Response Variability to Drug Testing in Two Models of Chemically Induced Colitis.

The lack of knowledge regarding the pathogenesis of IBD is a challenge for the development of more effective and safer therapies. Although in vivo preclinical approaches are critical for drug testing, none of the existing models accurately reproduce human IBD. Factors that influence the intra-individual response to drugs have barely been described. With this in mind, our aim was to compare the anti-inflammatory efficacy of a new molecule (MTADV) to that of corticosteroids in TNBS and DSS-induced colitis mice of both sexes in order to clarify further the response mechanism involved and the variability between sexes. The drugs were administered preventively and therapeutically, and real-time bioluminescence was performed for the in vivo time-course colitis monitoring. Morphometric data were also collected, and colonic cytokines and acute plasma phase proteins were analyzed by qRT-PCR and ELISA, respectively-bioluminescence images correlated with inflammatory markers. In the TNBS model, dexamethasone worked better in females, while MTADV improved inflammation in males. In DSS-colitis, both therapies worked similarly. Based on the molecular profiles, interaction networks were constructed to pinpoint the drivers of therapeutic response that were highly dependent on the sex. In conclusion, our results suggest the importance of considering sex in IBD preclinical drug screening.

The gestational power of mast cells in the injured tissue.

The inflammatory response expressed after wound healing would be the recapitulation of systemic extra-embryonic functions, which would focus on the interstitium of the injured tissue. In the injured tissue, mast cells, provided for a great functional heterogeneity, could play the leading role in the re-expression of extra-embryonic functions, i.e., coelomic-amniotic and trophoblastic-vitelline. Moreover, mast cells would favor the production of a gastrulation-like process, which in certain tissues and organs would induce the regeneration of the injured tissue. Therefore, the engraftment of mesenchymal stem cells and mast cells, both with an extra-embryonic regenerative phenotype, would achieve a blastema, from the repaired and regenerated injured tissue, rather than by fibrosis, which is commonly made through wound-healing.

Effects of nerve growth factor antagonist K252a on peritoneal mast cell degranulation: implications for rat postoperative ileus.

Stabilization of mast cell (MC) degranulation has been proposed to prevent postoperative ileus (POI). Nerve growth factor (NGF) mediates MC degranulation. The aim of the study was to evaluate whether NGF receptor antagonist K252a acts as a MC stabilizer in vitro and in vivo model of POI. Peritoneal mast cells (PMCs) were obtained from Sprague-Dawley rats and were incubated with K252a and exposed to NGF or Compound 48/80 (C48/80). MC degranulation was assessed by ß-hexosaminidase assay. POI was induced in rats by intestinal manipulation (IM). Rats were pretreated with K252a (100 µg/kg sc) 20 min prior to POI induction. At 20 min after IM, release of rat mast cell protease 6 (RMCP-6) was evaluated in peritoneal lavage. At 24 h, intestinal transit (IT) and gastric emptying (GE) were evaluated. Ileal inflammation was assessed by myeloperoxidase (MPO) activity, expression of IL-6, NGF, TrkA, RMCP-2 and 6, and MC density within the full-thickness ileum. C48/80 and NGF evoked degranulation of PMCs in a dose-dependent manner. K252a prevented NGF-evoked, but not C48/80-evoked, MC degranulation. IM evoked the release of peritoneal RMCP-6 and subsequently delayed IT and GE. IM increased MPO activity and expression of IL-6. In IM rats, K252a prevented upregulation of IL-6 expression and reduced TrkA. IT, GE, and inflammation were not affected by K252a. K252a inhibited NGF-evoked degranulation of PMCs in vitro. In vivo, K252a decreased IL-6 and PMC degranulation. This may be of relevance for the development of new therapeutic targets for POI.

Plasticity of dorsal root ganglion neurons in a rat model of post-infectious gut dysfunction: potential implication of nerve growth factor.

OBJECTIVE: Intestinal infections are suggested as a risk factor for the development of irritable bowel syndrome (IBS)-like visceral hypersensitivity. The mechanisms implicated might involve long-term changes in visceral afferents, with implication of nerve growth factor (NGF). We explored plastic changes in dorsal root ganglia (DRGs) receiving innervation from the gut and the potential implication of NGF in a rat model of IBS-like post-infectious gut dysfunction. MATERIALS AND METHODS: Rats were infected with Trichinella spiralis larvae. Thirty days post-infection, inflammatory markers, including interleukins (ILs) and mucosal mast cell infiltration (rat mast cell protease II [RMCPII]), and NGF and TrkA expression was determined in the jejunum and colon (RT-qPCR). In the same animals, morphometry (neuronal body size) and NGF content (immunofluorescence) were assessed in thoracolumbar DRG neurons. RESULTS: In infected animals, a low-grade inflammatory-like response, characterized by up-regulated levels of RMCPII and IL-6, was observed in the jejunum and colon. TrkA expression was increased in the jejunum, whereas the colon showed a slight reduction. NGF levels remained unaltered regardless the gut region. Overall, the mean cross-sectional area of DRG neurons was increased in T. spiralis-infected animals, with a reduction in both TrkA and NGF staining. CONCLUSIONS: Results suggest that during T. spiralis infection in rats, there is a remodeling of sensory afferents that might imply a NGF-mediated mechanism. Plastic changes in sensory afferents might mediate the long-lasting functional alterations that characterize this model of IBS. Similar mechanisms might be operating in patients with post-infectious-IBS.

Pharmacological characterization of alpha adrenoceptor-mediated motor responses in the rat colon.

BACKGROUND: Inhibitory neuromuscular transmission in the gastrointestinal tract is mediated by intrinsic nitrergic and purinergic neurons. Purines activate G protein-coupled receptor P2Y1 receptors, increasing intracellular Ca2+ that activates small conductance calcium-activated potassium (SKCa) channels. Little is known about the effect of adrenergic receptor activation on intestinal smooth muscle. In vascular tissue, stimulation of α-adrenoceptors causes smooth muscle contraction, while their effect on intestinal tissue is poorly understood. This study aimed to pharmacologically characterize the effect of α-adrenoceptor activation in the rat colon, which shares similar inhibitory pathways to the human colon. METHODS: Muscle bath experiments were performed with the rat proximal, mid, and distal colon oriented both circularly and longitudinally. RESULTS: The α1-adrenoceptor agonist phenylephrine (PE) (10-8-10-5 M) evoked concentration-dependent relaxations of the intestinal smooth muscle from all regions and orientations. However, in the mid-circular colon at low PE concentrations, a contraction sensitive to 10-5 M phentolamine (non-selective α-adrenoceptor blocker), the neural blocker tetrodotoxin (TTX; 10-6 M), and atropine (10-6 M) was recorded. PE-induced relaxations were insensitive to TTX (10-6 M) and the nonselective ß-adrenoceptor blocker propranolol (10-6 M). In contrast, PE-induced relaxations were blocked by phentolamine (10-5 M), prazosin (10-6 M) (α1-adrenoceptor blocker), and RS17053 (10-6 M) (α1A-blocker), but not by yohimbine (10-6 M) (α2-adrenoceptor blocker). Apamin (10-6 M), a SKCa channel blocker, abolished PE-induced relaxations. CONCLUSIONS: Contractile responses in the circular muscle of the mid colon could be attributed to α-adrenoceptors located on enteric cholinergic neurons. Stimulation of α1A-adrenoreceptors activates SKCa channels to cause smooth muscle relaxation, which constitutes a signaling pathway that shares similarities with P2Y1 receptors.

PAR-2-mediated control of barrier function and motility differs between early and late phases of postinfectious gut dysfunction in the rat.

Proteinase-activated receptor-2 (PAR-2) and mast cell (MC) mediators contribute to inflammatory and functional gastrointestinal disorders. We aimed to characterize jejunal PAR-2-mediated responses and the potential MC involvement in the early and late phases of a rat model of postinfectious gut dysfunction. Jejunal tissues of control and Trichinella spiralis-infected (14 and 30 days postinfection) rats, treated or not with the MC stabilizer, ketotifen, were used. Histopathology and immunostaining were used to characterize inflammation, PAR-2 expression, and mucosal and connective tissue MCs. Epithelial barrier function (hydroelectrolytic transport and permeability) and motility were assessed in vitro in basal conditions and after PAR-2 activation. Intestinal inflammation on day 14 postinfection (early phase) was significantly resolved by day 30 (late phase) although MC counts and epithelial permeability remained increased. PAR-2-mediated ion transport (Ussing chambers, in vitro) and epithelial surface PAR-2 expression were reduced in the early phase, with a trend toward normalization during the late phase. In control conditions, PAR-2 activation (organ bath) induced biphasic motor responses (relaxation followed by excitation). At 14 days postinfection, spontaneous contractility and PAR-2-mediated relaxations were enhanced; motor responses were normalized on day 30. Postinfectious changes in PAR-2 functions were not affected by ketotifen treatment. We concluded that, in the rat model of Trichinella spiralis infection, alterations of intestinal PAR-2 function and expression depend on the inflammatory phase considered. A lack of a ketotifen effect suggests no interplay between MCs and PAR-2-mediated motility and ion transport alterations. These observations question the role of MC mediators in PAR-2-modulating postinfectious gut dysfunction.

E. coli infection disrupts the epithelial barrier and activates intrinsic neurosecretory reflexes in the pig colon.

This study aims to assess the barrier integrity and possible activation of enteric neural pathways associated with secretion and motility in the pig colon induced by an enterotoxigenic Escherichia coli (ETEC) challenge. 50 Danbred male piglets were used for this study. 16 were challenged with an oral dose of the ETEC strain F4+ 1.5 × 109 colony-forming unit. Colonic samples were studied 4- and 9-days post-challenge using both a muscle bath and Ussing chamber. Colonic mast cells were stained with methylene blue. In control animals, electrical field stimulation induced neurosecretory responses that were abolished by tetrodotoxin (10-6M) and reduced by the combination of atropine (10-4M) and α-chymotrypsin (10U/mL). Exogenous addition of carbachol, vasoactive intestinal peptide, forskolin, 5-HT, nicotine, and histamine produced epithelial Cl- secretion. At day 4 post-challenge, ETEC increased the colonic permeability. The basal electrogenic ion transport remained increased until day 9 post-challenge and was decreased by tetrodotoxin (10-6M), atropine (10-4M), hexamethonium (10-5M), and ondansetron (10-5M). In the muscle, electrical field stimulation produced frequency-dependent contractile responses that were abolished with tetrodotoxin (10-6M) and atropine (10-6M). Electrical field stimulation and carbachol responses were not altered in ETEC animals in comparison with control animals at day 9 post-challenge. An increase in mast cells, stained with methylene blue, was observed in the mucosa and submucosa but not in the muscle layer of ETEC-infected animals on day 9 post-challenge. ETEC increased the response of intrinsic secretory reflexes and produced an impairment of the colonic barrier that was restored on day 9 post-challenge but did not modify neuromuscular function.

ICLAS LAQ Network for the Promotion of Animal Quality in Research.

ICLAS Laboratory Animal Quality Network (LAQN) programs currently consist of the Performance Evaluation Program (PEP), which focuses on microbial monitoring by and for laboratory animal diagnostic laboratories, and the Genetic Reference Monitoring Program (GENRef), which provides assay-ready reference DNA for genetic testing of mouse strains. Since 2008, PEP has grown to become a truly international program with participating laboratories in 5 continents. Launched in 2016, GENRef currently distributes DNA from 12 common inbred mouse strains for use in genetic monitoring of locally inbred colonies as well as for genetic testing of stocks, particularly genetically engineered stocks, of uncertain origins. GENRef has the capacity to include additional strains as well as additional species. PEP and GENRef provide the reagents at cost, as a resource to the international scientific community, in the interest of improving research quality in an environment of growing concern for research quality, rigor, and reproducibility.

Different responses of the blockade of the P2Y1 receptor with BPTU in human and porcine intestinal tissues and in cell cultures.

BACKGROUND: Gastrointestinal smooth muscle relaxation is accomplished by activation of P2Y1 receptors, therefore this receptor plays an important role in regulation of gut motility. Recently, BPTU was developed as a negative allosteric modulator of the P2Y1 receptor. Accordingly, the aim of this study was to assess the effect of BPTU on purinergic neurotransmission in pig and human gastrointestinal tissues. METHODS: Ca2+ imaging in tSA201 cells that express the human P2Y1 receptor, organ bath and microelectrodes in tissues were used to evaluate the effects of BPTU on purinergic responses. KEY RESULTS: BPTU concentration dependently (0.1 and 1 µmol L-1 ) inhibited the rise in intracellular Ca2+ evoked by ADP in tSA201 cells. In the pig small intestine, 30 µmol L-1 BPTU reduced the fast inhibitory junction potential by 80%. Smooth muscle relaxations induced by electrical field stimulation were reduced both in pig ileum (EC50  = 6 µmol L-1 ) and colon (EC50  = 35 µmol L-1 ), but high concentrations of BPTU (up to 100 µmol L-1 ) had no effect on human colonic muscle. MRS2500 (1 µmol L-1 ) abolished all responses. Finally, 10 µmol L-1 ADPßS inhibited spontaneous motility and this was partially reversed by 30 µmol L-1 BPTU in pig, but not human colonic tissue and abolished by MRS2500 (1 µmol L-1 ). CONCLUSIONS & INFERENCES: BPTU blocks purinergic responses elicited via P2Y1 receptors in cell cultures and in pig gastrointestinal tissue. However, the concentrations needed are higher in pig tissue compared to cell cultures and BPTU was ineffective in human colonic tissue.

Characterization of housing-related spontaneous variations of gut microbiota and expression of toll-like receptors 2 and 4 in rats.

Gut microbiota has been suggested as a key component of gut homeostasis, affecting immune responses within the gut. We determined changes in intestinal commensal bacteria and expression of toll-like receptors (TLR) 2 and 4 in rats bred under microbiologically controlled conditions (barrier), under standard conditions (conventional), and in barrier animals adapted to standard conditions (barrier/conventional). Cecal microbiota was analyzed by plate culture, and fluorescence in situ hybridization and microbial profiles were assessed by terminal restriction fragment length polymorphism. Cecal expression of TLR-2 and TLR-4 was determined by reverse transcription polymerase chain reaction (PCR). Total number of cecal bacteria was similar in the three groups. However, the barrier group showed a higher number of strict anaerobic bacteria (Bacteroides spp. and Clostridium spp.) while Bifidobacterium spp. were scarce. Re-housing the barrier-bred rats into conventional conditions led to a microbiota with intermediate characteristics between the barrier and conventional groups. Richness of the cecal microbial ecosystem was similar in the three groups, although a relative time-dependent variation, with highest homogeneity in the barrier group, was observed. Expression levels of TLR-2 and TLR-4 had no clear correlation with the microbiota. These results show that the relative composition of the cecal microbiota in rats varies spontaneously with changes in the environmental conditions, with minor impact in the expression of TLR-2 and TLR-4. These observations might be important in the understanding of variability in animal responses, particularly to immune-related stimuli, when assessed in the context of the environmental/microbiological conditions.

The asymmetric innervation of the circular and longitudinal muscle of the mouse colon differently modulates myogenic slow phasic contractions.

BACKGROUND: Neuromuscular transmission has been extensively studied in the circular layer of the mouse colon where a co-transmission of purines acting on P2Y1 receptors and NO has been previously described. However, the corresponding mechanisms in the longitudinal layer are less known. METHODS: Electrophysiological and myography techniques were used to evaluate spontaneous phasic contractions (SPC) and neural-mediated responses in the proximal, mid, and distal colon devoid of CD1 mice. Immunohistochemistry against c-kit and PDGFRα was performed in each colonic segment. KEY RESULTS: SPC were recorded in both muscle layers at a similar frequency being about four contractions per minute (c.p.m.) in the proximal and distal colon compared to the mid colon (2 c.p.m.). In non-adrenergic, non-cholinergic conditions, L-NNA (1 mmol/L) increased contractility in the circular but not in the longitudinal layer. In the longitudinal muscle, both electrophysiological and mechanical neural-mediated inhibitory responses were L-NNA and ODQ (10 µmol/L) sensitive. NaNP (1 µmol/L) caused cessation of SPC and the response was blocked by ODQ. Neither ADPßS (10 µmol/L) nor CYPPA (10 µmol/L), which both targeted the purinergic pathway, altered longitudinal contractions. PDGFRα + cells were located in both muscle layers and were more numerous compared with cKit + cells, which both formed a heterologous cellular network. A decreasing gradient of the PDGFRα labeling was observed along the colon. CONCLUSION: An inhibitory neural tone was absent in the longitudinal layer and neuronal inhibitory responses were mainly nitrergic. Despite the presence of PDGFRα + cells, purinergic responses were absent. Post-junctional pathways located in different cell types might be responsible for neurotransmitter transduction.

Mast cell-mediated splanchnic cholestatic inflammation.

INTRODUCTION: Splanchnic mast cells increase in chronic liver and in acute-on-chronic liver diseases. We administered Ketotifen, a mast cell stabilizer, and measured the mast cells in the splanchnic organs of cholestatic rats. MATERIAL AND METHODS: These groups were studied: sham-operated rats (S; n = 15), untreated microsurgical cholestasic rats (C; n = 20) and rats treated with Ketotifen: early (SK-e; n = 20 and CKe; n = 18), and late (SK-l; n = 15 and CK-l; n = 14). RESULTS: The cholestatic rats showed systemic and splanchnic impairments, such as ascites, portal hypertension, and biliary proliferation and fibrosis. The rats also showed a splanchnic increase of TNF-α, IL-1ß and MCP-1, and a reduction of IL-4, IL-10 and antioxidants. An increase of VEGF in the ileum and mesenteric lymphatic complex was associated with a liver reduction of TGF-ß1. Ketotifen reduces the degree of hepatic insufficiency and the splanchnic inflammatory mediators, as well as VEGF and TGF-ß1 levels. Ketotifen also reduces the connective tissue mast cells in the mesenteric lymphatic complex of cholestatic rats, while increases the hepatic mucosal mast cells. CONCLUSIONS: In cholestatic rats, Ketotifen improves liver function and ascites, and also reduces pro-inflammatory mediators in the splanchnic area. The decrease in connective tissue mast cells in the mesenteric lymphatic complex due to the administration of Ketotifen would lead to the improvement of the inflammatory splanchnic response, and consequently the abovementioned complications.

The nitric oxide donor LA-419 [S-(6-Nitro-oxi-hexahydro-furo[3,2-b]furan-3-1-il)thioacetate] prevents intestinal dysmotility, bacterial translocation, and inflammation in a rat model of enteritis.

Indomethacin induces a chronic model of inflammatory bowel disease (IBD) characterized by spontaneous relapses of inflammation, bacterial translocation, and long-lasting motor disturbances derived from cyclical up-regulated inducible nitric-oxide synthase (iNOS) and sustained down-regulated neuronal NOS (nNOS). The aims of this study were to evaluate whether LA-419 [S-(6-nitro-oxi-hexahydro-furo[3,2-b]furan-3-1-il)thioacetate], a NO-donor drug, could re-establish the normal expression of NOS and, hence, prevent the development of intestinal dysmotility, bacterial translocation, and relapses of inflammation associated to this model. Enteritis was induced in rats by administration of indomethacin with and without treatment with a novel NO-donor: LA-419 (0.5 mg/ml in the drinking water). Inflammatory reaction was evaluated by measuring blood leukocytes, serum tumor necrosis factor, and tissue myeloperoxidase. Intestinal motor activity was evaluated using strain-gauges. Ileal expression of iNOS and nNOS mRNA was determined by reverse transcription-polymerase chain reaction. Bacterial translocation was evaluated in cultures from mesenteric lymph nodes. The indomethacin-induced acute inflammatory reaction was associated with a rise in blood leukocytes and tumor necrosis factor. In the chronic stage, blood leukocyte monitoring allowed the selection of animals in active and inactive phases. Active phase was associated with iNOS up-regulation, high myeloperoxidase levels, hypomotility, and bacterial translocation. In contrast, inactive phase was associated with hypermotility and absence of bacterial translocation. LA-419 treatment restored nitric-oxide synthase isoenzyme expression and prevented the oscillation of both inflammatory and motor parameters that could be cyclically observed in inflamed rats. LA-419 also prevented intestinal dysmotility, bacterial translocation, and relapses of intestinal inflammation. LA-419 might be a novel therapeutic approach to prevent acute inflammatory relapses in patients with IBD.

Intestinal epithelial barrier dysfunction and dendritic cell redistribution during early stages of inflammation in the rat: role for TLR-2 and -4 blockage.

BACKGROUND: Dendritic cell (DC) redistribution during early stages of enteritis may be related to ileal barrier dysfunction. We used a rat model of ileitis to examine this hypothesis. METHODS: Sprague-Dawley rats were injected with indomethacin or saline and euthanized 2, 6, 12, or 24 hours later. Ileal segments and mesenteric lymph nodes were obtained for morphological, bacterial, or functional studies. To determine the role of Toll-like receptor (TLR)-2 and -4 blockages, rats were pretreated with normal IgG, anti-TLR-2, or anti-TLR-4 antibodies prior to indomethacin or saline, and ileal segments were collected 24 hours later. RESULTS: In control rats, CD103+DC were mainly located in the lamina propria (LP) and some expressed TLR-2. TLR-4+ cells with different morphology and distribution from CD103+DC were also detected. In indomethacin-treated rats at 6-24 hours, inflammation was evident as was redistribution of CD103+DC from LP to Peyer's patches. We also observed TLR-2+ monocyte depletion and changes in TLR-4 distribution. At 2-6 hours we detected opened tight junctions as well as abnormal trans- and para-epithelial enteric bacterial infiltration, while macromolecular permeability was not significantly enhanced until 24 hours. In the absence of indomethacin, anti-TLR-2 blockage induced a significant increase of LP CD103+DC, while in the presence of indomethacin, anti-TLR-2 or -4 blockages significantly inhibited (P < 0.05) the reduction of LP CD103+DC. CONCLUSIONS: During the early stages of indomethacin-induced ileitis, epithelial barrier damage and abnormal bacterial infiltration into the mucosa occurred in conjunction with initial redistribution of CD103+DC. Furthermore, we showed that TLR-2 and -4 blockade regulates CD103+DC distribution during early phases in this experimental model.

Prophylaxis with ketotifen in rats with portal hypertension: involvement of mast cell and eicosanoids.

BACKGROUND: Since we have previously shown an increase of mast cells in the small bowel and in the mesenteric lymph nodes in the rats with prehepatic portal hypertension, it can be hypothesized that this essential inflammatory cell would be involved in the pathogeny of the splanchnic changes related to portal hypertension. METHODS: To verify this hypothesis, we first studied mast cell infiltration in the ileum and in the mesenteric lymph nodes in sham-operated male Wistar rats (n=12) and in short-term prehepatic portal hypertensive rats (n=12), and the serum levels of rat mast cell protease II (RMCP-II) by ELISA. In a second set of experiments ketotifen, a mast cell stabilizer drug, was administered to sham-operated (n=10) and portal hypertensive (n=12) rats 24 hours before the intervention and prostanoids (PGE2, PGI2, TXB2) and leukotrienes (LTC4, LTB4) were assayed by RIA, mast cell infiltration in the ileum and in the mesenteric lymph nodes and the serum levels of RMCP-II were also studied, to show its effectiveness to prevent the mesenteric alterations produced by the inflammatory mediators released by the mast cell. RESULTS: Forty-eight hours after the intervention RMCP-II (P<0.05), PGE2 (P<0.001) and LTC4 serum levels decreased and mast cell number and RMCP-II levels increased in mesenteric lymph nodes in portal hypertensive rats. Prophylactic administration of ketotifen reduced portal pressure (P<0.001), serum levels of PGE2 (P<0.001) and RMCP-II (P<0.001) in mesenteric lymph nodes. CONCLUSIONS: In acute portal hypertension in the rat, the mast cell translocation from intestinal mucosa to mesenteric lymph nodes, where they are activated and degranulates, would represent a defence mechanism to avoid the activation of an acute and massive inflammatory response in this location. Prophylactic administration of ketotifen is able to reduce the splanchnic inflammatory changes related to acute portal hypertension in the rat.

Characterization of ileal dendritic cell distribution in a rat model of acute and chronic inflammation.

We examined ileal dendritic cell (DC) subpopulations in a rat model of indomethacin-induced enteritis to determine changes in phenotype and distribution associated with increased mucosal permeability during acute and chronic stages of inflammation. Sprague-Dawley rats were treated with indomethacin (7.5 mg/kg subcutaneously, 2 injections 48 h apart). Animals were killed at day 4 (acute stage) or at day 15 or 30 (chronic stages); control rats were injected with saline. DC distribution was evaluated by immunohistochemistry for CD103, CD11b, CD83, and CD163; inflammation was assessed by light microscopy; and permeability was determined by flux of horseradish peroxidase in Ussing chambers. In controls, both immature DC subpopulations, CD103+CD11b+CD163-CD83- and CD103+CD11b-CD163-CD83-, were observed in the lamina propria, and the CD11b- population also was present in Peyer's patches. In acute inflammation, permeability was increased (P<0.01), and inflamed areas with or without ulcers were observed. CD103+ and CD11b+ (CD83-) DCs were absent from inflamed areas, reduced in noninflamed tissues, but present in Peyer's patches. In the chronic stage at day 15, CD103+ and CD11b+ cells were located in inflamed and noninflamed areas and in Peyer's patches. In addition, CD83+ DCs were detected in inflamed areas. At day 30, when we observed a complete microscopic resolution of inflammation, numbers of CD103+ and CD11b+ DCs were increased, and there were CD83+ DCs beneath the epithelial cell layer. We conclude that antigen uptake in acute inflammation may activate resident immature DCs, inducing their migration to lymphoid tissue where they mature and then return to the intestine to play a role in the local inflammatory response.

Correlation between cyclical epithelial barrier dysfunction and bacterial translocation in the relapses of intestinal inflammation.

BACKGROUND: Although several factors have been implicated in the pathogenesis of inflammatory bowel disease (IBD), the mechanisms underlying the recurrent relapses have not yet been clarified. We hypothesized that epithelial barrier dysfunction, associated with intestinal motor disturbances, could play a key role in exacerbation of inflammatory illness due to an increased uptake of luminal antigen and bacterial translocation. METHODS: Indomethacin administration to rats induced a long-lasting oscillation of active and quiescent phases of inflammation associated with phases of hypo and hypermotility. Rats selected at either active or quiescent phase and from 2 to 60 days after indomethacin treatment were used. Short-circuit current; conductance and HRP flux were evaluated in small intestinal segments mounted in Ussing Chambers. Enterocyte endosomes containing HRP and ultrastructural damage were assessed by electron microscopy. Bacterial translocation was determined by cultures from mesenteric lymph nodes. RESULTS: Rats with induced enteritis in both phases demonstrated a long-lasting increase of epithelial paracellular permeability. In contrast, transcellular permeability was only disturbed during the active phases, coinciding with bacterial translocation and the increase in inflammatory parameters. Furthermore, although mithochondrial damage was observed throughout the inflammatory state, alterations were worse during the active phase. CONCLUSIONS: The sustained enhancement of paracellular permeability could facilitate the constant passage of luminal antigens through the mucosa, and hence, be the basis for chronicity. By contrast, transcellular permeability only increases during the active phases, when hypomotility and bacterial translocation are also present, suggesting this factor may play a critical role in the course of acute relapses in IBD.

Hepatic lipid metabolism changes in short- and long-term prehepatic portal hypertensive rats.

AIM: To verify the impairment of the hepatic lipid metabolism in prehepatic portal hypertension. METHODS: The concentrations of free fatty acids, diacylglycerol, triglycerides, and phospholipids were assayed by using D-[U-14C] glucose incorporation in the different lipid fractions and thin-layer chromatography and cholesterol was measured by spectrophotometry, in liver samples of Wistar rats with partial portal vein ligation at short- (1 mo) and long-term (1 year) (i.e. portal hypertensive rats) and the control rats. RESULTS: In the portal hypertensive rats, liver phospholipid synthesis significantly decreased (7.42 +/- 0.50 vs 4.70 +/- 0.44 nCi/g protein; P < 0.01) and was associated with an increased synthesis of free fatty acids (2.08 +/- 0.14 vs 3.36 +/- 0.33 nCi/g protein; P < 0.05), diacylglycerol (1.93 +/- 0.2 vs 2.26 +/- 0.28 nCi/g protein), triglycerides (2.40 +/- 0.30 vs 4.49 +/- 0.15 nCi/g protein) and cholesterol (24.28 +/- 2.12 vs 57.66 +/- 3.26 mg/g protein; P < 0.01). CONCLUSION: Prehepatic portal hypertension in rats impairs the liver lipid metabolism. This impairment consists in an increase in lipid deposits (triglycerides, diacylglycerol and cholesterol) in the liver, accompanied by a decrease in phospholipid synthesis.

Lipopolysaccharides facilitate colonic motor alterations associated to the sensitization to a luminal antigen in rats.

BACKGROUND/AIMS: Enteric dysbiosis is a risk factor for dietary proteins-associated intestinal alterations, contributing to the development of food allergies and the symptomatology of functional gastrointestinal disorders, mainly irritable bowel syndrome (IBS). We explored if a dysbiotic-like state, simulated by intraperitoneal administration of bacterial lipopolysaccharides (LPS), facilitates the sensitiza-tion to a luminal antigen, ovalbumin (OVA), in rats. METHODS: Rats were exposed to oral OVA for 1 week, alone or with LPS. Thereafter, colonic histology, goblet cell density, mucosal eosi-nophils and mucosal mast cell (MMC) and connective tissue mast cell (CTMC) were evaluated. Colonic expression (real-time quantitative polymerase chain reaction) of interleukins, IFN-α1 and integrins was assessed to determine local immune responses. Luminal and wall adhered microbiota were characterized by fluorescence in situ hybridization. Colonic contractility (in vitro) served to assess functional changes associated to OVA and/or LPS. RESULTS: Neither OVA nor LPS, alone or combined, lead to structural alterations, except for a reduced goblet cell density in OVA-LPS- treated rats. MMC density was unaffected, while CTMC counts increased within the submucosa of OVA-LPS-treated animals. Marginal immune activation (IFN-α1 up-regulation) was observed in OVA-LPS-treated rats. LPS induced a dysbiotic-like state characterized by decreased luminal bacterial counts, with a specific loss of clostridia. LPS facilitated Clostridium spp. wall adherence, an effect prevented by OVA. Colonic contractility was altered in OVA-LPS-treated animals, showing increased basal activity and enhanced motor responses to OVA. CONCLUSIONS: Changes in gut microbiota and/or direct effects of LPS might enhance/facilitate local neuroimmune responses to food antigens leading to motor alterations similar to those observed in IBS.

Changes in Epithelial Barrier Function in Response to Parasitic Infection: Implications for IBD Pathogenesis.

BACKGROUND AND AIMS: Mast cells [MCs] are implicated in epithelial barrier alterations that characterize inflammatory and functional bowel disorders. In this study, we describe mast cell proteinases [chymases and tryptases] and tight junction [TJ] proteins kinetics in a rat model of postinfectious gut dysfunction. METHODS: Jejunal tissues of control and -infected rats were used. Inflammation-related changes in MCs and the expression of TJ-related proteins were evaluated by immunostaining and reverse transcription-quantitative polymerase chain reaction. Epithelial barrier function was assessed in vitro (Ussing chambers) and in vivo. RESULTS: After infection, intestinal inflammation was associated with a generalized overexpression of MC chymases, peaking between Days 6 and 14. Thereafter, a mucosal MC hyperplasia and a late increase in connective tissue MC counts were observed. From Day 2 post-infection, TJ proteins occludin and claudin-3 expression was down-regulated whereas the pore-forming protein claudin-2 was overexpressed. The expression of proglucagon, precursor of the barrier-enhancing factor glucagon-like peptide-2, was reduced. These changes were associated with an increase in epithelial permeability, both in vitro and in vivo. CONCLUSIONS: Proteinases expression and location of mucosal and connective tissue MCs indicate a time-related pattern in the maturation of intestinal MCs following infection. Altered expression of TJ-related proteins is consistent with a loss of epithelial tightness, and provides a molecular mechanism for the enhanced epithelial permeability observed in inflammatory conditions of the gut.