Hyaluronate receptor CD44 is expressed by astrocytes in the adult chicken and in astrocyte cell precursors in early development of the chick spinal cord.

The role of the hyaluronate receptor, CD44, is well known in adult mammal astrocytes where it modulates neuron-glia interactions. However, no data exist regarding its expression in other vertebrates during their development. In order to detect the expression of CD44 in the chicken and its possible involvement in glial precursor migratory patterns during spinal cord development, a monoclonal antibody (MoAb) against the mammalian standard isoform, CD44-H, was used in immunohistochemical and immunoblot assays. With these methods, CD44 hyaluronate receptors were found on mature astrocyte membranes of adult chicken spinal cord. Astrocytes were identified using a MoAb against GFAP. During development, small clusters of CD44 labelled cells were seen lining the central canal starting from embryonic stage E10. These labelled cells were dispersed in the dorsal, lateral and ventral funiculi of the spinal cord in the subsequent stages. After stage E15, the CD44 labelled cells were identified as astrocytes because of their GFAP immunoreactivity. We conclude that CD44 receptors on immature astrocyte precursors should be considered as early astrocyte markers which have a possible role during cell migratory dispersal.

Phosphorylated and non-phosphorylated neurofilament epitopes are co-distributed in the fish Mauthner axon.

The neurofilament (NF) polypeptides of the fish giant Mauthner cell axons (MAs) and their degree of phosphorylation were investigated in the adult by means of immunoblot and immunohistochemical staining. Fasciculus longitudinalis medialis (FLM) axons of much smaller caliber, among which MAs run in the ventral spinal cord, were also analyzed in two teleost fish belonging to continuously growing species. To detect NF polypeptide subunits, commercially available monoclonal antibodies against mammalian NF-L (68 kDa), NF-M (160 kDa), phosphorylated (P) and non-phosphorylated (nP) NF-H (200 kDa) epitopes were used. These antibodies labelled bands of the identical molecular weight in fish cervical spinal cord total protein immunoblots. A peculiar NF composition of the MAs was observed, following immunohistochemical staining i.e. a low NF-M expression and a codistribution of P and (nP)-NF-H epitopes. Moreover, on the basis of immunoperoxidase staining in ultrathin longitudinal MA sections, we suggest that P NF-H are arranged in bundles, whilst (nP)-NF-H are likely to be free in the axoplasm. By contrast, FLM axons were found reactive with antibodies only against P NF-H. These results confirm that in carp and trout, NF have epitopes cross-reacting with monoclonal antibodies directed against the mammalian NF subunits. Furthermore, as regards the co-distribution of phosphorylated and non-phosphorylated NF-H epitopes in the M-cell axons, these might be considered as not yet completely mature axons taking into account that carp and trout belong to continuously growing species.

Non-specific pinocytosis by human endothelial cells cultured as multicellular aggregates: uptake of lucifer yellow and horse radish peroxidase.

We have analyzed the pattern of time-dependent and concentration-dependent incorporation of Lucifer Yellow CH (LY) and Horseradish Peroxidase (HRP) by human umbilical vein endothelial cells cultured on a non-adhesive substratum, where they they become organized into stable, multicellular aggregates. The data were compared with those previously obtained from low-density cultures of non-growing endothelial cells adherent to plastic. While the linear trend of the incorporation kinetics is preserved, the rate of uptake with both time and concentrations is highly dependent on the culture conditions, namely typology of cell-cell and cell-substrate interactions. An at least two-fold increase of the rate of uptake was observed with both markers in the aggregated cells. The extracellular concentration of LY required to saturate the binding capacity of the cell surface shifts from approximately 0.25 mg/ml, with the adherent cells, to approximately 0.5 mg/ml in the aggregated cells; the rate of uptake of three different forms of HRP shows, besides a sharp quantitative increase, also qualitative variations, testified by differential changes of their incorporation rates. These results are entirely consistent with the assumption that the association of the endothelial cells into multicellular aggregates increases the rate of pinocytic uptake by modifying the physicochemical properties of the cell surface, thereby increasing its differential affinity for the extracellular markers.

A quantitative assessment of non specific pinocytosis by human endothelial cells surviving in vitro.

Human umbilical vein endothelial cells have been assayed in vitro, 24 hrs. after plating, for non specific pinocytic activity. The culture conditions were designed to minimize the exogenous stimulations of pinocytosis, such as those possibly coming from mitotic induction and chemical and contact-dependent signaling. Two different markers were used: Lucifer Yellow CH (LY), and three different preparations of horseradish peroxidase, a multiple form (type II) composed of five different isoenzymes, and two purified acidic (type VIII) and basic (type IX) isoenzymes. The uptake of LY appears to depend on both fluid-phase incorporation and non specific adsorption to the cell surface, and it shows a linear monophasic dependence on time and a linear diphasic dependence on concentration. This probe is actively chased from the cells to an extent proportional to the amount incorporated. Therefore, the endocytic index obtained from the LY incorporation data is not a reliable estimate of extracellular fluid incorporation. The three different forms of HRP share an incorporation pattern linearly dependent on both time and concentration, consistent with the classical interpretation of a simple fluid-phase mechanism of intracellular uptake; however, the rates of uptake and chase activity of the pure isoenzymes are clearly different from that of the multiple form. The observed differences are related to possible local variations in the physicochemical properties of the cell surface, which may restrict the cell surface area suitable for fluid-phase uptake of differently charged macromolecular probes.

Biopsy diagnosis of a case of adult onset orthochromatic leukodystrophy. Clinical and brain biopsy findings.

We report the intra vitam histopathological findings on the brain of a female patient presenting an adult form of orthochromatic leukodystrophy. At 38 years of age the patient began to show progressive dementia and a pseudobulbar syndrome. The pedigree revealed an autosomal dominant pattern of inheritance. The CT scan showed a wide hypodensity of the anterior white matter. Biochemical investigations showed only a slight elevation of serum VLCFA and no alteration of urinary enzymatic activities. Cortical and subcortical biopsy specimens from the right frontal lobe showed: neuronal loss in the gray matter, accumulation of autofluorescent material within residual neurons and sudanophilic material within macrophages and astrocytes, sparing of axons. Electron microscopy showed lamination and fragmentation of the myelin and the presence of electrondense bodies and vesicular material into oligodendrocytes and astrocytes. We discuss the differential diagnosis of OLD forms with adult onset, namely between Löwenberg-Hill disease and the pure form of OLD with pigmented glial cells.

Morphometric analysis of Mauthner axon cytoskeletal components in adult and subadult fish.

A previous cytoskeletal analysis on trout MA during developmental stages demonstrated, during the subadult stages, neurofilaments (NF) as main components as expressed by the high values of neurofilament to microtubules (MT) ratio which was found to be of the order of 300:1. Since the MA cytoskeletal composition is not known in the adult fish, the MA cytoskeletal composition has been compared to other axons of much smaller diameter of the fasciculus longitudinalis medialis (flm) among which the MA run in the ventral spinal cord. The following parameters were measured on conventional electron microscopy in MA and flm axons cross sections micrographs by means of a computer linked graphic tablet (Apple II): axonal caliber, number of microtubules (MT), microtubular (MT/microns2) and neurofilament (NF/microns2) densities. The analysis of these parameters demonstrated that neurofilaments are the main architectural components in the adult and subadult fish MA and flm axons. However, MA cytoskeletal composition differs from the other flm axons because of its particular very high ratio of neurofilaments to microtubules. The inverse relationship of axonal caliber to microtubular density, previously found in the trout during developmental stages and suggested also for many other vertebrate species, was further confirmed for flm axons which, with calibers 10 times smaller than MA, exhibit a microtubular density 10 times larger.

Cytoskeletal components and calibers in developing fish Mauthner axon (Salmo gairdneri Rich.).

In developing axons of many vertebrates, microtubular density is inversely correlated with fiber caliber. It is suggested that microtubules are causally related to axonal caliber. For this reason, cytoskeletal analysis during development of the fish Mauthner axon, which displays a giant caliber, is of particular interest. The Mauthner axon originates from the Mauthner cell in the medulla and runs in the fasciculus longitudinalis medialis in the spinal cord. At embryonic, larval and postlarval stages in trout (Salmo gairdneri Rich.), the following parameters were measured on conventional electron micrographs of Mauthner axon cross sections; axonal caliber, number of microtubules per axons, and microtubular and neurofilament densities. Results at each stage point to an inverse correlation between axonal caliber (x) and microtubular density (y) expressed by the equation y = axb (R = 0.932). Furthermore, three periods of Mauthner axon development are identified on the basis of the cytoskeletal content: (1) embryonic; the Mauthner axon has small caliber with a high microtubule density, (2) elongation period (larval stages); the axon enlarges and a transient peak of microtubules, corresponding to the caliber increment, is observed, and (3) postlarval; the axon enlarges still further (greater than 500 microns 2) but has the lowest microtubular content. During this period neurofilaments are the main axonal component.

The response of ventral horn neurons to ricin. An experimental abiotrophy model.

The morphological alterations of anterior horn neurons of the spinal cord of rabbits produced by the administration of ricin conveyed via retrograde axonal flow are described. The differences with the primary response after axonal transection and the similarity with degenerative abiotrophic systemic lesions are discussed.

Preliminary histochemical and electron microscopic observations on the nervous system of hydra.

The nervous system of a primitive Coelenterate (Chlorohydra viridissima) has been studied using ultrastructural and histochemical techniques. The authors confirm the ultrastructural pattern of nerve cells described by Lentz and coworkers. Reserpine treatment fails to induce any reduction of catechol- and indoleamine content visible to histochemical observation. In vivo treatment with tetanus toxin does not induce behavioural changes and no specific binding of toxin is revealed by immunocytological analysis. This suggests that neuron tetanus toxin receptor sites are absent in hydra. Hydra nerve cells must therefore be considered as extremely primitive elements, which the Authors consider to support the hypothesis of neurons having originated as a gradual differentiation of myoepithelial cells, as proposed by Pantin (1956) and by Passano (1963).

The cerebrum of Dugesia gonocephala s.1. Platyhelminthes, Turbellaria, Tricladida. Morphological and functional observations.

Investigation of the cerebrum of Dugesia gonocephala s.1. using modern techniques has shown that the neurons of these animals situated at the bottom of the phylogenetic scale are basically the same as those of Vertebrates from the ultrastructural and molecular standpoints (tetanus toxin binding). There is no evidence of any biochemical glio-neuronal relationship, nor do the so-called supporting cells appear similar to the glial cells of the higher Vertebrates from this point of view.

Electron-microscopic visualization of binding of antibodies from sera of glioma patients on cultured glioma cells.

Several human cell lines (normal and neoplastic glia, cerebral metastases from adenocarcinoma, fibroblasts) were incubated with sera from patients with well and poorly differentiated glioma and with sera from healthy donors and then stained with PAP complex to define and localize the antibody reaction with cell surface antigens by means of electron microscopy. The sera of glioma patients proved to contain antibodies which bound the tumor-associated antigenic determinants on the cell membranes of gliomas and of cerebral metastases from adenocarcinoma in tissue cultures. Further, absorption testing of the reactive sera on normal brain, well-differentiated astrocytoma and cultured glioblastoma cells, together with cross-reactivity experiments suggests that at least two antigens or groups of antigens are expressed on the glioma cell surface: one shared by well and poorly differentiated glioma cells and the other by poorly differentiated glioma cells and the cells of cerebral metastases from adenocarcinoma.

Photosensitization and the nervous system in the planarian Dugesia gonocephala. A histochemical, ultrastructural and behavioral investigation.

Photosensitization phenomena may be induced in planarias by eosin and hematoporphyrin, and as a result, dopamine agonistic behavior ("screw-like hyperkinesia") is set up in the animal. Histochemical, ultrastructural and pharmacological investigations have shown that this hyperkinesia is of post-synaptic origin in eosin photosensitization, and of pre-synaptic origin in hematoporphyrin photosensitization. The authors suggest an hypothesis to explain the different activity of the two photosensitizers, and discuss the validity of the experiment with regard to human porphyria.

The pigmentary system of planaria. II. Physiology and functional morphology.

The pigmentary system of the planaria, Dugesia gonocephala s.l. (Platyhelminthes, Turbellaria, Tricladida), consists of granules contained in chromatophore cells distributed in the parenchyma tissue. The administration of MSH release-inhibiting Factor (M.I.F.) leads to an easily observable general decolouration of the animal due to the migration of the pigment granules towards the deeper-lying cell nucleus. In planarians bisected transversely through the pharyngeal region, the decolouration occurs only in the cephalic segment, and the caudal segment remains dark. When, however, the decapitated caudal segment regenerates a head region, a decolouration response occurs when exposed to M.I.F. The significance of these results is discussed, and an hypothesis on the hormonal regulation of the pigmentary system is proposed.

The pigmentary system of planaria. I. Morphology.

The pigmentary system of the planaria, Dugesia gonocephala s.l. (Platyhelminthes, Turbellaria, Tricladida), has been studied by light and electron microscopy. The system consists of granules contained in chromatophore-like cells embedded in the parenchyma. The cell processes penetrate between the muscle layers and extend to the sub-epidermal basal lamina. The nature of the pigment and the comparative anatomical significance of the chromatophore structure is discussed.

[Copper and nervous system. An experimental study (author's transl)]. / Cuivre et système nerveux. Etude expérimentale.

Sodium azide is known to produce alterations in mammalian copper proteins, thus rendering them unable to bind exogenous metal, which remains in the "labile pool" condition. Continuous administration of sodium azide at LD50 for 30 days causes copper accumulation in several tissues and even in the nervous system, with characteristic changes in neurones and glial cells, very much resembling the alterations observed in Wilson's disease. Dietary copper administration, on the contrary, though raising the level of tissue-bound metal, does not produce cellular damage. These findings allow us to suppose that sodium azide may alterate the coppper chelating proteins in the tissues, especially in the nervous system, thus causing the storage of cell-toxic "labile pool" metal. The pathogenesis of Wilson's disease and the problem of "pathoclisis" in the nervous system are debated.

Ultrastructural changes in human leukemic cell nuclei.

The ultrastructural changes of human leukemic cell nuclei have been investigated. Particular attention is paid to the alteration of the nuclear envelope and its constituents, i.e., the pores and the Zonula Nucleum Limitans which appear constantly involved in these pathologic processes. An alteration of the relationship between the components of the nuclear envelope and the chromatin itself may be responsible for the appearance of the most nuclear changes.