Selective tonicity-induced expression of the neutral amino-acid transporter SNAT2 in oligodendrocytes in rat brain following systemic hypertonicity.

Sodium-coupled neutral amino-acid transporter member 2 (SNAT2) belongs to the family of neutral amino-acid transporters. SNAT2 is encoded by the gene Slc38a2, whose expression was reported to increase in vitro in fibroblasts, endothelial and renal cells exposed to a hypertonic medium. SNAT2 tonicity-induced expression brings about cellular accumulation of amino-acid, which contributes to osmoadaptation to hypertonicity. Since brain osmoadaptation is observed in relationship to neurological disorders resulting from pathological osmotic imbalances in blood plasma, we have investigated, through immunocytochemistry, SNAT2 expression in brain of rats subjected to systemic hypertonicity. Following prolonged systemic hypertonicity (24 h), small, strongly immunolabeled elements were observed that were not present in sham-treated animals. They were evenly distributed in the gray matter, with a lower density in the forebrain and a higher density in the brain stem. However the highest density by far was observed in white matter, where they were frequently aligned in chain-like rows. These observations suggested an oligodendrocyte location that was further established by double immunofluorescent labeling, using the oligodendrocyte phenotypic markers 2'-3'-cyclic nucleotide 3'phosphodiesterase and carbonic anhydrase II. SNAT2-positive elements were found associated with oligodendrocyte cell bodies, while oligodendrocyte processes were devoid of labeling. A quantitative analysis performed in the cerebral cortex indicated that virtually all SNAT2-positive elements were associated with oligodendrocyte cell bodies and conversely that the overwhelming majority of oligodendrocytes showed SNAT2 immunolabeling. The tonicity-induced expression of SNAT2 was not observed following acute systemic hypertonicity (6 h). Our results suggest that the osmoadaptation of brain oligodendrocytes to hypertonicity relies upon amino-acid accumulation through the tonicity-induced expression of SNAT2. The possible significance of these findings in relationship to the selective loss of oligodendrocytes observed in osmotic demyelination syndrome is discussed.

In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant.

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

Large discrepancies in cellular distribution of the tonicity-induced expression of osmoprotective genes and their regulatory transcription factor TonEBP in rat brain.

Osmoprotective genes are tonicity-activated genes involved in cellular osmoadaptation to hypertonicity and considered to be regulated by a specific transcription factor called tonicity-responsive enhancer-binding protein (TonEBP). In the brain we had previously established that TonEBP was expressed and tonicity-induced in neurons only. Here we have compared in various brain regions of rats subjected to systemic hypertonicity, the cellular expression of TonEBP through immunocytochemistry and the cellular expression of osmoprotective genes, namely aldose reductase (AR), sodium-dependent myo-inositol transporter (SMIT), betaine/GABA transporter (BGT1) and taurine transporter (TauT), by in situ hybridization using non-radioactive digoxigenin-labeled riboprobes. In neurons where TonEBP was strongly tonicity-induced, AR-mRNA labeling was strongly increased in some subsets (e.g. hippocampus pyramidal cells, cerebellar Purkinje cells and neurons of the hypothalamic magnocellular nuclei) but remained undetectable in some other subsets (e.g. neurons in cerebral cortex). Tonicity-induced AR-mRNA labeling was observed only several hours after the tonicity-induced expression of TonEBP. SMIT-mRNA labeling was tonicity-induced as densely and evenly distributed dots in neuron poor regions (e.g. cerebral cortex layer I and hippocampus stratum lacunosum-moleculare). The tonicity-induced expression of SMIT-mRNA may thus occur in non-neuronal cells, presumably astrocytes, where TonEBP is neither significantly expressed, nor tonicity-induced. In neurons showing a strong tonicity-induced expression of TonEBP, no SMIT-mRNA labeling was observed. BGT1-mRNA and TauT-mRNA labeling could not be detected, even after systemic hypertonicity. The present work reveals large discrepancies between the cellular distribution of the tonicity-induced expression of osmoprotective genes and that of their regulatory transactivator TonEBP. Depending on the cell subsets and the osmoprotective genes, TonEBP may appear insufficient or conversely unnecessary for the tonicity-induced activation of an osmoprotective gene. Altogether our results show that brain cells, even from the same class, activate distinct osmoprotective genes through distinct activation processes to adapt to hypertonicity.

Differential cellular distribution of tonicity-induced expression of transcription factor TonEBP in the rat brain following prolonged systemic hypertonicity.

In a previous work performed on cerebral cortex and hippocampus we reported that tonicity-responsive enhancer binding protein (TonEBP), originally identified as a transactivator of osmoprotective genes involved in osmoadaptation of renal cells, was induced in neurons only, but to varying levels, following acute systemic hypertonicity. Whether or not this cellular specificity reflected a unique ability of neurons or a differential time course among brain cells for tonicity-induction of TonEBP was investigated throughout the brain in this study by subjecting the animals to prolonged systemic hypertonicity. In normal rats, TonEBP immunolabeling and TonEBP-mRNA in situ hybridization labeling showed a widespread, uneven and parallel distribution. TonEBP was expressed primarily in the cell nuclei of neurons, where it was heterogeneously distributed in a nucleoplasmic and a granular pool. In rats subjected to prolonged systemic hypertonicity, TonEBP labeling increased in the cell nuclei of neurons only. The tonicity-induced expression of TonEBP for a given cell group of neurons was rather uniform but varied greatly among neuronal cell groups and was positively correlated with the average size of the cell nuclei, as determined by quantitative analysis of digitized images. The detailed distribution of tonicity-induced expression of TonEBP is reported throughout the brain. In normal rats, a very minor proportion of non-neuronal cells, identified as a subset of astrocytes and possibly oligodendrocytes, showed faint nuclear immunolabeling, which however did not increase in hypertonic animals. Ependymocytes, capillary endothelial cells, and microglial cells showed no TonEBP labeling, even in hypertonic animals. Altogether our data indicate that neurons, albeit possibly to a varying extent, are the only brain cells able to use TonEBP-mediated processes for adaptation to a systemic hyperosmotic unbalance.

[The choroid plexuses: a dynamic interface between the blood and the cerebrospinal fluid]. / Les plexus choroïdes: une interface dynamique entre sang et liquide cephalo-rachidien.

The choroid plexuses form one of the interfaces that control the brain microenvironment by regulating the exchanges between the blood and the central nervous system. They appear early during brain development. Originating from four different areas of the neural tube, they protrude into the ventricular system of the brain. The choroidal mechanisms involved in the control of brain homeostasis include the structural properties of the epithelial cells that restrict diffusional processes, as well as specific exchange and secretion mechanisms. In addition to the anatomical and histological organization of the choroidal tissue, this review describes the mechanism of cerebrospinal fluid secretion which is the most studied function of the choroid plexus. Experimental evidence for an implication of the choroid plexuses in neuroprotective mechanisms and in the supply of biologically active polypeptides to the brain are also reviewed.

Individuality of Ag-selected and preimmune TCR repertoires.

We have combined flow cytometry and single-cell PCR to characterize the TCRBV repertoires selected by individual mice in a model CD8 response against a defined peptide/MHC complex (CW3 170-1 79/Kd). Ourresults established thatdifferent mice select individually distinct yet structurally similar CW3-specific repertoires. Repertoire selection appears to be flexible depending on the immunizing cell dose. Using a single-donor, matched-pair-recipient adoptive transfer strategy, we demonstrated that the CW3-specific TCR repertoires of normal mice are already distinct at the preimmune level. We combine our data with computer simulations to test models for the composition of an Ag-specific preimmune repertoire and its selection during an immune response.

Transplantation of fetal liver cells corrects accumulation of lipids in tissues and prevents fatal neuropathy in cholesterol-storage disease BALB/c mice.

The cholesterol storage disease (CSD) BALB/c mouse represents a model of the Niemann-Pick type C (NPC) disease. It is characterized by the accumulation of unesterified cholesterol within various tissues, resulting in fatal neurological lesions. Transplantation of 6x10(6) fetal liver cells from normal allogeneic CBA mice into lethally irradiated CSD mice led to reconstitution of the recipient mice with donor cells. As a result of this stable chimerism, deposition of lipids in tissues was decreased, neuropathy was prevented, and survival was significantly prolonged (over 190 days on average in transplanted mice versus 70 days in untreated mice). Foamy cells containing unesterified cholesterol, observed by filipin staining, were numerous in most tissues from untreated CSD mice; they were significantly fewer in CSD mice treated with fetal liver transplantation at the age of 36-45 days.

Vimentin and glial fibrillary acidic protein filaments in radial glia of the adult urodele spinal cord.

This work, based on Golgi impregnations, transmission electron microscopy and immunocytochemistry, demonstrates that the intermediate filaments found in the radial gliocytes of the adult newt spinal cord are both vimentin and glial fibrillary acidic protein (GFAP) structures. Gliocytes appeared as large, arboreous cells, with appendages extending peripherally. They were extensively immunolabelled with both anti-vimentin and anti-GFAP monoclonal antibody conjugates. Outstanding correspondence in cell configuration was found when Golgi-impregnated specimens were compared to the distribution of immunolabels. Electron micrographs showed cytoplasmic bundles of anti-vimentin decorated intermediate filaments occupying the radial projections. The presence of GFAP confirms the astroglial character of the radial glia in urodeles; the existence of vimentin suggests that the spinal cord of the adult animal retains immature astroglia, which should express enlarged capabilities of adaptation.

Transcription factor tonicity-responsive enhancer-binding protein (TonEBP) which transactivates osmoprotective genes is expressed and upregulated following acute systemic hypertonicity in neurons in brain.

Tonicity-responsive enhancer-binding protein (TonEBP) was initially identified as a transcription factor involved in adaptation of renal cells to hypertonicity by activation of osmoprotective genes encoding proteins for accumulation of compatible osmolytes. Since brain osmoadaptation is observed in relationship to neurological disorders resulting from pathological osmotic disbalances of blood plasma we have investigated through immunocytochemistry TonEBP expression in cerebral cortex and hippocampus of normal rat and rats submitted to an acute systemic hypertonicity or to a prolonged systemic hypotonicity. TonEBP-expressing cells were identified using double immunofluorescence and appropriate cell type markers. Their relative proportion was determined by quantitative image analysis. In normal rats TonEBP expressed primarily in neurons where it was strictly located in the cell nucleus but heterogeneously distributed into a nucleoplasmic pool and a granular pool. In animals made acutely hypertonic TonEBP labeling increased dramatically exclusively in the nuclei of neurons and reached a maximum within 1 h. In hypertonic animals TonEBP labeling covered the whole cell nucleus of virtually all neurons, appeared finely punctuated but was no more granular. Optical density of the labeling as determined by image analysis correlated linearly with the increased plasma osmolality. In animals made hypotonic for several days no conspicuous decrease of TonEBP labeling was observed. In normal animals a very minor proportion of non-neuronal cells showed a faint TonEBP nuclear labeling. This proportion increased slightly in hypertonic animals. Nevertheless these non-neuronal TonEBP-positive nuclei which belonged to oligodendrocytes and to a small subpopulation of astrocytes remained always very weakly labeled when compared with neuron nuclei. Brain capillary endothelial cells as well as microglial cells showed no TonEBP-labeling even in hypertonic animals. Our data demonstrate that in brain TonEBP is significantly expressed and tonicity-overexpressed in neurons and accordingly suggest that neurons only among brain cells accumulate compatible osmolytes through TonEBP-mediated activation of osmoprotective genes to adapt to acute systemic hypertonicity.

[Detection of circulating endothelial cells: a new diagnostic test of angina at rest]. / Détection des cellules endothéliales circulantes: un nouveau test diagnostique de l'angor de repos.

The diagnosis of spontaneous angina depends on the recording of per-critical electrocardiographic changes. There is no simple biological test to make its retrospective diagnosis. The attack is usually triggered by instability of an atheromatous plaque which fissures and liberates endothelial cells in the blod stream. The detection of these cells cold therefore be a biological sign of this condition. The technique of detection of circulating endothelial cells by immuno-magnetic method was used in 3 groups of patients admitted to hospital within 24 hours: group I comprised 11 patients with acute myocardial infarction, group II comprised 23 patients who had suffered from spontaneous angina with ST segment depression during the attack and significant coronary arterial stenosis, group III comprised 6 patients with chest pain for which coronary angiography is normal and provocative test of spasm is negative. Circulating endothelial cells were detected in all patients of group I (100%), in 18 of the 23 patients of group II (78%) and only in one of group III (18%). These results confer on this biological test for spontaneous angina a specificity and predictive positive value of 83 and 95% and a sensitivity and negative predictive value of 78 and 50%. Therefore the detection of circulating endothelial cells could be used as a simple and reliable test for retrospective diagnosis of spontaneous angina. The mediocre sensitivity and negative predictive value may be explained by a mechanism other than fissuration of atheromatous plaque in some cases of spontaneous angina.

Sialadenitis in nonobese diabetic mice: transfer into syngeneic healthy neonates by splenic T lymphocytes.

In the present study, we showed that both male and female nonobese diabetic (NOD) mice simultaneously develop frequent lymphocyte infiltrations in salivary submandibular glands (sialadenitis), very similar to those reported in the salivary glands of patients with Sjögren syndrome. These lesions were observed only in mice with pancreas exhibiting insulitis. The incidence of sialadenitis increased with the severity of insulitis. At the initial stage, small focal infiltrates were predominantly located around blood vessels. In older animals, inflammatory cells surrounded blood vessels and ducts. Most of the infiltrating cells proved to be L3T4+, whereas Lyt-2+ cells were comparatively few. Autoantibodies against duct epithelial cells were shown, but the degree of tissue invasion was not related to the existence of such antibodies. Antinuclear antibodies were also observed. These salivary gland infiltrates could be transferred in vivo to NOD neonates of both sexes by splenic T lymphocytes as well as by total spleen cells. These results suggest that sialadenitis in NOD mice is T cell mediated and may be related to insulitis.

Immunohistochemical localization of type IV collagen fibronectin and laminin in the juxtaglomerular apparatus of the rat kidney.

The juxtaglomerular apparatus (JGA) is a complex structure containing several components: the vessels, the extraglomerular mesangium and the distal tubule. These structures include cellular elements and an extracellular matrix (ECM). Collagenous (type IV collagen) and noncollagenous components of the basement membranes were studied. The localization of type IV collagen and of two extracellular glycoproteins (laminin and fibronectin) was investigated using immunofluorescent and immunoperoxidase labelled antibodies. Type IV collagen and laminin have the same localization on the JGA basement membranes. On the other hand, fibronectin is limited to the entrance of the glomerular stalk. On electron microscopy, type IV collagen is found in the basement membrane while fibronectin is restricted to certain areas of the extracellular matrix. These findings confirm data concerning the distribution of these three components in basement membranes and allow a better understanding of the histoarchitecture of the juxtaglomerular apparatus.

Immunologic phenotype of cultured endothelial cells: quantitative analysis of cell surface molecules.

Endothelial cells express a wide spectrum of surface molecules involved in multiple vascular functions. We quantitatively determined an extensive immunologic phenotype of endothelial cells through a large panel of antibodies directed against i) well-known endothelial molecules CD31, CD34, CD49b, e, f, CD51, CD54, CD55, CD62E and P, CD105, CD106, HLA class I and HLA class II; ii) molecules defined by monoclonal antibodies newly clustered during the 6th workshop of Human Leukocyte Differentiation Antigen (HLDA) CD109, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146 and CD147; iii) molecules defined by unclustered monoclonal antibodies. The expression of these molecules was quantified on human umbilical vein endothelial cells (HUVEC) cultured in resting conditions and after stimulation with IL-1beta (10 U/ml), TNF-alpha (10 ng/ml) and phorbol myristate acetate (60 ng/ml). Some molecules were constitutively expressed, and others were negative, which served to determine the basal phenotype. After cell stimulation, the molecules showed weak or strong expression modulation, leading to the definition of an activated phenotype. Changes in the kinetics and the amplitude of expression served to characterize poorly defined molecules and may be useful to determine their physiologic role. Also, we compared the phenotypes of endothelial cell lines EA.hy 926 and ECV 304 to that of HUVEC to assess their reliability as an endothelial cell model. Each cell line displayed a specific repertoire of molecules expressed at different levels, which could have significant implications for cell line behavior as endothelial cells.

Cyclosporine A nephrotoxicity: evidence for mesangial foam-cells in dogs.

Nephrotoxicity of cyclosporine A was studied in dog after 3 weeks' administration of the drug at high doses (20 mg/kg/day). Cyclosporine A concentrations measured in different organs revealed a high ratio between renal tissue and plasma. Renal histopathology showed non-specific tubular lesions and glomerular modifications. There were lipids in the mesangial cells which took on a foamy aspect. Ultrastructural study and lipid staining confirmed these findings, and non-specific esterase reaction revealed no macrophagic infiltration. Using anti-CSA antibodies it was not possible to demonstrate CSA in the mesangial cells. This alteration has never been described before in CSA therapy and its meaning is not clear; however, it does suggest that there occur modifications in the lipid metabolism or in the phagocytic function of the mesangial cells when cyclosporine A is administered over long periods.