Human Enterocytes Research Articles

Differences between peptide profiles of extensive hydrolysates and their influence on functionality for the management of cow's milk allergy: A short review.

Extensively hydrolyzed formulas (eHFs) are recommended for the dietary management of cow's milk protein allergy (CMPA) in non-exclusively breastfed infants. Studies show that peptide profiles differ between eHFs. This short review aims to highlight the variability in peptides and their ability to influence allergenicity and possibly the induction of tolerance by different eHFs. The differences between eHFs are determined by the source of the protein fraction (casein or whey), peptide size-distribution profile and residual β-lactoglobulin which is the most immunogenic and allergenic protein in bovine milk for human infants as it is not present in human breastmilk. These differences occur from the hydrolyzation process which result in variable IgE reactivity against cow's milk allergen epitopes by subjects with CMPA and differences in the Th1, Th2 and pro-inflammatory cytokine responses elicited. They also have different effects on gut barrier integrity. Results suggest that one particular eHF-casein had the least allergenic potential due to its low residual allergenic epitope content and demonstrated the greatest effect on restoring gut barrier integrity by its effects on mucin 5AC, occludin and Zona Occludens-1 in human enterocytes. It also increased the production of the tolerogenic cytokines Il-10 and IFN-γ. In addition, recent studies documented promising effects of optional functional ingredients such as pre-, pro- and synbiotics on the management of cow's milk allergy and induction of tolerance, in part via the induction of the production of short chain fatty acids. This review highlights differences in the residual allergenicity, peptide size distribution, presence of optional functional ingredients and overall functionality of several well-characterized eHFs which can impact the management of CMPA and the ability to induce immune tolerance to cow's milk protein.

The role of orally ingested milk fat globule membrane on intestinal barrier functions evaluated with a suckling rat pup supplementation model and a human enterocyte model

Milk fat globule membrane (MFGM), the membrane surrounding secreted fat droplets in milk, contains components involved in a wide range of bioprocesses including cell proliferation and differentiation. The intestine is relatively immature and permeable at birth. Since MFGM is partly resistant to digestion in infancy, we hypothesized that orally ingested MFGM promotes intestinal development by enhancing intestinal barrier functions in early life. An established suckling rat model was used; Sprague–Dawley rats were bred, and litters were culled to 10 pups/dam. Pups were supplemented orally with MFGM (0, 100, or 300 mg/kg/d) from postnatal day 1–20. Intestine samples were collected for histology, real-time quantitative PCR, immunoblotting, and immunohistochemistry analysis. Additionally, differentiated Caco-2 cells were used to assess effects of MFGM on the human intestinal barrier. Control and MFGM-supplemented rat pups showed similar growth. Intestinal differentiation and expression of tight junction proteins in jejunum and colon were significantly increased by orally ingested MFGM, and MFGM supplementation significantly activated PI3K/Akt/mTOR, mitogen-activated protein kinases, and myosin light chain kinase signaling pathways, suggesting that MFGM promotes intestinal development by triggering various signaling pathways. In human enterocytes (polarized Caco-2 cells), MFGM (400 µg/mL for 72 h) decreased permeability, as revealed by increased transepithelial electrical resistance. In Caco-2 cells, MFGM also enhanced expression of tight junction proteins, including claudin-4 and ZO-2. In conclusion, orally ingested MFGM may exert beneficial roles in intestinal development by activating various cell signaling pathways to upregulate tight junction proteins and thereby increasing intestinal barrier functions.

Protein oxidation marker, α-amino adipic acid, impairs proteome of differentiated human enterocytes: Underlying toxicological mechanisms

Protein oxidation and oxidative stress are involved in a variety of health disorders such as colorectal adenomas, inflammatory bowel's disease, neurological disorders and aging, among others. In particular, the specific final oxidation product from lysine, the α-amino adipic acid (α-AA), has been found in processed meat products and emphasized as a reliable marker of type II diabetes and obesity. Currently, the underlying mechanisms of the biological impairments caused by α-AA are unknown. To elucidate the molecular basis of the toxicological effect of α-AA, differentiated human enterocytes were exposed to dietary concentrations of α-AA (200 μM) and analyzed by flow cytometry, protein oxidation and proteomics using a Nanoliquid Chromatography-Orbitrap MS/MS. Cell viability was significantly affected by α-AA (p < 0.05). The proteomic study revealed that α-AA was able to alter cell homeostasis through impairment of the Na+/K+-ATPase pump, energetic metabolism, and antioxidant response, among other biological processes. These results show the importance of dietary oxidized amino acids in intestinal cell physiology and open the door to further studies to reveal the impact of protein oxidation products in pathological conditions.

Multidrug resistance-associated protein 2 (MRP2) is an efflux transporter of EGCG and its metabolites in the human small intestine

Green tea polyphenols have various beneficial effects on human health, such as antiobesity and anti-carcinogenesis. (-)-Epigallocatechin-gallate (EGCG) is one of the major potent green tea catechins; however, detailed mechanisms of EGCG transport and metabolism in the human small intestine remain unknown due to lack of a suitable model. We investigated metabolite profiles of EGCG in the fresh human duodenal biopsy, cryopreserved human duodenal mucosal enterocytes and Caco-2 cells, and found that EGCG was readily metabolized into methylated and sulphate conjugates, which are major metabolites in these models. Next, we examined possible efflux transporters of EGCG and its metabolites using specific inhibitors of MRP2, P-gp and BCRP in Caco-2 cell monolayers. MRP2 was thereby identified as an efflux transporter, and further analysis using MRP2-knockout Caco-2 cells and vesicular transport assays confirmed that MRP2 is a selective efflux transporter of EGCG and its metabolites. Assuming that functional inhibition of MRP2 would result in efficient uptake of EGCG, we screened for MRP2 functional blockade and identified quercetin, which led to increased intracellular accumulation and basal transport of EGCG in Caco-2 cells. This result suggested that co-administration of quercetin and EGCG would enable efficient transport of EGCG in the human intestine. Therefore, we performed co-oral administration of quercetin and EGCG in human subjects to examine whether this occurred in humans. These studies demonstrated that MRP2 is a selective transporter of EGCG and conjugates and Caco-2 is a model to examine transport mechanisms and metabolites of polyphenols in the human small intestine.

Regulation of nutrient and electrolyte absorption in human organoid-derived intestinal epithelial cell monolayers

Recently developed human intestinal epithelial 3D organoid cultures are a useful cell culture model to study intestinal transport physiology. From these, 2D monolayer cultures can be generated in which apical transporters are exposed to the medium, thereby better facilitating in vitro investigation of intestinal absorption processes. However, whether nutrient and electrolyte absorption can be physiologically regulated in human organoid-derived monolayers has not been determined. Constitutive nitric oxide (cNO) is known to regulate multiple gastrointestinal physiological functions. Previous studies using in vivo and in vitro mammalian animal models indicate that enhanced intracellular cNO differentially regulates the two primary apical Na transporters in small intestinal epithelial cells. Here, we generated human jejunal organoid-derived monolayers to determine whether apical nutrient and electrolyte transporter function is regulated by cNO in human enterocytes. Western blot analysis and immunocytochemical staining showed that organoid-derived 2D cultures express markers of enterocyte differentiation and form intact monolayers of apical-basal polarized epithelial cells. Uptake studies demonstrated that jejunal monolayers exhibit functional activity of Na-glucose cotransporter 1 (SGLT1; SLC5A1) and Na-H exchanger 3 (NHE3; SLC9A3). In response to physiological increases in cNO, the two primary apical Na transporters were differentially regulated in human intestinal organoid-derived monolayers, across multiple human specimens. An increase in cNO stimulated SGLT1, while NHE3 was inhibited. These results are similar to what is seen in vivo and in vitro in different animal intestinal models. Thus, human jejunal organoid-derived monolayers are an ideal in vitro model to better understand how intestinal nutrient absorption is regulated.

Silymarin‐mediated Natural Product–Drug Interactions involving Enteric Organic Anion Transporting Polypeptides

Organic anion transporting polypeptides (OATPs in humans; Oatps in rodents) represent a large family of uptake transporters important to drug disposition. Several factors can disrupt transport activity of OATPs/Oatps, including inhibition by natural products found in the diet and in dietary supplements. Silymarin, an extract of Silybum marianum (L.) Gaertn. seeds, is a widely consumed dietary supplement for hepatobiliary disorders that is reported to inhibit OATPs/Oatps. Although there is potential for high intestinal concentrations of flavonolignans after oral consumption, very little research has explored the risk of an enteric interactions between silymarin and OATP/Oatp substrates. Therefore, we hypothesize that clinically relevant doses of silymarin will inhibit enteric OATP/Oatp activity and pose a risk for decreased systemic drug concentrations. IC­50 data for the individual flavonolignans were collected using cells overexpressing OATPs. OATP2B1, the major OATP expressed in human enterocytes, was inhibited by silymarin (IC50: 3.9 µM). Further analysis of individual flavonolignans, which are the major constituents in silymarin revealed IC50 values below 10 µM (isosilybin A: 2.7 µM, isosilybin B: 9.4 µM, silychristin: 8.2 µM, silydianin: 5.5 µM). FDA guidance provides metrics for predicting the risk of an intestinal in vivo interaction from in vitro data (R value ≥ 11) based on the proportionality between the maximal extracellular inhibitor concentration and the IC50 data. For flavonolignans from silymarin, maximal intestinal concentrations are estimated to be considerably greater than the experimentally derived IC50 data. Consequently, silymarin doses as low as 140 mg produced R values for flavonolignans that exceeded the FDA threshold. These data suggest enteric OATPs/Oatps may be susceptible to silymarin‐mediated inhibition and potentially decrease systemic drug concentrations.

Cytotoxic Effects of Alternariol, Alternariol Monomethyl-Ether, and Tenuazonic Acid and Their Relevant Combined Mixtures on Human Enterocytes and Hepatocytes.

Alternariol (AOH), alternariol monomethyl-ether (AME), and tenuazonic acid (TeA) are major mycotoxins produced by fungi of the genus Alternaria and are common contaminants of food products such as fruits, vegetables, cereals and grains. Alternaria mycotoxins are known to cause relevant economic losses and to have a negative impact on human and animal health. EFSA stated in its scientific opinion that data on the toxicity of Alternaria mycotoxins in humans and livestock are generally lacking, precluding proper hazard characterization. This study aimed to fill some knowledge gaps by studying the in vitro cytotoxicity toward human intestinal epithelial cells (Caco-2) and hepatocytes (HepG2). Cytotoxic properties were assessed by flow cytometric analyses of remaining viable cells (i.e., propidium iodide negative) after mycotoxin exposure for 24–48 h versus solvent control. Treatment of cells with single doses of AOH, AME, and TeA resulted in a dose-dependent loss of cell viability for both cell lines. Half maximal effective concentrations (EC50) of the different mycotoxins were comparable for the two cell lines. On HepG2 cells, EC50 values varying between 8 and 16, 4 and 5, and 40 and 95 μg/mL were calculated for AOH, AME, and TeA, respectively. On Caco-2 cells, EC50 values of 19 μg/mL and varying between 6 and 23, and 60 and 90 μg/mL were calculated for AOH, AME, and TeA, respectively. A general relative cytotoxicity ranking of about 1 = 1 >>> 3 was obtained for AOH, AME, and TeA, respectively. Treatment of both cell lines with combined binary and ternary mixtures of AOH, AME, and TeA in a 1:1:3 ratio, also showed a dose-dependent decrease in cell viability. For both cell lines, the binary combination of especially AME and TeA (1:3 ratio) but also of AOH and AME (1:1 ratio) significantly increased the cytotoxicity compared to the single compound toxicity, although mainly at the highest concentrations tested. The ternary combinations of AOH, AME, and TeA induced only a slight increase in cytotoxicity compared to the single mycotoxins, again at the highest concentrations tested.

Protective effects of the postbiotic deriving from cow\u2019s milk fermentation with L. paracasei CBA L74 against Rotavirus infection in human enterocytes

Rotavirus (RV) is the leading cause of acute gastroenteritis-associated mortality in early childhood. Emerging clinical evidence suggest the efficacy of the postbiotic approach based on cow’s milk fermentation with the probiotic Lacticaseibacillus paracasei CBAL74 (FM-CBAL74) in preventing pediatric acute gastroenteritis, but the mechanisms of action are still poorly characterized. We evaluated the protective action of FM-CBAL74 in an in vitro model of RV infection in human enterocytes. The number of infected cells together with the relevant aspects of RV infection were assessed: epithelial barrier damage (tight-junction proteins and transepithelial electrical resistance evaluation), and inflammation (reactive oxygen species, pro-inflammatory cytokines IL-6, IL-8 and TNF-α, and mitogen-activated protein kinase pathway activation). Pre-incubation with FM-CBA L74 resulted in an inhibition of epithelial barrier damage and inflammation mediated by mitogen-activated protein kinase pathway activation induced by RV infection. Modulating several protective mechanisms, the postbiotic FM-CBAL74 exerted a preventive action against RV infection. This approach could be a disrupting nutritional strategy against one of the most common killers for the pediatric age.

A bovine miRNA, bta-miR-154c, withstands invitro human digestion but does not affect cell viability of colorectal human cell lines after transfection.

Colorectal cancer (CRC) is the third most frequent human cancer with over 1.3 million new cases globally. CRC is a complex disease caused by interactions between genetic and environmental factors; in particular, high consumption of red meat, including beef, is considered a risk factor for CRC initiation and progression. Recent data demonstrate that exogenous microRNAs (miRNAs) entering the body via ingestion could pose an effect on the consumer. In this study, we focused on bovine miRNAs that do not share a seed sequence with humans and mice. We identified bta‐miR‐154c, a bovine miRNA found in edible parts of beef and predicted via cross‐species bioinformatic analysis to affect cancer‐related pathways in human cells. When bovine tissue was subjected to cooking and a simulation of human digestion, bta‐miR‐154c was still detected after all procedures, albeit at reduced concentrations. However, lipofection of bta‐miR‐154c in three different colorectal human cell lines did not affect their viability as evaluated at various time points and concentrations. These data indicate that bta‐miR‐154c (a) may affect cancer‐related pathways in human cells, (b) can withstand digestion and be detected after all stages of an in vitro digestion protocol, but (c) it does not appear to alter epithelial cell viability after entering human enterocytes, even at supraphysiological amounts. Further experiments will elucidate whether bta‐miR‐154c exerts a different functional effect on the human gut epithelium, which may cause it to contribute to CRC progression through its consumption.

Divergent Impact of Enzyme Replacement Therapy on Human Cardiomyocytes and Enterocytes Affected by Fabry Disease: Correlation with Mannose-6-phosphate Receptor Expression.

Background: The impact of enzyme replacement therapy (ERT) on cardiomyocytes and intestinal cells, affected by Fabry disease (FD), is still unclear. Methods: Six patients with FD, including five family members with GLA mutation c.666delC and one with GLA mutation c.658C > T, manifesting cardiomyopathy and intestinal symptoms (abdominal pain, diarrhea and malabsorption) were included in the study. Clinical outcome, cardiac magnetic resonance (CMR), endomyocardial and gastro-intestinal biopsies were evaluated before and after 2 years of treatment with agalsidase-α (0.2 mg/kg every other week). Immunohistochemistry and Western blot assessments of mannose-6-phosphate receptors (IGF-II-R) on intestinal and myocardial frozen tissue were obtained at diagnosis and after 2 years of ERT. Results: After ERT left ventricular maximal wall thickness, ranging from pre (<10.5 mm) to mild (<15 mm) and moderate hypertrophy (16 mm), was not associated with significant changes at CMR. Degree of dyspnea, mean cardiomyocyte diameter and % vacuolated areas of cardiomyocytes, representing intracellular GL3, remained unmodified. In contrast, intestinal symptoms improved with disappearance of diarrhea, recovery of anemia and weight gain, correlating with near complete clearance of the enterocytes from GL3 inclusions. IGF-II-R expression was remarkably higher even at histochemistry in intestinal tissue compared with myocardium (p < 0.001) either at baseline and after ERT, thus justifying intestinal recovery. Conclusions: Human cells affected by FD may respond differently to ERT: while cardiomyocytes retain their GL3 content after 2 years of treatment, gastro-intestinal cells show GL3 removal with recovery of function. This divergent response may be related to differences in cellular turnover, as well as tissue IGF-II-R expression.

Anti-Inflammatory Effect of an O-2-Substituted (1-3)-β-D-Glucan Produced by Pediococcus parvulus 2.6 in a Caco-2 PMA-THP-1 Co-Culture Model.

Bacterial β-glucans are exopolysaccharides (EPSs), which can protect bacteria or cooperate in biofilm formation or in bacterial cell adhesion. Pediococcus parvulus 2.6 is a lactic acid bacterium that produces an O-2-substituted (1-3)-β-D-glucan. The structural similarity of this EPS to active compounds such as laminarin, together with its ability to modulate the immune system and to adhere in vitro to human enterocytes, led us to investigate, in comparison with laminarin, its potential as an immunomodulator of in vitro co-cultured Caco-2 and PMA-THP-1 cells. O-2-substituted (1-3)-β-D-glucan synthesized by the GTF glycosyl transferase of Pediococcus parvulus 2.6 or that by Lactococcus lactis NZ9000[pGTF] were purified and used in this study. The XTT tests revealed that all β-glucans were non-toxic for both cell lines and activated PMA-THP-1 cells’ metabolisms. The O-2-substituted (1-3)-β-D-glucan modulated production and expression of IL-8 and the IL-10 in Caco-2 and PMA-THP-1 cells. Laminarin also modulated cytokine production by diminishing TNF-α in Caco-2 cells and IL-8 in PMA-THP-1. All these features could be considered with the aim to produce function foods, supplemented with laminarin or with another novel β-glucan-producing strain, in order to ameliorate an individual’s immune system response toward pathogens or to control mild side effects in remission patients affected by inflammatory bowel diseases.

A New Butyrate Releaser Exerts a Protective Action against SARS-CoV-2 Infection in Human Intestine.

Butyrate is a major gut microbiome metabolite that regulates several defense mechanisms against infectious diseases. Alterations in the gut microbiome, leading to reduced butyrate production, have been reported in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. A new butyrate releaser, useful for all the known applications of butyrate, presenting physiochemical characteristics suitable for easy oral administration, (N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA), has been recently developed. We investigated the protective action of FBA against SARS-CoV-2 infection in the human small intestine and enterocytes. Relevant aspects of SARS-CoV-2 infection were assessed: infectivity, host functional receptor angiotensin-converting enzyme-2 (ACE2), transmembrane protease serine 2 (TMPRSS2), neuropilin-1 (NRP1), pro-inflammatory cytokines expression, genes involved in the antiviral response and the activation of Nf-kB nuclear factor (erythroid-derived 2-like) 2 (Nfr2) pathways. We found that FBA positively modulates the crucial aspects of the infection in small intestinal biopsies and human enterocytes, reducing the expression of ACE2, TMPRSS2 and NRP1, pro-inflammatory cytokines interleukin (IL)-15, monocyte chemoattractant protein-1 (MCP-1) and TNF-α, and regulating several genes involved in antiviral pathways. FBA was also able to reduce the number of SARS-CoV-2-infected cells, and ACE2, TMPRSS2 and NRP1 expression. Lastly, through the inhibition of Nf-kB and the up-regulation of Nfr2, it was also able to reduce the expression of pro-inflammatory cytokines IL-15, MCP-1 and TNF-α in human enterocytes. The new butyrate releaser, FBA, exerts a preventive action against SARS-CoV-2 infection. It could be considered as an innovative strategy to limit COVID-19.

An Integrated Strategy for Assessing the Metabolic Stability and Biotransformation of Macrocyclic Peptides in Drug Discovery toward Oral Delivery.

Macrocyclic peptides (MCPs) are an emerging class of promising drug modalities that can be used to interrogate hard-to-drug ("undruggable") targets. However, their poor intestinal stability is one of the major liabilities or obstacles for oral drug delivery. We therefore investigated the metabolic stability and biotransformation of MCPs via a systematic approach and established an integrated in vitro assay strategy to facilitate MCP drug discovery, with a focus on oral delivery liabilities. A group of diverse MCPs were incubated with representative matrices, including simulated intestinal fluid with pancreatin (SIFP), human enterocytes, liver S9 fractions, liver lysosomes, plasma, and recombinant enzymes. The results revealed that the stability and biotransformation of MCPs varied, with the major metabolic pathways identified in different matrices. Under the given conditions, the selected MCPs generally showed better stability in plasma compared to that in SIFP. Our data suggest that pancreatic enzymes act as the primary metabolic barrier for the oral delivery of MCPs, mainly through hydrolysis of their backbone amide bonds. Whereas in enterocytes, multiple metabolic pathways appeared to be involved and resulted in metabolic reactions such as oxidation and reduction in addition to hydrolysis. Further studies suggested that lysosomal peptidase cathepsin B could be a major enzyme responsible for the cleavage of side-chain amide bonds in lysosomes. Collectively, we developed and implemented an integrated assay for assessing the metabolic stability and biotransformation of MCPs for compound screening in the discovery stage toward oral delivery. The proposed question-driven assay cascade can provide biotransformation insights that help to guide and facilitate lead candidate selection and optimization.

Cover 1 - Manipulating Lipid Handling Through β-Oxidation in Human Enterocytes

Cover 1 - Manipulating Lipid Handling Through β-Oxidation in Human Enterocytes

A Planar Culture Model of Human Absorptive Enterocytes Reveals Metformin Increases Fatty Acid Oxidation and Export.

Background & AimsFatty acid oxidation by absorptive enterocytes has been linked to the pathophysiology of type 2 diabetes, obesity, and dyslipidemia. Caco-2 and organoids have been used to study dietary lipid-handling processes including fatty acid oxidation, but are limited in physiological relevance or preclude simultaneous apical and basal access. Here, we developed a high-throughput planar human absorptive enterocyte monolayer system for investigating lipid handling, and then evaluated the role of fatty acid oxidation in fatty acid export, using etomoxir, C75, and the antidiabetic drug metformin.MethodsSingle-cell RNA-sequencing, transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo absorptive enterocyte maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo absorptive enterocyte maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation was modulated pharmacologically and BODIPY (Thermo Fisher Scientific, Waltham, MA) (B)–labeled fatty acids were used to evaluate fatty acid handling via fluorescence and thin-layer chromatography.ResultsSingle-cell RNA-sequencing shows increasing expression of lipid-handling genes as absorptive enterocytes mature. Culture conditions promote ISC differentiation into confluent absorptive enterocyte monolayers. Fatty acid-handling gene expression mimics in vivo maturational states. The fatty acid oxidation inhibitor etomoxir decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 fatty acids, whereas the CPT1 agonist C75 and the antidiabetic drug metformin increased apical-to-basolateral export. Short-chain B-C5 was unaffected by fatty acid oxidation inhibition and diffused through absorptive enterocytes.ConclusionsPrimary human ISCs in culture undergo programmed maturation. Absorptive enterocyte monolayers show in vivo maturational states and lipid-handling gene expression profiles. Absorptive enterocytes create strong epithelial barriers in 96-Transwell format. Fatty acid export is proportional to fatty acid oxidation. Metformin enhances fatty acid oxidation and increases basolateral fatty acid export, supporting an intestine-specific role.

Regulation of citrulline synthesis in human enterocytes: Role of hypoxia and inflammation

AbstractIntensive care unit patients and chronic airway inflammatory disease are characterized by chronic systemic hypoxia and inflammation inducing a decrease in nitric oxide release due to impaired l‐arginine (ARG) homeostasis. As ARG is synthesized from circulating l‐citrulline (CIT), an alteration of CIT production in small intestine by ornithine carbamoyltransferase could be involved. Here, we posit that hypoxia and/or inflammation has effects on ornithine carbamoyltransferase regulation in enterocytes. A duodenal explant incubation model was used. Biopsy specimens taken from 25 selected patients were incubated for 6 h in 4 groups: control, inflammation, hypoxia, and hypoxia + inflammation. At the end of the incubation period, we measured CIT concentration in culture media, ornithine carbamoyltransferase activity, ornithine carbamoyltransferase protein and gene expression, protein expression of enzymes involved in the CIT production pathway, and expression of energy status proteins. Inflammation and/or hypoxia conditions did not affect CIT production. Ornithine carbamoyltransferase activity was increased in hypoxia conditions (p = 0.023). Expression of enzymes implicated in the CIT crossroads pathway and enzymes reflecting energy status variation was not affected by inflammation and hypoxia. Data sets were pooled to evaluate the variability of the four quartiles for each parameter. CIT production was found to increase over the quartiles whereas other parameters remained stable. Our results showed that intestinal CIT production is preserved during inflammation and/or hypoxia, thus confirming the significance of this metabolic pathway. This suggests that the CIT deficiency observed in clinical hypercatabolic states could be a consequence of high utilization for ARG synthesis.

Butyrate as a Bioactive Human Milk Protective Component Against Food Allergy

To understand the concentration of butyrate, a short chain fatty acid and metabolite derived from gut microbiota, in human milk and the mechanisms of immune tolerance.A total of 109 healthy mothers (aged 21–42 years) were enrolled after term singleton pregnancy in Italy.By using gas chromatography-mass spectrometry, the concentration of human milk butyrate was measured. By using in vivo and in vitro models, the tolerogenic mechanisms of human milk butyrate were evaluated.The median butyrate concentration of 0.75 mM was responsible for the maximum modulatory effects observed. In the mouse model, upregulation of several biomarkers important for gut barrier integrity by butyrate was seen. In animal models of food allergy, pretreatment with butyrate significantly reduced allergic response with inhibition of T helper 2 cytokine production and stimulation of tolerogenic cytokines. In human cell models, stimulation of human β defensin-3 and tight junctions’ expression in human enterocytes by butyrate were seen in peripheral blood mononuclear cells from children with food allergy; expression of interleukin 10, interferon γ and forkhead box P3 through epigenetic mechanisms occurred. Precursors of regulatory T cells and dendritic cells were promoted.Butyrate may have a pivotal role in protection against food allergy.Food allergy is a pervasive problem throughout the world. Breastfeeding is considered to have a protective effect against the development of food allergy, although previous data have revealed conflicting rates of efficacy. The underlying mechanism for food allergy protection has also been unclear. The immunoregulatory effectiveness of human milk may be secondary to butyrate concentration, as seen in this study. The variation of butyrate concentration in individual’s human milk may explain the inconsistent evidence for human milk’s protection against food allergy from previous studies. Butyrate supplementation to infants or lactating mothers is a potential strategy to prevent the development of food allergy.

Diosmectite inhibits the interaction between SARS-CoV-2 and human enterocytes by trapping viral particles, thereby preventing NF-kappaB activation and CXCL10 secretion

SARS-CoV-2 enters the intestine by the spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors in enterocyte apical membranes, leading to diarrhea in some patients. Early treatment of COVID-19-associated diarrhea could relieve symptoms and limit viral spread within the gastrointestinal (GI) tract. Diosmectite, an aluminomagnesium silicate adsorbent clay with antidiarrheal effects, is recommended in some COVID-19 management protocols. In rotavirus models, diosmectite prevents pathogenic effects by binding the virus and its enterotoxin. We tested the trapping and anti-inflammatory properties of diosmectite in a SARS-CoV-2 model. Trapping effects were tested in Caco-2 cells using spike protein receptor-binding domain (RBD) and heat-inactivated SARS-CoV-2 preparations. Trapping was assessed by immunofluorescence, alone or in the presence of cells. The effect of diosmectite on nuclear factor kappa B (NF-kappaB) activation and CXCL10 secretion induced by the spike protein RBD and heat-inactivated SARS-CoV-2 were analyzed by Western blot and ELISA, respectively. Diosmectite bound the spike protein RBD and SARS-CoV-2 preparation, and inhibited interaction of the spike protein RBD with ACE2 receptors on the Caco-2 cell surface. Diosmectite exposure also inhibited NF-kappaB activation and CXCL10 secretion. These data provide direct evidence that diosmectite can bind SARS-CoV-2 components and inhibit downstream inflammation, supporting a mechanistic rationale for consideration of diosmectite as a management option for COVID-19-associated diarrhea.

Simultaneous Evaluation of Membrane Permeability and UDP-Glucuronosyltransferase-Mediated Metabolism of Food-Derived Compounds Using Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells.

Pharmacokinetic prediction after oral ingestion is important for quantitative risk assessment of food-derived compounds. To evaluate the utility of human intestinal absorption prediction, we compared the membrane permeability and metabolic activities of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIECs) with Caco-2 cells or human primary enterocytes (hPECs). We found that membrane permeability in hiPSC-SIECs had better predictivity than that in Caco-2 cells against 21 drugs with known human intestinal availability (r = 0.830 and 0.401, respectively). Membrane permeability in hiPSC-SIECs was only 0.019-0.25-fold as compared with that in Caco-2 cells for 7 in 15 food-derived compounds, primarily those that were reported to undergo glucuronidation metabolism. The metabolic rates of the glucuronide conjugate were similar or higher in hiPSC-SIECs as compared with hPECs but lower in Caco-2 cells. Expression levels of UDP-glucuronosyltransferase (UGT) isoform mRNA in hiPSC-SIECs were similar or higher as compared with hPECs. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism. SIGNIFICANCE STATEMENT: Gastrointestinal absorption is an important step for predicting the internal exposure of food-derived compounds. This research revealed that human induced pluripotent stem cell-derived small intestinal cells (hiPSC-SIECs) had better predictivity of intestinal availability than Caco-2 cells; furthermore, the metabolic rates of UDP-glucuronosyltransferase (UGT) substrates of hiPSC-SIECs were closer to those of human primary enterocytes than those of Caco-2 cells. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism.

Protective effects elicited by cow milk fermented with L. Paracasei CBAL74 against SARS-CoV-2 infection in human enterocytes.

Fermented foods have been proposed in limiting SARS-CoV-2 infection. Emerging evidence suggest the efficacy of cow's milk fermented with the probiotic L. paracasei CBAL74 (FM-CBAL74) in preventing infectious diseases. We evaluated the protective action of FM-CBAL74 against SARS-CoV-2 infection in human enterocytes. Relevant aspects of SARS-CoV-2 infection were assessed: infectivity, host functional receptor angiotensin-converting enzyme-2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and pro-inflammatory cytokines expression (IL-6, IL-15, IL-1β, VEGFβ, TNF-α, MCP-1, CXCL1). Pre-incubation with FM-CBA L74 reduced the number of infected cells. The expression of ACE2 and the pro-inflammatory cytokines IL-6, VEGFβ, IL-15, IL-1β was downregulated by the pre-treatment with this fermented food. No effect on TMPRSS2, MCP-1, TNF-α and CXCL1 expression was observed. Modulating the crucial aspects of the infection, the fermented food FM-CBAL74 exerts a preventive action against SARS-CoV-2. These evidence could pave the way to innovative nutritional strategy to mitigate the COVID-19.