Glucocorticoid Receptor-Deficient Foxp3+ Regulatory T Cells Fail to Control Experimental Inflammatory Bowel Disease

Activation of the immune system increases systemic adrenal-derived glucocorticoid (GC) levels which downregulate the immune response as part of a negative feedback loop. While CD4+ T cells are essential target cells affected by GC, it is not known whether these hormones exert their major effects on CD4+ helper T cells, CD4+Foxp3+ regulatory T cells (Treg cells), or both. Here, we generated mice with a specific deletion of the glucocorticoid receptor (GR) in Foxp3+ Treg cells. Remarkably, while basal Treg cell characteristics and in vitro suppression capacity were unchanged, Treg cells lacking the GR did not prevent the induction of inflammatory bowel disease in an in vivo mouse model. Under inflammatory conditions, GR-deficient Treg cells acquired Th1-like characteristics and expressed IFN-gamma, but not IL-17, and failed to inhibit pro-inflammatory CD4+ T cell expansion in situ. These findings reveal that the GR is critical for Foxp3+ Treg cell function and suggest that endogenous GC prevent Treg cell plasticity toward a Th1-like Treg cell phenotype in experimental colitis. When equally active in humans, a rationale is provided to develop GC-mimicking therapeutic strategies which specifically target Foxp3+ Treg cells for the treatment of inflammatory bowel disease

Cell-Specific Immune Regulation by Glucocorticoids in Murine Models of Infection and Inflammation

Glucocorticoids (GC) are highly potent negative regulators of immune and inflammatory responses. Effects of GC are primarily mediated by the glucocorticoid receptor (GR) which is expressed by all cell types of the immune system. It is, therefore, difficult to elucidate how endogenous GC mediate their effects on immune responses that involve multiple cellular interactions between various immune cell subsets. This review focuses on endogenous GC targeting specific cells of the immune system in various animal models of infection and inflammation. Without the timed release of these hormones, animals infected with various microbes or challenged in inflammatory disease models succumb as a consequence of overshooting immune and inflammatory responses. A clearer picture is emerging that endogenous GC thereby act in a cell-specific and disease model-dependent manner, justifying the need to develop techniques that target GC to individual immune cell types for improved clinical application

Formation of 5-(hydroxymethyl)furfural during subcritical water extraction of natural matrices. Is it bioactivity-relevant?

Trabajo presentado al 10th International Symposium on Supercritical Fluids celebrado del 13 al 16 de mayo de 2012 en San Francisco (US).Subcritical water extraction (SWE) has demonstrated a great potential for the extraction of bioactive compounds from natural matrices. The application of high extraction temperatures and pressures, while keeping the water in the liquid state, allows the attainment of high extraction yields in fast extraction processes. Although the applications of SWE to obtain bioactives from natural matrices are increasing, the studies dealing with the processes that might take place during the extraction are still scarce. Recently, we demonstrated how under SWE conditions not only the solubility of the analytes might be improved but also neoformed antioxidants can be obtained. Indeed, it was observed how the occurrence of reactions like Maillard and caramelization reactions during SWE resulted on the formation of neoantioxidants. This fact can be advantageous although the possible toxicity of the compounds formed during these non-enzymatic reactions should be carefully considered. Formerly, we observed that during the extraction of olive tree leaves at high extraction temperatures (200ºC), more active extracts in terms of antioxidant and in-vitro antiproliferative activities were obtained. Chemical characterization of those extracts revealed not only the presence of phenolic bioactives but also 5-(hydroxymethyl)furfural (HMF), a compound related to Maillard reaction. For this reason, in this work, a more exhaustive study has been devised to observe the influence of the temperature in the formation of HMF in SWE extracts of olive leaves. The final aim was to determine the relevance of the HMF present on different SWE extracts in terms of bioactivity. Thus, extracts obtained at different extraction temperatures were produced (50, 75, 100, 125, 150, 175 and 200 ºC) and chemically characterized by LC-MS. HMF was quantified in the extracts by HPLC-DAD. Moreover, the antioxidant activity, the amount of total phenols (Folin method) as well as the anticancer activity of both, extracts and HMF standard, were determined.This work was financed thanks to AGL2008-05108-C03-01 and AGL2011-29857-C03-01 (Ministerio de Educacion y Ciencia), CSD2007-00063 FUN-CFOOD (Programa CONSOLIDER-INGENIO 2010) and ALIBIRD, S2009/AGR-1469 (Comunidad de Madrid) projects. M.H. would like to thank MICINN for a “Ramón y Cajal” research contract. M.C.P. thanks MICINN for her “Juan de la Cierva” contract.Peer Reviewe

Formation and relevance of 5-hydroxymethylfurfural in bioactive subcritical water extracts from olive leaves

Although subcritical water extraction (SWE) has already shown its great potential for the attainment of natural bioactive extracts, concerns still remain on possible unexpected reactions that can arise during the extraction process, usually taking place at high pressure and temperature. It is already well-known that different components might be formed during the SWE extraction protocol due e.g. to Maillard reaction, which can improve the bioactivity of the obtained extracts. On the other hand, the formation of other compounds derived from these reactions, such as 5-hydroxymethylfurfural (HMF), has raised some concerns, mainly related to its safety. In this work, the formation of HMF during subcritical water extraction, at different conditions, from olive leaves has been monitored by using liquid chromatography with mass spectrometry (LC-MS) and diode array detection (LC-DAD). The possible influence of this compound in the overall antioxidant and antiproliferative activities against colon cancer cells has been also studied. Results showed an increase of HMF formation when increasing the extraction temperature, being the maximum concentration achieved at 200 °C (3.17 μg. HMF/mg extract); nevertheless, the HMF contained in the olive leave extracts did not influence the antioxidant capacity or the antiproliferative activity of the natural extracts, thus demonstrating the safety of the SWE process. © 2012 Elsevier Ltd.This work has been financed by AGL2008-05108-C03-01 and AGL2011-29857-C03-01 (Ministerio de Educacion y Ciencia), CSD2007-00063 FUN-CFOOD (Programa CONSOLIDER-INGENIO 2010) and by ALIBIRD, S2009/AGR-1469 (Comunidad de Madrid) projects. M.H. would like to thank MICINN for a “Ramón y Cajal” research contract. M.C.P. thanks MICINN for her “Juan de la Cierva” contract.Peer Reviewe

Formación de 5-(hydroxymethyl)furfural durante la extracción de matrices naturales mediante SWE

2 páginas.-- Trabajo presentado al "Flucomp 2011: V Reunión de Expertos en Tecnologías de Fluidos Comprimidos" celebrada en Burgos (España) del 15 al 17 de junio de 2011.Peer reviewe

Metabolomic study of human HT29 colon cancer cells by CE-MS. A comparative study of metabolite purification strategies

Resumen del póster presentado al 28th International Symposium on Chromatography celebrado en Valencia (España) del 12 al 16 de septiembre de 2010.Capillary electrophoresis-mass spectrometry (CE-MS) is considered a complementary analytical tool to GC-MS and LC-MS for metabolomics since it provides fast separations with very high efficiencies and minimum sample volumes. CE-MS is particularly suitable for the separation of polar and charged compounds which might not be separated by LC or GC. In metabolomics, the presence of large molecular species such as proteins and/or high salt content, are potential interferences. Thus, when working with CE-MS, proteins can cause adsorption to the inner capillary wall, while high salt contents can lead important ESI ionization suppression and MS contamination. To avoid these interfering effects, sample preparation is often necessary for sensitive and reliable metabolite analysis. In the present work, a new CE-MS approach is presented to carry out the metabolome study of the cytoplasm fraction from human HT29 colon cancer cells. As a first step, four different metabolite purification strategies from HT29 cell culture cytoplasm were studied prior to its CE-MS analysis: two solid phase extractions (namely, using C18 stationary phase and polystyrene-divinylbencene sorbent), and two procedures to remove proteins (precipitation with methanol and ultracentrifugation with a 3 kDa size-exclusion membrane). The four different metabolite purification procedures were compared in terms of number of extracted metabolites and CE-MS peak intensities. It was observed that the different treatments allowed the CE-MS analysis of a large number of metabolites (namely, each procedure allowed the analysis of more than 80 different compounds) with different sensitivity and, more interestingly, with different selectivity. The best results in terms of number of metabolites extracted from the cytoplasm were obtained using SPE with polystyrene-divinylbencene sorbent and methanol precipitation. It was observed that approximately 40% of the detected species were different among the different treatments, what highlights the importance of the selection of an adequate extraction methodology in metabolomic studies. Once the best purification strategy was selected, the effect of dietary polyphenols on human HT29 colon cancer cells is now under study at our laboratory following a complete Foodomics approach.Peer Reviewe

Effect of rosemary polyphenols on human colon cancer cells: transcriptomic profiling and functional enrichment analysis

In this work, the effect of rosemary extracts rich on polyphenols obtained using pressurized fluids was investigated on the gene expression of human SW480 and HT29 colon cancer cells. The application of transcriptomic profiling and functional enrichment analysis was done via two computational approaches, Ingenuity Pathway Analysis and Gene Set Enrichment Analysis. These two approaches were used for functional enrichment analysis as a previous step for a reliable interpretation of the data obtained from microarray analysis. Reverse transcription quantitative-PCR was used to confirm relative changes in mRNA levels of selected genes from microarrays. The selection of genes was based on their expression change, adjusted p value, and known biological function. According to genome-wide transcriptomics analysis, rosemary polyphenols altered the expression of ~4 % of the genes covered by the Affymetrix Human Gene 1.0ST chip in both colon cancer cells. However, only ~18 % of the differentially expressed genes were common to both cell lines, indicating markedly different expression profiles in response to the treatment. Differences in induction of G2/M arrest observed by rosemary polyphenols in the two colon adenocarcinoma cell lines suggest that the extract may be differentially effective against tumors with specific mutational pattern. From our results, it is also concluded that rosemary polyphenols induced a low degree of apoptosis indicating that other multiple signaling pathways may contribute to colon cancer cell death.This work was supported by AGL2008-05108-C03-01, AGL2011-29857-C03-01 and 200970I083 projects (Ministerio de Ciencia e Innovación) and CSD2007-00063 FUN-C-FOOD program (Programa CONSOLIDER, Ministerio de Educacion y Ciencia).Peer Reviewe

Foodomics study of dietary polyphenols effect on colon cancer proliferation

Resumen del trabajo presentado al 4º Congreso BrMASS de la Sociedade Brasileira de Espectometria de Massas celebrado en Campinas (Brasil) del 10 al 13 de diciembre de 2011.Peer Reviewe

Effect of dietary polyphenols on K562 leukemia cells: A Foodomics approach

In this work, a global Foodomics strategy has been applied to study the antiproliferative effect of dietary polyphenols from rosemary on two human leukemia lines, one showing a drug-sensitive phenotype (K562), and another exhibiting a drug-resistant phenotype (K562/R). To this aim, whole-transcriptome microarray together with an MS-based nontargeted analytical approach (via CE-TOF MS and UPLC-TOF MS) have been employed to carry out transcriptomics and metabolomics analyses, respectively. Functional enrichment analysis was done using ingenuity pathway analysis (IPA) software as a previous step for a reliable interpretation of transcriptomic and metabolomic profiles. Rosemary polyphenols altered the expression of approximately 1% of the genes covered by the whole transcriptome microarray in both leukemia cell lines. Overall, differences in the transcriptional induction of a number of genes encoding phase II detoxifying and antioxidant genes, as well as differences in the metabolic profiles observed in the two leukemia cell lines suggest that rosemary polyphenols may exert a differential chemopreventive effect in leukemia cells with different phenotypes. IPA predictions on transcription factor analysis highlighted inhibition of Myc transcription factor function by rosemary polyphenols, which may explain the observed antiproliferative effect of rosemary extract in the leukemia cells. Metabolomics analysis suggested that rosemary polyphenols affected differently the intracellular levels of some metabolites in two leukemia cell sublines. Integration of data obtained from transcriptomics and metabolomics platforms was attempted by overlaying datasets on canonical (defined) metabolic pathways using IPA software. This strategy enabled the identification of several differentially expressed genes in the metabolic pathways modulated by rosemary polyphenols providing more evidences on the effect of these compounds. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Funded by: Ministerio de Ciencia e Innovación, Spain. Grant Numbers: AGL2008-05108-C03-01, AGL2008-05108-C03-02, AGL2011-29857-C03-01 and Ministerio de Educacion y Ciencia, Spain. Grant Number: CSD2007-00063 FUN-C-FOOD.Peer Reviewe