Extracellular vesicles and circulating nucleic acids最新文献

This report summarises the presentations and activities of the SELECTBIO Workshop on Rigor and Reproducibility in EV Research and Single EV Analysis held in San Diego, USA, in December 2021. The motivation for the session was the recognition that progress in the extracellular vesicle (EV) field is limited by the availability of rigorous and reproducible EV measurement tools. These tools are absolutely required for EVs to evolve from a research lab curiosity to something that will improve our ability to understand, diagnose, treat, and prevent disease. The program focused on guidelines for EV measurement and characterization as laid out in the recent MISEV2018 and MIFlowCyt-EV publications, their implementation in routine practice, and their continued evolution as new EV measurement technologies are introduced. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.

Previous studies have suggested that aberrant 5-hydroxymethylcytosines (5hmC) modifications are related to cancer pathobiology. Genome-wide profiling 5hmC in circulating cell-free DNA (cfDNA) using the highly sensitive chemical labeling-based 5hmC-Seal technique has been demonstrated to have the potential to be a robust epigenomic tool for cancer biomarker discovery. Prior studies have mostly focused on cfDNA-derived 5hmC-Seal data summarized in well-annotated genic features (e.g., gene bodies) or unbiased bins. Zhou et al. recently proposed long non-coding RNAs (lncRNAs) as an alternative molecular target for biomarker discovery using publicly available 5hmC-Seal data. Considering its potential clinical impact, we would like to comment on Zhou et al. and advocate more serious consideration of critical issues such as the availability of clinical information and technical variables, especially when performing secondary analysis using publicly available data, with the aim of improving data transparency and translatability.

Aim: A peripheral inflammatory response can drive neuroinflammation in a number of infections including human immunodeficiency virus (HIV). Monocyte/macrophage (M/Mφ) activation is a hallmark of acute HIV infection and a source of chronic inflammation in a subset of HIV-infected individuals. We sought to decrease peripheral inflammation and M/Mφ transmigration after HIV infection by engineering extracellular vesicles (EV) to antagonize a microRNA (miR) associated with inflammation. We hypothesized that induced pluripotent stem cell (iPSC)-derived monocyte EVs (mEVs), engineered to contain an antagomir to miR-155 (αmiR mEV) would target monocyte inflammation and influence neuroinflammation in an HIV-infected humanized mice.

Methods: mEVs were characterized by tetraspanins, nanoparticle tracking analysis, electron microscopy, and their preferential entry into circulating monocytes as well as testing for endogenous selected miRNAs. HIV-infected humanized mice were treated with control or antagomir155 mEVs. Plasma viral load was measured plus activation markers on lymphocytes and monocytes and the number of macrophages in the brain was quantified.

Results: mEVs preferentially entered peripheral monocytes. HIV infection increased C-C chemokine receptor type 5 (CCR5) and major histocompatibility complex, class II, DR (HLA-DR) expression on T cells and monocytes. Treatments with mEVs did not decrease plasma HIV viral load; however, mEVs alone resulted in a decrease in %CCR5+ and %HLA-DR+ on T cells and an increase in %CCR5+ monocytes. αmiR mEVs decreased %CCR5 on M/Mφ. The mEV-treated HIV-infected mice did not show an increase in macrophage transmigration to the brain.

Conclusion: mEVs alone caused an unexpected decrease in lymphocyte activation and increase in monocyte %CCR5; however, this did not translate to an increase in macrophage transmigration to the brain.

Aim: Targeting the modes of pathogen shedding/transmission via exosomes or extracellular vesicles has been envisioned as the best approach to control vector-borne diseases. This study is focused on altering exosomes stability to affect the pathogen transmission from infected to naïve recipient cells.

Methods: In this study, neuronal or arthropod exosomes were treated at different temperatures or with different salts or pH conditions to analyze their ability and efficiency in the transmission of tick-borne Langat virus (LGTV) from infected to naïve recipient cells.

Results: Quantitative real-time PCR (qRT-PCR) and immunoblotting analyses revealed that treatment of neuronal or tick exosomes at warmer temperatures of 37 °C or 23 °C, respectively, or with sulfate salts such as Magnesium or Ammonium sulfates or with highly alkaline pH of 9 or 11.5, dramatically reduced transmission of LGTV via infectious exosomes (human or tick cells-derived) to human neuronal (SH-SY5Y) cells or skin keratinocytes (HaCaT cells), respectively.

Conclusion: Overall, this study suggests that exosome-mediated viral transmission of vector-borne pathogens to the vertebrate host or the viral dissemination and replication within or between the mammalian host can be reduced by altering the ability of exosomes with basic changes in temperatures, salts or pH conditions.

Aim: Elevated brain deposits of amyloid beta (Aβ₄₀) contribute to neuropathology and cognitive dysfunction in Alzheimer's disease (AD). However, the role of the blood-brain barrier (BBB) as an interface for the transfer of Aβ₄₀ from the periphery into the brain is not well characterized. In addition, a substantial population of neural progenitor cells (NPCs) resides in close proximity to brain capillaries that form the BBB. The aim of this study is to understand the impact of brain endothelium-derived extracellular vesicles (EV) containing Aβ₄₀ on metabolic functions and differentiation of NPCs.

Methods: Endothelial EVs were derived from an in vitro model of the brain endothelium treated with 100 nM Aβ₄₀ or PBS. We then analyzed the impact of these EVs on mitochondrial morphology and bioenergetic disruption of NPCs. In addition, NPCs were differentiated and neurite development upon exposure to EVs was assessed using the IncuCyte Zoom live cell imaging system.

Results: We demonstrate that physiological concentrations of Aβ₄₀ can be transferred to accumulate in NPCs via endothelial EVs. This transfer results in mitochondrial dysfunction, disrupting crista morphology, metabolic rates, fusion and fission dynamics of NPCs, as well as their neurite development.

Conclusion: Intercellular transfer of Aβ₄₀ is carried out by brain endothelium-derived EVs, which can affect NPC differentiation and induce mitochondrial dysfunction, leading to aberrant neurogenesis. This has pathological implications because NPCs growing into neurons are incorporated into cerebral structures involved in learning and memory, two common phenotypes affected in AD and related dementias.

Aim: The fragmentation characteristics of cell-free DNA (cfDNA) are informative biomarkers in liquid biopsies, including non-invasive prenatal testing (NIPT), as they provide insights into the origins of the cfDNA. Viral infections by DNA viruses can contribute to the available cfDNA in these samples. Here, we characterize the fragment size distribution of viral cfDNA fragments obtained from available anonymous NIPT samples.

Methods: A viral database of 224 DNA viruses was generated from the NCBI RefSeq viral database. Paired-end cfDNA sequencing reads from 83.522 NIPT samples that did not map to any of the human chromosomes, or mitochondrial DNA of the human reference genome build GRCh38 (excluding alternative and unplaced contigs) were remapped to the generated viral database. Reads mapping to the 14 most abundant DNA viruses were selected, and fragment size distributions were analyzed in detail.

Results: Distinct fragmentation patterns were identified for several DNA viruses, most likely due to differences in viral tropism, chromatinization (binding of nucleosomes), and the topology of the viral DNA. In high viral load parvo B19 positive samples, the fragment size distribution differed between samples, potentially reflecting the state of the infection.

Conclusion: These findings outline the potential for liquid biopsies to elucidate the dynamics behind the viral infection, which may potentially have various clinical applications. Our data provide preliminary insights on the use of fragmentomics of viral cfDNA to distinguish between reactivation, reinfection, and primary infection and monitoring the state of viral infections.

Many researchers worldwide are currently trying to develop targeted molecular therapies such as nucleic acid medicines or antibody-drug conjugates for various diseases. Writing in Extracellular Vesicles and Circulating Nucleic Acids, Kim et al. summarized existing technologies for encapsulating therapeutic molecules into exosomes and introduced some human cell lines which are able to produce safe, effective therapeutic exosomes. Their review article offers the "magic bullet" for fighting threats to humanity such as the current coronavirus pandemic.