María Dolores Suárez

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

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

Experimental Ulcerative Colitis Impairs Antioxidant Defense System in Rat Intestine

Increasing attention has been given recently to the role of free radicals in the pathogenesis of ulcerative colitis, since the inflamed intestine is exposed to oxidative stress generated by infiltrating macrophages and neutrophils within the lamina propia. The overall goal of this study was to evaluate whether experimental ulcerative colitis induces significant changes in the antioxidant defense system in an experimental model induced by the intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid. Twenty rats were treated with 80 mg/kg body weight of trinitrobenzenesulfonic acid and 20 with the same volume of 0.9% NaCl. Rats were killed at one and two weeks after treatment to evaluate colon damage by light and electron transmission microscopy. The degree of tissue injury and inflammation was determined by measuring alkaline phosphatase, γ-glutamyltranspeptidase, and myeloperoxidase activities and prostaglandin E2 and leukotriene B4. Glutathione levels and the activity of the enzymes of the antioxidant defense system were determined. Enzymatic markers of colon injury showed higher activities in rats with ulcerative colitis. Concentrations of prostaglandin E2 and leukotriene B4 were higher in the groups treated for one week with trinitrobenzenesulfonic acid and markers decreased after two weeks of treatment. All antioxidant enzyme activities were higher at one and two weeks after treatment; however, a significant decrease in total glutathione content was also observed. In conclusion, ulcerative colitis induced by trinitrobenzenesulfonic acid damages the intestinal mucosa and is accompanied by a shift in the antioxidant enzyme activities, and low levels of glutathione. This deficiency in glutathione could be a target for new therapies to treat ulcerative colitis.

Effect of Dietary Nucleotides on Intestinal Repair in Rats with Experimental Chronic Diarrhea

Nucleic acid synthesis in tissues of rapid growth is preferentially done using dietary purines and pyrimidines via the salvage pathway. In the case of a low protein intake, dietary nucleotides may be semiessential for cell replication of gut, lymphocytes, and bone marrow, and especially in those intestinal diseases in which the mucosa is altered, dietary nucleotides may have a role in intestinal development. The effect of dietary nucleotides on intestinal weight and length, gut mucosal weight, intestinal protein and DNA contents, and lactase, maltase, and intestinal mucosal activities was assessed in a controlled way. Weanling (21-day-old) rats were separated into two groups of 36, each receiving blindly a basal diet containing glucose polymers (C) or a basal diet with lactose as the main carbohydrate (L) for 15 days. Those fed with L developed a syndrome of chronic diarrhea and malnutrition. Ten rats of each group were sacrificed at that time. The rest of the animals of each group were separated into two subgroups. The first was fed with the C diet and the second with the C diet supplemented with 50 mg/100 g of each of the following nucleotides: AMP, GMP, CMP, UMP, and IMP (CN). Thus the subgroups CC, CN, LC, and LN were formed. Rats were sacrificed after 4 weeks and gut separated into three segments corresponding to duodenum, jejunum, and ileum. Analysis of variance was used to compare the effect of diet or segments. DNA and lactase, maltase, and sucrase activities increased in the LN group with respect to LC especially in jejunum and ileum but there were not any differences between CC and CN.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary Monounsaturated n-3 and n-6 Long-Chain Polyunsaturated Fatty Acids Affect Cellular Antioxidant Defense System in Rats with Experimental Ulcerative Colitis Induced by Trinitrobenzene Sulfonic Acid

The intrarectal administration oftrinitrobenzene sulfonic acid in rats induces ulcerativecolitis, which results in histological alterations ofcolonic mucosa, severe modification of the cellularantioxidant defense system, and enhanced production ofinflammatory eicosanoids. This study evaluated theinfluence of different dietary fatty acids, ie,monounsaturated, n-3, and n-3 + n-6 polyunsaturatedfatty acids, on the recovery of the colonic mucosahistological pattern, the cellular antioxidant defensesystem of colon, and PGE2 and LTB4colonic mucosa contents in a model of ulcerative colitisinduced by intrarectal administration of trinitrobenzene sulfonicacid. Administration of dietary n-3 polyunsaturatedfatty acids led to a minimum stenosis score, a higherhistological recovery, lower colon alkaline phosphatase and gamma-glutamyltranspeptidase activities,and lower mucosal levels of PGE2 andLTB4 compared with the other two experimentalgroups. However, glutathione transferase, glutathionereductase, glutathione peroxidase, and catalase activities were lowerin the group treated with n-3 polyunsaturated fattyacids than in the groups fed with either themonounsaturated or the n-6 + n-3 polyunsaturatedenriched diet. We conclude that n-3 polyunsaturatedfatty acids can be administered to prevent inflammationin ulcerative colitis, but they cause a decrease in thecolonic antioxidant defense system, promoting oxidative injury at the site of inflammation.

Effect of flavonoids on rat splenocytes, a structure–activity relationship study

Flavonoids are polyphenols frequently consumed in the diet which have been suggested to exert a number of beneficial actions on human health, including intestinal anti-inflammatory activity. Their properties have been studied in numerous cell types, but little is known about their effect on leukocyte biology. We have selected 9 flavonoids (extended to 14 flavonoids plus the related polyphenol resveratrol in some cases) with different structural features to characterize their effects on leukocyte viability, proliferation, and expression of cyclooxygenase 2 (EC 1.14.99.1), inducible nitric oxide synthase (iNOS, EC 1.14.13.39) and proinflammatory cytokines (TNF-alpha, IFN-gamma, IL-2), as well as to elucidate the structural requirements in each case. Quiescent and concanavalin A-stimulated rat splenocytes were used as a model. Flavonoids (50 microM) had a dramatic inhibitory effect on cytokine secretion. Inducible nitric oxide synthase expression was also blocked largely by some flavonoids, especially quercetin, luteolin and apigenin, while cyclooxygenase 2 was downregulated only by apigenin, diosmetin and quercetin. Apigenin, luteolin, genistein and quercetin had substantial cytotoxic/proapoptotic effects, while chrysin, daidzein, hesperetin and kaempferol did not reduce cell viability. In contrast, all flavonoids had powerful antiproliferative effects. However, none of the compounds activated caspase 3 (EC 3.4.22.56), but actually lowered caspase 3 activation and expression in concanavalin A-stimulated cells. The activity of the quercetin metabolite isorhamnetin was generally lower than that of the parent compound. We conclude that flavonoids have powerful effects on lymphocytes with distinct structural requirements that may contribute to their intestinal anti-inflammatory activity. The bioactivity of orally administered flavonoids may be dampened by biotransformation in vivo, particularly in extraintestinal sites.

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 goats 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.

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

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

Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17.

Bovine glycomacropeptide (BGMP) is an inexpensive, non‐toxic milk peptide with anti‐inflammatory effects in rat experimental colitis but its mechanism of action is unclear. It is also unknown whether BGMP can ameliorate inflammation in proximal regions of the intestine. Our aim was therefore two‐fold: first, to determine the anti‐inflammatory activity of BGMP in the ileum; second, to characterise its mechanism of action.

Dietary trans fatty acids affect docosahexaenoic acid concentrations in plasma and liver but not brain of pregnant and fetal rats.

The aim of the present study was to investigate the maternal-fetal transport, incorporation, and effects on liver delta-6 fatty-acid desaturase activity of dietary trans fatty acids in pregnant rats. Three groups of six rats each were fed three experimental diets containing approximately 0%, 15%, and 30% of trans fatty acids but containing the same proportion of linoleic (18:2 n-6) and α-linolenic (18:3 n-3) acids for 10 wk. On d 20 of pregnancy, the animals from each group were killed. We determined the fatty acid profiles in plasma, brain, and liver microsomes of pregnant rats, as well as in placenta and fetal liver and brain. No changes were found in the number of fetuses of the pregnant rats. Trans fatty acids were incorporated in high concentrations in placenta and in maternal and fetal tissues, except brain, strongly elevating the linoleic acid proportion and lowering that of docosahexaenoic acid. The delta-6 fatty-acid desaturase activity in the liver microsomes of the pregnant rats was inhibited by trans isomers. In conclusion, high intakes of trans fatty acids partially inhibit liver delta-6 fatty-acid desaturase in pregnant rats, which may explain, in part, the low concentrations of docosahexaenoic acid in pregnant and fetal tissues. However, the fatty acid composition of both fetal and pregnant rat brain remains mostly unaffected regardless of the dietary trans fatty acid content.

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-1β 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-1β, 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.