Elaboración de blogs como herramienta virtual de aprendizaje y trabajo en equipo

Este proyecto de innovación se enmarca dentro de las actividades académicas no presenciales. Se trata de la elaboración de blogs como herramienta virtual de aprendizaje. Los blogs son un recurso de aprendizaje individual o grupal, de gran versatilidad y dinamización del aula. Además, promueve el desarrollo de competencias generales, específicas y transversales que los alumnos deben adquirir durante su formación. Creemos que su aplicación es especialmente interesante en asignaturas como “Bases Celulares de la Genética Humana” impartida en primero de Medicina, donde los alumnos se encuentran con una dificultad añadida a la complejidad de sus fundamentos: unos conceptos complejos con una terminología muy específica para asimilarlos. Los futuros médicos deberán ser capaces, de explicar con un lenguaje sencillo y comprensible a los pacientes la implicación de la genética en las patologías. Consideramos que el uso de blogs está especialmente indicado en este caso para facilitar la adquisición de esta competencia específica, además de apoyar el aprendizaje de parte de los contenidos de la asignatura y promover el trabajo en equipo. Esta herramienta aporta a los alumnos las competencias necesarias para su formación integral como profesional de la salud promoviendo el intercambio colaborativo. Es además, un recurso que puede ser utilizado tanto para la evaluación continua, por parte del profesor durante y a la finalización del diseño del blog, así como para la coevaluación entre los alumnos. Por tanto, esta propuesta facilita el aprendizaje creando espacios para ello. En definitiva, crea oportunidades de aprendizaje

Aplicación de la clase invertida en la asignatura Biología Celular e Histología

La clase invertida o clase al revés, también conocida por el anglicismo flipped classroom, es una metodología docente en la que, como su nombre indica, se invierten los procesos que clásicamente tienen lugar dentro y fuera del aula. De manera que se pasa de una clase centrada en el profesor, como transmisor de la información y con un alumno receptor pasivo de la información cuyo trabajo fuera del aula consiste en la realización de tareas, a un modelo en el que el alumno, debe recibir y asimilar la información que le es facilitada previamente a la clase. En el aula, los alumnos resuelven dudas y realizan tareas, en muchos casos en equipo, de manera que el trabajo en el aula pasa a estar centrado en el alumno, que debe ser responsable de su propio aprendizaje. Los profesores que ya aplican esta metodología en distintos niveles educativos, se muestran muy satisfechos con el aumento del rendimiento de los estudiantes, la gran aceptación que tiene esta metodología, el aumento del aprendizaje significativo, es decir, la capacidad de relacionar los conocimientos previos con los nuevos y ser capaces de aplicar lo aprendido para la resolución de problemas. La asignatura “Biología Celular e Histología” del Grado en Biología se imparte con carácter anual durante el primer curso del grado, con una carga docente de 12 ECTS. Cada año se matriculan en la asignatura más de 400 alumnos, 300 en primera matrícula. Los contenidos teóricos que deben adquirir los alumnos son extensos y complejos, lo que supone una notable carga de trabajo para los alumnos que, si se limitan a una mera asistencia pasiva a clase, no son capaces de obtener un aprovechamiento óptimo de los contenidos y por tanto obtener buenos resultados. Además de esos contenidos teóricos, los alumnos deben adquirir unos contenidos prácticos mediante 5 sesiones de laboratorio de Biología Celular y 9 sesiones de laboratorio de Histología. Existen varios profesores implicados en la docencia teórica de esta asignatura, ya que los alumnos matriculados se dividen en 6 grupos y en algunos casos, la docencia se divide entre dos profesores, uno encargado de la parte de Biología Celular y otro de la parte de Histología. Cada profesor tiene su propio perfil docente, pero todos basamos nuestras clases en una metodología tradicional de clase magistral, es decir, una docencia centrada en el profesor, como foco transmisor de información que el alumno debe asimilar. Todos utilizamos el Campus Virtual donde colocamos las presentaciones que utilizamos en clase y donde organizamos las sesiones de seminarios. En general, año tras año, los profesores de esta asignatura observamos una falta de implicación de los alumnos en su propio aprendizaje, de manera que reciben la información de manera pasiva, escuchando al profesor, en muchos casos sin ni siquiera tomar apuntes. Esta pasividad y escasa implicación del alumnado conlleva unos resultados reflejados en las calificaciones globales generalmente decepcionantes, y que a nuestro juicio son susceptibles de mejora aumentando la motivación del alumnado mediante nuevas metodologías docentes. Con este proyecto de innovación docente, hemos elaborado el material necesario para aplicar el método “flipped learning” o clase invertida en un bloque de temas de Biología Celular y en otro bloque de temas de Histología. Este tipo de metodología centrada en el alumno, permite que el estudiante procese y maneje la información suministrada de forma autónoma, antes de acudir a clase, a su ritmo, de manera que el tiempo de clase puede usarse de otras maneras, por ejemplo, para solucionar dudas, discutir a fondo los aspectos más complicados del tema, o para realizar tareas de aplicación de los conocimientos previamente aprendidos en casa. De esta manera se puede lograr un aprendizaje significativo alejado de la mera memorización de los contenidos expuestos, y en donde el alumno se sienta protagonista de su propio avance y capacitación teórica de la asignatura

Integrin and CD3/TCR activation are regulated by the scaffold protein AKAP450

During antigen recognition by T cells, membrane receptors and cytoskeletal molecules form a specialized structure at the T cell–antigen-presenting cell junction called the immune synapse (IS). We report a role for the scaffolding protein A-kinase anchoring protein-450 (AKAP450), a member of the A-kinase anchoring protein family, in IS formation and T-cell signaling in antigen- and superantigen-dependent T-cell activation. Suppression of AKAP450 by overexpression of a dominant-negative form or siRNA knockdown disrupted the positioning and conformational activation of lymphocyte function-associated antigen 1 at the IS and impaired associated signaling events, including phosphorylation of phospholipase C-γ1 and protein kinase C-θ. AKAP450 was also required for correct activation and phosphorylation of CD3, LAT, and Vav1, key T-cell receptor-activated intracellular signaling molecules. Consistently, antigen-triggered reorientation of the microtubule-organizing center at the IS and interleukin-2 secretion were diminished in AKAP450-disrupted T cells. These results indicate key roles for AKAP450 in the organization and activation of receptor molecules at the IS during T-cell signaling events.Ministerio de Educación y CienciaComunidad de MadridFundación CNIC del Instituto Carlos IIIRed RECAVADepto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

Atherosclerosis development in apolipoprotein E-null mice deficient for CD69

Aims: Atherosclerosis is a chronic inflammatory disease regulated by immune mechanisms. CD69 is a cell surface receptor rapidly induced after leukocyte activation at sites of chronic inflammation. Genetic disruption of CD69 in the mouse aggravates collagen-induced arthritis (CIA), and partial depletion of CD69-expressing cells with anti-CD69 monoclonal antibody (mAb) prevents CIA development in wild-type mice, suggesting that this receptor negatively modulates immune and inflammatory responses. It has been recently reported that CD69 is upregulated in a large subset of T cells in atherosclerosis-prone apolipoprotein E-null mice (apoE−/−). In this study, we investigated whether altering CD69 function affects atherosclerosis development. Methods and results: We studied native and diet-induced atherosclerosis in apoE−/− and doubly deficient apoE−/−CD69−/− mice and performed expression studies in tissues and primary cells derived from these animals. Plasma cholesterol level was unaffected by CD69 genetic inactivation. Although this genetic manipulation led to an elevated production of interferon γ and interleukin 10 by activated T cells, apoE−/− and apoE−/−CD69−/− mice fed control and high-fat diet exhibited atheromas of similar size and composition when analysed at different stages of the disease. Likewise, anti-CD69 mAb treatment had no effect on plasma cholesterol and atherosclerosis burden in fat-fed apoE−/− mice. Conclusion: In contrast to previous studies highlighting the protective function of CD69 against CIA, an autoimmune inflammatory disease, our results rule out a significant role for CD69 against atherosclerosis in apoE−/− mice, an experimental disease model featuring a local inflammatory response triggered and sustained by alterations in lipid homeostasis.Instituto de Salud Carlos III/Ministerio de Sanidad y ConsumoMinisterio de Educación y Ciencia/Fondo Europeo de Desarrollo RegionalMinisterio de Ciencia y Tecnología (MCYT). Programa Ramón y Cajal 2002Depto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

Impact of Genetic Variants of ATP Binding Cassette B1, AICAR Transformylase/IMP Cyclohydrolase, Folyl-polyglutamate Synthetase, and Methylenetetrahydrofolate Reductase on Methotrexate Toxicity

Objective: To analyze the effect of single nucleotide polymorphisms (SNPs) with well-known functional impact of methylenetetrahydrofolate reductase (MTHFR; rs1801131 and rs1801133), the membrane transporter ABCB1 (rs1045642), the AICAR transformylase/IMP cyclohydrolase (ATIC; rs2372536) and folyl-polyglutamate synthetase (FPGS; rs1544105), on liver and bone marrow toxicity of methotrexate (MTX). Patients and methods: We analyzed 1415 visits from 350 patients of the PEARL (Princesa Early Arthritis Register Longitudinal) study: (732 with MTX, 683 without MTX). The different SNPs were genotyped using speci c TaqMan probes (Applied Biosystems). Multivariate analyzes were performed using generalized linear models in which the dependent variables were the levels of serum alanine aminotransferase (liver toxicity), leukocytes, platelets or hemoglobin (hematologic toxicity) and adjusted for clinical variables (disease activity, etc.), analytical (renal function, etc.), sociodemographic (age, sex, etc.) and genetic variants of MTHFR, ABCB1, ATIC and FPGS. The effect of these variables on the MTX doses prescribed throughout follow-up was also analyzed through multivariate analysis nested by visit and patient. Results: When taking MTX, those patients carrying the CC genotype of rs1045642 in ABCB1 showed signi cantly higher GPT levels (7.1 2.0 U/L; P < .001). Carrying at least one G allele of rs1544105 in FPGS was associated with lower leukocyte ( 0.67 0.32; 0.038), hemoglobin ( 0.34 0.11 g/dL; P = .002), and platelet ( 11.8 4.7; P = .012) levels. The presence of the G allele of rs1544105 in FPGS, and the T allele of rs1801133 in MTHFR, was signi cantly associated with the use of lower doses of MTX. Discussion: Our data suggest that genotyping functional variants in FGPS and MTHFR enzymes and the transporter ABCB1 could help to identify patients with increased risk of MTX toxicity.Ministerio de Economía y Competitividad (MINECO)/Fondo Europeo de Desarrollo Regional (FEDER)Instituto de Salud Carlos IIIDepto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

CD69 Association with Jak3/Stat5 Proteins Regulates Th17 Cell Differentiation

This work was supported by grant SAF2008-02719 from the Spanish Ministry of Science and Innovation to P.M.; grant PI06/0937 from ISCIII-MSC to M.G.; and grants SAF2008-02635 from the Spanish Ministry of Science and Innovation, RECAVA [RD06/0014-0030] from the Instituto de Salud Carlos III, MEICA from the Genoma Espan˜a Foundation, and INSINET 01592006 from Comunidad de Madrid to F.S.-M. P.M. is an investigator for the Spanish Ministry of Science and Innovation Ramo´n y Cajal Program (RYC-2006). The CNIC is supported by the Ministry of Science and Innovation and the Pro CNIC Foundation.T-cell differentiation involves the early decision to commit to a particular pattern of response to an antigen. Here, we show that the leukocyte activation antigen CD69 limits differentiation into proinflammatory helper T cells (Th17 cells). Upon antigen stimulation in vitro, CD4+ T cells from CD69-deficient mice generate an expansion of Th17 cells and the induction of greater mRNA expression of interleukin 17 (IL-17), IL 23 receptor (IL-23R), and the nuclear receptor retinoic acid-related orphan receptor γt (RORγt). In vivo studies with CD69-deficient mice bearing OTII T-cell receptors (TCRs) specific for OVA peptide showed a high proportion of antigen-specific Th17 subpopulation in the draining lymph nodes, as well as in CD69-deficient mice immunized with type II collagen. Biochemical analysis demonstrated that the CD69 cytoplasmic tail associates with the Jak3/Stat5 signaling pathway, which regulates the transcription of RORγt and, consequently, differentiation toward the Th17 lineage. Functional experiments in Th17 cultures demonstrated that the selective inhibition of Jak3 activation enhanced the transcription of RORγt. Moreover, the addition of exogenous IL-2 restored Stat5 phosphorylation and inhibited the enhanced Th17 differentiation in CD69-deficient cells. These results support the early activation receptor CD69 as an intrinsic modulator of the T-cell differentiation program that conditions immune inflammatory processes.Depto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

Identification of Genes Responsive to Solar Simulated UV Radiation in Human Monocyte-Derived Dendritic Cells

Ultraviolet (UV) irradiation has profound effects on the skin and the systemic immune system. Several effects of UV radiation on Dendritic cells (DCs) functions have been described. However, gene expression changes induced by UV radiation in DCs have not been addressed before. In this report, we irradiated human monocyte-derived DCs with solar-simulated UVA/UVB and analyzed regulated genes on human whole genome arrays. Results were validated by RT-PCR and further analyzed by Gene Set Enrichment Analysis (GSEA). Solar-simulated UV radiation up-regulated expression of genes involved in cellular stress and inflammation, and down-regulated genes involved in chemotaxis, vesicular transport and RNA processing. Twenty four genes were selected for comparison by RT-PCR with similarly treated human primary keratinocytes and human melanocytes. Several genes involved in the regulation of the immune response were differentially regulated in UVA/UVB irradiated human monocyte-derived DCs, such as protein tyrosine phosphatase, receptor type E (PTPRE), thrombospondin-1 (THBS1), inducible costimulator ligand (ICOSL), galectins, Src-like adapter protein (SLA), IL-10 and CCR7. These results indicate that UV-exposure triggers the regulation of a complex gene repertoire involved in human-DC–mediated immune responses.Ministerio de Educación yCienciaPrograma de Estimulo para laTransferencia de Resultados de la Investigacion(PETRI)I.F. CantabriaDepto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

Synaptic Clusters of MHC Class II Molecules Induced on DCs by Adhesion Molecule–mediated Initial T-Cell Scanning

Initial adhesive contacts betweenT lymphocytes anddendritic cells (DCs) facilitate recognitionof peptide-MHC complexesbytheTCR. Inthisreport,westudiedthedynamicbehaviorofadhesionandAgreceptorsonDCsduring initialcontactswithT-cells.AdhesionmoleculesLFA-1-andICAM-1,3-GFPaswellasMHCclassII-GFPmoleculeswere veryrapidlyconcentratedattheDCcontactarea.BindingofICAM-3,andICAM-1toalesserextent, toLFA-1expressed bymaturebutnot immatureDC, inducedMHC-IIclusteringintotheimmunesynapse.Also, ICAM-3bindingtoDC inducedtheactivationoftheVav1-Rac1axis,aregulatorypathwayinvolvedinactincytoskeletonreorganization,which wasessentialforMHC-IIclusteringonDCs.OurresultssupportamodelinwhichICAM-mediatedMHC-IIclustering onDCconstitutesaprimingmechanismtoenhanceantigenpresentationtoT-cells.Ministerio de Educación, Cultura y DeporteFundación Juan March. Ayuda a la Investigación 2002Depto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

Comparative analysis of EV isolation procedures for miRNAs detection in serum samples

This work was supported by grant PIE13/00041 from Instituto de Salud Carlos III and co-funded by Fondo Europeo de Desarrollo Regional (FEDER) to MM, IG-A, FS-M and MY-M, and BFU2014-55478-R from Ministerio de Economía y Competitividad to MY-M.Extracellular vesicles (EVs) are emerging as potent non-invasive biomarkers. However, current methodologies are time consuming and difficult to translate to clinical practice. To analyse EV-encapsulated circulating miRNA, we searched for a quick, easy and economic method to enrich frozen human serum samples for EV. We compared the efficiency of several protocols and commercial kits to isolate EVs. Different methods based on precipitation, columns or filter systems were tested and compared with ultracentrifugation, which is the most classical protocol to isolate EVs. EV samples were assessed for purity and quantity by nanoparticle tracking analysis and western blot or cytometry against major EV protein markers. For biomarker validation, levels of a set of miRNAs were determined in EV fractions and compared with their levels in total serum. EVs isolated with precipitation-based methods were enriched for a subgroup of miRNAs that corresponded to miRNAs described to be encapsulated into EVs (miR-126, miR-30c and miR-143), while the detection of miR-21, miR-16-5p and miR-19a was very low compared with total serum. Our results point to precipitation using polyethylene glycol (PEG) as a suitable method for an easy and cheap enrichment of serum EVs for miRNA analyses. The overall performance of PEG was very similar, or better than other commercial precipitating reagents, in both protein and miRNA yield, but in comparison to them PEG is much cheaper. Other methods presented poorer results, mostly when assessing miRNA by qPCR analyses. Using PEG precipitation in a longitudinal study with human samples, we demonstrated that miRNA could be assessed in frozen samples up to 8 years of storage. We report a method based on a cut-off value of mean of fold EV detection versus serum that provides an estimate of the degree of encapsulation of a given miRNA.Depto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

Galectin-1: A Potential Biomarker Differentiating between Early Rheumatoid Arthritis and Spondyloarthritis

This research was funded by grants RD21/0002/0027 and PI21/00526 to IG-A, PI21/01583 to H.dF and PI21/01474 to SC from the Ministerio de Economía y Competitividad (Instituto de Salud Carlos III) and co-funded by European regional development fund (ERDF) “A way to make Europe”. The work of ER-V is currently funded by a grant Rio-Hortega CM19/00149 from the Ministerio de Economía y Competitividad (Instituto de Salud Carlos III) and co-funded by European regional development fund (ERDF) “A way to make Europe”Galectin-1 (Gal1) plays a regulatory role in the immune system. We have recently validated that Gal1 serum (sGal1) levels are increased in rheumatoid arthritis (RA) patients compared to healthy donors (HDs); however, there is no information on Gal1 in spondyloarthritis (SpA). Objective: To compare Gal1 levels in patients with SpA versus RA as a diagnostic biomarker. Methods: We studied sGal1 levels in HD (n = 52), SpA (n = 80) and RA patients (n = 64) who were randomly divided into discovery and validation sets. Synovial fluid (SF) from osteoarthritis (OA) (n = 28), peripheral SpA (n = 28) and RA (n = 28) were studied. In SpA patients, we analyzed the association between clinical parameters and sGal1 levels. Results: sGal1 levels were significantly lower in patients with SpA with respect to RA and similar to those of the HD. A cut-off of 20.50 ng/mL (sGal1) allowed one to differentiate RA patients from SpA and HD (Odd Ratio (OR) 8.23 and 12.64, respectively). Gal1 SF levels in SpA were slightly lower than OA patients and significantly lower than RA patients. No correlation was observed between sGal1 levels and clinical parameters in SpA patients. Conclusion: Gal1 could act as a diagnostic biomarker of RA and would allow one to distinguish SpA and RA patients.Depto. de Biología CelularFac. de Ciencias BiológicasTRUEpu