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In general, the metabolic roles played by vitamins are well known, but to date less is known about the anatomical localization of these substances in the mammalian brain. Using a highly specific antiserum directed against vitamin C, the distribution of vitamin C-immunoreactive structures in the monkey (Macaca fascicularis) brain was studied. Antibody avidity and specificity were checked using the ELISA method, and it was estimated that antibody avidity (IC50) was 10-7 M for anti-conjugated ascorbic acid antibodies. Thus, using an immunoperoxidase technique that increases the immuno-histochemical reaction product, no immunoreactive fibers containing vitamin C were found in the monkey brain, but a moderate density of vitamin C-immunoreactive cell bodies was observed in the primary somatosensory cortex (postcentral gyrus). These cell bodies were located in layers III-IV and were generally small, triangular, or round, showing no, or only one to two, short visible processes, or long dendrites. The anatomical distribution of vitamin C and those of other vitamins (folic acid, thiamine, riboflavin...) in the monkey brain are compared and discussed. The restricted distribution of vitamin C in the monkey brain suggests that this vitamin could be involved in very specific physiological mechanisms (e.g., antioxidative, neuromodulation, neuroprotection).

Endotherapia is a new therapeutic approach against chronic diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and rheumatoid arthritis. It combines the evaluation of circulating antibodies directed against specific self-antigens and self-antigens modified by free radicals. Here, we review the literature concerning this topic. In addition, our biological assays have allowed us to follow up the evolution of chronic diseases along time, checking remission phases. For more than 25 years, our scientific group has suggested that the chronicity of diseases may be due to nonpathological bacteria living in different biotopes. Endotherapia is the result of immunopathologic work addressing chronic incurable diseases with a multifactorial etiology. Several genes are susceptible to toxic environmental immunologic agents. Thus, the intestinal ecosystem plays an important role in these mechanisms. Therapy is a "tailor-made" combination (MG1/RA, GEMSP) of the small molecules (e.g., vitamins...) linked to poly-L-lysine (PLL). Each individual linkage to PLL offers great advantages owing to the chemical characteristics of PLL (e.g., non-immunogenic), increasing the half-life of the active PLL derivatives. Clinical trials have enabled us to confirm very good safety. Our results confirm the efficacy of the approach and we believe that it could be very promising for chronic diseases.

Introduction L'identification d'anticorps circulants dans la sclérose latérale amyotrophique (SLA), la maladie d'Alzheimer et la sclérose en plaques (SEP) a fait l'objet de nombreuses études sans apporter les arguments décisifs pour leur intérêt. Grâce à nos travaux antérieurs sur les sérums de patients atteints de maladie chronique, nous avons trouvé des anticorps circulants dirigés contre des néoantigènes liés à des évènements radicalaires. Les molécules du soi modifiées (néo-épitopes) ou démasquées ou synthétisées en grande quantité lors de certaines pathologies sont capables de déclencher une réponse immunitaire, après leur liaison aux protéines intracellulaires. Dans la présente note, nous rapportons des résultats obtenus par la même méthodologie sur des sérums de malades atteints d'affections citées plus haut. Pour la première fois il a été évalué si des réponses spécifiques étaient induites contre des néoantigènes issus de la voie l'indoleamine 2,3 dioxygénase (IDO), transformant le tryptophane en différents composés. De plus, nous rapportons la comparaison des réponses obtenues sur les acides aminés modifiés par le monoxyde d'azote (NO) et le NO2. Nous avons synthétisé in vitro des conjugués et recherché dans le sérum de malades la présence d'anticorps qui sont le reflet de ces modifications moléculaires. Cette approche permet de visualiser de façon indirecte les désordres moléculaires apparaissant dans l'évolution de ces maladies et donne des informations pertinentes sur les mécanismes physiopathologiques à l'origine de ces perturbations. Méthodes Dans le cadre de cette étude, nous avons utilisé 34 sérums de patients atteints de la SLA (34-62 ans), 48 sérums de patients atteints de la maladie d'Alzheimer ( > 60 ans), 23 sérums de patients atteints de la SEP de la forme rémittente (34-45 ans) et 20 patients atteints de la SEP de la forme progressive (25-78 ans). Une population de 19 sérums contrôle est appariée pour chaque pathologie. La synthèse des dérivés du tryptophane se fait par fixation de : la kynurénine, la 3 hydroxykynurénine, l'acide kynurénique, l'acide quinolinique, l'acide quinaldique, l'acide xanthrénique, l'acide picolinique, l'acide anthranilique et l'acide 3 hydroxyanthranilique, sur de l'albumine bovine (SAB) par une réaction à la carbodiimide. Les acides aminés et dérivés (tyrosine, phénylalanine, citrulline, tryptophane, asparagine, créatine, cystéine, histidine, arginine et albumine) sont liés à SAB par une réaction au glutaraldéhyde (G-SAB), puis nitrosylés en milieu acide, à l'exception de la NO2-tyrosine-G-SAB. Après dialyses, les conjugués sont adsorbés à des concentrations optimisées sur plaques MaxiSorb. La détection d'anticorps circulants sériques dirigés contre les dérivés du tryptophane et les néoantigènes nitrosylés ou nitrés est réalisée par la technique ELISA. Résultats Par une analyse immunochimique des réponses humorales, nos travaux ont permis l'identification d'anticorps circulants spécifiques dans le sérum de malades atteints de la SLA, de la maladie d'Alzheimer et de la SEP. Ces immunoglobulines rencontrées sont principalement d'isotypie A dans la SLA, la maladie d'Alzheimer et la SEP progressive. Par contre, dans la SEP de forme rémittente, elles sont d'isotypie M. Nous avons trouver des profils spécifiques pour chacune des pathologies étudiées. Conclusion Notre approche méthodologique de ces maladies chroniques a permis d'aborder de façon indirecte, par l'identification des anticorps circulants, des mécanismes mettant en jeu l'hyperproduction de NO et de NO2 mais aussi l'activation de la voie IDO. Il est connu que certains de ces composés résultants de la transformation du tryptophane, sont neurotoxiques (le 3 hydroxykynurénine et l'acide quinolinique).

In order to evaluate the effects of a new potential therapy in Multiple Sclerosis, we used a chronic Experimental Autoimmune Encephalomyelitis (EAE) model induced with a mixture of Myelin Oligodendrocyte Protein and Mycobacterium tuberculosis in 10 weeks old Lewis 1A female rats. A potential drug (GEMSP), was constituted of fatty acids linked to poly-lysine (PL), antioxidants linked to PL, scavengers linked to PL and amino acids linked to PL. Experiments were conducted on three groups: 1) rats with an EAE treated with GEMSP (40mg/kg/day); 2) rats with an EAE treated with NaCl (positive control); 3) rats without EAE (negative control) and treated with NaCl. The three groups were treated under the same conditions; it means that all the groups received two subcutaneous injections per day of 250µl of its respective solutions (six days a week, starting 9 days after induction of EAE). The usual clinical score (0-4) was evaluated six days a week from the first day until the day of perfusion (both included). Crises and the clinical scores were completely abolished by this potential therapy drug (GEMSP). Moreover, indirect immunocytochemical techniques were applied to evaluate leucocyte infiltration and demyelination in the rat CNS of the three groups mentioned above. Thus, the panleucocytic marker CD 45 shows a very large infiltration in the animals perfused belonging to the positive control group in the first crises; but at the end of the experiment (two months later) animals remained with clinical signs but in general no large infiltrations were found, since only some animals of the positive control group presented large infiltrations in some spinal cord levels and in the cerebellum. The anti-Myelin Basic Protein antibodies showed a lower immunoreactivity in the positive control group than in the other two groups (GEMSP and negative control). Moreover, data reported from histological techniques (Luxol Fast Blue) showed a disorganization in some levels of the spinal cord white matter only in the positive control group. Our studies clearly show that: 1) GEMSP was very active on leucocyte infiltration; 2) GEMSP may be effective on blood brain barrier rendering this less permeable to leucocyte infiltration, decreasing the leucocyte infiltration per se, and could play an immunomodulator role; 3) GEMSP abolish EAE crisis; 4) GEMSP seems to have a myelin protection

The first objective of this study was to identify specific circulating antibodies directed against tryptophan derivatives (TD), NO and NO2- modified amino acid residues in patient sera of amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD), remitting relapsing multiple sclerosis form (RR-MS) and secondary progressive form (SP-MS). The second objective was to determine immunoglobulin classes of identified circulating antibodies. For the antibody evaluation, we synthesized TD conjugates, NO- and NO2-conjugates in order to mimic metabolites induced during the pathogenic processes. The TD antigens were: tryptophan, kynurenin, 3OH-kynurenin, kynurenic acid, picolinic acid, xanthurenic acid, anthranilic acid, 3OH-anthranilic acid, quinolinic acid and quinaldic acid, linked to bovine serum albumin (BSA) using carbodiimide coupling reaction. Then, conjugates were purified and the coupling ratios were evaluated. NO- and NO2-conjugates were the following amino acid residues: nitrotyrosine, tyrosine, phenylalanine, citrulline, tryptophan, asparagine, creatin, cysteine, methionine, histidine, arginine, linked to BSA via glutaraldehyde reaction. After a reduction step and purification, a nitrosylation with NaNO2 was done, except for nitrotyrosine conjugate. All conjugates were coated on well plates for identification of circulating antibodies. High levels of circulating antibodies to TD conjugates, NO- and NO2-conjugates were found in ALS, AD, RR-MS, SP-MS in comparison of healthy control sera, using ELISA assays. Immunodetection of IgG, IgM and IgA classes directed against TD conjugates, NO- and NO2- amino acid residue conjugates was done. These studies shown that: 1) specific circulating antibodies directed against TD, NO- and NO2- modified components were found for each pathology with a specific pattern. The identification of specific antibodies indirectly reveals the stimulation of enzymatic and immunologic mechanism disorders associated with these diseases; 2) specific profiles of immunoglobulins were also observed for each disease. The identification of differential isotype classes to these neoantigens opens new perspectives for a better understanding of these autoimmune and neurodegenerative processes

International audience; Experimental Autoimmune Encephalomyelitis (EAE) was induced for the evaluation of a new drug candidate (GEM-SP) for multiple sclerosis. Using immunocytochemical techniques with a pan-leukocyte marker, "anti-CD 45", differential leukocyte infiltration was compared in different experimental groups: 1) EAE-immunized rats treated with GEM-SP; 2) EAE-immunized rats treated with NaCl; 3) EAE-immunized rats treated with free constituents (not linked to inert carrier protein) and the inert carrier protein of GEM-SP. The results were conclusive: a very high degree of infiltration was observed in groups 2 and 3. Compared with these, group 1 showed a very poor leukocyte infiltration. Thus, the effect of GEM-SP against leukocyte infiltration was very strong, suggesting a decrease of the blood brain barrier permeability. Moreover, the same composition of GEM-SP (non-linked) was poorly active against leukocyte infiltration. The effect of GEM-SP therefore on the blood brain barrier appears to be very effective, rendering it less permeable to leukocyte infiltration and decreasing leukocyte infiltration per se and/or it is also possible that GEM-SP could play an immunomodulator role. The present results suggest GEM-SP as a new potential drug candidate for the treatment of multiple sclerosis

International audience; Cette étude porte sur l'évaluation d'anticorps présents dans le sérum d'une population de 42 patientes ayant un cancer du sein dont le diagnostic a été établi il y a moins de 6 mois. Les échantillons ont été testés sur plus de 30 antigènes à l'aide de tests immuno-enzymatiques. Plus de 80 % des malades ont des titres élevés, voire très élevés en antiphosphatidylinositol (PtdIns) d'isotypie G. Ce résultat confirme nos données déjà publiées sur des patients porteurs de tumeurs malignes. Nous avons également trouvé des titres très élevés en anticorps d'isotypie A dirigés contre des préparations membranaires d'entérobactéries. Les paramètres cliniques et histologiques ont été mis en relation avec les titres des différents anticorps. On ne trouve pas de relation significative entre la taille tumorale, la présence ou non de ganglions, le type histologique et les titres en anti-PtdIns d'isotypie G. Quel que soit la valeur de ces paramètres, les anti-PtdIns sont toujours élevés. En revanche, les anticorps anti-résidu malondialdehyde, anti-NO-cystéine. anti-NO-phenylalanine, anti-NO-créatine, anti-NO-histidine d'isotypie M sont statistiquement inférieurs aux titres des contiôlcs, pour les patientes ayant des tumeurs T2. Les titres en anticorps dirigés contre les préparations membranaires de Klebsiella pneumoniae et Morganella enoigonji sont statistiquement plus élevés lorsque la tumeur est un carcinome infiltrant canalaire peu différencié par rapport au même type histologique plus différencié. Cette étude ouvre des perspectives nouvelles pour le suivi biologique dans le cancer du sein