The proximal promoter region of mTert is sufficient to regulate telomerase activity in ES cells and transgenic animals

BACKGROUND: The reverse transcriptase of telomerase (Tert) controls telomerase activity maintaining the end of linear chromosomes in eukaryotic cells. Telomerase function is highly active in undifferentiated multipotent stem cells, decreases with cell differentiation and is generally absent from most somatic cells in the adult. Its absence is responsible of telomeres shortening in such somatic cells. Using an in vivo transgenic model and an in vitro culture differentiation of adult stem cells, we examined the elements of the mouse Tert (mTert) promoter that control telomerase activity. RESULTS: Three constructs comprising 1, 2 or 5 kb of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (EGFP) were electroporated into embryonic stem (ES) cells. Transformed ES cells were able to mimic the expected mTert expression, which was associated to green fluorescence. One and 5 kb promoter produced the higher expression of EGFP, on ES cells. When ES cells were allowed to differentiate to embryoid bodies and to other cell types, they lost gradually the expression of mTert-EGFP as consequence of differentiation. No differences were found among the three constructs analyzed. We then generated transgenic mice with the three constructs. Expression of the reporter gene was monitored by reverse transcription-PCR analysis and EGFP visualization. The mRNA expression of the three constructs was lower than the endogenous mTert, but mimicked the endogenous mTert transcription pattern; however, no fluorescent expression of EGFP was detected in adult tissues. EGFP expression of the three constructs was visualized at the blastocysts stage and in new ES cells generated from them; in the germinal ring of E13 dpc foetuses; in ES-like colonies and in germinal stem cells generated from neonatal and adult testis cells; and in neuroesferes generated from E14 dpc foetuses' brain cells. CONCLUSION: The 1 kb promoter upstream of the initiating ATG codon of mTert contains all the regulatory elements to control telomerase expression in ES cells during in vitro loss of pluripotency. The transgenic mouse lines generated represent an appropriate system to analyze the expression of mouse Tert gene under physiological condition and during establishment of stem cell lines generated from embryonic or adult tissues

Diseño de un curso cero para el desarrollo de la competencia matemática y adquisición de conocimientos matemáticos para los alumnos de nuevo ingreso en titulaciones de Grado de Maestro y Pedagogía

Este proyecto continúa el trabajo realizado en el proyecto de innovación nº 21 (2014) y pretende el diseño de un curso cero de conocimientos matemáticos para los alumnos de nuevo ingreso para solventar las carencias detectadas en el proyecto anterior

Plasma mitochondrial DNA levels are inversely associated with HIV-RNA levels and directly with CD4 counts: potential role as a biomarker of HIV replication

[Abstract] Objectives. To evaluate plasma mitochondrial DNA (mtDNA) levels among HIV-infected patients and its potential role as a biomarker of residual viral replication. Methods. HIV-infected patients on follow-up at a reference hospital in north-west Spain were selected. DNA was isolated from plasma samples and mtDNA levels were assessed using a quantitative real-time PCR assay. HIV-RNA levels and CD4+ cell counts were evaluated in the same blood samples used for plasma mtDNA quantification. Epidemiological and clinical variables were included for the analysis. Results. A total of 235 HIV-infected patients were included. Mean plasma mtDNA levels were 217 ± 656 copies/μL for naive (31.9%) and 364 ± 939 copies/μL for HIV-infected patients receiving ART and with suppressed viraemia (P = 0.043). Among the latter, mean plasma mtDNA levels were 149 ± 440 copies/μL for those with low-level viraemia (LLV; HIV-RNA 20–200 copies/mL), 265 ± 723 copies/μL for those with detected-not-quantified (DNQ) viraemia (HIV-RNA <20 copies/mL) and 644 ± 1310 copies/μL for those with not-detected (ND) viraemia. Of note, a linear trend (P = 0.006) was observed among virologically suppressed (LLV, DNQ and ND) patients. ND patients had higher mtDNA levels compared with LLV patients (P = 0.057). Moreover, mtDNA levels were inversely associated with HIV-RNA levels (Spearman’s rho −0.191, P = 0.003) and directly associated with CD4+ counts (Spearman’s rho 0.131, P = 0.046). Conclusions. Increased plasma mtDNA levels are associated with lower HIV-RNA levels and higher CD4+ cell counts. Among ART-suppressed patients, mtDNA levels were significantly higher in those with complete virological suppression (ND) than in those with LLV. These data suggest that plasma mtDNA levels might serve as a biomarker of residual HIV replication.Instituto de Salud Carlos III; CPII14/00014Instituto de Salud Carlos III; PI10/02166Instituto de Salud Carlos III; PI13/02266Instituto de Salud Carlos III; FI14/00557Instituto de Salud Carlos III; CM15/00233Instituto de Salud Carlos III; PI16/02159Instituto de Salud Carlos III; MV16/02159Instituto de Salud Carlos III; PTA2013-8277-

A Novel Circulating MicroRNA for the Detection of Acute Myocarditis.

The diagnosis of acute myocarditis typically requires either endomyocardial biopsy (which is invasive) or cardiovascular magnetic resonance imaging (which is not universally available). Additional approaches to diagnosis are desirable. We sought to identify a novel microRNA for the diagnosis of acute myocarditis. To identify a microRNA specific for myocarditis, we performed microRNA microarray analyses and quantitative polymerase-chain-reaction (qPCR) assays in sorted CD4+ T cells and type 17 helper T (Th17) cells after inducing experimental autoimmune myocarditis or myocardial infarction in mice. We also performed qPCR in samples from coxsackievirus-induced myocarditis in mice. We then identified the human homologue for this microRNA and compared its expression in plasma obtained from patients with acute myocarditis with the expression in various controls. We confirmed that Th17 cells, which are characterized by the production of interleukin-17, are a characteristic feature of myocardial injury in the acute phase of myocarditis. The microRNA mmu-miR-721 was synthesized by Th17 cells and was present in the plasma of mice with acute autoimmune or viral myocarditis but not in those with acute myocardial infarction. The human homologue, designated hsa-miR-Chr8:96, was identified in four independent cohorts of patients with myocarditis. The area under the receiver-operating-characteristic curve for this novel microRNA for distinguishing patients with acute myocarditis from those with myocardial infarction was 0.927 (95% confidence interval, 0.879 to 0.975). The microRNA retained its diagnostic value in models after adjustment for age, sex, ejection fraction, and serum troponin level. After identifying a novel microRNA in mice and humans with myocarditis, we found that the human homologue (hsa-miR-Chr8:96) could be used to distinguish patients with myocarditis from those with myocardial infarction. (Funded by the Spanish Ministry of Science and Innovation and others.).Supported by a grant (PI19/00545, to Dr. Martín) from the Ministry of Science and Innovation through the Carlos III Institute of Health–Fondo de Investigación Sanitaria; by a grant from the Biomedical Research Networking Center on Cardiovascular Diseases (to Drs. Martín, Sánchez-Madrid, and Ibáñez); by grants (S2017/BMD-3671-INFLAMUNE-CM, to Drs. Martín and Sánchez-Madrid; and S2017/BMD-3867-RENIM-CM, to Dr. Ibáñez) from Comunidad de Madrid; by a grant (20152330 31, to Drs. Martín, Sánchez-Madrid, and Alfonso) from Fundació La Marató de TV3; by grants (ERC-2011-AdG 294340-GENTRIS, to Dr. Sánchez-Madrid; and ERC-2018-CoG 819775-MATRIX, to Dr. Ibáñez) from the European Research Council; by grants (SAF2017-82886R, to Dr. Sánchez-Madrid; RETOS2019-107332RB-I00, to Dr. Ibáñez; and SAF2017-90604-REDT-NurCaMeIn and RTI2018-095928-BI00, to Dr. Ricote) from the Ministry of Science and Innovation; by Fondo Europeo de Desarrollo Regional (FEDER); and by a 2016 Leonardo Grant for Researchers and Cultural Creators from the BBVA Foundation to Dr. Martín. The National Center for Cardiovascular Research (CNIC) is supported by the Carlos III Institute of Health, the Ministry of Science and Innovation, the Pro CNIC Foundation, and by a Severo Ochoa Center of Excellence grant (SEV-2015-0505). Mr. Blanco-Domínguez is supported by a grant (FPU16/02780) from the Formación de Profesorado Universitario program of the Spanish Ministry of Education, Culture, and Sports. Ms. Linillos-Pradillo is supported by a fellowship (PEJD-2016/BMD-2789) from Fondo de Garantía de Empleo Juvenil de Comunidad de Madrid. Dr. Relaño is supported by a grant (BES-2015-072625) from Contratos Predoctorales Severo Ochoa para la Formación de Doctores of the Ministry of Economy and Competitiveness. Dr. Alonso-Herranz is supported by a fellowship from La Caixa–CNIC. Dr. Caforio is supported by Budget Integrato per la Ricerca dei Dipartimenti BIRD-2019 from Università di Padova. Dr. Das is supported by grants (UG3 TR002878 and R35 HL150807) from the National Institutes of Health and the American Heart Association through its Strategically Focused Research Networks.S

CD69 controls inflammation in transplant rejection and cardiovascular diseases

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 17-12-2019Esta tesis tiene embargado el acceso al texto completo hasta el 17-06-2021El antígeno CD69 es una proteína transmembrana que se expresa rápidamente en leucocitos tras su activación. Más recientemente ha sido descrita como una molécula que regula negativamente la inflamación mediante la inhibición de la respuesta T efectora. Los ratones deficientes en CD69 son proclives a desarrollar respuestas autoinmunes de tipo Th17 y además tienen defectos en la diferenciación y función de las células T reguladoras, lo que lleva al desarrollo exacerbado de distintas enfermedades inflamatorias crónicas como artritis, colitis, asma, dermatitis o miocarditis autoinmune. Estudios previos demuestran que los ratones Cd69-/- son resistentes al crecimiento de tumores e infección por el virus Vaccinia. Sin embargo, la contribución específica de las células “Natural Killer” (NK) y el mecanismo por el que CD69 podría estar regulando su función se desconocía. En este trabajo hemos realizado un estudio fenotípico exhaustivo de estas células mediante citometría de masas (CyTOF). Este análisis reveló que las células NK Cd69-/- presentan alteraciones de expresión de los receptores Ly49H y Ly49G2 entre otros, implicados en las respuestas frente a MCMV, frente a tumores o en el reconocimiento de células alogénicas. Mediante la interacción con NKG2D, CD69 participa activamente en la reorganización de los receptores de las NKs en la membrana, jugando un papel esencial en su función efectora. Uno de los principales objetivos de este trabajo ha sido el análisis del papel del receptor CD69, expresado en linfocitos, en el desarrollo de aterosclerosis. Estudios previos demostraban que las LDL-oxidadas (LDLox) eran un ligando funcional de CD69 en linfocitos. En este trabajo hemos analizado el papel de esta molécula mediante el estudio de quimeras deficientes en el receptor de LDL (ldlr-/-) en el compartimento linfoide, en régimen de dieta grasa como modelo de aterosclerosis. En este modelo, la deficiencia de CD69 en linfocitos incrementa el ratio Th17/Treg lo que conlleva a un aumento en el tamaño de la placa de ateroma y de la progresión de la enfermedad cardiovascular. La microbiota intestinal modula la inflamación local y sistémica, siendo ciertas bacterias como SFB inductoras de respuestas Th17 que pueden migrar a la periferia afectando la inmunidad sistémica. Varias enfermedades autoinmunes como la escleroris múltiple, encefalomielitis autoinmune, artritis o diabetes tipo I han sido asociadas con la composición de la microbiota. En este estudio comprobamos que la microbiota es esencial en la generación de respuesta Th17 responsable de la inducción y progresión de miocarditis a cardiomiopatía dilatada. En condiciones libres de gérmenes, tanto ratones silvestres como deficientes en CD69 preservan la función cardiaca y no presentan infiltrado leucocitario en el corazón tras inducción de miocarditis autoinmune. Por último, CD69 regula las respuestas Th17 y la composición de la microbiota intestinalThe early lymphocyte activation antigen CD69 is a transmembrane protein that is induced early after activation of all bone marrow-derived cells except erythrocytes. It has been described as an anti-inflammatory molecule that negatively regulates inflammation. CD69 deficient mice display enhanced Th17 differentiation and defective Treg cell function presenting an exacerbated form of chronic inflammatory diseases like arthritis, colitis, allergic asthma, contact dermatitis or autoimmune myocarditis due to their inability to resolve inflammation or to maintain immune tolerance. Previous works have shown that Cd69-/- mice are resistant to tumor growth and vaccinia virus infection in part due to NK cells activity. However, the specific contribution of these cells and the mechanism by which CD69 could be regulating their function has not been elucidated. The activation of NK cells depends on the balance between activating/inhibitory signals. Thus, in this work we take advantage of the new Mass Cytometry (CyTOF) technology to perform an unbiased multiparametric phenotyping of Cd69-/- NK cells. By using different animal models such as anti-viral and anti-tumor immunity and graft vs host disease, we have studied the in vivo consequences of Cd69-/- NK cells phenotype. Our study revealed that Cd69-/- NK cells repertoire of inhibitory/activating receptors is altered, exhibiting high levels of non-self and missing self recognition receptors, due to modulation of NKG2D activity. Cd69-/- NK cells are then more efficient in the killing of allogenic and tumor cells, being Cd69-/- mice resistant to aGvHD. All these effects can be reproduced by an anti-CD69 mAb treatment, suggesting that it may be employed in the clinics to reduce GvH effects while potentially maintaining GvL effects. One of the main objectives of this work has been to analyze the role of CD69 expression in lymphocytes in atherosclerosis development. Using chimeric ldlr−/− mice subjected an HFD as atherosclerosis model, we show that the specific deletion of CD69 on the lymphoid compartment leads to an altered Th17/Treg equilibrium and a consequent increase in atheroma plaque size during HFD. Finally, it is well known that gut microbiota shapes local and systemic inflammation. Certain bacteria like SFB induce the accumulation of Th17 cells that might migrate to the periphery affecting systemic immunity. Indeed, several autoimmune disorders, such as multiple sclerosis, EAE, arthritis, type I diabetes or pancreatitis, have been associated with the gut microbiota composition. In this study we have seen that myocarditis induction and progression depends on the microbiota, as in germ free conditions both CD69 proficient and deficient mice preserve heart function and do not present leukocyte infiltration in the heart after EAM induction. Last, CD69 regulates Th17 responses and microbiota compositio

La interacción entre polifenoles y microbiota oral e intestinal en los trastornos del eje intestino-cerebro y la enfermedad de Alzheimer

Resumen del póster presentado a la 16ª Reunión de la Red Española de Bacterias Lácticas (RedBAL), celebrada en Madrid los días 11 y 12 de mayo de 2023.Debido a sus implicaciones para la salud, el microbioma humano es actualmente uno de los campos más dinámicos de investigación que integra las ciencias básicas y clínicas. Las alteraciones del estado de la microbiota, especialmente la microbiota intestinal, se asocian cada vez más con la incidencia de trastornos cognitivos y neuroinflamación en pacientes con enfermedad de Alzheimer (EA). El envejecimiento también se asocia a alteraciones de la microbiota y en la actualidad el ecosistema que representa la microbiota intestinal del anciano constituye una diana prometedora para múltiples estrategias dirigidas al envejecimiento saludable. Además, en los últimos años también se ha sugerido que algunas bacterias e infecciones orales aumentan el riesgo de desarrollar enfermedades neurodegenerativas, como la EA. Por tanto, existe un gran interés en la comprensión de cómo la microbiota regula la comunicación bidireccional entre el intestino y el cerebro como fundamento de las intervenciones basadas en el microbioma. La dieta se considera el principal factor modificable de las comunidades microbianas y, entre los componentes de la dieta, las investigaciones actuales sugieren que los compuestos fenólicos o polifenoles tienen un alto potencial de modular el microbioma, así como la fisiología y función cerebral. El objetivo global de la línea de investigación que enmarca este trabajo es comprender cómo la dieta, y especialmente los polifenoles presentes en alimentos vegetales, y otros factores del estilo de vida interactúan con el microbioma oral e intestinal en relación a la salud digestiva y el deterioro cognitivo. Para ello, como primera aproximación se está llevando a cabo un estudio prospectivo en pacientes diagnosticados de EA en fase temprana de la enfermedad (n=20) y su comparación con sujetos control de la misma edad, sexo y condición (n=20), reclutados en colaboración con el Servicio de Neurología del Hospital Infanta Sofía (Madrid). La finalidad de este estudio es establecer asociaciones entre las distintas variables de estudio (dieta y estilo de vida, microbioma oral e intestinal y metaboloma fecal, urinario, en plasma y suero), que permitan comprender mejor cómo el ecosistema oral e intestinal, y su potencial comunicación bidireccional, regula la función entre el intestino y el cerebro, como fase previa a definir los fundamentos de las intervenciones basadas en la microbiota. Por otro lado, y sabiendo que los efectos protectores de los polifenoles frente a enfermedades como la EA, podrían atribuirse a una acción directa sobre la respuesta del huésped (no dependiente de la microbiota), pero especialmente mediada por su conversión por la microbiota intestinal en metabolitos fenólicos microbianos biodisponibles, como segunda aproximación se ha llevado a cabo un estudio en un modelo de experimentación animal de taupatía (ratones ‘transgénicos’ P301S) en el que los grupos de ratones de tipo salvaje y ‘transgénicos’ se alimentaron con una dieta estándar y agua, suplementada o no, con una dieta rica en polifenoles durante 6 meses. El efecto protector de la dieta en relación a la EA se ha determinado en muestras de cerebro, intestino, sangre, orina y heces. Como tercera aproximación, se ha evaluado en modelos celulares y animales el efecto de metabolitos fenólicos microbianos con la finalidad de identificar su efecto protector frente a la neuroinflamación y la transmisión sináptica.MICINN (PID2019-108851RB-C21; PDC2022-133861-C21); Comunidad de Madrid (ALIBIRD2020-CM P2018/BAA-4343).N