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PubMed1985

Anti-intermediate filament antibodies, antikeratin antibody, and antiperinuclear factor in rheumatoid arthritis and infectious mononucleosis.

Sera from patients with rheumatoid arthritis (RA), patients with infectious mononucleosis (IM), and blood donors were tested by indirect immunofluorescence for the presence of antikeratin antibody (AKA), antibody to cytoskeletal intermediate filaments of prekeratin or vimentin type (AIFA) and antiperinuclear factor (APF). In 81.9% of the RA sera and 92.5% of the IM sera AIFA of IgM class was found at titres up to and in some cases exceeding 1/160. In blood donors the incidence of AIFA was 26%, at titres not exceeding 1/20. AKA and APF, always of IgG class, were found in 54.2% and 73.6% of rheumatoid sera. A weak correlation was found in RA between the incidence of AIFA and APF. AKA was not present in either IM or blood donor sera, and APF was found in only 2.5% and 3.2% of IM or blood donors respectively.

Antibodies, Antinuclear↗
PubMed1986

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.

Antigens, Protozoan↗
PubMed1994

Nucleotide sequence of an Onchocerca volvulus cDNA clone encoding an antigen homologous with the intermediate filament protein family.

A series of clones were isolated from an O. volvulus lambda gt11 cDNA library on the basis of strong recognition by sera from both Onchocerca volvulus or Wuchereria bancrofti patients. Several of these clones encoded a protein which clearly belonged to the intermediate filament (IF) class, and had most similarity with A1IF-A from Ascaris lumbricoides and a number of IF proteins from molluscs. Western blotting experiments implied that the first coiled domain appears to be markedly less antigenic in vivo than the remainder of the O. volvulus protein.

Amino Acid Sequence↗
PubMed1992

Different effects of pemphigus antibody and plasmin on the distribution of keratin intermediate filaments and desmoplakins between cultured oral and epidermal keratinocytes.

In order to clarify the molecular mechanism of blister formation in oral mucosa in pemphigus vulgaris (PV) comparing with that in epidermis, we analyzed the effects of PV serum on the distribution of keratin intermediate filaments (KIFs) and desmoplakins in oral as well as epidermal cultured keratinocytes by immunofluorescence microscopy using anti-keratin and anti-desmoplakin I/II monoclonal antibodies. After incubation with PV serum for 96 h at 37 degrees C, clusters of anti-keratin positive dots were formed around the nucleus in some of the keratinocytes from normal gingiva and soft palate but not in keratinocytes from tongue and skin, and desmoplakins also changed their distribution from linear arrangement at cell-cell contacts to clusters of dots around the nucleus in gingiva but not in epidermal keratinocytes. The dotted structures similar to those induced by pemphigus serum were formed also by incubation with human plasmin in gingival keratinocytes. However, no dot-formation of keratins was induced in these cells after incubation with trypsin. Furthermore, in epidermal keratinocytes, no keratin-dot formation was observed even after incubation with plasmin or trypsin. These results suggest that the dotted structures of KIFs caused by PV serum and plasmin might be a feature characteristic for the response of oral keratinocytes to PV serum and that there are some distinct differences in susceptibility to, and mode of, bulla formation between oral epithelium and epidermis.

Antibodies↗
PubMed1995

Behaviour of macroglial cells, as identified by their intermediate filament complement, during optic nerve regeneration of Xenopus tadpole.

Assessment of glial cell behaviour during optic nerve (ON) regeneration in Xenopus tadpoles is hampered by the lack of classical cellular markers that distinguish different glial cells in mammals. We thus have characterized the intermediate filament (IF) complement of tadpole glial cells and used it to follow the fate of glial cell subsets during the first 10 days after ON crush. Glial cells synthesize a restricted number of cytokeratin (CK) species and vimentin. This pattern remains essentially unchanged during metamorphosis and regeneration. However, vimentin turnover is specifically enhanced after injury. The expression of CKs and vimentin has been followed immunocytochemically in situ and in isolated cells recovered from dissociated ON segments. In the normal nerve, 79% of ramified glial cells express both CK and vimentin, 1% CK and 4% vimentin only, whereas 16% express neither IF protein. We tentatively classified CK expressing cells as mature astrocytes and those without IF proteins as oligodendrocytes. In the regenerating ON, the relative number of oligodendrocytes is decreased, while the astrocytic subset becomes accordingly larger but is decreased by day 10 already in favour of cells expressing vimentin only. Astrocytes invade the lesion site soon after crush, arrange into a central core within the distal nerve segment and establish a peripheral scaffold that is readily crossed by axons. Unlike mammalian astrocytes that remain absent from the lesion site but form a scar at some distance to it, amphibian astrocytes appear to provide active guidance to axons growing through the lesion site.

Animals↗
PubMed1990

Heterogeneity of intermediate filament expression in human testicular seminomas.

Testicular seminoma has in the past been considered to represent a germ cell tumor incapable of further differentiation. In recent years this view has been challenged on the basis of morphologic and chromosomal studies. Moreover, studies of intermediate filaments (IF) of seminoma cells have provided evidence of the capability of seminoma cells to differentiate in different directions. In the present study of the IF protein profile of 26 human testicular seminomas, using frozen as well as formalin-fixed, paraffin-embedded tissues, we report evidence of a heterogeneous differentiation potential inherent in these neoplasms. Thus, in 4 of the seminomas neither cytokeratins nor vimentin were detected; 3 showed vimentin positive cells but no cytokeratins; in 4 seminomas only cytokeratins were detected. In the remaining 15 cases both cytokeratins and vimentin were present, with occasional cells demonstrating coexpression of cytokeratin and vimentin. While the cytokeratins present were mostly of the "simple epithelial type", in 2 instances seminoma cells also contained cytokeratins 4 and 17, normally found in stratified and/or complex glandular epithelia. Furthermore, in 3 cases scattered tumor cells stained for desmin and in 2 other seminomas neurofilaments were identified. All of the cases showed variable positive staining for desmoplakins and desmoglein, indicative of the presence of desmosomes. It can therefore be concluded that, while some seminomas seem to be devoid of IFs, most of them show varied differentiation patterns usually with epithelial features but occasionally also with components commonly regarded as characteristic of myogenic or neurogenic differentiation. These observations may help to elucidate the relationship of seminomas to other germ cell tumors, and also contribute to our understanding of the histogenesis of these neoplasms.

Desmin↗
PubMed1998

Expression of the intermediate filament keratin gene, K15, in the basal cell layers of epithelia and the hair follicle.

The intermediate filament keratin, K15, is present in variable abundance in stratified epithelia. In this study we have isolated and characterized the sheep K15 gene, focusing on its expression in the follicles of sheep and mice. We show that K15 is expressed throughout the hair cycle in the basal layer of the outer root sheath that envelops the follicle. Strikingly, however, in large medullated wool follicles, a small group of basal outer root sheath cells located in the region thought to contain hair follicle stem cells are K15-negative. In the follicle bulb K15 is expressed in cells situated next to the dermal papilla but not in the inner bulb cells. Elsewhere, K15 is expressed at a low, variable level in the basal layer of the epidermis and sebaceous gland, often in a punctate pattern. In the esophagus of the sheep K15 expression is restricted to the basal layer, in contrast to human esophagus where it is expressed throughout the epithelium. Transgenic mouse lines established with a 15-kb sheep K15 gene construct exhibited faithful expression and showed no phenotypic consequences of K15 overexpression. An investigation of transgene expression showed that K15 is continuously expressed in outer root sheath cells during the hair cycle. Given its expression in the mitotically active basal cell layers of diverse epithelia and the follicle, K15 expression appears to signal an early stage in the pathway of keratinocyte differentiation that precedes the decision of a cell to become epidermal or hair-like.

Amino Acid Sequence↗
PubMed2000

Residues in the 1A rod domain segment and the linker L2 are required for stabilizing the A11 molecular alignment mode in keratin intermediate filaments.

Both analyses of x-ray diffraction patterns of well oriented specimens of trichocyte keratin intermediate filaments (IF) and in vitro cross-linking experiments on several types of IF have documented that there are three modes of alignment of pairs of antiparallel molecules in all IF: A11, A22 and A12, based on which parts of the major rod domain segments are overlapped. Here we have examined which residues may be important for stabilizing the A11 mode. Using the K5/K14 system, we have made point mutations of charged residues along the chains and examined the propensities of equimolar mixtures of wild type and mutant chains to reassemble using as criteria: the formation (or not) of IF in vitro or in vivo; and stabilities of one- and two-molecule assemblies. We identified that the conserved residue Arg10 of the 1A rod domain, and the conserved residues Glu4 and Glu6 of the linker L2, were essential for stability. Additionally, conserved residues Lys31 of 1A and Asp1 of 2A and non-conserved residues Asp/Asn9 of 1A, Asp/Asn3 of 2A, and Asp7 of L2 are important for stability. Notably, these groups of residues lie close to each other when two antiparallel molecules are aligned in the A11 mode, and are located toward the ends of the overlap region. Although other sets of residues might theoretically also contribute, we conclude that these residues in particular engage in favorable intermolecular ionic and/or H-bonding interactions and thereby may play a role in stabilizing the A11 mode of alignment in keratin IF.

Amino Acid Sequence↗
PubMed1989

Ultrastructure of parathyroid autografts in chronic renal failure including the occurrence of concentric membranous bodies and intermediate filaments.

The ultrastructure of long-term parathyroid autografts and their donor glands has been studied in nine patients with chronic renal failure. Autografts showed ultrastructural features of functional activity. Unusual findings included the presence of 'spironolactone-like' concentric membranous bodies derived from the endoplasmic reticulum in one hyperplastic autograft and prominent intracellular intermediate filaments (tonofilaments) in the donor parathyroid gland and recurrent autograft growths in another patient.

Adult↗
PubMed1986

Relationship between intermediate filaments and microfilaments in cultured fibroblasts: evidence for common foci during cell spreading.

Spreading and fully spread chick embryo fibroblasts (CEF) were examined by double-label fluorescence microscopy using the actin-specific probe rhodamine-phalloidin and an antibody directed against CEF intermediate filaments (IF). During midspreading, a striking relationship became discernible: statistical analysis showed that approximately half of the cell population exhibited one or more phase-dense, phalloidin-binding nodules that appeared to act as foci from which IF diverged. Coincidence between actin-containing structures and IF was not limited to these centers; IF could also frequently be seen running in close parallel arrays with stress fibers. Ultrastructural analysis confirmed the presence of non-membrane-bound out-pocketings along the length of stress fibers from which 10-nm IF diverged. These structures varied in size and shape, and displayed a dense, fine fibrillar appearance. IF and microfilaments (MF) were distinguished by size and by decoration of MF with myosin subfragment-1. Other IF-MF interactions were seen in cells of all stages: IF were observed to loop through stress fibers, most frequently at the cell margins. In colchicine-treated cells, IF became redistributed into cables that often ran parallel and appeared to merge with stress fibers. Cytochalasin D-treated CEF exhibited loose aggregates of actin-containing material that appeared to be associated with IF. These results suggest the possibility of an interaction between actin-containing structures and IF, particularly during cell spreading in cultured fibroblasts.

Actin Cytoskeleton↗
PubMed1991

Intermediate filament protein profiles of human testicular non-seminomatous germ cell tumors: correlation of cytokeratin synthesis to cell differentiation.

The patterns of cytoskeletal differentiation were studied in 20 testicular non-seminomatous germ cell tumors by immunohistochemistry, using diverse monoclonal antibodies specific for different intermediate filament (IF) proteins and for desmoplakin. Immunofluorescence and immunoperoxidase methods on both formalin-fixed and frozen tissues were applied, in some cases together with a gel electrophoretic analysis of IF proteins. The tumors examined included embryonal carcinoma (EC), endodermal sinus tumor (EST), choriocarcinoma and teratoma. Nine of the tumors were composed of only one histological type, the others showed mixed components. Cytokeratins 8 and 18 were identified in all these neoplasms, but their immunostaining was weak in ECs. Cytokeratin 19 was absent or very scarce in ECs, but strongly expressed in ESTs, choriocarcinomas and teratomas, thus allowing the identification of small EST and choriocarcinoma elements in ECs even when they were morphologically not obvious. Occasionally, some cells in ECs and ESTs also stained for cytokeratins 4 and/or 17, indicating potential for epithelial stratification. The majority of the germ cell tumors showed varied amounts of vimentin, often in co-existence with cytokeratins. Neurofilaments were demonstrated in scattered tumor cells in a single case of EST. In the teratomas studied, each type of tissue component present showed the expected IF protein. However, in many germ cell tumors some stromal cells and blood vessels contained, in addition to vimentin and desmin, also cytokeratins 8 and 18. This heterogeneity of the cytoskeletal profile of germ cell tumors is indicative of the varied differentiation potential inherent in these neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differentiation↗
PubMed1988

Intermediate filaments of hepatocytes and biliary epithelial cells in bile duct obstruction: transmission and scanning electron microscopy study.

The cytoskeletons of hepatocytes and biliary epithelial cells in bile duct ligated rate livers were investigated by transmission and scanning electron microscopy. The three dimensional organization of the intermediate filaments (IFs) of hepatocytes and biliary epithelial cells was clearly demonstrated by scanning electron microscopy. Cell borders and dilated bile canaliculi were well preserved after perfusion with detergent solution. A very dense filamentous network of IFs was seen throughout the cytoplasm, especially around the dilated bile canaliculi and at the cell borders. IFs in biliary epithelial cells were more numerous compared with hepatocytes. Morphometric analysis showed that the IFs of hepatocytes significantly (p greater than 0.001) increased in amount in bile duct ligated rats. The IFs of biliary epithelial cells showed no significant changes in bile duct ligated rats compared to controls. These results suggest that the increase in IFs in hepatocytes results from the adaptation of the hepatocytes to the stress imposed by bile duct ligation. It may be that the resulting intracanalicular pressure and back diffusion of bile induces a metaplastic change in hepatocytes so that they acquire more IFs to function like the bile duct epithelium to conduct bile flow.

Animals↗
PubMed1984

The intermediate filament proteins of rabbit normal epidermal Merkel cells are cytokeratins.

Four hundred Merkel cells (MC) have been studied by double-label immunofluorescence using: (1) a monoclonal antibody which has been previously demonstrated to react with MC and (2) antisera and monoclonal antibodies against the 5 types of intermediate filaments. It was demonstrated that MC did not react with vimentin, desmin, glial acidic fibrillary protein, or neurofilament antisera. A strong staining of MC was observed with 2 antisera and 2 monoclonal antibodies against keratin. The cytokeratin polypeptide pattern of MC is probably similar to that of simple epithelia. These findings attest to the epithelial nature of MC.

Animals↗
PubMed1996

Protein kinase C-delta associates with vimentin intermediate filaments in differentiated HL60 cells.

The subcellular localization of protein kinase C (PKC)-delta was determined in HL60 cells differentiated toward monocytes/macrophages by treatment with TPA. PKC-delta was detected in the nucleus and cytoplasm of differentiated HL60 cells and, more specifically, associated with structures resembling intermediate filaments. Indirect immunostaining revealed that PKC-delta colocalized with vimentin in the cytosol and perinuclear region of these cells. Immunoprecipitation studies showed that PKC-delta was in an active (autophosphorylated) state in differentiated HL60 cells and that vimentin immunoprecipitated from these cells was also phosphorylated. Treatment of HL60 cells with the PKC-specific inhibitor chelerythrine decreased the phosphorylation of vimentin. These data suggest that vimentin is a substrate for PKC-delta and that this PKC isoenzyme may play a specific role in the regulation of shape change and cell adhesion during HL60 differentiation.

Antibody Specificity↗
PubMed2003

Intermediate filament aggregation in fibroblasts of giant axonal neuropathy patients is aggravated in non dividing cells and by microtubule destabilization.

Giant axonal neuropathy (GAN) is a severe neurodegenerative disorder characterized by the accumulation of neurofilaments (NFs) in distended axons. GAN corresponds to a disorder of the cytoplasmic intermediate filaments (IFs), since an abnormal aggregation of different IFs has been reported in several cell types, including NFs in neurons and vimentin in fibroblasts. The recent identification of the defective protein, named gigaxonin, now renders possible investigations on the mechanisms that trigger the destabilization of IFs. Although gigaxonin domain organization suggests multiple protein-protein interactions, via the BTB and the Kelch domains, the low amino acid identity with other members of the BTB/Kelch subfamily did not allow hypothesis about its function. In the present work, we studied GAN primary fibroblasts, and show that vimentin aggregation suffers great variation on prolonged culture at confluence and in low serum condition. While neither the microfilament (MF) nor the microtubule (MT) networks are perturbed by vimentin destabilization, we found that the aggregates are in close proximity to the microtubule organizing centers (MTOCs). Moreover, we show that MTs depolymerization induces a total vimentin aggregation in GAN fibroblasts. The results, together with the recent finding of an interaction between gigaxonin and MAP1B, a MT associated protein, suggests that gigaxonin plays an important role in the crosstalk between the IF and MT networks. We found that, when overexpressed, gigaxonin is localized in the cytoplasm but does not colocalize with any of the cytoskeletal networks, suggesting that the presence of the binding partner is rate limiting for proper localization of gigaxonin.

Actin Cytoskeleton↗
PubMed2005

Intermediate filament proteins participate in signal transduction.

How timely transport of chemical signals between the distal end of long axonal processes and the cell bodies of neurons occurs is an interesting and unresolved issue. Recently, Perlson et al. presented evidence that cleavage products of newly synthesized vimentin, an intermediate filament (IF) protein, interact with mitogen-activated protein (MAP) kinases at sites of axon injury. These IF fragments appear to be required for the transport of these kinases to the cell body along microtubule tracks. The truncated vimentin is instrumental in signal propagation as it provides a scaffold that brings together activated MAP kinases (such as Erk 1 and Erk2), as well as importin beta and cytoplasmic dynein. The authors propose that this all-in-one transport complex has the extraordinary ability to travel towards the cell body and enter the nucleus where the kinases activate and influence gene expression so that a neuron can generate a timely response to injury.

Animals↗
PubMed1990

Fate of Mallory body-containing hepatocytes: disappearance of Mallory bodies and restoration of the hepatocytic intermediate filament cytoskeleton after drug withdrawal in the griseofulvin-treated mouse.

Mallory bodies are characteristic morphological features of alcoholic hepatitis in man and can be produced in the mouse by chronic griseofulvin intoxication. The appearance of Mallory bodies in hepatocytes is associated with derangement of the cytokeratin intermediate filament cytoskeleton, at least as revealed by immunofluorescence and suggested by immunoelectron microscopy. Immunohistochemical studies were performed to answer the question whether Mallory body formation and cytoskeleton alterations finally lead to cell death or are reversible phenomena. Chronically griseofulvin-intoxicated mice killed at different stages of recovery on a normal diet served as experimental animals. It could be shown that (a) Mallory bodies are very durable structures and are found for up to 6 mo after griseofulvin withdrawal as a result of persistence and neoformation; (b) new Mallory bodies can appear even several months after cessation of griseofulvin feeding; (c) Mallory body formation and cytoskeletal changes by themselves do not lead to irreversible cell damage; (d) the cytoskeletal changes are reversible within 7 mo after griseofulvin withdrawal; (e) a dissociation between disappearance of Mallory bodies and restoration of a regularly immunostained cytoplasmic cytokeratin meshwork is observed.

Animals↗