A Review Study of the Participation of Late Domains in Sorting and Transport of Viral Factors to Exosomes.

Cellular communication depends heavily on the participation of vesicular systems generated by most cells of an organism. Exosomes play central roles in this process. Today, these vesicles have been characterized, and it has been determined that the cargo they transport is not within a random system. In fact, it depends on various molecular signals and the recruitment of proteins that participate in the biogenesis of exosomes. It has also been shown that multiple viruses can recruit these vesicles to transport viral factors such as genomes or proteins. It has been shown that the late domains present in viral proteins are critical for the exosomal selection and biogenesis systems to recognize these viral proteins and introduce them into the exosomes. In this review, the researchers discuss the evidence related to the characterization of these late domains and their role in exosome recruitment during viral infection.

Clinical and immunological features associated to the development of a sustained immune humoral response in COVID-19 patients: Results from a cohort study.

Background: Until now, most of the research addressing long-term humoral responses in coronavirus disease 2019 (COVID-19) had only evaluated the serum titers of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgGs, without the assessment of the baseline antiviral clinical and immune profile, which is the aim of this study and may be the key factor leading to a broad and sustained antibody response. Methods: We included 103 patients with COVID-19. When the patients sought medical attention (baseline), a blood sample was drawn to perform immunophenotype of lymphocytes by flow cytometry. The patients were assessed 15 days after baseline and then every month until the third month, followed by a last visit 6 months after recruitment. We evaluated the anti-SARS-COV-2 IgG at all time points, and the serum levels of cytokines, chemokines, anti-cellular (AC) antibodies and neutrophil extracellular traps were also assessed during the follow-up. The primary outcome of the study was the presence of a sustained immune humoral response, defined as an anti-SARS-CoV-2 IgG titer >4.99 arbitrary units/mL in at least two consecutive measures. We used generalized lineal models to assess the features associated with this outcome and to assess the effect of the changes in the cytokines and chemokines throughout time on the development of a sustained humoral immune response. Results: At baseline the features associated to a sustained immune humoral response were the diagnosis of critical disease, absolute number of lymphocytes, serum IP-10, IL-4, IL-2, regulatory T cells, CD8+ T cells, and positive AC antibodies. Critical illness and the positivity of AC antibodies were associated with a sustained humoral immune response after 3 months, whilst critical illness and serum IL-13 were the explanatory variables after 6 months. Conclusion: A sustained immune humoral response is strongly related to critical COVID-19, which is characterized by the presence of AC antibodies, quantitative abnormalities in the T cell compartment, and the serum cytokines and chemokines during acute infection and throughout time.

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.

Conformational changes in myeloperoxidase induced by ubiquitin and NETs containing free ISG15 from systemic lupus erythematosus patients promote a pro-inflammatory cytokine response in CD4+ T cells.

BACKGROUND: Neutrophil extracellular traps (NETs) from patients with systemic lupus erythematosus (SLE) are characterized by lower ubiquitylation and myeloperoxidase (MPO) as a substrate. The structural and functional effect of such modification and if there are additional post-translational modifications (PTMs) are unknown. METHODS: To assess the expression and functional role of PTMs in NETs of patients with SLE; reactivation, proliferation and cytokine production was evaluated by flow cytometry using co-cultures with dendritic cells (DC) and CD4+ from SLE patients and healthy controls. The impact of ubiquitylation on MPO was assessed by molecular dynamics. The expression of ISG15 in NETs was evaluated by immunofluorescence and Western Blot. RESULTS: Fifteen patients with SLE and ten healthy controls were included. In the co-cultures of CD4+ lymphocytes with DC stimulated with ubiquitylated MPO or recombinant MPO, a higher expression of IFNγ and IL-17A was found in CD4+ from SLE patients (p < 0.05). Furthermore, with DC stimulated with ubiquitylated MPO a trend towards increased expression of CD25 and Ki67 was found in lupus CD4+ lymphocytes, while the opposite was documented in controls (p < 0.05). Through molecular dynamics we found the K129-K488-K505 residues of MPO as susceptible to ubiquitylation. Ubiquitylation affects the hydration status of the HEME group depending on the residue to which it is conjugated. R239 was found near by the HEME group when the ubiquitin was in K488-K505. In addition, we found greater expression of ISG15 in the SLE NETs vs controls (p < 0.05), colocalization with H2B (r = 0.81) only in SLE samples and increased production of IFNγ in PBMCs stimulated with lupus NETs compared to healthy controls NETs. CONCLUSION: The ubiquitylated MPO has a differential effect on the induction of reactivation of CD4+ lymphocytes in patients with SLE, which may be related to structural changes by ubiquitylation at the catalytic site of MPO. Besides a lower ubiquitylation pattern, NETs of patients with SLE are characterized by the expression of ISG15, and the induction of IFNγ by Th1 cells.

[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.

Inflammasomes and its importance in viral infections.

A complex interplay between pathogen and host determines the immune response during viral infection. A set of cytosolic sensors are expressed by immune cells to detect viral infection. NOD-like receptors (NLRs) comprise a large family of intracellular pattern recognition receptors. Members of the NLR family assemble into large multiprotein complexes, termed inflammasomes, which induce downstream immune responses to specific pathogens, environmental stimuli, and host cell damage. Inflammasomes are composed of cytoplasmic sensor molecules such as NLRP3 or absent in melanoma 2 (AIM2), the adaptor protein ASC (apoptosis-associated speck-like protein containing caspase recruitment domain), and the effector protein procaspase-1. The inflammasome operates as a platform for caspase-1 activation, resulting in caspase-1-dependent proteolytic maturation and secretion of interleukin (IL)-1ß and IL-18. This, in turn, activates the expression of other immune genes and facilitates lymphocyte recruitment to the site of primary infection, thereby controlling invading pathogens. Moreover, inflammasomes counter viral replication and remove infected immune cells through an inflammatory cell death, program termed as pyroptosis. As a countermeasure, viral pathogens have evolved virulence factors to antagonise inflammasome pathways. In this review, we discuss the role of inflammasomes in sensing viral infection as well as the evasion strategies that viruses have developed to evade inflammasome-dependent immune responses. This information summarises our understanding of host defence mechanisms against viruses and highlights research areas that can provide new approaches to interfere in the pathogenesis of viral diseases.