HIV-1 infection of the central nervous system causes HIV-associated neurocognitive disorders, even in aviremic patients. Although astrocyte malfunction was associated to these disorders, their implication is overshadowed by contributions of microglia and macrophages. Astrocytes are infected with HIV-1 in vivo and express a relevant amount of viral protein Nef. Nef was shown to stimulate its own release in exosomes from diverse cell types, which in turn have damaging effects on neighboring cells. Using immunoblotting and electron microscopy, we showed that human astrocytes expressing Nef.GFP similarly release Nef in exosomes. Importantly, Nef.GFP expression increases the secretion of exosomes from human astrocytes up to 5.5-fold, as determined by total protein content and nanoparticle tracking analysis. Protein analysis of exosomes and viruses separated on iodixanol gradient further showed that native or pseudotyped HIV-1-infected human astrocytes release exosomes, which contain Nef. Our results provide the basis for future studies of the damaging role of Nef-exosomes produced by HIV-infected astrocytes on the central nervous system.
COBISS.SI-ID: 33351641
Objective To address the role of translationally active HIV reservoir in chronic inflammation and non-AIDS related disorders, we first need a simple and accurate assay to evaluate viral protein expression in virally suppressed subjects. Design We optimized an HIV Nef enzyme-linked immunosorbent assay (ELISA) and used it to quantify plasma Nef levels as an indicator of the leaky HIV reservoir in an HIV-infected cohort. Methods This study accessed 134 plasma samples from a well-characterized cohort study of HIV-infected and uninfected adults in San Francisco (the SCOPE cohort). We optimized an ELISA for detection of plasma Nef in HIV-negative subjects and HIV-infected non-controllers, and evaluated its utility to quantify plasma Nef levels in a cross-sectional study of ART-suppressed and elite controller HIV-infected subjects. Results Here, we describe the performance of an optimized HIV Nef ELISA. When we applied this assay to the study cohort we found that plasma Nef levels were correlated with plasma HIV RNA levels in untreated disease. However, we were able to detect Nef in plasma of approximately half of subjects on ART or with elite control, despite the lack of detectable plasma HIV RNA levels using standard assays. Plasma Nef levels were not consistently associated with CD4+ T-cell count, CD8+ T-cell count, self-reported nadir CD4+ T-cell count or the CD4+/CD8+ T-cell ratio in HIV-infected subjects. Conclusion Since plasma HIV RNA levels are undetectable in virally suppressed subjects, it is reasonable to assume that viral protein expression in leaky reservoir, and not plasma virions, is the source of Nef accumulating in plasma. To examine this further, improvements of the assay sensitivity, by lowering the background through improvements in the quality of Nef antibodies, and detailed characterization of the HIV reservoirs are needed.
COBISS.SI-ID: 33608665
Determining the HIV-1 reservoir size in infected individuals is of great importance for improvement of their treatment. Plasma trans-activation response element (TAR) RNA has been suggested as one of the possible biomarkers. TAR RNA is produced during non-processive transcription in HIV-1 productively infected and latent T cells. Here, plasma samples and paired exosome samples of 55 subjects from the observational SCOPE cohort were analysed for the presence of TAR RNA. First, a PCR-based assay was optimized, which provided 100% specificity and 100% sensitivity in differentiating HIV-1 infected non-controllers from uninfected individuals. Next, TAR RNA was detected in the plasma of 63% of aviremic HIV-1-infected patients, who were either treated with antiretroviral therapy or were elite controllers. Although TAR RNA levels did not correlate with patient gender, age, CD4 levels, CD8 levels, they tended to correlate with CD4/CD8 ratio (P = 0.047). This study is the first to investigate plasma TAR RNA in a relatively large cohort of HIV- 1-infected patients. We additionally show that the TAR RNA molecules in the plasma of aviremic patients are not limited to exosomes.
COBISS.SI-ID: 33286873
Extracellular vesicles are a heterogeneous population of membrane vesicles, released from cells both in vivo and in vitro, with an important role in intercellular communication. They have been isolated from a variety of biological samples such as blood, bronchoalveolar lavage, synovial fluid, urine, amniotic fluid, semen, milk, saliva; and from cell cultures of most mammalian cells. Based on their size and the site of formation in the cell, they are classified as exosomes (30–100 nm), microvesicles (100–1,000 nm) and apoptotic bodies (1, 000–5,000 nm). Their protein, nucleic acid (miRNA) and lipid composition reflects the composition of the parental cell and depends on the current state of the cell. Consequently, the extracellular vesicles are an important source of novel biomarkers for diverse physiological conditions with important diagnostic and prognostic value, especially for disorders of the central-nervous system. Researchers also investigate the potential use of extracellular vesicles as targets for diagnostic and therapeutic purposes, but many obstacles still have to be overcome. Due to their great clinical potential, the interest in extracellular vesicle characterization, formation and role increased importantly in recent years, fueling also development of new methods for their isolation and analysis. In the present review we will describe the types of extracellular vesicles, methods for their isolation and analysis, their biological role and clinical potential.
COBISS.SI-ID: 32578521
Hypoxia-inducible factors (HIFs) belong to a family of transcription factors (TF) responsive to a low O2 availability, which is often a characteristic feature of solid tumors. The alpha subunit of the HIF heterodimer is O2 -sensitive, and once stabilized in hypoxia, it functions as a master regulator of various genes involved in hypoxia pathway. Changes in the HIF1A (hypoxia inducible factor 1, alpha subunit) nucleotide sequence or expression has been shown to be associated with development of several diseases. Due to increasing research interest in HIF1A gene a review of association studies was needed. We here reviewed published data on single nucleotide polymorphisms (SNPs) in HIF1A in various diseases; in total, 34 SNPs were tested for an association with 49 phenotypes, and the results were visualized using the Cytoscape software. Among all collected polymorphisms 16 SNPs showed significant associations with 40 different phenotypes, including six SNPs associated with 14 cancer types. Missense SNPs (rs11549465 and rs11549467) within the oxygen-dependent degradation domain were most frequently studied. The study provides a comprehensive tool for researchers working in this area and may contribute to more accurate disease diagnosis and identification of therapeutic targets.
COBISS.SI-ID: 3859080