Transforming growth factor-beta 1: histochemical localization with antibodies to different epitopes.

We have localized transforming growth factor-beta (TGF-beta) in many cells and tissues with immunohistochemical methods, using two polyclonal antisera raised to different synthetic preparations of a peptide corresponding to the amino-terminal 30 amino acids of TGF-beta 1. These two antibodies give distinct staining patterns; the staining by anti-CC(1-30) is intracellular. This differential staining pattern is consistently observed in several systems, including cultured tumor cells; mouse embryonic, neonatal, and adult tissues; bovine fibropapillomas; and human colon carcinomas. The extracellular staining by anti-CC(1-30) partially resembles that seen with an antibody to fibronectin, suggesting that extracellular TGF-beta may be bound to matrix proteins. The intracellular staining by anti-LC(1-30) is similar to that seen with two other antibodies raised to peptides corresponding to either amino acids 266-278 of the TGF-beta 1 precursor sequence or to amino acids 50-75 of mature TGF-beta 1, suggesting that anti-LC(1-30) stains sites of TGF-beta synthesis. Results from RIA and ELISAs indicate that anti-LC(1-30) and anti-CC(1-30) recognize different epitopes of this peptide and of TGF-beta 1 itself.

Immunohistochemical analysis of benign and malignant papillary lesions of the breast.

The histocytological diagnostic criteria and recently developed immunohistochemical procedures selective for either the epithelial or the myoepithelial mammary cells have been tested in a series of 60 cases of papillary lesions of the breast. These included 15 benign solitary intraductal papillomas, 41 papillary carcinomas (29 pure and 12 associated with other types of in situ or invasive ductal carcinoma), and four cases of "suspected" papillary carcinomas. Markers for epithelial cells (EMA) and for apocrine metaplasia (GCDFP-15) did not permit a distinction between benign and malignant papillary lesions; however, immunocytochemical staining for CEA using monoclonal antibodies, and for actin (a marker of the myoepithelial cells) was discriminative in this respect. Benign papillomas have a basal layer of actin-rich myoepithelial cells; the cytoplasm of the epithelial cells is CEA negative. Papillary carcinomas lack the myoepithelial layer, except in areas where multiple papillomas are present, associated with ductal or papillary cancer. CEA was detected in 85% of carcinomas. Two of the cases of "suspected carcinoma" lacked myoepithelial cells and were interpreted as carcinomas. It is concluded that the immunocytochemical methods for cell markers can offer valuable data in the study and diagnosis of papillary lesions of the breast; it is difficult, however, to be categorical in borderline cases since in our experience, the behavior of the malignant papillary lesions of the breast is usually favorable. Residual foci of multiple intraductal papillomas were found in seven cases of papillary carcinoma, supporting the pre-neoplastic potential of this condition.

Detection of human papillomavirus type 6/11 DNA in conjunctival papillomas by in situ hybridization with radioactive probes.

Twenty-three conjunctival papillomas and 28 conjunctival dysplasias were examined for human papillomavirus (HPV)-DNA sequences by in situ hybridization with nick-translated 35S-labeled HPV probes. Adjacent paraffin sections were hybridized with HPV type 2, 6, 16, and 18 probes at Tm - 17 degrees C. Fifteen tissues, all papillomas, displayed positive hybridization with the HPV-6 probe. Infection with HPV-6 (or the closely related HPV-11) appeared to be responsible for most of the conjunctival papillomas of children and young adults. The presence of genital tract HPV-6 in these lesions suggests that some of the infections were acquired during passage through an infected birth canal. The lack of hybridization in adult conjunctival dysplasias indicates either that HPVs are not associated with this condition or that the probes and the technique utilized were not adequate for demonstration of this association.

Pathologic findings from the National Surgical Adjuvant Breast Project (protocol no. 4). VI. Invasive papillary cancer.

Although papillary forms of intraductal cancer are a common component of amny mammary cancers, invasive papillary cancer of the breast is relatively rare. Only 35 examples were encountered in the specimens obtained from 1603 women participating in a prospective randomized study of invasive mammary cancer. Contingency-table analysis disclosed that invasive papillary cancer, as opposed to the non-papillary histologic types, is significantly more likely to show circumscription, cytoplasmic oxyphilia, microcalcification, intermediate histologic grade, moderate or marked mucin and an intraductal component of papillary type. Although patients who have invasive papillary cancer are frequently judged by clinical criteria to have regional nodal metastases, in pathologic analysis these metastases are not commonly found and, if present, involve fewer than four nodes. There is a significant frequency of marked sinus histiocytosis in regional nodes. The lesion occurs with a significantly high frequency among non-Caucasian and postmenopausal women. Only three patients with invasive papillary cancer experienced treatment failure after five years of observation. Life-table probabilities showed that the treatment failure rate was significantly lower for the group of patients who had any other histologic type, and in this regard invasive papillary cancer was similar to the tubular and mucinous varieties. Although apocrine metaplasia, fibrous supporting stalks and cellular differentiation have been espoused as important criteria distinguishing intraductal papi-loma from intraductal cancer, these features were also not uncommonly observed in the examples of invasive papillary cancer studied. It is concluded that invasive papillary cancer represents a unique histologic type of invasive mammary cancer attendant with a favorable prognosis.

Malignant progression of laryngeal papilloma associated with human papilloma virus type 6 (HPV-6) DNA.

A case of laryngeal squamous papilloma in the early stages of development showed histological features suggestive of virus infection. Five years later positive evidence of HPV-6 infection was obtained at a time when the lesion had developed into a squamous cell carcinoma. It is concluded that this case represents a complete example of the virus to papilloma to carcinoma sequence, and as far as is known, is the first reported case of its kind in the larynx.

Lysozyme and mucins in gastric adenomas.

A method for the simultaneous demonstration of lysozyme and mucins in 39 cases of gastric adenomas differentiated two intermediate cell types. The first was similar to a columnar cell comprising a single cell population which covered extensive areas of the adenomas. This cell type often showed supranuclear lysozyme reactivity and apical neutral mucins, sialomucins, and sulphomucins in variable amounts. The second cell type was found in 11 adenomas, located mainly in the fundal area. It seemed to be a transitional form between the goblet cell and the Paneth cell. This cell type was scattered among columnar cells, occasional Paneth-like cells, and small goblet cells. These two types of intermediate cells may be regarded as abnormally differentiated integral elements of gastric adenomas. They may be associated with the neck stem cells in the cytogenesis of gastric adenomas.

Sensitive detection of nucleic acids and protein of human papillomavirus type 6 in respiratory and genital tract papillomata.

We have developed a sensitive method to detect and localize HPV-6 viral DNA, mRNA and protein in biopsy specimens of genital and respiratory tract lesions by using in situ hybridization and immunoperoxidase assays on sections of plastic-embedded tissue. This modified in situ hybridization technique, using ultrathin sections and strand-specific 3H-labelled riboprobes, offers the advantages of superior morphological preservation and detection of viral genomes at low copy number with good resolution. This modified immunocytochemistry provides better sensitivity when compared to previous methods using paraffin-embedded materials. In respiratory tract lesions, immunoperoxidase assay detected only a few capsid antigen positive cells, while in the genital tract lesions, there were more capsid antigen positive cells. Southern transfer analyses and in situ hybridizations demonstrated the presence of more viral nucleic acids in genital tract papillomata than respiratory tract papillomata. Epithelial cells throughout the papillomata were infected by HPV-6 as evidenced by positive hybridization, with more viral DNA present in superficial cells. Our results suggest that genital tract epithelium is more permissive for HPV-6 replication than respiratory tract epithelium. Using stand-specific probes synthesized from subgenomic fragments of the HPV-6 genome in conjunction with nuclease digestions, we were able to demonstrate that HPV-6 transcripts specific to open reading frames (ORFs) E6, E7, E1, L1, and L2 occur in maturing superficial cells. In contrast, transcripts specific to ORFs E1, E2, E4, E5a, and E5b could be detected throughout the whole of the epithelium with more signals noted at the basal cell areas. In addition, the distribution of HPV-6 nucleic acids and protein in a carcinoma in situ of the larynx was analyzed. In comparison to benign respiratory tract papillomata, more viral DNA was found in the malignant lesion, but the pattern and distribution of transcription and capsid antigen was similar.

Determination of glial fibrillary acidic protein (GFAP) in human brain tumors.

Brain tumors have been tested for their glial fibrillary acidic protein (GFAP) content by means of the rocket electrophoresis technique. Meningiomas and neurinomas were low in GFAP. Metastases had a low level of GFAP except when contaminated with surrounding tissue. Non-nervous tumors such as myeloma, myeloplaxoma and adenocarcinoma gave negative results. More detailed correlations with histological observations have been looked for in glial tumors. Low levels of GFAP were always associated with signs of malignancy such as mitoses and giant or atypical cells, whereas high levels of GFAP were correlated with the presence of well-preserved astrocytes.

Differences of expression of cytokeratin polypeptides in various epithelial skin tumors.

In normal skin, cytokeratin polypeptides are expressed in different cell-type-specific patterns, in the keratinocytes of the different epidermal cell strata as well as in different lateral epithelial domains. Using light microscopically controlled microdissection of defined regions from frozen sections of biopsies, we have prepared cytoskeletons of various benign and malignant keratinocyte-derived tumors of human skin and analyzed their cytokeratin polypeptide patterns by two-dimensional gel electrophoresis. Premalignant fibroepitheliomas and basal cell epitheliomas display a relatively simple cytokeratin pattern (cytokeratins nos. 5, 14, 15, and 17). Pseudocarcinomatous hyperplasia, some squamous cell carcinomas, and a certain subtype of condylomata acuminata present a hair-follicle-like pattern (nos. 5, 6, 14, 16, 17). In addition to these components, variable, mostly low amounts of cytokeratins nos. 1 (Mr 68,000), and 11 are detected in most squamous cell carcinomas, in keratoacanthomas, verruca vulgaris, and another type of condylomata acuminata. In molluscum contagiosum, verruca plana, solar keratosis, and seborrheic keratosis, the cytokeratin expression is shifted more towards the normal epidermal pattern (polypeptides nos. 1, 2, 5, 10, 11, 14, 15 and traces of nos. 6 and 16 in the latter two tumors). No tumor-specific cytokeratins have been found. We conclude that keratinocyte-derived skin tumors contain various combinations of cytokeratins of the subset typical for normal keratinocytes of skin, but no cytokeratins typical for internal, simple epithelia. Different groups of tumors can be distinguished by their specific cytokeratin patterns. Possible applications of cytokeratin typing in clinical diagnosis are discussed.

Demonstration of papillomavirus antigen in paraffin sections of laryngeal papillomas.

A papillomavirus antigen has been identified in tissue sections of laryngeal papillomata removed at endoscopic operations. The demonstration of the papillomavirus antigen was achieved using the peroxidase-anti-peroxidase test of Sternberger after a broadly cross-reactive rabbit antiserum to the papillomavirus capsid antigen was incubated with the tissue sections. The antigen was detected in specimens from 7 of 15 patients with juvenile-onset laryngeal papillomas, but could not be demonstrated in the specimens from 6 patients with adult-onset papillomas. In all, 73 biopsy specimens from 21 patients were selected for study; 18% (11/61) of the sections from the juvenile-onset patients and 0% (0/12) of the sections from the adult-onset patient specimens exhibited the papillomavirus antigen.

In situ hybridization and immunohistochemical study of human papillomavirus infection in adult laryngeal papillomas.

Routinely processed paraffin sections from 20 patients with adult laryngeal papillomas were examined for the presence of human papillomavirus type 11 (HPV-11) DNA and its specific mRNA by in situ hybridization methods using 35S-labeled RNA probes. Immunohistochemical techniques were also used to identify papillomavirus genus-specific common antigen (pgs-antigen). HPV-11 DNA signals and/or papillomavirus genus-specific common antigen were detected in all eight samples of multiple laryngeal papilloma. On the other hand, in 12 samples of single laryngeal papilloma, neither papillomavirus genus-specific common antigen nor HPV-11 DNA were detected. Four patients were positive for both HPV-11 DNA and pgs-antigen. In three of these four patients, HPV-11 mRNA signals were also detected. These results provided direct evidence of the association of HPV and adult multiple laryngeal papilloma.

Laryngeal papillomatosis: clinical, histopathologic and molecular studies.

The clinical course and pathology of 57 patients with laryngeal papillomatosis were reviewed. Tissues from 26 patients were analyzed for human papillomavirus (HPV) DNA by Southern blot hybridization. Histopathologic evaluation of the papillomas showed no correlation with age of onset or clinical pattern of remission and recurrence. The pathology was characterized by abnormal squamous maturation with parakeratosis, retardation of superficial cell maturation, papillomatosis, and basal hyperplasia. HPV DNA was present in all lesions, with 92% containing either HPV-6 or 11. Latent HPV DNA was detected in clinically uninvolved tissues of 11 of 14 (78.5%) patients studied. There was no correlation between HPV type, histopathology and/or clinical pattern. Despite homogeneity of pathology, the clinical expression of laryngeal HPV infection varied widely. A mechanism for the pathogenesis of laryngeal papillomatosis, based on the concept of maturational arrest, is proposed.

Immunohistochemical localization of S100 protein in benign and malignant conditions of the breast.

A peroxidase--antiperoxidase technique for S100 protein has been applied to 122 breast lesions from 122 patients. These included 35 cases of fibrocystic disease, 16 cases of sclerosing adenosis, 24 cases of papilloma and papillomatosis, 43 intraduct carcinomas, and four intralobular carcinomas. In fibrocystic disease, S100 protein was demonstrable in large amounts in cells between the duct lining cells and the basement membrane of the ducts, being most pronounced in those exhibiting adenosis. Areas of epitheliosis showed scattered positive cells within the ducts with more strongly positive cells around these ducts. Apocrine metaplasia was moderately positive. No S100 protein was demonstrable in the epithelial lining cells of cysts or within the stroma. In sclerosing adenosis individual cells and groups of cells in the fibrous tissue were strongly positive. In papillomatosis and papilloma, the vascular core and epithelium failed to stain, but a discontinuous layer of cells between the epithelium and basement membrane was positive. In intraduct and intralobular carcinoma the tumor cells were uniformly negative, and wherever fibrocystic disease was also present, S100 protein was variably demonstrable. The study corroborated the view that fibrocystic disease and benign proliferative processes of the breast appear to contain cells that correspond to myoepithelial cells, and suggests that S100 protein may serve as a useful marker in the separation of benign proliferative breast lesions from in situ carcinoma.

Papillary adenoma of the stomach. Pathologic and immunohistochemical study.

A total of 13 gastric papillary adenomas composed of 8 papillary and 5 papillotubular adenomas were examined pathologically and immunohistochemically. They showed a dome-like or pedunculated appearance and were located at the antrum, except for one adenoma. Histologically, the adenoma cells showed atypia in varying degree and focal adenocarcinoma was noted in seven lesions. The number of goblet cells was apparently smaller in the papillary than in the tubular portion. Lysozyme was present at the supranuclear region in most papillary adenoma cells, whereas it was concentrated in Paneth's granules in tubular adenoma cells. No difference was found in the distribution and frequency of carcinoembryonic antigen, secretory component, and carbohydrate antigen CA 19-9 between papillary and tubular adenomas. Paucity of endocrine cells also characterized gastric papillary adenoma. Different phenotypic expressions might reflect the difference in histogenesis between papillary adenoma and tubular adenoma.

Analysis of glycoproteins from nasal polyps and papillomas by affinity chromatography.

Glycoproteins were isolated from the particulate fraction of four nasal polyps and three nasal papillomas by affinity chromatography on lectins conjugated with agarose (Concanavalin A [Con A], wheat germ agglutinin [WGA], Ricinus communis agglutinin [RCA], peanut agglutinin [PNA], and Dolichos biflorus agglutinin [DBA]). The glycoprotein mixtures so isolated were then analyzed by sodium dodecyl sulfate gel electrophoresis. Glycoprotein profiles of nasal polyps were similar to each other, but were distinctively different from those of nasal papillomas. Binding sites for Con A, WGA, and RCA isolated from nasal papillomas contained intense bands with a molecular weight less than 15,000 daltons, which were absent in nasal polyps. The major component of PNA-binding sites of nasal polyps is of a molecular weight of 65,000 daltons, which was not detected in nasal papillomas.

Tumor associated proteins of rat skin tumor induced by 7,12-dimethylbenz[a]anthracene.

The incidence of tumor and the time of expression, cellular localization and the molecular weight of tumor associated proteins of rat skin tumor induced by 7,12-dimethylbenz[a]anthracene (DMBA) with or without 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were studied. The time of the development of skin tumors in 0.1% DMBA-TPA treated rats was significantly shorter than that in rats which were treated with DMBA alone. In the complete carcinogenesis case, papillomas developed more slowly and were less common and also squamous cell carcinomas appeared much later. From the analysis of the proteins of each experimental group by SDS-PAGE and two dimensional gel electrophoresis, at least three tumor associated proteins were identified (54kd, pl = 5.66; 27kd, pl = 5.85; 11kd, pl = 4.90). Also these proteins were found in rat dorsal skin from 14 days gestation to 21 days postpartum, and disappeared after 28 days. In conclusions, two stage skin carcinogenesis could be successfully demonstrated in Sprague-Dawley rats and abnormal proteins were produced in DMBA or DMBA-TPA induced skin tumor. The tumor associated proteins of skin tumor induced by DMBA or DMBA-TPA were appeared at the late initiation stage or early promotion stage, and they were localized in plasma membrane and were glycoproteins that are thought to be related to the epidermal differentiation process.

Keratin expression in equine normal epidermis and cutaneous papillomas using monoclonal antibodies.

Keratin expressions in normal equine epidermis and experimentally induced equine papillomas were studied by immunohistochemical methods with three different human cytokeratin monoclonal antibodies, 34 beta B4 (directed against component 1), 34 beta E12 (directed against components 1, 5, 10, 11) and 35 beta H11 (directed against component 8). Staining patterns with 34 beta B4 and 34 beta E12 in the normal equine epidermis did not differ from those in the normal human epidermis. In the early developing papilloma, keratinocytes showed an abnormal suprabasal staining pattern and expressed an additional 56 kD keratin protein detected by 34 beta E12. In the advanced papilloma, cytolytic cells in the outer spinous and the granular layers did not stain positively with any of the three antibodies used. In both early and advanced papillomas, the expression of high molecular weight keratin proteins, as detected by 34 beta B4 and 34 beta E12, did not correlate with the degree of keratinization. By electron microscopy, keratinocytes in the advanced papilloma showed a marked decrease of tonofibrils and desmosome-tonofilament complex. These alterations may result from an abnormality in both proliferation and functional terminal differentiation of keratinocytes in the papilloma. There were obvious differences in staining patterns with 35 beta H11 between the normal human and equine epidermis; 54 kD keratin protein was expressed in suprabasal layers of the equine normal and papillomatous epidermis. Thus, this keratin protein may be regarded as a "permanent" marker for the equine epidermis.

[Steroid hormone receptors in malignant mammary gland tumors in dogs]. / Retseptory steroidnykh gormonov v zlokachestvennykh opukholiakh molochnykh zhelez sobak.

The content of estrogen (ER) and progesteron receptors (PR) were studied as related to histological type, the degree of malignancy and growth rate of spontaneous canine mammary carcinomas. No correlation was found between the ER and PR amount, histological type and degree of malignancy. Inverse correlation was detected between ER and PR and the growth rate. These results correspond to those obtained for human mammary tumours. Therefore canine mammary carcinomas can be considered as an adequate model for experimental therapy of this type of tumours.

Detection of human papillomavirus (HPV) structural antigens and DNA types in inverted papillomas and squamous cell carcinomas of the nasal cavities and paranasal sinuses.

To assess the suggested etiological role of human papillomavirus (HPV), biopsies from 14 patients operated on for an inverted papilloma (11 cases) and squamous cell carcinoma (3 cases) of the nasal cavity and paranasal sinuses were analysed for light microscopical evidence of HPV, by indirect immunoperoxidase (IP-PAP) to demonstrate HPV structural proteins, and using in situ DNA-hybridization to disclose the DNA of HPV types 6, 11 and 16. The majority of the inverted papillomas contained areas of metaplastic squamous cells, including koilocytes as well as dysplastic changes consistent with intra-epithelial neoplasia as described in uterine cervix. In 3 patients, frankly invasive squamous cell carcinomas were found, originating from dysplastic squamous epithelium. Of the 14 lesions, 7 (50%) expressed HPV antigens, usually confined to only a few cells close to the surface of the epithelium. None of the malignant lesions expressed HPV antigens. In situ DNA-hybridization disclosed HPV 11 DNA alone in 5 lesions, but none of the lesions contained HPV 6 DNA. HPV 16 DNA was found in 2 lesions as a single HPV type, and in 3 other lesions concomitant with HPV 11. All three carcinomas contained HPV 16 DNA. The HPV DNA distribution proved to be different from that found in the genital tract HPV lesions; HPV DNA was less abundant in the nasal papillomas, and it was also found in the basal and suprabasal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ectopic production of beta-human chorionic gonadotropin by inverted papilloma of the nose.

A series of 19 inverted papillomas (transitional type or Schneiderian papillomas) of the nose were examined using an immunogold-silver (IGS) technique and an indirect immunoperoxidase technique. We demonstrated beta-HCG production in all 19 specimens with both methods. The staining demonstrated by the IGS technique was of a greater intensity and in a larger number of cells than that observed by the indirect immunoperoxidase method.