This study aims to investigate whether human milk exosomes from gestational diabetes mellitus (GDM-EXO) and healthy (HEA-EXO) parturients differ in regulating intestinal development in offspring. The differential miRNAs associated with intestinal development in GDM-EXO and HEA-EXO were verified by using qPCR and their relationships with gut microbiota (GM) in infants were analyzed. C57BL/6J mice were gavaged with 50 mg/kg·BW HEA-EXO or GDM-EXO. The intestinal morphology, gut barriers, ZO-1 and Occludin, and GM were determined by histological staining, Western blotting, and 16S rDNA amplicon sequencing, respectively. Hsa-miR-19b-3p, hsa-miR-148a-3p, and hsa-miR-320a-3p were upregulated, and hsa-miR-429 was decreased in GDM-EXO compared to HEA-EXO. The GDM parturients' infants had increased intestinal Coriobacteriaceae, Clostridiaceae, Erysipelotrichaceae, Erysipelatoclostridiaceae, and fewer Lactobacillaceae than the healthy parturient's infants. The four differential miRNAs in GDM-EXO all correlated with the infants' GM. GDM-EXO- and HEA-EXO-fed mice had greater villus lengths, villus length-to-crypt depth ratios, goblet cell numbers, elevated ZO-1 and Occludin, and lower crypt depths than control mice. HEA-EXO-fed mice had better intestinal morphology and gut barrier integrity than GDM-EXO-fed mice. GDM-EXO-fed mice had significantly decreased Lachnospiraceae and Oscillospiraceae than HEA-EXO-fed mice. GDM-EXO demonstrate weaker ability to promote intestinal development in offspring than HEA-EXO.

The field of extracellular vesicles (EVs) is advancing rapidly, and this review aims to synthesize the latest research connected to EVs and the gastrointestinal tract. We will address new and emerging roles for EVs derived from internal sources such as the pancreas and immune system and how these miniature messengers alter organismal health or the inflammatory response within the GI tract. We will examine what is known about external EVs from dietary and bacterial sources and the immense anti-inflammatory, immune-modulatory, and proliferative potential within these nano-sized information carriers. EV interactions with the intestinal and colonic epithelium and associated immune cells at homeostatic and disease states, such as necrotizing enterocolitis (NEC) and inflammatory bowel disease (IBD) will also be covered. We will discuss how EVs are being leveraged as therapeutics or for drug delivery and conclude with a series of unanswered questions in the field.

The intricate interplay between human breast milk, nanoparticles, and macromolecules holds promise for innovative nutritional delivery strategies. Compared to bovine milk and infant formula, this study explores human breast milk's role in modulating intestinal permeability and its impact on nanoparticle and macromolecule transport. Comparative analysis with bovine milk and infant formula reveals significant elevations in permeability with human breast milk, accompanied by a decrease in transepithelial electrical resistance, suggesting enhanced paracellular transport. Mechanistically, human breast milk reduces Zonula occludens-1 levels, suggesting a regulatory role in intestinal barrier function. Through in vitro and ex vivo evaluations, we aim to understand better the mechanisms behind enhanced permeability and how human breast milk affects nanoparticle physicochemical properties, potentially modulating their behavior. Specifically, human breast milk improves the intestinal permeability of liposomes in a porcine intestinal model, with associated changes in the composition of milk proteins corona related to liposome charge. These findings underscore the unexploited potential of human breast milk in facilitating transport across the intestinal barrier, offering novel avenues for human nutritional delivery and therapeutic interventions.

Although the role of breast milk in promoting neonatal growth and maintaining intestinal homeostasis is well established, underlying mechanisms by which it protects the intestine from damage remain to be elucidated. Human breast milk-derived exosomes (HMDEs) are newly discovered active signaling vesicles with a diameter of 30-150 nm, which are key carriers of biological information exchange between mother and child. In addition, due to their ability to cross the gastrointestinal barrier, low immunogenicity, good biocompatibility and stability, HMDEs play an important role in regulating intestinal barrier integrity in newborns. In addition, HMDEs possess specific properties that are reformable and modifiable, offering promising strategies for the prevention and treatment of neonatal intestinal diseases. However, challenges such as purification, complex content, and quality control hinder their clinical application. This paper provides a comprehensive review of the biogenesis and properties of HMDEs, their isolation and purification, composition, and effects on neonatal intestinal barrier function, and further explores their potential biomedical applications. IMPACT: Breast milk helps maintain intestinal homeostasis in newborns and can prevent diseases, especially necrotizing enterocolitis (NEC). Breast milk contains abundant exosomes, which are important carriers of maternal and infant biological information exchange. Breast milk have the advantages of low immunogenicity, good biocompatibility and good stability, which helps to maintain the integrity of the intestinal barrier. Exosomes can be modified, which is expected to provide a more effective strategy for the prevention and treatment of intestinal diseases.

Human milk (HM) is the earliest form of extrauterine communication between mother and infant, that could promote early programming. The aim of this study is to look for specific biological processes, particularly those undergoing prematurity, modulated by proteins and miRNAs of HM that could be implicated in growth and development.

This is a prospective, observational, single center study in which we collected 48 human milk (HM) samples at two distinct stages of lactation: colostrum (first 72-96 h) and mature milk (at week 4 post-delivery) from mothers of very preterm newborns (<32 weeks) and term (≥37 and < 42 weeks). Qualitative and quantitative proteomic and transcriptomic analysis was done in our samples.

We performed isolation and characterization of HM extracellular vesicles (EVs) to carry out proteomic and transcriptomic analysis in colostrum (CM) and mature milk (MM). Proteomic analysis revealed a functional role of CM in immunological protection and MM in metabolic processes. TENA, TSP1 and OLF4, proteins with roles in immune response and inflammatory modulation, were upregulated in CM vs MM, particularly in preterm. HM modulation differed depending on gestational age (GA). The miRNAs identified in HM are implicated in structural functions, including growth and neurological development. miRNA-451a was differentially expressed between groups, and downregulated in preterm CM.

Because the particularities of each GA are reflected in the EVs content of HM, providing newborns with HM from their own mother is the optimal way for satisfying their specific needs. Although the role of the proteomic profile of CM and MM of different GA in relation to neurodevelopment has been previously described, this is the first study to show a complete functional characterization of HM (proteome, miRNA at the same time), unmasking the molecular mechanisms related to EVs signaling and their functional role in preterm.

Human milk extracellular vesicles (EVs) are crucial mother-to-baby messengers that transfer biological signals. These EVs are reported to survive digestion and transport across the intestine. The mechanisms of interaction between human milk EVs and the intestinal mucosa, including epithelial uptake remain unclear. Here, we studied the interaction of human milk EVs with the gut barrier components, including intestinal biofluids, enzymes, mucus and epithelium. Additionally, we probed the endocytic mechanisms mediating the EV intestinal uptake. Finally, using proteomic analysis, we determined the existence and identification of proteins enriched in the EV fraction transported across the intestinal epithelium. We show that human milk EVs are largely stable in the biochemical gut barriers and demonstrate high mucus diffusivity. EVs show a high level of epithelial cell uptake (∼70%) and efficient transport across Caco-2 monolayers. Whilst cell uptake of EVs was mediated by multiple routes, none of the pathway-specific inhibitors inhibited their epithelial translocation. Proteomic analysis of EVs transported across Caco-2 monolayers identified 14 enriched EV proteins that may facilitate intestinal transport. These findings significantly expand our understanding of the interactions between human milk EVs and the gut barriers, including their intestinal uptake.

Necrotizing enterocolitis (NEC) is a devastating disease affecting the gastrointestinal tract in premature infants with high mortality and morbidity. We aimed to analyze the research tendency and collaborations on models of NEC over the past three decades.

Bibliometric variables of included articles were retrieved from the Web of Science Core Collection from 1994 to 2023. Visualized studies were performed via VOSviewer software. Statistical analysis was applied by using GraphPad Prism and Microsoft Excel.

A total of 255 original articles from 17 countries were included in this study. The number of articles increased significantly from 22 (the year 1994-2003) to 161 (the year 2014-2023) (P < 0.0001). Collaborations in regions and countries have increased significantly over time (P < 0.0001). Developed regions contributed most of the research. While rat (56.08%) held the leading position in all types of models, followed by mouse (30.20%), notably, the proportion of mouse model has increased significantly from 4.55% to 41.36%.

This study might provide valuable insights into the model research of NEC. Research tendency has evolved to be collaborative and inclusive with more collaborations, broad model types, and studies from developing regions. However, the lack of an evident and robust pathophysiology mechanism will continue to make NEC a quite challenging case to decode, and research with strong evidence level and high quality is still required.

Necrotizing enterocolitis (NEC) is a severe inflammatory and necrotizing disease of the intestine that primarily affects the neonates, particularly premature infants. It has a high incidence of approximately 8.9% in extremely preterm infants, with a mortality rate ranging from 20 to 30%. In recent years, exosomes, particularly those derived from breast milk, have emerged as potential candidates for NEC therapy. Human breast milk-derived exosomes (BME) have been shown to enhance intestinal barrier function, protect intestinal epithelial cells from oxidative stress, promote the proliferation and migration of intestinal epithelial cells, and reduce the severity of experimental NEC models. As a subset of extracellular vesicles, BME possess the membrane structure, low immunogenicity, and high permeability, making them ideal vehicles for the treatment of NEC. Additionally, exosomes derived from various sources, including stem cells, intestinal epithelial cells, plants, and bacteria, have been implicated in the development and protection of intestinal diseases. This article summarizes the mechanisms through which exosomes, particularly BME, exert their effects on NEC and discusses the feasibility and obstacles associated with this novel therapeutic strategy.

Human milk contains extracellular vesicles (EVs) that carry bioactive molecules such as microRNA, to the newborn intestine. The downstream effects of EV cargo on signaling and immune modulation may shield neonates against inflammatory diseases, including necrotizing enterocolitis. Premature infants are especially at risk, while human milk-feeding may offer protection. The effect of gestational-age specific term and preterm EVs from transitional human milk was characterized on human intestinal epithelial cells (HIECs and Caco-2), primary macrophages, and THP-1 monocytes. We hypothesized that term and preterm EVs differentially influence immune-related cytokines and cell death. We found that preterm EVs were enriched in CD14 surface marker, while both term and preterm EVs increased epidermal growth factor secretion. Following inflammatory stimuli, only term EVs inhibited secretion of IL-6 in HIECs, and reduced expression of pro-inflammatory cytokine IL-1β in macrophages. Term and preterm EVs inhibited secretion of IL-1β and reduced inflammasome related cell death. We proposed that human milk EVs regulate immune-related signaling via their conserved microRNA cargo, which could promote tolerance and a homeostatic immune response. These findings provide basis for further studies into potential therapeutic supplementation with EVs in vulnerable newborn populations by considering functional, gestational age-specific effects. IMPACT: This study reveals distinct functional differences between term and preterm transitional human milk extracellular vesicles (EVs) highlighting the importance of gestational age in their bioactivity. Term EVs uniquely inhibited IL-6 secretion, IL-1β expression, and apoptosis following inflammatory stimuli. Both term and preterm human milk EVs reduced IL-1β secretion and inflammasome-induced cell death. Conserved human milk extracellular vesicle microRNA cargo could be a mediator of the anti-inflammatory effects, particularly targeting cytokine production, the inflammasome, and programmed cell death. These findings underscore the importance of considering gestational age in future research exploring the therapeutic potential of human milk extracellular vesicles to prevent or treat intestinal inflammatory diseases in neonates.

Mammalian milk contains milk-derived extracellular vesicles (MEVs), a group of biological nanovesicles that transport macromolecules. Their ability to cross the blood brain barrier and the presence of cargo capable of modifying gene function have led to the hypothesis that MEVs may play a role in brain function and development. Here, we investigated the uptake of MEVs by human microglia cells in vitro and explored the functional outcomes of MEV uptake. We examined the expression of the miR-148/152 family, highly abundant MEV microRNAs, that directly suppress the translation of DNA methyltransferase (DNMT) enzymes crucial for catalyzing DNA methylation modifications. We also measured phenotypic and inflammatory gene expression in baseline homeostatic and IFN-γ primed microglia to determine if MEVs induce anti-inflammatory effects. We found that MEVs are taken up and localize in baseline and primed microglia. In baseline microglia, MEV supplementation reduced miR-148a-5P levels, increased DNMT1 transcript, protein abundance, and enzymatic activity, compared to cells that did not receive MEVs. In primed microglia, MEV supplementation decreased miR-148a-5P levels and increased DNMT1 protein abundance, but DNMT1 transcript and enzymatic levels remained unchanged. Contrary to predictions, MEV supplementation failed to attenuate pro-inflammatory IL1β expression in primed microglia. This study provides the first evidence of MEV uptake by a brain macrophage, suggesting a potential role in regulating epigenetic machinery and neuroimmune modulation.

Extracellular vesicles (EVs) are naturally occurring non-replicating particles released from cells, known for their health-promoting effects and potential as carriers for drug delivery. Extensive research has been conducted on delivery systems based on culture-cell-derived EVs. Nevertheless, they have several limitations including low production yield, high expenses, unsuitability for oral administration, and safety concerns in applications. Conversely, food-derived EVs (FDEVs) offer unique advantages that cannot be easily substituted. This review provides a comprehensive analysis of the biogenesis pathways, composition, and health benefits of FDEVs, as well as the techniques required for constructing oral delivery systems. Furthermore, it explores the advantages and challenges associated with FDEVs as oral nanocarriers, and discusses the current research advancements in delivering active phytoconstituents. FDEVs, functioning as a nanocarrier platform for the oral delivery of active molecules, present numerous benefits such as convenient administration, high biocompatibility, low toxicity, and inherent targeting. Nevertheless, numerous unresolved issues persist in the isolation, characterization, drug loading, and application of FDEVs. Technical innovation and standardization of quality control are the key points to promote the development of FDEVs. The review aimed to provide frontier ideas and basic quality control guidelines for developing new functional food based on FDEVs oral drug delivery system.

Background: Human breast milk is a complex biological fluid containing multifaceted biological compounds that boost immune and metabolic system development that support the short- and long-term health of newborns. Recent literature suggests that human breast milk is a substantial source of nutrients, bioactive molecules, and exosomes. Objectives: This review examines the factors influencing exosomes noted in human milk and the impacts of exosomes on infant health. Furthermore, it discusses potential future prospects for exosome research in dietetics. Methods: Through a narrative review of the existing literature, we focused on exosomes in breast milk, exosome components and their potential impact on exosome health. Results: Exosomes are single-membrane extracellular vesicles of endosomal origin, with an approximate radius of 20-200 nm. They are natural messengers that cells secrete to transport a wide range of diverse cargoes, including deoxyribonucleic acid, ribonucleic acid, proteins, and lipids between various cells. Some studies have reported that the components noted in exosomes in human breast milk could be transferred to the infant and cause epigenetic changes. Thus, it can affect gene expression and cellular event regulation in several tissues. Conclusions: In this manner, exosomes are associated with several pathways, including the immune system, oxidative stress, and cell cycle, and they can affect the short- and long-term health of infants. However, there is still much to learn about the functions, effectiveness, and certain impacts on the health of human breast milk exosomes.

Mammalian milk exosomal miRNAs play an important role in maintaining intestinal immune homeostasis and protecting epithelial barrier function, but the specific miRNAs and whether miRNA-mediated mechanisms are responsible for these benefits remain a matter of investigation. This study isolated sheep milk-derived exosomes (sheep MDEs), identifying the enriched miRNAs in sheep MDEs, oar-miR-148a, and oar-let-7b as key components targeting TLR4 and TRAF1, which was validated by a dual-luciferase reporter assay. In dextran sulfate sodium-induced colitis mice, administration of sheep MDEs alleviated colitis symptoms, reduced colonic inflammation, and systemic oxidative stress, as well as significantly increased colonic oar-miR-148a and oar-let-7b while reducing toll-like receptor 4 (TLR4) and TNF-receptor-associated factor 1 (TRAF1) level. Further characterization in TNF-α-challenged Caco-2 cells showed that overexpression of these miRNAs suppressed the TLR4/TRAF1-IκBα-p65 pathway and reduced IL-6 and IL-12 production. These findings indicate that sheep MDEs exert gastrointestinal anti-inflammatory effects through the miRNA-mediated modulation of TLR4 and TRAF1, highlighting their potential in managing colitis.

With outstanding therapeutic potential in the tissue regeneration and anti-inflammation, mesenchymal stem cell-derived exosomes (MSC-EXOs) have emerged as a prominent therapeutic in recent. However, poor production yield and reproducibility have remained as significant challenges of their practical applications. To surmount these challenges, various alternative materials with stem cell-like functions, have been recently investigated, however, there has been no comprehensive analysis in these alternatives so far. Here, we discuss the recent progress of alternatives of MSC-EXOs, including exosomes and exosome-like nanovesicles from various biological sources such as plants, milk, microbes, and body fluids. Moreover, we extensively compare each alternative by summarizing their unique functions and mode of actions to suggest the expected therapeutic target and future directions for developing alternatives for MSC-EXOs.

Inflammation involves complex immune responses where cytokines such as TNF-α, IL-1, and IL-6 promote vasodilation and increased vascular permeability to facilitate immune cell migration to inflammation sites. Persistent inflammation is linked to diseases like cancer, arthritis, and neurodegenerative disorders. Although oral anti-inflammatory drugs are favored for their non-invasiveness and cost-effectiveness, their efficacy is often compromised due to gastrointestinal degradation and limited bioavailability. Recent advancements highlight the potential of extracellular vesicles (EVs) as nanocarriers that enhance drug delivery by encapsulating therapeutic agents, ensuring targeted release and reduced toxicity. These EVs, derived from dietary sources and cell cultures, exhibit excellent biocompatibility and stability, presenting a novel approach in anti-inflammatory therapies. This review discusses the classification and advantages of orally administered EVs (O-EVs), their mechanism of action, and their emerging role in treating inflammatory conditions, positioning them as promising vectors in the development of innovative anti-inflammatory drug delivery systems.

Small Extracellular Vesicles (sEVs) are nano-sized vesicles that are present in all biofluids including human milk (HM) playing a crucial role in cell-to-cell communication and the stimulation of the neonatal immune system. Oxylipins, which are bioactive lipids formed from polyunsaturated fatty acids, have gained considerable attention due to their potential role in mitigating disease progression and modulating the inflammatory status of breastfed infants. This study aims at an in-depth characterization of the oxylipin profiles of HM and, for the first time, of HM-derived sEVs (HMEVs) employing an ad-hoc developed and validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method.

The UPLC-MS/MS method covered a panel of 13 oxylipins for quantitation and 93 oxylipins for semi-quantitation. In 200 μL of HM and HMEV isolates of 15 individuals, 42 out of 106 oxylipins were detected in either HM or HMEVs, with 38 oxylipins being detected in both matrices. Oxylipins presented distinct profiles in HM and HMEVs, suggesting specific mechanisms responsible for the encapsulation of target molecules in HMEVs. Ten and eight oxylipins were quantified with ranges between 0.03 - 73 nM and 0.30 pM-0.07 nM in HM and HMEVs, respectively. The most abundant oxylipins found in HMEVs were docosahexaenoic acid derivatives (17-HDHA and 14-HDHA) with known anti-inflammatory properties, and linoleic acid derivatives (9-10-DiHOME and 12,13-DiHOME) in HM samples.

This is the first time a selective, relative enrichment of anti-inflammatory oxylipins in HMEVs has been described. Future studies will focus on the anti-inflammatory and pro-healing capacity of oxylipins encapsulated in HMEVs, with potential clinical applications in the field of preterm infant care, specifically the prevention of severe intestinal complications including necrotizing enterocolitis.

Breast milk, often referred to as "liquid gold," is a complex biofluid that provides essential nutrients, immune factors, and developmental cues for newborns. Recent advancements in the field of exosome research have shed light on the critical role of exosomes in breast milk. Exosomes are nanosized vesicles that carry bioactive molecules, including proteins, lipids, nucleic acids, and miRNAs. These tiny messengers play a vital role in intercellular communication and are now being recognized as key players in infant health and development. This paper explores the emerging field of milk exosomics, emphasizing the potential of exosome fingerprinting to uncover valuable insights into the composition and function of breast milk. By deciphering the exosomal cargo, we can gain a deeper understanding of how breast milk influences neonatal health and may even pave the way for personalized nutrition strategies.

Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes or ostomies. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super-resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β-casein and lactalbumin. HMEV and some dEV fractions contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin-mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.

Necrotizing enterocolitis (NEC) is a significant contributor to neonatal mortality. This study aimed to investigate the role of high levels of miR-375-3p in breast milk in the development of NEC and elucidate its mechanism.

Differential expression of miR-375-3p in the intestines of breast-fed and formula-fed mice was confirmed using real-time polymerase chain reaction (RT-PCR). NEC mice models were established, and intestinal injury was assessed using HE staining. RT-PCR and Western blot were conducted to examine the expression of miR-375-3p, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein β (YWHAB), as well as the inflammatory in IEC-6 cells, and intestinal tissues obtained from NEC mice and patients. Flow cytometry and cell counting kit-8 (CCK-8) were employed to elucidate the impact of miR-375-3p and YWHAB on cell apoptosis and proliferation.

Breastfeeding increases miR-375-3p expression in the intestines. The expression of miR-375-3p in NEC intestinal tissues exhibited a significant decrease compared to the healthy group. Additionally, the expression of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) was higher in the NEC group compared to the control group. Down-regulation of miR-375-3p inhibited IEC-6 cell proliferation, increased apoptosis, and elevated secretion of inflammatory factors. Bioinformatics revealed that YWHAB may be a target of miR-375-3p. RT-PCR and Western blot indicated a down-regulation of YWHAB expression in intestines of NEC patients and mice. Furthermore, YWHAB was found to be positively connected with miR-375-3p. Knockdown miR-375-3p down-regulated YWHAB expression in cells. Inhibition of YWHAB exhibited similar effects to miR-375-3p in IEC-6 cells. YWHAB plasmid partially reverse cellular functional impairment induced by miR-375-3p knockdown.

Breastfeeding elevated miR-375-3p expression in intestines in neonatal mice. MiR-375-3p leads to a decrease in apoptosis of intestinal epithelial cells, an increase in cell proliferation, and a concomitant reduction in the expression of inflammatory factors partly through targeting YWHAB.

Neonatal disorders, particularly those resulting from prematurity, pose a major challenge in health care and have a significant impact on infant mortality and long-term child health. The limitations of current therapeutic strategies emphasize the need for innovative treatments. New cell-free technologies utilizing extracellular vesicles (EVs) offer a compelling opportunity for neonatal therapy by harnessing the inherent regenerative capabilities of EVs. These nanoscale particles, secreted by a variety of organisms including animals, bacteria, fungi and plants, contain a repertoire of bioactive molecules with therapeutic potential. This review aims to provide a comprehensive assessment of the therapeutic effects of EVs and mechanistic insights into EVs from stem cells, biological fluids and non-animal sources, with a focus on common neonatal conditions such as hypoxic-ischemic encephalopathy, respiratory distress syndrome, bronchopulmonary dysplasia and necrotizing enterocolitis. This review summarizes evidence for the therapeutic potential of EVs, analyzes evidence of their mechanisms of action and discusses the challenges associated with the implementation of EV-based therapies in neonatal clinical practice.

Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes or ostomies. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super-resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β-casein and lactalbumin. HMEV and some dEV fractions contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin-mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.

Necrotizing enterocolitis (NEC) is one of the diseases in neonates, with a high morbidity and mortality rate, especially in preterm infants. This review aimed to briefly introduce the latest epidemiology, susceptibility factors, and clinical diagnosis and presentation of NEC. We also organized new prevention strategies by risk factors according to different pathogeneses and then discussed new treatment methods based on Bell's staging and complications, and the classification of mild to high severity based on clinical and imaging manifestations. Such a generalization will help clinicians and researchers to gain a deeper understanding of the disease and to conduct more targeted classification, grading prevention, and exploration. We focused on prevention and treatment of the early and suspected stages of NEC, including the discovery of novel biomarkers and drugs to control disease progression. At the same time, we discussed its clinical application, future development, and shortcomings.

Exosomes are extracellular vesicles with the diameter of 30 ~ 150 nm, and are widely involved in intercellular communication, disease diagnosis and drug delivery carriers for targeted disease therapy. Therapeutic application of exosomes as drug carriers is limited due to the lack of sources and methods for obtaining adequate exosomes. Milk contains abundant exosomes, several studies have shown that milk-derived exosomes play crucial roles in preventing and treating intestinal diseases. In this review, we summarized the biogenesis, secretion and structure, current novel methods used for the extraction and identification of exosomes, as well as discussed the role of milk-derived exosomes in treating intestinal diseases, such as inflammatory bowel disease, necrotizing enterocolitis, colorectal cancer, and intestinal ischemia and reperfusion injury by regulating intestinal immune homeostasis, restoring gut microbiota composition and improving intestinal structure and integrity, alleviating conditions such as oxidative stress, cell apoptosis and inflammation, and reducing mitochondrial reactive oxygen species (ROS) and lysosome accumulation in both humans and animals. In addition, we discussed future prospects for the standardization of milk exosome production platform to obtain higher concentration and purity, and complete exosomes derived from milk. Several in vivo clinical studies are needed to establish milk-derived exosomes as an effective and efficient drug delivery system, and promote its application in the treatment of various diseases in both humans and animals.

Exosomes, small tiny vesicle contains a large number of intracellular particles that employ to cause various diseases and prevent several pathological events as well in the human body. It is considered a "double-edged sword", and depending on its biological source, the action of exosomes varies under physiological conditions. Also, the isolation and characterization of the exosomes should be performed accurately and the methodology also will vary depending on the exosome source. Moreover, the uptake of exosomes from the recipients' cells is a vital and initial step for all the physiological actions. There are different mechanisms present in the exosomes' cellular uptake to deliver their cargo to acceptor cells. Once the exosomal uptake takes place, it releases the intracellular particles that leads to activate the physiological response. Even though exosomes have lavish functions, there are some challenges associated with every step of their preparation to bring potential therapeutic efficacy. So, overcoming the pitfalls would give a desired quantity of exosomes with high purity.

Milk contains bioactive compounds that have multiple essential benefits. Milk-derived small extracellular vesicles (M-sEVs) have emerged as novel bioactive milk components with various beneficial biological functions and broad applications. The M-sEVs from different mammalian sources have similar composition and bioactive functions. The digestive stability and biocompatibility of the M-sEVs provide a good foundation for their physiological functions. Evidence suggests that M-sEVs promote intestinal, immune, bone, neural, liver, and heart health and show therapeutic effects against cancer, indicating their potential for use in functional foods. In addition, M-sEVs can be developed as natural delivery carriers owing to their superior structural characteristics. Further studies are needed to elucidate the relationship between the specific components and functions of M-sEVs, standardize their extraction processes, and refine relevant clinical trials to advance the future applications of M-sEVs. This review summarizes the structure and composition of M-sEVs isolated from different milk sources and discusses several common extraction methods. Since the introduction of M-sEVs for digestion and absorption, studies have been conducted on their biological functions. Furthermore, we outline the theoretical industrial production route, potential application scenarios of M-sEVs, and the future perspectives of M-sEV research.

The growth and development of the human brain is a long and complex process that requires a precise sequence of genetic and molecular events. This begins in the third week of gestation with the differentiation of neural progenitor cells and extends at least until late adolescence, possibly for life. One of the defects of this development is that we know very little about the signals that modulate this sequence of events. The first 3 years of life, during breastfeeding, is one of the critical periods in brain development. In these first years of life, it is believed that neurodevelopmental problems may be the molecular causes of mental disorders. Therefore, we herein propose a new hypothesis, according to which the chemical signals that could modulate this entire complex sequence of events appear in this early period, and the molecular level study of human breast milk and colostrum of mothers who give birth to children in different gestation periods could give us information on proteins influencing this process. In this work, we collected milk and colostrum samples (term, late preterm and moderate/very preterm) and exosomes were isolated. The samples of exosomes and complete milk from each fraction were analyzed by LC-ESI-MS/MS. In this work, we describe proteins in the different fractions of mature milk and colostrum of mothers with term, late preterm, or very preterm delivery, which could be involved in the regulation of the nervous system by their functions. We describe how they differ in different types of milk, paving the way for the investigation of possible new neuroregulatory pathways as possible candidates to modulate the nervous system.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disorder occurring in newborns, with a mortality rate ranging from 20 % to 30 %. The existing therapeutic approaches for NEC are limited in their effectiveness. Various factors contribute to the development of NEC, including disruption of barrier function, dysregulation of the intestinal immune system, and abnormal colonization of the intestinal microbiota. Researchers have shown considerable interest in exploring the therapeutic potential of the constituents present in human breast milk (HBM) for treating NEC. HBM contains numerous bioactive components, such as exosomes, growth factors, and oligosaccharides. However, the precise mechanisms by which HBM exerts its protective effects against NEC remain incompletely understood. In this study, our objective was to comprehensively review the bioactive substances present in HBM, aiming to facilitate the development of novel therapeutic strategies for NEC.

Exosomes, a novel type of bioactive component in human milk (HM), affect infant development, growth, and health. Recent studies indicate that HM exosomes and miRNAs relate to gestational diabetes mellitus (GDM). However, the miRNAs profiles and functionalities of HM exosomes from GDM parturient remain unclear. This study aims to compare the differential miRNAs in HM exosomes from GDM and healthy parturient, and investigate the HM exosomes bioactivities in regulating hepatocyte proliferation and insulin sensitivity.

This study extracted HM exosomes from GDM (GDM-EXO) and healthy (NOR-EXO) parturient by ultracentrifugation, high-throughput sequenced and compared the exosomal miRNAs profiles, and explored the regulatory bioactivities on hepatocyte proliferation in HepG2 cells and Balb/c mice. As compared to NOR-EXO, GDM-EXO has similar morphology, size, concentration, and exosome-specific markers (CD9 and TSG101) expression. GDM-EXO and NOR-EXO specifically harbor 1299 and 8 miRNAs, respectively. Moreover, GDM-EXO had 176 upregulated and 47 downregulated miRNAs compared with NOR-EXO. Both GDM-EXO and NOR-EXO were absorbed in cultured HepG2 hepatocytes and mice liver. GDM-EXO inhibited hepatocytes proliferation by downregulating mammalian target of rapamycin (mTOR) possibly via exosomal miR-101-3p delivery.

HM exosomes from GDM and healthy parturient exhibit differential miRNAs profiles and distinct regulatory bioactivity on hepatocyte proliferation.

Inflammation is the defense mechanism of the immune system against harmful stimuli such as pathogens, toxic compounds, damaged cells, radiation, etc., and is characterized by tissue redness, swelling, heat generation, pain, and loss of tissue functions. Inflammation is essential in the recruitment of immune cells at the site of infection, which not only aids in the elimination of the cause, but also initiates the healing process. However, prolonged inflammation often brings about several chronic inflammatory disorders; hence, a balance between the pro- and anti-inflammatory responses is essential in order to eliminate the cause while producing the least damage to the host. A growing body of evidence indicates that extracellular vesicles (EVs) play a major role in cell-cell communication via the transfer of bioactive molecules in the form of proteins, lipids, DNA, RNAs, miRNAs, etc., between the cells. The present review provides a brief classification of the EVs followed by a detailed description of how EVs contribute to the pathogenesis of various inflammation-associated diseases and their implications as a therapeutic measure. The latter part of the review also highlights how EVs act as a bridging entity in blood coagulation disorders and associated inflammation. The findings illustrated in the present review may open a new therapeutic window to target EV-associated inflammatory responses, thereby minimizing the negative outcomes.

Maternal breast milk is the penultimate nutritional source for term and preterm neonates. Its composition is highly complex and includes multiple factors that enhance the development of nearly every neonatal organ system leading to both short- and long-term health benefits. Intensive research is focused on identifying breast milk components that enhance infant health. However, this research is complicated by the significant impact of maternal factors and the processing of pumped breast milk on bioactive ingredients. Optimizing enteral nutrition is particularly important for preterm neonates who miss the transplacental acquisition of nutrients in the third trimester of pregnancy and are at risk for illnesses associated with gut barrier dysfunction, including sepsis and necrotizing enterocolitis. In this review, we will discuss the health benefits of breast milk and its bioactive components.

Necrotizing enterocolitis (NEC) is a complex inflammatory disorder of the human intestine that most often occurs in premature newborns. Animal models of NEC typically use mice or rats; however, pigs have emerged as a viable alternative given their similar size, intestinal development, and physiology compared to humans. While most piglet NEC models initially administer total parenteral nutrition prior to enteral feeds, here we describe an enteral-feed only piglet model of NEC that recapitulates the microbiome abnormalities present in neonates that develop NEC and introduce a novel multifactorial definitive NEC (D-NEC) scoring system to assess disease severity.

Premature piglets were delivered via Caesarean section. Piglets in the colostrum-fed group received bovine colostrum feeds only throughout the experiment. Piglets in the formula-fed group received colostrum for the first 24 h of life, followed by Neocate Junior to induce intestinal injury. The presence of at least 3 of the following 4 criteria were required to diagnose D-NEC: (1) gross injury score ≥4 of 6; (2) histologic injury score ≥3 of 5; (3) a newly developed clinical sickness score ≥5 of 8 within the last 12 h of life; and (4) bacterial translocation to ≥2 internal organs. Quantitative reverse transcription polymerase chain reaction was performed to confirm intestinal inflammation in the small intestine and colon. 16S rRNA sequencing was performed to evaluate the intestinal microbiome.

Compared to the colostrum-fed group, the formula-fed group had lower survival, higher clinical sickness scores, and more severe gross and histologic intestinal injury. There was significantly increased bacterial translocation, D-NEC, and expression of IL-1α and IL-10 in the colon of formula-fed compared to colostrum-fed piglets. Intestinal microbiome analysis of piglets with D-NEC demonstrated lower microbial diversity and increased Gammaproteobacteria and Enterobacteriaceae.

We have developed a clinical sickness score and a new multifactorial D-NEC scoring system to accurately evaluate an enteral feed-only piglet model of NEC. Piglets with D-NEC had microbiome changes consistent with those seen in preterm infants with NEC. This model can be used to test future novel therapies to treat and prevent this devastating disease.

During pregnancy and lactation, mothers provide not only nutrients, but also many bioactive components for their offspring through placenta and breast milk, which are essential for offspring development. Extracellular vesicles (EVs) are nanovesicles containing a variety of biologically active molecules and participate in the intercellular communication. In the past decade, an increasing number of studies have reported that maternal-derived EVs play a crucial role in offspring growth, development, and immune system establishment. Hereby, we summarized the characteristics of EVs; biological functions of maternal-derived EVs during pregnancy, including implantation, decidualization, placentation, embryo development and birth of offspring; biological function of breast milk-derived EVs (BMEs) on infant oral and intestinal diseases, immune system, neurodevelopment, and metabolism. In summary, emerging studies have revealed that maternal-derived EVs play a pivotal role in offspring health. As such, maternal-derived EVs may be used as promising biomarkers in offspring disease diagnosis and treatment. However, existing research on maternal-derived EVs and offspring health is largely limited to animal and cellular studies. Evidence from human studies is needed.

In this Series paper, we examine how mother and baby attributes at the individual level interact with breastfeeding determinants at other levels, how these interactions drive breastfeeding outcomes, and what policies and interventions are necessary to achieve optimal breastfeeding. About one in three neonates in low-income and middle-income countries receive prelacteal feeds, and only one in two neonates are put to the breast within the first hour of life. Prelacteal feeds are strongly associated with delayed initiation of breastfeeding. Self-reported insufficient milk continues to be one of the most common reasons for introducing commercial milk formula (CMF) and stopping breastfeeding. Parents and health professionals frequently misinterpret typical, unsettled baby behaviours as signs of milk insufficiency or inadequacy. In our market-driven world and in violation of the WHO International Code for Marketing of Breast-milk Substitutes, the CMF industry exploits concerns of parents about these behaviours with unfounded product claims and advertising messages. A synthesis of reviews between 2016 and 2021 and country-based case studies indicate that breastfeeding practices at a population level can be improved rapidly through multilevel and multicomponent interventions across the socioecological model and settings. Breastfeeding is not the sole responsibility of women and requires collective societal approaches that take gender inequities into consideration.

Human breast milk is the optimal nutrition for all infants and is comprised of many bioactive and immunomodulatory components. The components in human milk, such as probiotics, human milk oligosaccharides (HMOs), extracellular vesicles, peptides, immunoglobulins, growth factors, cytokines, and vitamins, play a critical role in guiding neonatal development beyond somatic growth. In this review, we will describe the bioactive factors in human milk and discuss how these factors shape neonatal immunity, the intestinal microbiome, intestinal development, and more from the inside out.

MicroRNAs are non-coding segments of RNA involved in the epigenetic modulation of various biological processes. Their occurrence in biological fluids, such as blood, saliva, tears, and breast milk, has drawn attention to their potential influence on health and disease development. Hundreds of microRNAs have been isolated from breast milk, yet the evidence on their function remains inconsistent and inconclusive. The rationale for the current scoping review is to map the evidence on the occurrence, characterization techniques, and functional roles of microRNAs in breast milk. The review of the sources of this evidence highlights the need to address methodological challenges to achieve future advances in understanding microRNAs in breast milk, particularly their role in conditions such as neoplasms. Nonetheless, remarkable progress has been made in characterizing the microRNA profiles of human breast milk.

Exosomes were extracted from a variety of biological samples using several different purification processes, and our goal was to determine which method and sample were the most effective for exosome extraction.

We used ExoQuick-TC combined with ultrafiltration to separate and purify exosomes from the supernatant of gastric cancer cells, while we used the ExoQuick kit and ultracentrifugation to purify exosomes from human serum samples. Furthermore, exosomes were isolated and purified from human urine samples by diafiltration and from postparturition human breast milk samples by the filtration-polyethylene glycol precipitation method. The isolated exosomes were morphologically analyzed using a transmission electron microscope, the particle size was measured by NanoSight, and the protein content was analyzed by western blotting.

The isolated exosomes showed an obvious cup holder shape, with a clear outline and typical exosome morphological characteristics. The sizes of exosomes derived from gastric cancer cell supernatant, serum, urine, and milk were 65.8 ± 26.9 nm, 87.6 ± 50.9 nm, 197.5 ± 55.2 nm, and 184.1 ± 68.7 nm, respectively. Western blot results showed that CD9 and TSG101 on the exosomes were expressed to varying degrees based on the exosome source. Exosome abundance was higher in the serum, urine, and breast milk than in the supernatant. It is suggested that its exosomes can be extracted to obtain an excellent potential biological source of exosomes.

In this study, the extraction and separation methods of foreign bodies from different biological samples were obtained, and it was found that human breast milk was a potential excellent material for administration because of its high abundance.

Necrotizing enterocolitis (NEC) is one of the most severe diseases commonly afflicting premature infants. Our previous studies suggests that human milk-derived exosomes (HM-Exos) have a potential therapeutic effect on NEC. In this study, we investigate the potentially therapeutic role of HM-Exos in an NEC animal model via comprehensive lncRNA and mRNA expression profiles. A rat model of NEC was induced through hypoxia, hypothermia and formula feeds. We extracted exosomes from the colostrum of healthy lactating mothers and identified their functions in an NEC animal model. Furthermore, high-throughput lncRNA and mRNA sequencings were explored to find the underlying mechanisms. Although both exosomes from term human breast milk (Term-Exos) and exosomes from preterm human breast milk (Pre-Exos) alleviated the severity of NEC, Pre-Exos seemed to better promote the proliferation of intestinal epithelial cells in vivo. We identified a total of 44 differentially expressed lncRNAs and 88 differentially expressed mRNAs between Term-Exos and Pre-Exos. Further GO and KEGG pathway analysis showed that the lncRNA-mRNA network of HM-Exos was associated with the JAK-STAT signaling pathway, bile secretion and the AMPK signaling pathway, which are predicted to be involved in the proliferation of cells. Therefore, this study reveals for the first time the important roles of human milk derived lncRNAs and mRNAs in protecting against necrotizing enterocolitis. These results provide new insight into the development of NEC.