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.

A light microscopic investigation of the human midpalatal suture.

Biopsy samples of the human midpalatal suture, obtained from patients (age range: 10 and 30 yrs), were embedded in resin, cut with ultramicrotome and analyzed at light microscopy. The sutural connective tissue was made up of fibroblasts, collagen fibers, capillaries and nerve fibers. The sutural bone was made up of lamellar and bundle bone which alternated along both sides of the sutural connective tissue. No osteoblasts or osteoclasts were found, no signs of synostosis were ever detected. Our findings suggest that the lamellar bone replaces bundle bone when the suture is no longer involved in the growth of the palatal bones. The absence of bone remodelling shows that the sutures, at the time of sampling, were in a resting stage. Tissue architecture and cell types, so similar in samples from patients of such different ages, lead us to suppose that the sutures under examination are subject in time to very slow bone turnover.

A comparative light microscopic study of human midpalatal suture and periodontal ligament.

In order to provide information on the morphological features of both human midpalatal suture and periodontal ligament, bioptic samples were obtained from patients whose age was between 10 and 30 years. The samples were embedded in epoxy resin, sectioned, stained with toluidine blue and observed by light microscopy. The periodontal ligament and the midpalatal suture appeared very similar with regard to the histological characteristics of the connective tissue but differed in the nature of the bone tissue associated to it. The alveolar bone lining the periodontal ligament was a bundle bone whereas the bone lining the connective tissue of the sutures was both lamellar and bundle bone. The absence of osteoblasts, osteoclasts or undifferentiated cells in the sutures under examination suggests that they are subjected to a slow bone turnover during their life cycle. On the contrary, the presence of mesenchymal cells in the periodontal ligament supports the notion that these cells are precursors of osteogenic cells involved in the high rate of bone remodelling that characterizes the alveolar bundle bone. The present results are discussed taking into account physiologic and therapeutic aspects of the two structures under examination.

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.

[Necessity and validity of standard models for experimental preclinical evaluation of biomaterials. An example of biologic characterization of a hydroxyapatite-based implant material]. / Necessità e validità di modelli standard per la valutazione sperimentale preclinica dei biomateriali. Un esempio di caratterizzazione biologica di un materiale da impianto a base di idrossiapatite.

A large number of methods are now available for the preclinical screening of implantable materials concerning their biocompatibility and their ability to stimulate tissue formation. In vitro techniques represent a very useful tool, since this way we can realistically simulate the biological events which occur in vivo at the bone-implant interface. In the present study scanning electron microscopy and light microscopy observations were performed in order to assess the effect of an hydroxyapatite granulate on cell behaviour and morphology. Uptake of proteins to hydroxyapatite surface has been also investigated by comparing the amounts adsorbed after incubation with bovine serum albumin and bovine pancreaticamilase. According to our preliminary observation cells do not show signs of toxicity or inhibition of cell growth even after 14 days of co-culture with hydroxyapatite. Granules were covered by an uninterrupted cell layer by day seven. Even after two days micrographs show cells anchored and spread over the surface of the underlying granules, with a flattened and stellate shape. Such a morphology indicates a very high cellular activity, suggesting that the interaction with hydroxyapatite seriously increased metabolism. Measurements of protein adsorption on the hydroxyapatite surface show that changes in the size of particles affect the binding of proteins, while, in the case of granular hydroxyapatite, despite changes in size of granules, variations of protein adsorption were not observed, neither in relation to their different isoelectric point. Our preliminary results represent a good example of the opportunities presented by an experimental in vitro model.

Immunohistochemical study of the corpora cavernosa of the human clitoris.

The microarchitecture of the corpora cavernosa of the human clitoris was investigated by immunohistochemistry. The distribution pattern of the nerve network was demonstrated by S-100 and neuron specific enolase immunoreactivity. Vascular and nonvascular muscle cells were identified by desmin and/or vimentin expression, and fibroblasts and endothelial cells by vimentin immunoreactivity. The findings show that tissue organisation in the corpora cavernosa of the clitoris is essentially similar to that of the penis except for the absence of the subalbugineal layer interposed between the tunica albuginea and erectile tissue. This has functional implications, suggesting that the clitoral erection cycle differs from that of the penis.

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.

[The evaluation of acrylic resins for the study of nondecalcified human teeth with the light and electronic microscopes]. / Valutazione di alcune resine acriliche per lo studio dei denti umani non decalcificati al microscopio ottico ed elettronico.

Resin embedding of human teeth for light and transmission electron microscopic studies becomes difficult without previous decalcification. The limited and slow infiltration of the resin into hard tissues may cause problems during preparation and observation of the samples. Moreover the type of resin that is used may affect the morphologic preservation of both tissues and cellular elements. Recently there has been an increasing number of studies on the application of acrylic resins in light and electron microscopy, in order to overcome problems encountered with the use of epoxy resins still utilized in morphologic studies. We compared different acrylic resins (Technovit 7200 VLC, LR White, LR Gold, Bioacryl) in order to understand which one was more suitable for undecalcified human dental tissues under light and transmission electron microscope. Evaluation of such resins was performed using the following criteria: ease of cutting with ultramicrotome, soft and hard tissues infiltration, uptake of tissue stains for both light and electron microscopy, morphologic preservation and stability under electron beam. This study, carried out on the pulp area comprising predentin and dentin, showed excellent quality of Bioacryl and LR Gold, the two resins presenting, by far, the best results among all the different types tested. The optimal morphologic preservation obtained with such resins is indicated for light and electron microscopic studies, allowing their application in different fields of dental research.

[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.

Immunocompetent cell markers in human fetal astrocytes and neurons in culture.

During the past few years, evidence has accumulated that interaction with peripheral immune cells as well as immunoregulatory functions in the central nervous system (CNS) can be played by several types of brain resident cells. Since very little information is available in man, however, we investigated the presence of markers so far considered typical of immunocompetent cells in in vitro cultures of human fetal brain. Immunocytochemistry at the light, scanning, and transmission electron microscopic levels revealed positivity for a very restricted range of macrophage antigens in astrocytes, which, however, were incapable of phagocytosis. In particular, expression of the major histocompatibility complex-class II antigen HLA-DR was observed in the cytoplasm and on the cell surface of GFA-P+ astrocytes and increased with time in culture and cell passages. Among the T-lymphocyte markers tested, Thy.1 and CD4 were positive. Both neurons and astrocytes carried Thy.1 from early cell passages. Noteworthy was the presence of CD4, which serves as the receptor for AIDS virus, in neurons from the first 2 weeks, whereas astrocytes became positive after only 4-6 weeks. Even if most staining was in the cytoplasm, some was exposed on cell surface. Astrocytes were found positive for the B-lymphocyte marker CD21, the cellular receptor for Epstein-Barr virus, whereas CD24 was detected in both neurons and astrocytes. Both antigens are related to B-cell proliferation. Results are in favour of the hypothesis of human brain cells being actively involved in CNS immunological events.

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.

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.

Immunohistochemical study of neuropeptide Y-containing nerve fibers in the human clitoris and penis.

The occurrence and distribution of neuropeptide Y in the human clitoris and penis was investigated by light immunohistochemistry. Neuropeptide Y-containing nerve fibers were detected in the tunicae of arteries and veins as well as among trabecular smooth muscle. The distribution pattern of the peptide was similar in both organs although a higher density of immunoreactive nerve fibers was detected in the penis. The immunolocalization of neuropeptide Y was also compared with that of neuron-specific enolase, a neuronal marker which labels the entire nerve network. It is suggested that neuropeptide Y is involved in the physiology of the penis and the clitoris, affecting vascular and nonvascular smooth muscle activity.

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.

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.

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.