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Exosomes are derived from various sources, including primary and cultured cell lines and body fluids. It is now evident that they are important for communication between cells. They have, therefore, been proposed as potential carriers to deliver drugs to specific sites. In this study, we examined stability of exosomes derived from human saliva. Exosomes were stored at 4°C for up to 20 months and their membrane integrity assessed. Several exosomal markers, such as dipeptidyl peptidase IV (DPP IV; membrane marker) and programmed cell death 6-interacting protein (Alix, lumen marker), were retained intact after 20 months storage at 4°C. Moreover, intact exosomes could be isolated from whole saliva that had been stored at 4°C. Membrane disruption with detergents such as Triton X-100 and Nonidet P-40 caused partial solubilization of DPP IV and release of Alix into the supernatant. In contrast, sodium dodecyl sulfate treatment caused a complete disruption of the membrane. In addition, membrane stability was maintained after freezing and thawing. These results indicated that human saliva-derived exosomes are stable, maintaining their membrane integrity over a long storage period.

Exosomes are small extracellular vesicles containing microRNAs and mRNAs that are produced by various types of cells. We previously used ultrafiltration and size-exclusion chromatography to isolate two types of human salivary exosomes (exosomes I, II) that are different in size and proteomes. We showed that salivary exosomes contain large repertoires of small RNAs. However, precise information regarding long RNAs in salivary exosomes has not been fully determined. In this study, we investigated the compositions of protein-coding RNAs (pcRNAs) and long non-protein-coding RNAs (lncRNAs) of exosome I, exosome II and whole saliva (WS) by next-generation sequencing technology. Although 11% of all RNAs were commonly detected among the three samples, the compositions of reads mapping to known RNAs were similar. The most abundant pcRNA is ribosomal RNA protein, and pcRNAs of some salivary proteins such as S100 calcium-binding protein A8 (protein S100-A8) were present in salivary exosomes. Interestingly, lncRNAs of pseudogenes (presumably, processed pseudogenes) were abundant in exosome I, exosome II and WS. Translationally controlled tumor protein gene, which plays an important role in cell proliferation, cell death and immune responses, was highly expressed as pcRNA and pseudogenes in salivary exosomes. Our results show that salivary exosomes contain various types of RNAs such as pseudogenes and small RNAs, and may mediate intercellular communication by transferring these RNAs to target cells as gene expression regulators.

Small non-coding RNAs, such as microRNAs (miRNAs), are involved in diverse processes, including organ development and tissue differentiation. Exosomes are small membrane vesicles (30-100 nm in diameter) produced by numerous cells. Recently, exosomes have been shown to contain miRNAs. However, the small RNAs contained in exosomes are not fully characterized. In a previous study, we found at least two types of salivary exosome that are different in size and have different proteomes. Studies of salivary exosomal small RNAs are limited to miRNAs. In this study, we examined small RNA transcriptomes using next generation sequencing technology to elucidate a full transcriptome set of small RNAs expressed in the two types of salivary exosomes and in whole saliva (WS). Many types of small RNA, such as miRNA, piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA) and other small RNAs are contained in salivary exosomes and WS. Among these small RNAs we identified novel miRNA candidates.

Saliva contains a large number of proteins that participate in the protection of oral tissue. Exosomes are small vesicles (30-100 nm in diameter) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We have recently demonstrated that exosomes are present in human whole saliva. In this study, we found that whole saliva contained at least two types of exosomes (exosome I and exosome II) that are different in size and protein composition. Proteomic analysis revealed that both types of exosomes contained Alix, Tsg101 and Hsp70, all exosomal markers, immunoglobulin A and polymeric immunoglobulin receptor, whereas they had different protein compositions. Most of dipeptidyl peptidase IV known as CD26 in whole saliva, was present on the exosome II and metabolically active in cleaving chemokines (CXCL11 and CXCL12). Human whole saliva exosomes might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.

Saliva contains a large number of proteins that participate in the protection of oral tissue. We found, for the first time, small vesicles (30-130 nm in diameter) in human whole saliva. Vesicles from saliva were identified by electron microscopy after isolation by gel-filtration on Sepharose CL-4B. They resemble exosomes, which are vesicles with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We performed a biochemical characterization of these vesicles by amino acid sequence analysis and Western blot analysis. We found that they contain dipeptidyl peptidase IV (DPP IV), galectin-3 and immunoglobulin A, which have potential to influence immune response. The DPP IV in the vesicles was metabolically active in cleaving substance P and glucose-dependent insulinotropic polypeptide to release N-terminal dipeptides. Our results demonstrate that human whole saliva contains exosome-like vesicles; they might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.