Phenotypic heterogeneity influences apoptotic susceptibility to retinoic acid and cis-platinum of rat arterial smooth muscle cells in vitro: Implications for the evolution of experimental intimal thickening.

Rat aortic smooth muscle cells (SMCs) cultured from intimal thickening 15 days after endothelial injury (IT-15), unlike those of normal media, show a monolayered, epithelioid phenotype and high levels of cellular retinol binding protein-1 (CRBP). Epithelioid clones obtained from the normal media suggest a "mosaicism" of arterial SMCs. Intimal cell homeostasis from the balance of proliferation and apoptosis is critical for the progression of vascular lesions. All-trans retinoic acid (tRA) reduced [(3)H]thymidine incorporation and G(1)-->S phase progression of IT-15 and epithelioid clone but not of normal media and IT 60 days after injury (IT-60) SMCs. Hoechst staining, flow cytometry, and ligation-mediated polymerase chain reaction showed an increased susceptibility of IT-15 and epithelioid clone to tRA and cis-diaminedichloroplatinum II (CDDP)-induced apoptosis and cytotoxicity compared with normal media and IT-60 cells. The latter retained an increased susceptibility to tRA-induced apoptosis compared with normal media SMCs. tRA-induced apoptosis associated with an increased ratio of bax to bcl-2 by bax overexpression and cleavage of caspase-3. Anti-CRBP but not anti-IgG antibody prevented tRA-induced apoptosis and changes in related signaling molecules but not CDDP effects. Our findings support the relevant role of phenotypic heterogeneity in the determining proliferative as well as apoptotic behavior of arterial SMCs.

Characteristics of the effect of S-100 proteins on the assembly-disassembly of brain microtubule proteins at alkaline pH in vitro.

The ability of S-100 proteins to inhibit the assembly of brain microtubule proteins (MTPs) in the presence of microM levels of Ca2+ increases as a function of pH. This seems to be due to an increasingly larger inhibitory effect of S-100 on the nucleation and, probably, on the elongation of microtubules (MTs) as the pH raises. In the presence of microM Ca2+ levels, the ability of S-100 to disassemble MTs also increases linearly with the pH, suggesting that the larger inhibitory effect of S-100 on MTP assembly at alkaline than at acidic pH may depend on both a decrease in the assembly rate and an increase in the disassembly rate. Also, S-100 inhibits the assembly of phosphocellulose-purified tubulin to a larger and larger extent as the pH raises. S-100 brings about its effect on MT assembly-disassembly probably by sequestering soluble tubulin, though additional mechanisms cannot be excluded. The present data are briefly discussed in relation to the role attributed to changes in intracellular pH in the regulation of the state of assembly of cytoplasmic MTs.

Localization of the loosely bound nuclear proteins of rat brain: an immunocytochemical ultrastructural study.

Antibodies against the loosely bound subnuclear protein fraction (0.35 M NaCl-extractable subnuclear fraction) of rat brain were raised in rabbits, and the ultrastructural distribution of the antigenic determinants in rat cerebellum was studied using the unlabeled antibody peroxidase-antiperoxidase (PAP) method. A localization restricted to the nucleus was observed. Both neuronal and neuroglial nuclei exhibited antigens, whereas nuclei of pericytes and endothelial cells did not. The immunoreaction product was homogeneously distributed in dispersed chromatin and was absent from condensed chromatin, suggesting that the antigens were confined to the active regions of the genoma. The outer nuclear membrane and the nucleolus appeared to be free of the antigens, while a perinucleolar ring of immunoreaction was detectable. Liver preparations showed a nuclear reaction markedly weaker than the one for brain nuclei. Adsorption of the serum with isolated liver nuclei nullified the reactivity in liver tissue, whereas a sharp reaction was still observed in the cerebellum, indicating the subcellular reaction under examination to contain antigens specifically concentrated in the nervous system or unique to the brain.

Immunochemical and immunohistochemical detection of S-100-like immunoreactivity in spinach tissues.

S-100 proteins represent a group of closely related acidic, calcium binding proteins originally isolated from the mammalian nervous system and later detected in non-neural cell types and in a wide variety of vertebrate and invertebrate species. The present study used immunochemical and immunohistochemical methods to extend the investigation of S-100 during phylogenesis to plant tissues. The presence of S-100-like immunoreactive material was detected in extracts of spinach (Spinacia oleracea L.) terminal buds and young leaves by the ELISA method and by Western blotting using different anti-S-100 rabbit antisera. Using the PAP method, serial sections of young spinach leaves treated with the same antisera exhibited an immunoreaction product that was confined to the cytoplasm and nucleus (but absent from the vacuoles) in meristematic, epidermal, and parenchymal cells. The present data enlarge the field of investigation of S-100 proteins in the search of the function(s) of S-100 in biological organisms.

Localization of S-100 protein in Müller cells of the retina--2. Electron microscopical immunocytochemistry.

The cellular and subcellular distribution of S-100 protein was investigated at the ultrastructural level in the rat retina by the immunocytochemical PAP method. S-100 appeared to be localized in the cytoplasm and nucleus of Müller cells, offering conclusive evidence that in the mammalian retina, the protein is confined to glial cells. S-100, as a marker for Müller cells, may be a useful tool in order to study the cytoarchitecture of the retina in normal as well as in pathologic conditions. In addition, the retina may represent a suitable model for further investigation on the biologic role of S-100.

Localization of S-100 protein in Müller cells of the retina--1. Light microscopical immunocytochemistry.

S-100 is an acidic brain protein previously found to be present in glial cells of the brain and the nervous system of gut and respiratory tract. Immunocytochemistry at the light microscopical level localized immunoreactivity for S-100 in the Müller cells in the retina of rat, guinea pig, and Chinese hamster. The Müller cells represent the main glial component of the retina, with a structural role in the support and insulation of neurons and sensory elements. The use of S-100 protein as an immunocytochemical marker of Müller cells may be useful in the study of pathologic conditions of the retina where glial cell proliferation could reflect the index of neuronal injury.

Increase of S-100 immunoreactivity in the urinary bladder from patients with multiple sclerosis, an indication of peripheral neuronal lesion.

The Schwann cells in urinary bladder biopsies from multiple sclerosis patients and controls were examined by immunocytochemistry with an antiserum to S-100. S-100 immunoreactivity was found to be markedly increased in these tissues as compared with the controls, indicating a Schwann cell hyperplasia in the urinary bladder in multiple sclerosis. This finding suggests that local neuronal damage exists in the urinary bladder of patients with multiple sclerosis. Therefore, the concept of multiple sclerosis as a disease wholly of the central nervous system should be reexamined.

Immunocytochemical localization of ?-bungarotoxin receptors in the chick ciliary ganglion: Synaptic and extrasynaptic sites?

The immunocytochemical localization of nicotinic acetylcholine receptors in the chick ciliary ganglion was investigated at the ultrastructural level with a procedure utilizing binding of ?-bungarotoxin visualized by immunoperoxidase histology. Both ganglionic cell populations, i.e. the ciliary and choroid neurons, showed specific immunoreactivity for receptors. In both types of neurons evident stain was found on the postsynaptic membrane. Often the reaction product filled discrete regions of the synaptic cleft. Furthermore, specific staining was also observed on large areas of the neuronal surface devoid of presynaptic nerve endings. These data probably indicate the occurrence of both synaptic and extrasynaptic nicotinic receptors on the neuronal plasma membrane. Immunoreactivity was also observed on the membrane of the presynaptic nerve endings, and on the part of the satellite plasma membrane which is adjacent to the neuron. These last results are discussed in relation to the occurrence of possible artifacts or, alternatively, to the presence of presynaptic and glial receptors. Immunoreactivity at all sites was prevented almost completely by ganglion incubation in 1 mM d-tubocurarine prior to and during treatment with toxin.

Detection of S-100 labelled cells in nasopharyngeal carcinoma.

S-100 antigen containing cells with dendritic features, recognisable by morphological and immunohistochemical criteria as belonging to the Langerhans' or interdigitating reticulum cell type, have been found in undifferentiated nasopharyngeal carcinoma. The presence of these cells, which have a special function of antigen presentation in immune responses, may be involved in a possible modulation of nasopharyngeal carcinoma associated Epstein-Barr virus infection and host-tumour interactions.

S-100 protein in the brain of hypothyroid adult rats: an immunochemical and immunocytochemical study.

The levels and the distribution of S-100 protein were studied in the brain of hypothyroid adult rats. The concentration of the protein was significantly increased in the soluble fraction of hypothyroid rat brain as compared with controls (P less than 0.001). This finding probably reflects the increased number of astroglial cells, which has been widely reported in hypothyroid adult rats. The immunocytochemical ultrastructural distribution of the protein in the cerebellar cortex was not affected by thyroid deficiency. The protein was thus confined to the cytoplasm and the nucleus of the astroglial cells both in treated and untreated rats.

Glial-like cells in sympathetic neural crest derivatives during human embryogenesis. Detection by S-100 immunohistochemistry.

The present study investigates at the light and electron microscopic levels the possible presence and distribution during human development of glial-like satellite cells in sympathetic neural crest derivatives by S-100 immunohistochemistry. From the earliest stages investigated, immunostained cells were detected inside sympathetic migrating masses and at their periphery, where they constituted a continuous layer isolating sympathetic elements from mesenchymal cells. The detection and peculiar distribution of these glial-like cells in developing sympathetic tissue could open new perspectives in the study of events linked to the migration and differentiation of some neural crest derivatives.

Immunochemical detection of S-100 protein in non-nervous structures of the rabbit eye.

S-100 is a protein originally believed to be unique to the nervous system. We report here on its immunochemical detection in non-nervous structures of the rabbit eye, namely in the lens, cornea and iris, at a markedly higher concentration in the latter.

Immunochemical and immunocytochemical study of S-100 protein in rat adipocytes.

S-100 is a protein originally believed to be unique to the nervous system. We report here on the presence of S-100 in rat adipocytes, using immunohistochemical and immunochemical methods. We demonstrate that the protein in adipose tissue is present at a concentration comparable to that measured in the nervous tissue and is immunologically identical to brain S-100, indicating that the protein can no longer be regarded as being specific to the nervous system.

Comparative study by S-100 and GFAP immunohistochemistry of glial cell populations in the early stages of human spinal cord development.

The present study reports the presence and distribution of S-100-containing cells in the developing human spinal cord. From the earliest stages investigated, S-100-immunostained cells bordered the ventral third of the central canal or appeared dispersed in the basal lamina. On the other hand, radial glia processes were clearly depicted after GFAP immunolabelling. The presence and peculiar distribution of S-100-immunoreactive cells could be significant in the study of the early events of glial differentiation and migration.

Satellite cells in developing spinal ganglia. An immunohistochemical study.

The present immunohistochemical study investigates the presence and distribution of S-100-containing glial cells in the early stages of development in human spinal ganglia. From the earliest ages investigated immunoreactive cells could be detected in a continuous layer at the periphery as well as inside ganglionic rudiments in close relationship with neural elements, both at the light and ultrastructural levels. The possibility that these glial cells, exhibiting such a distinctive distribution, play a modulatory role on microenvironmental influences during maturation could be taken into account. Neither glial fibrillary acidic protein nor myelin basic protein could be detected at the ages investigated.

Human microglia cultures: a powerful model to study their origin and immunoreactive capacity.

In this paper, we report that pure cultures of human microglia were obtained from long-term astrocytic cultures of human fetal brain. After five to six months and repeated cell passages, macrophage-like cells started to spontaneously form in vitro, so that in two to three weeks the whole culture was populated by them. These cells were grown up to over 50 passages in culture and analyzed for morphology, specific marker positivity, growth rate and major histocompatibility complex (MHC) antigen expression with or without gamma-interferon (IFN) stimulation. We found that, regardless of embryonic age of original cultures (10-15 weeks of gestation), cultures showed a remarkable homogeneity and purity and over 90 stained for typical microglial markers. Under basal conditions, two cell subpopulations similar to those described in vivo, we observed: the reactive 'ameboid' type and the resting 'ramified' one, the latter increasing with time in vitro and cell passages. Both cell subpopulations were capable of active phagocytosis and of high-rate proliferation. They spontaneously expressed low levels of MHC class II antigens, but were negative for MHC class I. Stimulation with gamma-interferon lymphokine upregulated the MHC class II expression as well as the MHC class I heavy chain form in ameboid, 'reactive' cells but not in the ramified ones. We also found that beta 2 microglobulin, already expressed in basal conditions, was dissociated from HLA A-B-C molecules in lymphokine-stimulated cells at early passages. The physiological significance of these data, as well as the possible correlation with in vivo ontogenetic modifications, are also discussed.

Correlation between P19 presence and MHC class II expression in human fetal astroglial cells cocultured with HTLV-I donor cells.

The possibility of a direct infection of human brain by HTLV-I, has been studied using an in vitro model. Human fetal astroglial cells were cocultivated with irradiated HTLV-I donor cell line MT-2, and assayed for the presence of HTLV-I core protein p19 after 1 week. Fifty-six per cent of GFAP positive astrocytes showed the viral core protein p19 and increased expression of Class II MHC antigens. Electron microscopy of astroglial cells exposed to HTLV-I revealed the presence of vacuoli-like structures containing viral core protein p19. Cell intermediate filament cytoskeleton was also disorganized. Even if this study does not provide direct evidence for virus replication inside astroglial cells, all these findings suggest that HTLV-I can indeed enter the cell and exert a cytopathic effect. Therefore the results of the present study are consistent with the hypothesis that astroglial cells could be involved in demyelination processes occurring in the HTLV-I associated neurological disorders, such as human associated myelopathy and tropical spastic paraparesis.

Assessment of the acrylic resin Technovit 7200 VLC for studying the gingival mucosa by light and electron microscopy.

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extra cellular matrix. At the ultrastructural level, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.

A procedure for preparing undecalcified and unembedded bone sections for light microscopy.

We have developed a procedure for light microscopic investigation of undecalcified and unembedded bone sections. Biopsy samples of human metatarsus and femur and rat femur were fixed in aldehydes and sectioned with a cutting machine equipped with a diamond saw blade. Free sections 100-150 microns thick, stained with toluidine blue and von Kossa, did not show artifacts following the cutting, and the spatial relations of mineralized and nonmineralized components remained intact. Compact and trabecular bone, bone marrow and all cell types appeared well preserved and easily recognizable. Our procedure provides a simple and rapid method for preparing bone sections which undergo no chemical treatment other than fixation. This method is a useful alternative to standard histological protocols for studying bone specimens.

[The morphological and histochemical characteristics of the interprismatic structures and the human enamel. A light microscopy study (corrected)]. / Caratteristiche morfologiche ed istochimiche delle strutture interprismatiche dello smalto umano. Studio al microscopio ottico [corrected].

Inter-rod structures of human enamel were investigated at the light microscopic level in order to define their morphology as well as the presence and distribution of both organic and inorganic molecules inside them. The histologic procedure employed in the present study allowed us to obtain 100 (microns) thick sections from permanent teeth without previous decalcification and paraffin or resin embedding. Three inter-rod structures were identified in the innermost area of the enamel on the basis of their morphologic aspect, namely the endings of dentinal tubules, the spindles and structures with a spherical or ovoidal shape never described before and by us denominated spheroids. Both spindles and spheroids were connected to one or more dentinal tubules which cross the dentin-enamel junction. Measurements performed on all the specimens led to establish the real dimensions of the inter-rod structures. Histochemical staining carried out with selective dyes showed that spindles and spheroids were quite alike similar to each other as far as chemical composition and molecule distribution are concerned. Both structures contained calcium salts, glycosaminoglycans, glycoproteins, but not collagen. The endings of dentinal tubules among the rods showed histochemical characteristics similar to those of spindles and spheroids. On the basis of the above findings, spindles and spheroids could have a dentinal origin, and so likely derived from the metabolic activity of odontoblasts during the tooth-germ development. Whether they are embryonic vestigia without a functional role or are receptors which transmit signals to nerve endings in dentin and pulp remains to be clarified.