Redox control of indoleamine 2,3-dioxygenase expression and activity in human monocyte-derived dendritic cells is independent of changes in oxygen tension.

Dendritic cells (DCs) initiate adaptive immune responses to pathogens and tumours and maintain tolerance to self and innocuous antigens. These functions occur in organs and tissues exhibiting wide variations in nutrients, growth factors, redox and oxygen tension. Understanding how these microenvironmental factors influence DCs to affect immunological outcomes is of increasing relevance with the emerging success of DC-based cellular vaccines. In a previous study, we examined whether redox, an important environmental cue, could influence DC expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO). IDO-competent DCs promote long-term immune homoeostasis by limiting exaggerated inflammatory responses and directing regulatory T-cell effector function. To alter redox, we manipulated the activity of the cystine/glutamate antiporter, which functions to maintain intracellular and extracellular redox. The results of that study showed that redox perturbation strongly induced IDO expression and activity in DCs. While this study was performed using standard cell culture techniques with DCs cultured under 5% CO2 and 20% O2, it is clear that DCs capture and present antigens in inflamed tissues and secondary lymphoid organs which exhibit low oxygen tension (1-5% O2). Therefore, here we investigated whether oxygen tension influences DC expression of IDO in the context of homoeostatic and altered redox.

Evaluation of white water reuse in the bleaching process for reducing fresh water consumption.

The main objective of this work is to study the technical viability of using the effluent generated in paper machines (white water) in the wash presses of the bleaching stage, reducing fresh water consumption. As a case study, the industrial process of Ripasa S.A. Celulose e Papel was evaluated. White water rate is about 700 m3/h and it is not possible to reuse all this volume in the bleaching stage without causing operational problems (fouling in tubes and clogging in the screens). A mass balance of the bleaching unit was developed in an electronic spreadsheet in order to evaluate the possibility of reducing fresh water consumption, using only a fraction of the available white water in the wash presses. To achieve this objective some physical-chemistry properties of the white water stream and of other streams of the process were determined. The maximum concentration of some non-process elements (Si, Ca, Mn and Fe), which could accumulate in the process, were determined in order to establish some parameters to allow process integration of the streams involved, considering operational constraints. The results obtained have shown that it is possible to reduce approximately by 13% the consumption of fresh water and this methodology has been satisfactory.

Crystal and electronic structures of magnesium(II), copper(II), and mixed magnesium(II)-copper(II) complexes of the quinoline half of styrylquinoline-type HIV-1 integrase inhibitors.

A new target in AIDS therapy development is HIV-1 integrase (IN). It was proven that HIV-1 IN required divalent metal cations to achieve phosphodiester bond cleavage of DNA. Accordingly, all newly investigated potent IN inhibitors contain chemical fragments possessing a high ability to chelate metal cations. One of the promising leads in the polyhydroxylated styrylquinolines (SQLs) series is (E)-8-hydroxy-2-[2-(4,5-dihydroxy-3-methoxyphenyl)-ethenyl]-7-quinoline carboxylic acid (1). The present study focuses on the quinoline-based progenitor (2), which is actually the most probable chelating part of SQLs. Conventional and synchrotron low-temperature X-ray crystallographic studies were used to investigate the chelating power of progenitor 2. Mg2+ and Cu2+ cations were selected for this purpose, and three types of metal complexes of 2 were obtained: Mg(II) complex (4), Cu(II) complex (5) and mixed Mg(II)-Cu(II) complexes (6 and 7). The analysis of the crystal structure of complex 4 indicates that two tridentate ligands coordinate two Mg2+ cations, both in octahedral geometry. The Mg-Mg distance was found equal to 3.221(1) A, in agreement with the metal-metal distance of 3.9 A encountered in the crystal structure of Escherichia coli DNA polymerase I. In 5, the complex is formed by two bidentate ligands coordinating one copper ion in tetrahedral geometry. Both mixed Mg(II)-Cu(II) complexes, 6 and 7 exhibit an original arrangement of four ligands linked to a central heterometallic cluster consisting of three octahedrally coordinated magnesium ions and one tetrahedrally coordinated copper ion. Quantum mechanics calculations were also carried out in order to display the electrostatic potential generated by the dianionic ligand 2 and complex 4 and to quantify the binding energy (BE) during the formation of the magnesium complex of progenitor 2. A comparison of the binding energies of two hypothetical monometallic Mg(II) complexes with that found in the bimetallic magnesium complex 4 was made.

Long-circulating PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery.

PURPOSE: The aim of this study was to evaluate the ability of long-circulating PEGylated cyanoacrylate nanoparticles to diffuse into the brain tissue. METHODS: Biodistribution profiles and brain concentrations of [14C]-radiolabeled PEG-PHDCA, polysorbate 80 or poloxamine 908-coated PHDCA nanoparticles, and uncoated PHDCA nanoparticles were determined by radioactivity counting after intravenous administration in mice and rats. In addition, the integrity of the blood-brain barrier (BBB) after nanoparticles administration was evaluated by in vivo quantification of the diffusion of [14C]-sucrose into the brain. The location of fluorescent nanoparticles in the brain was also investigated by epi-fluorescent microscopy. RESULTS: Based on their long-circulating characteristics, PEGylated PHDCA nanoparticles penetrated into the brain to a larger extent than all the other tested formulations. Particles were localized in the ependymal cells of the choroid plexuses, in the epithelial cells of pia mater and ventricles, and to a lower extent in the capillary endothelial cells of BBB. These phenomena occurred without any modification of BBB permeability whereas polysorbate 80-coated nanoparticles owed, in part, their efficacy to BBB permeabilization induced by the surfactant. Poloxamine 908-coated nanoparticles failed to increase brain concentration probably because of their inability to interact with cells. CONCLUSIONS: This study proposes PEGylated poly (cyanoacrylate) nanoparticles as a new brain delivery system and highlights two requirements to design adequate delivery systems for such a purpose: a) long-circulating properties of the carrier, and b) appropriate surface characteristics to allow interactions with BBB endothelial cells.

Distinct mechanisms regulate slow-muscle development.

Vertebrate muscle development begins with the patterning of the paraxial mesoderm by inductive signals from midline tissues [1, 2]. Subsequent myotome growth occurs by the addition of new muscle fibers. We show that in zebrafish new slow-muscle fibers are first added at the end of the segmentation period in growth zones near the dorsal and ventral extremes of the myotome, and this muscle growth continues into larval life. In marine teleosts, this mechanism of growth has been termed stratified hyperplasia [3]. We have tested whether these added fibers require an embryonic architecture of muscle fibers to support their development and whether their fate is regulated by the same mechanisms that regulate embryonic muscle fates. Although Hedgehog signaling is required for the specification of adaxial-derived slow-muscle fibers in the embryo [4, 5], we show that in the absence of Hh signaling, stratified hyperplastic growth of slow muscle occurs at the correct time and place, despite the complete absence of embryonic slow-muscle fibers to serve as a scaffold for addition of these new slow-muscle fibers. We conclude that slow-muscle-stratified hyperplasia begins after the segmentation period during embryonic development and continues during the larval period. Furthermore, the mechanisms specifying the identity of these new slow-muscle fibers are different from those specifying the identity of adaxial-derived embryonic slow-muscle fibers. We propose that the independence of early, embryonic patterning mechanisms from later patterning mechanisms may be necessary for growth.

HIV-1 integrase: the next target for AIDS therapy?

HIV-1 is the aetiological agent of AIDS. Present treatment of AIDS uses a combination therapy with reverse transcriptase and protease inhibitors. Recently, the integrase (IN), the third enzyme of HIV-1 which is necessary for the integration process of proviral DNA into the host genome, has reached as a legitimate new drug target. Several families of inhibitors of the catalytic core domain of HIV-1 IN exhibiting submicromolar activities have now been identified. Our contribution in this field was related to the development of new polyhydroxylated styrylquinolines. The latter compounds have proved to be potent HIV-1 IN inhibitors, that block the replication of HIV-1 in cell culture, and are devoid of cytotoxicity. The crystal structure of the catalytically active core domain of a HIV-1 IN mutant has been determined. The active site region is identified by the position of two of the conserved carboxylate residues essential for catalysis, Asp64 and Asp116, which coordinate a Mg2+ ion, whereas the third catalytic residue, Glu152 does not participate in metal binding. However, a recent molecular dynamics simulation of the HIV-1 IN catalytic domain provides support to the hypothesis that a second metal ion is likely to be carried into the HIV-1 IN active site by the DNA substrate. The structure of a complex of the HIV-1 IN core domain with the inhibitor 5-CITEP has been recently reported. The inhibitor binds centrally in the active site of the IN and makes a number of close contacts with the protein, particularly with Lys156, Lys159 and Gln148, amino acids which were identified to be near the active site of the enzyme, through site-directed mutagenis and photo-crosslinking experiments. The exact mechanism by which HIV-1 IN inhibitors block the catalytic activity of the protein remains unknown. However, several putative pharmacophore components have been characterized. All these groups lie in a possible coordination to a divalent ion, supporting thus the hypothesis that the interaction causing the inhibition is mediated by one or two cations. Finally, among the HIV-1 IN inhibitors, three classes have proved to exhibit significant antiviral activities. Thus, it seems likely that the efficient use of HIV-1 IN as a target for rational design will become possible in the next future, possibly through the use of combination regimens including IN inhibitors.

Near infrared with principal component analysis as a novel analytical approach for nanoparticle technology.

PURPOSE: To progress in the characterization of a poly(MePEGcyanoacrylate-co-hexadecylcyanoacrylate) (poly(PEGCA-co-HDCA) copolymer and the nanoparticles formed from this copolymer. METHODS: Poly(PEGCA-co-HDCA) at a MePEG/hexadecyl ratio of 1:4 was investigated by 1H-NMR and near infrared spectroscopy. The nanoparticle suspensions, obtained by the methods of nanoprecipitation or emulsion--solvent evaporation, as well as the crude nanoparticles and their dispersion medium--were analyzed by MePEG measurement, 1H-NMR, and near infrared spectroscopy. RESULTS: The 1H-NMR results showed that the (poly(PEGCA-co-HDCA) copolymer obtained bore lateral hydrophilic MePEG chains and lateral hydrophobic hexadecyl chains in a final ratio of 1:4. However, this ratio, although reproducible from batch to batch, represented only a mean value for different molecular species. Indeed, our results demonstrated the formation of more hydrophobic poly(alkyl-cyanoacrylate) oligomers (with a higher content of hexadecyl chains) and other more hydrophilic oligomers (with a higher MePEG content). Only the more hydrophobic oligomers were able to form solid pegylated nanoparticles. As far as these nanoparticles were concerned, determination of their MePEG content allowed the calculation of a distance of 1.2 nm and 1.05 nm between 2 grafted MePEG chains at the nanoparticle surface, when obtained by nanoprecipitation and emulsion-solvent evaporation, respectively. Moreover, when the same copolymer batch was used, different nanoparticles were obtained according to the preparation method, as seen by near infrared spectroscopy. CONCLUSIONS: The nanoparticles obtained by nanoprecipitation or emulsion-solvent evaporation of poly(PEGCA-co-HDCA) 1:4 copolymer displayed a different supramolecular organization, as evidenced by the near infrared spectroscopy results. Moreover, these nanoparticles showed surface characteristics compatible with a long circulating carrier.

Design of folic acid-conjugated nanoparticles for drug targeting.

The new concept developed in this study is the design of poly(ethylene glycol) (PEG)-coated biodegradable nanoparticles coupled to folic acid to target the folate-binding protein; this molecule is the soluble form of the folate receptor that is overexpressed on the surface of many tumoral cells. For this purpose, a novel copolymer, the poly[aminopoly(ethylene glycol)cyanoacrylate-co-hexadecyl cyanoacrylate] [poly(H(2)NPEGCA-co-HDCA)] was synthesized and characterized. Then nanoparticles were prepared by nanoprecipitation of the obtained copolymer, and their size, zeta potential, and surface hydrophobicity were investigated. Nanoparticles were then conjugated to the activated folic acid via PEG terminal amino groups and purified from unreacted products. Finally, the specific interaction between the conjugate folate-nanoparticles and the folate-binding protein was evaluated by surface plasmon resonance. This analysis confirmed a specific binding of the folate-nanoparticles to the folate-binding protein. This interaction did not occur with nonconjugated nanoparticles used as control. Thus, folate-linked nanoparticles represent a potential new drug carrier for tumor cell-selective targeting.

Tautomers of styrylquinoline derivatives containing a methoxy substituent: computation of their population in aqueous solution and their interaction with RSV integrase catalytic core.

8-Hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethenyl]-7-quinoline carboxylic acid and 8-hydroxy-2-[2-(3-methoxy-4-hydroxyphenyl)ethenyl]-7-quinoline carboxylic acid inhibit the processing and strand transfer reactions catalyzed by HIV-1 integrase with an IC50 of 2 microM. Some of their spectral properties are briefly reported. Their fluorescence is so weak that it is of no use in an experimental determination of the binding to the protein and we resorted to computer simulation. Both styrylquinoline derivatives, in their monoanionic form, have several dozens of tautomers and each of these forms has four planar rotamers. In this work computer simulations have been performed to determine which tautomer is the most abundant in aqueous solution and which binds to the Rous sarcoma virus (RSV) integrase catalytic core. As the substituents on the quinoline moiety are the same as on salicylic acid, the energies of hydroxy benzoic acid tautomers were also computed both in vacuo and embedded in a continuous medium which had the dielectric constant of bulk water, using the recent CPCM technique. The CPCM method was then applied to the two integrase inhibitors to estimate the tautomer population in water. The binding site of the compounds on the RSV integrase catalytic core was determined through a docking protocol, consisting of coupling a grid search method with full energy minimization. The designed method is a way leading to identification of potent integrase inhibitors using in silico experiments.

Modeling of the inhibition of retroviral integrases by styrylquinoline derivatives.

Styrylquinoline derivatives, known to be potent inhibitors of HIV-1 integrase, have been experimentally tested for their inhibitory effect on the disintegration reaction catalyzed by catalytic cores of HIV-1 and Rous sarcoma virus (RSV) integrases. A modified docking protocol, consisting of coupling a grid search method with full energy minimization, has been specially designed to study the interaction between the inhibitors and the integrases. The inhibitors consist of two moieties that have hydroxyl and/or carboxyl substituents: the first moiety is either benzene, phenol, catechol, resorcinol, or salicycilic acid; the hydroxyl substituents on the second (quinoline) moiety may be in the keto or in the enol forms. Several tautomeric forms of the drugs have been docked to the crystallographic structure of the RSV catalytic core. The computed binding energy of the keto forms correlates best with the measured inhibitory effect. The docking procedure shows that the inhibitors bind closely to the crystallographic catalytic Mg(2+) dication. Additional quantum chemistry computations show that there is no direct correlation between the binding energy of the drugs with the Mg(2+) dication and their in vitro inhibitory effect. The designed method is a leading way for identification of potent integrase inhibitors using in silico experiments.

Structure-activity relationships and binding mode of styrylquinolines as potent inhibitors of HIV-1 integrase and replication of HIV-1 in cell culture.

Our prior studies showed that polyhydroxylated styrylquinolines are potent HIV-1 integrase (IN) inhibitors that block the replication of HIV-1 in cell culture at nontoxic concentrations. To explore the mechanism of action of these inhibitors, various novel styrylquinoline derivatives were synthesized and tested against HIV-1 IN and in cell-based assays. Regarding the in vitro experiments, the structural requirements for biological activity are a carboxyl group at C-7, a hydroxyl group at C-8 in the quinoline subunit, and an ancillary phenyl ring. However the in vitro inhibitory profile tolerates deep alterations of this ring, e.g. by the introduction of various substituents or its replacement by heteroatomic nuclei. Regarding the ex vivo assays, the structural requirements for activity are more stringent than for in vitro inhibition. Thus, in addition to an o-hydroxy acid group in the quinoline, the presence of one ortho pair of substituents at C-3' and C-4', particularly two hydroxyl groups, in the ancillary phenyl ring is imperatively required for inhibitory potency. Starting from literature data and the SARs developed in this work, a putative binding mode of styrylquinoline inhibitors to HIV-1 IN was derived.

Visualization of in vitro protein-rejecting properties of PEGylated stealth polycyanoacrylate nanoparticles.

The in vitro protein-rejecting properties of PEG-coated polyalkylcyanoacrylate (PACA) nanoparticles were for the first time visualized after freeze-fracture of the nanoparticles pre-incubated with fibrinogen as a model blood protein. The reduced protein association to the nanoparticles was evidenced also by two-dimensional PAGE after incubation of the nanoparticles with human plasma. In vivo experiments showed the 'stealth' long-circulating properties of the PEGylated nanoparticles after intravenous administration to mice. Thus, the images obtained after nanoparticle-protein incubation were predictive of the behavior observed in vivo. In conclusion, freeze-fracture analysis represents a novel and original qualitative approach to investigate the interactions between proteins and particulate systems.

Stealth PEGylated polycyanoacrylate nanoparticles for intravenous administration and splenic targeting.

The aim of the present work was to investigate the biodistribution characteristics of PEG-coated polycyanoacrylate nanoparticles prepared by the nanoprecipitation/solvent diffusion method using the previously synthesized poly(MePEGcyanoacrylate-hexadecylcyanoacrylate) copolymer. It was observed that [14C]-radiolabeled PEGylated nanoparticles remained for a longer time in the blood circulation after intravenous administration to mice, compared to the non-PEGylated poly(hexadecylcyanoacrylate) (PHDCA) nanoparticles. Furthermore, hepatic accumulation was dramatically reduced, whereas a highly increased spleen uptake was shown. The PEGylation degree of the polymer seemed not to affect the in vivo behavior of the nanoparticles, whereas previously obtained in vitro data have shown a modification of plasma protein adsorption depending on the density of PEG at the surface of the particles. Moreover, the study of the in vitro cytotoxicity of the nanoparticles revealed that the PEGylation of the cyanoacrylate polymer reduced its toxicity. These results open up interesting perspectives for the targeting of drugs to other tissues than the liver.

Styrylquinoline derivatives: a new class of potent HIV-1 integrase inhibitors that block HIV-1 replication in CEM cells.

On the basis of the fact that several polynucleotidyl transferases, related to HIV integrase, contain in their active site two divalent metal cations, separated by ca. 4 A, new potential HIV integrase inhibitors were designed, in which a quinoline substructure is linked to an aryl nucleus possessing various hydroxy substitution patterns, by means of an ethylenic spacer. Although the most active compounds contain the catechol structure, this group is not essential for the activity, since compound 21 that lacks such a moiety is a potent drug, implicating the presence of a different pharmacophore. The most promising styrylquinolines thus synthesized inhibit HIV-1 integrase in vitro at micromolar or submicromolar concentrations and block HIV replication in CEM cells, with no significant cellular toxicity in a 5-day period assay. These inhibitors are active against integrase core domain-mediated disintegration, suggesting that fragment 50-212 is their actual target. These new styrylquinolines may provide lead compounds for the development of novel antiretroviral agents for AIDS therapeutics, based upon inhibition of HIV integrase. They might also be used in the elucidation of the mechanism of inhibition of this enzyme; e.g., they could serve as candidates for cocrystallization studies with HIV integrase.

Pegylated nanoparticles from a novel methoxypolyethylene glycol cyanoacrylate-hexadecyl cyanoacrylate amphiphilic copolymer.

PURPOSE: The aim of this work was to develop PEGylated poly(alkylcyanoacrylate) nanoparticles from a novel methoxypolyethyleneglycol cyanoacrylate-co-hexadecyl cyanoacrylate copolymer. METHODS: PEGylated and non-PEGylated nanoparticles were formed by nanoprecipitation or by emulsion/solvent evaporation. Nanoparticles size, zeta potential and surface hydrophobicity were investigated. Surface chemical composition was determined by X-ray photoelectron spectroscopy. Nanoparticle morphology was investigated by transmission electron microscopy after freeze-fracture. Nanoparticles cytotoxicity was assayed in vitro, onto mouse peritoneal macrophages. Cell viability was determined through cell mitochondrial activity, by a tetrazolium-based colorimetric method (MTT test). Finally, the degradation of PEGylated and non-PEGylated poly(hexadecyl cyanoacrylate) nanoparticles was followed spectrophotometrically during incubation of nanoparticles in fetal calf serum. RESULTS: Monodisperse nanoparticles with a mean diameter ranging between 100 and 200 nm were obtained using nanoprecipitation or emulsion/solvent evaporation as preparation procedures. A complete physico-chemical characterization, including surface chemical analysis, allowed to confirm the formation of PEG-coated nanoparticles. The PEGylation of the cyanoacrylate polymer showed reduced cytotoxicity towards mouse peritoneal macrophages. Furthermore, the presence of the PEG segment increased the degradability of the poly(hexadecyl cyanoacrylate) polymer in presence of calf serum. CONCLUSIONS: We succeeded to prepare PEGylated nanoparticles from a novel poly(methoxypolyethyleneglycol cyanoacrylate-co-hexadecyl cyanoacrylate) by two different techniques. Physico-chemical characterization showed the formation of a PEG coating layer. Low cytotoxicity and enhanced degradation were also shown.

[Autism and children with Fragile X syndrome]. / L'autisme et les enfants porteurs d'un X fragile.

Many epidemiological studies have demonstrated the exceptionally high incidence of autism in children with the fragile X syndrome, and autism is often considered a "behavioral phenotype" of this syndrome. However, the discrepancies between the results of these studies disclosed strong effects of methodological flaws and demonstrated the need for gathering clinical data. Atypical "autistic-like" behaviors were then found to be common, early symptoms of the syndrome occurring against the background of early manifestations of mental retardation. These behaviors reflect these children's exquisite reactivity to change and contact with others. Avoidance of eye contact is the most significant feature. The appropriate diagnosis is not autism but phobia of social relationships. This highly specific vulnerability, which may be inherited, probably leads some of these patients to experience the fate of autistic children. It highlights the influence of environment on the clinical course and indirectly supports the role of early specialized care.