A dynamic model of the intestinal epithelium integrates multiple sources of preclinical data and enables clinical translation of drug-induced toxicity.

We have built a quantitative systems toxicology modeling framework focused on the early prediction of oncotherapeutic-induced clinical intestinal adverse effects. The model describes stem and progenitor cell dynamics in the small intestinal epithelium and integrates heterogeneous epithelial-related processes, such as transcriptional profiles, citrulline kinetics, and probability of diarrhea. We fitted a mouse-specific version of the model to quantify doxorubicin and 5-fluorouracil (5-FU)-induced toxicity, which included pharmacokinetics and 5-FU metabolism and assumed that both drugs led to cell cycle arrest and apoptosis in stem cells and proliferative progenitors. The model successfully recapitulated observations in mice regarding dose-dependent disruption of proliferation which could lead to villus shortening, decrease of circulating citrulline, increased diarrhea risk, and transcriptional induction of the p53 pathway. Using a human-specific epithelial model, we translated the cytotoxic activity of doxorubicin and 5-FU quantified in mice into human intestinal injury and predicted with accuracy clinical diarrhea incidence. However, for gefitinib, a specific-molecularly targeted therapy, the mice failed to reproduce epithelial toxicity at exposures much higher than those associated with clinical diarrhea. This indicates that, regardless of the translational modeling approach, preclinical experimental settings have to be suitable to quantify drug-induced clinical toxicity with precision at the structural scale of the model. Our work demonstrates the usefulness of translational models at early stages of the drug development pipeline to predict clinical toxicity and highlights the importance of understanding cross-settings differences in toxicity when building these approaches.

C1GalT1 expression reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression.

Although most cell membrane proteins are modified by glycosylation, our understanding of the role and actions of protein glycosylation is still very limited. ß1,3galactosyltransferase (C1GalT1) is a key glycosyltransferase that controls the biosynthesis of the Core 1 structure of O-linked mucin type glycans and is overexpressed by many common types of epithelial cancers. This study reports that suppression of C1GalT1 expression in human colon cancer cells caused substantial changes of protein glycosylation of cell membrane proteins, many of which were ligands of the galactoside-binding galectin-3 and the macrophage galactose-type lectin (MGL). This led to significant reduction of cancer cell proliferation, adhesion, migration and the ability of tumour cells to form colonies. Crucially, C1GalT1 suppression significantly reduced galectin-3-mediated tumour cell-cell interaction and galectin-3-promoted tumour cell activities. In the meantime, C1GalT1 suppression substantially increased MGL-mediated macrophage-tumour cell interaction and macrophage-tumour cell phagocytosis and cytokine secretion. C1GalT1-expressing cancer cells implanted in chick embryos resulted in the formation of significantly bigger tumours than C1GalT1-suppressed cells and the presence of galectin-3 increased tumour growth of C1GalT1-expressing but not C1GalT1-suppressed cells. More MGL-expressing macrophages and dendritic cells were seen to be attracted to the tumour microenvironment in ME C1galt1-/-/Erb mice than in C1galt1f/f /Erb mice. These results indicate that expression of C1GalT1 by tumour cells reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression. C1GalT1 overexpression in epithelial cancers therefore may represent a fundamental mechanism in cancer promotion and in reduction of immune response/surveillance in cancer progression.

A novel in vitro model of the small intestinal epithelium in co-culture with 'gut-like' dendritic cells.

Cross-talk between dendritic cells (DCs) and the intestinal epithelium is important in the decision to mount a protective immune response to a pathogen or to regulate potentially damaging responses to food antigens and the microbiota. Failures in this decision-making process contribute to the development of intestinal inflammation, making the molecular signals that pass between DCs and intestinal epithelial cells potential therapeutic targets. Until now, in vitro models with sufficient complexity to understand these interactions have been lacking. Here, we outline the development of a co-culture model of in vitro differentiated 'gut-like' DCs with small intestinal organoids (enteroids). Sequential exposure of murine bone marrow progenitors to Flt3L, granulocyte macrophage colony-stimulating factor (GM-CSF) and all-trans-retinoic acid (RA) resulted in the generation of a distinct population of conventional DCs expressing CD11b+SIRPα+CD103+/- (cDC2) exhibiting retinaldehyde dehydrogenase (RALDH) activity. These 'gut-like' DCs extended transepithelial dendrites across the intact epithelium of enteroids. 'Gut-like' DC in co-culture with enteroids can be utilized to define how epithelial cells and cDCs communicate in the intestine under a variety of different physiological conditions, including exposure to different nutrients, natural products, components of the microbiota, or pathogens. Surprisingly, we found that co-culture with enteroids resulted in a loss of RALDH activity in 'gut-like' DCs. Continued provision of GM-CSF and RA during co-culture was required to oppose putative negative signals from the enteroid epithelium. Our data contribute to a growing understanding of how intestinal cDCs assess environmental conditions to ensure appropriate activation of the immune response.

Interleukin-10 Deficiency Impacts on TNF-Induced NFκB Regulated Responses In Vivo.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that has a major protective role against intestinal inflammation. We recently revealed that intestinal epithelial cells in vitro regulate NFκB-driven transcriptional responses to TNF via an autocrine mechanism dependent on IL-10 secretion. Here in this study, we investigated the impact of IL-10 deficiency on the NFκB pathway and its downstream targets in the small intestinal mucosa in vivo. We observed dysregulation of TNF, IκBα, and A20 gene and protein expression in the small intestine of steady-state or TNF-injected Il10-/- mice, compared to wild-type C57BL6/J counterparts. Upon TNF injection, tissue from the small intestine showed upregulation of NFκB p65[RelA] activity, which was totally diminished in Il10-/- mice and correlated with reduced levels of TNF, IκBα, and A20 expression. In serum, whilst IgA levels were noted to be markedly downregulated in IL-10-deficient- mice, normal levels of mucosal IgA were seen in intestine mucosa. Importantly, dysregulated cytokine/chemokine levels were observed in both serum and intestinal tissue lysates from naïve, as well as TNF-injected Il10-/- mice. These data further support the importance of the IL-10-canonical NFκB signaling pathway axis in regulating intestinal mucosa homeostasis and response to inflammatory triggers in vivo.

Nfkb2 deficiency and its impact on plasma cells and immunoglobulin expression in murine small intestinal mucosa.

The alternative (noncanonical) nuclear factor-κB (NF-κB) signaling pathway predominantly regulates the function of the p52/RelB heterodimer. Germline Nfkb2 deficiency in mice leads to loss of p100/p52 protein and offers protection against a variety of gastrointestinal conditions, including azoxymethane/dextran sulfate sodium (DSS)-induced colitis-associated cancer and lipopolysaccharide (LPS)-induced small intestinal epithelial apoptosis. However, the common underlying protective mechanisms have not yet been fully elucidated. We applied high-throughput RNA-Seq and proteomic analyses to characterize the transcriptional and protein signatures of the small intestinal mucosa of naïve adult Nfkb2-/- mice. Those data were validated by immunohistochemistry and quantitative ELISA using both small intestinal tissue lysates and serum. We identified a B-lymphocyte defect as a major transcriptional signature in the small intestinal mucosa and immunoglobulin A as the most downregulated protein by proteomic analysis in Nfkb2-/- mice. Small intestinal immunoglobulins were dramatically dysregulated, with undetectable levels of immunoglobulin A and greatly increased amounts of immunoglobulin M being detected. The numbers of IgA-producing, cluster of differentiation (CD)138-positive plasma cells were also reduced in the lamina propria of the small intestinal villi of Nfkb2-/- mice. This phenotype was even more striking in the small intestinal mucosa of RelB-/- mice, although these mice were equally sensitive to LPS-induced intestinal apoptosis as their RelB+/+ wild-type counterparts. NF-κB2/p52 deficiency confers resistance to LPS-induced small intestinal apoptosis and also appears to regulate the plasma cell population and immunoglobulin levels within the gut.NEW & NOTEWORTHY Novel transcriptomic analysis of murine proximal intestinal mucosa revealed an unexpected B cell signature in Nfkb2-/- mice. In-depth analysis revealed a defect in the CD38+ B cell population and a gut-specific dysregulation of immunoglobulin levels.

Galectin-2 in Health and Diseases.

Galectin-2 is a prototype member of the galactoside-binding galectin family. It is predominately expressed in the gastrointestinal tract but is also detected in several other tissues such as the placenta and in the cardiovascular system. Galectin-2 expression and secretion by epithelial cells has been reported to contribute to the strength of the mucus layer, protect the integrity of epithelia. A number of studies have also suggested the involvement of galectin-2 in tissue inflammation, immune response and cell apoptosis. Alteration of galectin-2 expression occurs in inflammatory bowel disease, coronary artery diseases, rheumatoid arthritis, cancer, and pregnancy disorders and has been shown to be involved in disease pathogenesis. This review discusses our current understanding of the role and actions of galectin-2 in regulation of these pathophysiological conditions.

Soluble Non-Starch Polysaccharides From Plantain (Musa x paradisiaca L.) Diminish Epithelial Impact of Clostridioides difficile.

Clostridioides difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea. Adhesion of this Gram-positive pathogen to the intestinal epithelium is a crucial step in CDI, with recurrence and relapse of disease dependent on epithelial interaction of its endospores. Close proximity, or adhesion of, hypervirulent strains to the intestinal mucosa are also likely to be necessary for the release of C. difficile toxins, which when internalized, result in intestinal epithelial cell rounding, damage, inflammation, loss of barrier function and diarrhoea. Interrupting these C. difficile-epithelium interactions could therefore represent a promising therapeutic strategy to prevent and treat CDI. Intake of dietary fibre is widely recognised as being beneficial for intestinal health, and we have previously shown that soluble non-starch polysaccharides (NSP) from plantain banana (Musa spp.), can block epithelial adhesion and invasion of a number of gut pathogens, such as E. coli and Salmonellae. Here, we assessed the action of plantain NSP, and a range of alternative soluble plant fibres, for inhibitory action on epithelial interactions of C. difficile clinical isolates, purified endospore preparations and toxins. We found that plantain NSP possessed ability to disrupt epithelial adhesion of C. difficile vegetative cells and spores, with inhibitory activity against C. difficile found within the acidic (pectin-rich) polysaccharide component, through interaction with the intestinal epithelium. Similar activity was found with NSP purified from broccoli and leek, although seen to be less potent than NSP from plantain. Whilst plantain NSP could not block the interaction and intracellular action of purified C. difficile toxins, it significantly diminished the epithelial impact of C. difficile, reducing both bacteria and toxin induced inflammation, activation of caspase 3/7 and cytotoxicity in human intestinal cell-line and murine intestinal organoid cultures. Dietary supplementation with soluble NSP from plantain may therefore confer a protective effect in CDI patients by preventing adhesion of C. difficile to the mucosa, i.e. a "contrabiotic" effect, and diminishing its epithelial impact. This suggests that plantain soluble dietary fibre may be a therapeutically effective nutritional product for use in the prevention or treatment of CDI and antibiotic-associated diarrhoea.

Identifying key regulators of the intestinal stem cell niche.

The intestinal tract is lined by a single layer of epithelium that is one of the fastest regenerating tissues in the body and which therefore requires a very active and exquisitely controlled stem cell population. Rapid renewal of the epithelium is necessary to provide a continuous physical barrier from the intestinal luminal microenvironment that contains abundant microorganisms, whilst also ensuring an efficient surface for the absorption of dietary components. Specialised epithelial cell populations are important for the maintenance of intestinal homeostasis and are derived from adult intestinal stem cells (ISCs). Actively cycling ISCs divide by a neutral drift mechanism yielding either ISCs or transit-amplifying epithelial cells, the latter of which differentiate to become either absorptive lineages or to produce secretory factors that contribute further to intestinal barrier maintenance or signal to other cellular compartments. The mechanisms controlling ISC abundance, longevity and activity are regulated by several different cell populations and signalling pathways in the intestinal lamina propria which together form the ISC niche. However, the complexity of the ISC niche and communication mechanisms between its different components are only now starting to be unravelled with the assistance of intestinal organoid/enteroid/colonoid and single-cell imaging and sequencing technologies. This review explores the interaction between well-established and emerging ISC niche components, their impact on the intestinal epithelium in health and in the context of intestinal injury and highlights future directions and implications for this rapidly developing field.

Appearance of peanut agglutinin in the blood circulation after peanut ingestion promotes endothelial secretion of metastasis-promoting cytokines.

Peanut agglutinin (PNA) is a carbohydrate-binding protein in peanuts that accounts for ~0.15% peanut weight. PNA is highly resistant to cooking and digestion and is rapidly detectable in the blood after peanut consumption. Our previous studies have shown that circulating PNA mimics the actions of endogenous galactoside-binding protein galectin-3 by interaction with tumour cell-associated MUC1 and promotes circulating tumour cell metastatic spreading. The present study shows that circulating PNA interacts with micro- as well as macro-vascular endothelial cells and induces endothelial secretion of cytokines MCP-1 (CCL2) and IL-6 in vitro and in vivo. The increased secretion of these cytokines autocrinely/paracrinely enhances the expression of endothelial cell surface adhesion molecules including integrins, VCAM and selectin, leading to increased tumour cell-endothelial adhesion and endothelial tubule formation. Binding of PNA to endothelial surface MCAM (CD146), via N-linked glycans, and subsequent activation of PI3K-AKT-PREAS40 signalling is here shown responsible for PNA-induced secretion of MCP-1 and IL-6 by vascular endothelium. Thus, in addition to its influence on promoting tumour cell spreading by interaction with tumour cell-associated MUC1, circulating PNA might also influence metastasis by enhancing the secretion of metastasis-promoting MCP-1 and IL-6 from the vascular endothelium.

Long-Term Iron Deficiency and Dietary Iron Excess Exacerbate Acute Dextran Sodium Sulphate-Induced Colitis and Are Associated with Significant Dysbiosis.

BACKGROUND: Oral iron supplementation causes gastrointestinal side effects. Short-term alterations in dietary iron exacerbate inflammation and alter the gut microbiota, in murine models of colitis. Patients typically take supplements for months. We investigated the impact of long-term changes in dietary iron on colitis and the microbiome in mice. METHODS: We fed mice chow containing differing levels of iron, reflecting deficient (100 ppm), normal (200 ppm), and supplemented (400 ppm) intake for up to 9 weeks, both in absence and presence of dextran sodium sulphate (DSS)-induced chronic colitis. We also induced acute colitis in mice taking these diets for 8 weeks. Impact was assessed (i) clinically and histologically, and (ii) by sequencing the V4 region of 16S rRNA. RESULTS: In mice with long-term changes, the iron-deficient diet was associated with greater weight loss and histological inflammation in the acute colitis model. Chronic colitis was not influenced by altering dietary iron however there was a change in the microbiome in DSS-treated mice consuming 100 ppm and 400 ppm iron diets, and control mice consuming the 400 ppm iron diet. Proteobacteria levels increased significantly, and Bacteroidetes levels decreased, in the 400 ppm iron DSS group at day-63 compared to baseline. CONCLUSIONS: Long-term dietary iron alterations affect gut microbiota signatures but do not exacerbate chronic colitis, however acute colitis is exacerbated by such dietary changes. More work is needed to understand the impact of iron supplementation on IBD. The change in the microbiome, in patients with colitis, may arise from the increased luminal iron and not simply from colitis.

Canthin-6-one ameliorates TNBS-induced colitis in rats by modulating inflammation and oxidative stress. An in vivo and in silico approach.

Canthin-6-one (Cant) is an indole alkaloid found in several botanical drugs used as medicines, reported to be gastroprotective, anti-inflammatory, anti-microbial, anti-diarrheal and anti-proliferative. We aimed to explore Cant in the management of colitis using a trinitrobenzenesulfonic acid (TNBS)-induced rat model. Cant (1, 5 and 25 mg/kg) was administered by oral gavage to Wistar rats followed by induction of colitis with TNBS. Macroscopic and histopathological scores, myeloperoxidase (MPO), malondialdehyde (MDA) and reduced glutathione (GSH) were assessed in colon tissues. Pro- (TNF-α, IL-1ß and IL-12p70) and anti-inflammatory (IL-10) cytokines, and vascular endothelial growth factor (VEGF) were also quantified. Mitogen-activated protein kinase 14 (MAPK14) and Toll-like receptor-8 (TLR8), as putative targets, were considered through in silico analysis. Cant (5 and 25 mg/kg) reduced macroscopic and histological colon damage scores in TNBS-treated rats. MPO and MDA were reduced by up to 61.69% and 92.45%, respectively, compared to TNBS-treated rats alone. Glutathione concentration was reduced in rats administered with TNBS alone (50.00% of sham group) but restored to 72.73% (of sham group) with Cant treatment. TNF-α, IL-1ß, IL-12p70 and VEGF were reduced, and anti-inflammatory IL-10 was increased following Cant administration compared to rats administered TNBS alone. Docking ligation results for MAPK14 (p38α) and TLR8 with Cant, confirmed that these proteins are feasible putative targets. Cant has an anti-inflammatory effect in the intestine by down-regulating molecular immune mediators and decreasing oxidative stress. Therefore, Cant could have therapeutic potential for the treatment of inflammatory bowel disease and related syndromes.

A "Watch and Wait" Strategy Involving Regular Endoscopic Surveillance Is Safe for Many Patients with Small, Sporadic, Grade 1, Non-Ampullary, Non-Functioning Duodenal Neuroendocrine Tumours.

INTRODUCTION: Duodenal neuroendocrine tumours (d-NETs) are rare but are increasing in incidence. Current ENETS guidelines advocate resection of all localized d-NETs. However, "watch and wait" may be appropriate for some localized, small, grade 1, non-functioning, non-ampullary d-NETs. We evaluated whether patients with such d-NETs who chose "watch and wait" involving regular endoscopic surveillance had equivalent disease-related outcomes to patients undergoing endoscopic or surgical resection. METHODS: Retrospective review of patients with histologically confirmed d-NETs at Liverpool ENETS Centre of Excellence 2007-2020. RESULTS: Sixty-nine patients were diagnosed with d-NET of which 50 were sporadic, non-functioning, non-ampullary tumours. Patient treatment groups were similar in terms of age, gender, and tumour location and grade, but unsurprisingly, larger tumours (median diameter 17 mm [p < 0.0001]) were found in the surgically treated group. Five patients underwent surgical resection with no evidence of tumour recurrence or disease-related death. Twelve patients underwent endoscopic resection (ER), with 1 local recurrence detected during follow-up. Thirty patients (28 with d-NETs ≤10 mm) underwent "watch and wait" with resection only if tumours increased in size. The d-NETs in 28/30 patients remained stable or decreased in size over a median 27 months (IQR: 15-48, R: 3-98). In 7 patients, the d-NET was completely removed by avulsion during diagnostic biopsy and was not seen at subsequent endoscopies. Only 2 patients showed increased d-NET size during surveillance, of whom only one was fit for ER. No NET-related deaths were documented during follow-up. CONCLUSIONS: All of the localized, ≤10 mm, grade 1, non-functioning, non-ampullary d-NETs in this cohort behaved indolently with very low risks of progression and no tumour-related deaths. "Watch and wait," therefore, appears to be a safe alternative management strategy for selected d-NETs.

Dilodendron bipinnatum Radlk. extract alleviates ulcerative colitis induced by TNBS in rats by reducing inflammatory cell infiltration, TNF-α and IL-1ß concentrations, IL-17 and COX-2 expressions, supporting mucus production and promotes an antioxidant effect.

ETHNOPHARMACOLOGICAL RELEVANCE: Dilodendron bipinnatum (Sapindaceae) stem bark decoction and macerate were used to treat uterine inflammation, pain in general, dermatitis and bone fractures. These homemade preparations also have diuretic, stimulant, expectorants and sedative effects and are effective in treating worm infections in the Brazilian Pantanal population. Our previous research confirmed the anti-inflammatory activity of the hydroethanolic extract of inner stem bark of D. bipinnatum (HEDb). AIM: This work aimed to investigate the efficacy of HEDb in ameliorating experimental colitis in rats and to elucidate the possible mechanisms involved in the anti-ulcerative colitis properties of HEDb in rats and Caco-2 cell line. MATERIALS AND METHODS: The effects on cell viability, IL-8 and TNF-α in human colon adenocarcinoma (Caco-2) were determined by flow cytometer and ELISA. Wistar rats (n = 6-7) were orally gavaged with, vehicle (0.9% saline), HEDb at doses of 20, 100 or 500 mg/kg, or mesalazine at a dose of 500 mg/kg, at 48, 24 and 1 h prior to the administration of trinitrobenzene sulfonic acid via rectal administration to induce colitis. The anti-inflammatory effects of HEDb were assessed macroscopically, by myeloperoxidase (MPO) activity and for glutathione (GSH) concentration in the colon. Additionally, colonic histopathological analyses of UC severity were conducted by different staining methods (H&E, PAS and toluidine blue). Pro-inflammatory cytokines TNF-α and IL-1ß were quantified in colonic tissue by ELISA and colonic expressions of COX-2 and IL-17 were analyzed by western blotting. RESULTS: HEDb was shown to be non-cytotoxic with mean viability of 80% in Caco-2 cells. HEDb pre-treatments of 1, 5 or 20 µg/mL significantly reduced TNF-α production in Caco-2 cells by 21.8% (p < 0.05), 60.5 and 82.1% (p < 0.001) respectively following LPS treatment compared to LPS alone. However, no change in IL-8 production was observed. HEDb pre-treatment of rats subjected to TNBS significantly (p < 0.001) reduced colonic lesion score. Higher doses (100 and 500 mg/kg) caused a sharp downregulation of haemorrhagic damage, leukocyte infiltration, edema and restoration of mucus production. Moreover, mast cell degranulation was inhibited. Colonic MPO activity was reduced following all doses of HEDb, reaching 51.1% ± 1.51 (p < 0.05) with the highest dose. GSH concentration was restored by 58% and 70% following 100 and 500 mg/kg of HEDb, respectively. The oral treatment of HEDb at doses 20, 100 and 500 mg/kg decreased the concentrations of TNF-α and IL-1ß at all doses in comparison to vehicle treated control. In addition, HEDb inhibited the COX-2 and IL-17 expressions with maximal effect at 500 mg/kg (60.3% and 65% respectively; p < 0.001). In all trials, the effect of HEDb at all doses being 20, 100 and 500 mg/kg was statistically comparable to mesalazine (500 mg/kg). CONCLUSIONS: HEDb reduces colonic damage in the TNBS colitis model and relieves oxidative and inflammatory events, at least in part, by increasing mucus production, reducing leukocyte migration and reducing TNF-α (in vivo and in vitro), IL-1ß, IL-17 and COX-2 expression. Therefore, HEDb requires further investigation as a candidate for treating IBD.

Using systems medicine to identify a therapeutic agent with potential for repurposing in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) cause significant morbidity and mortality. Aberrant NF-κB signalling is strongly associated with these conditions, and several established drugs influence the NF-κB signalling network to exert their effect. This study aimed to identify drugs that alter NF-κB signalling and could be repositioned for use in IBD. The SysmedIBD Consortium established a novel drug-repurposing pipeline based on a combination of in silico drug discovery and biological assays targeted at demonstrating an impact on NF-κB signalling, and a murine model of IBD. The drug discovery algorithm identified several drugs already established in IBD, including corticosteroids. The highest-ranked drug was the macrolide antibiotic clarithromycin, which has previously been reported to have anti-inflammatory effects in aseptic conditions. The effects of clarithromycin effects were validated in several experiments: it influenced NF-κB-mediated transcription in murine peritoneal macrophages and intestinal enteroids; it suppressed NF-κB protein shuttling in murine reporter enteroids; it suppressed NF-κB (p65) DNA binding in the small intestine of mice exposed to lipopolysaccharide; and it reduced the severity of dextran sulphate sodium-induced colitis in C57BL/6 mice. Clarithromycin also suppressed NF-κB (p65) nuclear translocation in human intestinal enteroids. These findings demonstrate that in silico drug repositioning algorithms can viably be allied to laboratory validation assays in the context of IBD, and that further clinical assessment of clarithromycin in the management of IBD is required.This article has an associated First Person interview with the joint first authors of the paper.

Netazepide Inhibits Expression of Pappalysin 2 in Type 1 Gastric Neuroendocrine Tumors.

BACKGROUND & AIMS: In patients with autoimmune atrophic gastritis and achlorhydria, hypergastrinemia is associated with the development of type 1 gastric neuroendocrine tumors (gNETs). Twelve months of treatment with netazepide (YF476), an antagonist of the cholecystokinin B receptor (CCKBR or CCK2R), eradicated some type 1 gNETs in patients. We investigated the mechanisms by which netazepide induced gNET regression using gene expression profiling. METHODS: We obtained serum samples and gastric corpus biopsy specimens from 8 patients with hypergastrinemia and type 1 gNETs enrolled in a phase 2 trial of netazepide. Control samples were obtained from 10 patients without gastric cancer. We used amplified and biotinylated sense-strand DNA targets from total RNA and Affymetrix (Thermofisher Scientific, UK) Human Gene 2.0 ST microarrays to identify differentially expressed genes in stomach tissues from patients with type 1 gNETs before, during, and after netazepide treatment. Findings were validated in a human AGSGR gastric adenocarcinoma cell line that stably expresses human CCK2R, primary mouse gastroids, transgenic hypergastrinemic INS-GAS mice, and patient samples. RESULTS: Levels of pappalysin 2 (PAPPA2) messenger RNA were reduced significantly in gNET tissues from patients receiving netazepide therapy compared with tissues collected before therapy. PAPPA2 is a metalloproteinase that increases the bioavailability of insulin-like growth factor (IGF) by cleaving IGF binding proteins (IGFBPs). PAPPA2 expression was increased in the gastric corpus of patients with type 1 gNETs, and immunohistochemistry showed localization in the same vicinity as CCK2R-expressing enterochromaffin-like cells. Up-regulation of PAPPA2 also was found in the stomachs of INS-GAS mice. Gastrin increased PAPPA2 expression with time and in a dose-dependent manner in gastric AGSGR cells and mouse gastroids by activating CCK2R. Knockdown of PAPPA2 in AGSGR cells with small interfering RNAs significantly decreased their migratory response and tissue remodeling in response to gastrin. Gastrin altered the expression and cleavage of IGFBP3 and IGFBP5. CONCLUSIONS: In an analysis of human gNETS and mice, we found that gastrin up-regulates the expression of gastric PAPPA2. Increased PAPPA2 alters IGF bioavailability, cell migration, and tissue remodeling, which are involved in type 1 gNET development. These effects are inhibited by netazepide.

NF-κB2 signalling in enteroids modulates enterocyte responses to secreted factors from bone marrow-derived dendritic cells.

Alternative pathway NF-κB signalling regulates susceptibility towards developing inflammatory bowel disease (IBD), colitis-associated cancer and sepsis-associated intestinal epithelial cell apoptosis and shedding. However, the cell populations responsible for the perturbed alternative pathway NF-κB signalling in intestinal mucosal pathology remain unclear. In order to investigate the contribution of the epithelial compartment, we have tested whether NF-κB2 regulated transcription in intestinal epithelial cells controls the intestinal epithelial response to cytokines that are known to disrupt intestinal barrier permeability. Enteroids were generated from the proximal, middle and distal regions of small intestine (SI) from C57BL/6J wild-type mice and displayed region-specific morphology that was maintained during sub-culture. Enteroids treated with 100 ng/mL TNF were compared with corresponding regions of SI from C57BL/6J mice treated systemically with 0.33 mg/kg TNF for 1.5 h. TNF-induced apoptosis in all regions of the intestine in vitro and in vivo but resulted in Paneth cell degranulation only in proximal tissue-derived SI and enteroids. TNF also resulted in increased enteroid sphericity (quantified as circularity from two-dimensional bright field images). This response was dose and time-dependent and correlated with active caspase-3 immunopositivity. Proximal tissue-derived enteroids generated from Nfκb2-/- mice showed a significantly blunted circularity response following the addition of TNF, IFNγ, lipopolysaccharide (LPS) activated C57BL/6J-derived bone marrow-derived dendritic cells (BMDC) and secreted factors from LPS-activated BMDCs. However, Nfκb1-/- mouse-derived enteroids showed no significant changes in response to these stimuli. In conclusion, the selection of SI region is important when designing enteroid studies as region-specific identity and response to stimuli such as TNF are maintained in culture. Intestinal epithelial cells are at least partially responsible for regulating their own fate by modulating NF-κB2 signalling in response to stimuli known to be involved in multiple intestinal and systemic diseases. Future studies are warranted to investigate the therapeutic potential of intestinal epithelial NF-κB2 inhibition.

An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium.

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.

DNA extraction and amplicon production strategies deeply inf luence the outcome of gut mycobiome studies.

Microbial ecology studies are often performed through extraction of metagenomic DNA followed by amplification and sequencing of a marker. It is known that each step may bias the results. These biases have been explored for the study of bacterial communities, but rarely for fungi. Our aim was therefore to evaluate methods for the study of the gut mycobiome. We first evaluated DNA extraction methods in fungal cultures relevant to the gut. Afterwards, to assess how these methods would behave with an actual sample, stool from a donor was spiked with cells from the same cultures. We found that different extraction kits favour some species and bias against others. In terms of amplicon sequencing, we evaluated five primer sets, two for ITS2 and one for ITS1, 18S and 28S rRNA. Results showed that 18S rRNA outperformed the other markers: it was able to amplify all the species in the mock community and to discriminate among them. ITS primers showed both amplification and sequencing biases, the latter related to the variable length of the product. We identified several biases in the characterisation of the gut mycobiome and showed how crucial it is to be aware of these before drawing conclusions from the results of these studies.

Replication of Crohn's Disease Mucosal E. coli Isolates inside Macrophages Correlates with Resistance to Superoxide and Is Dependent on Macrophage NF-kappa B Activation.

Mucosa-associated Escherichia coli are increased in Crohn's disease (CD) and colorectal cancer (CRC). CD isolates replicate within macrophages but the specificity of this effect for CD and its mechanism are unclear. Gentamicin exclusion assay was used to assess E. coli replication within J774.A1 murine macrophages. E. coli growth was assessed following acid, low-nutrient, nitrosative, oxidative and superoxide stress, mimicking the phagolysosome. Twelve of 16 CD E. coli isolates replicated >2-fold within J774.A1 macrophages; likewise for isolates from 6/7 urinary tract infection (UTI), 8/9 from healthy subjects, compared with 2/6 ulcerative colitis, 2/7 colorectal cancer and 0/3 laboratory strains. CD mucosal E. coli were tolerant of acidic, low-nutrient, nitrosative and oxidative stress. Replication within macrophages correlated strongly with tolerance to superoxide stress (rho = 0.44, p = 0.0009). Exemplar CD E. coli HM605 and LF82 were unable to survive within Nfκb1-/- murine bone marrow-derived macrophages. In keeping with this, pre-incubation of macrophages with hydrocortisone (0.6 µM for 24 h) caused 70.49 ± 12.11% inhibition of intra-macrophage replication. Thus, CD mucosal E. coli commonly replicate inside macrophages, but so do some UTI and healthy subject strains. Replication correlates with resistance to superoxide and is highly dependent on macrophage NF-κB signalling. This may therefore be a good therapeutic target.

Stem cell models as an in vitro model for predictive toxicology.

Adverse drug reactions (ADRs) are the unintended side effects of drugs. They are categorised as either predictable or unpredictable drug-induced injury and may be exhibited after a single or prolonged exposure to one or multiple compounds. Historically, toxicology studies rely heavily on animal models to understand and characterise the toxicity of novel compounds. However, animal models are imperfect proxies for human toxicity and there have been several high-profile cases of failure of animal models to predict human toxicity e.g. fialuridine, TGN1412 which highlight the need for improved predictive models of human toxicity. As a result, stem cell-derived models are under investigation as potential models for toxicity during early stages of drug development. Stem cells retain the genotype of the individual from which they were derived, offering the opportunity to model the reproducibility of rare phenotypes in vitro Differentiated 2D stem cell cultures have been investigated as models of hepato- and cardiotoxicity. However, insufficient maturity, particularly in the case of hepatocyte-like cells, means that their widespread use is not currently a feasible method to tackle the complex issues of off-target and often unpredictable toxicity of novel compounds. This review discusses the current state of the art for modelling clinically relevant toxicities, e.g. cardio- and hepatotoxicity, alongside the emerging need for modelling gastrointestinal toxicity and seeks to address whether stem cell technologies are a potential solution to increase the accuracy of ADR predictivity in humans.