Gene therapy with mesenchymal stem cells expressing IFN-ß ameliorates neuroinflammation in experimental models of multiple sclerosis

[Background and Purpose]: Recombinant IFN‐ß is one of the first‐line treatments in multiple sclerosis (MS), despite its lack of efficacy in some patients. In this context, mesenchymal stem cells (MSCs) represent a promising therapeutic alternative due to their immunomodulatory properties and multipotency. Moreover, by taking advantage of their pathotropism, these cells can be genetically modified to be used as carriers for delivering or secreting therapeutic drugs into injured tissues. Here, we report the therapeutic effect of systemic delivery of adipose‐derived MSCs (AdMSCs), transduced with the IFN‐β gene, into mice with experimental autoimmune encephalomyelitis (EAE).[Experimental Approach]: Relapsing–remitting and chronic progressive EAE were induced in mice. Cells were injected i.v. Disease severity, inflammation and tissue damage were assessed clinically, by flow cytometry of spleens and histopathological evaluation of the CNS respectively.[Key Results]: Genetic engineering did not modify the biological characteristics of these AdMSCs (morphology, growth rate, immunophenotype and multipotency). Furthermore, the transduction of IFN‐ß to AdMSCs maintained and, in some cases, enhanced the functional properties of AdMSCs by ameliorating the symptoms of MS in EAE models and by decreasing indications of peripheral and central neuro‐inflammation.[Conclusion and Implications]: Gene therapy was found to be more effective than cell therapy in ameliorating several clinical parameters in both EAE models, presumably due to the continuous expression of IFN‐β. Furthermore, it has significant advantages over AdMSC therapy, and also over systemic IFN‐ß treatment, by providing long‐term expression of the cytokine at therapeutic concentrations and reducing the frequency of injections, while minimizing dose‐limiting side effects.This work was supported by Fondo de Investigaciones Sanitarias ISCIII (Spain) and Fondo Europeo de Desarrollo Regional (FEDER) from the European Union through the research grants PI12/01097 and PI15/00963 and ISCIII Red de Terapia Celular TerCel RD12/0019/0006 to F.M., by the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía‐FEDER/Fondo de Cohesion Europeo (FSE) de Andalucía through the research grants P09‐CTS‐04532, PI‐57069 and PAIDI‐Bio‐326 to F.M. and PI‐0160/2012 to K.B. M.J.P.‐M. has been supported by grants from Red Temática de Investigación Cooperativa Red Española de Esclerosis Múltiple REEM (RD07/0060 and RD12/0032). B.O. is financed by a contract from Excelent Project CTS‐7670/11 from Consejería de Economía, Innovación, Ciencia y Empleo (Junta de Andalucía)

TRAIL/TRAIL Receptor System and Susceptibility to Multiple Sclerosis

The TNF-related apoptosis inducing ligand (TRAIL)/TRAIL receptor system participates in crucial steps in immune cell activation or differentiation. It is able to inhibit proliferation and activation of T cells and to induce apoptosis of neurons and oligodendrocytes, and seems to be implicated in autoimmune diseases. Thus, TRAIL and TRAIL receptor genes are potential candidates for involvement in susceptibility to multiple sclerosis (MS). To test whether single-nucleotide polymorphisms (SNPs) in the human genes encoding TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 are associated with MS susceptibility, we performed a candidate gene case-control study in the Spanish population. 59 SNPs in the TRAIL and TRAIL receptor genes were analysed in 628 MS patients and 660 controls, and validated in an additional cohort of 295 MS patients and 233 controls. Despite none of the SNPs withstood the highly conservative Bonferroni correction, three SNPs showing uncorrected p values<0.05 were successfully replicated: rs4894559 in TRAIL gene, p = 9.8×10−4, OR = 1.34; rs4872077, in TRAILR-1 gene, p = 0.005, OR = 1.72; and rs1001793 in TRAILR-2 gene, p = 0.012, OR = 0.84. The combination of the alleles G/T/A in these SNPs appears to be associated with a reduced risk of developing MS (p = 2.12×10−5, OR = 0.59). These results suggest that genes of the TRAIL/TRAIL receptor system exerts a genetic influence on MS

Synthesis and pharmacological evaluation of sulfamide-based analogues of anandamide

Arachidonyl and linoleyl sulfamide derivatives have been synthesized and their potential cannabimi- metic properties evaluated in in vitro functional and binding assays. Replacement of the ethanolamide moiety of anandamide by –CH2NHSO2NH–R considerably reduces the CB1 receptor activity and only some of the compounds showed modest cannabinoid properties in binding assays. The new compounds were also tested as inhibitors of the FAAH enzyme but were inactive.Financial support from Spanish MCYT (SAF 2003-08003, SAF 2006-13391) FIS and Fundacio´n Eugenio Rodrı´guez Pascual is gratefully acknowledged.Peer reviewe

Guía de uso adecuado de modelos animals para el desarrollo de terapias en esclerosis múltiple

Introduction. The advance in the achievement of efective therapies for multiple sclerosis (MS), the defnition of appropriate therapeutic windows and to establish better diagnostic and prognostic biomarkers have become a challenging task for both researchers and clinicians. Some pitfalls in clinical trials might be related to lack of adequacy of the preclinical studies in MS experimental animal models. Aim. To standardize the methodological protocols of experimental models by developing a set of guidelines for preclinical studies by groups of experts from REEM (Spanish Network for MS). Development. A guide with recommendations for the application of MS models including a detailed assessment of appropriate experimental models taking into account the objective of the study that has been presented. Standards and quality criteria necessary in a preclinical study have been included. Conclusions. Standardized animal models of MS are essential to increase the success of the preclinical fndings in order to transfer them to the clinical practice. © 2012 Revista de Neurología.Peer Reviewe

The presence and suppressive activity of Myeloid-Derived Suppressor Cells are potentiated after Interferon-ß treatment in a murine model of Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the human central nervous system (CNS), mainly affecting young adults. Among the immunomodulatory disease modifying treatments approved up to date to treat MS, IFN-β remains to be one of the most widely prescribed for the Relapsing-Remitting (RR) variant of the disease, although its mechanism of action is still partially understood. RR-MS variant is characterized by phases with increasing neurological symptoms (relapses) followed by periods of total or partial recovery (remissions), which implies the existence of immunomodulatory agents to promote the relapsing-to-remitting transition. Among these agents, it has been described the immunosuppressive role of a heterogeneous population of immature myeloid cells, namely the myeloid-derived suppressor cells (MDSCs) during the clinical course of the experimental autoimmune encephalomyelitis (EAE), the most used MS model to study RRMS. However, it is still unknown how the current MS disease modifying treatments, e.g. IFN- β, affects to MDSCs number or activity. Our present results show that a single injection of IFN-β at the onset of the clinical course reduces the severity of the EAE, enhancing the presence of MDSCs within the smaller demyelinated areas. Moreover, the single dose of IFN-β promotes MDSC immunosuppressive activity both in vivo and in vitro, augmenting T cell apoptosis. Finally, we show that IFN-ß preserves MDSC immaturity, preventing their differentiation to mature and less suppressive myeloid cell subsets. Taking together, all these data add new insights into the mechanism of IFN-β treatment in EAE and point to MDSCs as a putative endogenous mediator of its beneficial role in this animal model of MS.This work was supported by the Spanish Ministerio de Economía, Industria y Competitividad-MINEICO (SAF2012-40023; SAF2016-77575-R; RD12-0032-12; RD16-0015-0019; PI15-00963; PI18/00357, partially financed by F.E.D.E.R.: European Union, “Una Manera de hacer Europa”), the Spanish Research Council/Consejo Superior de Investigaciones Científicas-CSIC (CSIC-2015201023), ADEM-TO (Spain) and ARSEP Foundation (France). DC, RL-G and IM-D were financed by SESCAM; CM-J holds a predoctoral Research Training contract from MINEICO (BES-2013-062630, –associated to SAF2012-40023 and PI15-00963) and is currently hired under SAF2016-77577-R. Dr. Clemente's group was sponsored by Aciturri Aeronáutica SLU, Vesuvius Ibérica LA and Fundación Galletas Coral

The presence and suppressive activity of myeloid-derived suppressor cells are potentiated after interferon-β treatment in a murine model of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the human central nervous system (CNS), mainly affecting young adults. Among the immunomodulatory disease modifying treatments approved up to date to treat MS, IFN-β remains to be one of the most widely prescribed for the Relapsing-Remitting (RR) variant of the disease, although its mechanism of action is still partially understood. RR-MS variant is characterized by phases with increasing neurological symptoms (relapses) followed by periods of total or partial recovery (remissions), which implies the existence of immunomodulatory agents to promote the relapsing-to-remitting transition. Among these agents, it has been described the immunosuppressive role of a heterogeneous population of immature myeloid cells, namely the myeloid-derived suppressor cells (MDSCs) during the clinical course of the experimental autoimmune encephalomyelitis (EAE), the most used MS model to study RRMS. However, it is still unknown how the current MS disease modifying treatments, e.g. IFN- β, affects to MDSCs number or activity. Our present results show that a single injection of IFN-β at the onset of the clinical course reduces the severity of the EAE, enhancing the presence of MDSCs within the smaller demyelinated areas. Moreover, the single dose of IFN-β promotes MDSC immunosuppressive activity both in vivo and in vitro, augmenting T cell apoptosis. Finally, we show that IFN-ß preserves MDSC immaturity, preventing their differentiation to mature and less suppressive myeloid cell subsets. Taking together, all these data add new insights into the mechanism of IFN-β treatment in EAE and point to MDSCs as a putative endogenous mediator of its beneficial role in this animal model of MS

Antiobesity designed multiple ligands: Synthesis of pyrazole fatty acid amides and evaluation as hypophagic agents

Searching for new antiobesity agents, a new series of fatty acid amide derivatives of 1,5-diarylpyrazole have been synthesized as dual peroxisome proliferator activated receptor alpha (PPARa)/cannabinoid receptor ligands. The compounds have been evaluated in vivo and in vitro as PPARa activators and as can- nabinoids in two tests of the mouse tetrad. In vivo, food intake studies have been performed with all the compounds. No significant cannabinoid activity has been found but some compounds behaved as potent PPARa activators. Several compounds showed anorexigenic properties reducing food intake in rats.This work has been supported by Grants from Proyectos de Excelencia de la Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía, Instituto de Salud Carlos III (FIS PI04/ 0834, CP04/0039, FIS 07/1226, Redes Temáticas RD06/001), Fun- dación Eugenio Rodríguez Pascual and Fundació Marató TV3. MCyT, proyectos: MAT2006-01997, SAF2006-13391-C03-02 and ‘‘Factoría de Cristalización” Consolider Ingenio 2010. M. Alva- rado is also grateful to the Xunta de Galicia (Spain) for a post- doctoral fellowship.Peer reviewe

Antiobesity designed multiple ligands: Synthesis of pyrazole fatty acid amides and evaluation as hypophagic agents

Searching for new antiobesity agents, a new series of fatty acid amide derivatives of 1,5-diarylpyrazole have been synthesized as dual peroxisome proliferator activated receptor alpha (PPARa)/cannabinoid receptor ligands. The compounds have been evaluated in vivo and in vitro as PPARa activators and as can- nabinoids in two tests of the mouse tetrad. In vivo, food intake studies have been performed with all the compounds. No significant cannabinoid activity has been found but some compounds behaved as potent PPARa activators. Several compounds showed anorexigenic properties reducing food intake in rats.This work has been supported by Grants from Proyectos de Excelencia de la Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía, Instituto de Salud Carlos III (FIS PI04/ 0834, CP04/0039, FIS 07/1226, Redes Temáticas RD06/001), Fun- dación Eugenio Rodríguez Pascual and Fundació Marató TV3. MCyT, proyectos: MAT2006-01997, SAF2006-13391-C03-02 and ‘‘Factoría de Cristalización” Consolider Ingenio 2010. M. Alva- rado is also grateful to the Xunta de Galicia (Spain) for a post- doctoral fellowship.Peer reviewe

Antiobesity designed multiple ligands: Synthesis of pyrazole fatty acid amides and evaluation as hypophagic agents

Searching for new antiobesity agents, a new series of fatty acid amide derivatives of 1,5-diarylpyrazole have been synthesized as dual peroxisome proliferator activated receptor alpha (PPARα)/cannabinoid receptor ligands. The compounds have been evaluated in vivo and in vitro as PPARα activators and as cannabinoids in two tests of the mouse tetrad. In vivo, food intake studies have been performed with all the compounds. No significant cannabinoid activity has been found but some compounds behaved as potent PPARα activators. Several compounds showed anorexigenic properties reducing food intake in rats

Computational and Biological Evaluation of <i>N</i>-octadecyl-<i>N</i>′-propylsulfamide, a Selective PPARα Agonist Structurally Related to <i>N</i>-acylethanolamines

<div><p>To further understand the pharmacological properties of N-oleoylethanolamine (OEA), a naturally occurring lipid that activates peroxisome proliferator-activated receptor alpha (PPARα), we designed sulfamoyl analogs based on its structure. Among the compounds tested, N-octadecyl-N′-propylsulfamide (CC7) was selected for functional comparison with OEA. The performed studies include the following computational and biological approaches: 1) molecular docking analyses; 2) molecular biology studies with PPARα; 3) pharmacological studies on feeding behavior and visceral analgesia. For the docking studies, we compared OEA and CC7 data with crystallization data obtained with the reference PPARα agonist GW409544. OEA and CC7 interacted with the ligand-binding domain of PPARα in a similar manner to GW409544. Both compounds produced similar transcriptional activation by <i>in vitro</i> assays, including the GST pull-down assay and reporter gene analysis. In addition, CC7 and OEA induced the mRNA expression of CPT1a in HpeG2 cells through PPARα and the induction was avoided with PPARα-specific siRNA. <i>In vivo</i> studies in rats showed that OEA and CC7 had anorectic and antiobesity activity and induced both lipopenia and decreases in hepatic fat content. However, different effects were observed when measuring visceral pain; OEA produced visceral analgesia whereas CC7 showed no effects. These results suggest that OEA activity on the PPARα receptor (e.g., lipid metabolism and feeding behavior) may be dissociated from other actions at alternative targets (e.g., pain) because other non cannabimimetic ligands that interact with PPARα, such as CC7, do not reproduce the full spectrum of the pharmacological activity of OEA. These results provide new opportunities for the development of specific PPARα-activating drugs focused on sulfamide derivatives with a long alkyl chain for the treatment of metabolic dysfunction.</p></div