Oenothein B in Eucalyptus Leaf Extract Suppresses Fructose Absorption in Caco-2 Cells

Inhibition of fructose absorption may suppress adiposity and adiposity-related diseases caused by fructose ingestion. Eucalyptus leaf extract (ELE) inhibits intestinal fructose absorption (but not glucose absorption); however, its active compound has not yet been identified. Therefore, we evaluated the inhibitory activity of ELE obtained from Eucalyptus globulus using an intestinal fructose permeation assay with the human intestinal epithelial cell line Caco-2. The luminal sides of a cell monolayer model cultured on membrane filters were exposed to fructose with or without the ELE. Cellular fructose permeation was evaluated by measuring the fructose concentration in the medium on the basolateral side. ELE inhibited 65% of fructose absorption at a final concentration of 1 mg/mL. Oenothein B isolated from the ELE strongly inhibited fructose absorption; the inhibition rate was 63% at a final concentration of 5 μg/mL. Oenothein B did not affect glucose absorption. In contrast, the other major constituents (i.e., gallic acid and ellagic acid) showed little fructose-inhibitory activity. To our knowledge, this is the first report that oenothein B in ELE strongly inhibits fructose absorption in vitro. ELE containing oenothein B can prevent and ameliorate obesity and other diseases caused by dietary fructose consumption.

AP-1 Recruits SMAP-1/SMAPs to the trans-Golgi Network to Promote Sorting in Polarized Epithelia

Coordinated AP-1 and clathrin coat assembly mediate secretory sorting on the trans-Golgi network (TGN) during conventional secretion. Here we found that SMAP-1/SMAPs deficiency caused the apical protein ERM-1 to accumulate on the basolateral side of the TGN. In contrast, the basolateral protein SLCF-1 appeared abnormally on the apical membrane. SMAP-1 colocalized with AP-1 on the TGN. The integrity of AP-1 is required for the subcellular presence of SMAP-1. Moreover, we found that the loss of SMAP-1 reduced clathrin-positive structures in the cytosol, suggesting that SMAP-1 has a regulatory role in clathrin assembly on the TGN. Functional experiments showed that overexpressing clathrin effectively alleviated exocytic defects due to the lack of SMAP-1, corroborating the role of SMAP-1 in promoting the assembly of clathrin on the TGN. Together, our results suggested that the AP-1 complex regulates the TGN localization of SMAP-1, promoting clathrin assembly to ensure polarized conventional secretion in C. elegans intestinal epithelia.

Metabolism of Selected 2-Arylbenzofurans in a Colon In Vitro Model System

2-arylbenzofurans represent a small group of bioactive compounds found in the plant family Moraceae. As it has not been investigated whether these substances are stable during passage through the gastrointestinal tract, their biological effects may be altered by the metabolism of intestinal microbiota or cells. The aim of the present study was to investigate and compare mulberrofuran Y (1), moracin C (2), and mulberrofuran G (3) in an in vitro model of human intestinal bacterial fermentation and in an epithelial model using the Caco-2 cell line. The analysis of compounds by LC-MS-Q-TOF showed sufficient stability in the fermentation model, with no bacterial metabolites detected. However, great differences in the quantity of permeation were observed in the permeability assay. Moreover, mulberrofuran Y (1) and moracin C (2) were observed to be transformed into polar metabolites by conjugation. Among the test compounds, mulberrofuran Y (1) was mostly stable and accumulated in endothelial cells (85.3%) compared with mulberrofuran G (3) and moracin C (2) (14% and 8.2%, respectively). Thus, only a small amount of mulberrofuran Y (1) was conjugated. Moracin C (2) and mulberrofuran G (3) were metabolized almost completely, with only traces of the unchanged molecule being found on the apical and cellular sides of the system. Only conjugates of mulberrofuran Y (1) and moracin C (2) were able to reach the basolateral side. Our results provide the basic description of bioavailability of these three compounds, which is a necessary characteristic for final evaluation of bio-efficacy.

1186. Increased Respiratory Syncytial Virus (RSV) Viral Replication Leads to Increased Cytokine Production and Polarized Interferon Response in Infant Mucosal Epithelium

Background RSV is the most frequent etiology of pediatric lower respiratory tract infection. Most children hospitalized for RSV are previously healthy without known risk factors. Children with mild disease managed as outpatients (OP) have higher viral loads than those hospitalized with severe disease. OP children have higher concentrations of the mucosal interferon (IFN), IFNλ2/3, and IP-10, but no differences in IFNλ1. We examined how RSV replication impacts cytokine production kinetics in the nasal mucosa. Methods Primary infant human nasal epithelial (iHNE) cells were collected from nasopharyngeal swabs and cultured on an air-liquid interface. Cultures were infected with 0.1 or 0.001 multiplicity of infection (MOI) of RSV-A, or mock infected. Concentrations of IFN-related (IFNα2, β, γ, λ1, λ2/3, and IP-10) and inflammatory (IL-1β, -6, -12, and TNFα) cytokines secreted to the apical and basolateral surfaces were quantified via immunoassay. Kinetics according to viral inocula were compared by ANOVA with Dunn post-hoc testing of the area under the curve (AUC) for each cytokine. Peak concentrations were compared according to MOI and secretion surface by 2-way ANOVA. Results AUC of IFNs in both surfaces of RSV infected cells were significantly higher than those of mock infected. The 0.1 MOI RSV inoculum resulted in significantly higher AUCs for all IFN cytokines on both surfaces than the 0.001 MOI. Peak IFNλ1 concentrations were higher on the apical than basolateral side; peak IFNλ2/3 concentrations were higher on the basolateral side than apical. AUCs of inflammatory cytokines in RSV infection were significantly higher on the basolateral, but not apical, surfaces than mock; all basolateral inflammatory cytokines were higher in the 0.1 MOI than the 0.001 MOI. Conclusion Higher RSV inoculum induces higher concentrations of IFN-related cytokines on both sides of epithelial cells, and higher concentrations of inflammatory cytokines on the basolateral side. Differential secretion of IFNλ1 and IFNλ2/3 to the apical and basolateral surfaces suggests they may play different roles in immune response during RSV infection. These data support viral replication as an important factor influencing RSV pathogenesis and severity through cytokine production. Disclosures Asuncion Mejias, MD, PhD, MsCS, Janssen (Grant/Research Support, Advisor or Review Panel member)Merck (Grant/Research Support, Advisor or Review Panel member)Roche (Advisor or Review Panel member)Sanofi (Advisor or Review Panel member) Octavio Ramilo, MD, Adagio (Consultant)Bill & Melinda Gates Foundation (Grant/Research Support)Janssen (Grant/Research Support)Lilly (Consultant)Merck (Consultant, Grant/Research Support)NIH (Grant/Research Support)Pfizer (Consultant)SANOFI (Board Member)

Parechovirus A Infection of the Intestinal Epithelium: Differences Between Genotypes A1 and A3

Human parechovirus (PeV-A), one of the species within the Picornaviridae family, is known to cause disease in humans. The most commonly detected genotypes are PeV-A1, associated with mild gastrointestinal disease in young children, and PeV-A3, linked to severe disease with neurological symptoms in neonates. As PeV-A are detectable in stool and nasopharyngeal samples, entry is speculated to occur via the respiratory and gastro-intestinal routes. In this study, we characterized PeV-A1 and PeV-A3 replication and tropism in the intestinal epithelium using a primary 2D model based on human fetal enteroids. This model was permissive to infection with lab-adapted strains and clinical isolates of PeV-A1, but for PeV-A3, infection could only be established with clinical isolates. Replication was highest with infection established from the basolateral side with apical shedding for both genotypes. Compared to PeV-A1, replication kinetics of PeV-A3 were slower. Interestingly, there was a difference in cell tropism with PeV-A1 infecting both Paneth cells and enterocytes, while PeV-A3 infected mainly goblet cells. This difference in cell tropism may explain the difference in replication kinetics and associated disease in humans.

Bacteriophage EK99P-1 Alleviates Enterotoxigenic Escherichia Coli K99-Induced Barrier Dysfunction and Inflammation

Bacteriophages have long been used as a potential alternative to antibiotics for livestock due to their ability to specifically kill ETEC, which are a major cause of diarrhea in piglets. However, the control of ETEC infection by phages within intestinal epithelial cells, and their relationship with host immune responses, remain poorly understood. In this study, we evaluated the effect of phage EK99P-1 against ETEC K99-infected IPEC-J2. Phage EK99P-1 prevented ETEC K99-induced barrier disruption by attenuating the increased permeability mediated by the loss of tight junction proteins such as ZO-1, occludin, and claudin-3. ETEC K99-induced inflammatory responses, such as IL-8 secretion, were decreased by treatment with phage EK99P-1. We used a IPEC-J2/PBMC Transwell co-culture system to investigate whether the modulation of barrier disruption and chemokine secretion by phage EK99P-1 in ETEC K99-infected IPEC-J2 would influence basolateral immune cells. The results showed that phage EK99P-1 reduced the mRNA expression of ETEC K99-induced pro-inflammatory cytokines, IL-1β and IL-8, from PBMC collected on the basolateral side. Together, these results suggest that phage EK99P-1 prevented ETEC K99-induced barrier dysfunction in IPEC-J2 and alleviated inflammation caused by ETEC K99 infection. Reinforcement of the intestinal barrier by phage EK99P-1 also modulates the immune cell inflammatory response.

Capsaicin Attenuates Lipopolysaccharide-Induced Inflammation and Barrier Dysfunction in Intestinal Porcine Epithelial Cell Line-J2

Capsaicin is a spicy, highly pungent, colorless, vanilloid compound found in chili peppers with anti-inflammatory, antioxidant, anti-cancer, and analgesic properties. However, the protective effects of capsaicin on the pig intestine during inflammation are yet to be explored. This study investigated the effects of capsaicin on the gut inflammatory response, intestinal epithelial integrity, and gene expression level of nutrient transporters in a model of lipopolysaccharide (LPS)-induced inflammation in non-differentiated intestinal porcine epithelial cell line-J2 (IPEC-J2). The results showed that the pre-treatment of cells with capsaicin (100 μM) significantly decreased the gene expression and secretion of proinflammatory cytokines induced by LPS through Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. In addition, pre-treatment of cells with capsaicin also increased both gene and protein abundance of tight junction proteins. Furthermore, pre-treatment cells with capsaicin significantly increased trans-epithelial electrical resistance (TEER) and decreased permeability of fluorescein isothiocyanate-dextran (FD4) from the apical side to the basolateral side compared with the control (P < 0.05). Additionally, pre-treatment of cells with capsaicin upregulated the mRNA abundance of nutrients transporters such as Na+/glucose cotransporter 1 (SGLT1). These results suggested that capsaicin could attenuate LPS-induced inflammation response through TLR4/NF-κB pathway and improve barrier integrity and glucose absorption.

Bacteriophage EK99P-1 Alleviates Enterotoxigenic Escherichia Coli K99-Induced Barrier Dysfunction and Inflammation

Background Bacteriophages have long been used as a potential alternative to antibiotics for livestock due to their ability to specifically kill enterotoxigenic Escherichia coli (ETEC), which are a major cause of diarrhea in piglets. However, the control of ETEC infection by phages within intestinal epithelial cells, and their relationship with host immune responses, remain poorly understood. Results In this study, we evaluated the effect of phage EK99P-1 against ETEC K99-infected porcine intestinal epithelial cell line (IPEC-J2). Phage EK99P-1 prevented ETEC K99-induced barrier disruption by attenuating the increased permeability mediated by the loss of tight junction proteins such as zonula occludens-1 (ZO-1), occludin, and claudin-3. ETEC K99-induced inflammatory responses, such as IL-8 secretion, were decreased by treatment with phage EK99P-1. We used a IPEC-J2/peripheral blood mononuclear cell (PBMC) Transwell co-culture system to investigate whether the modulation of barrier disruption and chemokine secretion by phage EK99P-1 in ETEC K99-infected IPEC-J2 would influence basolateral immune cells. The results showed that phage EK99P-1 reduced the mRNA expression of ETEC K99-induced pro-inflammatory cytokines, interleukin (IL)-1β and IL-8, from pPBMC collected on the basolateral side. Conclusion Together, these results suggest that phage EK99P-1 prevented ETEC K99-induced barrier dysfunction in IPEC-J2 and alleviated inflammation caused by ETEC K99 infection. Reinforcement of the intestinal barrier by phage EK99P-1 also modulates the immune cell inflammatory response.

Oral intake of rice overexpressing ubiquitin ligase inhibitory pentapeptide prevents atrophy in denervated skeletal muscle

We previously reported that intramuscular injections of ubiquitin ligase CBLB inhibitory pentapeptide (Cblin; Asp-Gly-pTyr-Met-Pro) restored lost muscle mass caused by sciatic denervation. Here, we detected Cblin on the basolateral side of Caco-2 cells after being placed on the apical side, and found that cytochalasin D, a tight junction opener, enhanced Cblin transport. Orally administered Cblin was found in rat plasma, indicating that intact Cblin was absorbed in vitro and in vivo. Furthermore, transgenic Cblin peptide-enriched rice (CbR) prevented the denervation-induced loss of muscle mass and the upregulation of muscle atrophy-related ubiquitin ligases in mice. These findings indicated that CbR could serve as an alternative treatment for muscle atrophy.

Effects of Absorption Kinetics on the Catabolism of Melatonin Released from CAP-Coated Mesoporous Silica Drug Delivery Vehicles

Melatonin (MLT) is a pineal hormone involved in the regulation of the sleep/wake cycle. The efficacy of exogenous MLT for the treatment of circadian and sleep disorders is variable due to a strong liver metabolism effect. In this work, MLT is encapsulated in mesoporous silica (AMS-6) with a loading capacity of 28.8 wt%, and the mesopores are blocked using a coating of cellulose acetate phthalate (CAP) at 1:1 and 1:2 AMS-6/MLT:CAP ratios. The release kinetics of MLT from the formulations is studied in simulated gastrointestinal fluids. The permeability of the MLT released from the formulations and its 6-hydroxylation are studied in an in vitro model of the intestinal tract (Caco-2 cells monolayer). The release of MLT from AMS-6/MLT:CAP 1:2 is significantly delayed in acidic environments up to 40 min, while remaining unaffected in neutral environments. The presence of CAP decreases the absorption of melatonin and increases its catabolism into 6-hydroxylation by the cytochrome P450 enzyme CYP1A2. The simple confinement of melatonin into AMS-6 pores slightly affects the permeability and significantly decreases melatonin 6-hydroxylation. Measurable amounts of silicon in the basolateral side of the Caco-2 cell monolayer might suggest the dissolution of AMS-6 during the experiment.