Exosomes are nano-vesicles present in the circulation that are involved in cell-to-cell communication and regulation of different biological processes. MicroRNAs (miRNAs) are part of their cargo and are potential biomarkers. Methods of exosome isolation and the inter-individual and intra-individual variations in circulating miRNA exosomal cargo have been poorly investigated. This study aims for comparing two exosome isolation methods and to assess the stability of eleven plasma exosomal miRNAs over time. In addition to evaluate miRNA variability of both kits, the effect of freezing plasma before exosome isolation or freezing isolated exosomes on miRNA stability was also evaluated. MiRNA levels were tested in 7 healthy subjects who underwent four different blood extractions obtained in 4 consecutive weeks. One of the isolation kits displayed generally better amplification signals, and miRNAs from exosomes isolated after freezing the plasma had the highest levels. Intra-subject and inter-subject coefficients of variance were lower for the same isolation kit after freezing plasma. Finally, miRNAs that showed an acceptable expression level were stable across the consecutive extractions. This study shows for the first time the stability over time of miRNAs isolated from circulating plasma exosomes, establishing a key step in the use of exosomal miRNAs as biomarkers.

Obstructive sleep apnea (OSA) is associated with increased risk for end-organ morbidities, which can collectively be viewed as accelerated aging. Vascular senescence is an important contributor to end-organ dysfunction. Exosomes are released ubiquitously into the circulation, and transfer their cargo to target cells facilitating physiological and pathological processes. Plasma exosomes from 15 patients with polysomnographically diagnosed OSA at baseline (OSA-T1) after 12 months of adherent continuous positive airway pressure (CPAP) treatment (OSA-T2), 13 untreated OSA patients at 12-month intervals (OSA-NT1, OSA-NT2), and 12 controls (CO1 and CO2) were applied on naïve human microvascular endothelialcells-dermal (HMVEC-d). Expression of several senescence gene markers including p16 (CDKN2A), SIRT1, and SIRT6 and immunostaining for β-galactosidase activity (x-gal) were performed. Endothelial cells were also exposed to intermittent hypoxia (IH) or normoxia (RA) or treated with hydrogen peroxide (H2O2), stained with x-gal and subjected to qRT-PCR. Exosomes from OSA-T1, OSA-NT1, and OSA-NT2 induced significant increases in x-gal staining compared to OSA-T2, CO1, and CO2 (p-value < 0.01). p16 expression was significantly increased (p < 0.01), while SIRT1 and SIRT6 expression levels were decreased (p < 0.02 and p < 0.009). Endothelial cells exposed to IH or to H2O2 showed significant increases in x-gal staining (p < 0.001) and in senescence gene expression. Circulating exosomes in untreated OSA induce marked and significant increases in senescence of naïve endothelial cells, which are only partially reversible upon long-term adherent CPAP treatment. Furthermore, endothelial cells exposed to IH or H2O2 also elicit similar responses. Thus, OSA either directly or indirectly via exosomes may initiate and exacerbate cellular aging, possibly via oxidative stress-related pathways.

Autophagy is a dynamic cellular mechanism involved in protein and organelle turnover through lysosomal degradation. Autophagy regulation modulates the pathologies associated with many neurodegenerative diseases. Using sheep naturally infected with scrapie as a natural animal model of prion diseases, we investigated the regulation of autophagy in the central nervous system (CNS) during the clinical phase of the disease. We present a gene expression and protein distribution analysis of different autophagy-related markers and investigate their relationship with prion-associated lesions in several areas of the CNS. Gene expression of autophagy markers ATG5 and ATG9 was downregulated in some areas of scrapie brains. In contrast, ATG5 protein accumulates in medulla oblongata and positively correlates with prion deposition and scrapie-related lesions. The accumulation of this protein and p62, a marker of autophagy impairment, suggests that autophagy is decreased in the late phases of the disease. However, the increment of LC3 proteins and the mild expression of p62 in basal ganglia and cerebellum, primarily in Purkinje cells, suggests that autophagy machinery is still intact in less affected areas. We hypothesize that specific cell populations of the CNS may display neuroprotective mechanisms against prion-induced toxicity through the induction of PrPSc clearance by autophagy.

Scrapie is a transmissible spongiform encephalopathy (TSE), or prion disease, of sheep and goats. As no simple diagnostic tests are yet available to detect TSEs in vivo, easily accessible biomarkers could facilitate the eradication of scrapie agents from the food chain. To this end, we analysed by quantitative reverse transcription PCR a selected set of candidate microRNAs (miRNAs) from circulating blood plasma of naturally infected, classical scrapie sheep that demonstrated clear scrapie symptoms and pathology. Significant scrapie-associated increase was repeatedly found for miR-342-3p and miR-21-5p. This is the first demonstration, to our knowledge, of circulating miRNA alterations in any animal suffering from TSE. Genome-wide expression studies are warranted to investigate the true depth of miRNA alterations in naturally occurring TSEs, especially in presymptomatic animals, as the presented study demonstrates the potential feasibility of miRNAs as circulating TSE biomarkers.

Lung diseases (LD) are one of the most common causes of death worldwide. Although it is known that chronic airway inflammation and excessive tissue repair are processes associated with LD such as asthma, chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF), their specific pathways remain unclear. Extracellular vesicles (EVs) are heterogeneous nanoscale membrane vesicles with an important role in cell-to-cell communication. EVs are present in general biofluids as plasma or urine but also in secretions of the airway as bronchoalveolar lavage fluid (BALF), induced sputum (IS), nasal lavage (NL) or pharyngeal lavage. Alterations of airway EV cargo could be crucial for understanding LD. Airway EVs have shown a role in the pathogenesis of some LD such as eosinophil increase in asthma, the promotion of lung cancer in vitro models in COPD and as biomarkers to distinguishing IPF in patients with diffuse lung diseases. In addition, they also have a promising future as therapeutics for LD. In this review, we focus on the importance of airway secretions in LD, the pivotal role of EVs from those secretions on their pathophysiology and their potential for biomarker discovery.

Sleep is very important for overall health and quality of life, while sleep disorder has been associated with several human diseases, namely cardiovascular, metabolic, cognitive, and cancer-related alterations. Obstructive sleep apnea (OSA) is the most common respiratory sleep-disordered breathing, which is caused by the recurrent collapse of the upper airway during sleep. OSA has emerged as a major public health problem and increasing evidence suggests that untreated OSA can lead to the development of various diseases including neurodegenerative diseases. In addition, OSA may lead to decreased blood oxygenation and fragmentation of the sleep cycle. The formation of free radicals or reactive oxygen species (ROS) can emerge and react with nitric oxide (NO) to produce peroxynitrite, thereby diminishing the bioavailability of NO. Hypoxia, the hallmark of OSA, refers to a decline of tissue oxygen saturation and affects several types of cells, playing cell-to-cell communication a vital role in the outcome of this interplay. Red blood cells (RBCs) are considered transporters of oxygen and nutrients to the tissues, and these RBCs are important interorgan communication systems with additional functions, including participation in the control of systemic NO metabolism, redox regulation, blood rheology, and viscosity. RBCs have been shown to induce endothelial dysfunction and increase cardiac injury. The mechanistic links between changes of RBC functional properties and cardiovascular are largely unknown. Extracellular vesicles (EVs) are secreted by most cell types and released in biological fluids both under physiological and pathological conditions. EVs are involved in intercellular communication by transferring complex cargoes including proteins, lipids, and nucleic acids from donor cells to recipient cells. Advancing our knowledge about mechanisms of RBC-EVs formation and their pathophysiological relevance may help to shed light on circulating EVs and to translate their application to clinical practice. We will focus on the potential use of RBC-EVs as valuable diagnostic and prognostic biomarkers and state-specific cargoes, and possibilities as therapeutic vehicles for drug and gene delivery. The use of RBC-EVs as a precision medicine for the diagnosis and treatment of the patient with sleep disorder will improve the prognosis and the quality of life in patients with cardiovascular disease (CVD).

Inflammatory cell counts from sputum induction (SI) are currently the most effective non-invasive way to assess bronchial inflammation and predict therapeutic responses in patients with asthma. 1,2 However, there is currently a high risk of contagion from patients infected with SARS-CoV-2, 3 since SI involves the generation of aerosols, cough manoeuvres and the handling of samples. There is therefore a need to establish safety protocols for medical procedures performed in routine clinical practice in a return to normality during the current COVID-19 pandemic.

Scrapie and bovine spongiform encephalopathy are fatal neurodegenerative diseases caused by the accumulation of a misfolded protein (PrP(res)), the pathological form of the cellular prion protein (PrP(C)). For the last decades, prion research has greatly progressed, but many questions need to be solved about prion replication mechanisms, cell toxicity, differences in genetic susceptibility, species barrier or the nature of prion strains. These studies can be developed in murine models of transmissible spongiform encephalopathies, although development of cell models for prion replication and sample titration could reduce economic and timing costs and also serve for basic research and treatment testing. Some murine cell lines can replicate scrapie strains previously adapted in mice and very few show the toxic effects of prion accumulation. Brain cell primary cultures can be more accurate models but are difficult to develop in naturally susceptible species like humans or domestic ruminants. Stem cells can be differentiated into neuron-like cells and be infected by prions. However, the use of embryo stem cells causes ethical problems in humans. Mesenchymal stem cells (MSCs) can be isolated from many adult tissues, including bone marrow, adipose tissue or even peripheral blood. These cells differentiate into neuronal cells, express PrP(C) and can be infected by prions in vitro. In addition, in the last years, these cells are being used to develop therapies for many diseases, including neurodegenerative diseases. We review here the use of cell models in prion research with a special interest in the potential use of MSCs.