Carlos J. Aranda

Dose-dependent antiinflammatory effect of ursodeoxycholic acid in experimental colitis.

The denomination of inflammatory bowel disease comprises a group of chronic inflammatory diseases of the digestive tract, ulcerative colitis and Crohns disease being the most important conditions. Bile acids may play a role both in etiology and pharmacology of this disease. Thus, although deoxycholic acid is regarded as a proinflammatory agent ursodeoxycholic acid, which is currently being used to treat certain types of cholestasis and primary biliary cirrhosis, because of their choleretic, cytoprotective and immunomodulatory effects, it has been reported to exert an anti-inflammatory activity. We aim to confirm and characterize the intestinal antiinflammatory activity of ursodeoxycholic acid. The experimental model trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats has been used. Animal status was characterized by a number of macroscopic and biochemical parameters. Oral administration of ursodeoxycholic acid was able to ameliorate experimental colonic inflammation. This occurred only at a relatively high dose (50 mg/kg day), whereas ursodeoxycholic acid was without significant effect at doses of 10 and 25 mg/kg day. The therapeutic effect was evidenced, among others, by a higher body weight recovery, a diminished affected to total mucosal area and lower alkaline phosphatase activity in treated vs. control (TNBS treated) animals. These results indicate that, at the appropriate dose, ursodeoxycholic acid is a potentially useful drug to reduce intestinal inflammation and could be envisaged to be incorporated in the treatment of inflammatory bowel diseases.

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-1β, 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.

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-1β 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.

The nutritional supplement Active Hexose Correlated Compound (AHCC) has direct immunomodulatory actions on intestinal epithelial cells and macrophages involving TLR/MyD88 and NF-κB/MAPK activation.

Active Hexose Correlated Compound (AHCC) is an immunostimulatory nutritional supplement. AHCC effects and mechanism of action on intestinal epithelial cells or monocytes are poorly described. AHCC was added to the culture medium of intestinal epithelial cells (IEC18 and HT29 cells) and monocytes (THP-1 cells) and assessed the secretion of proinflammatory cytokines by ELISA. Inhibitors of NFκB and MAPKs were used to study signal transduction pathways while TLR4 and MyD88 were silenced in IEC18 cells using shRNA. It was found that AHCC induced GROα and MCP1 secretion in IEC18 and IL-8 in HT29 cells. These effects depended on NFκB activation, and partly on MAPKs activation and on the presence of MyD88 and TLR4. In THP-1 cells AHCC evoked IL-8, IL-1β and TNF-α secretion. The induction of IL-8 depended on JNK and NFκB activation. Therefore, AHCC exerts immunostimulatory effects on intestinal epithelial cells and monocytes involving TLR4/MyD88 and NFκB/MAPK signal transduction pathways.

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-1β, 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.

Biosimilars: Concepts and controversies

Graphical abstract Figure. No Caption available. &NA; Biosimilars are copies of reference biological drugs, developed as the patents for original biologicals expire. They are thus developed to replicate an original biological medicine just a generics are intended to replicate a chemically‐synthesized medicine; however, there are important technical and regulatory differences between the two. Unlike chemical drugs, molecular identity cannot generally be established for any two biological drugs. Accordingly, their pharmacological properties cannot be assumed to be the same. This is due to the complexity of the production of biologicals and to the presence of minor natural variations in the molecular structure (collectively known as microheterogeneity). Further, biological production yields slightly different versions of the drug over time, particularly when changes are introduced in the production process. In this case the prechange and postchange versions of the biological are analyzed in what is called a comparability exercise. The comparable versions thus validated are considered not to have any significant differences at the clinical level. Likewise, biosimilars are not identical copies but comparable versions of the original biological drug, also validated through a comparability exercise, although of a much broader scope. Although current knowledge about biosimilars has increased significantly, they still arise a number of controversies and misconceptions, particularly regarding issues like extrapolation of indications, immunogenicity and substitution. This review deals with concepts and controversies in the biosimilar field.

Calprotectin protects against experimental colonic inflammation in mice

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.

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.

Interaction of glucocorticoids with FXR/FGF19/FGF21-mediated ileum-liver crosstalk

At high doses, glucocorticoids (GC) have been associated with enhanced serum bile acids and liver injury. We have evaluated the effect of GC, in the absence of hepatotoxicity, on FXR/FGF91(Fgf15)/FGF21-mediated ileum-liver crosstalk. Rats and mice (wild type and Fxr-/-, Fgf15-/- and int-Gr-/- strains; the latter with GC receptor (Gr) knockout selective for intestinal epithelial cells), were treated (i.p.) with dexamethasone, prednisolone or budesonide. In both species, high doses of GC caused hepatotoxicity. At a non-hepatotoxic dose, GC induced ileal Fgf15 down-regulation and liver Fgf21 up-regulation, without affecting Fxr expression. Fgf21 mRNA levels correlated with those of several genes involved in glucose and bile acid metabolism. Surprisingly, liver Cyp7a1 was not up-regulated. The expression of factors involved in transcriptional modulation by Fxr and Gr (p300, Drip205, CBP and Smrt) was not affected. Pxr target genes Cyp3a11 and Mrp2 were not up-regulated in liver or intestine. In contrast, the expression of some Pparα target genes in liver (Fgf21, Cyp4a14 and Vanin-1) and intestine (Vanin-1 and Cyp3a11) was altered. In mice with experimental colitis, liver Fgf21 was up-regulated (4.4-fold). HepG2 cells transfection with FGF21 inhibited CYP7A1 promoter (prCYP7A1-Luc2). This was mimicked by pure human FGF21 protein or culture in medium previously conditioned by cells over-expressing FGF21. This response was not abolished by deletion of a putative response element for phosphorylated FGF21 effectors present in prCYP7A1. In conclusion, GC interfere with FXR/FGF19-mediated intestinal control of CYP7A1 expression by the liver and stimulate hepatic secretion of FGF21, which inhibits CYP7A1 promoter through an autocrine mechanism.