Local proliferation of brain macrophages in central nervous system tissue cultures.

Mouse central nervous system tissue cultured for different lengths of time was analyzed for the proliferation of macrophages. These cells were identified and characterized by ultrastructural features, cell surface determinants and their ability to ingest latex particles and bacteria. Under the experimental conditions chosen brain macrophages were derived from perivascular cells which in short term cultures remained attached to blood vessels and later differentiated into brain macrophages with a typical ultrastructural appearance. Identical results were obtained when intravascular cells were largely removed by extensive saline perfusion before culturing. Macrophages assembled around stab wounds of the central nervous system or obtained from peritoneal lavage showed comparable cytological characteristics and cell membrane determinants.

The use of lectins to study normal differentiation and malignant transformation.

Lectins are polypeptides that specifically recognize carbohydrate residues of glycoproteins and glycolipids. They can be extracted from plants, invertebrates and vertebrates. The binding between lectins and carbohydrate moieties can be blocked by the inhibitory sugar for which the lectin is specific. In analogy to antibodies, lectins can be used to analyse cell surface determinants. Thus, differentiation of cells, in particular of immunocytes, has been studied and lectin receptors are now important markers for distinguishing different developmental stages of T-lymphocytes. In consequence, premature, but already committed, T-cells can be eliminated from human bone marrow by means of lectins prior to transplantation in order to avoid graft-versus-host reactions. Moreover, it has been shown that malignant cells can be distinguished from their non-malignant counterparts by the profile of lectin receptors on their surface. This had led to the use of lectin binding sites as tumour markers in lymphomas and carcinomas.

Autoradiographic study of early neurogenesis in rat neocortex.

The early neurogenesis of rat neocortex was analysed by means of light and electron microscopic autoradiography. It was found that the very first preneurons originate probably as early as ED 11. They are the horizontal cells of Cajal-Retzius. The peak of their formation is on ED 13 (surface index estimated on ED 17 after injection of 3H-thymidine on ED 13:21, after injection on ED 12:4, after injection on ED 15:5), WHereas no Cajal-Retzius cells could be found to have originated after ED 15. Theses cells are the developmentally most advanced of the neocortex. The cells second in date of origin and maturation are preneurons which presumably correspond to the presumptive neurons of Layer VII (VI b), and begin to originate on ED 12. The end of their formation could not be defined owing to a lack of ultrastructural differences to other, younger preneurons in later gestational stages. These two cell types are the first cellular components of the primordial plexiform layer (Marin-Padilla, 1978) or pallial anlage (Rickmann, 1977), demonstrating an outside-in gradient within this layer, and are separated by the formation of the cortical plate. This could be proven by their simultaneous labelling above and below the cortical plate after administration of 3H-thymidine before ED 15. These results confirm the hypothesis of a dual origin of the mammalian neocortex (Marin-Padilla, 1978).

Varying expressions of lectin receptors within embryonic cell layers of murine cerebral cortex.

The ability of prenatal cerebral tissue to bind different lectins was analyzed using cryostat sections of mouse brains. It was shown that the immature cells within the embryonic cell layers possess receptors for different lectins in varying amounts. Of all lectins tested, only PNL, RCL and LPL were bound on the outer cell membranes to a considerable degree. Although the labeling patterns of PNL and RCL are similar, the latter is additionally well detectable on the wall of cerebral blood vessels. Cells of the ventricular layer are moderately labeled by PNL, which recognizes beta-D-galactosyl (1-3)-N-acetyl-D-galactosamine, but heavily labeled by LPL which binds to terminal sialic acid residues. Cells of the intermediate layer, on the other hand, are heavily stained PNL and only faintly by LPL. Hence it is suggested that the process of migration might be correlated to the removal of terminal sialic acid moieties from cell surface glycoconjugates, resulting in an exposure of the penultimate galactosyl residues.

The median ventricular formation. A distinct structure at the mesencephalic apex.

The prenatal ontogenesis of the median ventricular formation (MVF)--a cell group at the seam between both sides of the mesencephalic roof--was analyzed ultrastructurally, autoradiographically and for the expression of intracytoplasmatic structures, i.e. glial filament antigen. As compared with other regions of the mesencephalic roof it was found that from embryonal day 12 onwards DNA synthesis of ventricular cells in the dorsal midline is significantly reduced. This reduction is more pronounced at later developmental stages. On the other hand, the MVF gains drastically in width during ontogenesis. It was shown that this increase may be caused by an immigration of postmitotic neighbouring ventricular cells. The characteristic morphological feature of MVF cells is their extension from the ventricular lining to the pial basement membrane. Their dorsal processes are joined to a thin fibre bundle and predominantly display microtubules as well as filaments and glycogen within their electrolucent cytoplasm. They also contain intracellular structures that react with antibodies against glial filaments as revealed by an enzyme-coupled immunolabelling. The perikarya of MVF cells, on the other hand, are almost all situated at the same level within the ventral third of the mesencephalic roof, thus bulging concentrically at the lateral sides of the MVF. Characteristically, a subfraction of MVF cells exhibits vast amounts of rough ER. The nature and function of the MVF cells is discussed in the light of the concept of guidance of preneurons by radial glia (Sidman and Rakic 1973).

[Changes in the cerebellum of the rat after actinomycin during the postnatal period]. / Veränderungen des Kleinhirns der Ratte durch Aktinomycin während der Postnatalzeit

A single dose of actinomycin was applied to young Wistar albino rats in the critical phase of their cerebellar development. The morphological alterations of the cerebellar cortex were studied by means of light and electron microscopy on several postnatal days. The cell types of the cerebellar cortex reacted in different ways toward the noxious substance according to their stage of development. The acute alterations consisted of an edematous reaction of the neuronal and glial perikarya (light degeneration) and a shrinkage of the neurons (dark degeneration). A massive intercellular edema and a rarefaction of glia cells as well as the Purkinje cell fibres proved to be the long-term damage. This pattern of the alteration was discussed regarding the chemodifferentiation of the cells of the cerebellar cortex, the onset of cerebellar function on day 14, and the establishment of a neuroglial functional unit.

Developmental modulation of neuronal cell surface determinants.

Proceeding from the hypothesis that cellular differentiation processes are correlated with structural changes of the cell membrane, the expression of antigen and lectin receptors, as well as lectin-like molecules during migration and differentiation of pre- and perinatal neurons in the cerebral cortex, was analysed. It could be shown that a number of cell surface structures exist throughout the whole pre- and perinatal period, e.g. receptors for Robinia pseudoacacia lectin (RPL), pokeweed lectin (PWL) and concanavalin A (ConA) and the Ia and H-2-D/K antigens of the major histocompatibility complex (MHC). The expression of other cell surface structures, for example receptors for peanut and Limulus polyphemus lectin (LPL) and of Thy-1, is determined by the developmental stage; i.e. in the perinatal period higher amounts are found than in early prenatal stages. Binding sites for Phaseolus vulgaris lectin, PWL and anti-Thy-1.2 are not only demonstrated on perikaryal membranes, but additionally on diverse tangential or radial fibre structures. While on cells of the ventricular layer - the proliferating cell compartment - peanut lectin (PNL) receptors are observed in low density, LPL receptors in high density, in the migration zone, i.e. the intermediate layer, receptors are found predominantly for PNL and only few cells carry a significant number of LPL-binding sites. After the preneurons have migrated through the intermediate layer and the neighbouring cortical plate, remaining at the pia-near border of the latter, LPL receptors are again expressed on the cell surface of the now bipolar preneurons, while PNL receptors cannot be demonstrated any more. Experimental evidence is put forward indicating the possibility that this modulation of exposed carbohydrate residues on the cell surface might be mediated by a membrane-associated enzyme system on the same single molecule. For the investigation of neuronal cell interaction the ability of disintegrated suspended preneurons was used to reaggregate spontaneously in vitro and to build histiotypic cell formations within these reaggregates. It was found that this reaggregation of suspended neuronal single cells is dependent on the presence of ionized calcium, on the temperature, and on the conditions that influence the frequency of cell contacts. Furthermore, the structures expressed on the cell surface of preneurons during the pre- and perinatal period were investigated in regard to their influence on the reaggregation of these cells by means of a blockade by monoclonal antibodies or saccharides, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Prenatal differentiation of colliculus superior in the rat.

Up till emybryonic day (ED) the 13 the prenatal histogenesis of rat mesencephalic roof is characterized by an exclusive proliferation of ventricular cells. Then the first differentiating postmitotic cells are found above the ventricular layer, so that proliferation and differentiation occur simultaneously. On ED 15 a lamination typical for embyronic CNS with ventricular, subventricular, intermediate layers and stratum zonale can be observed in the area of presumptive colliculus superior. A definite demarcation between ventricular and subventricular layers, however, is only possible by means of autoradiograpahic methods. At the end of gestation the lamination of adult colliculus superior is already noticeable. During early prenatal development, cells which originate on one single day can autoradiographically be demonstrated in different cell layers and particularly over the whole width of the intermediate layer, while towards the end of gestation time of cell origin and location inside the mesencephalic roofs are closely correlated: cells that originate on ED 13 can be found predominantly in deeper cell layers (stratum griseum profundum, lower stratum griseum intermedium), while cells originating on EDs 15 and 17 are on ED 21 situated in the upper cell layers (upper stratum griseum intermedium, stratum griseum superficiale). This results in an inside-outside gradient of cell origin, which corresponds to an inside-outside gradient of cell differentiation. Besides, a rostro-caudal and a lateromedial gradient can be observed. On ED 13 a median ventriculaar formation (MVF) can be distinguished displaying a significantly lower mitotic activity than the cells of the neighbouring ventricular layer as could be demonstrated by autoradiographic investigation. This difference is even more pronounced in latter embryonic stages. From ED 17 on the cells of this particular structure are arranged in a typical onion-like pattern. Towards the end of gestation the MVF expands considerably as compared with the surrounding ventricular layer. As long-time experiments with 3H-thymidine reveala this increase in area is brought about by the immigration of numerous ventricular cells which originated on earlier embryonic days. The apical processes of the cells of the MVF form a thick fibre bundle which can be followed upwards to the stratum zonale. Ulstructurally these strictly parallel arranged fibers mainly resembly astroglial processes, but possess, in contrast to the latter, numerous microtubules. Because of their morphological characteristics a classification of these cells as tanycytes appears justified. The MVF is compared with similar structures in other regions of the CNS and its possible function as a guide for migrating preneurons is discussed.

Interaction between intestinal and peripheral blood mononuclear cells.

Effective methods are available to isolated immunocytes from human intestinal mucosa. Isolated mononuclear cells from the mucosa of patients with Crohn's disease and normal controls were classified according to ultrastructure and cell-surface profile by the use of a panel of monoclonal antibodies. In both compartments of the mucosa, i.e., the lamina propria and the epithelial layer, no differences were found between intestinal tissue from control patients and unaffected mucosa of patients with Crohn's disease. Inflamed mucosa was characterized by an accumulation of surface-Ig+ cells and, to a lesser degree, of monocytes and granulocytes. The predominant T cell phenotypes were a T4 cell in the lamina propria and a T8 cell in the epithelial layer. Most of the isolated immunocytes were found to be HLA/DR. Analysis of the functional properties of mucosa-associated T cells in a pokeweed-stimulated autologous B cell assay revealed that T cells originating from normal mucosa enhance the generation of IgA-producing B cells, whereas those from chronically inflamed mucosa do not. In contrast, peripheral T cells from patients with Crohn's disease favour expression of the IgA isotype, as compared with peripheral T cells from normal controls. Removal of Fc + T4 cells resulted in a significantly reduced number of IgA B cells. These findings point to an alteration of isotypic immunoregulation in Crohn's disease. It is postulated that a shift from IgA to IgG and IgM in the local immune response could result in antibody-dependent cytotoxicity for enterocytes.

Terminal galactosyl residues of cell-surface glycoconjugates exposed on both human and murine immature T- and B-cells.

Exposure of terminal galactosyl residues on cell-surface molecules as detected by their ability to bind peanut lectin (PNL) is found to be characteristic for immature cortical human and murine thymic lymphocytes. While in prenatal mice PNL staining is found to be uniformly distributed among all thymic lymphocytes, in adult thymic a cortico-medullary gradient is detectable concerning the PNL-binding capacity of thymic cortical lymphocytes, a phenomenon that appears to be correlated to their maturational degree. In secondary lymphatic tissue, i.e. lymph nodes and spleen of man, mouse and rat, strongly labeled cells are found exclusively in germinal centers. Ultrastructurally, these cells could be identified as centrocytes and centroblasts. These observations suggest that exposed galactosyl moieties of cell-surface glycoconjugates are expressed by undifferentiated lymphocytes of both T- and B-cell lineage. Furthermore, it could be shown that PNL-binding properties of immature cells are not restricted to lymphatic tissue but can also be demonstrated on various embryonic cells of non-lymphatic origin in distinct developmental stages. Thus, they might have a fundamental significance in the course of maturation processes.

Differences of glycoconjugates exposed on hypernephroma and normal kidney cells.

We analyzed the outer cell membranes of normal kidney cells, hypernephroma cells, nude mouse transplants of hypernephroma tissue, and cell lines derived from hypernephroma using horseradish peroxidase-coupled lectins of different specificity, in order to investigate their binding to cryostat sections or the cell surfaces of suspended single cells. We showed that hypernephroma cells express higher amounts of receptors for Robinia pseudoacacia lectin, soy bean lectin and Phaseolus vulgaris lectin compared to those prepared from normal kidneys. In contrast, Helix pomatia lectin is bound to connective tissue elements of both malignant and non-malignant tissue exclusively. L-fucose and D-galactose residues are detectable on malignant cells, but not on their non-malignant counterparts. However, the expression of the latter varies considerably among cells from different hypernephromas. No significant difference could be demonstrated between hypernephroma, hypernephroma cell lines and hypernephroma transplants in regard to the carbohydrate profile on the cell surfaces.

Evidence for the presence of Thy-1 on cultured thymic epithelial cells of mice and rats.

Thymic tissue of C57BL/6 mice and DA rats was cultured. After 6--8 weeks, cultures were analyzed for their capacity to absorb anti-Thy-1 serum, and for the expression of Thy-1 on the surface of different cell types by means of the indirect peroxidase labeling method. Three main cell types were identified: epithelium-like cells, fibrocyte-like cells and macrophages, but no lymphocytes were found. The presence of Thy-1 on cultured nonlymphocytic thymus-derived cells was demonstrated by their ability to absorb the cytotoxic activity of the appropriate anti-Thy-1 sera. Electron microscopical analyses of labeling experiments revealed that Thy-1 was predominantly expressed on epithelium-like cells with preference for their cell protrusions. The possible role of Thy-1 expression, both on thymocytes and thymus epithelium for cellular interaction, is discussed.

Subcapsular thymic lymphoblasts expose receptors for soy bean lectin.

In the course of analysing thymic cellular constituents concerning the expression of lectin receptors and defined lymphocyte differentiation antigens, respectively, a subpopulation of thymic lymphocytes was detected exposing both peanut lectin (PNL) and soy bean lectin (SBL) receptors on their surfaces, while the majority of cortical thymic lymphocytes were found to be PNL positive, but SBL negative. This subset of SBL+PNL+thymic lymphocytes in the adult populates the area beneath the thymic capsule, forming a narrow rim only, while in perinatal thymus a corresponding subcapsular cell layer comprises almost one third of the thymic tissue and in early ontogenesis the vast majority of thymic lymphoid cells are PNL+SBL+. By ultrastructural analysis of double labelled cell suspensions as well as of cells separated by affinity chromatography, it could be shown that PNL/SBL+ cells frequently exhibit morphological features of lymphoblastic cells. Thus this subcapsularly located subset seems to represent a compartment of proliferating immature cells, descended from bone marrow and foetal liver prethymic cells, respectively, and giving rise to PNL/SBL- cortical thymic lymphocytes.

Lectin-defined cell surface glycoconjugates of pancreatic cancer cells and their nonmalignant counterparts.

Alterations of cell surface carbohydrates of human pancreatic cancer cells from long-term cultures (COLO 357, RPMI 7451, PC 103, PC 107) were assessed ultrastructurally by use of an array of lectin-enzyme conjugates, and compared with lectin-defined changes of glycoconjugates on human pancreatic tissue sections of normal and various pathological conditions. Ulex europeus and, to a lesser degree, Lotus tetragonolobus lectin binding indicate that L-fucose-containing glycoconjugates are expressed predominantly on pancreatic cancer cell surfaces, but not, or restricted to intracytoplasmic structures, on nonmalignant pancreas cells. A comparable binding pattern to pancreatic carcinoma cells is found for Phaseolus vulgaris lectin. This is in contrast to the results with soy bean lectin, the reactivity of which was not restricted to cancer cell surfaces, and with Helix pomatia lectin, which did not bind to pancreatic cancer cells at all, although the latter three lectins possess similar sugar specificities. Between the long-term-cultured malignant pancreas cells differences were observed concerning the binding of wheat germ and pokeweed lectin. Besides, qualitative assets of lectin-binding absorption analyses elaborated quantitative differences in the expression of lectin-defined glycoconjugates on pancreatic cancer cell surfaces.

The intrathymic microenvironment: expression of lectin receptors and lectin-like molecules of differentiation antigens and MHC gene products.

The expression of MHC products, differentiation antigens and lectin receptors has been investigated in the various cell types populating different compartments of the thymus. The ultrastructural classification of suspended thymic epithelial cells was facilitated by using a technique that preserves cortical nursing cells or medullary epithelial cell clusters. A subset of peanut lectin positive lymphocytes could be distinguished by their ability to bind soybean lectin also. This subset corresponds to the large proliferating lymphocytes that populate the area between the thymic capsule and the cortex. Ia and H-2 D/K antigens could be detected on nearly all epithelial and lymphoid cells. Expression of H-2 antigens, however, is more pronounced on medullary epithelial cells. T-cell differentiation antigens such as Thy-1 and Lyt-1 could be demonstrated not only on lymphocytes, but, interestingly enough, on cortical epithelial cells as well. These latter cells, in addition, exhibit a cell membrane-bound lectin with a specificity for D-galactose which might well be the structure responsible for binding the galactosyl residues of the peanut lectin receptor of thymic lymphocytes. Binding sites for a large set of lectins could be demonstrated on both, thymic lymphocytes and epithelium. The intrathymic differentiation pathway of T-lineage cells is discussed with regard to those lymphocytic and epithelial cell surface structures considered to enable cellular interaction.