We were the first to show that extracellular vesicles, in addition to two already described mechanisms, that is fusion of a multivesicular body (MVB) with the plasma membrane and outward invagination of the plasma membrane, can also be released from an alternative intracellular source. This is a CD9+CD81+, MVB-like organelle, which is not of endosomal origin but originates from the plasma membrane. Once infected by HIV-1, microglia abundantly produce protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking, and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with a green fluorescent protein (Nef.GFP) to identify Nef.GFP-associated cellular compartments, examine their mobility and Nef release from cultured cells. Immunoblotting revealed that Nef.GFP confined to subcellular fractions with a buoyant density similar to organelles, positive for protein LAMP1, but it segregated from dextran-laden and LysoTracker-laden endo/lysosomes in live cells. Confocal microscopy showed Nef.GFP-positive vesicle-like structures were smaller than dextran-laden vesicles and in contrast to those displayed slow and non-directional mobility. Ionomycin-evoked elevation in intracellular free Ca2+ concentration negligibly affected the mobility of Nef.GFP structures but it inhibited the release of those structures from the cell. Nef.GFP+ structures significantly co-localized with membrane sites immunopositive for the tetraspanins CD9 and CD81.
COBISS.SI-ID: 33745113
This paper is an important update of Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines, proposed for the field in 2014, based on the evolution of the knowledge in the last four years. The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations and to characterize them properly. Ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond a mere description of the function in a crude, potentially contaminated, and heterogeneous preparation. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
COBISS.SI-ID: 34072025
We were the first to show that Nef is present in the plasma of half of the HIV infected individuals, in which effective antiretroviral therapy maintains the level of plasma HIV RNA below the clinically detectable level. This implies that translationally active viral reservoirs, which do not produce viruses but release Nef, are present in the body. Nef is a known HIV pathogenic factor, which might contribute to chronic inflammation and to non-AIDS related disorders, observed in effectively treated individuals. Plasma Nef could, therefore, serve as a biomarker to identify individuals, which would benefit from the change in the therapy. Because of Nef's toxicity, Nef-vesicles could also serve as a good therapeutic target. To easily and accurately evaluate Nef expression, we optimized an HIV Nef enzyme-linked immunosorbent assay and used it to quantify plasma Nef levels as an indicator of the leaky HIV reservoir. We included 134 plasma samples from a well-characterized cohort study of HIV-infected and uninfected adults in San Francisco (the SCOPE cohort). We found that plasma Nef levels correlated with plasma HIV-RNA levels in untreated disease. However, we were able to detect Nef in plasma of approximately half of the treated subjects and in elite controlers, despite the lack of detectable plasma HIV-RNA levels. Plasma Nef levels were not consistently associated with neither CD4+, CD8+, self-reported nadir CD4+ T-cell counts, nor CD4+/CD8+ T-cell ratio in HIV-infected subjects.
COBISS.SI-ID: 33608665