and genital tract as well as various glands, liver and kidney. Examples of positive reactions were shown in a squamous cell carcinoma, a basalioma and a pleomorphic adenoma of the parotis. It is concluded that the immunohistochemical analysis of intermediate filament proteins has diagnostic potential in clinical pathology and may help to elucidate histogenesis and differentiation of tumors and possibly also prognosis of tumor growth. It is further suggested to use antibodies recognizing different subsets of proteins of the cytokeratin family in order to distinguish between different types of carcinomas.     

Cytokeratin polypeptides expression in different epithelial elements of human salivary glands

Summary Immunofluorescent labeling of human salivary glands was carried out with a battery of monoclonal antibodies reactive with specific cytokeratin polypeptides. All the epithelial elements of the glands were positively labelled by a broad-spectrum cytokeratin antibody (KG 8.13) and by antibody Ks 18.18, which reacts with cytokeratin No. 18 exclusively. Labelling of frozen sections with antibody KM 4.62, which is reactive with the 40 Kd (No. 19) cytokeratin, was confined to the ductal system and apparently absent from the acini. Antibody KA-1, reactive with polypeptides 4, 5 and 6 stained both the myoepithelial cells and the basal cells of the large ducts. Antibody KS 8.58, however, reacted with the basal cells exclusively. It is thus proposed that the combined use of the various monoclonal antibodies may provide a most useful probe in studies on epithelial cell diversity in normal salivary glands as well as in pathological disorders of that gland.     

Rat antikeratin monoclonal antibodies are specific for stratified squamous epithelia

Seven monoclonal antibodies of rat origin were generated against human epidermal keratins and were assayed by immunoblot and by immunoperoxidase staining of frozen sections from various human epithelial tissues. By immunoblotting, the antibodies recognized two different subsets of high molecular weight cytokeratin polypeptides. Unlike the majority of the reported mouse monoclonal antibodies obtained to similar immunogens, these rat monoclonal antibodies showed an unexpected degree of specificity, i.e., they stained only stratified squamous epithelia and not simple epithelia nor urothelia. The potential of using rats and the appropriate immunogen to generate more tissue-specific anticytokeratin monoclonal antibodies is discussed.     

Primary culture of human oral epithelial cells. Growth requirements and expression of differentiated characteristics

Epithelial cell cultures were initiated from explants of normal human oral mucosa. Growth parameters, cell type, and degree of maturation/cytodifferentiation were assessed by morphological and surface topographical criteria (light and scanning electron microscopy) together with immunofluorescence studies with a panel of antibodies to cytokeratins and extracellular matrix components. The effects of different media formulations were compared. Whereas stromal cell over-growth soon became apparent in media containing 10% serum, in low serum (0.5%) media containing insulin, hydrocortisone, epidermal growth factor (EGF), and/or cholera toxin (CT), epithelial growth was maintained with minimal or absent stromal cell contamination. Cell proliferation, maturation, and differentiation were modulated by EGF and CT: cultures maintained on EGF showed optimal growth but cells typically displayed only limited differentiation. By contrast, CT promoted considerably more cytodifferentiation but at the expense of proliferative capacity. Both factors together were complementary, resulting in maintenance of cells of a more mature phenotype of high proliferative capacity. Cytokeratins of normal oral epithelium in situ demonstrated characteristic changes in patterns of expression associated with differentiation. In culture, proliferative epithelial cells expressed keratins typical of the basal layer, whereas the most differentiated cells were identified by their strong reactivity with antibodies to epidermal keratins. Less mature cells showed expression of keratins associated with nonstratified epithelia. In cultures maintained with CT but no EGF, there was a tendency for weaker expression of basal type keratins, further suggesting that these cells were maintaining a more differentiated phenotype. Extracellular matrix components (fibronectin, laminin, collagen type IV) were not expressed by any epithelial cells in culture. Irrespective of medium composition, cultures did not survive beyond 100 days (5 or 6 subcultivations) before undergoing an irreversible 'crisis' of growth arrest and onset of degenerative changes.     

Alterations in the expression of two epidermal differentiation antigens in human epidermal disorders

The expression of an epidermal keratin subunit and a specific antigen of the keratinocyte membrane, two differentiation antigens in normal human epidermis, was studied in benign and malignant epidermal lesions by use of monoclonal antibodies KL1 (anti 55-57 Kd keratins) and KL3 (anti keratinocyte membrane antigen). In normal human epidermis, KL1 labelled all keratinocytes from the suprabasal layers, KL3 stained the intercellular spaces in all epidermal layers with a fluorescence intensity increasing from the basal to the more upper layers and recognized a keratinocyte membrane antigen as demonstrated in electron microscopy. Frozen or deparaffinized sections of basal cell carcinomas (BCC), squamous cell carcinomas (SCC) malignant melanomas, warts, and skin biopsies from benign lesions (psoriasis, lichen planus, bullous pemphigoid, lupus erythematodes, pemphigus, vasculitis) were tested with either KL1 or KL3 by indirect immunofluorescence and/or immunoperoxidase. Benign and malignant lesions in which modifications of the keratinization process and cell differentiation are known to occur (BCC, SCC, warts, psoriasis) showed the most severe alterations as compared to normal epidermis. With KL1 we observed an irregular staining of basal cells; a reorganization of keratin filaments and variable staining intensities within tumoral cells which did not express high MW keratins. With KL3 drastic alterations in the epidermal intercellular patterns and loss of reactivity of tumoral cells were noted. Conversely, the positivity of epidermal basal cells with KL1, in some cases, was the only modification noted in other skin lesions.     

体外培养人骨髓间充质干细胞向表皮细胞分化及表皮细胞角蛋白的表达

背景：动物实验已经证实骨髓间充质干细胞体外诱导可分化为表皮细胞。 目的：观察体外培养条件下人骨髓间充质干细胞向表皮细胞的分化及表皮细胞角蛋白表达。   方法：采用Ficoll-Paque密度梯度离心法提取人胚胎骨髓间充质干细胞,以免疫细胞化学及流式细胞仪测定细胞表面CD33、CD34标记物进行鉴定。取第3代骨髓间充质干细胞以30%条件培养基诱导其向表皮细胞分化,免疫细胞化学染色观察诱导后细胞形态与细胞角蛋白水平变化。 结果与结论：采用密度梯度离心法从人胚胎骨髓中分离培养得到细胞成分均一的骨髓间充质干细胞。骨髓间充质干细胞经体外诱导后,出现细胞角蛋白19表达阳性细胞,说明骨髓间充质干细胞在体外诱导后可能发生向表皮细胞分化。     

Trichoblastic carcinoma ("malignant trichoblastoma") with lymphatic and hematogenous metastases.

We report an aggressively behaving malignant trichogenic tumor arising in a trichoblastoma (TB) with widespread lymphatic and hematogenous metastases in a 55-year-old man with a concomitant B-cell chronic lymphocytic leukemia. The primary tumor had been present and unchanged for as long as 40 years before excision. Typical trichogenic TB with dystrophic calcification and even ossification was still present peripheral to the malignant transformation. The malignant neoplasm consisted of basaloid cells, spindle cells arranged in fascicles and densely packed rounded nests or "cell balls." The metastases consisted of immature basaloid cells and cell balls, and the recurrences became successively more undifferentiated. The residual TB reacted with antibodies to cytokeratin (CK) 6, 8, 14, and 17 and focally to S-100; the malignant primary tumor reacted uniformly with antibodies to vimentin and only focally with antibodies to CK and S-100. The metastatic tumor had lost epidermal CK expression but maintained expression of S-100 in paraffin-embedded tissues. Trichoblastic differentiation was confirmed in frozen tissues with antibodies to hair keratins. No expression of p53 or bcl-2 was identified, but p-glycoprotein (MDR-1 gene related) was expressed by primary and metastatic tumor cells. We believe that this neoplasm is best classified as a trichoblastic carcinoma arising in a TB in association with a B-cell chronic lymphocytic leukemia. This case illustrates that TBs have the potential for malignant transformation and aggressive behavior.     

Changing patterns of keratin expression during progression of cervical intraepithelial neoplasia.

The expression of keratins in normal cervical epithelia, metaplastic epithelium, and cervical intraepithelial neoplasia (CIN) grades I, II, and III is investigated with a panel of keratin polypeptide-specific monoclonal antibodies. This approach allowed the detection of individual keratins 4, 7, 8, 10, 13, 14, 18, and 19 at the single-cell level. By using an antibody recognizing keratins 5 and 8 (RCK 102) and two antibodies specific for keratin 8 (CAM 5.2 and M 20), it was also possible to derive information on the distribution of keratin 5. Our results show that during immature squamous metaplasia there is an acquisition of keratins typical of squamous epithelium, ie, keratins 4, 5, 13, and 14. This process continues during further differentiation to mature squamous metaplasia. In premalignant lesions the expression pattern of the progenitor reserve cells and immature squamous metaplastic epithelium is partly conserved. However, in most cases an induction in the expression of the keratins 4, 13, and 14 was observed. Furthermore, CIN III shows a more extensive expression of keratins typical of simple epithelia, ie, keratins 8 and 18, as compared to CIN I and CIN II.     

Effect of retinoid deficiency on keratin expression in mouse bladder

Twelve to sixteen weeks following treatment of CF-1 mice with a vitamin A-deficient diet, characteristic signs of retinoid deficiency including body wasting, poor hair coat, altered gait, decreased mobility, and xerophthalmia were observed. Histological examination of tissue sections from these mice revealed dramatic changes in the urinary tract epithelium. The normal transitional epithelium was replaced by a stratified squamous epithelium that resembled hyperproliferative epidermis. Using two-dimensional gel electrophoresis, a number of new proteins were found to be synthesized in vitamin A-deficient bladder when compared to tissue from control bladders. Using antikeratin antibodies in immunoblot experiments, we found that at least some of the newly synthesized proteins were keratins. These proteins, which comprise the intermediate filaments of the cytoskeleton, are known to be specific markers of epithelial differentiation. Of particular interest was the appearance of a Mr 67,000 basic and Mr 61,000 acidic keratin pair, characteristic of terminally differentiating murine epidermal cells. Unexpectedly, several other keratins, previously associated only with hyperproliferative epidermis, were also expressed in the tissue. These results demonstrate that vitamin A deficiency in the mouse leads to the appearance of a squamous metaplasia in the urinary tract epithelium that is characterized by the expression of distinct epidermal keratins.     

Mutated c-Ha-ras oncogene alters cytokeratin expression in the human breast epithelial cell line MCF-10A.

MCF-10A, a spontaneously immortalized human breast epithelial cell line, has been transformed by transfection with the mutated c-Ha-ras oncogene obtained from T24 carcinoma cells. The pattern of cytokeratin expression was studied in MCF-10A cells in comparison with plasmid transfected or MCF-10Aneo cells, normal ras proto oncogene transfected or MCF-10AneoN cells, and transformed or MCF-10AneoT cells. Cytokeratin expression was studied by western immunoblot of total cellular proteins separated by two-dimensional gel electrophoresis. Blots with cytokeratin specific AE1 and AE3 antibodies showed identical molecular weight species of cytokeratins in MCF-10A, MCF-10Aneo, MCF-10AneoN, and MCF-10AneoT cells; however, in MCF-10AneoT cells, the intensity of immunostaining and the number of immunoreactive phosphorylated polypeptides keratins 7, 8, 15, and 16 was decreased. It was concluded that c-Ha-ras oncogene-induced transformation alters quantitatively the cytokeratin pattern of human breast epithelial cells and that these changes could explain some of the morphologic alterations observed in c-Ha-ras transformed cells.     

Notch Signaling May Be Involved in the Abnormal Differentiation of Epidermal Keratinocytes in Psoriasis

Localization of each keratin isoform differs among epidermal layers. Proliferating basal cells synthesize keratin 14 (K14) and suprabasal cells express keratin 10 (K10) in normal skin. Notch signaling is essential for keratinocyte differentiation. Notch1 is expressed in all epidermal layers, Notch2 in the basal cell layer and Notch3 in basal cell and spinous cell layers in normal epidermis. It has been poorly elucidated how localization and expression levels of Notch molecules are related to epidermal molecular markers K10 and K14 in psoriatic skin with abnormal differentiation of epidermal tissue. This study aimed to investigate the relationship between abnormal differentiation of epidermal cells in psoriatic skin and expression of Notch molecules. We investigated keratins (K14 and K10) and Notches (1, 2, 3 and 4) using immunohistochemistry in psoriatic skin (n=30) and normal skin (n=10). In normal skin, K14 and K10 were discretely observed in the basal cell layer and suprabasal layer, respectively. In psoriatic skin, K14 was expressed in the pan epidermal layer while it and K10 were co-expressed in some middle suprabasal layer cells. Notch1, 2, 3, and 4 localized in all epidermal layers in normal skin. In psoriatic skin, Notch1, 2, and 4 mainly localized in suprabasilar layers and Notch3 is lacalized in pan epidermal, suprabasilar, and basilar layers. Protein and mRNA of Notch1, 2, and 3 isoforms decreased in psoriatic epidermis compared with normal epidermis. These data suggest that decrements in these Notch molecules might cause aberrant expression of K10 and K14 leading to anomalous differentiation of the epidermis in psoriatic lesions.     

Hsp40 regulates the amount of keratin proteins via ubiquitin-proteasome pathway in cultured human cells

Keratins represent important structural components of intermediate filament proteins. Their expression profiles are remarkably tissue-specific. Recent data have shown that keratins associate with many proteins including heat shock proteins (HSP). We recently identified cell-specific keratin and HSP expression. We aimed to gain further insight into the regulation of keratins by specific inhibition through knockdown of Hsp40 in human keratinocyte cells. Keratin-HSP interaction in HaCaT cell lysate was evaluated by immunoprecipitation followed by Western blotting. Immunofluorescence, was used to examine the co-localization of keratins and Hsp40. Hsp40 depletion led to an increase in the levels of keratin proteins (K5, K14, K10) and a decrease in keratin ubiquitination without influencing keratin gene expression. Our results demonstrate direct or indirectly association of Hsp40 and imply that expressed keratin proteins were regulated by Hsp40 depending on the ubiquitin-proteasome pathway in HaCaT. Furthermore, the K10 differentiation marker was increased by knockdown of Hsp40. The results presented in this study indicate that Hsp40 is related to the differentiation exchange of keratin pairs.     

Keratin 17 expression as a marker for epithelial transformation in viral warts.

The profile of keratin expression in benign warts from various cutaneous and mucosal sites along with dysplastic warts and squamous cell carcinomas has been examined using a panel of monospecific antibodies to epithelial keratins. Viral warts and verrucous keratoses from immunosuppressed renal transplant recipients show a spectrum of squamous atypia from benign lesions, from minimal changes to full thickness dysplasia. Changes associated with malignancy include loss of differentiation-specific keratins 1 and 10 together with expansion of basal cell epitopes and inappropriate expression of simple epithelial keratins 8, 18, and 19 in advanced squamous cell carcinoma. This late expression of keratins 8 and 18 contrasts with early expression of keratin 17 in all dysplastic lesions examined. Keratin 17 is found suprabasally in hyperproliferative lesions, including benign warts, but marked basal plus suprabasal expression is seen increasingly in malignantly transformed epidermis. These findings were not specific to immunosuppression, as shown by identical findings in control squamous cell carcinoma from nonimmunosuppressed individuals. Keratin 17 expression may prove prognostically helpful when assessing dysplasia in epidermal tumors.     

Cytokeratins, smooth muscle actin and vimentin in human normal salivary gland and pleomorphic adenomas. Immunohistochemical studies with particular reference to myoepithelial and basal cells.

The distribution of immunostaining in normal major salivary gland and in 12 pleomorphic adenomas was studied using monospecific monoclonal antibodies to a number of cytokeratins, including cytokeratin 14, to smooth muscle actin and vimentin. A number of these antibodies enabled a distinction to be made between structural components of the normal gland, and to relate this to the different structures of pleomorphic adenomas. In the normal gland, the luminal duct cells expressed cytokeratins 7, 8, 18 and 19. Three antibodies were of particular value for the characterization of normal myoepithelial and basal cells; while the antibody to smooth muscle actin and the cytokeratin antibody Ks8.12 mutually exclusively stained the myoepithelial (basket) cells and the basal duct (light) cells, respectively, the recently established monospecific antibodies to cytokeratin 14 showed specific immunostaining with both cell types. These three antibodies left luminal cells virtually unstained. Ck 13 was found occasionally in single luminal excretory duct cells. Antibodies to cytokeratins 1/2, 10 and 10/11 did not show any staining in the normal gland. In the pleomorphic adenomas, the staining pattern of the two-layered tubular formation resembled that of the normal gland ducts: tumour luminal cells showed the characteristic, although more irregular, expression of cytokeratins 7, 8, 18 and 19; the outer cells resembled normal ductal basal cells with their anti-cytokeratin 14/Ks8.12-epitope staining and in that they virtually lacked staining for smooth muscle actin. Trabecular formations and cells in myxoid areas were reactive with Ks8.12 and for cytokeratin 14, occasionally also for cytokeratins 7, 18 and 19. Epidermoid cell islets expressed mainly cytokeratin 14 and inconsistently the squamous epithelial cytokeratin 13 and the epidermal cytokeratin 10/11. Vimentin was found in cells of myxoid areas. The results support the postulate that some of the normal duct basal cells act as reserve cells and can give rise to tumour formation with a primitive myxoid or trabecular pattern and a more differentiated tubular or epidermoid configuration.     

The intermediate filament cytoskeleton of cutaneous neuroendocrine carcinoma (Merkel cell tumour)

Summary The presence and distribution of cytokeratins, neurofilament proteins, vimentin and glial fibrillary acidic protein were studied in 10 cutaneous neuroendocrine carcinomas (CNEC) by immunohistochemical techniques, using specific antibodies. In all cases tumour cells were specifically stained with antibodies to mouse liver cytokeratin component D in paraffin-embedded formalin-fixed and frozen sections. Moreover, one- and two-dimensional SDS-polyacrylamide gel electrophoresis of high salt/detergent resistant cytoskeletal residues from tumour material, isolated by microdissection from frozen sections, revealed the presence of cytokeratin components 8 and 18 which are characteristic constitutents of cytokeratin filaments of simple epithalia. Neurofilament proteins were detected by immunocytochemistry in tumour cells from 2 patients, from whom frozen material was available, and their presence was also positively identified in cytoskeletal residues by immunoblotting using specific antibodies. Glial fibrillary acidic protein and vimentin could not be demonstrated in tumour cells. Our studies did not confirm the suggested origin of CNEC from epidermal Merkel cells.This work was supported in part by Fonds zur Förderung der wissenschaftlichen Forschung, Grant P4708 to H.D.     

Keratin expression in anal carcinoma: an immunohistochemical study

The keratin expression of 40 frozen tissue specimens of anal carcinoma was investigated using a panel of 17 monoclonal antibodies to 14 individual keratins. The tumours were divided into three histological subgroups showing pure squamous, squamous and basaloid, or squamous and glandular differentiation. A further assessment of the tumour grade was made. The overall profile was of expression of keratins 4, 13, 17, 18 and 19 across the majority of the tumours, with the minority expressing keratins 1 and 10, and keratin 7. Dedifferentiation was associated with loss of expression of keratinocyte keratins, particularly the cornification markers keratin 1 and 10, and K6 and 16 associated with keratinocyte hyperproliferation and differentiation. This correlated with acquisition of the simple epithelial keratins 7 and 8. Compared with the tumours as a whole, well differentiated squamous tumours (the most easily identifiable histological group) showed consistent positivity for keratins 6 and 16, expressed suprabasally, while simple keratins 18 and 19 were also found. Independent of grade, mixed tumours showed more widespread positivity for simple epithelial keratins 7, 8 and 18 and loss of expression of cornification keratins 1 and 10 and K6 and 16 compared with pure squamous tumours. The relatively limited keratin profile of pure squamous tumours, predominantly consisting of keratinocyte keratins, suggests that these malignancies are less likely to originate from the region of the anal canal where the keratin profile is heterogeneous, i.e. the anal transitional zone.     

Do epidermal cells produce thymic hormones in vivo? An immunohistochemical study using anti-thymic hormone antibodies

Cultured human epidermal cells have been shown to produce soluble factors endowed with T cell differentiating activities. In addition, the presence of thymopoietin and FTS/thymulin-like factors has been reported in normal human and mouse epidermis using immunohistochemical methods and anti-thymic hormone antibodies. The present study was conducted to re-evaluate the presence of thymic hormones in normal epidermal cells using a panel of monoclonal antibodies (mAbs) and rabbit antisera to several well characterized thymic factors. The reactivity of the following antibodies was tested by indirect immunofluorescence on human and mouse tissue sections: a) two anti-FTS/thymulin mAbs; b) one anti-FTS/thymulin rabbit antiserum; c) one anti-thymopoietin rabbit antiserum; d) one anti-thymosin alpha 1 mAb. Our results show that: 1) all five antibodies reacted with human and/or mouse thymic epithelial cells; 2) none of the 3 antithymic hormone mAbs (2 anti-FTS/thymulin and 1 anti-thymosin alpha 1 mAbs) reacted with normal skin; 3) only 2 out of 5 antibodies, namely the anti-FTS and anti-thymopoietin antisera cross-reacted with mouse and human epidermis and labeled keratinocytes, as previously reported; these latter 2 antibodies also stained nude mouse epidermal cells and labeled non-thymic, non-epidermal normal mouse epithelial tissues, suggesting that the cross-reactive epitope is common to a number of epithelial cells; 4) the antigen defined by the anti-FTS and anti-thymopoietin antisera was not related to keratins, since absorption experiments using purified human epidermal keratins failed to abolish staining of the epidermis. We conclude from this study that epidermal cells do not produce in vivo the well characterized thymic hormones: FTS/thymulin, thymopoietin and thymosin alpha 1. The precise nature of the antigenic structure recognized within epidermis by the anti-FTS and anti-thymopoietin antibodies remains to be defined.