Svante Stenman

Changes in the matrix proteins, fibronectin and collagen, during differentiation of mouse tooth germ.

Abstract The distribution of the matrix protein fibronectin was studied by indirect immunofluorescence in differentiating mouse molars from bud stage to the stage of dentin and enamel secretion, and compared to that of collagenous proteins procollagen type III and collagen type I. Fibronectin was seen in mesenchymal tissue, basement membranes, and predentin. The dental mesenchyme lost fibronectin staining when differentiating into odontoblasts. Fibronectin was not detected in mineralized dentin. Epithelial tissues were negative except for the stellate reticulum within the enamel organ. Particularly intense staining was seen at the epithelio-mesenchymal interface between the dental epithelium and mesenchyme. Fibronectin may here be involved in anchorage of the mesenchymal cells during their differentiation into odontoblasts. Procollagen type III was lost from the dental mesenchyme during odontoblast differentiation but reappeared with advancing vascularization of the dental papilla. Similarly, procollagen type III present in the dental basement membrane during the bud and cap stages disappeared from the cuspal area along with odontoblast differentiation. Weak staining was seen in predentin but not in mineralized dentin. The staining with anti-collagen type I antibodies was weak in dental mesenchyme but intense in predentin as well as in mineralized dentin.

Fibronectin in the development of embryonic chick eye

Abstract 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 Descemets 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.

Activation of complement by cytoskeletal intermediate filaments.

INTEREST in cytoplasmic intermediate (10 nm) filaments has grown since the successful immunohistochemical differentiation of these filaments from microtubules and microfilaments1,2. Despite the widespread occurrence of intermediate filaments in various cell types, little is known about their function1,3,4. A cytoskeletal nucleus-anchoring role has been suggested based on their intracellular distribution, their connections to the Plasma membrane and nucleus, and their marked insolubility in detergents and salt solutions5,6. The presence of autoantibodies against intermediate filaments in the sera of patients7,8 suggests that these filaments can be involved in pathogenetic processes. We report here that in studies on possible pathogenetic mechanisms involving intermediate filaments, antibody-independent binding of complement components to intermediate filaments has been observed. Initial observations were made using cultured human embryonal fibroblasts, as cytoplasmic filaments present in these cells have been characterised by using both immunological markers and drugs capable of selectively altering the morphology of microfilaments, intermediate filaments and microtubules1,7. Subsequent studies showed complement binding to similar cytoplasmic structures of other cultured cells and also cells in tissue sections.

Fibronectin in synovial fluid and tissue in rheumatoid arthritis

Abstract. Fibronectin is a glycoprotein found in body fluids, loose connective tissue matrix and in basement membranes. Fibronectin in rheumatoid arthritis synovial fluid was immunologically indistinguishable from the plasma form, as shown by double‐diffusion analysis. Fibronectin isolated from rheumatoid synovial fluid by affinity chromatography on gelatin‐Sepharose had a polypeptide pattern similar to that of plasma fibronectin in SDS‐polyacrylamide gel electrophor‐esis. In fifty‐one patients with rheumatoid arthritis and related diseases fibronectin concentrations in synovial fluid were 445 ±103 μg/ml (mean ±SD) and within normal range, 335±52 μg/ml, in plasma. Immuno‐fluorescence staining showed a prominent increase of fibronectin in the proliferating synovial connective tissue in rheumatoid arthritis as compared to normal synovial membrane. The results suggest an increased local production of fibronectin in rheumatoid synovial tissue.

Fluorescent antibodies and lectins stain intracellular structures in fixed cells treated with nonionic detergent.

Nonionic detergent (NP40) treatment of paraformaldehyde-fixed normal and SV40-transformed human fibroblasts resulted in intracellular penetration of two chosen fluorescent antibodies and Concanavalin A (Con A). After the detergent treatment nuclear SV40 T antigen, cytoplasmic fibronectin glycoprotein and Con A binding sites could be visualized in fluorescence microscopy. The lowest NP40 concentration which made fixed cells permeable was 0.05%. The morphology of cells was preserved better by this new method than by conventional fixation methods, such as acetone treatment. In scanning electron microscopy the surface of the fixed NP40-treated cells had only small rugosities and fine pores. The subsurface cytoskeleton especially was well preserved and had a more distinct fine structure. The improved morphology made it possible to detect a similar distribution of fibronectin and Con A binding sites in the perinuclear endoplasmic reticulum regions.

Enhancement of radiation effects by alpha interferon in the treatment of small cell carcinoma of the lung

The effects on lung tissue and tumor of natural human alpha interferon (IFN) and radiotherapy were investigated in a multimodality treatment program for selected patients with small cell carcinoma of the lung (SCLC). Interferon was given first as a single agent, then concomitantly with radiotherapy to 12 previously untreated patients with limited disease. At disease progression outside the chest, interferon was discontinued and combination chemotherapy was initiated. In the first series, 7 patients received a high interferon induction dose (800 X 10(6) IU i.v. over 5 days) followed by low-dose maintenance therapy (6 X 10(6) IU i.m. TIW), median total dose 1380 X 10(6) IU (range 794-2074). At local progression, split-course radiotherapy, 55 Gy/20 F/7 wk, was added to interferon therapy. In the second series, 5 patients received low-dose interferon from the start (6 X 10(6) IU i.m. daily) combined with twice-a-day fractionated radiotherapy 44 Gy/40 F/4 wk. Median total dose of interferon in this series was 698 X 10(6) IU (range 354-828). Tumor response and normal tissue reactions were evaluated by monthly chest X rays, 3-monthly CT scans, restaging bronchoscopies and by serial respiratory function tests. Autopsy specimens from both lungs within and outside the radiation field were systematically evaluated when available. After the completion of radiotherapy, there were 4/7 CR in the high-dose IFN group compared to 3/5 CR in the low-dose IFN group. Rapid shrinkage of huge tumor masses was observed. At 2 months post radiotherapy radiological grade III fibrosis occurred in 4/7 patients in the high-dose and 1/5 patients in the low-dose group. Lung function studies showed a significant decrease in diffusing capacity and in lung volumes. Seven patients died within 12 months from start of interferon treatment, one of them from treatment complication. At autopsy the tumor area was in most cases replaced by severe fibrosis. Outside the radiation field lung fibrosis was mild. Our results suggest enhancement of radiation effect by interferon with a possible dose and/or schedule dependence of interferon and radiotherapy and call for more clinical studies of IFN and radiotherapy in combination.

Characterization of human smooth muscle autoantibodies reacting with cytoplasmic intermediate filaments.

Abstract Human smooth muscle antibodies (SMA) reacting with cytoplasmic intermediate filaments were characterized by the indirect immunofluorescence technique. These antibodies were noted to comprise a major type of SMA. When cultured human embrynic fibroblasts treated with a microtubulus-disrupting drug, vinblastine, were used, the SMA reacting with intermediate filaments (IMF-SMA), could be readily distinguished from other types of SMA by the immunofluorescence staining pattern. Absorption studies with intermediate filaments prepared from cultured human embryonic fibroblasts and from bovine smooth muscle tissue indicated that there are more than one autoantigen in the intermediate filaments of smooth muscle. The major subunit protein of intermediate filaments having a molecular weight of 55,000 was enriched in the antigenically active preparations of intermediate filaments. Five of 26 patients positive for IMF-SMA had signs of liver diseases and only two had chronic hepatitis.

Circulating antibodies to connective tissue microfibrils and dermal immunoglobulin deposits in leprosy

Abstract 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.

Depression of RNA synthesis in the prematurely condensed chromatin of pulverized HeLa cells

Chromosome pulverization is a premature condensation of interphase chromatin (PCC) in fused cells, apparently related to the presence of metaphase chromosomes in the same cytoplasm. The RNA synthesis in such chromatin in fused cells produced by treatment with Sendai virus was investigated. When 3H-uridine was administered before cell fusion, all PCC cells had incorporated the isotope, but when uridine was administered after virus treatment, the incorporation in PCC cells was strongly reduced. Fusion as such, or the virus infection, were excluded as a cause of the inhibition of RNA synthesis. The phenomenon appears analogous to the inhibition of RNA synthesis occurring during the metaphase coiling of chromosomes or in heterochromatin.

Intermediate filaments in enucleation of human fibroblasts

Abstract 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.