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Cardiotrophin-1 (CT-1) is a recently isolated cytokine belonging to the interleukin-6 cytokine family. In the present study we show that CT-1 activates its receptor expressed at the surface of a human neural cell line by recruiting gp130 and gp190/leukemia inhibitory factor receptor β, as shown by analyzing their tyrosine phosphorylation level. Neutralizing antibody directed against gp130 and reconstitution experiments performed in the COS-7 cell line demonstrate that gp130-gp190 heterocomplex formation is essential for CT-1 signaling. Analysis of the subsequent activation events revealed that CT-1 induces and utilizes Jak1-, Jak2-, and Tyk2-associated tyrosine kinases, which are in turn relayed by STAT-3 transcription factor. Cross-linking of iodinated CT-1 to the cell surface led to the identification of a third α component in addition to gp130 and gp190, with an apparent molecular mass of 80 kDa. Removal of N-linked carbohydrates from the protein backbone of the α component resulted in a protein of 45 kDa. Our results provide evidence that the CT-1 receptor is composed of a tripartite complex, a situation similar to the high affinity receptor for ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) associates with an alpha subunit (CNTFRalpha) of the receptor complex to initiate signal transduction by facilitating heterodimerization of the gp130 transducing protein and the leukemia inhibitory factor receptor (LIFR) beta. CNTFRalpha is anchored to the membrane by a glycosylphosphatidylinositol linkage; however, a soluble form of the alpha subunit can still bind CNTF to recruit the signal transducing components of the receptor complex. In the present study we show that alanine substitution for residues Thr268 and Asp269 of the CNTFRalpha subunit results in a mutated receptor subunit (R3), which can bind CNTF with an affinity similar to that of the wild type CNTFRalpha but, when expressed as a soluble receptor subunit, lowers the binding of CNTF to its tripartite receptor. In addition, CNTFR3alpha inhibits the proliferation of the TF1 hematopoietic cell line triggered by CNTF plus soluble wild type CNTFRalpha but not by IL-6 or oncostatin M. Similarly, CNTFR3alpha specifically antagonizes the induction of gp130 and LIFRbeta tyrosine phosphorylation observed in response to CNTF and wild type soluble CNTFRalpha in the HepG2 hepatoma cell line, as well as the subsequent events leading to haptoglobin synthesis. Positions 268 and 269 of CNTFRalpha appear to be critical for its interaction with gp130 and LIFRbeta, whereby alanine substitution of the residues at these positions results in antagonism of the CNTF-induced response.

gp130 transducing receptor is involved in the formation of high affinity receptors for the cytokines of the interleukin-6 (IL-6) family. Recruitment of gp130 by IL-6 associated to its receptor leads to the dimerization of the transducing component. In the present study we did characterize the B-S12 monoclonal antibody raised against gp130 and able to elicit IL-6 type biological activities. B-S12 antibody triggered strongly the proliferation of TF1 and XG1 hematopoietic cell lines and was able to increase the synthesis of acute phase proteins in HepG2 hepatoma cell line. B-S12 also behaved as a synergistic factor with granulocyte-macrophage colony-stimulating factor for both proliferation and differentiation of CD34-positive hematopoietic cell progenitors. By using a symmetric enzyme-linked immunosorbent assay, allowing the detection of dimeric forms of soluble gp130, we found that addition of B-S12 to gp130 led to its dimerization. Analysis of the tyrosine phosphorylation events in gp130 and Jak kinase family members revealed that B-S12 quickly induced the phosphorylation of gp130 in a neural derived cell line, and that Jak1 and Jak2 were also recruited. In conclusion, we show that gp130 cross-linking with the B-S12 monoclonal antibody was sufficient to generate functional IL-6 type responses in hematopoietic, neural, and hepatic cells.

We have previously shown that pure oligodendrocyte (OL) secondary cultures derived from newborn rat brain, in which cells form myelin-like membranes, can be used as a model to investigate the putative role of growth factors in myelin repair. After disruption of these membranes by lysophosphatidylcholine (LPC), a 3 day treatment with 10 ng/ml basic fibroblast growth factor (bFGF) induced reconstruction of myelin figures, albeit less compacted than in untreated controls. Here we show that in LPC treated cultures: 1) bFGF can not prevent OL from LPC-induced cell death; 2) platelet-derived growth factor (PDGF) pretreatment although preventing some cell death does not improve recovery compared to delayed treatment; 3) PDGF is as potent as bFGF in terms of O-2A progenitor proliferation; 4) PDGF is far more effective than bFGF, inducing the reappearance of more myelin-like structures with a better compaction; 5) there is no potentiation between these growth factors; and 6) after withdrawal of bFGF the compaction of myelin figures partly increases. These results indicate that PDGF, probably by inducing O-2A progenitors to proliferate and then allowing them to differentiate into mature myelinating OL, is a better candidate than bFGF to participate in myelin repair mechanisms in the central nervous system.

Resume Nous avons teste l'action d'acides gras polyinsatures (AGPI) des series n-6 (acide arachidonique et acide γ linolenique) et n-3 (acide eicosopentaenoique et acide docosahexaenoique), fortement representes dans le systeme nerveux central (SNC), sur la croissance neuritique de cellules PC12. Un systeme d'analyse d'image pilote par ordinateur a ete utilise pour mesurer la longueur des neurites. Les cellules PC12 ont la propriete d'adopter une morphologie neuronale, avec emission de neurites, quand elles sont en presence du nerve growth factor. Nous montrons que ces AGPI induisent une croissance neuritique importante (+ 45 % comparativement au temoin sans AGPI) a une concentration de 10−7 M pour les AGPI n-6 et a 10−5 M pour les AGPI n-3. Cette action decroit dans le temps plus rapidement pour les AGPI n-6 (+ 10 % a J3) que pour les AGPI n-3 (+ 26 % a J3). Les resultats sont discutes selon l'importance de ces AGPI dans le SNC durant l'ontogenese et les maladies neurodegeneratives.

To investigate the mechanisms involved in graft survival, a rat cell line (PC12) that differentiates into sympathetic-like neurons by exposure to trophic factors has been grafted into rat striatum and hippocampus, two structures which differ in their amounts of trophic factors. Our results show that grafted PC12 cells behave differently depending on the area of implantation; they display a differentiated morphology in the hippocampus and proliferate as a tumor in the striatum. A qualitatively similar immunological reaction occurs in both structures, characterized by the invasion of T and B lymphocytes, macrophage-like cells and by the expression of major histocompatibility complex class I and II antigens around the graft.

Alzheimer's disease (AD) is a degenerative disorder of the central nervous system accompanied by several immunological disturbances and a number of common features features exist between AD and Down's syndrome (DS). High resolution two-dimensional electrophoresis of lymphocyte proteins demonstrates an actin abnormality in AD and DS: a double actin spot instead of the single spot observed in controls. This dual form was studied by pulse-chase experiments and seems to be related to extracellular factors which influence the post-translational modification of actin. These results agree with the immunological disturbances observed in AD and DS, and with the well established hypothesis that AD is a systemic as well as cerebral disease.

We had demonstrated that platelet-derived growth factor (PDGF) enhanced the reconstruction of myelin-like membranes after their disruption by lysophosphatidylcholine (LPC) in vitro. To investigate its role in vivo, a demyelinating lesion of the corpus callosum was induced in adult Wistar rats by a stereotaxic microinjection of 1 microl LPC, then 63 pairs of rats received either 1 microg PDGF, or its vehicle buffer which were injected above LPC. The effects of PDGF were significant after 2 weeks: the number of oligodendrocytes (OL) expressing 2',3'-cyclic nucleotide 3'-phosphodiesterase in the lesion increased by 49%, mature OL labelled by in situ hybridization for myelin basic protein-mRNA increased by 27% (P

Cardiotrophin-1 (CT-1) is a recently isolated cytokine belonging to the interleukin-6 cytokine family. In the present study, we show that CT-1 binds to hepatocyte-derived cell lines of rat and human origin with high (Kd = 600-800 pM) and low (Kd approximately 3-6 nM) binding affinities. Treatment of HepG2 cells with CT-1 resulted in the induction of tyrosine phosphorylation of both transducing receptor subunits, gp130 and LIF receptor, and this phosphorylation was completely inhibited by a neutralizing anti-gp130 mAb. Addition of CT-1 to HepG2 or H35 cell cultures induced a dose-dependent production of several acute phase proteins (haptoglobin, fibrinogen, alpha1-acid glycoprotein, alpha2-macroglobulin). Moreover, the use of a neutralizing mAb to gp130 in cultures of HepG2 cells grown in the presence of CT-1, inhibited the induction of acute phase protein secretion, indicating an absolute requirement of gp130 in the formation of a functional CT-1 receptor. Altogether, these results suggest that CT-1 could play an important role in the regulation of hepatocyte metabolism in inflammatory responses.

Oncostatin M (OSM) mediates its bioactivities through two different heterodimer receptors. They both involve the gp130-transducing receptor, which dimerizes with either leukemia inhibitory receptor beta or with OSM receptor beta (OSMRbeta) to generate, respectively, type I and type II OSM receptors. Co-precipitation of gp130-associated proteins, flow cytometry, polymerase chain reaction, and tyrosine phosphorylation analyses allowed the characterization of both types of OSM receptors expressed on the surface of different cell lines. It also allowed the detection of a large size protein, p250, that specifically associates to the type II OSM receptor components and that is tyrosine-phosphorylated after the activation peak of the gp130.OSMRbeta heterocomplex. The restricted expression of type I OSM receptor by the JAR choriocarcinoma cell line, and type II receptor by the A375 melanoma cell line, permitted the characterization of their signaling machineries. Both type I and type II OSM receptors activated Jak1, Jak2, and Tyk2 receptor-associated tyrosine kinases. The information is next relayed to the nucleus by the STAT3 transcriptional activator, which is recruited by both types of OSM receptors. In addition, STAT5b was specifically activated through the gp130.OSMRbeta type II heterocomplex. The signaling pathway differences observed between the common type I LIF/OSM receptor and the specific type II OSM receptor might explain some of the bioactivities specifically displayed by OSM.

Cardiotrophin-1 (CT-1) is a recently isolated cytokine belonging to the interleukin-6 cytokine family. In the present study we show that CT-1 activates its receptor expressed at the surface of a human neural cell line by recruiting gp130 and gp190/leukemia inhibitory factor receptor beta, as shown by analyzing their tyrosine phosphorylation level. Neutralizing antibody directed against gp130 and reconstitution experiments performed in the COS-7 cell line demonstrate that gp130-gp190 heterocomplex formation is essential for CT-1 signaling. Analysis of the subsequent activation events revealed that CT-1 induces and utilizes Jak1-, Jak2-, and Tyk2-associated tyrosine kinases, which are in turn relayed by STAT-3 transcription factor. Cross-linking of iodinated CT-1 to the cell surface led to the identification of a third alpha component in addition to gp130 and gp190, with an apparent molecular mass of 80 kDa. Removal of N-linked carbohydrates from the protein backbone of the alpha component resulted in a protein of 45 kDa. Our results provide evidence that the CT-1 receptor is composed of a tripartite complex, a situation similar to the high affinity receptor for ciliary neurotrophic factor.

Gp130 transducing protein was shown to be involved in the formation of the high affinity receptors for interleukin 6 (IL-6), interleukin-11 (IL-11), leukemia inhibitory factor, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1. In the present study we have characterized the functional properties of antibodies directed against this protein and identified a group of monoclonal antibodies able to antagonize the biological activities of all the cytokines belonging to the IL-6 cytokine family. The B-R3 pan-blocking antibody weakly interfered with the binding of the radiolabeled ligands (with the exception of OSM, whose binding was abrogated in the presence of B-R3 monoclonal antibody) but inhibited the gp130 homodimerization or its association with gp190/leukemia inhibitory factor receptor, as well as the subsequent tyrosine phosphorylation events. In addition we identified antibodies that were able to neutralize only one single cytokine of the IL-6 family. This was the case for the B-K5 antibody, which antagonized the binding of OSM to gp130 but did not interfere with the signals provided by the related cytokines triggering the proliferation of the TF1 erythroleukemia cell line or the induction of haptoglobin synthesis in the HepG2 hepatoma cell line. Similarly, we also characterized two additional antibodies B-P8 and B-P4, which inhibited the TF1 cell proliferation observed in the presence of CNTF and IL-11, respectively. B-P8 antibody only faintly interfered with the binding of the gp130-ligands and might modulate the signal transduction pathways. This study indicates that in addition to functional site(s) required by the whole family of IL-6 type cytokines to transduce the signal insight the cell, specific cognate functional sites were recruited by OSM, CNTF, or IL-11.

The implantation of polymer devices in the brain that release neuroactive drugs locally and in a controlled manner is gaining increasing interest. The fates and tissue reactions of poly(epsilon-caprolactone), ethylcellulose, and polystyrene microspheres, prepared by the solvent evaporation method, radiosterilized by gamma-irradiation, and stereotactically implanted in rat brain have been studied by routine staining and immunohistochemistry. During the first few days after implantation, a nonspecific astrocytic brain tissue reaction was observed along with a macrophagous-microglial cell reaction typically found following any damage in the central nervous system, except in the presence of certain foreign body giant cells. Nine months into the experiment, microspheres appeared to be engulfed by histiocytic cells. The microsphere cluster was surrounded by a sheath composed of collagen and astrocytic cells. No necrosis was observed, suggesting the absence of toxicity. In some animals, however, an hydrocephalus developed as a result of obstruction of the medial ventricle by some microspheres.

An inflammatory reaction, essential for defence against infection and for wound repair, may also induce irreversible tissue damage. It appears that the central nervous system has developed its own immunosuppressive strategy in order to limit the destructive effects of inflammation. To clarify this point, we have characterized in one unique model of inflammation induced in the rat by intracerebral lipopolysaccharide injection the kinetics of the inflammatory reaction, the participation of immunitary and glial cells and of three growth factors. Among these molecules, brain-derived neurotrophic factor mRNA expression was found decreased following LPS injection. No striking differences were observed in the brain parenchyma after stab lesion or inflammatory lesion apart from an increase in the number of monocytes/macrophages recruited early to the lesion area. Macrophages were later accumulated around the lesion when astroglia and microglia reactions occurred. Some of the macrophages and microglia expressed major histocompatibility complex class II antigens on their surface whereas no T or B lymphocytes were observed in the brain parenchyma. However, a subpopulation of CD3- and CD4-negative CD8-positive cells, likely natural killer cells, was observed around the lesion site; this recruitment was inhibited by the highest dose of LPS. This study therefore supports the hypothesis of a suppression of some aspects of cell-mediated immunity in the brain, mechanisms which need to be further characterized.

The discovery of reliable peripheral markers would be of great interest for the diagnosis of Alzheimer's disease. Interestingly specific lesions of Alzheimer's disease were found in the olfactory areas of the brain. The loss of the detection and the identification capabilities in olfaction suggest a defect in the olfactory mucosa. The ultrastructural study of biopsy of olfactory mucosa from patients suspected to suffer of Alzheimer's disease revealed a degeneration of sustentacullar and olfactory cells as well as an architectural disorganization. No neuropathological characteristic lesion such as Paired Helicoidal Filaments or amyloid fibrils could be evidenced. Immunohistochemical analysis achieved with a polyclonal antiserum raised against A4 protein (fragment 1-28) showed a specific staining in the sustencellular and of the outer part of the olfactory epithelium corresponding to the mucus area, whereas appropriate controls were negative. Our study raised the question of the amyloid protein origin in the olfactory mucosa. Analysis of some more patients will enable us to determine the diagnostic value of this study in Alzheimer's disease.

We have tested the action of three n-6 polyunsaturated fatty acids, either free or in the form of ethyl esters, on the neurite outgrowth in two neuronal models: a rat pheochromocytoma cell line (PC12) and embryonic chick motoneurons, after 7 days in culture. An inverted microscope coupled with the ‘VIDS 4’ software was used for measuring the neurite length. Free fatty acids were found to be cytotoxic at 10 −3 M and the maximal increase of the neurite length was obtained at 10 −5 M. In contrast, fatty acids in the form of ethylesters were not cytotoxic and at 10 −3 M induced the maximal increase in the neurite length. This increase (1.2 to 2 fold) significantly differed from the control and was dose-dependent. These results were discussed in relation to the action of fatty acids on enzyme activation and membrane fluidity.

The therapeutic application of neuroactive molecules in neuroscience is limited, due to the problems posed by the administration of these drugs (peripheral metabolism, systemic effect and passage of the blood-brain barrier). One solution is the implantation in the brain of biodegradable polymer devices with controlled release of a neuroactive drug. The biodegradation and tissue reaction of the copolymer poly(D,L-lactide-co-glycolide) microspheres prepared by the solvent evaporation method, radiosterilized and stereotactically implanted in the rat brain were studied by routine staining, immunohistochemistry and transmission electronic microscopy. The brain tissue reaction observed was a non-specific astrocytic proliferation and a macrophagous-microglial cell reaction, typically found following damage to the central nervous system. Some foreign-body giant cells were observed and the inflammatory and macrophagous reaction decreased dramatically after 1 month and almost ended after 2 months when the microspheres were totally biodegraded. The copolymer poly(D,L-lactide-co-glycolide) microspheres may be considered biocompatible to the brain tissue.

Regeneration of central nervous system (CNS) axons has been studied in the cholinergic septo-hippocampal system using various 'bridges' able to support fiber growth. In this study, a pure Schwann cell (Sc) suspension labeled with bisbenzimide (Hoechst 33342) was grafted in the lesioned septo-hippocampal pathway. At 2 weeks post-grafting, acetyl-cholinesterase (AChE)-positive fibers invaded the graft and grew in association with the Hoechst-labeled Sc, some of which expressed the low-affinity nerve growth factor receptor (NGF-R). At 2 months and 4 months post-grafting, the dorsal hippocampus was reinnervated with an apparently normal innervation pattern. Analysis of fiber growth in the hippocampus at four months post-grafting revealed a significant increase of reinnervation in the grafted animals (2 mm) compared to the non-grafted ones. No difference was observed in the number of cholinergic septal neurons expressing the NGF-R. These results demonstrate that a Sc suspension grafted into the lesioned septo-hippocampal system, integrates well into the host tissue, and supports axonal CNS outgrowth, implying that Sc by themselves provide an adequate environment for regeneration to occur.

The discovery of a reliable peripheral marker would be of a great interest for the early diagnosis of the Alzheimer's Disease. The olfactory deficit and the major histologic changes of the olfactory-related areas of the brain occurring during this disease raised the possibility that the olfactory epithelia could be one of the way of entry of a possible process that still has to be identified. We have developed an instrument and a technique of biopsy of the human olfactory mucosa to search for the presence of characteristics lesions on patients suffering of an Alzheimer's Disease. These small specimens have been prepared for electronic microscopy. The ultrastructural study of a sample of olfactory mucosa has been realised in 9 cases (5 Alzheimer's-4 controls) revealing in 4 patients suspected of an Alzheimer's Disease a complete architectural disorganisation with a destruction of the dendrite of the olfactory cells and a severe degeneration of the sustentacular cells. We did not find any characteristic changes such as Paired Helicoidal Filaments or amyloid fibrils. These results do not presuppose of their eventual presence at a precocious stage of the disease. Further ultrastructural and immunochemical studies carried out with patients at various stages of the disease are necessary in order to confirm this hypothesis.

Recent advances suggested that proteases and their inhibitors could be implicated in the genesis and/or maturation of insoluble deposits associated with Alzheimer's disease (AD). This study was designed to measure the level of α1-antichymotrypsin (ACT) and α1-antitrypsin (AT) in cerebrospinal fluid (CSF) of patients with AD and nondemented humans at various ages. Our analysis failed to demonstrate a significant relationship between inhibitor content and disease. However, a positive correlation was observed between age and the ACT level for the normal control group. Such observation suggests a specific association of ACT with the mechanisms of brain aging.

Intravascular malignant lymphomatosis (IML), so-called malignant angioendotheliomatosis, was found in lumbosacral spinal cord and nerve roots of a 78-year-old women who died one month after the onset of symptoms. With regard to the majority of the 37 reviewed neurological cases in the literature, this report is unusual in that the disease was exclusively localized in the spinal cord and systemic involvement was absent. The usual clinical hallmark of the disease is a subacute dementia or encephalopathy, often associated with focal neurological signs, culminating in death within several months. The pathological features of IML characteristically include multiple small foci of necrosis of the whole brain, caused by occlusion of small vessels by noncohesive neoplastic cells and secondary changes of the vascular wall. All organs may be involved, especially the skin and the adrenals, sometimes with tumoral formations. Despite the fact that lymphoid tissues are usually spared, recent reports and the present case strongly suggest a lymphoid rather than endothelial origin of the malignant cells. The pathogenesis of this mainly intravascular lymphoma remains obscure.

A 70 year-old woman presented with a progressive supranuclear ophthalmoplegia, with "apraxia" of eyelid opening, axial akinesia and dementia. CT scan showed a mild cortico-subcortical atrophy and there was a high level of immunoglobulins, with an oligoclonal pattern, without cell reaction in the CSF. The patient died two years after the onset. Post-mortem examination, limited to CNS, showed subacute encephalitis confined to the tectal, pretectal, subthalamic areas and to Ammon's horns. These changes and their location were strongly suggestive of polioencephalomyelitis with or without cancer, in which such a prevalent midbrain involvement has been exceptionally described.

The mouse monoclonal antibody ME 101 raised against human peripherin, an intermediate filament protein (IFP) specific to well defined neuronal populations, recognizes all the major classes of vertebrate IFP in immunoblotting assays. Desmin, GFAP, vimentin, peripherin and the lightest neurofilament protein (NF-L) were cleaved into carboxy- and amino-terminal halves by N-chlorosuccinimide at their unique trytophan residue. Whereas the antibody directed against the epitope common to every IFP (intermediate filament antigen or IFA) and located on the carboxy-terminal end of the rod domain recognizes the carboxy-terminal half, the ME 101 antibody, as the present study illustrates, recognizes specifically the amino-terminal half. From the amino acid sequence data of IFP, it is deduced that the cognate epitope is localized on the amino-terminal part of coil la.

Using direct immunofluorescence on 5-µm sections from frozen brain specimens obtained shortly after death, the different complement factors of the classical pathway (C1, C3, C4) were demonstrated in the brain amyloid lesions of four patients who had Alzheimer’s disease.