Immunophenotype and Transcriptome Profile of Patients With Multiple Sclerosis Treated With Fingolimod: Setting Up a Model for Prediction of Response in a 2-Year Translational Study.

BACKGROUND: Fingolimod is a functional sphingosine-1-phosphate antagonist approved for the treatment of multiple sclerosis (MS). Fingolimod affects lymphocyte subpopulations and regulates gene expression in the lymphocyte transcriptome. Translational studies are necessary to identify cellular and molecular biomarkers that might be used to predict the clinical response to the drug. In MS patients, we aimed to clarify the differential effects of fingolimod on T, B, and natural killer (NK) cell subsets and to identify differentially expressed genes in responders and non-responders (NRs) to treatment. MATERIALS AND METHODS: Samples were obtained from relapsing-remitting multiple sclerosis patients before and 6 months after starting fingolimod. Forty-eight lymphocyte subpopulations were measured by flow cytometry based on surface and intracellular marker analysis. Transcriptome sequencing by next-generation technologies was used to define the gene expression profiling in lymphocytes at the same time points. NEDA-3 (no evidence of disease activity) and NEDA-4 scores were measured for all patients at 1 and 2 years after beginning fingolimod treatment to investigate an association with cellular and molecular characteristics. RESULTS: Fingolimod affects practically all lymphocyte subpopulations and exerts a strong effect on genetic transcription switching toward an anti-inflammatory and antioxidant response. Fingolimod induces a differential effect in lymphocyte subpopulations after 6 months of treatment in responder and NR patients. Patients who achieved a good response to the drug compared to NR patients exhibited higher percentages of NK bright cells and plasmablasts, higher levels of FOXP3, glucose phosphate isomerase, lower levels of FCRL1, and lower Expanded Disability Status Scale at baseline. The combination of these possible markers enabled us to build a probabilistic linear model to predict the clinical response to fingolimod. CONCLUSION: MS patients responsive to fingolimod exhibit a recognizable distribution of lymphocyte subpopulations and a different pretreatment gene expression signature that might be useful as a biomarker.

Mechanisms of action of cannabidiol in adoptively transferred experimental autoimmune encephalomyelitis.

Cannabidiol (CBD) is one of the most important compounds in Cannabis sativa, lacks psychotropic effects, and possesses a high number of therapeutic properties including the amelioration of experimental autoimmune encephalomyelitis (EAE). The aim of this study was to analyse the relative efficacy of CBD in adoptively transferred EAE (at-EAE), a model that allows better delineation of the effector phase of EAE. Splenocytes and lymph nodes from mice with actively induced EAE were cultured in the presence of MOG35-55 and IL-12 and inoculated intraperitoneally in recipient female C57BL/6J mice. The effects of CBD were evaluated using clinical scores and magnetic resonance imaging (MRI). In the central nervous system, the extent of cell infiltration, axonal damage, demyelination, microglial activation and cannabinoid receptors expression was assessed by immunohistochemistry. Lymph cell viability, apoptosis, oxidative stress and IL-6 production were measured in vitro. Preventive intraperitoneal treatment with CBD ameliorated the clinical signs of at-EAE, and this improvement was accompanied by a reduction of the apparent diffusion coefficient in the subiculum area of the brain. Inflammatory infiltration, axonal damage, and demyelination were reduced, and cannabinoid receptor expression was modulated. Incubation with CBD decreased encephalitogenic cell viability, increasing early apoptosis and reactive oxygen species (ROS) and decreasing IL-6 production. The reduction in viability was not mediated by CB1, CB2 or GPR55 receptors. CBD markedly improved the clinical signs of at-EAE and reduced infiltration, demyelination and axonal damage. The CBD-mediated decrease in the viability of encephalitogenic cells involves ROS generation, apoptosis and a decrease in IL-6 production and may contribute to the therapeutic effect of this compound.

Combined use of pharmacophoric models together with drug metabolism and genotoxicity "in silico" studies in the hit finding process.

In this study we propose a virtual screening strategy based on the generation of a pharmacophore hypothesis, followed by an in silico evaluation of some ADME-TOX properties with the aim to apply it to the hit finding process and, specifically, to characterize new chemical entities with potential to control inflammatory processes mediated by T lymphocytes such as multiple sclerosis, systemic lupus erithematosus or rheumatoid arthritis. As a result, three compounds with completely novel scaffolds were selected as final hits for future hit-to-lead optimization due to their anti-inflammatory profile. The biological results showed that the selected compounds increased the intracellular cAMP levels and inhibited cell proliferation in T lymphocytes. Moreover, two of these compounds were able to increase the production of IL-4, an immunoregulatory cytokine involved in the selective deviation of T helper (Th) immune response Th type 2 (Th2), which has been proved to have anti-inflammatory properties in several animal models for autoimmune pathologies as multiple sclerosis or rheumatoid arthritis. Thus our pharmacological strategy has shown to be useful to find molecules with biological activity to control immune responses involved in many inflammatory disorders. Such promising data suggested that this in silico strategy might be useful as hit finding process for future drug development.

Synthesis, structural analysis, and biological evaluation of thioxoquinazoline derivatives as phosphodiesterase 7 inhibitors.

PDE7 inhibitors regulate pro-inflammatory and immune T-cell functions, and are a potentially novel class of drugs especially useful in the treatment of a wide variety of immune and inflammatory disorders. Starting from our lead family of thioxoquinazolines, we designed, synthesized, and characterized a novel series of thioxoquinazoline derivatives. Many of these compounds showed inhibitory potencies at sub-micromolar levels against the catalytic domain of PDE7A1 and at the micromolar level against PDE4D2. Cell-based studies showed that these compounds not only increased intracellular cAMP levels, but also had interesting anti-inflammatory properties within a therapeutic window. The in silico data predict that these compounds are capable of the crossing the blood-brain barrier. The X-ray crystal structure of the PDE7A1 catalytic domain in complex with compound 15 at a resolution of 2.4 A demonstrated that hydrophobic interactions at the active site pocket are a key feature. This structure, together with molecular modeling, provides insight into the selectivity of the PDE inhibitors and a template for the discovery of new PDE7 or PDE7/PDE4 dual inhibitors.

The Th17 lineage: Answers to some immunological questions

En los últimos años se han estudiado exhaustivamente las funciones ylas rutas de desarrollo del subtipo de células T helper especializado en la producción de IL-17 (Th17). Este linaje celular de células efectoras desempeñaun papel decisivo tanto en la respuesta inmune a agentes infecciosos, comoen inmunopatologías. Al igual que para los subtipos Th1 y Th2, la definiciónde Th17 está dirigida por citocinas y factores de transcripción específicos. Lacombinación de TGF-β e IL-6, y los factores de transcripción RORγt, RORαy Stat3 son esenciales para comprometer el subtipo Th17. IL-23 juega un papelclave en la estabilización del fenotipo y de la actividad patogénica de célulasproductoras de IL-17. La citocina IL-21 producida por células Th17 participaen un mecanismo de retroalimentación para favorecer el desarrollo de células productoras de IL-17, mientras que las citocinas IL-27, IL-4, IFN-γ, IL-25e IL-2 limitan el fenotipo Th17. Las células T reguladoras CD4+CD25+Foxp3+(Treg) siguen una ruta de desarrollo divergente al establecimiento de las células IL-17, aunque ambas alternativas son gobernadas por TGF-β, el cual dirige el destino de células CD4+naïve hacia uno u otro de estos subtipos celulares mutuamente excluyentes dependiendo de la presencia de IL-6. Además, datos recientes indican que células Treg ya establecidas pueden modificar su programa genético para convertirse en células Th17. En esta revisiónse resumen y analizan los datos disponibles actualmente acerca de la biología de las células Th17 (AU)

In recent years the function and developmental pathway for the Thelper subset specialized in IL-17 production (Th17) have been exhaustively studied. This lineage of effector cells plays a decisive role in the immune response to infectious agents, as well as in immunopathologies. Similar to the Th1 and Th2 subsets, the Th17 definition is orchestrated by specific cytokines and transcription factors. A combination of TGF-β plus IL-6, and the transcription factors RORγt, RORα and Stat3 are essential forTh17 commitment. IL-23 plays a key role in the stabilization of the phenotype and in the promotion of the pathogenic activity of IL-17-producercells. The IL-21 cytokine produced by Th17 cells participates in a feedbackmechanism to favour this phenotype, while IL-27, IL-4, IFN-γ, IL-25 andIL-2 cytokines limit the Th17 response. CD4+CD25+Foxp3+regulator cells(Treg) follow a development pathway divergent to Th17 establishment,although both alternatives are governed by TGF-β that directs the fateof naïve CD4+cells to each of these mutually exclusive T cell subsets depending on the presence of IL-6. Furthermore, recent data indicate that preestablished Treg cells can switch its genetic program to become IL-17-producer cells. In this review we summarize and discuss the current available data about the biology of Th17 cells (AU)

Effect of pH, EDTA, and anions on heavy metal toxicity toward a bioluminescent cyanobacterial bioreporter.

The bioavailability and therefore toxicity of a metal depends on the chemical species present in a particular environment. We evaluated the effect of a series of factors that could potentially modify metal speciation on the toxicity of Hg, Cu, Zn, and Cd toward a recombinant strain of the freshwater cyanobacterium Anabaena sp. PCC 7120 with cloned lux operon of luminescent terrestrial bacterium Photorhabdus luminescens. The strain, denoted as Anabaena CPB4337, showed a high constitutive luminescence with no need to add exogenous aldehyde. The tested factors were pH, EDTA (as organic ligand), and anions PO(4)(3-), CO(3)(2-), and Cl(-). Chemical modeling and correlation analyses were used to predict metal speciation and link it with toxicity. In general, metal toxicity significantly correlated to the predicted metal free-ion concentration, although Zn-EDTA complexes and certain Hg chloro-complexes could also exhibit some toxicity to cyanobacteria. An interesting feature of metal toxicity to strain Anabaena CPB4337 was that low amounts of PO(4)(3-) and CO(3)(2-) increased metal toxicity; this effect could not be related to significant changes in metal speciation and could be attributed to a modulating effect of these anions on metal/uptake toxicity. The combination of toxicity studies that take into account a range of factors that might modulate metal toxicity with chemical modeling to predict changes in metal speciation might be useful for interpreting complex toxicity data. Finally, this cyanobacterial bioreporter, due to its ecological relevance as a primary producer, could be used as a tool for toxicity assessment in freshwater environments.

Beta interferon restricts the inflammatory potential of CD4+ cells through the boost of the Th2 phenotype, the inhibition of Th17 response and the prevalence of naturally occurring T regulatory cells.

Beta-interferon (IFN-beta) is a valuable therapy for multiple sclerosis (MS) which is also effective in the animal model of experimental autoimmune encephalomyelitis (EAE). However, the accurate mechanisms to explain its anti-inflammatory activity in the disease are not fully revealed. Available data support that T lymphocytes are among the main cell targets of IFN-beta. We have found that in vitro anti-CD3 stimulation of uncommitted murine naïve T cells under IFN-beta treatment results in skewing the T cell differentiation process towards the T2 phenotype, in a prevention from apoptosis of naturally occurring CD4+ T regulatory cells (nTreg) in correlation with an increase in Bcl-XL expression, and in a decrease of IL-17 expression. Elimination of nTreg from the primary culture of naïve CD4+ cells abolished the down-regulation of IL-17 driven by IFN-beta, what suggests the interaction between Th17 and nTreg subsets. Experiments in EAE induced in SJL mice, showed in vivo evidence for the accumulation of spleen CD4+CD25+GITR+Foxp3+ cells after IFN-beta treatment. On the other hand, treated animals showed a striking decrease of IL-17 expression by peripheral CD4+ cells (Th17) and MBP-specific spinal cord cells. Both the in vivo and in vitro results point out new targets through which IFN-beta could exert its therapeutic action.