MicroRNA-181a regulates IFN-γ expression in effector CD8+ T cell differentiation

CD8+ T cells are key players in immunity against intracellular infections and tumors. The main cytokine associated with these protective responses is interferon-γ (IFN-γ), whose production is known to be regulated at the transcriptional level during CD8+ T cell differentiation. Here we found that microRNAs constitute a posttranscriptional brake to IFN-γ expression by CD8+ T cells since the genetic interference with the Dicer processing machinery resulted in the overproduction of IFN-γ by both thymic and peripheral CD8+ T cells. Using a gene reporter mouse for IFN-γ locus activity, we compared the microRNA repertoires associated with the presence or absence of IFN-γ expression. This allowed us to identify a set of candidates, including miR-181a and miR-451, which were functionally tested in overexpression experiments using synthetic mimics in peripheral CD8+ T cell cultures. We found that miR-181a limits IFN-γ production by suppressing the expression of the transcription factor Id2, which in turn promotes the Ifng expression program. Importantly, upon MuHV-4 challenge, miR-181a-deficient mice showed a more vigorous IFN-γ+ CD8+ T cell response and were able to control viral infection significantly more efficiently than control mice. These data collectively establish a novel role for miR-181a in regulating IFN-γ-mediated effector CD8+ T cell responses in vitro and in vivo.info:eu-repo/semantics/publishedVersio

Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis

Funding Information: This work was supported by European Regional Development Fund (FEDER) through the Operational Program for Competitiveness Factors (COMPETE) [HealthyAging2020 CENTRO-01-0145-FEDER-000012-N2323, POCI-01-0145-FEDER-016385, POCI-01-0145-FEDER-007440 to CNC.IBILI, POCI-01-0145-FEDER-007274 to i3S/INEB and NORTE-01-0145-FEDER-000012 to T.L.L.]; national funds through the Portuguese Foundation for Science and Technology (FCT) [PTDC/SAU-ORG/119296/2010, PTDC/ NEU-OSD/0312/2012, PESTC/ SAU/UI3282/2013-2014, MITP-TB/ECE/0013/ 2013, FCT-UID/NEU/04539/2013], PD/BD/52294/2013 to T.M.R.R., SFRH/ BD/85556/2012 (co-financed by QREN) to V.C.S]; Lisboa Portugal Regional Operational Programme (LISBOA 2020) and Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement; and by INFARMED Autoridade Nacional do Medicamento e Produtos de Saúde, I.P. [FIS-FIS-2015-01_CCV_20150630-157]. Publisher Copyright: © 2017 The Author.Aims Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs). Methods and results Exosomes secreted by H9c2 myocardial cells and primary cardiomyocytes, cultured either in control or ischaemic conditions were isolated and added to ECs. We show that ischaemic exosomes, in comparison with control exosomes, confer protection against oxidative-induced lesion, promote proliferation, and sprouting of ECs, stimulate the formation of capillary-like structures and strengthen adhesion complexes and barrier properties. Moreover, ischaemic exosomes display higher levels of metalloproteases (MMP) and promote the secretion of MMP by ECs. We demonstrate that miR-222 and miR-143, the relatively most abundant miRs in ischaemic exosomes, partially recapitulate the angiogenic effect of exosomes. Additionally, we show that ischaemic exosomes stimulate the formation of new functional vessels in vivo using in ovo and Matrigel plug assays. Finally, we demonstrate that intramyocardial delivery of ischaemic exosomes improves neovascularization following MI. Conclusions This study establishes that exosomes secreted by cardiomyocytes under ischaemic conditions promote heart angiogenesis, which may pave the way towards the development of add-on therapies to enhance myocardial blood supply.publishersversionpublishe

Usefulness of bone turnover markers as predictors of mortality risk, disease progression and skeletal-related events appearance in patients with prostate cancer with bone metastases following treatment with zoledronic acid: TUGAMO study

Owing to the limited validity of clinical data on the treatment of prostate cancer (PCa) and bone metastases, biochemical markers are a promising tool for predicting survival, disease progression and skeletal-related events (SREs) in these patients. The aim of this study was to evaluate the predictive capacity of biochemical markers of bone turnover for mortality risk, disease progression and SREs in patients with PCa and bone metastases undergoing treatment with zoledronic acid (ZA). Methods: This was an observational, prospective and multicenter study in which ninety-eight patients were included. Patients were treated with ZA (4mg every 4 weeks for 18 months). Data were collected at baseline and 3, 6, 9, 12, 15 and 18 months after the beginning of treatment. Serum levels of bone alkaline phosphtase (BALP), aminoterminal propeptide of procollagen type I (P1NP) and beta-isomer of carboxiterminal telopeptide of collagen I (b-CTX) were analysed at all points in the study. Data on disease progression, SREs development and survival were recorded. Results: Cox regression models with clinical data and bone markers showed that the levels of the three markers studied were predictive of survival time, with b-CTX being especially powerful, in which a lack of normalisation in visit 1 (3 months after the beginning of treatment) showed a 6.3-times more risk for death than in normalised patients. Levels of these markers were also predictive for SREs, although in this case BALP and P1NP proved to be better predictors. We did not find any relationship between bone markers and disease progression. Conclusion: In patients with PCa and bone metastases treated with ZA, b-CTX and P1NP can be considered suitable predictors for mortality risk, while BALP and P1NP are appropriate for SREs. The levels of these biomarkers 3 months after the beginning of treatment are especially importantThis study was supported by Novartis Oncology Spai

Running title: Non-toxic broad anti-tumor activity of an EGFR×4-1BB bispecific trimerbod

32 p.-4 fig.Purpose: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell–mediated antitumor response. Systemic administration of anti-4-1BB–agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity.Experimental Design: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo.Results: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non–small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer.Conclusions: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.This work was supported by grants from the European Union [IACT Project (602262), H2020-iNEXT (1676)]; the Spanish Ministry of Science, Innovation and Universities and the Spanish Ministry of Economy and Competitiveness (SAF2017-89437-P, CTQ2017-83810-R, RTC-2016-5118-1, RTC-2017-5944-1), partially supported by the European Regional Development Fund; the Carlos III Health Institute (PI16/00357), co-founded by the Plan Nacional de Investigación and the European Union; the CRIS Cancer Foundation (FCRIS-IFI-2018); and the Spanish Association Against Cancer (AECC, 19084). C. Domínguez-Alonso was supported by a predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universities (PRE2018-083445). M. Zonca was supported by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness, co-founded by the European Social Fund (PTQ-16-08340).Peer reviewe

Assessment of a New ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in Patients with Non–Small Cell Lung Carcinoma: the ROSING Study

Introduction: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. Methods: A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). Results: An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). Conclusions: The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm

Búsqueda de biomarcadores moleculares y estructurales en el trombo y a nivel circulante, útiles para el diagnóstico, estratificación y pronóstico en el ictus isquémico agudo

La inclusión de biomarcadores en la toma de decisiones en el ictus isquémico es crucial para seleccionar a los pacientes para el tratamiento de revascularización, optimizar la asignación de recursos y predecir posibles complicaciones. Sin embargo, ninguna molécula ha demostrado suficiente sensibilidad, especificidad, accesibilidad, precisión y rentabilidad para su generalización en el ictus isquémico. Así, en la práctica clínica actual las decisiones se basan en marcadores diagnósticos y pronósticos basados en características del paciente, presentación clínica y marcadores de imagen que evalúan la oclusión arterial y la viabilidad del tejido cerebral. Este trabajo se centra en identificar biomarcadores relacionados con la formación del trombo responsable de la oclusión vascular en el ictus isquémico, así como en los procesos de isquemia, daño peri-isquemia y remodelación en el tejido cerebral. Se examinan varios marcadores accesibles a través de determinaciones analíticas de rutina, proteínas específicas que desempeñan un papel crítico en la inflamación y la hemostasis como la calprotectina, metaloproteasas e inhibidores de la fibrinólisis, y elementos encontrados en el trombo oclusivo (fibrina, plaquetas, eritrocitos) así como su relación con los marcadores de inflamación y otros componentes del trombo. El trabajo también explora el papel del Factor XIII en la formación de trombos y su resistencia al tratamiento fibrinolítico, convirtiéndolo en un posible objetivo terapéutico para tratamientos futuros. El estudio examina la relación entre la inflamación y la hemostasis en el ictus isquémico y su efecto combinado en el pronóstico, con varios marcadores que muestran una correlación entre un estado inflamatorio más elevado dentro de las primeras 24 horas del ictus y peores resultados a los tres meses. El estudio se enfoca en la presencia de factores hemostáticos en el trombo y su interrelación con otros componentes del sistema fibrinolítico endógeno en pacientes con ictus isquémico. Se describe una asociación significativa entre niveles bajos de TAFI en el trombo y una mayor mortalidad a los 3 meses del ictus. También se observa una correlación entre la presencia de MMP10 y la inactivación del TAFI en el trombo, lo que sugiere un posible papel en la modulación de la inflamación y fibrinolisis. El estudio también revela una agregación de marcadores de activación leucocitaria y elementos de la coagulación en el trombo, en concreto del Factor XIII de la coagulación (FXIII). El FXIII es una proteína importante en la formación de coágulos sanguíneos y su estabilización. En este estudio se encontró que la presencia de FXIII durante la polimerización de fibrina in vitro resultó en una mayor densidad de fibras y una menor permeabilidad del coágulo. El FXIII también puede participar en la unión de otras proteínas al coágulo, como FVW, NE y NETs, y puede estar implicado en la retracción del coágulo. Se investigó la inhibición del FXIII como una posible terapia para prevenir la formación de coágulos sanguíneos o promover la lisis de coágulos ya formados. Los experimentos demostraron que la inhibición del FXIII puede retrasar la formación del coágulo, hacerlo más pequeño y más fácil de lisar. También se encontró que la inhibición del FXIII puede aumentar la eficacia de la lisis con tPA

Casuística de códigos ictus atendidos por 061 Aragón en el período 2010-2016. Factores que influyen en los tiempos de respuesta y de acceso a la fibrinólisis.

Objetivo. Estudiar los tiempos de respuesta en la atención al código ictus por unidades asistenciales del 061 ARAGÓN, analizando los factores implicados y su relación con el acceso al tratamiento fibrinolítico en la fase hiperaguda. Pacientes y métodos. Estudio descriptivo transversal sobre la asistencia extrahospitalaria a partir del registro de casos atendidos por unidades asistenciales del 061 ARAGÓN a pacientes con código ictus durante el período 2010-2016. Resultados. Se recogieron 1.743 pacientes con código ictus (54,6%, varones), con una edad media de 72,83 ± 13,1 años. El número de ictus atendidos en 2015 y 2016 (372 y 366, respectivamente) fue mayor que la media de 201 ictus anuales en el resto de los años. El 27,2% de los pacientes fueron atendidos entre las 08:00 y las 11:59 h, intervalo horario con mayor frecuentación. Respecto al tiempo que se tardó en atender al paciente, la media fue de 71,93 ± 33,64 minutos, con ma­yor tiempo de respuesta en Teruel. Cuando se analizó la influencia del intervalo horario sobre el porcentaje de casos tratados con fibrinólisis, se observó una mayor tasa de fibrinólisis cuando se activó entre las 12:00 y las 15:59 h (28,1%). Conclusión. En el 55,3% de los pacientes, el tiempo de respuesta del 061 fue mayor de 60 minutos, pero este tiempo no se vio condicionado por la hora de activación. Sin embargo, sí había diferencias en el porcentaje de casos de fibrinólisis en los diferentes intervalos horarios, lo que sugiere que factores distintos al tiempo de respuesta del 061 influyen en la indicación del tratamiento fibrinolítico

microRNAs regulate TAL1 expression in T-cell acute lymphoblastic leukemia.

The transcription factor TAL1 is a proto-oncogene whose aberrant expression in committed T-cell precursors is associated with the development of T-cell acute lymphoblastic leukemia (T-ALL). The mechanisms leading to aberrant activation of TAL1 in T-ALL patients who lack chromosomal rearrangements involving the TAL1 locus remain largely unknown. We hypothesized that TAL1 levels decrease during normal T-cell development at least in part due to miRNA-dependent silencing, in which case TAL1 over-expression in some T-ALL cases could be the consequence of deregulated miRNA expression. By performing computational prediction of miRNAs that bind to the human TAL1 mRNA we compiled a list of miRNAs that are candidates to regulate TAL1. Using a luciferase reporter system and mutagenesis assays we confirmed the miRNA-TAL1 mRNA interactions and selected candidate miRNAs: miR-101, miR-520d-5p, miR-140-5p, miR-448 and miR-485-5p. Over-expression of these microRNAs in different T-ALL cell lines consistently resulted in the down-regulation of TAL1 protein. In accordance, inhibition of miR-101 and miR-520d-5p promoted TAL1 protein expression. Importantly, we found that miR-101, miR-140-5p, miR-448 and miR-485-5p were down-regulated in T-ALL patient specimens and T-ALL cell lines. Our results show for the first time the existence of epigenetic regulation of TAL1 by specific miRNAs which may contribute, at least in part, to the ectopic expression of TAL1 in some T-ALL cases.These studies were financed by Liga Portuguesa Contra o Cancro (Terry Fox Award) and by Fundacao para a Ciencia e a Tecnologia (project PTDC/BIM-ONC/1548/2012). NCC received an FCT-SFRH PhD fellowship. JTB is supported by an FCT Consolidation Grant. The Cell Division and Cancer group of the CNIO is funded by the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2012-38215: Consolider-Ingenio 2010 Programme SAF2014-57791-REDC; Red Tematica CellSYS BFU2014-52125-REDT), and the Comunidad de Madrid (OncoCycle Programme S2010/BMD-2470).S

CM-352 Efficacy in a mouse model of anticoagulant-associated intracranial hemorrhage

Background: Intracranial hemorrhage (ICH) is one of the major devastating complications of anticoagulation. Matrix metalloproteinase (MMP) inhibition has been proposed as a novel pharmacological approach for ICH treatment. Objectives: We evaluated the effects of CM-352 (MMP-fibrinolysis inhibitor) in an experimental ICH model associated with oral anticoagulants as compared with clinically used prothrombin complex concentrate (PCC). Methods: ICH was induced by collagenase injection into the striatum of wild type (C57BL/6J) anticoagulated mice (warfarin or rivaroxaban) and Mmp10 -/- mice. Hematoma volume and neurological deficits were measured 24 hours later by diaminobenzidine staining and different behavioral tests. Circulating plasminogen activator inhibitor-1 (PAI-1) activity and interleukin-6 (IL-6) were measured in plasma samples and local inflammation was assessed by neutrophil infiltration. Finally, fibrinolytic effects of MMP-10 and rivaroxaban were evaluated by thromboelastometry and thrombin-activatable fibrinolysis inhibitor (TAFI) activation assays. Results: Only PCC reduced hemorrhage volume and improved functional outcome in warfarin-ICH, but both PCC and CM-352 treatments diminished hemorrhage volume (46%, p < 0.01 and 64%, p < 0.001, respectively) and ameliorated functional outcome in rivaroxaban-ICH. We further demonstrated that CM-352, but not PCC, decreased neutrophil infiltration in the hemorrhage area at 24 hours. The effect of CM-352 could be related to MMP-10 inhibition since Mmp10 -/- mice showed lower hemorrhage volume, better neurological score, reduced IL-6 levels and neutrophil infiltration, and increased PAI-1 after experimental ICH. Finally, we found that CM-352 reduced MMP-10 and rivaroxaban-related fibrinolytic effects in thromboelastometry and TAFI activation. Conclusion: CM-352 treatment, by diminishing MMPs and rivaroxaban-associated fibrinolytic effects, might be a novel antihemorrhagic strategy for rivaroxaban-associated ICH