Efectos moleculares de los productos finales de glicación avanzada en la célula endotelial humana

En la diabetes se producen alteraciones que pueden llevar a la pérdida de la función endotelial y a las distintas enfermedades cardiovasculares a través de varios mecanismos moleculares, siendo el estrés oxidativo uno de los más importantes. Durante la diabetes se producen glicaciones no enzimáticas en las proteínas y éstas se han asociado con un aumento del estrés oxidativo. La glicación más abundante en las proteínas son los productos de Amadori, tanto in vitro como in vivo. El aumento de estrés oxidativo vascular puede estar mediado por la NADPH oxidasa, principal productora de especies reactivas del oxígeno (ROS) en los vasos. Así, nuestro objetivo ha sido determinar si los productos de Amadori (albúmina glicada) pueden producir disfunción endotelial, estudiando la producción extra e intracelular de ROS inducida por estos productos, además de las rutas y mecanismos que median estos efectos. En nuestros experimentos, el tratamiento de las células endoteliales de cordón umbilical (HUVEC) con un producto de Amadori, como es la albúmina glicada (gHSA), a bajas concentraciones, aumentó la producción de aniones superóxido extracelularmente de manera dependiente de la concentración a través de la NADPH oxidasa. El aumento de la producción alcanzó un máximo a las 4 horas y se mantuvo al menos hasta las 12 horas. El aumento de ROS también se produjo a nivel intracelular. El incremento en la producción de ROS inducido por la gHSA podría deberse a un incremento en la expresión de las principales subunidades de la NADPH oxidasa en la célula endotelial humana, la Nox4 y la p22phox, observado tanto a nivel del ARN mensajero como de la proteína. La gHSA indujo la activación del NF-kB y ésta lleva al aumento de la expresión de Nox4. La inhibición de las cascadas de fosforilación mediadas por la PI3K o la MEK 1/2 no tuvo efecto en la producción de ROS por las HUVEC. Por otra parte, la inhibición de la AP-1 aumentó la producción de ROS en HUVEC tratadas con la albúmina pero no con la albúmina glicada. El tratamiento con el inhibidor de AP- 1 aumentó la expresión de las subunidades de NADPH oxidasa, Nox4 y p22phox, pero disminuyó la expresión de eNOS. El tratamiento con albúmina glicada desacopló la eNOS en las HUVEC y esta acción podría explicar el efecto diferencial en la producción de ROS en presencia del inhibidor de AP-1. Por otra parte, en las HUVEC, la gHSA indujo la expresión de ICAM-1 y VCAM-1, además de tener un papel regulador en la expresión de MCP-1. Estos datos demostraron que la gHSA puede ser un modulador importante de la actividad inflamatoria endotelial y que esta modulación puede ejercerse de forma indirecta, a través de la inducción de respuestas oxidativas. Los resultados de este estudio explican parte de los mecanismos por los que la albúmina glicada, un producto de Amadori, afecta al estrés oxidativo y al estado inflamatorio de la célula endotelial y en consecuencia, puede ser causa de la disfunción endotelial relacionada con la diabetes. Conocer estas rutas de señalización intracelular permitiría saber más sobre como se inicia la disfunción endotelial relacionada con la diabetes y como, en el futuro, se podría avanzar en el tratamiento de estos pacientes

Eosinophils in the Gastrointestinal Tract: Key Contributors to Neuro-Immune Crosstalk and Potential Implications in Disorders of Brain-Gut Interaction

Disorders of brain-gut interaction; Intestinal eosinophils; Neuro-immune interactionTrastorns de la interacció cervell-intestí; Eosinòfils intestinals; Interacció neuroimmuneTrastornos de la interacción cerebro-intestino; Eosinófilos intestinales; Interacción neuroinmuneEosinophils are innate immune granulocytes actively involved in defensive responses and in local and systemic inflammatory processes. Beyond these effector roles, eosinophils are fundamental to maintaining homeostasis in the tissues they reside. Gastrointestinal eosinophils modulate barrier function and mucosal immunity and promote tissue development through their direct communication with almost every cellular component. This is possible thanks to the variety of receptors they express and the bioactive molecules they store and release, including cytotoxic proteins, cytokines, growth factors, and neuropeptides and neurotrophines. A growing body of evidence points to the eosinophil as a key neuro-immune player in the regulation of gastrointestinal function, with potential implications in pathophysiological processes. Eosinophil–neuron interactions are facilitated by chemotaxis and adhesion molecules, and the mediators released may have excitatory or inhibitory effects on each cell type, with physiological consequences dependent on the type of innervation involved. Of special interest are the disorders of the brain–gut interaction (DBGIs), mainly functional dyspepsia (FD) and irritable bowel syndrome (IBS), in which mucosal eosinophilia and eosinophil activation have been identified. In this review, we summarize the main roles of gastrointestinal eosinophils in supporting gut homeostasis and the evidence available on eosinophil–neuron interactions to bring new insights that support the fundamental role of this neuro-immune crosstalk in maintaining gut health and contributing to the pathophysiology of DBGIs.This study was funded in part by the Fondo Europeo de Desarrollo Regional (FEDER), the Fondo de Investigación Sanitaria, the Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), the Instituto de Salud Carlos III, Subdirección General de Investigación Sanitaria, and the Ministerio de Economía y Competitividad: FI12/00254 (E.S-R.), CD15/00010 (B.K.R.-J.), FI20/00256 (M.A-B.), PI19/01643 (B.L.), PI17/0190 (J.S.), CP18/00116 (C.M), CPII16/00031 and PI19/01643 (M.V.), CIBEREHD CB06/04/0021 (J.S., R.F., M.V.)

Modeling the Number of People Infected With SARS-COV-2 From Wastewater Viral Load in Northwest Spain

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract] The quantification of the SARS-CoV-2 RNA load in wastewater has emerged as a useful tool to monitor COVID–19 outbreaks in the community. This approach was implemented in the metropolitan area of A Coruña (NW Spain), where wastewater from a treatment plant was analyzed to track the epidemic dynamics in a population of 369,098 inhabitants. Viral load detected in the wastewater and the epidemiological data from A Coruña health system served as main sources for statistical models developing. Regression models described here allowed us to estimate the number of infected people (R2 = 0.9), including symptomatic and asymptomatic individuals. These models have helped to understand the real magnitude of the epidemic in a population at any given time and have been used as an effective early warning tool for predicting outbreaks in A Coruña municipality. The methodology of the present work could be used to develop a similar wastewater-based epidemiological model to track the evolution of the COVID–19 epidemic anywhere in the world where centralized water-based sanitation systems exist.This work was supported by EDAR Bens S.A., A Coruña, Spain [grant references INV04020, INV12120 and INV05921 to MP], the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 funded by the ISCIII, Spain - General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe” [grant numbers PI15/00860 to GB, PI17/01482 and PI20/00413 to MP], the GAIN, Xunta de Galicia, Spain [grant number IN607A 2016/22 to GB, ED431C-2016/015 and ED431C-2020/14 to RC, ED431C 2017/58 to SL, ED431G 2019/01 to RC and SL, and ED431C 2017/66 to MCV], MINECO, Spain [grant number MTM2017-82724-R to RC], Ministerio de Ciencia e Innovación, Spain [grant number PID2020-113578RB-100 to RC], and the Spanish Network for Research in Infectious Diseases [REIPI RD16/0016/006 to GB]. The work was also supported by the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union's Horizon 2020 - Research and Innovation Framework Programme under grant agreement no 871029. SR-F was financially supported by REIPI RD16/0016/006, KC-P by IN607A 2016/22 and the Spanish Association against Cancer (AECC) and JAV by IN607A 2016/22. Funding for open access charge: Universidade da Coruña/CISUGEDAR Bens S.A.; INV04020EDAR Bens S.A.; INV12120EDAR Bens S.A.; INV05921Xunta de Galicia; IN607A 2016/22Xunta de Galicia; ED431C-2016/015Xunta de Galicia; ED431C-2020/14Xunta de Galicia; ED431C 2017/58Xunta de Galicia; ED431G 2019/01Xunta de Galicia; ED431C 2017/6

Wastewater early warning system for SARS-CoV-2 outbreaks and variants in a Coruña, Spain

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract]: Wastewater-based epidemiology has been widely used as a cost-effective method for tracking the COVID-19 pandemic at the community level. Here we describe COVIDBENS, a wastewater surveillance program running from June 2020 to March 2022 in the wastewater treatment plant of Bens in A Coruña (Spain). The main goal of this work was to provide an effective early warning tool based in wastewater epidemiology to help in decision-making at both the social and public health levels. RT-qPCR procedures and Illumina sequencing were used to weekly monitor the viral load and to detect SARS-CoV-2 mutations in wastewater, respectively. In addition, own statistical models were applied to estimate the real number of infected people and the frequency of each emerging variant circulating in the community, which considerable improved the surveillance strategy. Our analysis detected 6 viral load waves in A Coruña with concentrations between 103 and 106 SARS-CoV-2 RNA copies/L. Our system was able to anticipate community outbreaks during the pandemic with 8-36 days in advance with respect to clinical reports and, to detect the emergence of new SARS-CoV-2 variants in A Coruña such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529 and BA.2) in wastewater with 42, 30, and 27 days, respectively, before the health system did. Data generated here helped local authorities and health managers to give a faster and more efficient response to the pandemic situation, and also allowed important industrial companies to adapt their production to each situation. The wastewater-based epidemiology program developed in our metropolitan area of A Coruña (Spain) during the SARS-CoV-2 pandemic served as a powerful early warning system combining statistical models with mutations and viral load monitoring in wastewater over time.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Funding for open access charge: Universidade da Coruña/CISUG. This work was supported by EDAR Bens S.A., A Coruña, Spain [grant references INV04020, INV12120, INV05921, and INV148721 to MP], by the National Plan for Scientific Research, Development and Technological Innovation funded by the Institute of Health Carlos III (ISCIII), Spain—General Subdirection of Assessment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe” [grant references PI15/00860 to GB, PI17/01482, and PI20/00413 to MP], by the Galician Innovation Agency (GAIN) (Xunta de Galicia, Spain) [grant references IN607A 2016/22 to GB, ED431C-2016/015 and ED431C-2020/14 to RC, ED431C 2021/53 to SL and ED431G 2019/01 and COV20/00604 to RC and SL, by Ministry of Economic Affairs and Digital Transformation (MINECO), Spain [grant references MTM2017-82724-R to RC], by the Spanish Network for Research in Infectious Diseases [REIPI RD16/0016/0006 to GB], by the “Innova Saúde” Program, (INNOVAMICROLAB project) co-founded by the Galician Healthcare Service (SERGAS) and the Spanish Ministry of Science and Innovation, and by the Spanish Network of Research in Infectious Diseases (CIBERINFEC, ISCIII), and by the European Virus Archive Global (EVA-GLOBAL) project that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 871029. SR-F was financially supported by REIPI RD16/0016/006, KC-P by IN607A 2016/22 and the Spanish Association against Cancer (AECC) and JAV by IN607A 2016/22. DP was funded by grant EPICOVIGAL FONDO SUPERA-COVID19 from Banco Santander-CSIC-CRUE, Spain, and grant CT850A-2 from (Health Knowledge Agency) ACIS SERGAS from the Consellería de Sanidade of Xunta de Galicia, Spain.EDAR Bens S.A.; INV04020EDAR Bens S.A.; INV12120EDAR Bens S.A.; INV05921EDAR Bens S.A.; INV148721Xunta de Galicia; IN607A 2016/22Xunta de Galicia; ED431C-2016/015Xunta de Galicia; ED431C-2020/14Xunta de Galicia; ED431C 2021/53Xunta de Galicia; ED431G 2019/01Xunta de Galicia; COV20/0060

Mucosal Plasma Cell Activation and Proximity to Nerve Fibres Are Associated with Glycocalyx Reduction in Diarrhoea-Predominant Irritable Bowel Syndrome: Jejunal Barrier Alterations Underlying Clinical Manifestations

Intestinal barrier dysfunction; Intestinal glycocalyx; Mucosal nerve fibresDisfunción de la barrera intestinal; Glicocálix intestinal; Fibras nerviosas de la mucosaDisfunció de la barrera intestinal; Glicocàlix intestinal; Fibres nervioses de la mucosaIrritable bowel syndrome (IBS) is a disorder of brain-gut interaction characterised by abdominal pain and changes in bowel habits. In the diarrhoea subtype (IBS-D), altered epithelial barrier and mucosal immune activation are associated with clinical manifestations. We aimed to further evaluate plasma cells and epithelial integrity to gain understanding of IBS-D pathophysiology. One mucosal jejunal biopsy and one stool sample were obtained from healthy controls and IBS-D patients. Gastrointestinal symptoms, stress, and depression scores were recorded. In the jejunal mucosa, RNAseq and gene set enrichment analyses were performed. A morphometric analysis by electron microscopy quantified plasma cell activation and proximity to enteric nerves and glycocalyx thickness. Immunoglobulins concentration was assessed in the stool. IBS-D patients showed differential expression of humoral pathways compared to controls. Activation and proximity of plasma cells to nerves and IgG concentration were also higher in IBS-D. Glycocalyx thickness was lower in IBS-D compared to controls, and this reduction correlated with plasma cell activation, proximity to nerves, and clinical symptoms. These results support humoral activity and loss of epithelial integrity as important contributors to gut dysfunction and clinical manifestations in IBS-D. Additional studies are needed to identify the triggers of these alterations to better define IBS-D pathophysiology.This study was funded in part by Fondo Europeo de Desarrollo Regional (FEDER), Fondo de Investigación Sanitaria and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Instituto de Salud Carlos III, Subdirección General de Investigación Sanitaria, Ministerio de Economía y Competitividad: CP18/00116 (C.M.), PI19/01643 (B.L.); PI17/01443 (D.G.); PI15/00301 (C.A.-C.), PI17/0190 (J.S.), PI19/01643 & CPII16/00031, (M.V.); CIBEREHD CB06/04/0021 (F.A., C.A.-C., J.S., M.V.); Ministerio de Educación, Dirección General de Investigación: SAF 2016-76648-R (F.A.); Agència de Gestió d’Ajuts Universitaris i de Recerca, de la Generalitat de Catalunya: 2014 SGR 1285 (F.A.); Vall d’Hebron Institut de Recerca, Programa de becas predoctorales Amics de Vall d’Hebron: PRED-VHIR-2016-34 (C.P.-C.), PRED-VHIR-2014-018 (M.F.), the Swedish Research Council dnr 2019-00653 (J.-P.G.M.), and the European Union’s Horizon research and innovation programme 2020, grant no. 848228 (E.E., A.R.-U., B.L., C.A.-C., J.S.)

Kpi, a chaperone-usher pili system associated with the worldwide-disseminated high-risk clone Klebsiella pneumoniae ST-15.

Control of infections caused by carbapenem-resistant Klebsiella pneumoniae continues to be challenging. The success of this pathogen is favored by its ability to acquire antimicrobial resistance and to spread and persist in both the environment and in humans. The emergence of clinically important clones, such as sequence types 11, 15, 101, and 258, has been reported worldwide. However, the mechanisms promoting the dissemination of such high-risk clones are unknown. Unraveling the factors that play a role in the pathobiology and epidemicity of K. pneumoniae is therefore important for managing infections. To address this issue, we studied a carbapenem-resistant ST-15 K. pneumoniae isolate (Kp3380) that displayed a remarkable adherent phenotype with abundant pilus-like structures. Genome sequencing enabled us to identify a chaperone-usher pili system (Kpi) in Kp3380. Analysis of a large K. pneumoniae population from 32 European countries showed that the Kpi system is associated with the ST-15 clone. Phylogenetic analysis of the operon revealed that Kpi belongs to the little-characterized γ2-fimbrial clade. We demonstrate that Kpi contributes positively to the ability of K. pneumoniae to form biofilms and adhere to different host tissues. Moreover, the in vivo intestinal colonizing capacity of the Kpi-defective mutant was significantly reduced, as was its ability to infect Galleria mellonella The findings provide information about the pathobiology and epidemicity of Kpi+K. pneumoniae and indicate that the presence of Kpi may explain the success of the ST-15 clone. Disrupting bacterial adherence to the intestinal surface could potentially target gastrointestinal colonization.The authors thank Dr Fidel Madrazo for help with transmission electron microscopy and confocal laser-scanning microscopy assays. This research was supported by Projects p-01216A and IJCI-2016-29524 (to A.P.), funded by the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) and the Minestry of Economy and Competetiveness (MINECO), respectively. It was also supported by Projects PI11/01034 (to M.P.), PI14/00059 and PI17/1482 (to M.P. and A.B.), and PI18/00501 (to G.B.), included in the National Plan for Scientific Research, Development and Technological Innovation 2013-2016 and funded by the Instituto de Salud Carlos III (ISCIII) and Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/006) cofinanced by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014-2020. Grant BFU2016-77835-R of the MINECO (to A.R.) also supported this research. E.G. was financially supported by the SEIMC project. J.C.V.-U. was financially supported by the PFIS (Contratos Predoctorales de Formación en Investigación en Salud) program (F18/00315); J.A.V. was financially supported by IN607A 2016/22; M.M.-G. was financially supported by a Clara Roy grant (SEIMC); A.B. was financially supported by the Miguel Servet program (ISCIII, Spain); B.K.R.-J. was financially supported by Marie S. Curie Action SaPhaDe project (MSCA-IF-GF-836754); and A.P. was financially supported by the Juan de la Cierva program (MINECO, IJCI-2016-29524).S