Expression of intermediate filament proteins in normal and diseased thyroid glands.

A total of 67 samples from normal and pathological thyroid glands were stained (as formalin fixed paraffin sections) with a panel of monoclonal antibodies directed against intermediate filament proteins. The study confirmed previous reports of cytokeratin and vimentin coexpression in primary thyroid carcinomas, but coexpression was also detected in normal thyroid and in a range of benign conditions including follicular adenomas, Hashimoto's thyroiditis, and diffuse hyperplasia (thyrotoxicosis). Prekeratin expression was found (using antibodies recognising higher molecular weight cytokeratins) predominantly in areas of squamous change, independent of the underlying thyroid pathology. This study does not therefore support previous findings that prekeratin expression provides a reliable means of distinguishing follicular pattern papillary carcinoma from follicular carcinoma with its poorer prognosis or that it helps distinguish benign from malignant papillary lesions. No evidence of desmin or neurofilament expression was seen, and in particular, neurofilaments could not be detected in any of the cases of medullary carcinoma studied.     

Monoclonal antibodies to human intermediate filament proteins. II. Distribution of filament proteins in normal human tissues.

Monoclonal antibodies generated against different human intermediate filament (IF) proteins were assayed on fixed, embedded tissue by the biotin-avidin-immunoperoxidase method for evaluation of the tissue specificity of these antibodies. An antibody (43 beta E8) made to fibroblast IF protein stains mesenchymal tissue such as endothelium, histiocytes, stromal fibroblasts, and Schwann cells but does not stain epithelium, skeletal muscle, lymphocytes, or neurons. Three different anti-cytokeratin antibodies decorate epithelium in three unique patterns. One (35 beta H11) stains all nonsquamous epithelium but fails to recognize squamous epithelium. Antibody 34 beta E12 stains the full thickness of squamous epithelium and ductular epithelium but does not react with hepatocytes, pancreatic acinar cells, proximal renal tubules, or endometrial glands. Antibody 34 beta B4 stains only the suprabasal portion of squamous epithelium. None of these three anti-cytokeratin antibodies reacts with nerve or mesenchymal tissue. Two anti-neurofilament antibodies recognize only neurons, failing to react with epithelial or mesenchymal tissue. We conclude that these anti-intermediate filament antibodies can be used as tissue-specific markers. Neoplasms retain the same intermediate filament patterns as the normal parental tissue; therefore, these antibodies can be used as diagnostic aids in surgical pathology.     

Distribution of fibronectin in normal and diseased human kidneys.

The distribution of fibronectin in 42 renal biopsies was investigated using an indirect immunoperoxidase technique on paraffin sections of formalin-fixed material. Biopsies were obtained from normal human kidneys and from patients with a variety of glomerular disorders. In normal glomeruli, fibronectin was present in Bowman's capsule, in the mesangium, and outlining peripheral capillary loops. A significant increase in fibronectin was observed in most types of glomerulopathy with a distribution closely related to the histopathological diagnosis. Fibronectin was diminished or absent in segmental scars, old diabetic nodules, and hyalinised glomeruli.     

Intermediate filament expression by normal and diseased human corneal epithelium

Cicatricial conjunctivitis may be a sequel to systemic disorders (eg, Stevens-Johnson syndrome, cicatricial pemphigoid) or local disorders such as chemical burns. The cicatrisation is often associated with corneal epithelial changes that cause visual loss. These have been attributed to encroachment of the conjunctival epithelium over the cornea. However, the epithelial anomalies are poorly understood. We investigated the corneal epithelial changes in cicatricial conjunctivitis with an immunohistochemical study of intermediate filaments in normal and pathological specimens. Our results show that the normal corneal epithelium is immunoreactive for cytokeratin 3 (CK 3) but not cytokeratin 19 (CK 19), whereas normal conjunctival epithelium is CK 3 negative and CK 19 positive. Conjunctiva artificially transposed over the cornea (after therapeutic conjunctival flap reconstruction) retained the normal pattern of conjunctival cytokeratin expression (CK 3 negative, CK 19 positive). Conversely, the entire corneal epithelium exhibited the normal cytokeratin pattern (CK 3 positive, CK 19 negative) in 82% of Stevens-Johnson, 80% of cicatricial pemphigoid, and 69% of chemical burns specimens. The findings suggest that conjunctival encroachment is not responsible for the changes at the corneal surface in cicatricial conjunctivitis and that the abnormal corneal epithelium is derived from native corneal cells in these diseases.     

Distribution of interleukin-6 in normal and diseased human kidney

Distribution of interleukin 6 (IL-6) in normal and diseased kidneys was examined by an immunohistochemical method. Four specimens of normal kidney tissue and 47 specimens obtained from diseased kidneys by biopsy were studied by indirect immunofluorescence microscopy using specific monoclonal antibody to human IL-6. In the normal kidney, IL-6 was distributed in the glomerular mesangial area and in vascular walls. In the diseased kidneys, IL-6 was observed in the glomerulus in various patterns. The extent of the distribution of IL-6 correlated with the average number of glomerular cells. When sclerosis appeared, the expression of IL-6 decreased. Occasionally, IL-6 was found in the area of synechiae or crescents as well as in the parietal epithelial cells of the glomerulus with synechiae and/or crescents. In the interstitium, IL-6 was seen in atrophic tubules, and the distribution correlated with the extent of tubular atrophy. The binding of anti-IL-6 monoclonal antibody to the renal sections was completely inhibited by preincubation of the antibody with recombinant IL-6. These results suggests that IL-6 expression is a good marker for glomerular cell proliferation and that IL-6 may be involved in the formation of synechiae or crescents and in tubulointerstitial injury.     

Monoclonal antibodies to human intermediate filament proteins. III. Analysis of tumors

A panel of monoclonal antibodies to human intermediate filament proteins was tested on an unselected series of 246 neoplasms. The antibody panel includes two different anti-cytokeratin antibodies, an anti-vimentin antibody, and an anti-neurofilament antibody (Gown and Vogel, Am J Pathol 114:309, 1984). The studies were done on Carnoy's or methacarn-fixed, paraffin-embedded tissue. When used as a panel, they can unequivocally distinguish carcinomas, melanomas, and lymphomas. All carcinomas react with at least one of the anti-cytokeratin antibodies, and carcinomas can be subtyped based upon the pattern of reactivity with the two anti-cytokeratin antibodies. Melanomas react only with the anti-vimentin antibody, and lymphomas react with none of the antibodies. Neural and neuroendocrine tumors can be identified with the anti-neurofilament antibody. A minority of neoplasms, including lymphomas, seminomas, and some sarcomas, do not react with any of the antibodies. These antibodies are reliable diagnostic reagents that are useful in distinguishing different categories of human tumors.     

Distribution of intermediate filament proteins in developing and adult salivary glands in man

Summary Adult and developing salivary glands were investigated using five monoclonal antibodies against cytokeratins (CKs) and vimentin. Acinar cells displayed mainly CK 18 whereas CKs 7, 17 and 19 were only detected in duct and myoepithelial cells. All epithelial and myoepithelial cells were unreactive for one vimentin antibody (Vim 9) whereas with the other (Vim 24), myoepithelial cells and basal cells of excretory ducts were stained. Fetal cells showed the CK pattern of duct cells. At gestational week 18, a reaction for both vimentin antibodies could be found in basal cells of terminal tubules. Although vim 9 reactivity has been shown for a number of salivary neoplasms, it has not been detected in any adult epithelial salivary tissue. The finding of this reactivity in the fetal gland indicates that the expression of this intermediate filament protein in certain salivary neoplasms may be a sign of dedifferentiation resulting in the expression of a filament pattern found in an earlier stage of gland development.     

Expression of intermediate filament proteins in fetal and adult human kidney: modulations of intermediate filament patterns during development and in damaged tissue.

The expression of intermediate filament proteins, particularly individual cytokeratins (CKs), vimentin, and glial filament protein, was immunohistochemically investigated using frozen sections and Carnoy-fixed, paraffin-embedded tissue from normal fetal and adult human kidneys as well as from pathologically altered kidneys. In fetal kidneys, the co-expression of CKs and vimentin was detected in the visceral and parietal epithelium of the glomerulus, the proximal tubules, the thin loops of Henle, and the collecting ducts. In contrast, in the tubules of normal adult kidneys, the presence of vimentin and CKs was nearly always mutually exclusive. While CKs 8 and 18 were present in all tubular epithelia, CKs 19 and 7 each exhibited a distinctive distribution pattern, there being a striking alteration between positive and negative segments and, not infrequently, intratubular heterogeneities. In certain segments, particular cell types (e.g., "plica cells," intercalated cells) could thus be recognized. In tubular epithelia altered by various injurious conditions, novel or enhanced expression of vimentin, CK 19 and CK 7, and, less frequently, CK 17 and glial filament protein was noted in certain segments. The increase in intermediate filament protein expression in altered (particularly proximal) tubules appeared to parallel the reduction in the degree of differentiation. Vimentin was never detected in distal tubules. The present results reveal a considerable similarity between the intermediate filament patterns in non-neoplastic proximal tubules of fetal and damaged kidney tissue and those in clear-cell and chromophilic renal cell carcinomas. They also serve to illustrate that the analysis of both fetal development and reactive cell changes may significantly contribute to our understanding of differentiation phenomena in malignant tumors.     

Antibodies to intermediate filament proteins in the diagnosis and classification of human tumors

Immunohistochemistry of intermediate filaments (IF) is a new and important way to evaluate the epithelial, mesenchymal, muscular, glial, or neural differentiation in tumors. This is based on the stable cell-type-specific expression of IF proteins in normal and neoplastic tissues. Immunohistochemical studies with antibodies to intermediate filaments have also given new perspectives in the histogenesis and biologic nature of many tumors. This article reviews both the recent findings and the authors' experience in the use of intermediate filament antibodies in tumor diagnosis and classification.     

Expression of vimentin and cytokeratin types of intermediate filament proteins in developing and adult human kidneys

Expression of cytokeratins and vimentin in developing and adult human kidney was studied by the indirect immunofluorescence technique using monoclonal and conventional antiintermediate filament antibodies. The undifferentiated cells of the metanephric mesenchyme expressed vimentin but not cytokeratins, whereas only cytokeratins could be visualized in the cells of induced renal vesicles. At the S-shaped body stage of nephrogenesis, the presumptive visceral and parietal cells of the developing glomeruli did not contain either detectable vimentin or cytokeratins, whereas cells of the tubular pole of the S-shaped body were stained within antibodies to cytokeratins. At a later stage the cuboidal visceral epithelial cells of the presumptive glomeruli were brightly positive for vimentin, but not for cytokeratins, and maintained their expression of vimentin during later stages of glomerulogenesis and in adult kidneys. Only some of the cells of the parietal epithelium, but not other glomerular elements, showed cytokeratin-specific staining. In fetal kidneys, the epithelial cells of proximal and distal tubules and of collecting ducts showed a cytoplasmic staining with two monoclonal antibodies to cytokeratins (PKK1 and PKK2), reacting with different cytokeratin polypeptides. In adult kidneys, a basolateral fluorescence in proximal and distal tubules was obtained with PKK1 antibodies, whereas PKK2 stained only the cells of the collecting ducts. Vimentin was not found in tubular epithelial cells at any developmental stage, whereas the cells of collecting ducts showed a transient expression of vimentin in fetal kidneys. Our results show a developmental stage-dependent pattern in the expression of the intermediate filament antigens in the cells of the kidney and show the exceptional expression of only vimentin in the visceral epithelial cells of human glomeruli.     

Expression of intermediate filament proteins in adrenal cortex and related tumours

The intermediate filament profile of adrenal cortex and its related tumours has been evaluated. Most adrenocortical cells contained cytokeratin 8 and 18 as demonstrated by monoclonal antibodies CAM 5.2, M20, M9 and RGE53. Cytokeratin immunoreactivity was not confined to a functional zone of the adrenal cortex. Only a small number of the adrenocortical cells showed vimentin immunoreactivity. From normal adrenal cortex through adenomas, to carcinomas, there is a progressive decrease or even loss of cytokeratin immunoreactivity and an increase in vimentin immunoreactivity. Aberrant cytokeratin expression was not found in adrenocortical adenomas and carcinomas with the antibodies used. Awareness of the possible absence of cytokeratin immunoreactivity in adrenocortical carcinomas is important whenever antibodies to cytokeratins and vimentin are used for diagnostic purposes in poorly differentiated neoplasms.     

Complexity of expression of intermediate filament proteins, including glial filament protein, in endometrial and ovarian adenocarcinomas.

The expression patterns of intermediate filament proteins of primary and metastatic endometrial (n = 18) and ovarian (n = 24) adenocarcinomas were analyzed by immunocytochemistry using a panel of specific antibodies and by gel electrophoresis of cytoskeletal preparations, followed by immunoblotting. All cells of all endometrial adenocarcinomas studied contained the "simple epithelial"-type cytokeratins (CKs) 8, 18, and (mostly) 19, with variable numbers of cells also positive for CK 7 and vimentin. In addition, most of these tumors contained individual cells or groups of cells that were positive for the stratification-related CKs 4, 5, 6, 13, 14, and 17. The latter CKs were often associated with squamous cell foci, but were also found in some single (nonsquamous) tumor cells, indicative of early stages of squamous cell differentiation. Ovarian carcinomas of various histologic types and grades contained predominantly CKs 7, 8, 18, and 19. Serous, endometrioid, and anaplastic tumors, but not mucinous and clear cell tumors, also contained minor amounts of stratification-related CKs in variable combinations, mostly including CK 4. In all tumor types except mucinous tumors, vimentin was consistently detected in variable proportions of tumor cells which, however, were rather low in anaplastic carcinomas. Surprisingly, glial filament protein was detected in a minor proportion (< or = 20%) of tumor cells in seven of 14 serous and endometrioid ovarian carcinomas and in three of 18 endometrial carcinomas. These different intermediate filament expression patterns of müllerian duct-type carcinomas, only partly related to the morphologic appearance of the specific type of tumor, might reflect the multipotentiality of differentiation of müllerian duct-derived epithelia. Cytoskeletal features of potential diagnostic value, especially in metastatic carcinomas, are discussed.     

Expression of intermediate filament proteins in thyroid gland and thyroid tumors

The presence of intermediate filament proteins of cytokeratin/prekeratin type and vimentin type was evaluated in non-neoplastic thyroid glands and in different types of thyroid neoplasms. Follicular epithelium of both normal and goitrous thyroids showed a strong reaction with anticytokeratin antibodies that widely cross-react with various simple epithelia. On the other hand, in normal thyroid, there were only occasionally (in one of 12 cases) solitary cells reacting with antibodies to epidermal prekeratin. In nodular goiters, such cells were often seen (eight of 18), especially among the lining cells of cysts, and in chronic thyroiditis in all (12 of 12) cases. Only the stromal cells and intraluminal macrophages reacted with antibodies to vimentin. Neoplastic cells of papillary carcinomas showed a positive staining reaction both with antibodies to cytokeratins and to epidermal prekeratin. Follicular carcinoma cells, although positive for cytokeratins, could generally not be stained with antibodies to epidermal prekeratin. Medullary carcinoma cells also showed cytokeratin positivity and, only occasionally, positivity for epidermal prekeratin. Anaplastic carcinomas were also reactive with antibodies to cytokeratin but, for the most part, were negative for epidermal prekeratin. Interestingly, some neoplastic cells of all types of thyroid carcinomas also appeared to contain vimentin, as shown with both polyclonal and monoclonal antivimentin antibodies. In contrast to carcinomas, the intermediate filaments of thyroid sarcomas and lymphomas were only of vimentin type. Furthermore, it was found that the papillary structures in benign goiters were only reactive with cytokeratin antibodies and lacked, in contrast to papillary carcinomas, epidermal prekeratin-like immunoreactivity. Hence, the analysis of intermediate filament proteins of thyroid tumors can be utilized to differentiate between papillary and follicular carcinomas and between benign and malignant papillary lesions as well as between anaplastic thyroid carcinomas and sarcomas or lymphomas.     

Malignant fibrous histiocytoma. Heterogeneous patterns of intermediate filament proteins by immunohistochemistry

Patterns of intermediate filament expression of 10 malignant fibrous histiocytomas (MFHs) were immunohistochemically evaluated using acetone-fixed frozen sections. Seven cases represented the storiform-pleomorphic subtype, 2 were of myxoid type, and 1 was of giant-cell type. All cases had been studied by electron microscopy, and no proof for the diagnoses of liposarcoma, rhabdomyosarcoma, and leiomyosarcoma could be obtained. All tumors showed prominent vimentin immunoreactivity in the tumor cells. Cytokeratin-positive neoplastic cells were found in 2 cases, and in the majority of tumor cells in 1 of these. The 68k neurofilament-positive cells were found in 2 cases. Desmin was not found beyond doubt in the neoplastic cells in any cases, and all cases were negative for glial fibrillary acidic protein. The expression of several types of intermediate filament indicates divergent differentiation properties in MFH and may suggest the heterogeneity of this entity, but more cases should be studied to elaborate any possible consistent patterns of intermediate filament expression in different types of MFH. The expression of multiple types of intermediate filament proteins in MFH can alternatively signify random activation of the corresponding genes in the primitive tumor cells. The complex patterns of intermediate filament proteins in morphologically defined MFHs should be taken into account in the practical immunohistologic analysis of tumors.     

Nonspecific alkaline phosphatase activity in normal and diseased human breast.

Nonspecific alkaline phosphatase activity was identified in human normal and diseased breasts with the use of the calcium-cobalt, the lead-nitrate, and the azo-dye methods. The results varied not only with the staining method, but also with the functional status of the breast structures. In normal, dysplastic, and fibroadenomatous tissues there was a strong parallelism between myoepithelial and capillary enzyme activities. The calcium-cobalt method was the only technique which allowed staining of carcinoma cells; cancer stromal enzyme activity was evidenced only with the use of the same method. Our findings suggest that nonspecific alkaline phosphatase activity probably reflects a functional status of the labelled structures; the enzyme activity of myoepithelial cells is variable and not really specific.     

Heterogeneity and co-expression of intermediate filament proteins in adenoid cystic carcinoma of salivary glands.

Among a series of 76 adenoid cystic carcinomas (ACC), 51 cases with cibriform or trabecular patterns were selected for an immunohistochemical study. These tumors were composed of three types of cells: basaloid, myoepithelial and ductal luminal acinous or tubular cells with a variable amount of each cell type from one case to another. Monoclonal antibodies (MoAb) against keratins (PKK1, KL1, K8.12, K8.13, K4.62 anti-cytokeratins antibodies) and against vimentin were used. Tubular cells were characteristically marked by anti-cytokeratins MoAb, but with a great heterogeneity of keratin distribution. In myoepithelial cells, keratin was absent or slightly positive and vimentin was present, with co-expression of the two types of filaments in some cells. Like myoepithelial cells, basaloid cells were positive to anti-vimentin antibody and negative or slightly positive to anti-keratin antibodies, with sometimes a co-expression of vimentin and keratin filaments in the same cell. Histogenesis of adenoid cystic carcinomas was discussed. Progenitor intercalated duct reserve cells may change into ductal luminal and myoepithelial tumor cells. Otherwise, basaloid cells, arising also from intercalated duct reserve cells, are able to acquire some secretory organites.     

Unexpected immunoreactivities of intermediate filament antibodies in human brain and brain tumors.

Immunoreactivities of 35 different monoclonal antibodies (MAbs) that detect intermediate filaments were studied systematically on serial cryostat sections of 14 well-defined human gliomas (five astrocytomas, three oligodendrogliomas, six glioblastomas) and on normal brain. Glial fibrillary acidic protein (GFAP), vimentin, desmin, neurofilaments, and broad-specificity keratin MAbs, as well as MAbs that recognize several or only single keratin polypeptides, were used. Unexpected reactivities were surprisingly frequent. As these may lead to diagnostic confusion and misinterpretation on this material, the authors investigated these phenomena more thoroughly. Four major sources of artifactual staining were found: 1) positive staining attributable to the rabbit gamma G immunoglobulins used in the alkaline phosphatase anti-alkaline phosphatase technique; 2) certain desmin and keratin MAbs cross-reacted with astrocytic glia and with other brain-specific epitopes; 3) technical difficulties; 4) some MAbs directed against neurofilaments and keratins showed unexpected reactivities only on individual anaplastic gliomas. The implications of these findings for intermediate filament typing of neuropathologic material are discussed.     

Occurrence of autoantibodies to intermediate filament proteins in human visceral leishmaniasis and their induction by experimental polyclonal B-cell activation.

Fifteen sera of patients with visceral leishmaniasis were investigated for the occurrence of autoantibodies. They were found in high incidence and titre, and with specificity to the intermediate filament (INFIL) proteins vimentin (12 out of 15 with a titre higher than 1:10) and keratin (9 out of 15 with a titre higher than 1:10) as well as to speckled anti-nuclear antigens (ANA). Additionally, supernatants of Leishmania major and Leishmania donovani cultures containing soluble parasite-derived antigens were mitogenic to cultures of mononuclear cells (MNC) obtained from healthy donors without specific antibodies to leishmanial antigens. The activation of MNC resulted in significant immunoglobulin production, some of which demonstrated autoantibody specificity to INFIL. The co-operation of monocytes, T cells and B cells was required in order to obtain maximal stimulation. The importance of polyclonal B-cell activation for the genesis and occurrence of autoantibodies in visceral leishmaniasis is discussed.