Nowadays new sophisticated techniques of molecular biology based on the principles of hybridization between nucleic acids, allow a correct diagnosis of genital HPV infection. In the present paper, beside traditional diagnostic methods, we used In Situ Hybridization (ISH) and Polymerase Chain Reaction (PCR) to detect the presence of HPV types 6, 11, 16, 18, 31 and 33. We tested ten patients affected by cervical lesions of high histological atypias associated with HPV, who underwent surgical conization. Types 6 and 11, at low risk of evolution, are less frequent than 31 and 33, at medium grade of evolution, and than 16 and 18 which are at high risk of evolution.

A total of 55 formalin-fixed and paraffin-embedded specimens of normal, inflamed and neoplastic uterine cervix have been studied in order to correlate the epithelial changes with the expression of alpha-smooth-muscle actin in stromal cells. This actin isoform is typical of smooth-muscle cells, but appears also temporarily in fibroblasts during wound healing and permanently during fibrocontractive diseases and stromal reaction to epithelial tumors. While positive stromal cells were absent in normal and inflamed cervix, they accumulated in relation to neoplastic tissues. The number of alpha-smooth-muscle actin positive cells and the intensity of stain were related to the increasing grading of cervical intra-epithelial neoplasia. Our results suggest that alpha-smooth-muscle actin is a marker of stromal-cell reaction to the development of neoplastic lesions. The evaluation of alpha-smooth-muscle actin in stromal cells of the uterine cervix may be a useful adjunct to diagnostic criteria of cervical intra-epithelial neoplasia and may help understanding of the mechanisms of mesenchymal-epithelial interactions during neoplasia.

The distribution of cytokeratins Nos. 19 (CK 19), 14, 16 and 17 (CK2-27), and 8 and 18 (CK 60-61) in 96 oral mucosal biopsies taken from women with genital HPV infections were studied by immunohistochemistry, using polyclonal antibody CK 19, as well as monoclonal antibodies CK 2-27 and CK 60-61. White staining of the buccal mucosa after acetic acid application, which recently was shown to be affected mostly by smoking and age, could not be explained by differences in cytokeratin pattern. In HPV DNA-positive biopsies, the staining with CK 19 antibody in the basal cell layer was more intense than in HPV DNA-negative biopsies. The staining with CK 2-27 antibody was seen in 76% and 91% of the basal and superficial layers, respectively, even though these low molecular weight cytokeratins should be found mainly from the basal and parabasal cells. CK 60-61 staining was almost similar to that seen recently in normal genital mucosa. When trying to distinguish oral HPV infections from normal mucosa, CK 2-27 and CK 60-61 stainings were of no diagnostic value. The more efficient expression of CK 19 in HPV DNA-positive samples suggests that viral infection might accelerate the production of low molecular weight cytoskeletal protein. This could be interpreted as evidence that HPV might disturb the keratinocyte differentiation in the basal cells. As a result of the present study, CK 19 staining in oral mucosa needs to be further studied in regard to viral infections, because it may help to better understand the interaction between a virus and a host cell.

We investigated the subcellular distribution of S-100b protein in primary cultures of Schwann cells. The subcellular localization of the protein in cells fixed and then permeabilized is similar, if not identical, to that seen in Schwann cells in peripheral nerves, i.e., S-100b protein is found in the cytoplasm and associated with membranes and filamentous structures. In cells either fixed in the presence of Triton X-100 or exposed to Triton X-100 for a short time before fixation (Triton cytoskeletons), the immune reaction product is considerably less intense, and the protein is associated with filaments running parallel to the long axis of the cell as well as in a submembranous position. Including CaCl2 in the buffer during fixation in the presence of Triton X-100 does not result in any increase in the intensity of the immune reaction product in Triton cytoskeletons, suggesting that, within the limits of the technique employed, no binding of additional S-100b protein to the Triton X-100-resistant material can be induced. On the other hand, including EGTA results in a substantial decrease in the intensity of the immune reaction product in Triton cytoskeletons. Altogether, these findings suggest that a remarkable fraction of S-100b protein in cultured Schwann cells is associated with elements of the cytoskeleton and that Ca2+ exerts some regulatory role in the association of S-100b protein with the cytoskeleton.

Filaggrin is a protein normally present in the granular and horny layer of stratified squamous epithelia. We studied the presence of this protein in 83 benign lesions and in 73 cases of malignant epithelial tumours of the oral cavity and investigated its possible role as an immunohistochemical marker. The immunohistochemical technique was based on the P.A.P. method. The results in benign lesions show a distribution of filaggrin similar to that observed in the normal mucosa. By contrast, an irregular distribution of filaggrin is observed in areas of leukoplakia with parakeratosis and in papillomas. In malignant lesions the expression of this protein is closely related to the degree of differentiation of the cellular elements, being positive in more differentiated and negative in anaplastic areas. Therefore in some types of benign lesions filaggrin testifies an alteration of the normal process of keratinization. Filaggrin is more significant in malignant lesions in which its presence if any permits an evaluation of the degree of differentiation of the tumour.

Synthetic peptides corresponding to amino acid sequences of amino terminal non-alpha helical domains of human cytokeratin 18 and to low molecular weight human neurofilament subunit were used to obtain monospecific antisera. The results of our immunohistochemical investigations confirmed in general the data previously published on the distribution of cytokeratin 18 in human, rat, and calf tissues. The reactivity of the antiserum was abolished after formalin fixation of specimens. Immunolocalization of the neurofilament subunit using our monospecific antiserum was quite variable from species to species in cells of the central and peripheral nervous systems, and also varied as the result of the tissue fixation procedures. In particular, formalin fixation destroyed the immunoreactivity of the recognized epitope. We discuss the advantages and limits of the use of synthetic peptides as immunogens to produce polyclonal antibodies against intermediate filament proteins, with particular attention to the epitope masking phenomena in cytokeratin polypeptides and the phosphorylation of epitopes in neurofilament subunits.

A series of 23 punch biopsies proved to contain human papillomavirus (HPV) type 16 and with established clinical course (including HPV-NCIN, HPV-CIN I, and HPV-CIN II lesion), and 18 additional biopsies of HPV 6-, 11-, 16- or 18-induced genital lesions were analyzed immunohistochemically for expression of cytokeratin No. 19 polypeptide. An immunoperoxidase-ABC technique was used with a polyclonal antibody raised against a synthetic nonapeptide corresponding to the residues 2-10 of the NH2-end, non-alpha-helical region. This polyclonal cytokeratin No. 19 antibody stained mainly (but not exclusively) the basal cells of the normal exocervical epithelium (heterogeneous pattern). Basal cell staining was intense slightly more frequently in HPV-CIN than HPV-NCIN lesions, i.e., ++ or more in 14/24 (58.3%) versus 8/17 (47.0%), respectively. The difference was more marked in the staining of the superficial cells, 70.8 and 58.8% showing intense expression of cytokeratin No. 19, respectively. In 6 (21.4%) of the 28 HPV 16 lesions, basal cell layer was intensely stained, as contrasted to none of the 13 HPV 6, 11 or 18 lesions. The most distinct feature was the well-defined granular staining pattern of the superficial layer in 8 out of 10 HPV 6/11 lesions, as contrasted to the homogeneous pattern in 24 out of 28 HPV-16-infected lesions. In superficial cells, regressed lesions exhibited intense staining in 9/13 (69.2%), as compared with only 4/10 (40%) of the progressed lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

The diagnosis of orbital rhabdomyosarcoma (RMS) in childhood gives rise to several clinical and anatomo-pathological problems. Antibodies recognizing structural proteins and cytoskeletal components have been shown to increase the diagnostic accuracy of different neoplastic lesions. In this study we examined anatomo-clinically and, where possible, by means of immunohistochemistry and electron microscopy, a series of 14 cases of orbital RMS in childhood. In the 12 cases studied by immunohistochemistry, desmin was always present, although showing variable patterns, and alpha-sarcomeric actin was found in 10 cases. alpha-Smooth muscle actin was always absent. The other markers tested (myoglobin, polyclonal actin, vimentin and enolase) proved unreliable for several reasons. We conclude that antibodies against desmin and alpha-sarcomeric actin are useful for the diagnostic definition of RMS. In addition, immunohistochemical analysis supplies data regarding the degree of tumor differentiation and may be applied to monitor radio- and chemotherapy.

In this study antibodies specific for different intermediate-sized proteins (cytokeratins and neurofilaments) have been tested on a series of neuroendocrine (NE) lung tumors in order to evaluate their diagnostic validity. In particular we used a panel of polyclonal anti-neurofilament 200-kilodalton subunits whose reactivity against phospho-dependent epitopes was known. At least one NF subunit was constantly present and in all cases coexpression of cytokeratins and neurofilaments was confirmed. However, in cases of carcinoid tumor (CT) the results were homogeneous, while the cases of small cell lung carcinoma (SCLC) showed a much wider range of immunostaining. Our investigation confirms the hypothesis that the phosphorylation state is a significant determinant of immunohistochemical properties of neurofilaments. This might explain the large number of negative results obtained in previous investigations on NE tumors. The phosphorylation of neurofilaments may also be considered an indication of the degree of differentiation of the tumor.

A series of 64 punch biopsies collected from women prospectively followed-up for cervical Human papillomavirus (HPV) infections (with and without CIN), and 38 control biopsies (normal epithelia, and classical CIN) were analysed for expression of filaggrin (a histidine-rich protein constituent of keratohyalin granules) using the ABC technique and polyclonal antibody. HPV typing was completed using the in situ hybridization technique with DNA probes for HPV 6, 11, 16, 18 and 31. Three patterns of filaggrin distribution were differentiated: pattern I, all layers above the basal cells stained irregularly, and pattern III, scattered superficial cells stained positive. There was a significant difference between HPV-noCIN and HPV-CIN lesions in their filaggrin patterns, pattern I being present in the majority (77.7%) of HPV-noCIN lesions, as contrasted to HPV-CIN lesions, where pattern III was the predominant one (43.5%), followed by pattern II (32.6%). In HPV-CIN CIN as well as in CIN lesions, pattern I was inversely related to the grade of CIN, being entirely absent in HPV-CIN III and CIN III. A significant difference exists between CIN and HPV-CIN lesions, concerning the presence of pattern III (4.3% and 43.5%, respectively,P< 0.001). The difference was less dramatic with regard to pattern I (30.4% and 21.7%, respectively,P< 0.05). In the lesions containing HPV 6, 11 or 31 DNA, filaggrin distribution was shown to be more close to that of the normal epithelium (I 36.7%, and II 34.7%), while in the HPV 16 and 18-infected cases, pattern III was the predominant one (46.7%). The assessment of filaggrin pattern in HPV lesions might be of help in evaluating the severity of the disturbance of keratinocyte differentiation induced by the progression of HPV infections.

Normal bladder urothelium and large spectrum bladder lesions have been investigated by immunohistochemistry with monoclonal antibodies of variable specificity (SK 56-23, a large spectrum antibody; SK 60-61, which reacts with cytokeratin polypeptides no. 8 and 18 of Moll's catalogue; SK 2-27, specific for polypeptides no. 14, 16 and 17). The normal urothelial pattern is in agreement with previous reports. In pathological conditions, modified immunostaining has been demonstrated in almost all cases. In detail, the cytoskeletal pattern detected in transitional cell papilloma seems to discriminate between types which are otherwise histologically similar. We also observed a correlation between higher degrees of malignancy and loss of specialization, as demonstrated by the increasing positivity for SK 60-61, which as a rule specifically stains "umbrella" cells, and SK 2-27, an antibody exclusively detected in cells of the basal layer. These findings indicate that the cytokeratin pattern may constitute a modern new tool for the pathologist in the diagnosis of urothelial proliferative disorders.

A series of 74 punch biopsies, derived from 513 women prospectively followed for cervical human papillomavirus (HPV) infections (including HPV-NCIN, HPV-CIN I, HPV-CIN II, and HPV-CIN III lesions), and 43 control cases (consisting of normal epithelia, nonspecific cervicitis, and classical CIN lesions) were analysed for expression of cytokeratin polypeptides using the ABC technique and monoclonal antibodies SK 56-23 (wide-spectrum antibody), SK 60-61 (specific for keratins 8 and 18), and SK 2-27 (detecting keratins 14, 16, and 17). HPV typing was carried out using the in situ hybridization technique with DNA probes for HPV 6, 11, 16, 18, and 31. All layers of the exocervical epithelium were regularly stained with the antibody SK 56-23, and the staining pattern remained unaltered in all cervical lesions studied. In contrast to the normal exocervical epithelium, which remained negative with SK 60-61, positive staining was observed in 3 of 15 cervicitis cases and 6 of 23 classical CIN lesions. Interestingly, the majority (69 of 74, 93.2%) of both HPV-NCIN and HPV-CIN lesions showed positive staining with this antibody either in all layers or in suprabasal cells. Antibody SK 2-27 stained the basal cells of the normal exocervical epithelium with remarkable specificity. In 18 of 19 HPV-NCIN lesions, basal cells could not be stained by SK 2-27 monoclonal, but the suprabasal cells were stained instead. In HPV-CIN, but not in classical CIN, this antibody demonstrated the presence of the epitope typical of the cytokeratins 14, 16, and 17 in all layers of the epithelium, the highest frequency (9 of 12, 75%) being found in HPV 16-induced lesions. These disturbances of cytokeratin patterns in cervical epithelium could be associated with cell transformation by HPV, leading to development of HPV-CIN, and could be specific for this virus. The present data are of interest in assessing the stage of maturation of the squamous cells in progressing cervical HPV infections.

Spermatozoa of a phasmid insect, Bacillus rossius, contain, in the whole length of the tail, two longitudinal crystalline bodies flanking the axoneme and made up of a texture of 10-nm filaments. These filaments are resistant to nonionic detergent extraction (Triton X-100) and appear particularly evident after deep-etching. SDS-polyacrylamide gel electrophoresis, immunoblotting, and immunochemical methods, at both the light and the electron microscopic level, show that they share antigenic determinants with the vertebrate cytokeratins. All these properties suggest that these filaments are members of the cytokeratin family. The occurrence of keratin in invertebrates is discussed.

ABSTRACT. The presence in insect tracheal taenidia of a protein having an immunochemical determinant common to vertebrate keratins is suggested. Taenidia react positively with a specific anti keratin serum both at the fluorescent and electron microscope level. In tracheal preparations, immunoblotting shows specificity for three polypeptides having molecular weights ranging from 62 to 43 kd, two of them corresponding to prekeratins. In the region of taenidia reacting with the antisera, 7–8 nm thick filaments are present. The occurrence of a keratin‐like protein in insects, and the role played in the tracheal intima are discussed.

The intermediate filament cytoskeleton of various types of human soft tissue tumors was analyzed by immunofluorescence microscopy with the use of specific antibodies against cytokeratins, vimentin, and desmin, as well as by one- and two-dimensional gel electrophoresis of high-salt buffer- and detergent-resistant cytoskeletal preparations. All leiomyomas as well as a leiomyosarcoma contained desmin. Leiomyomas of both gastrointestinal and uterine derivation and the retroperitoneal leiomyosarcoma showed strong reaction for desmin in the smooth muscle cells, but the latter two exhibited also vimentin staining. In embryonal rhabdomyosarcomas, desmin prevailed in the large, apparently well-differentiated rhabdomyoblasts; whereas the smaller, less differentiated tumor cells preferentially contained vimentin. Cells of malignant fibrous histiocytomas were characterized by their content of vimentin as the only intermediate filament protein present. In alveolar soft part sarcoma, a rare tumor of hitherto unknown histogenesis, vimentin and desmin co-existed within the same tumor cells, indicating, together with chemical determinations, the myogenic derivation of this neoplasm. The results show that immunologic and biochemical analysis of proteins associated with the intermediate filament cytoskeleton is a useful adjunct in the diagnosis of diverse neoplasms, particularly those with equivocal histologic features, and thus aids in the histogenetic classification of soft tissue tumors.