Size Determination and Phenotypic Analysis of Urinary Extracellular Vesicles using Flow Cytometry.

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of different sizes ranging from 40-100 nm in diameter such as in exosomes to 100-1000 nm in microvesicles. They can also contain different molecules that can be used as biomarkers for the prognosis and diagnosis of many diseases. Many techniques have been developed to characterize these vesicles. One of these is flow cytometry. However, there are no existing reports to show how to quantify the concentration of EVs and differentiate them by size, along with biomarker detection. This work aims to describe a procedure for the isolation, quantification, and phenotypification of urinary extracellular vesicles, uEVs, using a conventional cytometer for the analysis without any modification to its configuration. The method's limitations include staining a maximum of four different biomarkers per sample. The method is also limited by the amount of EVs available in the sample. Despite these limitations, with this protocol and its subsequent analysis, we can obtain more information on the enrichment of EVs markers and the abundance of these vesicles present in urine samples, in diseases involving kidney and brain damage.

[Regulatory B cells (Bregs) role in allergic diseases]. / Participación de los linfocitos B reguladores (Breg) en las enfermedades alérgicas.

Immune tolerance, both to exogenous antigens and autoantigens, is essential for restraining undesired inflammatory responses that might result in severe damage to body tissues or cause chronic diseases. During the past few decades, different cell populations and molecules by them secreted have been associated with suppressing and regulatory mechanisms of immune responses. Although B cells typically acquire relevance as precursors of antibody-producing cells, they can also develop potent regulatory functions through the production of soluble molecules or by establishing direct cellular interactions mediated by different surface proteins implicated in signal transduction. While most studies of regulatory B cells define the role of these lymphocytes in autoimmune diseases, evidence of their importance and mechanisms of action in allergic diseases has accumulated in recent years. As a result, regulatory B cells appear to be relevant elements for the establishment or loss of allergen tolerance in different allergic diseases, although they still have been little explored.

La tolerancia inmunológica, tanto a los antígenos exógenos como a los autoantígenos, es esen-cial para restringir las respuestas inflamatorias no deseadas que pudieran derivar en daño grave a los tejidos del organismo o provocar enfermedades crónicas. Durante las últimas décadas, diversas poblaciones celulares y moléculas secretadas por estas se han asociado con mecanismos supresores y reguladores de las respuestas inmunes. Aunque las células B adquieren relevancia típicamente como precursores de células productoras de anticuerpos, también son capaces desarrollar potentes funciones reguladoras a través de la producción de moléculas solubles o mediante el establecimiento de interacciones celulares directas mediadas por diferentes proteínas de superficie implicadas en transducción de señales. Si bien la mayoría de los estudios de células B reguladoras definen el papel de estos linfocitos en enfermedades autoinmunes, en años recientes se ha acumulado evidencia de su importancia y mecanismos de acción en enfer-medades alérgicas. Las células reguladoras B parecen ser elementos relevantes en el establecimiento o pérdida de la tolerancia a alérgenos en diferentes enfermedades alérgicas, si bien aún han sido poco explorados.