Fermín Sánchez de Medina

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.

Intestinal inflammation and mucosal barrier function.

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

Anti‐inflammatory effect of diosmectite in hapten‐induced colitis in the rat

Diosmectite is a natural silicate effectively used in the treatment of infectious diarrhoea. Its antidiarrhoeal properties involve adsorption of toxins and bacteria and modifications of the rheological characteristics of gastrointestinal mucus. Hence, the aim of this study was to test the intestinal anti‐inflammatory activity of diosmectite. Diosmectite (500 mg kg−1 day−1, p.o.) was administered as a post‐treatment to rats with chronic trinitrobenzene sulphonic acid colitis. Colonic status was checked 1 and 2 weeks after colitis induction by macroscopic, histological and biochemical examination. Diosmectite post‐treatment resulted in amelioration of the morphological signs (intestinal weight, macroscopic damage, necrosed area, histology) and biochemical markers (myeloperoxidase activity, glutathione levels, MUC2 expression, inducible nitric oxide synthase and interleukin‐1β (IL‐1β) and leukotriene B4 synthesis), as well as in the reduction of the severity of diarrhoea. The effect of the clay was comparable to that of sulphasalazine (50 mg kg−1 day−1). Diosmectite exhibited a dose‐dependent capacity to adsorb proteins in vitro as well as a dose‐dependent inhibitory effect on the basolateral secretion of IL‐8 by lipopolysaccharide (LPS)‐stimulated HT29 cells. Diosmectite had a dose‐dependent inhibitory effect on IL‐1β production by LPS‐stimulated THP‐1 cells. The effect of diosmectite on MUC2 was post‐transcriptional, since mRNA levels were unaffected. However, diosmectite is able to upregulate MUC2 mRNA levels in HT29‐MTX cells. Diosmectite has anti‐inflammatory activity administered as a post‐treatment. Possible mechanisms include adsorption of luminal antigens, increase of colonic mucin levels and possibly a direct modulatory action of cytokine production by mucosal cells.

Serum amino acid changes in rats with thioacetamide-induced liver cirrhosis

To date, no attempt has been made to study alterations occurring in the amino acid profile in chronic models of thioacetamide-induced liver cirrhosis. In this work, changes in serum amino acids and proteins in rats with thioacetamide-induced liver cirrhosis are reported, together with changes in enzyme activities in the liver and serum. Seventeen female Wistar rats were used. Eight rats were given 300 mg thioacetamide/l in drinking water for 4 months and nine rats were given water ad libitum during the same time-period. Significant increases in glycine, alanine, serine, methionine, glutamate, ornithine, phenylalanine, tyrosine, histidine and proline were observed in rats with the resulting experimental liver cirrhosis. Threonine, taurine, glutamine, lysine and citrulline tended to increase while isoleucine, leucine, aspartate, arginine and tryptophan tended to decrease. Total and nonessential amino acids increased significantly in cirrhotic animals. Total essential and aromatic amino acids tended to increase in the thioacetamide-treated group, whereas branched chain amino acids tended to decrease in the same group. Regarding serum proteins, a decrease in albumin concentration in the thioacetamide-treated animals was the only change detected. The liver enzyme activities under observation (aspartate and alanine aminotransferases, glutamate dehydrogenase and threonine deaminase) were lower in the thioacetamide group. Decreases were significant for both transaminases and threonine deaminase. Results for serum activities showed that transaminases did not change in thioacetamide-treated rats in comparison with controls. In contrast, alkaline phosphatase rose dramatically in cirrhotic rats. We conclude that the serum amino acid pattern in this chronic model of liver cirrhosis resembles in part that of the corresponding human disease.

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.

Chemoprevention, chemotherapy, and chemoresistance in colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death in industrialized countries. Chemoprevention is a promising approach, but studies demonstrating their usefulness in large populations are still needed. Among several compounds with chemopreventive ability, cyclooxygenase inhibitors have received particular attention. However, these agents are not without side effects, which must be weighed against their beneficial actions. Early diagnosis is critical in the management of CRC patients, because, in early stages, surgery is curative in >90% of cases. If diagnosis occurs at stages II and III, which is often the case, neoadjuvant chemotherapy and radiotherapy before surgery are, in a few cases, recommended. Because of the high risk of recurrence in advanced cancers, chemotherapy is maintained after tumor resection. Chemotherapy is also indicated when the patient has metastases and in advanced cancer located in the rectum. In the last decade, the use of anticancer drugs in monotherapy or in combined regimens has markedly increased the survival of patients with CRC at stages III and IV. Although the rate of success is higher than in other gastrointestinal tumors, adverse effects and development of chemoresistance are important limitations to pharmacological therapy. Genetic profiling regarding mechanisms of chemoresistance are needed to carry out individualized prediction of the lack of effectiveness of pharmacological regimens. This would minimize side effects and prevent the selection of aggressive, cross-resistant clones, as well as avoiding undesirable delays in the use of the most efficient therapeutic approaches to treat these patients.

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.

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.

Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-κB signal transduction pathways

Background and purpose:  Bovine glycomacropeptide (BGMP) is a natural milk peptide that is produced naturally in the gastrointestinal tract during digestion. Glycomacropepide has intestinal anti‐inflammatory activity, but the mechanism of action is unknown. Here we have characterized the effects of BGMP on monocytes.