New mechanisms involved in the development of cardiovascular disease in chronic kidney disease.

Chronic kidney disease (CKD) is a pathology with a high worldwide incidence and an upward trend affecting the elderly. When CKD is very advanced, the use of renal replacement therapies is required to prolong its life (dialysis or kidney transplantation). Although dialysis improves many complications of CKD, the disease does not reverse completely. These patients present an increase in oxidative stress, chronic inflammation and the release of extracellular vesicles (EVs), which cause endothelial damage and the development of different cardiovascular diseases (CVD). CKD patients develop premature diseases associated with advanced age, such as CVD. EVs play an essential role in developing CVD in patients with CKD since their number increases in plasma and their content is modified. The EVs of patients with CKD cause endothelial dysfunction, senescence and vascular calcification. In addition, miRNAs free or transported in EVs together with other components carried in these EVs promote endothelial dysfunction, thrombotic and vascular calcification in CKD, among other effects. This review describes the classic factors and focuses on the role of new mechanisms involved in the development of CVD associated with CKD, emphasizing the role of EVs in the development of cardiovascular pathologies in the context of CKD. Moreover, the review summarized the EVs' role as diagnostic and therapeutic tools, acting on EV release or content to avoid the development of CVD in CKD patients.

Efecto de vesículas extracelulares derivadas de células madre mesenquimales humanas precondicionadas en hipoxia sobre la osteoblastogénesis y la adipogénesis in vitro / Effect of stem cell-derived extracellular vesicles human mesenchymal cells preconditioned in hypoxia on osteoblastogenesis and adipogenesis in vitro

Objetivos: las células madre mesenquimales (MSC) se caracterizan por su actividad antiinflamatoria, inmunosupresora y sucapacidad de diferenciación. Esto las convierten en una interesante herramienta terapéutica en terapia celular y medicinaregenerativa. En parte, el efecto terapéutico de las MSC, está mediado por la secreción de vesículas extracelulares (EV). Elprecondicionamiento en hipoxia de las MSC puede mejorar la capacidad regenerativa de las EV secretadas. En este con-texto, el objetivo del estudio ha sido evaluar si EV derivadas de MSC humanas cultivadas en hipoxia y normoxia afectan ala osteoblastogénesis y adipogénesis de las MSC.Material y métodos: se aislaron EV de MSC mantenidas 48 h en normoxia o hipoxia (3 % O2) mediante ultrafiltración ycromatografía de exclusión por tamaño. Las EV fueron caracterizadas por “Western blot”, microscopía electrónica y análisisde seguimiento de nanopartículas. En cultivos de MSC se evaluó el efecto de las EV sobre la viabilidad por ensayo con MTT,la migración por “Oris assay” y la diferenciación a osteoblastos y adipocitos.Resultados: las EV aumentaron la viabilidad y migración, pero no hubo diferencias entre las derivadas de normoxia ehipoxia. Las EV, principalmente las derivadas de hipoxia, aumentaron la mineralización y la expresión de genes osteoblás-ticos. Sin embargo, no afectaron significativamente a la adipogénesis.Conclusiones: las EV derivadas de MSC en hipoxia no afectan a la adipogénesis, pero tienen una mayor capacidad de inducirla osteoblastogénesis. Por lo tanto, podrían potencialmente ser utilizadas en terapias de regeneración ósea y tratamientosde patologías óseas como la osteoporosis.(AU)

Nuevos mecanismos implicados en el desarrollo de la enfermedad cardiovascular en la enfermedad renal crónica / New mechanisms involved in the development of cardiovascular disease in chronic kidney disease

La enfermedad renal crónica (ERC) tiene una alta incidencia mundial y una tendencia ascendente que afecta principalmente a personas de edad avanzada. Cuando la ERC está muy avanzada se requiere el uso de terapias renales sustitutivas para prolongar la vida (diálisis o trasplante renal) y, pese a que la diálisis mejora muchas complicaciones de la ERC, la enfermedad no revierte de manera completa. Estos pacientes presentan un aumento del estrés oxidativo, inflamación crónica y aumento de la liberación de vesículas extracelulares (VE), que provocan daño endotelial y el desarrollo de distintas enfermedades cardiovasculares (ECV). De hecho, los pacientes con ERC desarrollan de forma prematura enfermedades asociadas a una edad avanzada, como es el caso de las ECV. Las VE desempeñan un papel muy importante en el desarrollo de ECV en pacientes con ERC, ya que su número aumenta en el plasma y su contenido se modifica. Las VE de pacientes con ERC generan disfunción endotelial, senescencia y calcificación vascular. Además, los miRNA libres o transportados en las VE junto a otros componentes vehiculados en estas VE promueven disfunción endotelial, eventos trombóticos y calcificación vascular en los pacientes con ERC, entre otros efectos. En esta revisión se describen los factores clásicos y el papel de nuevos mecanismos que intervienen en el desarrollo de la ECV asociada a la ERC, con especial hincapié en el papel de las VE en el desarrollo de enfermedades cardiovasculares en un contexto de ERC. Además, se expone el papel de las VE como herramienta diagnóstica y como diana terapéutica, actuando sobre su liberación o contenido para intentar evitar el desarrollo de ECV en enfermos renales crónicos. (AU)

Chronic kidney disease (CKD) is a pathology with a high worldwide incidence and an upward trend affecting the elderly. When CKD is very advanced, the use of renal replacement therapies is required to prolong its life (dialysis or kidney transplantation). Although dialysis improves many complications of CKD, the disease does not reverse completely. These patients present an increase in oxidative stress, chronic inflammation and the release of extracellular vesicles (EVs), which cause endothelial damage and the development of different cardiovascular diseases (CVD). CKD patients develop premature diseases associated with advanced age, such as CVD. EVs play an essential role in developing CVD in patients with CKD since their number increases in plasma and their content is modified. The EVs of patients with CKD cause endothelial dysfunction, senescence and vascular calcification. In addition, miRNAs free or transported in EVs together with other components carried in these EVs promote endothelial dysfunction, thrombotic and vascular calcification in CKD, among other effects. This review describes the classic factors and focuses on the role of new mechanisms involved in the development of CVD associated with CKD, emphasizing the role of EVs in the development of cardiovascular pathologies in the context of CKD. Moreover, the review summarized the EVs’ role as diagnostic and therapeutic tools, acting on EV release or content to avoid the development of CVD in CKD patients. (AU)

Isolation and characterization of exosome-enriched urinary extracellular vesicles from Dent's disease type 1 Spanish patients.

BACKGROUND AND OBJECTIVES: Dent's disease type 1 (DD1) is a rare X-linked hereditary pathology caused by CLCN5 mutations that is characterized mainly by proximal tubule dysfunction, hypercalciuria, nephrolithiasis/nephrocalcinosis, progressive chronic kidney disease, and low-weight proteinuria, the molecular hallmark of the disease. Currently, there is no specific curative treatment, only symptomatic and does not prevent the progression of the disease. In this study we have isolated and characterized urinary extracellular vesicles (uEVs) enriched in exosomes that will allow us to identify biomarkers associated with DD1 progression and a better understanding of the pathophysiological bases of the disease. MATERIALS AND METHODS: Through a national call from the Spanish Society of Nephrology (SEN) and the Spanish Society of Pediatric Nephrology (AENP), urine samples were obtained from patients and controls from different Spanish hospitals, which were processed to obtain the uEVS. The data of these patients were provided by the respective nephrologists and/or extracted from the RENALTUBE registry. The uEVs were isolated by ultracentrifugation, morphologically characterized and their protein and microRNA content extracted. RESULTS: 25 patients and 10 controls were recruited, from which the urine was processed to isolate the uEVs. Our results showed that the relative concentration of uEVs/mL is lower in patients compared to controls (0.26 × 106 uEVs/mL vs 1.19 × 106 uEVs/mL, p < 0.01). In addition, the uEVs of the patients were found to be significantly larger than those of the control subjects (mean diameter: 187.8 nm vs 143.6 nm, p < 0.01). Finally, our data demonstrated that RNA had been correctly extracted from both patient and control exosomes. CONCLUSIONS: In this work we describe the isolation and characterization of uEVs from patients with Dent 1 disease and healthy controls, that shall be useful for the subsequent study of differentially expressed cargo molecules in this pathology.

Nuevos conocimientos sobre el papel clave de las vesículas extracelulares en la enfermedad de hígado graso no alcohólica / New insights into the key role of extracellular vesicles in non-alcoholic fatty liver disease

La enfermedad del hígado graso no alcohólico (EHGNA) es la enfermedad hepática crónica más común del mundo. La EHGNA se considera la manifestación hepática del síndrome metabólico al estar directamente asociada con la obesidad, la resistencia a la insulina, la diabetes mellitus tipo 2 y las complicaciones cardiovasculares. Pese a su prevalencia, los factores que desencadenan la progresión de la EHGNA a la esteatohepatitis no alcohólica, la cirrosis y el carcinoma hepatocelular son poco conocidos. Actualmente, no existe tratamiento eficaz ni hay disponible un método fiable para su diagnóstico y estatificación más allá de la biopsia hepática altamente invasiva.Recientemente, las vesículas extracelulares (VEs) han emergido como posibles biomarcadores para el diagnóstico de la EHGNA. Las VEs son vesículas derivadas de las células que contienen proteínas y ácidos nucleicos, entre otros componentes, que interactúan y desencadenan una gran variedad de respuestas en células diana próximas o distantes. Varios mecanismos implicados en la progresión de la EHGNA, como la inflamación, la fibrosis y la angiogénesis, relacionados con la lipotoxicidad, desencadenan la secreción de VEs por las células hepáticas. En esta revisión nos centraremos en las VEs secretadas por los hepatocitos (Hep-VEs) como mensajeros del interactoma entre las diferentes células hepáticas en la patogénesis de la EHGNA, así como en su papel como biomarcadores no invasivos para su diagnóstico y estratificación. Además, destacaremos las investigaciones disponibles hasta la fecha, las limitaciones actuales y las futuras directrices para su implementación en un entorno clínico como biomarcadores o dianas terapéuticas de la enfermedad hepática. (AU)

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. NAFLD, the hepatic manifestation of the metabolic syndrome, closely associates with insulin resistance, type 2 diabetes mellitus, obesity and cardiovascular disease. Until now, the specific factors involved in the progression of NAFLD from fatty liver to non-alcoholic steatohepatitis, cirrhosis and, ultimately hepatocellular carcinoma have not been totally elucidated.Also, patients have to face the lack of efficient or personalized treatments, as well as the absence of reliable diagnosis or staging methods beyond the highly invasive liver biopsy. In the last years, extracellular vesicles (VEs) are considered as potential biomarkers for the diagnosis many diseases including NAFLD. VEs are released by different cells types into the circulation and contain nucleic acids and proteins, among other components of their, that interact with surrounding or distant target cells, thereby triggering a plethora of responses. During NAFLD progression, several processes such as inflammation, fibrosis and angiogenesis, all related to MS-associated lipotoxicity, lead to VEs release by liver cells. In this review we will focus in the role of hepatocyte-derived VEs (Hep-VEs) and their interactions with non-parenchymal liver cells populations during NAFLD pathogenesis, as well as in their role as non-invasive biomarkers for disease diagnosis and progression. We will highlight the recent work currently available on VEs in the context of NAFLD, the current limitations and future directions for the implementation of VEs as biomarkers or targets of liver disease in the clinical setting. (AU)

Vesículas extracelulares en la comunicación hospedador-helminto: aplicaciones biomédicas / Extracellular vesicles in the host-helminth communication: biomedical applications

Las vesículas extracelulares constituyen un nuevo mecanismo de comunicación intercelular, junto a los clásicos mediados por contacto directo entre células y por la emisión de moléculas. Su estudio ha despertado considerable interés desde su descubrimiento en 1983 en reticulocitos. El término incluye diferentes tipos de vesículas que varían en tamaño y origen, considerándose fundamentalmente exosomas, microvesículas y cuerpos apoptóticos.Estas estructuras se encuentran formadas por una membrana lipídica, donde se enclavan diversos tipos de receptores, y pueden transportar diversas moléculas como azúcares, proteínas y ácidos nucleicos, así como metabolitos. Se han descrito en todos los reinos de la naturaleza (participando en diversos tipos de interacciones, tanto intercelulares como inter-específicas), así como en todo tipo de fluidos biológicos, constituyendo parte de lo que se conoce como “biopsia líquida”. De hecho, su presencia en muestras procedentes tanto de procesos fisiológicos como en procesos patológicos las ha hecho excelente biomarcadores. Su papel en salud y enfermedad está siendo ampliamente investigado.En este contexto, el estudio de las vesículas extracelulares producidas por parásitos, y en concreto por helmintos, constituye un campo de investigación en expansión, de gran interés biomédico, como es el control de las infecciones causadas por estos. De hecho, dichas vesículas pueden ser utilizadas para realizar un diagnóstico rápido y específico, así como constituir herramientas para vacunación, y ser posibles dianas de nuevos tratamientos.La capacidad de las vesículas extracelulares de modular la respuesta inmunitaria, abre asimismo nuevas posibilidades de su uso frente a enfermedades autoinmunes. (AU)

Extracellular vesicles participate in intercellular communications, altogether with classic mechanisms like direct contact between cells and the secretion of mediators. They have attracted considerable interest since their discovery in reticulocytes in 1983. The term includes different types of vesicles that vary in size and origin, with exosomes, microvesicles and apoptotic bodies as the major ones.These structures are sorrounded by a lipid membrane, where various types of receptors are located, and can carry different cargo molecules, including sugars, proteins, nucleic acids and metabolites. They have been described in all kingdoms in nature (participating in both intercellular and inter-specific communications), in all types of biological fluids (as part of liquid biopsy). In fact, their presence in samples from both physiological and pathological processes has suggested them as excellent biomarkers. Their role in health and disease is being widely investigated.In this context, the study of extracellular vesicles produced by parasites, and specifically by helminths, constitutes a growing field of research, with great biomedical interest, mainly in the control of infections caused by them. In fact, these vesicles can be used to generate rapid and specific diagnosis systems, to produce new tools for vaccination, and to identify targets for new treatments.The ability of extracellular vesicles to modulate the immune response also opens new possibilities for their use against autoimmune diseases. (AU)

Nuevos conocimientos sobre el papel clave de las vesículas extracelulares en la enfermedad de hígado graso no alcohólica / New insights into the key role of extracellular vesicles in non-alcoholic fatty liver disease

La enfermedad del hígado graso no alcohólico (EHGNA) es la enfermedad hepática crónica más común del mundo. La EHGNA se considera la manifestación hepática del síndrome metabólico al estar directamente asociada con la obesidad, la resistencia a la insulina, la diabetes mellitus tipo 2 y las complicaciones cardiovasculares. Pese a su prevalencia, los factores que desencadenan la progresión de la EHGNA a la esteatohepatitis no alcohólica, la cirrosis y el carcinoma hepatocelular son poco conocidos. Actualmente, no existe tratamiento eficaz ni hay disponible un método fiable para su diagnóstico y estatificación más allá de la biopsia hepática altamente invasiva.Recientemente, las vesículas extracelulares (VEs) han emergido como posibles biomarcadores para el diagnóstico de la EHGNA. Las VEs son vesículas derivadas de las células que contienen proteínas y ácidos nucleicos, entre otros componentes, que interactúan y desencadenan una gran variedad de respuestas en células diana próximas o distantes. Varios mecanismos implicados en la progresión de la EHGNA, como la inflamación, la fibrosis y la angiogénesis, relacionados con la lipotoxicidad, desencadenan la secreción de VEs por las células hepáticas. En esta revisión nos centraremos en las VEs secretadas por los hepatocitos (Hep-VEs) como mensajeros del interactoma entre las diferentes células hepáticas en la patogénesis de la EHGNA, así como en su papel como biomarcadores no invasivos para su diagnóstico y estratificación. Además, destacaremos las investigaciones disponibles hasta la fecha, las limitaciones actuales y las futuras directrices para su implementación en un entorno clínico como biomarcadores o dianas terapéuticas de la enfermedad hepática. (AU)

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. NAFLD, the hepatic manifestation of the metabolic syndrome, closely associates with insulin resistance, type 2 diabetes mellitus, obesity and cardiovascular disease. Until now, the specific factors involved in the progression of NAFLD from fatty liver to non-alcoholic steatohepatitis, cirrhosis and, ultimately hepatocellular carcinoma have not been totally elucidated.Also, patients have to face the lack of efficient or personalized treatments, as well as the absence of reliable diagnosis or staging methods beyond the highly invasive liver biopsy. In the last years, extracellular vesicles (VEs) are considered as potential biomarkers for the diagnosis many diseases including NAFLD. VEs are released by different cells types into the circulation and contain nucleic acids and proteins, among other components of their, that interact with surrounding or distant target cells, thereby triggering a plethora of responses. During NAFLD progression, several processes such as inflammation, fibrosis and angiogenesis, all related to MS-associated lipotoxicity, lead to VEs release by liver cells. In this review we will focus in the role of hepatocyte-derived VEs (Hep-VEs) and their interactions with non-parenchymal liver cells populations during NAFLD pathogenesis, as well as in their role as non-invasive biomarkers for disease diagnosis and progression. We will highlight the recent work currently available on VEs in the context of NAFLD, the current limitations and future directions for the implementation of VEs as biomarkers or targets of liver disease in the clinical setting. (AU)

Exosomes and cystic echinococcosis: systematic review / Exosomas y equinococosis quística: revisión sistemática

SUMMARY: Exosomes are small and single-membrane secreted organelles, up to 200 nm in diameter that have the same topology as the cell, but are enriched in proteins, nucleic acids, lipids, and glycoconjugates. It can be found in any type of body fluids such as plasma, urine, saliva, sperm, bile, etc. On the other hand, cystic Echinococcosis (CE) has been studied from different points of view, among others, from genomics and proteomics, and the presence of CE exosomes in humans and other intermediate hosts has been reported in very few articles. The aim of this review was to report the evidence available regarding exosomes and CE. Systematic review. Data sources: Trip Database, SciELO, WoS, MEDLINE, EMBASE and SCOPUS. Eligibility criteria: Studies related to CEin any type of host and state of the parasite, without language restriction, published between 1966-2021. Variables: Year of publication, geographical origin, species isolated, location of exosomes. Forty-two studies were initially identified. After scrutinizing titles and abstracts, checking duplications and in-depth analysis of the studies selected, 12 articles including human, bovine, sheep, dog samples were also included. All were case reports or case series. The highest proportion of articles was published between 2019 and 2021 (58.3 %). Publications were predominantly from China (58.3 %). Evidence about exosomes and CE is scarce and reduced range of articles and cases.

RESUMEN: Los exosomas son orgánulos pequeños y secretados por una sola membrana, de hasta 200 nm de diámetro que tienen la misma topología que la célula, pero están enriquecidos en proteínas, ácidos nucleicos, lípidos y glicoconjugados. Se puede encontrar en cualquier tipo de fluidos corporales como plasma, orina, saliva, esperma, bilis, etc. Por otro lado, la equinococosis quística (CE) ha sido estudiada desde diferentes puntos de vista, entre otros, desde la genómica y proteómica, y la presencia de exosomas de CE en humanos y otros huéspedes intermediarios se ha informado en muy pocos artículos. El objetivo de esta revisión fue informar la evidencia disponible con respecto a los exosomas y la CE. Revisión sistemática. Fuentes de datos: Trip Database, SciELO, WoS, MEDLINE, EMBASE y SCOPUS. Criterios de elegibilidad: Estudios relacionados con la EC en cualquier tipo de hospedador y estado del parásito, sin restricción de idioma, publicados entre 1966-2021. Variables: año de publicación, origen geográfico, especie aislada, ubicación de exosomas. Se identificaron inicialmente 42 estudios. Después de examinar los títulos y resúmenes, verificar las duplicaciones y analizar en profundidad los estudios seleccionados, se incluyeron 12 artículos que incluían muestras de humanos, bovinos, ovinos y caninos. Todos fueron informes de casos o series de casos. La mayor proporción de artículos se publicó entre 2019 y 2021 (58,3 %). Las publicaciones fueron predominantemente de China (58,3 %). La evidencia sobre exosomas y CE es escasa y la gama de artículos y casos es reducida.

Vesículas extracelulares en la comunicación hospedador-helminto: aplicaciones biomédicas / Extracellular vesicles in the host-helminth communication: biomedical applications

Las vesículas extracelulares constituyen un nuevo mecanismo de comunicación intercelular, junto a los clásicos mediados por contacto directo entre células y por la emisión de moléculas. Su estudio ha despertado considerable interés desde su descubrimiento en 1983 en reticulocitos. El término incluye diferentes tipos de vesículas que varían en tamaño y origen, considerándose fundamentalmente exosomas, microvesículas y cuerpos apoptóticos. Estas estructuras se encuentran formadas por una membrana lipídica, donde se enclavan diversos tipos de receptores, y pueden transportar diversas moléculas como azúcares, proteínas y ácidos nucleicos, así como metabolitos. Se han descrito en todos los reinos de la naturaleza (participando en diversos tipos de interacciones, tanto intercelulares como inter-específicas), así como en todo tipo de fluidos biológicos, constituyendo parte de lo que se conoce como “biopsia líquida”. De hecho, su presencia en muestras procedentes tanto de procesos fisiológicos como en procesos patológicos las ha hecho excelente biomarcadores. Su papel en salud y enfermedad está siendo ampliamente investigado. En este contexto, el estudio de las vesículas extracelulares producidas por parásitos, y en concreto por helmintos, constituye un campo de investigación en expansión, de gran interés biomédico, como es el control de las infecciones causadas por estos. De hecho, dichas vesículas pueden ser utilizadas para realizar un diagnóstico rápido y específico, así como constituir herramientas para vacunación, y ser posibles dianas de nuevos tratamientos. La capacidad de las vesículas extracelulares de modular la respuesta inmunitaria, abre asimismo nuevas posibilidades de su uso frente a enfermedades autoinmunes.(AU)

Extracellular vesicles participate in intercellular communications, altogether with classic mechanisms like direct contact between cells and the secretion of mediators. They have attracted considerable interest since their discovery in reticulocytes in 1983. The term includes different types of vesicles that vary in size and origin, with exosomes, microvesicles and apoptotic bodies as the major ones. These structures are sorrounded by a lipid membrane, where various types of receptors are located, and can carry different cargo molecules, including sugars, proteins, nucleic acids and metabolites. They have been described in all kingdoms in nature (participating in both intercellular and inter-specific communications), in all types of biological fluids (as part of liquid biopsy). In fact, their presence in samples from both physiological and pathological processes has suggested them as excellent biomarkers. Their role in health and disease is being widely investigated. In this context, the study of extracellular vesicles produced by parasites, and specifically by helminths, constitutes a growing field of research, with great biomedical interest, mainly in the control of infections caused by them. In fact, these vesicles can be used to generate rapid and specific diagnosis systems, to produce new tools for vaccination, and to identify targets for new treatments. The ability of extracellular vesicles to modulate the immune response also opens new possibilities for their use against autoimmune diseases.(AU)

Retos y oportunidades en el estudio de vesículas extracelulares: contexto institucional a nivel mundial y situación actual en Colombia / Challenges and opportunities in the study of extracellular vesicles: Global institutional context and national state of the art

Resumen En la última década se ha incrementado el número de estudios y publicaciones sobre las vesículas extracelulares y los exosomas. En Colombia, ha habido interés y avances en su estudio, lo que se evidencia en el aumento de publicaciones y proyectos de investigación. Sin embargo, este es un campo de investigación aún en desarrollo, con desafíos analíticos y limitaciones técnicas, por lo cual, en el planteamiento de los proyectos de investigación y desarrollo, es necesario considerar cuál es el estado del campo científico a nivel mundial en cuanto a la nomenclatura y la clasificación de las vesículas extracelulares, las técnicas, recursos, requisitos y especificaciones de calidad y las instituciones que regulan el campo. La respuesta a esta pregunta permitirá desarrollar estudios que cumplan con los estándares internacionales, y las exigencias y recomendaciones institucionales. Sin embargo, la información científica disponible se encuentra dispersa y no todos los aspectos son tratados a cabalidad. En este actualización se condensa la información disponible y se presentan los términos oficiales para denominar las vesículas extracelulares y la nomenclatura aceptada actualmente, así como la evolución del campo, la homogenización de los parámetros experimentales, el establecimiento de autoridades científicas, instituciones y recursos, y las recomendaciones que se han generado a nivel mundial para el desarrollo de investigaciones en vesículas extracelulares, incluidos su aislamiento, caracterización y estudio funcional. Por último, se analiza el contexto nacional de una forma crítica, teniendo en cuenta las fortalezas institucionales, los errores usualmente cometidos, y las técnicas y tecnologías analíticas disponibles.

Abstract In the last decade, the number of studies and publications on extracellular vesicles (EV) and exosomes has boomed. Colombia has displayed interest and progress in their study as shown in the increase of research project publications and products. However, this research field is still developing and has its own analytical challenges and technical limitations. For planning research projects and developing EV studies it is necessary to consider what is the state of the scientific field worldwide concerning EV nomenclature and classification, available techniques, resources, requirements and quality specifications, and the institutions that regulate the field. Answering this question will elicit EV studies that comply with international standards and respond to institutional demands and recommendations. However, the scientific information available is scattered and not all the aspects are considered in full. In this update, the available information is condensed and the official terms and currently defined nomenclature is presented, as well as the evolution of the field, the homogenization of the experimental parameters, the establishment of scientific authorities, institutions, and resources, and the recommendations generated worldwide for their development and research including their isolation, characterization, and functional studies. Finally, I analyzed the national context in a critical way, considering institutional strengths, common mistakes, and available analytical techniques and technologies.

Bases inmunológicas de la enfermedad neumocócica y el candidato vacunal PVC7-TT / Immunological bases of pneumococcal disease and vaccine candidate PVC7-TT

Introducción: Las enfermedades infecciosas del tracto respiratorio se encuentran entre las primeras causas de entidades respiratorias en edades extremas de la vida. Objetivo: Describir las bases inmunológicas de la enfermedad y el nuevo candidato vacunal conjugado antineumocócico PCV7-TT desarrollado en Cuba. Métodos: Se realizó una búsqueda en las bases de datos Medline, Pubmed, SciELO, LILACS, Cochrane Library y Web of Science, de documentos publicados entre mayo del 2018 y marzo del 2020. Se seleccionaron los 64 artículos de mayor relevancia y novedad. Resultados: Streptococcus pneumoniae es el agente etiológico de la enfermedad neumocócica; se le atribuye alrededor de un millón de defunciones anuales, principalmente en países en vías de desarrollo. Es un coco Gram-positivo, anaerobio facultativo y encapsulado que se encuentra dividido en 48 serogrupos y 97 serotipos tipificados. Presenta varios factores de virulencia que garantizan su mecanismo de patogenicidad; uno de los más importantes es el polisacárido capsular que constituye la diana de las vacunas antineumocócicas conjugadas y no conjugadas existentes. En el presente artículo se consideró la proteína de superficie C del neumococo como un posible candidato en la investigación y desarrollo de vacunas preventivas. Asimismo, las vesículas extracelulares podría ser un posible candidato para adyuvante vacunal con fines preventivos y terapéuticos. Conclusiones: El neumococo es un problema de salud a nivel global y el uso de vacunas conjugadas antineumocócicas constituye la herramienta más eficaz para su prevención. El candidato vacunal PCV7-TT desarrollado en Cuba es seguro, bien tolerado, inmunogénico y no inferior a las vacunas actualmente registradas(AU)

Introduction: Infectious diseases of the respiratory tract are among the leading causes of respiratory conditions in patients at extreme ages. Objective: Describe the immunological bases of the disease and the new conjugate pneumococcal vaccine candidate PCV7-TT developed in Cuba. Methods: A search was conducted in the databases Medline, Pubmed, SciELO, LILACS, Cochrane Library and Web of Science for documents published from May 2018 to March 2020. The 64 most relevant and novel papers were selected. Results: Streptococcus pneumoniae is the causative agent of pneumococcal disease, a condition causing about one million deaths a year worldwide, mainly in developing countries. It is a Gram-positive facultative anaerobic encapsulated coccus divided into 48 serogroups and 97 typified serotypes. Several virulence factors ensure its pathogenicity mechanism. One of the most important of these is the capsular polysaccharide constituting the target of the existing conjugate and non-conjugate pneumococcal vaccines. The study considered pneumococcal surface protein C as a possible candidate for the research and development of preventive vaccines. On the other hand, extracellular vesicles could be a possible vaccine adjuvant candidate for preventive and therapeutic use. Conclusions: Pneumococcus is a global health problem, and the use of conjugate pneumococcal vaccines is the most effective tool for its prevention. The vaccine candidate PCV7-TT developed in Cuba is safe, well-tolerated, immunogenic and not inferior to the vaccines so far registered(AU)

Exosomas: terapia celular regenerativa libre de células / Exosomes: "cell-free" regenerative cell therapy

La terapia celular basada en células mesenquimales/estromales se aplica ampliamente en la medicina moderna, aun cuando no todos los mecanismos de supervivencia y diferenciación están identificados. Sin embargo, hace pocos años se comenzaron a encontrar elementos extracelulares que generan nuevos paradigmas. En el presente trabajo se explican las principales características y funciones atribuidas a los exosomas, nanopartículas constituidas por microvesículas secretadas por las células con efecto en la matriz extracelular, y su repercusión como alternativa hacia una medicina regenerativa libre de células. Estas estructuras participan de forma notoria y crucial en la comunicación intercelular, lo que ha supuesto un cambio en el concepto de las funciones y el papel que desempeñan estas vesículas en los organismos vivos, en particular en la restauración de tejidos dañados y la respuesta inflamatoria e inmunológica. Se comentan algunos ejemplos de la repercusión biotecnológica de los exosomas en empresas y el mercado biofarmaceútico(AU)

Mesenchymal/stromal cell ;based therapy is widely applied in modern medicine, even though not all survival and differentiation mechanisms are identified. However, a few years ago, extracellular elements began to be found that generate new paradigms. The present work explains the main characteristics and functions attributed to exosomes, nanoparticles made up of microvesicles secreted by with an effect on the extracellular matrix, and their impact as an alternative towards cell-free regenerative medicine. These structures participate, notoriously and critically, in intercellular communication, which has led to a change in the concept of the functions and role that these vesicles play within living organisms, particularly in the restoration of damaged tissues and the inflammatory and immunological response. Some examples of the exosomes' biotechnological impact on companies and the biopharmaceutical market are discussed(AU)

Purificação e expressão de vesículas extracelulares humanas e a correlação com a toxoplasmose / Purification and expression of human extracellular vesicles and the correlation with toxoplasmosis

O objetivo deste estudo foi isolar e caracterizar as vesículas extracelulares (EVs) humanas e compará-las entre grupos de indivíduos com ou sem toxoplasmose, uma zoonose de distribuição mundial e de importância na saúde pública por seu papel na gestação e na co-infecção com o vírus da imunodeficiência humana (HIV). Oitenta e três amostras clínicas foram divididas em: grupo I, 20 plasmas de indivíduos saudáveis e gestantes (soronegativos para toxoplasmose); grupo II, 21 plasmas de pacientes com toxoplasmose sintomática; grupo III, 26 amostras de líquido cefalorraquidiano (LCR) de pacientes com toxoplasmose cerebral e co-infecção por HIV (TC/aids); e grupo IV, 16 amostras de LCR de pacientes sem toxoplasmose, mas com aids e outras infecções oportunistas (OI/aids). Após a recuperação das EVs por ultracentrifugação as amostras seguiram para análise de tamanho e concentração por Análise de Rastreamento de Nanopartícupas (NTA); morfologia, por Microscopia Eletrônica de Transmissão (TEM); perfil proteico, por SDS-PAGE/Imunoblot e expressão gênica de microRNAs (miRNAs), por PCR em tempo real (qPCR). A concentração das EVs-plasma do grupo II (2,4x1010 EVs/mL) foi estatisticamente maior do que as do grupo I (5,9x109 EVs/mL). Já nas EVs-LCR a concentração foi semelhante nos dois grupos, TC/aids (2,9x109 EVs/mL) e OI/aids (4,8x109 EVs/mL). Apenas a concentração das EVs foi capaz de distinguir os pacientes com...(AU)

This study aimed to isolate and characterize EVs and compare them between groups of individuals with or without toxoplasmosis, a worldwide distribution zoonosis, that plays an important role during pregnancy and co-infection with the human immunodeficiency virus (HIV). Eighty-three samples was divided as: group I, 20 plasma of health individuals and pregnant women (negatives for toxoplasmosis); group II, 21 plasma of toxoplasmosis patients; group III, 26 samples of cerebrospinal fluid (CSF) of patients with cerebral toxoplasmosis/aids (TC/AIDS) e group IV, 16 samples of CSF of patients without toxoplasmosis, but with AIDS and others opportunistic infections (OI/AIDS). After EV isolation by ultracentrifugation, size and concentration of were measured by Nano Tracking Analisys (NTA). In addition, morphology by Transmission Electronic Microscopy (TEM), protein profile by SDS-PAGE/Imunoblot and miRNAs expression, by Real time PCR (qPCR) were performed. Concentration of plasma EVs from group II patients (2.4x1010 EVs/mL) was statistically higher than those from group I (5.9x109 EVs/mL). Concentration od CSF EVs was similar in both groups, TC/AIDS (2.9x109 EVs/mL) and OI/AIDS (4,8x109 EVs/mL). Therefore, only the concentration was able to distinguish patients with toxoplasmosis than health individuals. Exosomes were also confirmed by TEM and CD-63 and CD-9 tetraspanina detection by immunoblot. EVs-plasma from group II had higher expression of miR-125b (4.9 times) and miR-146a (4.1 times) from control group...(AU)

Purificação e caracterização de vesículas extracelulares de taquizoítos de toxoplasma gondii / Purification and characterization of extracellular vesicles of taquizoítos of toxoplasma gondii

Toxoplasma gondii, protozoário causador da toxoplasmose, é transmitido dos animais para os seres humanos pela ingestão de carne contaminada ou por oocistos liberados nas fezes de felinos no ambiente. Nos seres humanos, a infecção é normalmente assintomática, mas em casos em que a infecção primária ocorre durante a gravidez ou a reativação da infecção latente ocorre em pacientes imunosuprimidos pode ser grave. Nas últimas décadas tem se estudado pequenas estruturas secretadas pelas células procarióticas e eucarióticas denominadas de vesículas extracelulares (EVs). As EVs podem transportar biomarcadores de doenças, macromoléculas biorreativas contribuindo para a patogênese e sendo uma forma de diagnóstico não invasivo. Estas pequenas estruturas podem ser isoladas por ultracentrifugação, cromatografia e observadas por microscopia eletrônica. Elas participam na comunicação entre as células, na transferência de proteínas, lipídios e ácidos nucléicos. Diante do exposto, o presente estudo teve como objetivo estabelecer um protocolo para isolar e caracterizar vesículas extracelulares produzidas e excretadas por taquizoítos da cepa RH de T. gondii. Taquizoítos provenientes de culturas de células VERO foram isolados dos sobrenadantes das culturas e centrifugados em cinco séries de lavagens. A seguir, foram incubados em meio de cultura por 24 horas para secreção das EVs. Em seguida, investigou-se o tamanho e concentração das EVs isoladas por análise de varredura de partículas (NTA) no equipamento NanoSight. Paralelamente, a morfologia e a liberação das EVs também foram investigadas por microscopia eletrônica de transmissão e de varredura. Então, as EVs foram purificadas por cromatografia em gelexclusão em alíquotas de 1 mL (24-32 frações) e imuno-selecionadas por ELISA utilizando um "pool" de soros reagente para toxoplasmose. A seguir foi investigado a presença de miRNA nas EVs; e finalmente, foi avaliado o perfil proteico das EVs de T. gondii para verificar por testes sorológicos se estas partículas poderiam ser reconhecidas pelo sistema imune hospedeiro. As análises realizadas por NTA permitiram determinar que cerca de 1 x 106 taquizoítos secretaram de 4 a 8 x 108 EVs/mL num período de 24 horas de incubação em meio de cultura celular. Adicionalmente, estas vesículas apresentaram morfologia e tamanho de 165-175 nm de diâmetro, tamanho correspondente às microvesículas. Estes resultados também foram confirmados pela avaliação das imagens fornecidas pelas microscopias eletrônicas de transmissão e varredura. Tambem foi possível determinar a presenção de small RNAs e micro RNAs através de uma corrida eletroforética micro fluídica. As análises por SDS-PAGE mostram que as proteínas que compõem as EVs apresentaram um perfil eletroforético com espectro de 15 a 70 kDa. Soros de camundongos cronicamente infectados (com 2 diferentes cepas de T. gondii) e soros humanos reconheceram distintos padrões eletroforéticos no immunoblotting. As vesículas liberadas por T. gondii podem ser um mecanismo importante pelo qual os parasitas apresentam seus antígenos ao hospedeiro e, portanto, podem ter um papel importante na patogênese da toxoplasmose

Toxoplasma gondii, a protozoan that causes toxoplasmosis, is transmitted from animals to humans through ingestion of infected meat or oocysts released by felines into the environment. In humans, infection is usually asymptomatic, but in cases where primary infection occurs during pregnancy or the reactivation of latent infection occurs in immunosuppressed patients it can be serious. In the last decades, small structures secreted by prokaryotic and eukaryotic cells called extracellular vesicles (EVs) were studied. These small structures can be isolated by ultracentrifugation, chromatography and examined by electron microscopy. EVs participate in the communication between cells, transfer of proteins, lipids and nucleic acids. In addition, them can carry disease biomarkers, bioreactive macromolecules contributing to the pathogenesis of diseases. In view of the above, the present study aimed to establish a protocol to isolate and characterize extracellular vesicles produced and excreted by tachyzoites of T. gondii RH strain. To achieve this goal, tachyzoites from VERO cell cultures were isolated from the culture supernatants and centrifuged in five sets of washes. Next, they were incubated in culture medium for 24 hours for secretion of the EVs. The size and concentration of the isolated EVs by particle scan analysis (NTA) were investigated on the NanoSight (NTA) equipment. In parallel, the morphology and the release of the EVs were also investigated by transmission and scanning electron microscopy. Then, EVs were purified by gel-exclusion chromatography in 1 mL aliquots (24-32 fractions) and immuno-selected by ELISA using a pool of reagent sera for toxoplasmosis. Next, the presence of miRNA in the EVs was investigated; and finally, the protein profile of T. gondii EVs was evaluated and verify by serological tests if these particles could be recognized by the host immune system. The results showed that the protocol for recovery of T. gondii EVs was established from 1 to 1010 tachyzoites obtained from cultured cells. Analyzes performed by NTA allowed us to determine that about 1 x 106 tachyzoites secreted 4 to 8 x 108 EVs / mL within 24 hours of incubation in culture medium. Additionally, these vesicles presented morphology and size of 165-175 nm of diameter, corresponding microvesicles size. These results were also confirmed by the evaluation of images provided by transmission and scanning electron microscopy. The miRNA purifications from the EVs and subsequent microfluidic electrophoretic run confirmed the presence of smallRNA and miRNA in the vesicles. SDS-PAGE analyzes show that the proteins carried by the EVs showed an electrophoretic profile with a spectrum of 15 to 70 kDa. Sera from chronically infected mice (with 2 different strains of T. gondii) and humam serum recognized distinct electrophoretic patterns in immunoblotting

Vesículas extracelulares derivadas de macrófagos alteram o potencial de invasão, proliferação e migração de linhagens celulares do carcinoma de células escamosas de língua oral / Macrophage-derived extracellular vesicles alter the potential for invasion, proliferation, and migration of oral tongue squamous cell carcinoma cell lines

O câncer é composto pelas células malignas em proliferação associadas às diferentes células circunjacentes, formando o microambiente tumoral (TME), onde há uma constante troca de informações. Uma das formas de comunicação entre os diferentes tipos celulares do TME se dá por meio da liberação de vesículas extracelulares (EVs), um campo de estudo ainda pouco explorado. O presente estudo se propôs a avaliar os efeitos das EVs liberadas por macrófagos do TME, células altamente plásticas em seu fenótipo (M1 ­ perfil antitumoral; M2 ­ perfil pró-tumoral), em diferentes linhagens do carcinoma de células escamosas de língua oral (CCELO) no tocante à capacidade invasiva, proliferativa e migratória. Foi observado que as amostras de EVs extraídas dos macrófagos eram relativamente puras em EVs, porém subtipo inespecíficas. No ensaio de invasão em miomas, quando colocadas as células inflamatórias em cocultura com as células HSC-3, as células M1 inibiram a invasão e M2 aumentaram a capacidade invasiva das células malignas. Por outro lado, o tratamento com M1 EVs aumentou a capacidade invasiva das células HSC-3 e o tratamento com EVs de M2 inibiu a invasão dessas células, sendo observado um perfil semelhante nas células SCC-25 e SAS quando submetidas aos mesmos tratamentos. Na análise do marcador Ki-67 nos miomas, tanto as células HSC-3 quanto SCC-25 e SAS apresentaram o mesmo padrão de proliferação independentemente do tratamento utilizado, quando comparados com os respectivos controles negativos. Quando analisada a proliferação das células malignas no IncuCyte®, tratadas com EVs dos diferentes tipos de macrófagos em diferentes concentrações, foi identificado um aumento na capacidade proliferativa de células HSC-3 e SAS tratadas com M1 EVs em um padrão dose dependente. Um aumento da capacidade proliferativa seguindo um padrão dose dependente também ocorreu quando as células SAS foram tratadas com M2 EVs. Nos demais ensaios de proliferação no IncuCyte® também foram identificados efeitos na capacidade proliferativa, no entanto um padrão dose dependente não foi observado. No ensaio de migração no IncuCyte®, foram identificadas diferenças significativas na capacidade migratória de células SCC-25 e SAS tratadas com diferentes tipos de EVs nas diferentes concentrações, quando comparadas ao controle negativo. Os achados deste estudo sugerem que as EVs derivadas de macrófagos são fatores importantes na tumorigênese do CCELO, bem como abre discussões sobre os diferentes efeitos das células inflamatórias no TME a depender do tipo de comunicação celular executada (AU).

Cancer is an entity composed of proliferating malignant cells associated with the different types surrounding cells, forming the tumor microenvironment (TME), where there is a constant exchange of information. One of the ways of communicating between different types of TME cells is through the release of extracellular vesicles (EVs), a field of study that remains poorly understood. The aim of the present study was to evaluate the effects of EVs released from TME macrophages, which are cells highly plastic in their phenotype (M1 showing an anti-tumor profile and M2 exhibiting a pro-tumor profile) in different cell lines of tongue squamous cells carcinoma (TSCC) regarding to invasive, proliferative and migratory capacity. It was observed that EVs samples obtained from macrophages were relatively pure in EVs, although they were non-specific subtypes. In the myoma invasion assay, it was observed that when inflammatory cells were co-cultured with HSC-3 cells, M1 cells inhibited invasion and M2 increased the invasive ability of the malignant cells. On the other hand, treatment with M1 EVs increased the invasive capacity of HSC-3 cells, and treatment with M2 EVs inhibited the invasion of these malignant cells, and a similar profile was observed in SCC-25 and SAS cells when they were submitted to the same treatments. In the analysis of the Ki-67 marker in myomas, HSC-3, SCC-25 and SAS cells showed the same proliferation pattern regardless the type of the treatment used when compared to the respective negative controls. When it was analyzed the proliferation of malignant cells in IncuCyte® treated with EVs derived from different types of macrophages at different concentrations, an increase in the proliferative ability of HSC-3 and SAS cells treated with M1 EVs was observed in a dosedependent pattern. An increase in proliferative ability in dose-dependent profile was also observed when SAS cells were treated with M2 EVs. In the other proliferation assays performed in IncuCyte®, effects on proliferative capacity were also highlighted, however a dose-dependent pattern was not observed. In the IncuCyte® migration assay, significant differences were observed in the migration capacity of SCC-25 and SAS cells treated with different types of EVs at different concentrations when compared to the negative control. The findings of this study suggest that macrophages-derived EVs are pivotal factors in TSCC tumorigenesis, as well as permits discussions on the different effects of inflammatory cells on TME depending on the type of cell communication performed (AU).

Therapeutic application of extracellular vesicles in acute and chronic renal injury. / Uso terapéutico de las vesículas extracelulares en insuficiencia renal aguda y crónica.

A new cell-to-cell communication system was discovered in the 1990s, which involves the release of vesicles into the extracellular space. These vesicles shuttle bioactive particles, including proteins, mRNA, miRNA, metabolites, etc. This particular communication has been conserved throughout evolution, which explains why most cell types are capable of producing vesicles. Extracellular vesicles (EVs) are involved in the regulation of different physiological processes, as well as in the development and progression of several diseases. EVs have been widely studied over recent years, especially those produced by embryonic and adult stem cells, blood cells, immune system and nervous system cells, as well as tumour cells. EV analysis from bodily fluids has been used as a diagnostic tool for cancer and recently for different renal diseases. However, this review analyses the importance of EVs generated by stem cells, their function and possible clinical application in renal diseases and kidney transplantation.

[Cardiovascular risk study in patients with renin-angiotensin system blockade by means of the proteone of circulating extracellular vesicles]. / Estudio del riesgo cardiovascular en pacientes con bloqueo del sistema renina-angiotensina a través del proteoma de las vesículas extracelulares circulantes.

INTRODUCTION: Extracellular vesicles (EVs) are released to the bloodstream by certain cell types due to transport, activation and cell death processes. Blood count of EVs from platelet and endothelial origin has been proved to be a cardiovascular risk biomarker. Thus, EVs proteome might reflect the underlying cellular processes in hypertensive patients with albuminuria. MATERIAL AND METHODS: Protein content of circulating EVs was analyzed by liquid chromatography coupled to mass spectrometry. EVs were isolated by an ultracentrifugation protocol optimized in order to avoid contamination by blood plasma proteins. Purity of the isolated fraction was verified by electronic and confocal microscopy, and by flow cytometry. RESULTS: We hereby show a method to isolate circulating EVs from hypertensive patients with/without albuminuria with high yield and purity. Besides, we provide a reference proteome of the EVs of these patients, composed of 2,463 proteins, and prove that the proteins carried by these vesicles are associated with crucial processes involved in the inherent cardiovascular risk. CONCLUSION: The proteome of circulating EVs is an interesting source of indicators in the evaluation of cardiovascular risk in hypertensive patients with renin-angiotensin system blockage.

Challenges and perspectives to enhance cattle production via in vitro techniques: focus on epigenetics and cell-secreted vesicles / Desafios e perspectivas na produção in vitro de embriões bovinos: enfoque epigenético e em vesículas secretadas por células

This review aim to present some clinical problems found in IVP-derived animals focusing on NT procedures and to discuss the possible role of epigenetics in such process. Also, as cell-secreted vesicles have been reported as possible regulators of important physiological reproductive processes such as folliculogenesis and fertilization, it is also presented herein a new perspective of manipulating the pre-implantation period trough effector molecules contained in such vesicles.

Nesta revisão, apresentamos alguns problemas clínicos encontrados nos animais derivados de PIV, principalmente derivados de transferência de núcleo, e discutimos o possível papel da epigenética em tais processos. Além disso, uma vez que vesículas secretadas por células têm sido descritas como possíveis reguladores de processos reprodutivos fisiológicos importantes, tais como a foliculogênese e a fertilização, estas são aqui apresentadas como uma possível nova ferramenta para a manipulação do período embrionário pré-implantacional através de moléculas efetoras, contidas em tais vesículas.