Calprotectin protects against experimental colonic inflammation in mice.

BACKGROUND AND PURPOSE: Calprotectin is a heterodimer composed of two myeloid-related proteins, S100A8 and S100A9, that is abundant in neutrophils and monocytes/macrophages. Faecal levels of calprotectin are used routinely to monitor inflammatory bowel disease activity. EXPERIMENTAL APPROACH: We aimed to assess the role of calprotectin in intestinal inflammation, using the dextran sulfate sodium model of colitis in mice. Calprotectin was administered (50 or 100 µg·day-1 ) by the intrarectal or by i.p. injection (50 µg·day-1 only). The condition of the mice was characterized by morphological and biochemical methods. KEY RESULTS: Intrarectal calprotectin protected significantly against colitis, as shown by lower levels of macroscopic and microscopic damage, colonic myeloperoxidase activity and decreased expression of TNFα and toll-like receptor 4. IL-17 production by spleen and mesenteric lymph node cells was reduced. Calprotectin had no effect on body weight loss or colonic thickening. There were no effects of calprotectin after i.p. injection. Calprotectin had virtually no effects in control, non-colitic mice. Calprotectin had almost no effect on the colonic microbiota but enhanced barrier function. Treatment of rat IEC18 intestinal epithelial cells in vitro with calprotectin induced output of the chemokines CXL1 and CCL2, involving the receptor for advanced glycation end products- and NFκB. CONCLUSION AND IMPLICATIONS: Calprotectin exerted protective effects in experimental colitis when given by the intrarectal route, by actions that appear to involve effects on the epithelium.

A synbiotic composed of Lactobacillus fermentum CECT5716 and FOS prevents the development of fatty acid liver and glycemic alterations in rats fed a high fructose diet associated with changes in the microbiota.

We investigated the effect of a high fructose diet (HFD) on Sprague Dawley rats and the impact of a synbiotic composed of Lactobacillus fermentum CECT5716 and fructooligosaccharides. Feeding the HFD for 5 weeks resulted in liver steatosis and insulin resistance but not obesity. These changes were associated with increased production of short-chain fatty acids and increased Bacteroidetes in feces, with an augmented Bacteroidetes/Firmicutes ratio, among other changes in the microbiota. In addition, barrier function was weakened, with increased LPS plasma levels. These data are consistent with increased fructose availability in the distal gut due to saturation of absorptive mechanisms, leading to dysbiosis, endotoxemia, hepatic steatosis, and insulin resistance. Treatment with the synbiotic prevented some of the pathological effects, so that treated rats did not develop steatosis or systemic inflammation, while dysbiosis and barrier function were greatly ameliorated. In addition, the synbiotic had hypolipidemic effects. The synbiotic composed by L. fermentum CECT5716 and fructooligosaccharides has beneficial effects in a model of metabolic syndrome induced by a HFD, suggesting it might be clinically useful in this type of condition, particularly considering that high fructose intake has been related to metabolic syndrome in humans.

The Bisphosphonate Pamidronate is an Intestinal Antiinflammatory Agent in Rat and Mouse Experimental Colitis.

BACKGROUND: Statins have antiinflammatory effects at the cardiovascular level because of inhibition of prenylation, which also probably underlies their therapeutic effects in preclinical models of inflammatory bowel disease. Another inhibitor of prenylation, namely alendronate, reduces colitis in rodents. In this study, we aim to explore the therapeutic potential of second-generation, nitrogen-containing bisphosphonates in 3 preclinical models of colitis. METHODS: The trinitrobenzenesulfonic acid and dextran sulfate sodium models of rat colitis and the adoptive lymphocyte transfer model of colitis in mice were used. Pamidronate, alendronate, and ibandronate were tested. Treatments were administered in equimolar doses through the oral or intraperitoneal route. The effect of pamidronate on prenylation and cytokine release was assessed in vivo and in vitro. RESULTS: Pretreatment with pamidronate, but not with ibandronate or alendronate, improves chemically induced trinitrobenzenesulfonic acid and dextran sulfate sodium colitis in rats. Moreover, this beneficial effect is extended to lymphocyte transfer colitis. Pamidronate has no effect on intestinal epithelial cells in vitro in terms of cytokine/chemokine release, but enhances IFN-γ, IL-6, and IL-10 production by T cells in coculture. Pamidronate also exerts a direct immunomodulatory effect on T cells, favoring Th1 differentiation and impairing Th17 polarization. CONCLUSIONS: Pamidronate presents antiinflammatory and immunomodulatory properties in 3 different models of experimental colitis in rodents. This effect requires oral administration and may involve T cells in the gut mucosa, although the exact mechanism is unclear.

The glucocorticoid budesonide has protective and deleterious effects in experimental colitis in mice.

Glucocorticoids are widely used for the management of inflammatory bowel disease, albeit with known limitations for long-term use and relevant adverse effects. In turn, they have harmful effects in experimental colitis. We aimed to explore the mechanism and possible implications of this phenomenon. Regular and microbiota depleted C57BL/6 mice were exposed to dextran sulfate sodium (DSS) to induce colitis and treated with budesonide. Colonic inflammation and animal status were compared. In vitro epithelial models of wound healing were used to confirm the effects of glucocorticoids. Budesonide was also tested in lymphocyte transfer colitis. Budesonide (1-60µg/day) exerted substantial colonic antiinflammatory effects in DSS colitis. At the same time, it aggravated body weight loss, increased rectal bleeding, and induced general deterioration of animal status, bacterial translocation and endotoxemia. As a result, there was an associated increase in parameters of sepsis, such as plasma NOx, IL-1ß, IL-6, lung myeloperoxidase and iNOS, as well as significant hypothermia. Budesonide also enhanced DSS induced colonic damage in microbiota depleted mice. These effects were correlated with antiproliferative effects at the epithelial level, which are expected to impair wound healing. In contrast, budesonide had significant but greatly diminished deleterious effects in noncolitic mice or in mice with lymphocyte transfer colitis. We conclude that budesonide weakens mucosal barrier function by interfering with epithelial dynamics and dampening the immune response in the context of significant mucosal injury, causing sepsis. This may be a contributing factor, at least in part, limiting clinical usefulness of corticoids in inflammatory bowel disease.

Germ-free and Antibiotic-treated Mice are Highly Susceptible to Epithelial Injury in DSS Colitis.

BACKGROUND AND AIMS: Intestinal microbiota is required to maintain immune homeostasis and intestinal barrier function. At the same time, intraluminal bacteria are considered to be involved in inflammatory bowel disease and are required for colitis induction in animal models, with the possible exception of dextran sulphate sodium [DSS] colitis. This study was carried out to ascertain the mechanism underlying the induction of colitis by DSS in the absence of bacteria. METHODS: Conventional and germ-free [GF] Naval Medical Research Institute [NMRI] mice were used, plus conventional mice treated with an antibiotic cocktail to deplete the intestinal microbiota ['pseudo-GF' or PGF mice]. The differential response to DSS was assessed. RESULTS: Conventional mice developed DSS-induced colitis normally, whereas GF mice showed only minimal inflammation [no colonic thickening, lower myeloperoxidase activity, IL-6, IL-17, TNF-α, and IFN-γ secretion by splenocytes and mesenteric cell cultures, etc.]. However, these mice suffered enhanced haemorrhage, epithelial injury and mortality as a consequence of a weakened intestinal barrier, as shown by lower occludin, claudin 4, TFF3, MUC3, and IL-22. In contrast, PGF mice had a relatively normal, albeit attenuated, inflammatory response, but were less prone to haemorrhage and epithelial injury than GF mice. This was correlated with an increased expression of IL-10 and Foxp3 and preservation barrier-related markers. CONCLUSIONS: We conclude that enteric bacteria are essential for the development of normal DSS-induced colitis. The absence of microbiota reduces DSS colonic inflammation dramatically but it also impairs barrier function, whereas subtotal microbiota depletion has intermediate effects at both levels.

Fructooligosaccharides exert intestinal anti-inflammatory activity in the CD4+ CD62L+ T cell transfer model of colitis in C57BL/6J mice.

PURPOSE: Fructooligosaccharides (FOS) are used as functional foods due to their prebiotic effects. Intestinal anti-inflammatory activity has been established in most, but not all, studies in animal models of colitis, using mainly chemically induced inflammation. Our goal was to test the effect of FOS (degree of polymerization 2-8) in the chronic, lymphocyte-driven CD4+ CD62L+ T cell transfer model of colitis. METHODS: Colitis was induced by transfer of CD4+ CD62L+ T cells to C57BL/6J Rag1(-/-) mice. FOS (75 mg day(-1)) was administered by gavage as a post-treatment. Three groups were established: non-colitic (NC), colitic control (C, CD4+ CD62L+ transferred mice treated with vehicle) and colitic+FOS (C+FOS, similar but treated with FOS). Mice were killed after 13 days. RESULTS: Treatment of mice with FOS ameliorated colitis, as evidenced by an increase in body weight, a lesser myeloperoxidase and alkaline phosphatase activities, a lower secretion of proinflammatory cytokines by mesenteric lymph node cells ex vivo (IFN-γ, IL-17, and TNF-α), and a higher colonic expression of occludin (C+FOS vs. C, p < 0.05). Increased relative abundance of lactic acid bacteria was observed in FOS-treated mice (p < 0.05). CONCLUSIONS: FOS exert intestinal anti-inflammatory activity in T lymphocyte-dependent colitis, suggesting it may be useful in the management of inflammatory bowel disease in appropriate conditions.

Expression of glucose transporters in the prelaminar region of the optic-nerve head of the pig as determined by immunolabeling and tissue culture.

BACKGROUND: To develop the use of cultured tissue of the prelaminar optic nerve of the pig to explore possible alterations of the astrocyte-axon metabolic pathways in glaucoma, we map the distribution of the glucose transporters GLUT1 and GLUT3 in fresh and cultured tissue. METHODS: We monitor cell survival in cultures of the prelaminar optic-nerve tissue, measuring necrosis and apoptosis markers biochemically as well as morphologically, and establish the presence of the glucose transporters GLUT1 and GLUT3. We map the distribution of these transporters with immunolabeling in histological sections of the optic nerve using confocal and electronic transmission microscopy. RESULTS: We find that the main death type in prelaminar culture is apoptosis. Caspase 7 staining reveals an increment in apoptosis from day 1 to day 4 and a reduction from day 4 to day 8. Western blotting for GLUT1 shows stability with increased culture time. CLSM micrographs locate GLUT1 in the columnar astrocytes and in the area of axonal bundles. Anti-GLUT3 predominantly labels axonal bundles. TEM immunolabeling with colloidal gold displays a very specific distribution of GLUT-1 in the membranes of vascular endothelial cells and in periaxonal astrocyte expansions. The GLUT-3 isoform is observed with TEM only in axons in the axonal bundles. CONCLUSIONS: Tissue culture is suitable for apoptosis-induction experiments. The results suggest that glucose is transported to the axonal cleft intracytoplasmically and delivered to the cleft by GLUT1 transporters. As monocarboxylate transporters have been reported in the prelaminar region of the optic-nerve head, this area is likely to use both lactate and glucose as energy sources.

A new therapeutic association to manage relapsing experimental colitis: Doxycycline plus Saccharomyces boulardii.

Immunomodulatory antibiotics have been proposed for the treatment of multifactorial conditions such as inflammatory bowel disease. Probiotics are able to attenuate intestinal inflammation, being considered as safe when chronically administered. The aim of the study was to evaluate the anti-inflammatory effects of doxycycline, a tetracycline with immunomodulatory properties, alone and in association with the probiotic Saccharomyces boulardii CNCMI-745. Doxycycline was assayed both in vitro (Caco-2 epithelial cells and RAW 264.7 macrophages) and in vivo, in the trinitrobenzenesulfonic acid (TNBS) model of rat colitis and the dextran sodium sulfate (DSS) model of mouse colitis. In addition, the anti-inflammatory effect of the association of doxycycline and the probiotic was evaluated in vitro and in vivo in a DSS model of reactivated colitis in mice. Doxycycline displayed immunomodulatory activity in vitro, reducing IL-8 production by intestinal epithelial cells and nitric oxide by macrophages. Doxycycline administration to TNBS-colitic rats (5, 10 and 25 mg/kg) ameliorated the intestinal inflammatory process, being its efficacy comparable to that previously showed by minocycline. Doxycycline treatment was also effective in reducing acute intestinal inflammation in the DSS model of mouse colitis. The association of doxycycline and S. boulardii helped managing colitis in a reactivated model of colitis, by reducing intestinal inflammation and accelerating the recovery and attenuating the relapse. This was evidenced by a reduced disease activity index, colonic tissue damage and expression of inflammatory mediators. This study confirms the intestinal anti-inflammatory activity of doxycycline and supports the potential use of its therapeutic association with S. boulardii for the treatment of inflammatory bowel diseases, in which doxycycline is used to induce remission and long term probiotic administration helps to prevent the relapses.

Intestinal anti-inflammatory activity of apigenin K in two rat colitis models induced by trinitrobenzenesulfonic acid and dextran sulphate sodium.

Flavonoids are polyphenolic compounds that are widespread in nature, and consumed as part of the human diet in significant amounts. The aim of the present study was to test the intestinal anti-inflammatory activity of apigenin K, a soluble form of apigenin, in two models of rat colitis, namely the trinitrobenzenesulfonic acid (TNBS) model and the dextran sulphate sodium (DSS) model. Apigenin K (1, 3 and 10 mg/kg; by the oral route; n 4-6 per group) was administered as a pre-treatment to rats with TNBS and DSS colitis, and colonic status was checked by macroscopic and biochemical examination. Apigenin K pre-treatment resulted in the amelioration of morphological signs and biochemical markers in the TNBS model. The results demonstrated a reduction in the inflamed area, as well as lower values of score and colonic weight:length ratio compared with the TNBS group. Myeloperoxidase (MPO) activity was reduced by 30 % (P< 0·05). Moreover, apigenin K pre-treatment ameliorated morphological signs and biochemical markers in the DSS model. Thus, macroscopic damage was significantly reduced and the colonic weight:length ratio was lowered by approximately 10 %, while colonic MPO and alkaline phosphatase activities were decreased by 35 and 21 %, respectively (P< 0·05). Apigenin K pre-treatment also tended to normalise the expression of a number of colonic inflammatory markers (e.g. TNF-α, transforming growth factor-ß, IL-6, intercellular adhesion molecule 1 or chemokine (C-C motif) ligand 2). In conclusion, apigenin K is found to have anti-inflammatory effects in two preclinical models of inflammatory bowel disease.

Rutin has intestinal antiinflammatory effects in the CD4+ CD62L+ T cell transfer model of colitis.

Rutin, one of the most abundant flavonoids in nature, has been shown to exert intestinal antiinflammatory effects in experimental models of colitis. Our aim was to study the antiinflamatory effect of rutin in the CD4+ CD62L+ T cell transfer model of colitis, one of the closest to the human disease. Colitis was induced by transfer of CD4+ CD62L+ T cells to Rag1(-/-) mice. Rutin was administered by gavage as a postreatment. Treatment with rutin improved colitis at the dose of 57mg/kg/day, while no effect was noted with 28.5mg/kg/day. Therapeutic benefit was evidenced by a reduced disease activity index, weight loss and damage score, plus a 36% lower colonic myeloperoxidase and a 54% lower alkaline phosphatase activity. In addition, a decreased secretion of proinflammatory cytokines (IFNγ and TNFα) by mesenteric lymph node cells was observed ex vivo. The colonic expression of proinflammatory genes, including IFNγ, TNFα, CXCL1, S100A8 and IL-1ß, was significantly reduced by more than 80% with rutin as assessed by RT-qPCR. Flavonoid treated mice exhibited decreased activation of splenic CD4+ cells (STAT4 phosphorylation and IFNγ expression) and reduced plasma cytokine levels. This effect was also apparent in mucosal lymphocytes based on reduced STAT4 phosphorylation. The protective effect was comparable to that of 3mg/kg/day budesonide. Rutin had no effect on splenocytes or murine T cells in vitro, while its aglycone, quercetin, exhibited a concentration dependent inhibition of proinflammatory cytokines, including IFNγ. Rutin but not quercetin showed vectorial basolateral to apical transport in IEC18 cells, associated with reduced biotransformation. We conclude that rutin exerts intestinal antiinflammatory activity in chronic, T lymphocyte dependent colitis via quercetin release and actions involving mucosal and lymph node T cells. Our results suggest that rutin may be useful in the management of inflammatory bowel disease in appropriate dosage conditions.

Active hexose correlated compound exerts therapeutic effects in lymphocyte driven colitis.

Active hexose correlated compound (AHCC) is a commercial extract of Basidiomycetes fungi enriched in oligosaccharides that is used as a human nutritional supplement for various purposes in humans. Our aim was to study the anti-inflammatory effect of AHCC in the CD4+ CD62L(+) T cell transfer model of colitis, considered one of the closest to the human disease. Colitis was induced by transfer of CD4(+) CD62L(+) T cells to recombination activating gene 1(-/-) mice. AHCC (75 mg/d) was administered by gavage as a post-treatment. Three groups were established: noncolitic, colitic (CD4(+) CD62L(+) transferred mice treated with vehicle), and AHCC (colitic treated with AHCC). AHCC improved colitis, as evidenced by a 24% lower colonic myeloperoxidase and a 21% lower alkaline phosphatase activity. In addition, a decreased secretion of proinflammatory genes assessed by RT-qPCR was observed, particularly TNF-α and IL-1ß. Ex vivo mesenteric lymph node cells obtained from AHCC treated mice exhibited a fully normalized production of IL-6, IL-17, and IL-10 (p < 0.05). Also, AHCC treated mice exhibited decreased STAT4 and IκB-α phosphorylation in splenic CD4(+) cells. Our data provide validation of AHCC colonic anti-inflammatory activity in a chronic, T cell driven model of inflammatory bowel disease.

Intestinal anti-inflammatory effects of oligosaccharides derived from lactulose in the trinitrobenzenesulfonic acid model of rat colitis.

Intestinal microbiota modulation is becoming an interesting approach to manage inflammatory bowel disease and can be achieved by the administration of prebiotics. Previous studies showed the intestinal anti-inflammatory effects of the prebiotic lactulose. The aim of the present study was to test the preventative effects of oligosaccharides derived from lactulose with prebiotic properties (OsLu) in the trinitrobenzenesulfonic acid model of rat colitis and compare them with those of lactulose. Both treatments modified bacterial profile in intestinal contents, increasing the bifidobacteria and lactobacilli counts and up-regulating the production of short-chain fatty acids, although OsLu generated a larger amount. OsLu also inhibited to a greater extent different pro-inflammatory markers such as interleukins (IL) 1, 6, 12, and 23 and chemokines (MCP-1 and CINC-1). However, both prebiotics equally restored colonic epithelial integrity, evaluated both with a histological score (OsLu, 9.8 ± 2.2; and lactulose, 12.1 ± 2.1, vs colitic control, 27.3 ± 3.3) and by measuring several key proteins of the mucosal barrier (MUC-2, MUC-3, and TTF-3). OsLu effect was also associated with an inhibition of iNOS expression and a reduction of Th17 cell activity in the inflamed tissue that facilitated the intestinal mucosa barrier recovery. In conclusion, OsLu showed a better anti-inflammatory profile than lactulose in this model of experimental colitis.

Prebiotic oligosaccharides directly modulate proinflammatory cytokine production in monocytes via activation of TLR4.

SCOPE: Prebiotic oligosaccharides are currently used in a variety of clinical settings for their effects on intestinal microbiota. Here, we have examined the direct, microbiota independent, effects of prebiotics on monocytes and T lymphocytes in vitro. METHODS AND RESULTS: Prebiotics generally evoked cytokine secretion (TNF-α, IL-6, and IL-10) by mouse splenocytes but inhibited LPS -induced IFN-γ and IL-17 release. Inulin was found to enhance LPS-induced IL-10 secretion. Splenocytes from TLR4(-/-) (where TLR is Toll-like receptor) mice showed a markedly depressed response. Conversely, in both basal and LPS-stimulated conditions, prebiotic inhibition of IFN-γ levels was preserved. These results suggested a predominant effect on monocytes via TLR4 ligation and possible inhibition of T cells. Hence, we studied the modulation of primary rat monocytes and T lymphocytes, focusing on fructooligosaccharides (FOS) and inulin. In monocytes, FOS and inulin induced TNF-α, growth-regulated oncogene α, and IL-10, but not IL-1ß release. The NF-κB inhibitor Bay 11-7082 fully prevented these effects. Pharmacological evidence also indicated a significant involvement of mitogen-activated protein kinase and phosphatidylinositol-3-kinase. There was little effect on T cells. FOS and inulin also generally increased TNF-α, IL-1ß, and IL-10, but not IL-8, in human peripheral blood monocytes. CONCLUSION: We conclude that prebiotics may act as TLR4 ligands or as indirect TLR4 modulators to upregulate cytokine secretion in monocytes.

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.

Negative clinical outcomes of medication resulting in emergency department visits.

PURPOSE: The results of analyses of patients' health problems related to medication use have been highly variable due to various factors, such as different study methodology, diverse variables determined, fields of study. The aim of our study was to determine the prevalence and preventability of negative clinical outcomes of medication (NCOMs). METHODS: This was a cross-sectional study performed in the emergency departments (EDs) of nine Spanish hospitals during a 3-month period. A two-stage probabilistic sampling method was used , and a systematic appraisal tool was used to identify the NCOMs based on information gathered through patient interview and review of the medical records. Case evaluations were conducted in two phases by pharmacists and physicians. The prevalence and preventability of NCOM were calculated. A homogeneity test was performed to assess potential differences in the prevalence for each hospital. RESULTS: A total of 4,611 patients were included in the study. The overall prevalence of NCOMs was 35.7 % [95 % confidence interval (CI) 33.3-38.1]. These NCOMs could be divided into three categories: ineffectiveness (18.2 %; 95 % CI 16.2-20.1), necessity (14.9 %; 95 % CI 13.4-16.6), and lack of safety (2.4 %; 95 % CI 1.9-2.8). About 81 % (95 % CI 80.1-82.3) of the NCOMs could have been prevented. CONCLUSIONS: NCOMs provoked approximately one-third of visits to the EDs, and a high percentage of these were preventable. Implementation of strategies for patient safety and pharmaceutical care could help to prevent these problems and optimize the use of medications.

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.

Active colitis exacerbates immune response to internalized food antigens in mice.

BACKGROUND: Previous studies have indicated that colitis increases intestinal permeability to food antigens. This condition also generates an immunoreactive milieu in the gut, which may exacerbate or counteract allergy reactions. This, along with the fact that both colitis and allergy are being codiagnosed more frequently, means the scientific interest on the immune relation between these pathologies is increasing. We evaluated the immune response to an internalized food antigen that was initiated during a concomitant active intestinal inflammatory response. METHODS: An ovalbumin (OVA)-induced immune response was analyzed in healthy mice and in mice suffering from colitis induced by the administration of dinitrofluorobenzene/dinitrosulfonic acid (DNFB/DNS) at the moment of OVA challenge. The OVA-induced clinical score and allergy response both in plasma and in splenocyte cultures from these animals were compared. RESULTS: Although no differences were observed in the allergy clinical score, the concomitant active colitis led to an increase in the immune response to OVA antigen, as shown by increased spleen size and OVA-induced splenocyte proliferation, exacerbated expression of total and OVA-specific IgG1 levels, increased colonic IL-4 expression and OVA-induced IL-4 and IL-5 cytokine expression in spleen cells. CONCLUSIONS: Our results indicate that animals with active colitis undergo an exacerbated immune response to an internalized antigen. This finding could be relevant for the allergy management of patients presenting simultaneously with chronic colitis.

Novel effects of the cannabinoid inverse agonist AM 251 on parameters related to metabolic syndrome in obese Zucker rats.

BACKGROUND AND OBJECTIVE: Recent research suggests that cannabinoid receptor CB1 antagonists can affect appetite and body weight gain, although their influence on other parameters related to metabolic syndrome is not well documented. The present study was designed to assess the effects of chronic treatment with the CB1 receptor inverse agonist AM 251 (3 mg/kg for 3 weeks) in obese and lean Zucker rats on parameters related to metabolic syndrome. MATERIALS AND METHODS: Four groups of rats were used: lean Zucker rats, untreated obese Zucker rats, AM 251-treated obese Zucker rats and a pair-fed obese Zucker rat experimental group which received the same amount of food as that consumed by the animals treated with AM251. Food intake, body weight gain, energy expenditure, plasma biochemical parameters, leptin, insulin and hepatic status markers were analysed. RESULTS: Daily injection of AM 251 in obese Zucker rats produced a marked and sustained decrease in daily food intake and body weight and a considerable increase in energy expenditure in comparison with untreated obese Zucker rats. AM 251 administration to obese rats significantly reduced plasma levels of glucose, leptin, AST, ALT, Gamma GT, total bilirubin and LDL cholesterol whereas HDL cholesterol plasma levels increased. The results also showed a decrease in liver/weight body ratio and total fat content in the liver. The main effects of AM251 (3 mg/kg) found in this study were not observed in pair-fed obese animals, highlighting the additional beneficial effects of treatment with AM 251. The results obtained in obese rats can be interpreted as a decrease in leptin and insulin resistance, thereby improving glucose and lipid metabolism, alleviating the steatosis present in the metabolic syndrome and thus favourably modifying plasma levels of hepatic biomarkers. CONCLUSION: Our results indicate that the cannabinoid CB1 inverse agonist AM 251 represents a promising therapeutic strategy for the treatment of obesity and metabolic syndrome.

Effects of peroxisome proliferator-activated receptor-ß activation in endothelin-dependent hypertension.

AIMS: We analysed the chronic effects of the peroxisome proliferator-activated receptor ß/δ (PPAR-ß) agonist GW0742 on the renin-independent hypertension induced by deoxycorticosterone acetate (DOCA)-salt. METHODS AND RESULTS: Rats were treated for 5 weeks with: control-vehicle, control-GW0742 (5 or 20 mg kg(-1) day(-1)), DOCA-vehicle, DOCA-GW0742 (5 or 20 mg kg(-1) day(-1)), DOCA-GSK0660 (1 mg kg(-1) day(-1)), and DOCA-GSK0660-GW0742. Rats receiving DOCA-vehicle showed increased systolic blood pressure, left ventricular and kidney weight indices, endothelin-1 (ET-1), and malondialdehyde plasma levels, urinary iso-PGF2α excretion, impaired endothelium-dependent relaxation to acetylcholine, and contraction to ET-1 when compared with controls. Aortic reactive oxygen species content, NADPH oxidase activity, and p47(phox), p22(phox), NOX-4, glutathione peroxidase 1, hemeoxygenase-1, and preproET-1 expression were increased, whereas catalase and regulators of G protein-coupled signalling proteins (RGS)5 expression were decreased in the DOCA-vehicle group. GW0742 prevented the development of hypertension in a dose-dependent manner but the reduction of renal and cardiac hypertrophy, systemic and vascular oxidative stress markers, and improvement of endothelial dysfunction were only observed after the higher dose. GW0742, at 20 mg kg(-1) day(-1), attenuated ET-1 contraction by increasing RGS5 expression and restored the intracellular redox balance by reducing NADPH-oxidase activity, and by increasing the antioxidant genes expression. The PPAR-ß antagonist GSK0660 prevented all vascular changes induced by GW0742 but not its antihypertensive effects. CONCLUSION: Vascular protective effects of GW0742 operate via PPAR-ß by interference with the ET-1 signalling as a result of increased expression of RGS5 and up-regulation of antioxidant genes and via PPAR-ß-independent mechanisms to decrease blood pressure.

Intestinal anti-inflammatory activity of hydroalcoholic extracts of Phlomis purpurea L. and Phlomis lychnitis L. in the trinitrobenzenesulphonic acid model of rat colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Different species from genus Phlomis, frequently native from the the eastern Mediterranean zone, have been used in traditional medicine as an anti-inflammatory remedy. Among other constituents, they contain polyphenols that show antioxidant properties, which are interesting for the treatment of inflammatory pathologies associated with oxidative stress in humans, such as inflammatory bowel disease (IBD). The aim of this study was to evaluate the intestinal anti-inflammatoy effect of hydroalcoholic extracts of Phlomis lychnitis and P. purpurea in the trinitrobenzenesulphonic acid (TNBS) model of rat colitis, a well characterized experimental model with some resemblance to human IBD. MATERIALS AND METHODS: Hydroalcoholic extracts of both plants were characterized by determining their polyphenolic content and then assayed in the TNBS model of rat colitis. For this purpose, female Wistar rats were assigned to seven groups (n=10): healthy control, untreated TNBS-colitis and five TNBS- colitis groups treated with Phlomis lychnitis (10 and 20mg/kg), P. purpurea (10 and 25mg/kg) and sulphasalazine (200mg/kg), as a positive control. Treatments started the same day of TNBS colitis induction, and rats were sacrificed one week later. Colonic inflammation was evaluated both histologically and biochemically. RESULTS: The histological (macroscopic and microscopic) analysis of colonic samples revealed that both extracts showed an anti-inflammatory effect, which was confirmed biochemically by a decreased colonic MPO activity, a maker of neutrophil infiltration, an increased colonic glutathione content, which counteracts the oxidative status associated with the inflammatory process, and a down-regulated iNOS expression. However, only the extract of P. purpurea reduced the expression of the proinflammatory cytokines IL-1ß and IL-17, the chemokines CINC-1 and MCP-1, as well as the adhesion molecule ICAM-1, ameliorating the altered immune response associated with the colonic inflammation. Furthermore, both P. lychnitis and P. purpurea extracts were able to significantly increase the expression of markers of epithelial integrity such as MUC-2, MUC-3 and villin, thus revealing an improvement in the altered colonic permeability that characterizes colonic inflammation. CONCLUSIONS: Both extracts showed intestinal anti-inflammatory activity in the TNBS model of rat colitis, thus confirming their traditional use in digestive inflammatory complaints. In addition to their antioxidant properties, other mechanisms can contribute to this beneficial effect, like an improvement in the intestine epithelial barrier and a downregulation of the immune response.