Characterization of Extracellular Vesicles from Human Saliva: Effects of Age and Isolation Techniques.

Salivary extracellular vesicles (EVs) represent an attractive source of biomarkers due to the accessibility of saliva and its non-invasive sampling methods. However, the lack of comparative studies assessing the efficacy of different EV isolation techniques hampers the use of salivary EVs in clinical settings. Moreover, the effects of age on salivary EVs are largely unknown, hindering the identification of salivary EV-associated biomarkers across the lifespan. To address these questions, we compared salivary EV concentration, size mode, protein concentration, and purity using eight EV isolation techniques before and after magnetic bead immunocapture with antibodies against CD9, CD63, and CD81. The effects of age on salivary EVs obtained with each isolation technique were further investigated. Results showed higher expression of CD63 on isolated salivary EVs compared to the expression of CD81 and flotillin-1. Overall, magnetic bead immunocapture was more efficient in recovering salivary EVs with Norgen's Saliva Exosome Purification Kit and ExoQuick-TC ULTRA at the cost of EV yield. Regardless of age, Invitrogen Total Exosome Isolation Solution showed the highest level of protein concentration, whereas Izon qEVOriginal-70nm columns revealed the highest purity. This study provides the first comprehensive comparison of salivary EVs in younger and older adults using different EV isolation techniques, which represents a step forward for assessing salivary EVs as a source of potential biomarkers of tissue-specific diseases throughout the life cycle.

Salivary lactoferrin is associated with cortical amyloid-beta load, cortical integrity, and memory in aging.

BACKGROUND: Aging is associated with declining protective immunity and persistent low-grade inflammatory responses, which significantly contribute to Alzheimer's disease (AD) pathogenesis. Detecting aging-related cerebral vulnerability associated with deterioration of the immune system requires from non-invasive biomarkers able to detect failures in the brain-immunity connection. Reduced levels of salivary lactoferrin (sLF), an iron-binding protein with immunomodulatory activity, have been related to AD diagnosis. However, it remains unknown whether decreased sLF is associated with increased cortical amyloid-beta (Aß) load and/or with loss of cortical integrity in normal aging. METHODS: Seventy-four cognitively normal older adults (51 females) participated in the study. We applied multiple linear regression analyses to assess (i) whether sLF is associated with cortical Aß load measured by 18F-Florbetaben (FBB)-positron emission tomography (PET), (ii) whether sLF-related variations in cortical thickness and cortical glucose metabolism depend on global Aß burden, and (iii) whether such sLF-related cortical abnormalities moderate the relationship between sLF and cognition. RESULTS: sLF was negatively associated with Aß load in parieto-temporal regions. Moreover, sLF was related to thickening of the middle temporal cortex, increased FDG uptake in the posterior cingulate cortex, and poorer memory. These associations were stronger in individuals showing the highest Aß burden. CONCLUSIONS: sLF levels are sensitive to variations in cortical Aß load, structural and metabolic cortical abnormalities, and subclinical memory impairment in asymptomatic older adults. These findings provide support for the use of sLF as a non-invasive biomarker of cerebral vulnerability in the general aging population.