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ABSTRACT: In this study, a scanning microscopic computer-assisted image analysis system was used for the immunocytochemical characterisation of collagen types I, III and V in normal human fibroblasts from pulp and gingival explants, using specific purified antibodies and peroxidase labeling. The culture conditions were standardized in order to evaluate simultaneously the expression of the three antigens in four different culture passages of the two fibroblast types. The optical density values of immunostaining intensities were quantified, the integrated optical density per cell was calculated, and the results were analyzed by a variance test. It was found that all three collagen types were present in the tissues, and in both gingival and pulp fibroblasts after three to nine culture passages. A non-parametric statistical analysis of the staining intensity variances revealed significant differences between antigenic levels depending on the tissue origin of the fibroblasts and an effect of culture passages. The results seemed to justify application of this technique at the light microscope level for the evaluation of collagen production, the principal function of fibroblasts, but the tissue origin and number of culture passages should be taken into consideration for in vitro biocompatibility testing of dental materials.
Journal de biologie buccale 10/1992; 20(3):169-74.
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ABSTRACT: The differentiation of myofibroblastic cells from normal human gingival fibroblasts in vitro has been established by transmission electron microscopy and quantitated by immunohistochemistry, using antigelsolin monoclonal antibodies. Untreated control cultures were compared to cultures exposed to Helium-Neon (He-Ne) laser irradiation. A direct and massive transformation of the cultured fibroblasts into myofibroblasts was observed as early as 24 hours after laser treatment, whereas control cultures were comprised of only resting fibroblasts and active fibroblasts. This in vitro induction of myofibroblasts may be analogous to that which occurs in vivo. Therefore we undertook a similar study using biopsies from gingival tissues after wisdom tooth extraction. Myofibroblasts were present in the connective tissue of laser-treated gums 48 hours after irradiation, but not in untreated contralateral control tissues. These data provide evidence that the primary biologic effect of the Helium-Neon laser on connective tissue is the rapid generation of myofibroblasts from fibroblasts. The induction of a phenotype with contractile properties may have clinical significance in the acceleration of the wound-healing process.
American Journal Of Pathology 08/1990; 137(1):171-8. · 4.89 Impact Factor
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ABSTRACT: The present study reveals the dynamic distribution of membrane laminin receptors induced by laminin binding in a rat rhabdomyosarcoma cell line RMS S4. The treatment of the cells with soluble laminin did not modify cell adhesion to laminin-coated substrates in in vitro attachment assays. Fluorescent labeling of membrane-bound laminin revealed that occupied receptors were induced to cluster and cap. New free membrane binding sites were made evident after capping of bound laminin by a double labeling technique. Cytochalasin D (CD) treatment prevented the capping process. The adhesion of CD-treated cells to laminin-coated substrates was inhibited by cell preincubation with soluble laminin. Cycloheximide treatment had no effect on the ability of RMS S4 cells to adhere to adsorbed laminin after preincubation in the presence of soluble laminin. These results taken as a whole suggest that free receptors may arise from an intracellular pool that could be maintained by membrane receptor recycling. Since capping and motility seem related events, migration of RMS S4 cells on laminin was studied in the agarose drop assay. Immobilized laminin stimulated basic cell motility by more than 200%. E8 laminin fragment retained partially the motility stimulating property of laminin while P1 pepsinic fragment had no effect. The presence of constantly available receptors at the cell surface could be determinant in the ability of cells to migrate on laminin substrates.
Experimental Cell Research 01/1990; 185(2):482-95. · 3.58 Impact Factor
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ABSTRACT: The authors have developed a new methodology for characterizing in situ the cell cycle of human mammary epithelial cell lines. Using a SAMBA 200 cell image processor (scanning cytometry), 15 densitometric and textural parameters were computed on each Feulgen-stained nucleus. Parameters computed from the grey level cooccurrence and run-length section matrices allowed assessment of the chromatin pattern. Multiparametric analysis of data defined: 1) the relative position of each cell; 2) the relative positions of groups of cells, each group corresponding to a definite phase of the cell cycle; and 3) the function of these parameters best separating these phases. Files then were constructed for each phase: G0/G1, S, G2/ and M. Using these three files as a reference to classify cells, it was possible to ascertain the phase of the cell cycle for each cell of a population. The MDA AG human cell line synchronized by mitotic selection was used as a model to develop this method. The criteria used to assign cells to G0/G1, S, or G2 was DNA content. Classification in M phase was achieved by visual identification of mitotic cells. This method was checked on unsynchronized MDA AG and then applied to other human cell lines (MDA MB231, MCF-7, T47D C111). Comparison of results obtained by scanning cytometry and flow cytometry showed the proportion of cells assigned to G0/G1, S, and G2/M by the two methods to be similar. This new method removes some of the limitations of flow cytometry by 1) allowing visual verification of each cell analyzed; 2) lowering the number of cells required for study; 3) discriminating between G2 and M; and 4) preserving cell topography.
Cytometry 06/1989; 10(3):263-72.
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ABSTRACT: A new methodology was developed to study dynamic processes topographically in biological systems by means of a graph-theoretical method. It is based upon order parameters obtained from a minimal spanning tree analysis coupled with computer simulations. The method was used to analyse the heterogeneous behavior of two neoplastic cell lines after treatment with laminin. The laminin-induced cell detachment was quantitated and shown to be inversely related to cell population density and thus to cellular interactions. Our statistical analysis is a very powerful tool to obtain information from seemingly disorderly heterogeneous biological models.
Analytical cellular pathology: the journal of the European Society for Analytical Cellular Pathology 05/1989; 1(2):123-32.
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ABSTRACT: The biosynthesis of the basement-membrane glycoprotein laminin in the mouse teratocarcinoma cell line PFHR9 was studied by immunoelectron microscopy and pulse-chase experiments using monoclonal and polyclonal antibodies. By immunoelectron microscopy, most of the protein was found to be aggregated on the outer cell surface. Cytoplasmic stainings were rare and were located next to the intracellular side of the plasma membrane. Sequential immunoprecipitations of cell extracts with a monoclonal antibody (4C12) sensitive to the laminin native conformation and with a polyclonal antibody enables laminin, the B1 subunit and a 410 kDa molecule to be distinguished. Most of the laminin is of the A(B1B2) type, and the 410 kDa molecule appears to be a B1B2 heterodimer. The assembly of laminin from subunits is completed in less than 1 h, and B chains are incorporated via the formation of the B heterodimers. The B2 and A chains are not found as free forms, so their levels appear to be the rate-limiting factors for the assembly of the dimers and laminin respectively. The formation of an uncross-linked A(B1B2) complex as a short-lived intermediate in the biosynthetic process is possible. Together with immunoelectron microscopy, the present study suggests that the protein is rapidly exported after assembly to accumulate on the outer side of the cell membrane. The biosynthesis of laminin in the PFHR9 cell line appears to be similar to that in other matrix-producing cell lines.
Biochemical Journal 04/1988; 250(3):843-52. · 4.90 Impact Factor
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ABSTRACT: Biosynthesized by epithelial, endodermal and Swann type cells, laminin (Lam) is a cross shaped multifunctional glycoprotein formed by the multimeric assembly of subunits which result from the activation of several genes. In vivo, depending on its location, because of its adhesive properties and multivalent affinities, Lam is in association as a part of supramolecular complexes together with compounds of the plasma, the basement membrane and the cell coat. In the basement membrane (MB) Lam has structural and functional roles. It may also be adsorbed on the cell coat or secreted. It is interacting with epithelial cells by the way of a plasma membrane receptor and has a role to play in cellular differentiation and proliferation. Lam is a molecular link of epithelial cells to MB. These features implicate the molecule in organogenesis, embryogenesis and post-traumatic healing. As a structural component of MB and as an attachment factor, Lam is involved: 1 in tumoral invasion which allows metastatic spreading, 2 in homing because metastatic cell display an increased receptivity to the molecule. The study of Lam expression, Lam receptivity and their factors of control should lead to a better understanding of the biochemical and molecular basis of differentiation, embryogenesis, organogenesis and metastasis.
Pathologie Biologie 11/1986; 34(8):955-63. · 1.53 Impact Factor
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ABSTRACT: The distribution of laminin was studied in 98 breast carcinomas with antilaminin and the avidin-biotin-peroxidase complex method. Laminin was observed within vascular and epithelial basement membranes. Laminin displayed a continuous linear pattern in intraductal carcinomas, and it was heterogeneously distributed, with a discontinuous linear pattern, in invasive carcinomas. No intracellular laminin staining was detected. Electron microscopic study showed laminin immunostaining in the lamina densa of basement membranes in nonneoplastic breast tissue. In tumors, laminin immunostaining frequently revealed multilayered basement membranes and abnormal multilayered basement membranes in blood vessels in the tumor stroma. These data suggest that laminin immunostaining, as a new approach to the heterogeneous basement membrane changes occurring in carcinomas, should permit better understanding of cell diffusion processes and of stroma-tumor cell interactions. The consistent extracellular distribution of laminin in contact with the stroma indicates that the latter plays an important role in the assembly of basement membrane components.
Human Pathlogy 05/1986; 17(4):355-65. · 2.88 Impact Factor
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ABSTRACT: An estrogen receptor immunocytochemical assay (ER-ICA) was applied to 130 malignant breast carcinomas and the results were compared to those of steroid binding assays performed on cytosol extracts of the same tumors. Also laminin (lam) distribution was studied in the same tumors. A semi quantitative analysis and a computerized image analysis system (SAMBA 200 TITN) were used to evaluate the positive ER and lam immunostaining. Positive ER immunostaining was always located in the nucleus of tumor cells and of normal cells in adjacent breast tissue. When immunohistochemical staining was correlated to biochemical assay there was a 88% correlation staining intensity and percentage of positive cells significantly increased (p less than 0.01) with cytosolic ER levels. Lam was observed within vascular and epithelial basement membranes (BMs). Lam staining displayed a continuous linear pattern in intraductal carcinomas or was heterogeneously distributed with a discontinuous linear pattern in invasive carcinomas. No intracellular lam staining was detected. In tumors, laminin immunostaining revealed often multilayered BMs and abnormal multilayered BMs in blood vessels in the tumor stroma. These results indicated that (ER-ICA) is to date the most reliable histochemical method for ER detection and correlated in 88% of the cases with ER biochemical assay ER-ICA provides additional information for heterogeneous ER distribution within tumors ER-ICA as a qualitative method is unable to replace the quantitative ER determination obtained with biochemical assay ER-ICA based on ER antigenic site detection is complementary to biochemical assay based on ER functional site determination laminin immunostaing constitutes a new approach to the heterogeneous BM changes occurring in carcinomas, and permits a better understanding of cell diffusion processes and of stroma-tumor cells interactions: the consistent extracellular lam distribution in contact with the stroma, indicates that the latter plays an important role in the assembly of BM components the SAMBA 200 permits a reliable accurate evaluation of the percentage of the immunostained cells and surfaces.
Bulletin du cancer 02/1986; 73(6):651-64. · 0.67 Impact Factor
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ABSTRACT: An immunoelectron microscopic study was carried out on human placenta and decidua with the use of preembedding, the avidin-biotin-peroxidase complex technique, and rabbit anti-murine laminin antibody. Laminin was detected in the lamina lucida of basement membrane of placental villi, amniotic membranes, umbilical cord, endometrial glands, and blood vessels. No positive laminin immunostaining was observed in intracytoplasmic organelles. However, positive immunostaining surrounded decidual cells as a more or less continuous linear membrane. It is suggested that laminin, as a component of this basement membrane-like material that has already been reported in decidual cells, may be related to the hormonal stimulation occurring during pregnancy and trophoblastic attachment.
American Journal of Obstetrics and Gynecology 04/1985; 151(6):822-6. · 3.47 Impact Factor
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ABSTRACT: Laminin distribution in tissues from surgically removed thyroids consisting in normal gland (5), Basedow's disease (5), thyroiditis (5), follicular adenomas (8), papillary carcinomas (8), follicular carcinomas (6) was studied using rabbit laminin antibody raised against murine laminin. Immunoperoxidase technique (Avidin-Biotin-Peroxidase Complex) was performed on (1) paraffin sections of fixed tissue (2) frozen sections for light microscopy examination. Vibratome thick sections (100 micron) and pre-embedding technique were used for electron microscopy study. Positive staining, was obtained only on frozen and vibratome sections and was found within basement membranes but never in epithelial cell cytoplasm. Laminin had a similar distribution in follicular adenomas, Basedow's disease and normal tissue. Nests of damaged cells in Hashimoto's thyroiditis lacked positive laminin immunostaining. In papillary carcinomas positive staining was found beneath the epithelial cells along the cores. In well differentiated follicular carcinomas the perifollicular laminin staining was preserved, whereas in poorly differentiated follicular carcinomas laminin staining was barely visible or absent.
Bulletin du cancer 02/1985; 72(1):6-15. · 0.67 Impact Factor
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ABSTRACT: A wide range of normal human tissue samples including cervix, endometrium, thyroid, pancreas, parotid, breast, placenta, gastric mucosae, striated muscle were compared with tumorous and non tumorous disorders (thyroiditis, Graves disease, follicular adenoma, thyroid carcinomas, breast cystic disease, fibroadenoma, adenosis, breast carcinomas) using anti-laminin and Avidin Biotin Peroxidase complex method on frozen sections (light microscopy study) and vibratome cut 100 micrometer-thick-sections (electron microscopy study). It was shown that laminin was located in the lamina densa of basement membranes (BM) in normal human tissue and visible on BM like structures around decidua cells, BM were abnormally thick and often multilayered but continuous and laminin positive in intraductal breast carcinomas and well differentiated follicular carcinomas of thyroid, in invasive carcinomas laminin immunostaining displayed an heterogeneous pattern with disruptions and even may completely disappeared, in tumor stroma, blood vessels BM had a laminin abnormal staining with a multilayered pattern. Since laminin is involved in cell attachment to basement membrane through specific receptors to laminin and to biochemical components of modified interstitium found in tumorous disorders, laminin immunohistochemical detection constitutes a valuable method for a better understanding of tumor cells diffusion and metastases development.
Annales de Pathologie 02/1985; 5(2):77-84. · 0.25 Impact Factor
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ABSTRACT: An immunohistochemical study was carried out on a human breast carcinoma series with the aim to delineate the technical approach to distinguish the epithelial cells from stromal nonepithelial cells in the primary tumor and in cultured tumor cells. Frozen sections from 30 unfixed breast carcinomas were incubated with mouse monoclonal antiepithelial cell membrane antibodies and then with a biotinilizated antimouse antibody and the avidin biotin peroxidase complex (ABC). Thick (100 micron) sections from fresh, unfixed tissue were similarly treated prior to the araldite embedding procedure for EM study. In all cases the epithelial tumor cells were immunostained in contrast to nonepithelial stromal cells, particularly the fibroblasts. Because standard light microscopy fails to determine the real nature of the fusiforme cultured cells, it is suggested that these techniques represent a reliable method for mammary epithelial cell identification and may further be applied on cultured tissue.
Cancer Detection and Prevention 02/1985; 8(1-2):77-85. · 2.52 Impact Factor
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ABSTRACT: Since early antiquity malignant tumors have been recognized and characterized by: 1) their particular local-regional invasive properties. Developing irregularly from the primary tumor mass, the invasion of adjacent tissues often displays classical crab-like images which distinguish, macroscopically and microscopically, malignant tumors from benign tumors. 2) their metastatic capacities. Even though they have been taught since the time of Hippocrates, the specific clinical characteristics of malignant tumors have only recently been apprehended and studied on cellular and biochemical levels. The development of the metastatic phenomenon is a complex chain of events limited to only a few tumor subpopulations. Like every biological study knowledge has progressed over the past years only by concentration of efforts on some of these stages: Invasion associated with disorganization and destruction of basement membranes; Migration of metastatic tumor cells in the stroma and bridging of vascular basal membranes; Colonization of target tissues by recognition and penetration of specific vascular membranes, establishment and concentric development within these tissues. These three stages result from successive interactions between multiple cellular and biochemical phenomena. This review is a schema of our present knowledge of tumor invasion, and more precisely of that concerning the relations between tumor cells and extracellular matrices, particularly basal membranes, leading to a definition of the cellular phenotype with a high metastatic potential.
Bulletin du cancer 02/1985; 72(5):367-76. · 0.67 Impact Factor
