Antibodies to cytoplasmic microfilaments, intermediate filaments (vimentin filaments), and microtubules which comprise the cytoskeleton of the cell were assayed in sera from 23 patients with HBsAg-negative chronic active hepatitis and 1 with HBsAg-positive chronic active hepatitis, 15 patients with primary biliary cirrhosis, 20 patients with alcoholic liver disease, and 32 healthy controls. The cytoskeleton antibodies were assayed by indirect immunofluorescence technique using vinblastine-treated cultured human embryonic fibroblasts as substrates. There was a significantly increased incidence of cytoskeleton antibodies in patients with liver disease as compared to the control group. Antibodies to microfilaments were found frequently in sera from patients with chronic active hepatitis (67%) and primary biliary cirrhosis (53%) but were rare in sera from patients with alcoholic liver disease (25%) and in control sera (3%). On the other hand, antibodies to microtubules were found in 50% of sera from patients with alcoholic liver disease but in only 7 to 13% of sera from other groups. Intermediate filament antibodies of IgG or IgA class were found only in patient sera whereas intermediate filament antibodies of IgM class were found in the majority of sera in all groups including control sera. The highest titers of intermediate filament antibodies were seen in primary biliary cirrhosis and chronic active hepatitis. The production of cytoskeleton antibodies may be due to the reorganization or destruction of cytoskeletal structures in the liver.

In the present paper results from our studies on the expression and regulation of the differentiated normal and transformed epithelial and fibroblastic phenotypes are reviewed. The expression of the extracellular matrix- and basement membrane-associated proteins, fibronectin and laminin, and different intermediate filament proteins were studied in different fused cells. Heterokaryons and cytoplasmic hybrids (cybrids) were formed by fusing normal or malignant epithelial cells with normal fibroblasts or malignant glial cells. No differences were observed in the expression of these phenotypic markers between unfused parental cells and the corresponding homokaryons. Thus, the fusion process itself does not cause changes in the expression of these phenotypic markers. In heterokaryons formed after fusion of normal or malignant epithelial cells with normal fibroblasts or malignant glial cells, two or even three different types of intermediate filaments could be co-expressed. Thus, no suppression of the expression of the various intermediate filaments is caused by the non-homologous parental cell. Expression of the extracellular matrix proteins, fibronectin and laminin, on the other hand, could be extinguished by cells and cytoplasts not expressing fibronectin matrix, such as transformed fibroblasts and HeLa cells. These results support the role of transacting regulatory factors controlling the expression of the different extracellular matrix proteins. Alternatively, the results can be explained by changes in the distribution and concentration of the extracellular matrix receptor proteins in the fused cells.

Expression of intermediate filaments was studied immunohistologically in oat-cell (6 cases), epidermoid (9 cases), adeno- (7 cases), large-cell anaplastic (3 cases), and bronchioalveolar carcinoma (3 cases), of lung. Affinity-purified antibodies against epithelial (anti-keratin), neural (anti-neurofilament), muscle (anti-desmin) and mesenchymal (anti-vimentin) intermediate filaments were used. In indirect immunofluorescence microscopy of oat-cell carcinomas a positive cytoplasmic fluorescence was seen only with antibodies against neural intermediate filaments, neurofilaments, while no staining of the tumor cells was observed with antibodies against other types of intermediate filaments. On the other hand, all the epidermoid, adeno, anaplastic, and bronchioalveolar carcinomas showed constantly a strong reaction with anti-keratin antibodies in a varying number of cells but no decoration with anti-neurofilament antibodies. The results show that expression of neural intermediate filaments is a major distinguishing feature between oat-cell carcinomas and other lung cancers and suggest that anti-neurofilament antibodies can be used as a diagnostic aid in the surgical pathologic study of pulmonary neoplasms.

The expression of intermediate filaments of the keratin- and the vimentin-type was studied in heterokaryons of human fibroblasts and amnion epithelial cells by immunofluorescence microscopy. Fibroblasts and their homokaryons showed a fibrillar, vimentin-specific fluorescence throughout the cytoplasm but were negative when stained for keratin. Amnion epithelial cells and their homokaryons, on the other hand, showed a keratin-specific fibrillar staining, and only some of them contained also detectable vimentin. When suspended epithelial cells were fused with adherent fibroblasts, keratin fibrils spread within 3 h into the fibroblasts, intermixing with the vimentin fibrils. 1-3 d after fusion, both vimentin and keratin filaments were expressed as typical fibrillar cytoplasmic arrays, and the distribution of keratin in heterokaryons resembled closely that of vimentin. A typical cell-to-cell arrangement of keratin fibrils, seen in cultures of amnion epithelial cells, could also be found between heterokaryons. Treatment of the cultures with vinblastine sulphate induced coiling of the vimentin filaments in both homo- and heterokaryons, whereas the keratin organization was only slightly affected. Our results show that both vimentin and keratin filaments are incorporated into the cytoskeleton of heterokaryons formed between fibroblasts and epithelial cells, and that they behave in the same way as in their parental cells. Both epithelial and fibroblastic characteristics thus appear to the coexpressed in such heterokaryons.

The pericellular fibronectin matrix of human fibroblasts is lost when they are fused with normal or malignant cells, which do not produce fibronectin matrix. In the present study we have investigated whether also the cytoplasmic fraction of fibronectin-negative HeLa or MDCK cells can cause this effect. Enucleated epithelial cells (cytoplasts) were therefore fused with normal human fibroblasts. Fibronectin expression of the resulting cytoplasmic hybrids (cybrids) was studied by the indirect immunofluorescence technique. Three hours after fusion cybrids formed between fibroblasts and enucleated epithelial cells showed fibronectin matrix expression clearly weaker than that seen on the intact fibroblasts. An accumulation of fibronectin matrix was observed in the cybrids, analogously to the intact fibroblasts, and already 12–24 h after fusion the cybrids showed a fibronectin matrix expression similar to that of the fibroblasts. No fibronectin matrix was detected in the epithelial cells or their cytoplasts. Our results indicate that cytoplasmic factors from epithelial cells are able to cause an initial suppression in the formation of fibronectin matrix. However, the cytoplasts are not capable of causing a long-term effect on the fibroblasts.

Thrombi and thrombus formation in nonbacterial thrombotic endocarditis (NBTE) were studied using light microscopy and immunohistology. Samples from vegetations on cardiac valves were taken at autopsy from five patients with NBTE and adenocarcinoma as an underlying disease. Morphological studies disclosed proliferative changes underneath the thrombi. In immunofluorescence microscopy, focal deposits of immunoglobulins and complement components Clq and C3 were found. The results suggest that immune complexes, elicited by the underlying malignant process, may play important role in the pathogenesis of the thrombus formation in NBTE.

ABSTRACT The occurrence of different types of intermediate filaments in primary cultures of cells and in cultured cell lines was studied by the indirect immunofluorescence (IFL) technique. The antibodies used were spontaneous monoclonal human antibodies of immunoglobulin M (IgM) class against vimentin-type intermediate filaments (fibroblast 58 × 103 mol. wt subunit protein), and experimental rabbit antibodies of IgG class against vimentin, desmin (muscle 55 × 103 mol. wt subunit protein), glial fibrillary acidic protein (GFA), 68 × 103 mol. wt neurofilament polypeptide and human keratin polypeptides. Cultured fibroblasts from different species, and both aortic and venous endothelial cells, showed a fibrillar cytoplasmic fluorescence when stained with antibodies against vimentin. On the other hand, in cultures of chicken embryonal fibroblasts, cells showing a bright desminspecific fluorescence, but lacking vimentin-βpecific staining, were seen even after several subcultivations. The presence of both desmin and vimentin polypeptides in these cultures was also confirmed by polyacrylamide gel electrophoresis. In chicken fibroblast cultures the 2 types of intermediate filaments were not expressed simultaneously in individual cells, whereas in baby hamster kidney (BHK-21) cells and human rhabdomyosarcoma cells a variable co-staining with anti-vimentin and anti-desmin antibodies could be seen. In contrast, cultured human fibrosarcoma cells and simian virus 40-transformed human fibroblasts showed only vimentin-specific fibrillar fluorescence. Glial cells from mouse embryonic spinal cord appeared to express only GFA-containing intermediate filaments in a primary culture, whereas both subcultured mouse glial cells and a cultured glioma cell-line also showed vimentin-specific staining. On the other hand, neuronelike cells in the primary cultures could only be stained with the antibodies to 68 × 103 mol. wt neurofilament polypeptide. Interestingly, clones of mouse neuroblastoma (C1 300) cells contained only vimentin-type intermediate filaments, whereas rat pheochromocytoma (PC 12) cells contained both vimentin- and neurofilament-specific fluorescence. Two types of intermediate filaments were also seen in cultured epithelial cells. In primary cultures of human amnion epithelial cells a fibrillar keratin-specific fluorescence was seen in all cells but only a few of the cells also showed vimentin-specific fluorescence as distinct juxta-nuclear aggregates. On the other hand, subcultured amnion epithelial cells and various epithelial cell lines contained both keratin and vimentin fibrils. Our results show that cultured fibroblasts contain only vimentin-type intermediate filaments and that differentiated cells in primary culture contain primarily tissue-specific intermediate filaments. On the other hand, all proliferating cultured cells appeared to contain vimentin-type filaments in addition to tissue-specific intermediate filaments. This suggests that vimentin expression is connected with the adaptation of cells to culture conditions.

We report here that we have found an accumulation of condensed mitotic figures in cultures of various human haematopoietic cell lines treated with diazepam (40–80 µg ml−1). The mode of mitosis arresting activity of diazepam was analysed with cultured human fibroblasts. Unlike conventional anti-mitotic drugs, diazepam did not affect the microtubular integrity at the concentrations used. Instead, it seems to block mitosis by inhibiting the deviation of centrioles in the prometaphase.

Fibronectin in human solid tumors was studied by indirect immunofluorescence staining of biopsy material. Altogether 73 tumors were examined, comprising 12 sarcomas, 3 melanomas I reticulum cell sarcoma, 39 carcinomas, 6 benign soft‐tissue tumors and 12 benign epithelial tumors. In all sarcomas the individual tumor cells were surrounded by a network of fibronectin which was continuous with the stroma. The distribution of fibronectin was similar in the benign soft‐tissue tumors. In contrast, no fibronectin was detected in the individual carcinoma cells or in their periphery. However, the reactive connective tissue stroma of carcinomas was strongly positive for fibronectin. This was true also for the stroma of benign epithelial tumors. These results show that, contrary to the situation in cell culture, in vivo sarcoma cells and benign soft‐tissue tumor cells contain fibronectin in their pericellular matrix. On the other hand, fibronectin can be used to distinguish carcinomas from sarcomas in vivo .

Enucleation of cultured human fibroblasts was used to study the interaction between the nucleus and the cytoskeletal intermediate filaments. In enucleated cells the filaments stained for immuno-fluorescence were seen as fibrillar cytoplasmic arrays similar to those of intact cells. Typical reorganization of the filaments into coiling bundles occurred in cells treated with vinblastine either before or after enucleation. The cytoplasmic stalk connecting the extruding nucleus to the rest of the cell contained filaments, whereas the karyoplasts lacked them. The present results indicate that the filaments are essential for the anchorage of the nucleus and that the weakest point in this system is between the nucleus and the intermediate filaments.

ABSTRACT. Fibronectin is a polymorphic glycoprotein of plasma, other body fluids and connective tissue, and it occurs in an insoluble and a soluble form. Insoluble fibronectin is found associated with basement membranes and in loose connective tissue matrix as well as in the pericellular matrix formed around cultured adherent cells, such as endothelial, fibroblastic and smooth muscle cells. In these positions fibronectin apparently functions as a substrate for cell attachment and as a scaffold for cell migration and movement. Soluble fibronectin, present e.g. in the circulation (300 μg/ml) exhibits some important interactions with other proteins. It is covalently cross-linked to fibrin during thrombus formation and binds to collagen. Fibronectin is released from platelets during their aggregation and soluble fibronectin potentiates the action of plasminogen activator. We have detected fibronectin in the sub-endothelium, in the matrix of smooth muscle cells of the media and in the adventitia of arteries. By using immunohistological techniques we have further found that fibronectin is prominent in atherosclerotic lesions of the intima, especially in developing fibrous plaques. Fibronectin was also prominent in experimentally induced atherosclerotic lesions. These findings suggest that fibronectin is an indicator of connective tissue formation in atherosclerotic processes and that the protein can have a role in their pathogenesis.

A disease characterized by massive tumorous cutaneous hyalinosis has been studied histologically, immunologically, and biochemically. The precipitated hyalin material differed from amyloid in being Congo-red-negative and ultrastructurally nonfibrillary. In lipoid proteinosis, massive hyalin deposits have not been encountered and the clinical course is distinct from the course of massive cutaneous hyalinosis. The clinical and histologic pictures of both adult and juvenile forms of colloid milium differed from that found in our patient, although the colloid milium in adult form is ultrastructurally also nonfibrillary like the hyalin from our patient. A strong humoral immune response to components of the cytoskeleton of fibroblasts and especially to keratin was found in our patient.

Fibronectin is a polymorphic glycoprotein of plasma, other body fluids and connective tissue, and it occurs in an insoluble and a soluble form. Insoluble fibronectin is found associated with basement membranes and in loose connective tissue matrix as well as in the pericellular matrix formed around cultured adherent cells, such as endothelial, fibroblastic and smooth muscle cells. In these positions fibronectin apparently functions as a substrate for cell attachment and as a scaffold for cell migration and movement. Soluble fibronectin, present e.g. in the circulation (300 micronm/ml) exhibits some important interations with other proteins. It is covalently cross-linked to fibrin during thrombus formation and binds to collagen. Fibronectin is released from platelets during their aggregation and soluble fibronectin potentiates the action of plasminogen activator. We have detected fibronectin in the sub-endothelium, in the matrix of smooth muscle cells of the media and in the adventitia of arteries. By using immunohistological techniques we have further found that fibronectin is prominent in atherosclerotic lesions of the intima, especially in developing fibrous plaques. Fibronectin was also prominent in experimentally induced atherosclerotic lesions. These findings suggest that fibronectin is an indicator of connective tissue formation in atherosclerotic processes and that the protein can have a role in their pathogenesis.

Cultured human glioma cells were studied by double indirect immunofluorescence technique using antisera against intermediate filaments and glial fibrillary acidic protein. With both antisera cytoplasmic fibrillar fluorescence was seen. Perinuclear bundles of intermediate-sized filaments, induced by vinblastine treatment, were strongly stained with both antisera. The degree of codistribution of the two types of antigenic determinants varied considerably from cell to cell. These results suggest that two types of filament-related antigenic determinants can be present in the same cell, and also that glial fibrillary acidic protein-related filaments may possess functional similarities to the intermediate filaments found in other cells. Glial fibrillary acidic protein remains as a useful and specific antigenic marker for the study of glial cells in vitro.

Subepidermal fibrillar deposits containing IgM immunoglobulin occur in apparently normal skin from leprosy patients. The distribution of the deposits is similar to that of the microfibrillar epidermis anchoring system which consists of elastic fibers. The deposits may be related to the presence of a novel autoantibody reacting with connective tissue microfibrils and the microfibrillar part of elastic fibers. The antibodies were of IgM class and were found in 67 of 97 leprosy patients. They occurred in higher titer in patients classified as borderline lepromatous as compared to borderline tuberculoid patients.

The time course of appearance and distribution of fibronectin in the developing eye have been studied in chick embryos by indirect immunofluorescence. At the 12-somite stage, fibronectin was detected as a layer under the ectodermal cells overlying the forebrain vesicle; it was also present in the head mesenchyme. During formation of the lens placode and its invagination, a zone containing fibronectin persisted around the lens as a component of the capsule. The fibronectin-containing layer was separated from the corneal epithelial cells during the formation of the acellular stroma. The migrating corneal endothelial cells were seen posterior to the fibronectin layer. The secondary stroma was strongly positive for fibronectin. Fibronectin disappeared from the cornea starting from its posterior part along with the corneal condensation. In the newborn chicken cornea, fibronectin was present only in Descemet's membrane. In addition, the embryonic vitreous body had a network of fibronectin-containing material. The distribution of fibronectin in the developing cornea, as well as other data available on this glycoprotein, is consistent with the proposed role of fibronectin in positioning and migration of cells and in organization of the extracellular matrix.