Rigorous Process for Isolation of Gut-Derived Extracellular Vesicles (EVs) and the Effect on Latent HIV

Area of Science:

• Microbiology
• Cell Biology
• Biochemistry

Background:

• The human gastrointestinal tract harbors trillions of microorganisms.
• These microbes secrete molecules like extracellular vesicles (EVs) and extracellular condensates (ECs).
• These secretions can influence host physiological and pathophysiological activities, but pure isolation methods are lacking.

Purpose of the Study:

• To develop an efficient protocol for isolating pure and functional EVs from colonic content (ColEVs).
• To characterize the purity, toxicity, and functionality of isolated ColEVs.
• To establish a robust tool for GI-derived EV research.

Main Methods:

• Utilized high-resolution particle purification liquid chromatography (PPLC) with a gradient-bead-column.
• Integrated polyvinylpolypyrrolidone (PVPP)-mediated extraction to remove impurities.
• Assessed ColEVs' purity, non-toxicity, cellular internalization, and functional activation of the HIV LTR promoter.

Main Results:

• PVPP facilitated the isolation of pure, non-toxic, and functionally active ColEVs.
• Isolated ColEVs were internalized by cells and activated the HIV LTR promoter.
• ColEVs isolated without PVPP showed reductase activity, indicating potential for MTT reduction.

Conclusions:

• The developed PPLC and PVPP protocol provides a robust method for purifying GI-derived EVs.
• This method enhances the rigor of research involving microbial-contaminated samples.
• The protocol supports translational applications of GI-derived EVs in diagnostics and therapeutics, offering insights into disease pathogenesis.