Adenylyl cyclase 6 is involved in the hyposecretory status of experimental colitis.

One of the cardinal symptoms of intestinal inflammation is diarrhea. Acute intestinal inflammation is associated with inhibition of ion absorption and increased secretion, along with fluid leakage due to epithelial injury and changes in permeability. However, in the chronic situation, a downregulation of both absorptive and secretory transport has been reported. We investigated how experimental colitis reduces cAMP levels in intestinal epithelial cells through modulation of adenylyl cyclases (AC). Primary colonic epithelial cells obtained from rats with trinitrobenzenesulfonic acid colitis and non-colitic controls were analyzed for AC expression by RT-qPCR and Western blot, following a preliminary microarray analysis. AC6 and AC5 were found to be expressed in colonocytes, and downregulated by inflammation, with the former exhibiting considerably higher mRNA levels in both cases. To test the hypothesis that inflammatory cytokines may account for this effect, Caco 2 cells were treated with IL-1ß, TNF-α, or IFN-γ. All three cytokines inhibited forskolin evoked short-circuit currents in Ussing chambers and lowered intracellular cAMP, but failed to alter AC6 mRNA levels. AC5/AC6 expression was however inhibited in mouse jejunal organoids treated with IFN-γ and TNF-α, but not IL-1ß. Gene knockdown of AC6 resulted in a significant decrease of ion secretion in T84 cells. We conclude that the disturbances in ion secretion observed in rat TNBS colitis are associated with low intracellular levels of cAMP in the epithelium, which may be explained in part by the downregulation of AC5/AC6 expression by proinflammatory cytokines.

The role of molecular remodeling in differential regulation of tight junction permeability.

Tight junctions create a paracellular barrier that is essential for survival of complex organisms. In many cases tight junctions define separate, generally sterile, tissue compartments. In the skin and gut, tight junctions must also seal the paracellular space to prevent microbiota from accessing the internal milieu. This is a relatively simple task in the integument, where an absolute barrier is effective. However, intestinal epithelial tight junctions are charged with the far more complex task of supporting paracellular transport of water, ions, and nutrients while providing a barrier to microbial translocation. The delicate nature of this balance, which is disrupted in disease, makes the intestine a unique organ in which to explore the complexities of tight junction permeability and barrier regulation. Here we review recent progress in understanding the molecular determinants of barrier function and events responsible for regulation, and dysregulation, of tight junction permeability.

Validation of bovine glycomacropeptide as an intestinal anti-inflammatory nutraceutical in the lymphocyte-transfer model of colitis.

Milk κ-casein-derived bovine glycomacropeptide (GMP) exerts immunomodulatory effects. It exhibits intestinal anti-inflammatory activity in chemically induced models of colitis. However, to validate its clinical usefulness as a nutraceutical, it is important to assess its effects in a model with a closer pathophysiological connection with human inflammatory bowel disease. Therefore, in the present study, we used the lymphocyte-transfer model of colitis in mice and compared the effects of GMP in this model with those obtained in the dextran sulphate sodium (DSS) model. GMP (15 mg/d) resulted in higher body-weight gain and a reduction of the colonic damage score and myeloperoxidase (MPO) activity in Rag1(-/-) mice with colitis induced by the transfer of naïve T cells. The colonic and ileal weight:length ratio was decreased by approximately 25%, albeit non-significantly. GMP treatment reduced the percentage of CD4⁺ interferon (IFN)-γ⁺ cells in mesenteric lymph nodes (MLN). The basal production of IL-6 by MLN obtained from the GMP-treated mice ex vivo was augmented. However, concanavalin A-evoked production was similar. The colonic expression of regenerating islet-derived protein 3γ, S100A8, chemokine (C-X-C motif) ligand 1 and IL-1ß was unaffected by GMP, while that of TNF-α and especially IFN-γ was paradoxically increased. In the DSS model, GMP also reduced the activity of colonic MPO, but it failed to alter weight gain or intestinal weight:length ratio. GMP augmented the production of IL-10 by MLN cells and was neutral towards other cytokines, except exhibiting a trend towards increasing the production of IL-6. The lower effect was attributed to the lack of the effect of GMP on epithelial cells. In conclusion, GMP exerts intestinal anti-inflammatory effects in lymphocyte-driven colitis.

Intestinal inflammation and the enterocyte transportome.

Diarrhoea is a hallmark of intestinal inflammation. The mechanisms operating in acute inflammation of the intestine are well characterized and are related to regulatory changes induced by inflammatory mediators such as prostaglandins, cytokines or reactive oxygen species, along with leakage due to epithelial injury and changes in permeability. In chronic colitis, however, the mechanisms are less well known, but it is generally accepted that both secretory and absorptive processes are inhibited. These disturbances in ionic transport may be viewed as an adaptation to protracted inflammation of the intestine, since prolonged intense secretion may be physiologically unacceptable in the long term. Mechanistically, the changes in transport may be due to adjustments in the regulation of the different processes involved, to broader epithelial alterations or frank damage, or to modulation of the transportome in terms of expression. In the present review, we offer a summary of the existing evidence on the status of the transportome in chronic intestinal inflammation.

Better together: circulating tumor cell clustering in metastatic cancer.

Circulating tumor cells (CTCs) are vital components of liquid biopsies for diagnosis of residual cancer, monitoring of therapy response, and prognosis of recurrence. Scientific dogma focuses on metastasis mediated by single CTCs, but advancement of CTC detection technologies has elucidated multicellular CTC clusters, which are associated with unfavorable clinical outcomes and a 20- to 100-fold greater metastatic potential than single CTCs. While the mechanistic understanding of CTC cluster formation is still in its infancy, multiple cell adhesion molecules and tight junction proteins have been identified that underlie the outperforming attributes of homotypic and heterotypic CTC clusters, such as cell survival, cancer stemness, and immune evasion. Future directions include high-resolution characterization of CTCs at multiomic levels for diagnostic/prognostic evaluations and targeted therapies.

Reversible Ponceau staining as a loading control alternative to actin in Western blots.

It is becoming standard practice to measure a housekeeping gene, typically actin, in Western blots, as it is the rule in RNA blots. We have applied reversible Ponceau staining to check equal loading of gels and measured actin in parallel under different conditions. Our results show that densitometric analysis is comparable with both techniques. Therefore, routine quantitation of Ponceau staining before antibody probing is validated as an alternative to actin blotting.

Automated microfluidic platform for dynamic and combinatorial drug screening of tumor organoids.

Three-dimensional (3D) cell culture technologies, such as organoids, are physiologically relevant models for basic and clinical applications. Automated microfluidics offers advantages in high-throughput and precision analysis of cells but is not yet compatible with organoids. Here, we present an automated, high-throughput, microfluidic 3D organoid culture and analysis system to facilitate preclinical research and personalized therapies. Our system provides combinatorial and dynamic drug treatments to hundreds of cultures and enables real-time analysis of organoids. We validate our system by performing individual, combinatorial, and sequential drug screens on human-derived pancreatic tumor organoids. We observe significant differences in the response of individual patient-based organoids to drug treatments and find that temporally-modified drug treatments can be more effective than constant-dose monotherapy or combination therapy in vitro. This integrated platform advances organoids models to screen and mirror real patient treatment courses with potential to facilitate treatment decisions for personalized therapy.

Hippo-YAP1 Is a Prognosis Marker and Potentially Targetable Pathway in Advanced Gallbladder Cancer.

Gallbladder cancer is an aggressive disease with late diagnosis and no efficacious treatment. The Hippo-Yes-associated protein 1 (YAP1) signaling pathway has emerged as a target for the development of new therapeutic interventions in cancers. However, the role of the Hippo-targeted therapy has not been addressed in advanced gallbladder cancer (GBC). This study aimed to evaluate the expression of the major Hippo pathway components mammalian Ste20-like protein kinase 1 (MST1), YAP1 and transcriptional coactivator with PDZ-binding motif (TAZ) and examined the effects of Verteporfin (VP), a small molecular inhibitor of YAP1-TEA domain transcription factor (TEAD) protein interaction, in metastatic GBC cell lines and patient-derived organoids (PDOs). Immunohistochemical analysis revealed that advanced GBC patients had high nuclear expression of YAP1. High nuclear expression of YAP1 was associated with poor survival in GBC patients with subserosal invasion (pT2). Additionally, advanced GBC cases showed reduced expression of MST1 compared to chronic cholecystitis. Both VP treatment and YAP1 siRNA inhibited the migration ability in GBC cell lines. Interestingly, gemcitabine resistant PDOs with high nuclear expression of YAP1 were sensitive to VP treatment. Taken together, our results suggest that key components of the Hippo-YAP1 signaling pathway are dysregulated in advanced gallbladder cancer and reveal that the inhibition YAP1 may be a candidate for targeted therapy.

Human Organoids Share Structural and Genetic Features with Primary Pancreatic Adenocarcinoma Tumors.

Patient-derived pancreatic ductal adenocarcinoma (PDAC) organoid systems show great promise for understanding the biological underpinnings of disease and advancing therapeutic precision medicine. Despite the increased use of organoids, the fidelity of molecular features, genetic heterogeneity, and drug response to the tumor of origin remain important unanswered questions limiting their utility. To address this gap in knowledge, primary tumor- and patient-derived xenograft (PDX)-derived organoids, and 2D cultures for in-depth genomic and histopathologic comparisons with the primary tumor were created. Histopathologic features and PDAC representative protein markers (e.g., claudin 4 and CA19-9) showed strong concordance. DNA- and RNA-sequencing (RNAseq) of single organoids revealed patient-specific genomic and transcriptomic consistency. Single-cell RNAseq demonstrated that organoids are primarily a clonal population. In drug response assays, organoids displayed patient-specific sensitivities. In addition, the in vivo PDX response to FOLFIRINOX and gemcitabine/abraxane treatments were examined, which was recapitulated in vitro with organoids. This study has demonstrated that organoids are potentially invaluable for precision medicine as well as preclinical drug treatment studies because they maintain distinct patient phenotypes and respond differently to drug combinations and dosage. IMPLICATIONS: The patient-specific molecular and histopathologic fidelity of organoids indicate that they can be used to understand the etiology of the patient's tumor and the differential response to therapies and suggests utility for predicting drug responses.

Intestinal inflammation and mucosal barrier function.

Intestinal mucosal barrier function is the capacity of the intestine to provide adequate containment of luminal microorganisms and molecules while preserving the ability to absorb nutrients. The central element is the epithelial layer, which physically separates the lumen and the internal milieu and is in charge of vectorial transport of ions, nutrients, and other substances. The secretion of mucus-forming mucins, sIgA, and antimicrobial peptides reinforces the mucosal barrier on the extraepithelial side, while a variety of immune cells contributes to mucosal defense in the inner side. Thus, the mucosal barrier is of physical, biochemical, and immune nature. In addition, the microbiota may be viewed as part of this system because of the mutual influence occurring between the host and the luminal microorganisms. Alteration of the mucosal barrier function with accompanying increased permeability and/or bacterial translocation has been linked with a variety of conditions, including inflammatory bowel disease. Genetic and environmental factors may converge to evoke a defective function of the barrier, which in turn may lead to overt inflammation of the intestine as a result of an exacerbated immune reaction toward the microbiota. According to this hypothesis, inflammatory bowel disease may be both precipitated and treated by either stimulation or downregulation of the different elements of the mucosal barrier, with the outcome depending on timing, the cell type affected, and other factors. In this review, we cover briefly the elements of the barrier and their involvement in functional defects and the resulting phenotype.

Nondigestible oligosaccharides exert nonprebiotic effects on intestinal epithelial cells enhancing the immune response via activation of TLR4-NFκB.

SCOPE: Prebiotic effects of non absorbable glucids depend mainly on digestion by the colonic microbiota. Our aim was to assess nonprebiotic, direct effects of 4 prebiotics, namely fructooligosaccharides, inulin, galactooligosaccharides, and goat's milk oligosaccharides on intestinal epithelial cells. METHODS AND RESULTS: Prebiotics were tested in intestinal epithelial cell 18 (IEC18), HT29, and Caco-2 cells. Cytokine secretion was measured by ELISA and modulated with pharmacological probes and gene silencing. Prebiotics induced the production of growth-related oncogene, (GROα), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 2 (MIP2) in IEC18 cells, with an efficacy that was 50-80% that of LPS. Prebiotics did not change RANTES expression, which was robustly induced by LPS in IEC18 cells. Cytokine secretion was suppressed by Bay11-7082, an inhibitor of IκB-α phosphorylation. The response was markedly decreased by Myd88 or TLR4 gene knockdown. Prebiotics also elicited cytokine production in HT29 but not in Caco-2 cells, consistent with reduced and vestigial expression of TLR4 in these cell lines, respectively. Prebiotic-induced MCP-1 secretion was reduced also in colonic explants from TLR4 KO mice compared with the controls. CONCLUSIONS: We conclude that prebiotics are TLR4 ligands in intestinal epithelial cells and that this may be a relevant mechanism for their in vivo effects.

Dose-dependent antiinflammatory effect of ursodeoxycholic acid in experimental colitis.

The denomination of inflammatory bowel disease comprises a group of chronic inflammatory diseases of the digestive tract, ulcerative colitis and Crohn's disease being the most important conditions. Bile acids may play a role both in etiology and pharmacology of this disease. Thus, although deoxycholic acid is regarded as a proinflammatory agent ursodeoxycholic acid, which is currently being used to treat certain types of cholestasis and primary biliary cirrhosis, because of their choleretic, cytoprotective and immunomodulatory effects, it has been reported to exert an anti-inflammatory activity. We aim to confirm and characterize the intestinal antiinflammatory activity of ursodeoxycholic acid. The experimental model trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats has been used. Animal status was characterized by a number of macroscopic and biochemical parameters. Oral administration of ursodeoxycholic acid was able to ameliorate experimental colonic inflammation. This occurred only at a relatively high dose (50 mg/kg day), whereas ursodeoxycholic acid was without significant effect at doses of 10 and 25 mg/kg day. The therapeutic effect was evidenced, among others, by a higher body weight recovery, a diminished affected to total mucosal area and lower alkaline phosphatase activity in treated vs. control (TNBS treated) animals. These results indicate that, at the appropriate dose, ursodeoxycholic acid is a potentially useful drug to reduce intestinal inflammation and could be envisaged to be incorporated in the treatment of inflammatory bowel diseases.

Tissue-nonspecific alkaline phosphatase is activated in enterocytes by oxidative stress via changes in glycosylation.

BACKGROUND: Intestinal inflammation produces an induction of alkaline phosphatase (AP) activity that is attributable in part to augmented expression, accompanied by a change in isoform, in epithelial cells. METHODS: This study focuses on induction of AP in intestinal epithelial cells in vitro. RESULTS: Treatment with the oxidants H2O2, monochloramine, or tButOOH increases AP activity in vitro in Caco-2, HT29, and IEC18 cells. We selected IEC18 cells for further testing. Basal AP activity in IEC18 cells is of the tissue-nonspecific (bone-liver-kidney) type, as indicated by Northern and Western blot analysis. Oxidative stress augments AP activity and the sensitivity of the enzyme to levamisole, homoarginine, and heat in IEC18 cells. Increased immunoreactivity to tissue-nonspecific AP antibodies suggests an isoform shift from liver to either kidney or bone type. This effect occurs without changes at the mRNA level and is sensitive to tunicamycin, an inhibitor of N-glycosylation, and neuraminidase digestion. Saponin and deoxycholate produce similar effects to oxidants. Butyrate but not proinflammatory cytokines or LPS can induce a similar effect but without toxicity. The AP increase is not prevented by modulators of the MAPK, NF-κB, calcium, and cyclic adenosine monophosphate (cAMP) pathways, and is actually enhanced by actinomycin D via higher cell stress. CONCLUSIONS: Oxidative stress causes a distinct increase in enterocyte AP activity together with cell toxicity via changes in the glycosylation of the enzyme that correspond to a shift in isotype within the tissue-nonspecific paradigm. We speculate that this may have physiological implication for gut defense.

Molecular bases of impaired water and ion movements in inflammatory bowel diseases.

The intestine is dedicated to the absorption of water and nutrients. Fine tuning of this process is necessary to maintain an adequate balance and inflammation disrupts the equilibrium. This review summarizes the current evidence in this field. Classical mechanisms proposed include alteration of epithelial integrity, augmented secretion, and reduced absorption. In addition, intestinal inflammation is associated with defects in epithelial barrier function. However, our understanding of the phenomenon has been complicated by the fact that ionic secretion is in fact diminished in vivo, even after inflammation has subsided. Inhibited ionic secretion can be reversed partially or totally in vitro by maneuvers such as blockade of inducible nitric oxide synthase or removal of the submucosal layer. Disturbances in ionic absorption are less well characterized but clearly involve both electroneutral and electrogenic Na(+) absorption. Altered ionic transport is associated with changes in the expression and function of the transporters, including the Na(+)/K(+) ATPase, the sodium/potassium/chloride cotransporter 1 (NKCC1), the sodium/hydrogen exchanger 3 (NHE3), and the epithelial sodium channel (ENaC), as well as to the modulation of intracellular signaling. Further investigation is needed in this area in order to provide an integrated paradigm of ionic transport in the inflamed intestine. In particular, we do not know exactly how diarrhea ensues in inflammation and, consequently, we do not have specific pharmacological tools to combat this condition effectively and without side effects. Moreover, whether transport disturbances are reversible independently of inflammatory control is unknown.