REVIEW

Perspectives on Bovine Milk-Derived Extracellular Vesicles for Therapeutic Applications in Gut Health

Daye Mun1https://orcid.org/0000-0002-3470-7632, Sangnam Oh2,*https://orcid.org/0000-0002-2428-412X, Younghoon Kim1,*https://orcid.org/0000-0001-6769-0657
Author Information & Copyright
1Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Korea
2Department of Functional Food and Biotechnology, Jeonju University, Jeonju 55069, Korea
*Corresponding author : Sangnam Oh, Department of Functional Food and Biotechnology, Jeonju University, Jeonju 55069, Korea, Tel: +82-63-220-3109, Fax: +82-63-220-2054, E-mail: osangnam@jj.ac.kr
*Corresponding author : Younghoon Kim, Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Korea, Tel: +82-2-880-4808, Fax: +82-2-873-2271, E-mail: ykeys2584@snu.ac.kr

© Korean Society for Food Science of Animal Resources. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Jan 16, 2022 ; Revised: Feb 10, 2022 ; Accepted: Feb 11, 2022

Published Online: Mar 01, 2022

Abstract

Extracellular vesicles (EVs) are nanosized vesicles secreted from cells into the extracellular environment and are composed of a lipid bilayer that contains cargos with biological activity, such as lipids, proteins, mRNAs, and noncoding microRNAs (miRNAs). Due to their biological activity and their role in cell-to-cell communication, interest in EVs is rapidly increasing. Bovine milk is a food consumed by people of all ages around the world that contains not only a significant amount of nutrients but also EVs. Milk-derived EVs also exhibit biological activity similar to other source-derived EVs, and studies on bovine milk EVs have been conducted in various research fields regarding sufficient milk production. In particular, not only are the effects of milk EVs themselves being studied, but the possibility of using them as drug carriers or biomarkers is also being studied. In this review, the characteristics and cargo of milk EVs are summarized, as well as their uptake and stability, efficacy and biological effects as carriers, and future research directions are presented.

Keywords: bovine milk; extracellular vesicles; gut health; therapeutics; carrier

Introduction

Extracellular vesicles (EVs) are nanosized, nonreplicating biological vesicles that are released from cells into extracellular space (Pegtel and Gould, 2019; van Niel et al., 2018). EVs contain cargoes of mRNAs, noncoding microRNAs (miRNAs), proteins, and lipids within a lipid bilayer, which are transported from donor cells to recipient cells and play an important role in intercellular interactions (Veziroglu and Mias, 2020). The structure and microscopic images of EVs are shown in Fig. 1. Recently, EVs have been classified into several subtypes, including ectosomes, exosomes, and apoptotic bodies, depending on their biogenesis, size, and release pathways (Zaborowski et al., 2015). However, there is no well-established separation method that clearly distinguishes among these subtypes, and with no consensus on specific markers, sufficient evidence is still needed for characterization and should be used with caution (Théry et al., 2018).

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Fig. 1. Structure and microscopic images of milk EVs. (A) Milk EVs structure. Adapted from Adriano et al. (2021) with CC BY-NC-ND. (B) Scanning electron microscopy (left) and transmission electron microscopy (right) images of bovine colostrum exosomes. Adapted from Yun et al. (2020) with permission of Elsevier. (C) Bovine colostrum exosomal protein profiles. Adapted from Yun et al. (2020) with permission of Elsevier. (D) Internalization of fluorescently labeled bovine colostrum microvesicles. Adapted from Maburutse et al. (2017) with CC-BY-NC.
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Bovine milk is one of the world’s most consumed foods, is high in nutritional value and contains a large amount of physiologically functional substances (Haug et al., 2007; Scholz-Ahrens et al., 2020). Most of high abundance nutrients, such as protein and fat, in milk have already been characterized, and research on EVs, which are minor components of milk, has rapidly increased over the past decade as the functionalities of EVs and their cargos are being revealed (Ong et al., 2021). In addition, since milk is a food that can be consumed by humans, it is safe and the production volume is abundant, so the mass production of EVs is possible (Somiya et al., 2018). Therefore, studies on the function of milk-derived EVs are being conducted in various research fields (Aarts et al., 2021; Adriano et al., 2021). The objective of this review is to summarize the recent findings in understanding the cargo, uptake and stability, potential use as a carrier, and therapeutic effects of bovine milk-derived EVs.

Cargo of Bovine Milk-Derived Extracellular Vesicles

Noncoding miRNAs

miRNAs are noncoding RNAs of approximately 18 to 24 nucleotides that bind to a specific region of the mRNA and regulate it translation. Chen et al. (2010) found for the first time that substantial amounts of miRNAs are present in bovine milk and identified 245 miRNAs in raw milk and found that they are altered during lactation. miRNAs known to be involved in immune regulation were confirmed to be expressed both in bovine colostrum and mature milk exosomes. When miRNAs from colostrum and mature milk exosomes were compared, the ratio of the 10 most highly expressed miRNAs was different (Yun et al., 2021). Izumi et al. (2012) published a study comparing miRNAs from bovine colostrum and mature milk using microarray and qPCR and found that there are more miRNAs related to immunity or development, including miR-15b, miR-27b, miR-106b, and miR-223, in colostrum than in mature milk. The same researchers also found that miRNAs present in milk exosomes could be taken up by human macrophages (Izumi et al., 2015). Benmoussa et al. (2020) reported that miRNAs in milk EVs isolated from differential ultracentrifugation exhibited distinct compositions. In particular, the general length (around 22 nt) of miRNA sequence was abundant at milk EVs isolated by ultracentrifugation at 100,000×g, but the heterogeneous iso-miRNA was more abundant in milk EVs separated by ultracentrifugation at 12,000×g and 35,000×g. Additionally, when milk EVs were incubated with HeLa cells, miR-223, known to regulate immunity, was increased. To examine the possibility of using miRNAs as biomarkers, miRNAs from cow milk infected with Staphylococcus aureus were analyzed, and 14 or 18 miRNAs, including bta-miR-142-5p and bta-miR-223, were found to be differentially expressed in infection conditions (Cai et al., 2018; Sun et al., 2015). The composition and ratio of exosomal miRNAs from cow milk of two different breeds were also different. In Holstein milk, miRNAs related to milk synthesis and Doğu Anadolu Kirmizisi’s milk were expressed in milk fat and milk protein metabolism, respectively (Özdemir, 2020). Changes in feed and differences in breed of cattle have also been shown to alter milk miRNAs. Four miRNAs were upregulated and 5 miRNAs were downregulated when a fiber-rich diet was replaced with a nonforage fiber source diet (Quan et al., 2020). Inducing stress by relocation of the cow group have been shown to affect the composition of milk exosomal miRNAs. In particular, whether miRNAs and target genes such as miR-142a, miR-135, and miR-320a would be useful as potential biomarkers on response of mild stress has been postulated (Colitti et al., 2019). The functions and target miRNAs from milk-derived EVs are summarized in Table 1.

Table 1. Summary of articles on miRNAs of bovine milk-derived EVs
Description Targeted miRNAs References
Identification of miRNA in bovine colostrum and mature milk and proposal of potential biomarkers for quality control of raw milk and dairy foods. miR-26a, miR-26b, miR-200c, miR-21, miR-30d, miR-99a, and miR-148a could potentially be used as biomarkers for quality control in raw milk and dairy products (Chen et al., 2010)
Identification and comparison of exosomal miRNA composition in human, caprine, and bovine colostrum and mature milk. miR-30a-5p, miR-22-3p, and miR-26a are highly conserved in colostrum and mature milk of three mammals, including humans, caprine, and bovines. (Yun et al., 2021)
Identification and comparing miRNAs in bovine colostrum and mature milk using microarray and qPCR analysis. miR-15b, miR-27b, miR-34a, miR-106b, miR-130a, miR-155, and miR-223 are related to immunity or development and are more expressed in colostrum than mature milk. (Izumi et al., 2012)
Identification of miRNA and mRNA of bovine milk exosomes and confirmation of uptake into human macrophages. Among the analyzed miRNAs, 14 bovine-specific miRNAs (miR-2478, miR-2412, miR-2305, miR-2881, miR-2328*, miR-2888, miR-2304, miR-2391, miR-2892, miR-2887, miR-2316, miR-2374, miR-2291, and miR-2284l) were recognized. (Izumi et al., 2015)
Comparison of miRNA distribution in milk EVs according to different ultracentrifugation. The top 4 miRNAs of P12K and P35K were bta-let7a, bta-miR-30a, bta-miR-21, and bta-let7b, and bta-mir-10a and bta-mir-10b were most expressed in P70K and P100K, respectively. (Benmoussa et al., 2020)
Identification and characterization of milk exosomal miRNAs in cows with mastitis. Eighteen miRNAs, including four novel miRNAs (bta-let-7b, bta-miR-103, bta-miR-142-3p, bta-miR-142-5p, bta-miR-1468, bta-miR-146a, bta-miR-146b, bta-miR-147, bta-miR-221, bta-miR-223, bta-miR-2284w, bta-miR-2285b, bta-miR-23a, bta-miR-423-5p, etc.), had differential expression, and miR-223 and miR-142-5p were considered for use as potential biomarkers for the diagnosis of mastitis. (Cai et al., 2018)
Profiling of expression of milk exosomal miRNA in cows infected with Staphylococcus aureus. According to the presence or absence of infection, 14 miRNAs (bta-miR-142-5p, bta-miR-296-5p, bta-miR-223, bta-miR-1246, bta-miR-183, bta-miR-502b, bta-miR-378b, bta-miR-2285 g-3p, bta-miR-99a-5p, bta-miR-181b, bta-miR-101, bta-miR-10a, bta-miR-99b, and bta-miR-2419-5p) were expressed differently, in particular bta-miR-142-5p, and -223 were suggested as potential biomarkers for detection of infection. (Sun et al., 2015)
Confirmation of changes in the expression of bovine milk EVs-miRNA as a result of changes in feed. When fed on a nonforage fiber sources diet, 4 miRNAs (bta-miR-29c, bta-miR-760-3p, bta-miR-383, and bta-miR-11973) among milk EVs miRNAs were upregulated, and 5 miRNAs (bta -miR-328, bta-miR-10167-3p, bta-miR-103, bta-miR-885, and bta-miR-302d) were downregulated. (Colitti et al., 2019)

EV, extracellular vesicles.

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Proteins and lipids

In milk-derived EVs, not only miRNAs but also proteins play a role in cell-to-cell communication and physiological activity (Fig. 1C). According to Reinhardt et al. (2012), a total of 2,107 proteins were identified in milk-derived EVs, of which 1,002 could be converted to David Resources. The same researchers also analyzed milk proteins from healthy cattle and from cattle infected with S. aureus. Interestingly, 2,350 proteins were identified in milk exosomes, and proteins related to host immunity and inflammation, including the heat shock protein family, protocadherin gamma family, and acute phase protein, were differentially expressed in S. aureus infection (Reinhardt et al., 2013). As a result of proteomics analysis of exosomes isolated from bovine colostrum and mature milk, a total of 9,430 proteins were detected in four samples, and 1,264, 1,404, 963, and 1,306 proteins were identified in colostrum and mature milk exosomes at 24 h, 48 h, and 72 h, respectively (Samuel et al., 2017). A total of 162 proteins were identified from exosomes and EV-containing pellets isolated through acid precipitation and ultracentrifugation, 43 of which were unique (Brown et al., 2020). When human and bovine milk exosomal proteins were analyzed and compared using the iTRAQ-coupled LC-MS/MS method, a total of 920 exosomal proteins were identified, and 575 proteins were differentially expressed. In particular, it was found that the proteins from human and bovine milk exosomes were clearly distinguishable (Yang et al., 2017). When the milk EV proteins from bovine leukemia virus-infected cattle were compared to that of uninfected cattle, a total of 1,330 proteins were detected in the milk EVs of infected cattle, of which 118 were uniquely expressed in the infected cattle. In addition, 26 proteins were expressed at significantly higher or lower levels than those of uninfected cattle. Through these studies, the possible use of proteins in EVs as a biomarker for specific diseases was revealed (Rahman et al., 2021). As a result of analyzing the lipids of EVs isolated by sequential ultracentrifugation and density gradient centrifugation through mass spectrometry-based lipid analysis, 8 major lipid classes were detected, and more than 200 fatty acid variations were identified (Grossen et al., 2021). Although it has been revealed through several previous studies that the lipids of EVs play an important role, there have been few lipidomics studies of bovine milk-derived EVs. This is because, due to the difficulty in reliably separating milk-derived EVs from milk lipids, there is still a limitation to the lipid analysis of milk EVs itself (Feng et al., 2021; Ong et al., 2021).

Uptake and Stability of Bovine Milk-Derived Extracellular Vesicles

As the physiological effects of milk-derived EVs were revealed, various experiments were conducted to confirm whether they could be leveraged as therapies. In particular, it was confirmed that EVs and their cargo were not degraded in the harsh digestive environment encountered when milk EVs were ingested (Benmoussa et al., 2016; Izumi et al., 2012; Shandilya et al., 2017; Zeng et al., 2019). It was also confirmed in vitro (Hansen et al., 2020; Izumi et al., 2015; Maburutse et al., 2017; Rani et al., 2017; Roerig et al., 2021; Wolf et al., 2015), in vivo (Kirchner et al., 2020; Manca et al., 2018), or under both conditions (López de Las Hazas et al., 2022; Munagala et al., 2016) that these EVs and their cargo are taken up (Fig. 1D). Additionally, a study examining whether the integrity of EVs is affected during the processing of raw milk has also been reported (Kleinjan et al., 2021; Shome et al., 2021). A summary of these papers is shown in Table 2.

Table 2. Summary of articles on the uptake and stability of bovine milk-derived EVs
Description References
Bovine milk-derived EVs are uptaken into the intestinal cell model (Caco-2) and have more beneficial features than liposomes. (Roerig et al., 2021)
EVs-encapsulated miRNA was stable in both in vitro and in vivo digestion, and it was confirmed that cellular uptake was possible and reached each organ upon oral administration to mice. (López de Las Hazas et al., 2022)
The drug-loaded bovine milk exosomes showed higher efficiency and targetability compared to the free drug in both cell and mouse tests. (Munagala et al., 2016)
Fluorophore-labeled bovine milk exosomes and miRNAs are uptaken by human colon carcinoma cells and rat small intestinal cells, which are mediated by endocytosis. (Wolf et al., 2015)
Milk exosomes crossed intestinal epithelial cells in in vitro culture, and milk exosomal miRNAs can resist in vitro digestion and cross the intestinal barrier. (Rani et al., 2017)
When fluorophore-labeled bovine milk exosomes and miRNA were orally administered, it was expressed in the organs of mice and pigs, confirming that interspecies transfer was possible. (Manca et al., 2018)
Postprandial blood analysis of newborn calves showed that bovine colostrum-derived EVs were transferred into the circulation but not miRNAs. (Kirchner et al., 2020)
Confirmation of uptake of bovine milk EVs into intestinal epithelial cells through fluorophore-conjugated lactadherin labeling. (Hansen et al., 2020)
Confirmation of bovine milk exosome uptake into differentiated THP-1 cells by flow cytometry and fluorescence microscopy. (Izumi et al., 2015)
Confirmation of uptake of PKH-labeled bovine colostrum-derived EVs into RAW 264.7 cells (Maburutse et al., 2017)
Resistance of miRNA and mRNA in milk with acidic conditions and RNase treatment. (Izumi et al., 2012)
Bovine milk exosomal long noncoding RNAs are protected from degradation during in vitro digestion system. (Zeng et al., 2019)
Bovine milk exosomal small interfering RNA resists in vitro digestive conditions and is uptaken into Caco-2 cells. (Shandilya et al., 2017)
bta-miR-223 and bta-miR-125b present in commercial cow milk are resistant in a computer-controlled gastrointestinal model (TIM-1). (Benmoussa et al., 2016)
The integrity and molecular structure of EVs in bovine milk are affected by Industrial processing. (Kleinjan et al., 2021)
Processing in commercial dairy plants have no statistical effect on the abundance value of miRNAs in dairy products. (Shome et al., 2021)

EV, extracellular vesicles.

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Drug Delivery as Biological Carrier of Bovine Milk-Derived Extracellular Vesicles

Milk EVs may play an important role in drug delivery because of their beneficial properties, such as mass production due to the industrial scale of milk, passage of biological barriers, resistance in the digestive tract, etc., and supporting studies have been reported (Adriano et al., 2021; Kandimalla et al., 2021b; Sedykh et al., 2020; Zhong et al., 2021). An approach to efficiently treat diseases such as cancer by loading small interfering RNA or drugs into bovine milk EVs is being performed. The related papers are shown in Table 3.

Table 3. Summary of articles on the evaluation of bovine milk-derived EVs as carriers
Description References
Confirmation of cellular and intestinal uptake of milk exosomes loaded with curcumin, which is poorly absorbed. (Komine-Aizawa et al., 2020)
By attaching hyaluronan, a CD44-specific ligand, to doxorubicin-loaded milk exosome, it enables specific transport of CD44 overexpressing cancer cells and induces tumor cell death. (Li et al., 2020)
Development of a pH- and light-sensitive drug delivery system based on milk exosomes for highly active anticancer treatment of oral squamous carcinoma. (Zhang et al., 2020)
Ultrasonically loaded bcl-2 siRNA into bovine milk exosomes crossed the cell membrane and inhibited tumor growth. (Tao et al., 2020)
Development of efficient and nontoxic lactoferrin-poly-l-lysine mediated loading method for siRNA into bovine milk exosome (Shandilya et al., 2020)
The therapeutic efficacy of Paclitaxel was significantly increased by loading Paclitaxel, an anticancer drug with low water solubility and strong toxicity, in bovine colostrum-derived exosomes. (Kandimalla et al., 2021a)
Oral administration and confirmation of uptake of bovine milk EVs loaded with locked nucleic acid antisense oligonucleotides. (Grossen et al., 2021)
Epicatechin gallate-loaded bovine milk exosomes have a neuroprotective role in rotenone-induced Parkinson’s disease. (Luo et al., 2021)
Developed efficient oral delivery platform of siRNA by coating hydrophilic polyethylene glycol (PEG) on the surface of bovine milk exosomes and loading siRNA with cationic chemical transfection. (Warren et al., 2021)
Evaluation of uptake in hepatic and intestinal cell lines of milk exosomes loaded with exogenous hsa-miR148a-3p. (del Pozo-Acebo et al., 2021)

EV, extracellular vesicles.

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Therapeutic Effects of Bovine Milk-Derived Extracellular Vesicles

Immune system

Milk and milk-derived EVs are important for the development of the immune system of calves, and EVs express a large number of immune-related miRNAs and proteins and exert immunomodulatory functions. Matic et al. (2020) reported that bovine milk exosomes promoted the proliferation of RAW 264.7 cells, decreased cisplatin-induced cytotoxicity, and regulated the expression of proteins related to the cell cycle and proliferation. Additionally, a previous study reported that the pretreatment with milk EVs in LPS-treated RAW 264.7 cells alleviated the production of inflammatory cytokines and decreased NK-κB activity (Ascanius et al., 2021). According to Pieters et al. (2015), commercial cow milk contains exosomes that are stable under low pH and boiling-freezing conditions and can carry bioactive immunoregulatory TGF-β. In addition, cow milk exosomes alone could not regulate immune cells but enhanced the production of interferon-γ cells by NK cells and γδT cells in inflammatory conditions (Komine-Aizawa et al., 2020). A previous study examined whether agricultural dust-derived inflammatory responses were modulated by bovine milk-derived EVs in a mouse model. When mice were fed a diet containing milk EVs and then exposed to organic dust, the inflammatory response was improved, and M1 polarization of macrophages was promoted, suggesting that milk-derived exosomes promote immune responses in inflammatory conditions (Nordgren et al., 2019).

Gut health

In addition to regulating the immune system, milk-derived EVs are absorbed in the intestine and exert beneficial effects on intestinal health. In particular, they not only affect intestinal epithelial cells and the microbiota but may also have a protective effect in diseases that occur in the intestine. When milk EVs were fed to mice for 8 weeks, expression levels of genes important for the integrity of the mucus layer, including Muc2, RegIIIγ, and Myd88, were increased (Tong et al., 2020). Additionally, when oxidative stress was induced in intestinal crypt epithelial cells pretreated with bovine exosomes, superoxide dismutase, glutathione peroxidase activity and heme-oxygenase 1 (HO-1) protein levels was increased. In addition, ROS levels and the activity of adenosine deaminase and xanthine oxidase were decreased, suggesting that milk EVs could enhance the energy state of damaged cells to exert a protective effect against oxidative stress (Wang et al., 2021a; Wang et al., 2021b). Maghraby et al. (2021) reported that malnutrition-associated intestinal dysfunction was significantly alleviated by milk EV-induced intestinal stem cell proliferation and villus architecture restoration. Necrotizing enterocolitis (NEC), a type of colitis that can occur in premature infants, is characterized by significant intestinal damage and decreased mucin production. Treatment of milk exosomes with experimental NEC pups can alleviate the symptoms of NEC by increasing the expression of goblet cells and regulating genes involved in protein synthesis (Li et al., 2019). In addition, previous studies reported that treatment using cow milk-derived EVs alleviated colitis in both a DSS-induced colitis model and a genetic mouse model of ulcerative colitis (Benmoussa et al., 2019; Reif et al., 2020; Stremmel et al., 2020; Tong et al., 2021).

Regulation of the gut microbiota

Milk-derived EVs have been reported to exert a beneficial effect on intestinal health, which may be due to milk EVs bringing positive changes to the gut microbiota. Therefore, studies are being conducted to determine how milk EVs affect the intestinal microbiota. Yu et al. (2019) proposed that when exosomes isolated from milk were cocultured with bacteria, they promoted the growth of Escherichiacoli K-12 MG1655, a commensal bacterium, and Lactobacillus plantarum WCFS1, a probiotic, and influenced gene expression. Feeding C57BL/6 mice a diet containing milk exosomes and RNA was shown to affect the cecum microbiota composition in mice, a non-bovine species (Zhou et al., 2019). Similarly, when C57BL/6 mice were fed milk EVs containing 1.0×1010 particles per gram body weight, they had an effect on the gut microbiota and serum metabolites. In particular, among the intestinal microbiota, Akkermansia and Muribaculum, which are known to be beneficial, increased, and Desulfovibrio, which is known to be harmful, decreased. In addition, serum metabolite analysis revealed that milk EVs affected lipid and amino acid metabolism, especially anti-inflammatory factors such as lysophosphatidylcholines, deoxycholic acid, eicosapentaenoic acid, related to immune and metabolic diseases (Du et al., 2021). In addition to previous studies showing that milk EVs affect the intestinal microbiota and bacteria, studies have also investigated how these changes may affect intestinal immunity and colitis. When milk EVs were administered to mice, as in previous results, they had an effect on intestinal microbes and their metabolites, short-chain fatty acids, and the expression of genes related to intestinal immunity was increased (Tong et al., 2020). The same researchers reported that milk EVs alleviated ulcerative colitis by altering the gut microbiome and modulating gut immunity. Feeding milk EVs to an experimental mouse model of ulcerative colitis restricted the TLR4 signaling pathway and NLRP3 activation and restored DSS-induced intestinal dysbiosis (Tong et al., 2021).

Other Functions in Human Health

Previous studies have consistently reported that milk-derived EVs display beneficial biological activity in immune and intestinal health, as well as in various other conditions. Milk is a nutritionally rich source of calcium and protein that is known to support bone health (Pirilä et al., 2011; Uenishi et al., 2007). Several studies reported on the effect of EVs derived from milk on bone health. Milk EVs promote osteoblastogenesis, increase the number of osteocytes, and have been shown to increase bone mineral density (Oliveira et al., 2016; Oliveira et al., 2017; Oliveira et al., 2020; Yun et al., 2020). They exert beneficial effect on reproduction (Sadri et al., 2020), cardiac fibrosis (Zhang et al., 2021), arthritis (Arntz et al., 2015), cancer (Fonseka et al., 2021; Samuel et al., 2021), melanogenesis (Bae and Kim, 2021), and scar-free wound healing (Ahn et al., 2021). Milk EVs have been reported to exhibit biological effects in various case, but precise mechanistic studies and human trials are essential to define a strategy to leverage milk EVs as therapeutics.

Conclusion and Perspectives

Based on current studies, bovine milk-derived EVs are highly stable and absorbed in the body and are full of bioactive substances with multiple functions. Therefore, there are expectations for further therapeutic utilization. However, there are still goals that need to be achieved for success. Until now, due to the development of NGS and MS technology, the global investigation of cargo in milk EVs has been conducted in various ways. However, to administer it to the human body, it is necessary to analyze all components precisely whether there is any component to induce any adverse. To this end, it is necessary to develop more efficient technical methods to isolate EVs for clinical trial and a technology that can fully analyze their cargo. In particular, for the role of transporting drugs or nucleic acids for the treatment of diseases, a precise study on whether there is any problem in the long-term and high-dose application of milk EVs itself is required. In addition, it is necessary to investigate the detailed mechanisms of the biological effects revealed hitherto and to conduct further studies on whether therapeutic clinical application is possible.

Conflicts of Interest

The authors declare no potential conflicts of interest.

Acknowledgements

This research was supported by a National Research Foundation of Korea Grant, funded by the Korean government (MEST) (NRF-2018R1D1A3B07050304 to SO and 2021R1A2C3011051 to YK).

Author Contributions

Conceptualization: Mun D, Oh S, Kim Y. Data curation: Mun D, Oh S, Kim Y. Formal analysis: Mun D, Oh S, Kim Y. Investigation: Mun D, Oh S, Kim Y. Writing - original draft: Mun D, Oh S, Kim Y. Writing - review & editing: Mun D, Oh S, Kim Y.

Ethics Approval

This article does not require IRB/IACUC approval because there are no human and animal participants.

References

1.

Aarts J, Boleij A, Pieters BCH, Feitsma AL, Van Neerven RJJ, Ten Klooster JP, M’Rabet L, Arntz OJ, Koenders MI, van de Loo FAJ. 2021; Flood control: How milk-derived extracellular vesicles can help to improve the intestinal barrier function and break the gut-joint axis in rheumatoid arthritis. Front Immunol. 12:703277

2.

Adriano B, Cotto NM, Chauhan N, Jaggi M, Chauhan SC, Yallapu MM. 2021; Milk exosomes: Nature’s abundant nanoplatform for theranostic applications. Bioact Mater. 6:2479-2490

3.

Ahn G, Kim YH, Ahn JY. 2021; Multifaceted effects of milk-exosomes (mi-exo) as a modulator of scar-free wound healing. Nanoscale Adv. 3:528-537

4.

Arntz OJ, Pieters BCH, Oliveira MC, Broeren MGA, Bennink MB, de Vries M, van Lent PLEM, Koenders MI, van den Berg WB, van der Kraan PM, van de Loo FAJ. 2015; Oral administration of bovine milk derived extracellular vesicles attenuates arthritis in two mouse models. Mol Nutr Food Res. 59:1701-1712

5.

Ascanius SR, Hansen MS, Ostenfeld MS, Rasmussen JT. 2021; Milk-derived extracellular vesicles suppress inflammatory cytokine expression and nuclear factor-κb activation in lipopolysaccharide-stimulated macrophages. Dairy. 2:165-178

6.

Bae IS, Kim SH. 2021; Milk exosome-derived microrna-2478 suppresses melanogenesis through the Akt-GSK3β pathway. Cells. 10:2848

7.

Benmoussa A, Diallo I, Salem M, Michel S, Gilbert C, Sévigny J, Provost P. 2019; Concentrates of two subsets of extracellular vesicles from cow’s milk modulate symptoms and inflammation in experimental colitis. Sci Rep. 9:14661

8.

Benmoussa A, Laugier J, Beauparlant CJ, Lambert M, Droit A, Provost P. 2020; Complexity of the microRNA transcriptome of cow milk and milk-derived extracellular vesicles isolated via differential ultracentrifugation. J Dairy Sci. 103:P16-29

9.

Benmoussa A, Lee CHC, Laffont B, Savard P, Laugier J, Boilard E, Gilbert C, Fliss I, Provost P. 2016; Commercial dairy cow milk microRNAs resist digestion under simulated gastrointestinal tract conditions. J Nutr. 146:2206-2215

10.

Brown BA, Zeng X, Todd AR, Barnes LF, Winstone JMA, Trinidad JC, Novotny MV, Jarrold MF, Clemmer DE. 2020; Charge detection mass spectrometry measurements of exosomes and other extracellular particles enriched from bovine milk. Anal Chem. 92:3285-3292

11.

Cai M, He H, Jia X, Chen S, Wang J, Shi Y, Liu B, Xiao W, Lai S. 2018; Genome-wide microRNA profiling of bovine milk-derived exosomes infected with Staphylococcus aureus. Cell Stress Chaperones. 23:663-672

12.

Chen X, Gao C, Li H, Huang L, Sun Q, Dong Y, Tian C, Gao S, Dong H, Guan D, Hu X, Zhao S, Li L, Zhu L, Yan Q, Zhang J, Zen K, Zhang CY. 2010; Identification and characterization of microRNAs in raw milk during different periods of lactation, commercial fluid, and powdered milk products. Cell Res. 20:1128-1137

13.

Colitti M, Sgorlon S, Licastro D, Stefanon B. 2019; Differential expression of miRNAs in milk exosomes of cows subjected to group relocation. Res Vet Sci. 122:148-155

14.

del Pozo-Acebo L, Hazas MCL, Tomé-Carneiro J, Gil-Cabrerizo P, San-Cristobal R, Busto R, García-Ruiz A, Dávalos A. 2021; Bovine milk-derived exosomes as a drug delivery vehicle for miRNA-based therapy. Int J Mol Sci. 22:1105

15.

Du C, Quan S, Nan X, Zhao Y, Shi F, Luo Q, Xiong B. 2021; Effects of oral milk extracellular vesicles on the gut microbiome and serum metabolome in mice. Food Funct. 12:10938-10949

16.

Feng X, Chen X, Zheng X, Zhu H, Qi Q, Liu S, Zhang H, Che J. 2021; Latest trend of milk derived exosomes: Cargos, functions, and applications. Front Nutr. 8:747294

17.

Fonseka P, Kang T, Chee S, Chitti SV, Sanwlani R, Ang CS, Mathivanan S. 2021; Temporal quantitative proteomics analysis of neuroblastoma cells treated with bovine milk-derived extracellular vesicles highlights the anti-proliferative properties of milk-derived extracellular vesicles. Cells. 10:750

18.

Grossen P, Portmann M, Koller E, Duschmalé M, Minz T, Sewing S, Pandya NJ, van Geijtenbeek SK, Ducret A, Kusznir EA, Huber S, Berrera M, Lauer ME, Ringler P, Nordbo B, Jensen ML, Sladojevich F, Jagasia R, Alex R, Gamboni R, Keller M. 2021; Evaluation of bovine milk extracellular vesicles for the delivery of locked nucleic acid antisense oligonucleotides. Eur J Pharm Biopharm. 158:198-210

19.

Hansen MS, Gadegaard ISE, Arnspang EC, Blans K, Nejsum LN, Rasmussen JT. 2020; Specific and non-invasive fluorescent labelling of extracellular vesicles for evaluation of intracellular processing by intestinal epithelial cells. Biomedicines. 8:211

20.

Haug A, Høstmark AT, Harstad OM. 2007; Bovine milk in human nutrition: A review. Lipids Health Dis. 6:25

21.

Izumi H, Kosaka N, Shimizu T, Sekine K, Ochiya T, Takase M. 2012; Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions. J Dairy Sci. 95:P4831-4841

22.

Izumi H, Tsuda M, Sato Y, Kosaka N, Ochiya T, Iwamoto H, Namba K, Takeda Y. 2015; Bovine milk exosomes contain microRNA and mRNA and are taken up by human macrophages. J Dairy Sci. 98:P2920-2933

23.

Kandimalla R, Aqil F, Alhakeem SS, Jeyabalan J, Tyagi N, Agrawal A, Yan J, Spencer W, Bondada S, Gupta RC. 2021a; Targeted oral delivery of paclitaxel using colostrum-derived exosomes. Cancers. 13:3700

24.

Kandimalla R, Aqil F, Tyagi N, Gupta R. 2021b; Milk exosomes: A biogenic nanocarrier for small molecules and macromolecules to combat cancer. Am J Reprod Immunol. 85e13349

25.

Kirchner B, Buschmann D, Paul V, Pfaffl MW. 2020; Postprandial transfer of colostral extracellular vesicles and their protein and miRNA cargo in neonatal calves. PLOS ONE. 15e0229606

26.

Kleinjan M, van Herwijnen MJ, Libregts SFWM, van Neerven RJJ, Feitsma AL, Wauben MHM. 2021; Regular industrial processing of bovine milk impacts the integrity and molecular composition of extracellular vesicles. J Nutr. 151:1416-1425

27.

Komine-Aizawa S, Ito S, Aizawa S, Namiki T, Hayakawa S. 2020; Cow milk exosomes activate NK cells and γδT cells in human PBMCs in vitro. Immunol Med. 43:161-170

28.

Li B, Hock A, Wu RY, Minich A, Botts SR, Lee C, Antounians L, Miyake H, Koike Y, Chen Y, Zani A, Sherman PM, Pierro A. 2019; Bovine milk-derived exosomes enhance goblet cell activity and prevent the development of experimental necrotizing enterocolitis. PLOS ONE. 14e0211431

29.

Li D, Yao S, Zhou Z, Shi J, Huang Z, Wu Z. 2020; Hyaluronan decoration of milk exosomes directs tumor-specific delivery of doxorubicin. Carbohydr Res. 493:108032

30.

López de Las Hazas MC, del Pozo-Acebo L, Hansen MS, Gil-Zamorano J, Mantilla-Escalante DC, Gómez-Coronado D, Marín F, Garcia-Ruiz A, Rasmussen JT, Dávalos A. 2022; Dietary bovine milk miRNAs transported in extracellular vesicles are partially stable during GI digestion, are bioavailable and reach target tissues but need a minimum dose to impact on gene expression. Eur J Nutr. 62:1043-1056

31.

Luo S, Sun X, Huang M, Ma Q, Du L, Cui Y. 2021; Enhanced neuroprotective effects of epicatechin gallate encapsulated by bovine milk-derived exosomes against Parkinson’s disease through antiapoptosis and antimitophagy. J Agric Food Chem. 69:5134-5143

32.

Maburutse BE, Park MR, Oh S, Kim Y. 2017; Evaluation and characterization of milk-derived microvescicle isolated from bovine colostrum. Korean J Food Sci Anim Resour. 37:654-662

33.

Maghraby MK, Li B, Chi L, Ling C, Benmoussa A, Provost P, Postmus AC, Abdi A, Pierro A, Bourdon C, Bandsma RHJ. 2021; Extracellular vesicles isolated from milk can improve gut barrier dysfunction induced by malnutrition. Sci Rep. 11:7635

34.

Manca S, Upadhyaya B, Mutai E, Desaulniers AT, Cederberg RA, White BR, Zempleni J. 2018; Milk exosomes are bioavailable and distinct microRNA cargos have unique tissue distribution patterns. Sci Rep. 8:11321

35.

Matic S, D’Souza DH, Wu T, Pangloli P, Dia VP. 2020; Bovine milk exosomes affect proliferation and protect macrophages against cisplatin-induced cytotoxicity. Immunol Invest. 49:711-725

36.

Munagala R, Aqil F, Jeyabalan J, Gupta RC. 2016; Bovine milk-derived exosomes for drug delivery. Cancer Lett. 371:48-61

37.

Nordgren TM, Heires AJ, Zempleni J, Swanson BJ, Wichman C, Romberger DJ. 2019; Bovine milk-derived extracellular vesicles enhance inflammation and promote M1 polarization following agricultural dust exposure in mice. J Nutr Biochem. 64:110-120

38.

Oliveira MC, Arntz OJ, Blaney Davidson EN, van Lent PLEM, Koenders MI, van der Kraan PM, van den Berg WB, Ferreira AVM, van de Loo FAJ. 2016; Milk extracellular vesicles accelerate osteoblastogenesis but impair bone matrix formation. J Nutr Biochem. 30:74-84

39.

Oliveira MC, Di Ceglie I, Arntz OJ, van den Berg WB, van den Hoogen FHJ, Ferreira AVM, van Lent PLEM, van de Loo FAJ. 2017; Milk-derived nanoparticle fraction promotes the formation of small osteoclasts but reduces bone resorption. J Cell Physiol. 232:225-233

40.

Oliveira MC, Pieters BCH, Guimarães PB, Duffles LF, Heredia JE, Silveira ALM, Oliveira ACC, Teixeira MM, Ferreira AVM, Silva TA, van de Loo FAJ, Macari S. 2020; Bovine milk extracellular vesicles are osteoprotective by increasing osteocyte numbers and targeting rankl/opg system in experimental models of bone loss. Front Bioeng Biotechnol. 8:891

41.

Ong SL, Blenkiron C, Haines S, Acevedo-Fani A, Leite JAS, Zempleni J, Anderson RC, McCann MJ. 2021; Ruminant milk-derived extracellular vesicles: A nutritional and therapeutic opportunity?. Nutrients. 13:2505

42.

Özdemir S. 2020; Identification and comparison of exosomal microRNAs in the milk and colostrum of two different cow breeds. Gene. 743:144609

43.

Pegtel DM, Gould SJ. 2019; Exosomes. Annu Rev Biochem. 88:487-514

44.

Pieters BCH, Arntz OJ, Bennink MB, Broeren MGA, van Caam APM, Koenders MI, van Lent PLEM, van den Berg WB, de Vries M, van der Kraan PM, van de Loo FAJ. 2015; Commercial cow milk contains physically stable extracellular vesicles expressing immunoregulatory TGF-β. PLOS ONE. 10e0121123

45.

Pirilä S, Taskinen M, Viljakainen H, Kajosaari M, Turanlahti M, Saarinen-Pihkala UM, Mäkitie O. 2011; Infant milk feeding influences adult bone health: A prospective study from birth to 32 years. PLOS ONE. 6e19068

46.

Quan SY, Nan XM, Wang K, Zhao YG, Jiang LS, Yao JH, Xiong BH. 2020; Replacement of forage fiber with non-forage fiber sources in dairy cow diets changes milk extracellular vesicle-miRNA expression. Food Funct. 11:2154-2162

47.

Rahman MM, Takashima S, Kamatari YO, Badr Y, Kitamura Y, Shimizu K, Okada A, Inoshima Y. 2021; Proteomic profiling of milk small extracellular vesicles from bovine leukemia virus-infected cattle. Sci Rep. 11:2951

48.

Rani P, Vashisht M, Golla N, Shandilya S, Onteru SK, Singh D. 2017; Milk miRNAs encapsulated in exosomes are stable to human digestion and permeable to intestinal barrier in vitro. J Funct Foods. 34:431-439

49.

Reif S, Elbaum-Shiff Y, Koroukhov N, Shilo I, Musseri M, Golan-Gerstl R. 2020; Cow and human milk-derived exosomes ameliorate colitis in dss murine model. Nutrients. 12:2589

50.

Reinhardt TA, Lippolis JD, Nonnecke BJ, Sacco RE. 2012; Bovine milk exosome proteome. J Proteomics. 75:1486-1492

51.

Reinhardt TA, Sacco RE, Nonnecke BJ, Lippolis JD. 2013; Bovine milk proteome: Quantitative changes in normal milk exosomes, milk fat globule membranes and whey proteomes resulting from Staphylococcus aureus mastitis. J Proteomics. 82:141-154

52.

Roerig J, Schiller L, Kalwa H, Hause G, Vissiennon C, Hacker MC, Wölk C, Schulz-Siegmund M. 2021; A focus on critical aspects of uptake and transport of milk-derived extracellular vesicles across the Caco-2 intestinal barrier model. Eur J Pharm Biopharm. 166:61-74

53.

Sadri M, Shu J, Kachman SD, Cui J, Zempleni J. 2020; Milk exosomes and miRNA cross the placenta and promote embryo survival in mice. Reproduction. 160:501-509

54.

Samuel M, Chisanga D, Liem M, Keerthikumar S, Anand S, Ang CS, Adda CG, Versteegen E, Jois M, Mathivanan S. 2017; Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth. Sci Rep. 7:5933

55.

Samuel M, Fonseka P, Sanwlani R, Gangoda L, Chee SH, Keerthikumar S, Spurling A, Chitti SV, Zanker D, Ang CS, Atukorala I, Kang T, Shahi S, Marzan AL, Nedeva C, Vennin C, Lucas MC, Cheng L, Herrmann D, Pathan M, Chisanga D, Warren SC, Zhao K, Abraham N, Anand S, Boukouris S, Adda CG, Jiang L, Shekhar TM, Baschuk N, Hawkins CJ, Johnston AJ, Orian JM, Hoogenraad NJ, Poon IK, Hill AF, Jois M, Timpson P, Parker BS, Mathivanan S. 2021; Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis. Nat Commun. 12:3950

56.

Scholz-Ahrens KE, Ahrens F, Barth CA. 2020; Nutritional and health attributes of milk and milk imitations. Eur J Nutr. 59:19-34

57.

Sedykh S, Kuleshova A, Nevinsky G. 2020; Milk exosomes: Perspective agents for anticancer drug delivery. Int J Mol Sci. 21:6646

58.

Shandilya S, Rani P, Onteru SK, Singh D. 2017; Small interfering RNA in milk exosomes is resistant to digestion and crosses the intestinal barrier in vitro. J Agric Food Chem. 65:9506-9513

59.

Shandilya S, Rani P, Onteru SK, Singh D. 2020; Natural ligand-receptor mediated loading of siRNA in milk derived exosomes. J Biotechnol. 318:1-9

60.

Shome S, Jernigan RL, Beitz DC, Clark S, Testroet ED. 2021; Non-coding RNA in raw and commercially processed milk and putative targets related to growth and immune-response. BMC Genomics. 22:749

61.

Somiya M, Yoshioka Y, Ochiya T. 2018; Biocompatibility of highly purified bovine milk-derived extracellular vesicles. J Extracell Vesicles. 7:1440132

62.

Stremmel W, Weiskirchen R, Melnik BC. 2020; Milk exosomes prevent intestinal inflammation in a genetic mouse model of ulcerative colitis: A pilot experiment. Inflamm Intest Dis. 5:117-123

63.

Sun J, Aswath K, Schroeder SG, Lippolis JD, Reinhardt TA, Sonstegard TS. 2015; MicroRNA expression profiles of bovine milk exosomes in response to Staphylococcus aureus infection. BMC Genomics. 16:806

64.

Tao H, Xu H, Zuo L, Li C, Qiao G, Guo M, Zheng L, Leitgeb M, Lin X. 2020; Exosomes-coated bcl-2 siRNA inhibits the growth of digestive system tumors both in vitro and in vivo. Int J Biol Macromol. 161:470-480

65.

Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, Antoniou A, et al. 2018; Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 7:1535750

66.

Tong L, Hao H, Zhang X, Zhang Z, Lv Y, Zhang L, Yi H. 2020; Oral administration of bovine milk-derived extracellular vesicles alters the gut microbiota and enhances intestinal immunity in mice. Mol Nutr Food Res. 64e1901251

67.

Tong L, Hao H, Zhang Z, Lv Y, Liang X, Liu Q, Liu T, Gong P, Zhang L, Cao F, Pastorin G, Lee CN, Chen X, Wang JW, Yi H. 2021; Milk-derived extracellular vesicles alleviate ulcerative colitis by regulating the gut immunity and reshaping the gut microbiota. Theranostics. 11:8570-8586

68.

Uenishi K, Ishida H, Toba Y, Aoe S, Itabashi A, Takada Y. 2007; Milk basic protein increases bone mineral density and improves bone metabolism in healthy young women. Osteoporos Int. 18:385-390

69.

van Niel G, D’Angelo G, Raposo G. 2018; Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 19:213-228

70.

Veziroglu EM, Mias GI. 2020; Characterizing extracellular vesicles and their diverse RNA contents. Front Genet. 11:700

71.

Wang L, Shi Z, Wang X, Mu S, Xu X, Shen L, Li P. 2021a; Protective effects of bovine milk exosomes against oxidative stress in IEC-6 cells. Eur J Nutr. 60:317-327

72.

Wang L, Wang X, Shi Z, Shen L, Zhang J, Zhang J. 2021b; Bovine milk exosomes attenuate the alteration of purine metabolism and energy status in IEC-6 cells induced by hydrogen peroxide. Food Chem. 350:129142

73.

Warren MR, Zhang C, Vedadghavami A, Bokvist K, Dhal PK, Bajpayee AG. 2021; Milk exosomes with enhanced mucus penetrability for oral delivery of siRNA. Biomater Sci. 9:4260-4277

74.

Wolf T, Baier SR, Zempleni J. 2015; The intestinal transport of bovine milk exosomes is mediated by endocytosis in human colon carcinoma caco-2 cells and rat small intestinal iec-6 cells. J Nutr. 145:2201-2206

75.

Yang M, Song D, Cao X, Wu R, Liu B, Ye W, Wu J, Yue X. 2017; Comparative proteomic analysis of milk-derived exosomes in human and bovine colostrum and mature milk samples by iTRAQ-coupled LC-MS/MS. Food Res Int. 92:17-25

76.

Yu S, Zhao Z, Xu X, Li M, Li P. 2019; Characterization of three different types of extracellular vesicles and their impact on bacterial growth. Food Chem. 272:372-378

77.

Yun B, Kim Y, Park DJ, Oh S. 2021; Comparative analysis of dietary exosome-derived microRNAs from human, bovine and caprine colostrum and mature milk. J Anim Sci Technol. 63:593-602

78.

Yun B, Maburutse BE, Kang M, Park MR, Park DJ, Kim Y, Oh S. 2020; Short communication: Dietary bovine milk-derived exosomes improve bone health in an osteoporosis-induced mouse model. J Dairy Sci. 103:7752-7760

79.

Zaborowski MP, Balaj L, Breakefield XO, Lai CP. 2015; Extracellular vesicles: Composition, biological relevance, and methods of study. Bioscience. 65:783-797

80.

Zeng B, Chen T, Xie MY, Luo JY, He JJ, Xi QY, Sun JJ, Zhang YL. 2019; Exploration of long noncoding RNA in bovine milk exosomes and their stability during digestion in vitro. J Dairy Sci. 102:6726-6737

81.

Zhang C, Lu X, Hu J, Li P, Yan J, Ling X, Xiao J. 2021 Bovine milk exosomes alleviate cardiac fibrosis via enhancing angiogenesis in vivo and in vitro. J Cardiovasc Trans Res.

82.

Zhang Q, Xiao Q, Yin H, Xia C, Pu Y, He Z, Hu Q, Wang J, Wang Y. 2020; Milk-exosome based pH/light sensitive drug system to enhance anticancer activity against oral squamous cell carcinoma. RSC Advances. 10:28314-28323

83.

Zhong J, Xia B, Shan S, Zheng A, Zhang S, Chen J, Liang XJ. 2021; High-quality milk exosomes as oral drug delivery system. Biomaterials. 277:121126

84.

Zhou F, Paz HA, Sadri M, Cui J, Kachman SD, Fernando SC, Zempleni J. 2019; Dietary bovine milk exosomes elicit changes in bacterial communities in C57BL/6 mice. Am J Physiol Gastrointest Liver Physiol. 317:G618-G624

Call for Special Section


We are pleased to invite you a paper for Food Science of Animal Resources for a special issue “New concept of probiotics for human and animal health”. Both research and review articles are welcome for possible publication in this issue. Both research and review articles are welcome for possible publication in this section. All manuscript will be peer-reviewed before their acceptance for publication.

Edited by Dr. Seok-Seong Kang
- Deadline for manuscript submissions: 30 June 2022
- We offer 50% reduction of the article processing charge and provide the fast review process (approximately 1 month).

The specific topics of interest for the special issue include:
 • New probiotics and alternative probiotics such as postbiotics
 • Probiotics/postbiotics for human and animal health
 • Probiotics/postbiotics and metabolic disorders
 • Probiotics/postbiotics and gut microbiome balance
 • The next generation of probiotics


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