Abstract
Aim:To confirm the presence of extracellular vesicles (EVs) in cell-free saliva (CFS) of children with asthma and describe the isolated EV population.Methods:A pooled sample of CFS EVs isolated from 180 participants using ExoQuick-TC was examined in downstream analyses. Transmission electron microscopy (TEM) was used to confirm the presence of EVs. Nanoparticle tracking analysis (NTA) and single particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence were used for sizing, counting, and phenotyping of EVs. Capillary immunoassays were used for protein quantitation.Results:TEM confirmed the presence of EVs of diverse sizes, indicating the prep contained a heterogeneous population of EVs. Capillary immunoassays confirmed the presence of EV-associated proteins (CD9, CD63, CD81, ICAM-1, and ANXA5) and indicated limited cellular contamination. As others have also reported, there were discrepancies in the EV sizing and enumeration across platforms. Fluorescent NTA detected particles with a mode diameter of ~90 nm, whereas SP-IRIS reported sizes of ~55–60 nm that more closely approximated the TEM results. Consistent with protein immunoassay results, SP-IRIS with fluorescence showed that the majority of these EVs were CD9- and CD63-positive, with little expression of CD81.Conclusion:EVs from CFS can be isolated using a high-throughput method that can be scaled to large epidemiological studies. To our knowledge, we are the first to characterize CFS EVs from patients with asthma. The use of CFS EVs as potential novel biomarkers in asthma warrants further investigation and opens a new avenue of research for future studies.
Highlights
Asthma is the most common noncommunicable childhood disease, affecting approximately 8% of children worldwide and 12%-15% of urban children in the United States[1,2]
Extracellular vesicles (EVs) from cell-free saliva (CFS) can be isolated using a high-throughput method that can be scaled to large epidemiological studies
Evidence of EVs in human saliva of children with asthma. This analysis includes 180 children with asthma aged 4 to 14 years who were enrolled in School Inner-City Asthma Study (SICAS) and provided a saliva sample at the study baseline visit
Summary
Asthma is the most common noncommunicable childhood disease, affecting approximately 8% of children worldwide and 12%-15% of urban children in the United States[1,2]. While the clinical presentations may appear similar, evidence suggests that asthma may not be a single disease but rather encompasses a group of heterogeneous endotypes with various etiologies and prognoses[3,4]. Saliva, regarded as the “mirror of the body”, is an accessible biofluid that harbors constituents that provide sources for monitoring of health and disease states[13,14] including asthma[15,16,17,18,19,20,21]. To the best of our knowledge, no study has investigated the characteristics of saliva EVs among patients with asthma. The aim of the present study is to describe the EVs isolated from cell-free saliva (CFS) of children with asthma
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