Effect of In Vitro Enzyme Digestion and Bile Treatment on Milk Extracellular Vesicles Stability.

SCOPE: Milk extracellular vesicles (EVs) are nanosized particles with potential immune bioactivities. This study examines their fate during in vitro infant gastrointestinal digestion (GI). METHODS AND RESULTS: Bovine milk is digested using the in vitro INFOGEST method, adjusted for the infant. To unravel the contribution of digestive enzymes from bile, milk is treated with digestive enzymes, bile, or a combination of both. EVs are collected posttreatment using differential ultracentrifugation. EVs characterization includes electrophoresis, immunoblotting, nanoparticle tracking analysis, and atomic force microscopy. EVs protein markers programmed cell death 6-interacting protein (ALIX), tumor susceptibility gene 101 (TSG101), cluster of differentiation 9 (CD9), and xanthine dehydrogenase (XDH) are detected after gastric digestion (G60), but their signal intensity is significantly reduced by intestinal conditions (p < 0.05). Enzyme digestion, compared to bile treatment (I60 + bile), results in a significant reduction of signal intensities for TSG101 and CD9 (p < 0.05). Nanoparticle tracking analysis shows a significant reduction (p < 0.05) of EV numbers at the end of the intestinal phase. EVs are detected by atomic force microscopy at the end of the intestinal phase, showing that intact EVs can survive upper gut digestion. CONCLUSION: Intact EVs can be found at the end of the intestinal phase. However, digestive enzymes and bile reduce the quantity and characteristics of EVs, with digestive enzymes playing a larger role.

An extensive study of potential inhibitors of extracellular vesicles release in triple-negative breast cancer.

BACKGROUND: Cancer cells release heterogeneous populations of extracellular vesicles (EVs) that transmit aggressive phenotypic traits to recipient cells. We aimed to establish if the heterogenous EVs population or a sub-population is responsible, if we could block undesirable cell-to-cell communication by EVs, and, if some EVs continued to be released, would their undesirable influences on recipient cells continue. METHODS: Three triple-negative breast cancer (TNBC) cell lines were used. Non-toxic concentrations of calpeptin, Y27632, manumycin A, GW4869 and combinations thereof were tested to block EVs. Ultracentrifugation-based methods collected EVs, which were then characterised by nanoparticle tracking analysis, immunoblotting, and transmission electron microscopy. A quick screening flow cytometry method evaluated EVs in solution. The influences of EVs on recipient cells' migration was investigated. RESULTS: All EV sub-populations were apparently involved in transmitting undesirable phenotypic characteristics. All compounds/combinations significantly (64-98%) reduced EVs' release. Our quick screening broadly reflected our more comprehensive EVs analysis. The 2-36% of EVs that continued to be released caused less transmission to recipient cells, but not on a comparable scale to the reduction of EVs release achieved. CONCLUSION: Up to 98% inhibition of EVs' release was achieved. To prevent the transmission of undesirable phenotypic traits by EVs, their total inhibition may be necessary.

Doxorubicin Loading into Milk and Mesenchymal Stem Cells' Extracellular Vesicles as Drug Delivery Vehicles.

Extracellular vesicles (EVs) have great potential as drug delivery vehicles. While mesenchymal/stromal stem cell (MSC) conditioned medium (CM) and milk are potentially safe and scalable sources of EVs for this purpose, the suitability of MSC EVs and milk EVs as drug delivery vehicles has never been compared and so was the objective of this study. Here EVs were separated from MSCs' CM and from milk and were characterised by nanoparticle tracking analysis, transmission electron microscopy, total protein quantification, and immunoblotting. An anti-cancer chemotherapeutic drug, doxorubicin (Dox), was then loaded into the EVs by one of three methods: by passive loading or by active loading by either electroporation or sonication. Dox-loaded EVs were analysed by fluorescence spectrophotometer, high-performance liquid chromatography (HPLC), and imaging flow cytometer (IFCM). Our study showed that EVs were successfully separated from the milk and MSC CM, with significantly (p < 0.001) higher yields of milk EVs/mL starting material compared to MSC EVs/mL of starting material. Using a fixed amount of EVs for each comparison, electroporation achieved significantly more Dox loading when compared to passive loading (p < 0.01). Indeed, of 250 µg of Dox made available for loading, electroporation resulted in 90.1 ± 12 µg of Dox loading into MSC EVs and 68.0 ± 10 µg of Dox loading into milk EVs, as analysed by HPLC. Interestingly, compared to the passive loading and electroporation approach, after sonication significantly fewer CD9+ EVs/mL (p < 0.001) and CD63+ EVs/mL (p < 0.001) existed, as determined by IFCM. This observation indicates that sonication, in particular, may have detrimental effects on EVs. In conclusion, EVs can be successfully separated from both MSC CM and milk, with milk being a particularly rich source. Of the three methods tested, electroporation appears to be superior for achieving maximum drug loading while not causing damage to EV surface proteins.

An investigation of extracellular vesicles in bovine colostrum, first milk and milk over the lactation curve.

Extracellular vesicles (EVs) in milk have claimed benefits ranging from conveying immunological privilege to infants to being suitable as natural delivery vehicles for therapeutic drugs. However, a longitudinal study of bovine EVs quantities and characteristics in colostrum (COL), first milk (FM) and throughout the lactation curve of mature milk (MM) had never been performed and so was our aim. COL, FM and 9 months of MM samples were collected. Caseins -overlapping size with EVs- were removed. EVs were collected by density gradient ultracentrifugation and characterised by SDS-PAGE, Bradford assay, nanoparticle tracking analysis, immunoblotting, imaging flow cytometry analysis, and transmission electron microscopy. COL and FM had substantially more EVs than MM, with COL enriched in small EVs. No significant differences were observed between months 1-9 of MM. Altogether, although COL and FM are particularly rich sources of EVs, mature milk throughout the lactation curve is also an abundant source of intact EVs.

Maternal supplementation with a casein hydrolysate and yeast beta-glucan from late gestation through lactation improves gastrointestinal health of piglets at weaning.

Improving maternal nutrition during pregnancy/lactation is a promising strategy to maximise the intestinal health of piglets undergoing abrupt weaning under commercial production conditions. This experiment investigated the effects of maternal supplementation of a casein hydrolysate and yeast ß-glucan (CH-YBG) from day 83 of gestation until weaning (day 28) on sow faecal microbial populations and measures of piglet gastrointestinal health parameters at weaning. Sows (n = 10 sows/group) were assigned to: (1) control diet, and (2) control diet + CH-YBG. Maternal supplementation increased the abundance of the phylum Firmicutes, including members Lactobacillus in the sows faeces, with a concomitant increase in the caecal abundance of Lactobacillus in the weaned piglets compared to the controls. Piglets weaned from the supplemented sows had increased villus height in the duodenum (P < 0.05) and increased villus height to crypt depth ratio in the jejunum, as well as a decreased expression of the proinflammatory cytokine genes (IL6/TNF/TGFB), the tight junction gene CLDN3 and the mucin gene MUC2 in the duodenum/jejunum compared to the controls (P < 0.05). In conclusion, maternal CH-YBG supplementation during pregnancy/lactation improved microbial, structural, and inflammatory measures of gastrointestinal health of piglets at weaning. This is a promising strategy to alleviate the challenges that occur with early abrupt weaning in commercial pig production.

Maternal and/or direct supplementation with a combination of a casein hydrolysate and yeast ß-glucan on post-weaning performance and intestinal health in the pig.

A 2 × 2 factorial experiment was conducted to investigate the effect of maternal supplementation from day 83 of gestation and/or direct supplementation from weaning of a bovine casein hydrolysate plus a yeast ß-glucan (CH-YBG) on pig performance and intestinal health on day ten post-weaning. Twenty cross bred gilts (Large White × Landrace) were randomly assigned to one of two dietary groups (n = 10 gilts/group): basal diet (basal sows) and basal diet supplemented with CH-YBG (supplemented sows) from day 83 of gestation until weaning (2g/sow/day). At weaning, 120 pigs (6 pigs/sow) were selected. The two dam groups were further divided, resulting in four experimental groups (10 replicates/group; 3 pigs/pen) as follows: 1) BB (basal sows + basal pigs); 2) BS (basal sows + supplemented pigs); 3) SB (supplemented sows + basal pigs); 4) SS (supplemented sows + supplemented pigs). Supplemented pigs were offered 0.5g CH-YBG/kg of feed for 10 days post-weaning. On day 10 post-weaning, 1 pig/pen was humanely sacrificed and samples were taken from the gastrointestinal tract for analysis. Pigs weaned from supplemented sows (SS, SB) had reduced faecal scores and incidence of diarrhoea (P<0.05) compared to pigs weaned from basal sows (BB, BS), with SS pigs not displaying the transient rise in faecal scores seen in the other three groups from day 3 to day 10 post-weaning (P<0.05). Pigs weaned from supplemented sows had reduced feed intake (P<0.05), improved feed efficiency (P<0.05), increased butyrate concentrations (P<0.05), increased abundance of Lactobacillus (P<0.05) and decreased abundance of Enterobacteriaceae and Campylobacteraceae (P<0.05) compared to pigs weaned from basal sows. In conclusion, maternal supplementation increased the abundance of Lactobacillus and decreased the abundance of Enterobacteriaceae and Campylobacteraceae while also increasing butyrate concentrations. The combination of maternal and direct supplementation led to pigs having the lowest faecal scores compared to all other groups.

Extracellular vesicle separation from milk and infant milk formula using acid precipitation and ultracentrifugation.

Separation of highly enriched extracellular vesicles (EVs) fractions from milk is desirable for quantification, cargo analysis, functional characterization, and investigation as delivery vehicles for nutrients and/or therapeutics. However, a rigorous, reproducible protocol is lacking. This protocol considers a crucial aspect typically overlooked, i.e., that caseins are of similar size to, but more abundant than, EVs in milk. Our protocol combines acid pre-treatment and gradient ultracentrifugation, producing EV-enriched fractions suitable for downstream orthogonal characterization approaches. For complete details on the use and execution of this protocol, please refer to Mukhopadhya et al. (2021).

The NR4A agonist, Cytosporone B, attenuates pro-inflammatory mediators in human colorectal cancer tissue ex vivo.

Inflammation is a pivotal pathological factor in colorectal cancer (CRC) initiation and progression, and modulating this inflammatory state has the potential to ameliorate disease progression. NR4A receptors have emerged as key regulators of inflammatory pathways that are important in CRC. Here, we have examined the effect of NR4A agonist, Cytosporone B (CsnB), on colorectal tissue integrity and its effect on the inflammatory profile in CRC tissue ex vivo. Here, we demonstrate concentrations up 100 µM CsnB did not adversely affect tissue integrity as measured using transepithelial electrical resistance, histology and crypt height. Subsequently, we reveal through the use of a cytokine/chemokine array, ELISA and qRT-PCR analysis that multiple pro-inflammatory mediators were significantly increased in CRC tissue compared to control tissue, which were then attenuated with the addition of CsnB (such as IL-1ß, IL-8 and TNFα). Lastly, stratification of the data revealed that CsnB especially alters the inflammatory profile of tumours derived from males who had not undergone chemoradiotherapy. Thus, this study demonstrates that NR4A agonist CsnB does not adversely affect colon tissue structure or functionality and can attenuate the pro-inflammatory state of human CRC tissue ex vivo.

Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk.

Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM.

Evaluation of the in vitro effects of the increasing inclusion levels of yeast ß-glucan, a casein hydrolysate and its 5 kDa retentate on selected bacterial populations and strains commonly found in the gastrointestinal tract of pigs.

Previously, the 5 kDa retentate (5kDaR) of a casein hydrolysate (CH) and yeast ß-glucan (YBG) were identified as promising anti-inflammatory dietary supplements for supporting intestinal health in pigs post-weaning. However, their direct effects on intestinal bacterial populations are less well-known. The main objectives of this study were to determine if the increasing concentrations of the CH, 5kDaR and YBG individually, can: (1) alter the bacterial and short-chain fatty acid profiles in a weaned pig faecal batch fermentation assay, and (2) directly influence the growth of selected beneficial (Lactobacillus plantarum, L. reuteri, Bifidobacterium thermophilum) and pathogenic (Enterotoxigenic Escherichia coli, Salmonella Typhimurium) bacterial strains in individual pure culture growth assays. The potential of CH as a comparable 5kDaR substitute was also evaluated. The 5kDaR increased lactobacilli counts and butyrate concentration in the batch fermentation assay (P < 0.05) and increased L. plantarum (linear, P < 0.05), L. reuteri (quadratic, P < 0.05) and B. thermophilum (linear, P < 0.05) counts and reduced S. typhimurium (quadratic, P = 0.058) counts in the pure culture growth assays. CH increased butyrate concentration (P < 0.05) in the batch fermentation assay. YBG reduced Prevotella spp. counts (P < 0.05) and butyrate concentration (P < 0.05) in the batch fermentation assay. Both CH and YBG had no major effects in the pure culture growth assays. In conclusion, the 5kDaR had the most beneficial effects associated with increased counts of Lactobacillus and Bifidobacterium genera and butyrate production and reduced S. typhimurium counts in vitro indicating its potential to promote gastrointestinal health.

Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation.

MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary.

Anti-Inflammatory Effects of Pomegranate Peel Extracts on In Vitro Human Intestinal Caco-2 Cells and Ex Vivo Porcine Colonic Tissue Explants.

The aim of this study was to determine the anti-inflammatory potential of pomegranate peel extracts (PPE) prepared from waste material of pomegranate juice production both in vitro on Caco-2 cells and ex vivo using porcine colonic tissue explants. Caco-2 cells were stimulated in vitro by TNF and colonic tissue explants were stimulated ex vivo with lipopolysaccharide (LPS). Both tissues were co-treated with PPE at 0, 1.0, 2.5, 5.0, 10 and 25 µg/mL. The secretion of CXCL8 in the supernatant of both experiments was determined by enzyme linked immunosorbent assay (ELISA) and the relative expression of inflammatory cytokines were evaluated in the colonic tissue by quantitative polymerase chain reaction (QPCR). The 2.5 to 25 µg/mL of PPE suppressed CXCL8 (p < 0.001) in the Caco-2 cells, whereas CXCL8 production was suppressed by only 5 and 25 µg/mL (p < 0.01) of PPE in the colonic explants. The 5 µg/mL of PPE also suppressed the expression of IL1A (p < 0.05), IL6 (p < 0.01) and CXCL8 (p < 0.05) in LPS challenged colonic tissues compared to controls. In conclusion, the 5 µg/mL of PPE consistently elicits strong anti-inflammatory activity. These results support the potential of bioactive compounds from the waste peel of pomegranate in terms of their anti-inflammatory activity in cells and tissues of the gastrointestinal tract.

A combination of yeast beta-glucan and milk hydrolysate is a suitable alternative to zinc oxide in the race to alleviate post-weaning diarrhoea in piglets.

Zinc oxide (ZnO) is currently used as a dietary supplement to support gut homeostasis during the standard 'abrupt' weaning practices in commercial pig production. However, a replacement is urgently required as a ban on ZnO usage is imminent. The objective of this study was to explore the potential of a bovine casein hydrolysate (5kDaR) and yeast ß-glucan, and their combination, as an alternative to ZnO. Eighty 21d old male piglets received a basal diet or supplemented with 5kDaR and yeast ß-glucan alone or in combination, or ZnO from the day of weaning and were monitored for 10 days (n = 8/group; dietary groups: control diet; control diet + 5kDaR; control diet + yeast ß-glucan; control diet + 5kDaR + yeast ß-glucan; control diet + ZnO). Individually, supplement yeast ß-glucan or 5kDaR did not improve gut health. In contrast, the yeast ß-glucan + 5kDaR combination supplement supported a healthy gut, indicated by healthy faecal scores and improved growth parameters; similar to ZnO inclusion (P > 0.05). There was no negative effect on the gut microbiota with yeast ß-glucan + 5kDaR supplementation; while ZnO negatively affected the Bifidobacterium spp. abundance (P < 0.05). The inflammatory NFκB pathway was suppressed by yeast ß-glucan + 5kDaR supplementation, similar to ZnO (P > 0.05). In conclusion, the dietary supplement yeast ß-glucan + 5kDaR restored homeostasis of the newly weaned piglet gut similar to the widely used ZnO, and can potentially replace ZnO.

Breed differences in humoral and cellular responses of lambs to experimental infection with the gastrointestinal nematode Teladorsagia circumcincta.

While Texel lambs have increased resistance to infection with the gastrointestinal nematode Teladorsagia circumcincta compared to Suffolk lambs, the underlying resistance mechanisms are still unknown. The aim of this study was to compare parasitological, humoral and cellular responses of Texel and Suffolk lambs over time following a single experimental infection with T. circumcincta. Gastrointestinal nematode free (but not naïve) lambs received a single oral dose of 3 × 104 infective T. circumcincta larvae. The variables examined included worm burden, mucosal and serum IgA, abomasal mast cells and eosinophils, haematological parameters and plasma pepsinogen. Texel lambs had significantly lower worm burden on day 14 and lower plasma pepsinogen concentration from day 14 onwards than Suffolks and their response in mucosal IgA to infection occurred earlier. The results from the study suggest that an earlier local IgA response in the Texel contributes to the resistant characteristics of the breed, while the increased level of plasma pepsinogen in the Suffolk lambs implies greater abomasal tissue damage arising from the nematode infection.

Anti-inflammatory effects of a casein hydrolysate and its peptide-enriched fractions on TNFα-challenged Caco-2 cells and LPS-challenged porcine colonic explants.

Bioactive milk peptides are reported to illicit a range of physiological benefits and have been proposed as potential functional food ingredients. The objective of this study was to characterize the anti-inflammatory properties of sodium caseinate (NaCAS), its enzyme hydrolysate (EH) and peptide-enriched fractions (5 kDa retentate [R], 1 kDaR and 1 kDa permeate [P]), both in vitro using a Caco-2 cell line, and also ex vivo using a porcine colonic tissue explant system. Caco-2 cells were stimulated with tumour necrosis factor alpha (TNFα) and co-treated with casein hydrolysates for 24 h. Following this, interleukin (IL)-8 concentrations in the supernatant were measured using enzyme-linked immunosorbent assay. Porcine colonic tissue was stimulated with lipopolysaccharide and co-treated with casein hydrolysates for 3 h. The expression of a panel of inflammatory cytokines was measured using qPCR. While dexamethasone reduced the IL-8 concentration by 41.6%, the 1 kDaR and 1 kDaP fractions reduced IL-8 by 68.7% and 66.1%, respectively, relative to TNFα-stimulated Caco-2 cells (P < 0.05). In the ex vivo system, only the 1 kDaR fraction elicited a decrease inIL1-α,IL1-ß,IL-8,TGF-ß andIL-10 expression (P < 0.05). This study provides evidence that the bioactive peptides present in the 1 kDaR fraction of the NaCAS hydrolysate possess anti-inflammatory properties in vitro and ex vivo. Further in vivo analysis of the anti-inflammatory properties of the 1 kDaR is proposed.