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International audience; Background: The revolution in cancer genomics shows that the dominant mutations are CG->TA transitions. The sources of these mutations are probably two host cell cytidine deaminases APOBEC3A and APOBEC3B. The former in particular can access nuclear DNA and monotonously introduce phenomenal numbers of C->T mutations in the signature 5'TpC context. These can be copied as G->A transitions in the 5'GpA context.Methods: DNA hypermutated by an APOBEC3 enzyme can be recovered by a technique called 3DPCR, which stands for differential DNA denaturation PCR. This method exploits the fact that APOBEC3-edited DNA is richer in A+T compared with the reference. We explore explicitly 3DPCR error using cloned DNA.Results: Here we show that the technique has a higher error rate compared with standard PCR and can generate DNA strands containing both C->T and G->A mutations in a 5'GpCpR context. Sequences with similar traits have been recovered from human tumour DNA using 3DPCR.Conclusions: Differential DNA denaturation PCR cannot be used to identify fixed C->T transitions in cancer genomes. Presently, the overall mutation frequency is ∼10(4)-10(5) base substitutions per cancer genome, or 0.003-0.03 kb(-1). By contrast, the 3DPCR error rate is of the order of 4-20 kb(-1) owing to constant selection for AT DNA and PCR-mediated recombination. Accordingly, sequences recovered by 3DPCR harbouring mixed C->T and G->A mutations associated with the 5'GpC represent artefacts.

International audience; Inorganic arsenic, a carcinogenic environmental contaminant, exerts immunosuppressive effects on human T lymphocytes. In particular, interleukin-2 (IL2) secretion and T cell proliferation are reduced when peripheral blood mononuclear cells (PBMC) from individuals chronically exposed to arsenic are stimulated ex vivo with lectins such as phytohemaglutinin (PHA). However, it is not clear whether the metalloid directly acts on T cells or blocks monocyte-dependent accessory signals activated by PHA. We report that in vitro pre-treatment of PBMC with sodium arsenite (NaAs) reduces IL2 secretion and T cell proliferation induced by PHA, but does not prevent expression of monocyte-derived cytokines (IL1, IL6, TNFα) functioning as lymphocyte-activating factors. In addition, we found that NaAs delays induction of IL2 and IL2 receptor α chain (IL2RA) mRNA levels in human primary isolated T cells activated by PHA. Kinetic analysis showed that NaAs pre-treatment first inhibits, but thereafter markedly increases, induction of IL2 and IL2RA mRNA when T cells are stimulated with PHA for 8 h and 72 h, respectively. We conducted whole genome microarray-based analysis of gene expression in primary T cell cultures derived from independent donors. NaAs systematically and significantly up-regulated a set of 35 genes, including several immune and stress genes, such as IL13, granulocyte-macrophage colony stimulating factor, lymphotoxin α and heme oxygenase-1 (HO-1). Up-regulation of HO-1, a stress and immunosuppressive protein, was rapidly detectable, both in T cells and in PBMC treated with NaAs. Inhibition of the immunosuppressive activity of HO-1 in PBMC however failed to prevent NaAs-dependent inhibition of T cell proliferation induced by PHA. Our findings demonstrate that, at least in vitro, inorganic arsenic acts directly on human T cells and impairs their activity, probably independently of HO-1 expression and monocyte-related accessory signals.

International audience; We recently determined in rats that iontophoretic application of bicuculline or gabazine [two GABAa antagonists] and kainic acid (a glutamate agonist) in the sublaterodorsal nucleus (SLD) induces with a very short latency a paradoxical sleep-like state. From these results, we proposed that GABAergic and glutamatergic inputs to the SLD paradoxical sleep (PS)-executive neurons gate the onset of PS [R. Boissard et al. (2002) Eur. J. Neurosci., 16, 1959-1973]. We therefore decided to determine the origin of the GABAergic and non-GABAergic inputs to the SLD combining ejection of a retrograde tracer [cholera-toxin B subunit (CTb)] with glutamate decarboxylase (GAD) immunohistochemistry. The presence of GAD-immunoreactive neurons in the SLD was confirmed. Then, following CTb ejections centred on the SLD, combined with GAD and CTb immunohistochemistry, double-labelled cells were observed in the mesencephalic and pontine reticular nuclei and to a lesser extent the parvicellular reticular nucleus. A large number of GAD-negative retrogradely labelled cells was also seen in these structures as well as in the primary motor area of the frontal cortex, the central nucleus of the amygdala, the ventral and lateral bed nucleus of the stria terminalis, the lateral hypothalamic area, the lateral and ventrolateral periaqueductal grey and the lateral paragigantocellular reticular nucleus. From these results, we propose that the activation of PS-executive neurons from the SLD is due to the removal of a tonic inhibition from GABAergic neurons localized in the SLD, and the mesencephalic and pontine reticular nuclei. Strong non-GABAergic inputs to the SLD could be excitatory and responsible for the tonic glutamatergic input on the PS-on neurons we have previously described. They could also terminate on SLD GABAergic interneurons and be indirectly responsible for the inhibition of the PS-on neurons during waking and slow-wave sleep.

International audience; The aim of this study was to examine the afferents to the rat locus coeruleus by means of retrograde and anterograde tracing experiments using cholera-toxin B subunit and phaseolus leucoagglutinin. To obtain reliable injections of cholera-toxin B in the locus coeruleus, electrophysiological recordings were made through glass micropipettes containing the tracer and the noradrenergic neurons of the locus coeruleus were identified by their characteristic discharge properties. After iontophoretic injections of cholera-toxin B into the nuclear core of the locus coeruleus, we observed a substantial number of retrogradely labeled cells in the lateral paragigantocellular nucleus and the dorsomedial rostral medulla (ventromedial prepositus hypoglossi and dorsal paragigantocellular nuclei) as previously described. We also saw a substantial number of retrogradely labeled neurons in (1) the preoptic area dorsal to the supraoptic nucleus, (2) areas of the posterior hypothalamus, (3) the Kölliker-Fuse nucleus, (4) mesencephalic reticular formation. Fewer labeled cells were also observed in other regions including the hypothalamic paraventricular nucleus, dorsal raphe nucleus, median raphe nucleus, dorsal part of the periaqueductal gray, the area of the noradrenergic A5 group, the lateral parabrachial nucleus and the caudoventrolateral reticular nucleus. No or only occasional cells were found in the cortex, the central nucleus of the amygdala, the lateral part of the bed nucleus of the stria terminalis, the vestibular nuclei, the nucleus of the solitary tract or the spinal cord, structures which were previously reported as inputs to the locus coeruleus. Control injections of cholera-toxin B were made in areas surrounding the locus coeruleus, including (1) Barrington's nucleus, (2) the mesencephalic trigeminal nucleus, (3) a previously undefined area immediately rostral to the locus coeruleus and medial to the mesencephalic trigeminal nucleus that we named the peri-mesencephalic trigeminal nucleus, and (4) the medial vestibular nucleus lateral to the caudal tip of the locus coeruleus. These injections yielded patterns of retrograde labeling that differed from one another and also from that obtained with cholera-toxin B injection sites in the locus coeruleus. These results indicate that the area surrounding the locus coeruleus is divided into individual nuclei with distinct afferents. These results were confirmed and extended with anterograde transport of cholera-toxin B or phaseolus leucoagglutinin. Injections of these tracers in the lateral paragigantocellular nucleus, preoptic area dorsal to the supraoptic nucleus, the ventrolateral part of the periaqueductal gray, the Kölliker-Fuse nucleus yielded a substantial to large number of labeled fibers in the nuclear core of the locus coeruleus.(ABSTRACT TRUNCATED AT 400 WORDS)

International audience; The organization of efferent projections from the spinal trigeminal nucleus oralis (Sp5O) to the spinal cord in the rat was studied using the anterograde tracer Phaseolus vulgaris leucoagglutinin. Sp5O projections to the spinal cord are restricted to the cervical cord. No labeled terminal can be detected in the thoracic and lumbar cord. The organization of these projections happens to critically depend on the dorso-ventral location of the injection site. On the one hand, the dorsal part of the Sp5O projects to the medial part of the dorsal horn (laminae III-V) at the C1 level, on the ipsilateral side, and to the ventral horn, on both sides but mainly on the ipsilateral one. Ipsilateral labeled terminals are distributed throughout laminae VII to IX but tend to cluster around the dorso-medial motor nuclei, especially at C3-C5 levels. Within the contralateral ventral horn, label terminals are found particularly in the region of the ventro-medial motor nucleus. This projection extends as far caudally as C3 or C4 level. On the other hand, the ventral part of the Sp5O projects to the lateral part of the dorsal horn (laminae III-V) at the C1 level, on the ipsilateral side, and to the ventral horn, on both sides but mainly on the contralateral one. Contralateral labeled terminals are distributed within the region of the dorso- and ventro-medial motor nuclei at C1-C4 levels whereas they are restricted to the dorso-medial motor nucleus at C5-C8 levels. These findings suggest that Sp5O is involved in the coordination of neck movements and in the modulation of incoming sensory information at the cervical spinal cord.

International audience; Rabies virus (RV) has widely been used as a trans-synaptic retrograde tracer to analyze chains of connected neurons. The use of antibodies directed against the viral nucleoprotein enables viral nucleocapsids to be visualized within the cell soma, as well as within the thickest main dendrites. However, through this approach it is often difficult to accurately define post-synaptic elements (thin dendrites and/or dendritic spines). This limitation can now easily been circumvented by taking advantage of antibodies directed against a soluble viral phosphoprotein that spreads throughout the cytoplasm of the infected neuron, thereby producing Golgi-like immunofluorescent labeling of first-order projection neurons that are infected with RV. Furthermore, when combined with anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biotinylated dextran amine (BDA), this procedure to detect RV facilitates the accurate visualization of both the pre- and post-synaptic elements. Finally, this method of viral detection is sufficiently sensitive to detect weakly labeled second-order neurons, which can then be further characterized neurochemically. Several examples are provided to illustrate why retrograde trans-synaptic tracing using RV can be regarded as an important breakthrough in the analysis of brain circuits, providing an unprecedented level of resolution.

International audience; Labelling of human lactotransferrin with fluorescein 5'-isothiocyanate (FITC) in an equimolar ratio inhibits the binding of the protein to phytohaemagglutinin-activated human peripheral-blood lymphocytes. Therefore it can be assumed that FITC reacts at, or near, the receptor-binding site. Three FITC-labelled peptides have been purified from a tryptic digest of the FITC-labelled lactotransferrin. The determination of their amino acid sequence and their localization on the primary structure of the protein permitted the identification of two FITC-accessible areas in the N-terminal lobe and one in the C-terminal lobe. In fact, only 10% of the total FITC was conjugated to one lysine residue (Lys579) of the C-terminal lobe, whereas most (80%) of the FITC was conjugated to three close lysine residues [Lys263 (65% of total fluorescence), Lys280 and Lys282 (15% of total fluorescence)] located in beta-turn structures, of the N-terminal domain I of human lactotransferrin. The results obtained show that the receptor-binding site should be located in the vicinity of the FITC-accessible Lys263, Lys280 and Lys282, and corroborate our preliminary results reporting the involvement of the N-terminal domain I in the binding of human lactotransferrin to mitogen-stimulated lymphocytes [Rochard, Legrand, Mazurier, Montreuil & Spik (1989) FEBS Lett. 255, 201-204]. In any case, FITC labelling is not suitable for studying the binding of lactotransferrin to activated lymphocytes and its use may lead to erroneous interpretations of cell binding experiments.

International audience; The Escherichia Coli bacterial extract (OM-89) is used in the treatment of rheumatoid arthritis (RA). We evaluated the immunological changes induced by oral administration of OM-89 in 12 RA patients (polyclonal T cell reactivity to PHA, T cell precursor frequencies specific for OM-89 and Tetanus toxoid (TT), a control antigen and the release of Th1 (IFN-gamma, TNF-alpha), Th2 (IL-4) and T regulatory 1 cell (Tr1) (IL-10) cytokines in the supernatants of PBMC cultures. Stimulation index in response to PHA decreased at month 3 as well as T cell precursor frequencies specific for TT with similar trends for OM-89-specific T cell precursor frequencies. OM-89 induced a strong production of IL-10, a significant decrease in IL-4 production while TNF-alpha and IFN-gamma production tended to decrease during the study. Our results suggest that OM-89 has immunomodulatory properties by inducing changes in PBMC cytokines release suggestive of an induced Tr1 response to OM-89.

We previously reported the existence of pharmacologically related gastrin/CCKB type receptors (CCKB-R) in a variant of Jurkat T lymphoblastoid cells (JK(CD3−CD4+)). We studied here the expression of mRNAs encoding CCKA and CCKB receptors in various human white cells by means of Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Using CCKB-R specific primers, we detected a significant expression of CCKB-R mRNA in JK(CD3−CD4+) cells. These transcripts were also expressed, at a lower level, in two other Jurkat clones (JK(CD3+CD4−) and JK(CD3+CD4+)), in peripheral blood lymphocytes (PBL) and in purified CD4+ and CD8+ lymphocytes. Activation of Jurkat cells and PBL by T cells mitogenic lectins (jacalin, phytohemaglutinin) did not modify CCKB-R mRNA expression. In all these cells, using CCKA-R specific primers, we could not amplify any specific cDNA fragment corresponding to this receptor. Neither CCKB-R nor CCKA-R mRNAs could be detected in monocytic cells. Our data show for the first time a constitutive expression of CCKB-R transcripts in lymphoid cells. Moreover, the modulation of immunocyte functions by cholecystokinin-related peptides could occur through CCKB-R rather than CCKA-R and affect lymphocytes rather than monocytes.

International audience; Fluorescein isothiocyanate derivatization of human lactotransferrin on Lys-264 as well as covalent addition of sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionate (SASD)* on Lys-74 inhibits the binding of the glycoprotein to both human PHA-activated lymphocytes and non-activated platelets. This suggests that the cell binding site of lactotransferrin is located in the vicinity of the lysine residues 74 & 264 and does not occur either through electrostatic or lectin interactions. In contrast, the derivatization of lactotransferrin using sulfosuccinimidyl 6-(4'-azido-2'-nitrophenyl-amino) hexanoate (sulfo-SANPAH), on Lys-281 does not modify the binding parameters of lactotransferrin to the cells. Molecular modeling showed the position of SASD, sulfo-SANPAH and fluorescein molecules at the surface of the protein and suggested that SASD and fluorescein could mask the two loop-containing regions of human lactotransferrin (residues 28-34 and 38-45). Elsewhere, a 6 kDa peptide covering the peptide chain from residues 4 to 52 was isolated and its inhibitory effect on the binding of lactotransferrin to both human PHA-activated lymphocytes and non-activated platelets was demonstrated. Inhibition of ADP-induced platelet aggregation by lactotransferrin (50% inhibition = 10 nM) was also found with the N-t fragment of lactotransferrin (residues 3-281; 50% inhibition = 2 microM) and with two synthetic peptides: KRDS tetrapeptide (50% inhibition = 350 microM) and CFQWQRNMRKVRGPPVSC octodecapeptide (50% inhibition = 20 microM) corresponding to the lactotransferrin amino acid sequence 39-42 and 20-37, respectively.

International audience; Fluorescein isothiocyanate derivatization of the human lactotransferrin on Lys-264 inhibits the binding of the protein of human PHA-activated lymphocytes [Legrand, D., Mazurier, J., Maes, P., Rochard, E., Montreuil, J., & Spik, G. (1991) Biochem. J. 276, 733-738], indicating that part of the receptor-binding site is located in the N-terminal domain I of lactotransferrin. In the present study, a 6-kDa peptide (residues 4-52) was isolated from the N-terminal lobe of human lactotransferrin which inhibited the binding of the protein to its cell receptor. In addition, lactotransferrin was derivatized using sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) and sulfosuccinimidyl 6-((4'-azido-2'-nitrophenyl)amino)hexanoate (sulfo-SANPAH), two heterobifunctional reagents generally used for receptor-ligand cross-linking. The azide group of these two reagents was inactivated by photolysis, and only the succinimidyl ester group was allowed to react with lysine residues of the protein. The binding of the derivatized lactotransferrins to the human lymphocyte receptor was assayed. SASD, which binds to Lys-74, was able to inhibit the binding of lactotransferrin to the cell receptor, in contrast to Lys-281-binding sulfo-SANPAH. Molecular modeling showed the position of SASD, sulfo-SANPAH, and fluorescein molecules at the surface of the protein and suggested that SASD and fluorescein could mask residues 4-6 and two loop-containing regions of human lactotransferrin (residues 28-34 and 38-45). The comparison of the primary and tertiary structures of human lactotransferrin and serotransferrin, which bind to specific cell receptors, shows that the above-mentioned regions, which are likely involved in protein-receptor interactions, possess specific structural features.

International audience; Several lines of evidence have recently suggested the occurrence of a specific lactotransferrin receptor in the small intestinal brush-border membrane in several animal species, which is thought to be involved in lactotransferrin-mediated intestinal iron absorption. We report here for the first time the isolation and partial characterization of this receptor from mouse intestinal brush border. The receptor has been purified to homogeneity by affinity chromatography on an immobilized human lactotransferrin column. The purified receptor was found to be active in that it binds iron-free and iron-saturated lactotransferrin with a Kd of 0.1 microM. Anti-receptor antibodies were prepared, and the receptor was further isolated by immunoaffinity chromatography in higher yield but in a denatured form. The purified receptor was revealed by sodium dodecyl sulfate-polyacrylamide electrophoresis to be a protein of about Mr = 130,000, consisting of a single polypeptide chain. The isoelectric point was determined to be 5.8. The receptor was further shown to bear concanavalin A and phytohemagglutinin L binding glycans. Digestion by N-glycanase and endo-N-acetyl-beta-D-glucosaminidase B led to a decrease of Mr = 25,000, while the endo-N-acetyl-beta-D-glucosaminidase H was uneffective, suggesting that the lactotransferrin receptor is mainly glycosylated by bi- and triantennary glycans. To gain further insight into the interaction of the receptor with lactotransferrin, namely, the number of ligand molecules bound per molecule of receptor, mouse lactotransferrin was cross-linked to its membrane-bound enterocyte receptor by use of radiolabeled sulfosuccinimidyl 3-[[2-(p-azidosalicylamido)ethyl]dithio]propionate (SASD).(ABSTRACT TRUNCATED AT 250 WORDS)

International audience; In the resting rate, the human peripheral blood lymphocytes did not show detectable surface and intracellular receptors for human lactotransferrin. However, both types of lactotransferrin receptors were expressed during stimulation of lymphocytes with phytohemagglutinin. The appearance of receptors was time-dependent and the number of receptors reached a plateau after at least two days of mitogen stimulation. These results suggest that the presence of surface receptors on mitogen-stimulated lymphocytes is not consecutive to a modification of subcellular distribution but to an induction of biosynthesis of the receptors. As measured by incorporation of [3H]thymidine into DNA, addition of human lactotransferrin in a serum-free medium increased the proliferative activity of phytohemagglutinin-stimulated lymphocytes. Optimal enhancement of [3H]thymidine incorporation was obtained by adding 30% iron-saturated lactotransferrin at a concentration of 0.17 microM. Therefore, the role of lactotransferrin in the response of lymphocytes to mitogen stimulation appears to be similar to that previously described for serotransferrin. The lactotransferrin receptor was visualized using 125I-labeled lactotransferrin on nitrocellulose paper after electroblotting of the Triton X-100 extract of the phytohemagglutinin-stimulated lymphocytes as two protein bands of 100 and 110 kDa molecular mass. Purification of the lactotransferrin receptor from the Triton-X-100-soluble extract of stimulated lymphocytes was performed by antiligand-affinity chromatography. The binding of lactotransferrin to the purified receptors was reversible and dependent on concentration and pH.

International audience; Human lactotransferrin receptors have been recently characterized on mitogen-stimulated human lymphocytes [(1989) Eur. J. Biochem. 179, 481-487]. In order to define the lactotransferrin recognition site by these receptors, the binding to lymphocytes of several tryptic fragments, isolated from human lactotransferrin by mild tryptic hydrolysis [(1984) Biochim. Biophys. Acta 787, 90-96], has been investigated. The 30 kDa N-tryptic fragment (residues 4-281) and the re-associated N,C-tryptic complex bind to lactotansferrin lymphocyte receptor with a dissociation constant of 44 nM and 39 nM, respectively, similar to the value obtained for the native lactotransferrin (Kd = 46 nM). However, neither the N-terminal domain II (residues 91-257) nor the 50 kDa C-tryptic fragment (residues 282-703) are recognized. These results suggest that the binding site of human lactotransferrin by the lymphocyte receptor is located in the N-terminal lobe and more precisely in the N-terminal domain I (residues 4-90 and/or 258-281).