Guidelines for the purification and characterization of extracellular vesicles of parasites

Abstract

Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite‐parasite and parasite‐host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite‐infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells

Taenia solium cysticerci's extracellular vesicles Attenuate the AKT/mTORC1 pathway for Alleviating DSS‐induced colitis in a murine model

“…EV isolation from serum‐free culture medium was performed strictly as per recent MISEV guidelines and protocol as described elsewhere, that was submitted to EVTRACK (EV230582) database and outlined in Figure 1a (Fernandez‐Becerra, 2023 ; Coakley et al., 2017 ; Kuipers et al., 2020 ; Nowacki et al., 2015 ; Simbari et al., 2016 ; Thery et al., 2018 ; Tkach & Thery, 2016 ; Welsh et al., 2024 ). In brief, cellular debris from the collected media was removed by spinning at 300× g at 4°C for 10 min.…”

Exploring Peripheral Blood-Derived Extracellular Vesicles as Biomarkers: Implications for Chronic Chagas Disease with Viral Infection or Transplantation

“…In total, 30 fractions were obtained (1 mL each fraction). All fractions were screened using CL-ELISA, following the methods outlined in a prior publication by our group [ 53 , 56 ].…”

“…All antibodies were diluted at 1:500 in TBS with 5% nonfat milk. After incubating for 1 h at RT, the NM underwent TBS-Tween washing and was then exposed to anti-rabbit IgG-peroxidase conjugate or anti-mouse Ig-peroxidase (both from Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) at a 1:5000 dilution in TBS with 5% nonfat milk for 1 h. After five additional washes in TBS-Tween, the peroxidase signals were detected by introducing a chemiluminescent (ECL) solution (A-38555, Thermo Fisher Scientific) and incubated in the dark for 15 min, with images acquired using an Odyssey C imaging system (Li-Cor) [ 56 ].…”

“…A chemiluminescent substrate was added to the wells, and the plates were read at approximately 425 nm using an appropriate detection instrument. The chemiluminescent reaction generated light in proportion to the presence of the target antigens bound to the antibodies immobilized on the plate [ 56 ].…”