Maternal protein restriction induces gastrointestinal dysfunction and enteric nervous system remodeling in rat offspring.

Early-life adversity is a major risk factor for the development of diseases later in life. Maternal protein restriction (MPR) is associated with morbidities in offspring affecting multiple organs, but its impact on the gastrointestinal (GI) tract remains poorly studied. Using a rat model, we examined the consequences of MPR on GI function and on the enteric nervous system (ENS) in the offspring at postnatal d 35 under basal state and following a water avoidance stress (WAS). Compared with control rats, MPR rats exhibited greater colonic motility, permeability, and corticosteronemia. In contrast to controls, MPR rats presented a blunted functional and corticosteronemic response to WAS. Furthermore, MPR rats showed an increased proportion of choline acetyltransferase-immunoreactive (ChAT-IR) neurons and a reduced level of autophagy in colonic myenteric neurons. In ENS cultures, corticosterone treatment increased the proportion of ChAT-IR neurons and reduced autophagy level in enteric neurons. Inhibition of autophagy in ENS cultures resulted in a higher vulnerability of enteric neurons to a cellular stress. Altogether, this study suggests that MPR induced GI dysfunction and ENS alterations in offspring rats and that MPR-induced increased corticosteronemia might be involved in ENS remodeling and altered responsiveness of the gut to stressors later in life.-Aubert, P., Oleynikova, E., Rizvi, H., Ndjim, M., Le Berre-Scoul, C., Grohard, P. A., Chevalier, J., Segain, J.-P., Le Drean, G., Neunlist, M., Boudin, H. Maternal protein restriction induces gastrointestinal dysfunction and enteric nervous system remodeling in rat offspring.

Perinatal supplementation of 4-phenylbutyrate and glutamine attenuates endoplasmic reticulum stress and improves colonic epithelial barrier function in rats born with intrauterine growth restriction.

Intrauterine growth restriction (IUGR) can affect the structure and function of the intestinal barrier and increase digestive disease risk in adulthood. Using the rat model of maternal dietary protein restriction (8% vs. 20%), we found that the colon of IUGR offspring displayed decreased mRNA expression of epithelial barrier proteins MUC2 and occludin during development. This was associated with increased mRNA expression of endoplasmic reticulum (ER) stress marker XBP1s and increased colonic permeability measured in Ussing chambers. We hypothesized that ER stress contributes to colonic barrier alterations and that perinatal supplementation of dams with ER stress modulators, phenylbutyrate and glutamine (PG) could prevent these defects in IUGR offspring. We first demonstrated that ER stress induction by tunicamycin or thapsigargin increased the permeability of rat colonic tissues mounted in Ussing chamber and that PG treatment prevented this effect. Therefore, we supplemented the diet of control and IUGR dams with PG during gestation and lactation. Real-time polymerase chain reaction and histological analysis of colons from 120-day-old offspring revealed that perinatal PG treatment partially prevented the increased expression of ER stress markers but reversed the reduction of crypt depth and goblet cell number in IUGR rats. In dextran sodium sulfate-induced injury and recovery experiments, the colon of IUGR rats without perinatal PG treatment showed higher XBP1s mRNA levels and histological scores of inflammation than IUGR rats with perinatal PG treatment. In conclusion, these data suggest that perinatal supplementation with PG could alleviate ER stress and prevent epithelial barrier dysfunction in IUGR offspring.

Oral sodium butyrate impacts brain metabolism and hippocampal neurogenesis, with limited effects on gut anatomy and function in pigs.

Butyrate can improve gut functions, whereas histone deacetylase inhibitors might alleviate neurocognitive alterations. Our aim was to assess whether oral butyrate could modulate brain metabolism and plasticity and if this would relate to gut function. Sixteen pigs were subjected to sodium butyrate (SB) supplementation via beverage water or water only [control (C)]. All pigs had blood sampled after 2 and 3 wk of treatment, and were subjected to a brain positron emission tomography after 3 wk. Animals were euthanized after 4 wk to sample pancreas, intestine, and brain for gut physiology and anatomy measurements, as well as hippocampal histology, Ki67, and doublecortin (DCX) immunohistochemistry. SB compared with C treatment triggered basal brain glucose metabolism changes in the nucleus accumbens and hippocampus ( P = 0.003), increased hippocampal granular cell layer volume ( P = 0.006), and neurogenesis (Ki67: P = 0.026; DCX: P = 0.029). After 2 wk of treatment, plasma levels of glucose, insulin, lactate, glucagon-like peptide 1, and peptide tyrosine tyrosine remained unchanged. After 3 wk, plasma levels of lactate were lower in SB compared with C animals ( P = 0.028), with no difference for glucose and insulin. Butyrate intake impacted very little gut anatomy and function. These results demonstrate that oral SB impacted brain functions with little effects on the gut.-Val-Laillet, D., Guérin, S., Coquery, N., Nogret, I., Formal, M., Romé, V., Le Normand, L., Meurice, P., Randuineau, G., Guilloteau, P., Malbert, C.-H., Parnet, P., Lallès, J.-P., Segain, J.-P. Oral sodium butyrate impacts brain metabolism and hippocampal neurogenesis, with limited effects on gut anatomy and function in pigs.

Hydrocortisone prevents immunosuppression by interleukin-10+ natural killer cells after trauma-hemorrhage.

OBJECTIVE: Trauma induces a state of immunosuppression, which is responsible for the development of nosocomial infections. Hydrocortisone reduces the rate of pneumonia in patients with trauma. Because alterations of dendritic cells and natural killer cells play a central role in trauma-induced immunosuppression, we investigated whether hydrocortisone modulates the dendritic cell/natural killer cell cross talk in the context of posttraumatic pneumonia. DESIGN: Experimental study. SETTINGS: Research laboratory from an university hospital. SUBJECTS: Bagg Albino/cJ mice (weight, 20-24 g). INTERVENTIONS: First, in an a priori substudy of a multicenter, randomized, double-blind, placebo-controlled trial of hydrocortisone (200 mg/d for 7 d) in patients with severe trauma, we have measured the blood levels of five cytokines (tumor necrosis factor-α, interleukin-6, interleukin-10, interleukin-12, interleukin-17) at day 1 and day 8. In a second step, the effects of hydrocortisone on dendritic cell/natural killer cell cross talk were studied in a mouse model of posttraumatic pneumonia. Hydrocortisone (0.6 mg/mice i.p.) was administered immediately after hemorrhage. Twenty-four hours later, the mice were challenged with Staphylococcus aureus (7 × 10 colony-forming units). MEASUREMENTS AND MAIN RESULTS: Using sera collected during a multicenter study in patients with trauma, we found that hydrocortisone decreased the blood level of interleukin-10, a cytokine centrally involved in the regulation of dendritic cell/natural killer cell cluster. In a mouse model of trauma-hemorrhage-induced immunosuppression, splenic natural killer cells induced an interleukin-10-dependent elimination of splenic dendritic cell. Hydrocortisone treatment reduced this suppressive function of natural killer cells and increased survival of mice with posthemorrhage pneumonia. The reduction of the interleukin-10 level in natural killer cells by hydrocortisone was partially dependent on the up-regulation of glucocorticoid-induced tumor necrosis factor receptor-ligand (TNFsf18) on dendritic cell. CONCLUSIONS: These data demonstrate that trauma-induced immunosuppression is characterized by an interleukin-10-dependent elimination of dendritic cell by natural killer cells and that hydrocortisone improves outcome by limiting this immunosuppressive feedback loop.

Visceral adipose tissue and leptin increase colonic epithelial tight junction permeability via a RhoA-ROCK-dependent pathway.

Proinflammatory cytokines produced by immune cells play a central role in the increased intestinal epithelial permeability during inflammation. Expansion of visceral adipose tissue (VAT) is currently considered a consequence of intestinal inflammation. Whether VAT per se plays a role in early modifications of intestinal barrier remains unknown. The aim of this study was to demonstrate the direct role of adipocytes in regulating paracellular permeability of colonic epithelial cells (CECs). We show in adult rats born with intrauterine growth retardation, a model of VAT hypertrophy, and in rats with VAT graft on the colon, that colonic permeability was increased without any inflammation. This effect was associated with altered expression of tight junction (TJ) proteins occludin and ZO-1. In coculture experiments, adipocytes decreased transepithelial resistance (TER) of Caco-2 CECs and induced a disorganization of ZO-1 on TJs. Intraperitoneal administration of leptin to lean rats increased colonic epithelial permeability and altered ZO-1 expression and organization. Treatment of HT29-19A CECs with leptin, but not adiponectin, dose-dependently decreased TER and altered TJ and F-actin cytoskeleton organization through a RhoA-ROCK-dependent pathway. Our data show that adipocytes and leptin directly alter TJ function in CECs and suggest that VAT could impair colonic epithelial barrier.

Connecting metabolism to intestinal barrier function: The role of leptin.

Structure and function of the intestinal epithelial barrier (IEB) are dependent upon the integrity of junctional protein structures sealing the apical surface between epithelial cells. Tight junctions (TJ) and the surrounding apical F-actin cytoskeleton are involved in the regulation of paracellular permeability. The regulation of actin cytoskeleton organization by RhoA/Rho-kinase (ROCK) pathway plays an important role in TJ assembly and function. There is mounting evidence that the adipocyte-derived hormone leptin exerts pleiotropic effects on the intestinal epithelium including nutrient absorption, epithelial growth, inflammation and injury. Leptin activates multiple cell signaling pathways in intestinal epithelial cells (IEC) that can explain these pleiotropic effects. However, these pathways are also involved in the primary role of leptin that is the regulation of energy and glucose metabolism homeostasis. In this commentary, we examine how the interplay between leptin signaling pathways that regulate cell metabolism could impact upon IEB function.

Toll-like receptor-4 agonist in post-haemorrhage pneumonia: role of dendritic and natural killer cells.

Haemorrhage-induced immunosuppression has been linked to nosocomial infections. We assessed the impact of monophosphoryl lipid A, a Toll/interleukin-1 receptor-domain-containing adaptor protein inducing interferon-biased Toll-like receptor-4 agonist currently used as a vaccine adjuvant in humans, on post-haemorrhage susceptibility to infection. We used a mouse model of post-haemorrhage pneumonia induced by methicillin-susceptible Staphylococcus aureus. Monophosphoryl lipid A was administered intravenously after haemorrhage and before pneumonia onset. Haemorrhage altered survival rate, increased lung damage (neutrophil accumulation, oedema and cytokine release) and altered the functions of dendritic and natural killer cells. Here, we show that monophosphoryl lipid A decreased systemic dissemination of S. aureus and dampened inflammatory lung lesions. Monophosphoryl lipid A partially restored the capacity for antigen presentation and the transcriptional activity in dendritic cells. Monophosphoryl lipid A did not restore the interferon-γ mRNA but prevented interleukin-10 mRNA overexpression in natural killer cells compared with untreated mice. Ex vivo monophosphoryl lipid A-stimulated dendritic cells or natural killer cells harvested from haemorrhaged animals were adoptively transferred into mice undergoing post-haemorrhage pneumonia. Stimulated dendritic cells (but not stimulated natural killer cells) improved the survival rate compared with mice left untreated. In vivo depletion of natural killer cells decreased survival rate of monophosphoryl lipid A-treated mice. Dendritic and natural killer cells are critically involved in the beneficial effects of monophosphoryl lipid A within post-haemorrhage pneumonia.

Perinatal undernutrition alters intestinal alkaline phosphatase and its main transcription factors KLF4 and Cdx1 in adult offspring fed a high-fat diet.

Nutrient restriction during gestation and/or suckling is associated with an increased risk of developing inflammation, obesity and metabolic diseases in adulthood. However, the underlying mechanisms, including the role of the small intestine, are unclear. We hypothesized that intestinal adaptation to the diet in adulthood is modulated by perinatal nutrition. This hypothesis was tested using a split-plot design experiment with 20 controls and 20 intrauterine growth-retarded (IUGR) rats aged 240 days and randomly assigned to be fed a standard chow or a high-fat (HF) diet for 10 days. Jejunal tissue was collected at necropsy and analyzed for anatomy, digestive enzymes, goblet cells and mRNA levels. Cecal contents and blood serum were analyzed for alkaline phosphatase (AP). IUGR rats failed to adapt to HF by increasing AP activity in jejunal tissue and cecal content as observed in controls. mRNA levels of transcription factors KLF4 and Cdx1 were blunted in jejunal epithelial cell of IUGR rats fed HF. mRNA levels of TNF-α were lower in IUGR rats. They also displayed exacerbated aminopeptidase N response and reduced jejunal goblet cell density. Villus and crypt architecture and epithelial cell proliferation increased with HF in both control and IUGR rats. Serum AP tended to be lower, and serum levamisole inhibition-resistant AP fraction was lower, in IUGR than controls with HF. Serum fatty acids and triglycerides were higher in IUGR rats and higher with HF. In conclusion, the adult intestine adapts to an HF diet differentially depending on early nutrition, jejunal AP and transcription factors being blunted in IUGR individuals fed HF.

Escherichia coli-induced productions of pro-inflammatory cytokines are regulated by MAP kinases and G-protein but not by Akt: Relationship with phylogenetic groups and resistance patterns.

INTRODUCTION: We investigated the role of PI3-K, MAP kinases, and heterotrimeric G proteins in inducing cytokines production in human whole blood cultures stimulated by viable Escherichia coli (E. coli) clinical strains. MATERIALS AND METHODS: We used eight E. coli strains that belong to different phylogenetic groups and presented by different antibiotic resistance patterns. Whole blood from healthy volunteers was incubated at 37°C for 150min, with lipopolysaccharide (LPS) from E. coli O111:B4 or selected viable E. coli clinical strains, with or without SB202190 (p38 inhibitor), PD98059 (ERK inhibitor), PTX (pertussis toxin; heterotrimeric G proteins inhibitor), wortmaninn (PI3-K inhibitor). The TNF-α, IL-1ß, IL-10 and IFN-γ concentrations were measured in culture supernatants (ELISA). RESULTS: IL-10 and IFN-γ were not detectable. Susceptible strains induced higher TNF-α and IL-1ß productions than ß-lactam resistant strains (p<0.05), with no difference between phylogenetic groups. A transformed strain carrying a plasmid-mediated AmpC-ß-lactamase gene (CMY-2) induced lower TNF-α and IL-1ß production than the parent wild type strain (p<0.05). SB202190 (p38 inhibitor) and PD98059 (ERK inhibitor) reduced TNF-α concentrations by, respectively, 80% (p<0.05) and 50% (p<0.05). Wortmaninn (PI3-K inhibitor) had no significant effect. PTX (heterotrimeric G proteins inhibitor) altered TNF-α production after viable bacteria stimulation (1.7-fold increase; p<0.05) but not after LPS (TLR-4) stimulation. Regarding IL-1ß, wortmaninn, SB202190 and PTX had no significant effect whereas PD98059 significantly decreased production in whole cell cultures (p<0.05). CONCLUSION: Susceptible strains induce greater TNF-α and IL-1ß productions than resistant strains. ERK kinase plays a major role in viable E. coli strains inducing TNF-α and IL-1ß production. E. coli exerts an effect on the pertussis toxin-sensitive G-protein through a TLR-4-independent mechanism.

Is Crohn's creeping fat an adipose tissue?

BACKGROUND: In human pathology, the "creeping fat" (CF) of the mesentery is unique to Crohn's disease (CD). CF is usually referred to as an ectopic extension of mesenteric adipose tissue (MAT). However, since no animal model developing CF has ever been established, very little is known about this type of fat-depot expansion and its role in the development of the disease. METHODS: We developed and standardized an experimental protocol in mice that reproducibly induces CF development when a severe colonic inflammation is obtained by intracolonic instillation of DNBS. RESULTS: Macro-microscopic observations revealed a fatty appearance of CF. Yet when compared to MAT from the same animals, CF contains very little triglycerides, few adipocytes, and we observed a very low expression and protein levels of both adipose markers (hormone-sensitive lipase, perilipin) and adipocytokines (leptin, adiponectin). The decreased expression of perilipin in CF was also observed by immunohistochemistry. Conversely, the expression of proinflammatory and fibrous markers (Pref-1) was much higher in CF than in MAT. These observations were fully consistent with those made on CF recovered from five CD patients and compared with subcutaneous and mesenteric fat from the same patients. CONCLUSIONS: Altogether, this work reports an original experimental mice model of CF. In this model we establish for the first time that CF only occurs in severe colonic inflammation and shows an inflammatory, fibrous but not an adipose pattern.

CpG-ODN and MPLA prevent mortality in a murine model of post-hemorrhage-Staphyloccocus aureus pneumonia.

Infections are the most frequent cause of complications in trauma patients. Post-traumatic immune suppression (IS) exposes patients to pneumonia (PN). The main pathogen involved in PN is Methicillin Susceptible Staphylococcus aureus (MSSA). Dendritic cells () may be centrally involved in the IS. We assessed the consequences of hemorrhage on pneumonia outcomes and investigated its consequences on DCs functions. A murine model of hemorrhagic shock with a subsequent MSSA pneumonia was used. Hemorrhage decreased the survival rate of infected mice, increased systemic dissemination of sepsis and worsened inflammatory lung lesions. The mRNA expression of Tumor Necrosis Factor-alpha (TNF-α), Interferon-beta (IFN-ß) and Interleukin (IL)-12p40 were mitigated for hemorrhaged-mice. The effects of hemorrhage on subsequent PN were apparent on the pDCs phenotype (reduced MHC class II, CD80, and CD86 molecule membrane expression). In addition, hemorrhage dramatically decreased CD8(+) cDCs- and CD8(-) cDCs-induced allogeneic T-cell proliferation during PN compared with mice that did not undergo hemorrhage. In conclusion, hemorrhage increased morbidity and mortality associated with PN; induced severe phenotypic disturbances of the pDCs subset and functional alterations of the cDCs subset. After hemorrhage, a preventive treatment with CpG-ODN or Monophosphoryl Lipid A increased transcriptional activity in DCs (TNF-α, IFN-ß and IL-12p40) and decreased mortality of post-hemorrhage MSSA pneumonia.

Short-chain fatty acids regulate the enteric neurons and control gastrointestinal motility in rats.

BACKGROUND & AIMS: Little is known about the environmental and nutritional regulation of the enteric nervous system (ENS), which controls gastrointestinal motility. Short-chain fatty acids (SCFAs) such as butyrate regulate colonic mucosa homeostasis and can modulate neuronal excitability. We investigated their effects on the ENS and colonic motility. METHODS: Effects of butyrate on the ENS were studied in colons of rats given a resistant starch diet (RSD) or intracecal perfusion of SCFAs. Effects of butyrate were also studied in primary cultures of ENS. The neurochemical phenotype of the ENS was analyzed with antibodies against Hu, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) and by quantitative polymerase chain reaction. Signaling pathways involved were analyzed by pharmacologic and molecular biology methods. Colonic motility was assessed in vivo and ex vivo. RESULTS: In vivo and in vitro, RSD and butyrate significantly increased the proportion of ChAT- but not nNOS-immunoreactive myenteric neurons. Acetate and propionate did not reproduce the effects of butyrate. Enteric neurons expressed monocarboxylate transporter 2 (MCT2). Small interfering RNAs silenced MCT2 and prevented the increase in the proportion of ChAT- immunoreactive neurons induced by butyrate. Butyrate and trichostatin A increased histone H3 acetylation in enteric neurons. Effects of butyrate were prevented by inhibitors of the Src signaling pathway. RSD increased colonic transit, and butyrate increased the cholinergic-mediated colonic circular muscle contractile response ex vivo. CONCLUSION: Butyrate or histone deacetylase inhibitors might be used, along with nutritional approaches, to treat various gastrointestinal motility disorders associated with inhibition of colonic transit.

Degraded carrageenan causing colitis in rats induces TNF secretion and ICAM-1 upregulation in monocytes through NF-kappaB activation.

Carrageenan (CGN) is a high molecular weight sulphated polysaccharide derived from red seaweeds. In rodents, its degraded forms (dCGN) can induce intestinal inflammation associated with macrophage recruitment and activation. The aim of this study was: 1) to analyze the size-dependent effects of dCGN on colon inflammation in vivo, and 2) to correlate these effects with monocyte/macrophage proliferation, cytokine production and expression of various cell surface antigens including ICAM-1 adhesion molecule. Peripheral blood monocytes (PBM) and THP-1 monocytic cells were cultured in the presence of either 10 or 40 kDa, dCGN. The 40 kDa, but not the 10 kDa dCGN, induced colitis in in vivo. Degraded CGN inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-alpha expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-kappaB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype.

Butyrate utilization by the colonic mucosa in inflammatory bowel diseases: a transport deficiency.

The short-chain fatty acid butyrate, which is mainly produced in the lumen of the large intestine by the fermentation of dietary fibers, plays a major role in the physiology of the colonic mucosa. It is also the major energy source for the colonocyte. Numerous studies have reported that butyrate metabolism is impaired in intestinal inflamed mucosa of patients with inflammatory bowel disease (IBD). The data of butyrate oxidation in normal and inflamed colonic tissues depend on several factors, such as the methodology or the models used or the intensity of inflammation. The putative mechanisms involved in butyrate oxidation impairment may include a defect in beta oxidation, luminal compounds interfering with butyrate metabolism, changes in luminal butyrate concentrations or pH, and a defect in butyrate transport. Recent data show that butyrate deficiency results from the reduction of butyrate uptake by the inflamed mucosa through downregulation of the monocarboxylate transporter MCT1. The concomitant induction of the glucose transporter GLUT1 suggests that inflammation could induce a metabolic switch from butyrate to glucose oxidation. Butyrate transport deficiency is expected to have clinical consequences. Particularly, the reduction of the intracellular availability of butyrate in colonocytes may decrease its protective effects toward cancer in IBD patients.

Down-regulation of the monocarboxylate transporter 1 is involved in butyrate deficiency during intestinal inflammation.

BACKGROUND & AIMS: Butyrate oxidation is impaired in intestinal mucosa of patients with inflammatory bowel diseases (IBD). Butyrate uptake by colonocytes involves the monocarboxylate transporter (MCT) 1. We aimed to investigate the role of MCT1 in butyrate oxidation deficiency during colonic inflammation. METHODS: Colonic tissues were collected from patients with IBD or healthy controls and from rats with dextran sulfate sodium (DSS)-induced colitis. The intestinal epithelial cell line HT-29 was treated with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). MCT1 expression was analyzed by real-time reverse-transcription polymerase chain reaction, Western blot, and immunofluorescence. Butyrate uptake and oxidation in HT-29 cells was assessed using [(14)C]-butyrate. The mechanism of MCT1 gene regulation was analyzed by nuclear run-on and reporter gene assays. RESULTS: MCT1 messenger RNA (mRNA) and protein levels were markedly decreased in inflamed colonic mucosa of IBD patients and rats. In HT-29 cells, down-regulation of MCT1 mRNA and protein abundance by IFN-gamma and TNF-alpha correlated with a decrease in butyrate uptake and subsequent oxidation. IFN-gamma and TNF-alpha did not affect MCT1 mRNA stability but rather down-regulated gene transcription. We demonstrate that the cytokine response element is located in the proximal -111/+213 core region of the MCT1 promoter. CONCLUSIONS: The data suggest that butyrate oxidation deficiency in intestinal inflammation is a consequence of reduction in MCT1-mediated butyrate uptake. This reinforces the proposition that butyrate oxidation deficiency in IBD is not a primary defect.

Urotensin II is a new chemotactic factor for UT receptor-expressing monocytes.

Urotensin II (U-II), a vasoactive cyclic neuropeptide which activates the G protein-coupled receptor UT receptor, exerts various cardiovascular effects and may play a role in the pathophysiology of atherosclerosis. In this study, we report that the UT receptor is expressed and functional on human PBMC and rat splenocytes. PBMC surface expression of the UT receptor was mainly found in monocytes and NK cells, also in a minority of B cells, but not in T cells. Stimulation of monocytes with LPS increased UT receptor mRNA and protein expression. Cloning and functional characterization of the human UT receptor gene promoter revealed the presence of NF-kappaB-binding sites involved in the stimulation of UT receptor gene expression by LPS. Activation of the UT receptor by U-II induced chemotaxis with maximal activity at 10 and 100 nM. This U-II effect was restricted to monocytes. Analysis of the signaling pathway involved indicated that U-II-mediated chemotaxis was related to RhoA and Rho kinase activation and actin cytoskeleton reorganization. The present results thus identify U-II as a chemoattractant for UT receptor-expressing monocytes and indicate a pivotal role of the RhoA-Rho kinase signaling cascade in the chemotaxis induced by U-II.

Intestinal translocation capabilities of wheat allergens using the Caco-2 cell line.

Because intestinal absorption of food protein can trigger an allergic reaction, the effect of wheat proteins on intestinal epithelial cell permeability was evaluated and the abilities of these proteins in native or pepsin-hydrolyzed state to cross the epithelial cell monolayer were compared. Enterocytic monolayers were established by culturing Caco-2 cells, a model of enterocytes, on permeable supports that separate the apical and basal compartments. Proteins were added into the apical compartment, and the transepithelial resistance (TER) was measured; proteins that crossed the cell monolayer were detected in the basal medium by ELISA. Wheat proteins did not alter the cell monolayer. TER and Caco-2 cell viability were conserved, and the passage of dextran was prevented. Native and pepsin-hydrolyzed forms of omega5-gliadin and lipid transfer proteins were detected in the basal medium. The results suggest that these two major allergens in food allergy to wheat were able to cross the cell monolayer by the transcellular route.

Altered lipid, apolipoprotein, and lipoprotein profiles in inflammatory bowel disease: consequences on the cholesterol efflux capacity of serum using Fu5AH cell system.

Epidemiological data suggest a link between chronic inflammation condition and atherosclerosis. Infection and inflammation can also impair lipoprotein metabolism and produce a wide variety of changes in plasma concentrations of lipids and lipoproteins. Twenty-one patients with inflammatory bowel diseases (IBDs) and 28 healthy subjects were recruited. Serum concentrations of lipids, lipoproteins, apolipoproteins, leptin, ghrelin, and inflammation markers (C-reactive protein and serum amyloid A) were measured, and subjects' lipoproteins were characterized. The ability of patients with serum IBD to efflux free cell cholesterol was measured. Serum cholesterol, high-density lipoprotein cholesterol, apolipoprotein (apo) A-I, apoC-II, apoC-III bound to apoB, phospholipid, and phospholipids not bound to apoB levels were significantly lower, whereas serum triglyceride, serum amyloid A, and C-reactive protein levels were significantly higher in patients with active IBD. Apolipoprotein A-I immunoreactivity (pre-beta small particles and small alpha-high-density lipoprotein particles) is decreased in patients with IBD. In contrast, apoE immunoreactivity (slow/small apoE containing lipoprotein particles [LpE particle]) increased in these patients. The efflux capacity of serum from patients with IBD using [(3)H]-cholesterol-labeled Fu5AH cells was reduced (P < .005). Our results demonstrate that, in subjects with active IBD, inflammation leads to alterations in lipid, apolipoprotein, and lipoprotein profiles and reduced cholesterol efflux. These changes are similar to those proposed to promote atherogenesis and may contribute to the development of cardiovascular events.

The VSL# 3 probiotic mixture modifies microflora but does not heal chronic dextran-sodium sulfate-induced colitis or reinforce the mucus barrier in mice.

The mucus layer covering the epithelium is one of the main lines of defense of the colonic barrier. Both mucus gel and mucin expressions are altered during colonic inflammation and could be involved in epithelial repair. We postulated that modulating colonic mucus and mucins by probiotic supplementation could contribute to healing inflammatory mucosa. Our aim in this study was to determine whether probiotics could repair dextran-sodium sulfate (DSS)-induced chronic colitis in mice, and whether modifications of the colonic mucins could be involved. For that purpose, the VSL#3 probiotic mixture of 8 lactic acid bacteria probiotic strains was administered daily for 2 wk to mice with a mucosa impaired by a mild DSS treatment, and to mice with a normal mucosa. Probiotic strains survived in the gastrointestinal tract, increased the cecal concentrations of bifidobacteria, and modified cecal microflora metabolic activity in both DSS-treated and healthy mice. However, probiotic supplementation did not reverse the inflammation induced by DSS at either the macroscopic or histological level. Concurrently, probiotics did not modify the colonic mucus barrier, in terms of either mucin gene expression or adherent mucus layer thickness. In conclusion, the modification of microflora by supplementation with the VSL#3 probiotic mixture did not help to repair the colonic barrier breakdown caused by DSS treatment. The potential healing roles of mucins were neither confirmed nor invalidated by this study.

Rho kinase blockade prevents inflammation via nuclear factor kappa B inhibition: evidence in Crohn's disease and experimental colitis.

BACKGROUND & AIMS: Rho proteins are involved in the regulation of several cellular functions. Data from in vitro studies suggest that RhoA could be involved in the inflammatory response. We investigated the role of RhoA and its downstream effector Rho kinase in intestinal inflammation. METHODS: Activation of RhoA was assessed by pull-down assays. A specific inhibitor of Rho kinase, Y-27632, was used to examine the role of Rho kinase in inflammatory response in vivo and in vitro by molecular biology and by immunological and biochemical approaches. RESULTS: Increased activation of RhoA was found in inflamed intestinal mucosa of patients with Crohn's disease and of rats with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Oral administration of Y-27632 in rats significantly reduced the colonic inflammation. In vitro, activation of RhoA alone was sufficient to induce tumor necrosis factor production. Y-27632 inhibited production of tumor necrosis factor-alpha and interleukin-1 beta by lamina propria and peripheral blood mononuclear cells. Rho kinase inhibition prevented nuclear factor kappa B activation and I-kappa B phosphorylation and degradation. We showed that Rho kinase associates with and activates I-kappa B kinase alpha and that Y-27632 prevents I-kappa B kinase activation. CONCLUSIONS: Our study provides the first evidence that Rho kinase activates I-kappa B kinase and, thus, nuclear factor kappa B, suggesting a key role of Rho kinase in inflammatory responses and intestinal inflammation. Specific inhibition of Rho kinase may be a promising approach for the treatment of patients with Crohn's disease.