Probiotic Sonicates Selectively Induce Mucosal Immune Cells Apoptosis Through Ceramide Generation Via Neutral Sphingolyelinase.

Background: Probiotics appear to be beneficial in inflammatory bowel disease, but their mechanism of action is incompletely understood. We investigated whether probiotic-derived sphingomyelinase mediates this beneficial effect. Methodology/Principal Findings: Neutral sphingomyelinase (NSMase) activity was measured in sonicates of the probiotic L.brevis (LB)and S. thermophilus (ST) and the non-probiotic E. coli EC) and E. faecalis (EF). Lamina propria mononuclear cells (LPMC) were obtained from patients with Crohn"s disease (CD) and Ulcerative Colitis (UC), and peripheral blood mononuclear cells (PBMC) from healthy volunteers, analysing LPMC and PBMC apoptosis susceptibility, reactive oxygen species (ROS) generation and JNK activation. In some experiments, sonicates were preincubated with GSH or GW4869, a specific NSMase inhibitor. NSMase activity of LB and ST was 10-fold that of EC and EF sonicates. LB and ST sonicates induced significantly more apoptosis of CD and UC than control LPMC, whereas EC and EF sonicates failed to induce apoptosis. Pre-stimulation with anti-CD3/CD28 induced a significant and time-dependent increase in LB-induced apoptosis of LPMC and PBMC. Exposure to LB sonicates resulted in JNK activation and ROS production by LPMC. NSMase activity of LB sonicates was completely abrogated by GW4869, causing a dose-dependent reduction of LB -induced poptosis. LB and ST selectively induced immune cell apoptosis, an effect dependent on the degree of cell activation and mediated by bacterial NSMase. Conclusions: These results suggest that induction of immune cell apoptosis is a mechanism of action of some probiotics and that NSMase-mediated ceramide generation contributes to the therapeutic effects of probiotics.

Mononuclear cell transcriptome response after sustained virgin olive oil consumption in humans: an exploratory nutrigenomics study

Virgin olive oil (VOO) is considered to be one of the main components responsible for the health benefits of the Mediterranean diet, particularly against atherosclerosis where peripheral blood mononuclear cells (PBMNCs) play a crucial role in atherosclerosis development and progression. The objective of this article was to identify the PBMNC genes that respond to VOO consumption in order to ascertain the molecular mechanisms underlying the beneficial action of VOO in the prevention of atherosclerosis. Gene expression profiles of PBMNCs from healthy individuals were examined in pooled RNA samples by microarrays after 3 weeks of moderate and regular consumption of VOO, as the main fat source in a diet controlled for antioxidant content. Gene expression was verified by qPCR. The response to VOO consumption was confirmed for individual samples (n = 10) by qPCR for 10 upregulated genes (ADAM17, ALDH1A1, BIRC1, ERCC5, LIAS, OGT, PPARBP, TNFSF10, USP48, and XRCC5). Their putative role in the molecular mechanisms involved in atherosclerosis development and progression is discussed, focusing on a possible relation with VOO consumption. Our data support the hypothesis that 3 weeks of nutritional intervention with VOO supplementation, at doses common in the Mediterranean diet, can alter the expression of genes related to atherosclerosis development and progression.

Characterization of human gene expression changes after olive oil ingestion: an exploratory approach

Olive oil consumption is protective against risk factors for cardiovascular and cancer diseases. A nutrigenomic approach was performed to assess whether changes in gene expression could occur in human peripheral blood mononuclear cells after oli ve oil ingestion at postprandial state. Six healthy male volunteers ingested, at fasting state, 50 ml of olive oil. Prior to intervention a 1-week washout period with a controlled diet and sunflower oil as the only source of fat was followed. During the 3 days before and on the intervention day, a very low-phenolic compound diet was followed. At baseline (0 h) and at post-ingestion (6 h), total RNA was isolated and gene expression (29,082 genes) was evaluated by microarray. From microarray data, nutrient-gene interactions were observed in genes related to metabolism, cellular processes, cancer, and atherosclerosis (e.g. USP48 by 2.16; OGT by 1.68-fold change) and associated processes such as inflammation (e.g. AKAP13 by 2.30; IL-10 by 1.66-fold change) and DNA damage (e.g. DCLRE1C by 1.47; POLK by 1.44- fold change). When results obtained by microarray were verified by qRT-PCR in nine genes, full concordance was achieved only in the case of up-regulated genes. Changes were observed at a real-life dose of olive oil, as it is daily consumed in some Mediterranean areas. Our results support the hypothesis that postprandial protective changes related to olive oil consumption could be mediated through gene expression changes.

Cell-Cell interaction in erythropoiesis: role of human monocytes

Erythroid burst forming units (BFU-E) are proliferative cells present in peripheral blood and bone marrow which may be precursors of the erythroid colony forming cell found in the bone marrow. To examine the possible role of monocyte-macrophages in the modulation of erythropoiesis, the effect of monocytes on peripheral blood BFU-E proliferation in response to erythropoietin was investigated in the plasma clot culture system. Peripheral blood mononuclear cells from normal human donors were separated into four fractions. Fraction-I cells were obtained from the interface of Ficoll-Hypaque gradients (20-30% monocytes; 60-80% lymphocytes); fraction-II cells were fraction-I cells that were nonadherent to plastic (2-10% monocytes; 90-98% lymphocytes); fraction-III cells were obtained by incubation of fraction-II cells with carbonyl iron followed by Ficoll-Hypaque centrifugation (>99% lymphocytes); and fraction-IV cells represented the adherent population of fraction-II cells released from the plastic by lidocaine (>95% monocytes). When cells from these fractions were cultured in the presence of erythropoietin, the number of BFU-E-derived colonies was inversely proportional to the number of monocytes present (r = ¿0.96, P < 0.001). The suppressive effect of monocytes on BFU-E proliferation was confirmed by admixing autologous purified monocytes (fraction-IV cells) with fraction-III cells. Monocyte concentrations of ¿20% completely suppressed BFU-E activity. Reduction in the number of plated BFU-E by monocyte dilution could not account for these findings: a 15% reduction in the number of fraction-III cells plated resulted in only a 15% reduction in colony formation. These results indicate that monocyte-macrophages may play a significant role in the regulation of erythropoiesis and be involved in the pathogenesis of the hypoproliferative anemias associated with infection and certain neoplasia in which increased monocyte activity and monopoiesis also occur.

Combined intermittent hypoxia and surface muscle electrostimulation as a method to increase peripheral blood progenitor cell concentration.

Background: Our goal was to determine whether short-term intermittent hypoxia exposure, at a level well tolerated by healthy humans and previously shown by our group to increase EPO and erythropoiesis, could mobilizehematopoietic stem cells (HSC) and increase their presence in peripheral circulation. Methods: Four healthy male subjects were subjected to three different protocols: one with only a hypoxic stimulus (OH), another with a hypoxic stimulus plus muscle electrostimulation (HME) and the third with only muscle electrostimulation (OME). Intermittent hypobaric hypoxia exposureconsisted of only three sessions of three hours at barometric pressure 540 hPa (equivalent to an altitude of 5000 m) for three consecutive days, whereas muscular electrostimulation was performed in two separate periods of 25 min in each session. Blood samples were obtained from an antecubital vein on three consecutive days immediately before the experiment and 24 h, 48 h, 4 days and 7 days after the last day of hypoxic exposure. Results: There was a clear increase in the number of circulating CD34+ cells after combined hypobaric hypoxia and muscular electrostimulation. This response was not observed after the isolated application of the same stimuli. Conclusion: Our results open a new application field for hypobaric systems as a way to increase efficiency in peripheral HSC collection.

Combined intermittent hypoxia and surface muscle electrostimulation as a method to increase peripheral blood progenitor cell concentration.

Background: Our goal was to determine whether short-term intermittent hypoxia exposure, at a level well tolerated by healthy humans and previously shown by our group to increase EPO and erythropoiesis, could mobilizehematopoietic stem cells (HSC) and increase their presence in peripheral circulation. Methods: Four healthy male subjects were subjected to three different protocols: one with only a hypoxic stimulus (OH), another with a hypoxic stimulus plus muscle electrostimulation (HME) and the third with only muscle electrostimulation (OME). Intermittent hypobaric hypoxia exposureconsisted of only three sessions of three hours at barometric pressure 540 hPa (equivalent to an altitude of 5000 m) for three consecutive days, whereas muscular electrostimulation was performed in two separate periods of 25 min in each session. Blood samples were obtained from an antecubital vein on three consecutive days immediately before the experiment and 24 h, 48 h, 4 days and 7 days after the last day of hypoxic exposure. Results: There was a clear increase in the number of circulating CD34+ cells after combined hypobaric hypoxia and muscular electrostimulation. This response was not observed after the isolated application of the same stimuli. Conclusion: Our results open a new application field for hypobaric systems as a way to increase efficiency in peripheral HSC collection.

Differential methylation of TCF7L2 promoter in peripheral blood DNA in newly diagnosed, drug-naïve patients with type 2 diabetes

TCF7L2 is the susceptibility gene for Type 2 diabetes (T2D) with the largest effect on disease risk that has been discovered to date. However, the mechanisms by which TCF7L2 contributes to the disease remain largely elusive. In addition, epigenetic mechanisms, such as changes in DNA methylation patterns, might have a role in the pathophysiology of T2D. This study aimed to investigate the differences in terms of DNA methylation profile of TCF7L2 promoter gene between type 2 diabetic patients and age- and Body Mass Index (BMI)- matched controls. We included 93 type 2 diabetic patients that were recently diagnosed for T2D and exclusively on diet (without any pharmacological treatment). DNA was extracted from whole blood and DNA methylation was assessed using the Sequenom EpiTYPER system. Type 2 diabetic patients were more insulin resistant than their matched controls (mean HOMA IR 2.6 vs 1.8 in controls, P<0.001) and had a poorer beta-cell function (mean HOMA B 75.7 vs. 113.6 in controls, P<0.001). Results showed that 59% of the CpGs analyzed in TCF7L2 promoter had significant differences between type 2 diabetic patients and matched controls. In addition, fasting glucose, HOMA-B, HOMA-IR, total cholesterol and LDL-cholesterol correlated with methylation in specific CpG sites of TCF7L2 promoter. After adjustment by age, BMI, gender, physical inactivity, waist circumference, smoking status and diabetes status uniquely fasting glucose, total cholesterol and LDL-cholesterol remained significant. Taken together, newly diagnosed, drug-naïve type 2 diabetic patients display specific epigenetic changes at the TCF7L2 promoter as compared to age- and BMI-matched controls. Methylation in TCF7L2 promoter is further correlated with fasting glucose in peripheral blood DNA, which sheds new light on the role of epigenetic regulation of TCF7L2 in T2D.

Evaluación del uso de exosomas como aloantígenos para la inducción de tolerancia en trasplante

La consecución de tolerancia aloespecífica es de mucha relevancia en trasplante. Las células dendríticas (DC) son las principales responsables de la inducción de la respuesta inmune frente a las moléculas de histocompatibilidad (MHC) del donante, provocando el rechazo del injerto. Sin embargo las DC son también responsables de la inducción de tolerancia. Diversos modelos animales de alotrasplante han mostrado la tolerización del injerto mediante DC diferenciadas in vitro en condiciones tolerogénicas (tDC). En humanos, las fuentes de aloantígenos potencialmente utilizables en terapia son, entre otras, los cuerpos apoptóticos y los exosomas. Éstos expresan antígenos MHC de forma abundante y su composición es relativamente uniforme, lo que supone una ventaja frente a otras fuentes. En este proyecto, se ha evaluado la obtención de exosomas secretados por una línea de linfocitos T y por células dendríticas derivadas de médula ósea. Se ha caracterizado la captura de exosomas derivados de linfocitos T por células dendríticas humanas derivadas de sangre periférica y su presentación a linfocitos T autólogos. Por otra parte, se ha comenzado a desarrollar los experimentos para estudiar la inducción de tolerancia en un modelo de trasplante renal en rata. Se han generado células dendríticas tolerógenicas derivadas de médula ósea (tolDC), en presencia de dexametasona. Las tolDC expresan menos moléculas de histocompatibilidad y de coestimulación e inducen una menor proliferación en reacciones mixtas leucocitaras, comparadas con las células dendríticas maduras. Por último, se han caracterizado los exosomas de plasma humano con el fin de estudiar su posible uso como aloantígenos. El análisis proteómico revela la presencia de proteínas relacionadas con el sistema inmune, la coagulación, la señalización celular y moléculas implicadas en el transporte y metabolismo de nutrientes. El estudio de la captura por diferentes líneas celulares sugiere que deben existir mecanismos específicos para su internalización.

Propietats protectores de la lipoproteïna de alta densitat (HDL) sobre l'acció inflamatòria de la lipoproteïna de baixa densitat electronegativa (LDL(-)) en monòcits humans

Electronegative low-density lipoprotein (LDL(-)) is a modified fraction of LDL present in peripheral blood whose proportion is elevated in subjects with increased cardiovascular risk. LDL(-) has been shown to have an inflammatory effect on human endothelial cells and mononuclear blood cells. On the other hand, high-density lipoprotein (HDL) is known to have a protective effect against cardiovascular disease, partly mediated by its anti-inflammatory properties. The objective of the current work is to study the putative protective properties of HDL towards the inflammatory effect of LDL(-) in human monocytes, in order to elucidate the mechanisms behind their interaction. Total LDL and HDL were isolated by ultracentrifugation and LDL(-) was obtained from total LDL by anion exchange chromatography. HDL and LDL(-) were incubated together and then re-isolated, and their characteristics were compared to those of untreated lipoproteins. The inflammatory activity of the lipoproteins was determined by incubating monocytes with lipoproteins and measuring cytokine release from the cultured monocytes. The biochemical composition and electrophoretic mobility of the lipoproteins were also determined before and after their interaction. Incubation of HDL with LDL(-) reduced the inflammatory effect of LDL(-) and, in turn, HDL gained inflammatory properties. This indicates a transfer of inflammatory potential taking place during the interaction of LDL(-) and HDL. Additionally, LDL(-) lost non-esterified fatty acids (NEFAs) while HDL gained the same. We conclude that a transfer of NEFAs takes place between LDL(-) and HDL. These observations suggest that NEFAs play a role in the inflammatory effect mediated by LDL(-).

Blood lymphocyte subsets in rats with adjuvant arthritis

To determine the phenotype of peripheral blood lymphocytes during the time-course of adjuvant arthritis (AA) to detect alterations that could be involved in the pathogenesis of the arthritic process. METHODS--Phenotype analysis was performed on days 7, 14, 21, 28, 42, 56 and 70 after arthritis induction using monoclonal antibodies to CD5, CD4 and CD8 subsets, and flow cytometry. The proportion of activated lymphocytes and lymphocytes was also assessed with monoclonal antibodies to IL-2R (CD25), to Ia antigen and by polyclonal antibodies to rat Ig. RESULTS--Adjuvant arthritis produced leukocytosis with neutrophilia. Rats with AA showed a marked increase in the number of both CD4+ and CD8+ cells. The ratio CD4/CD8 decreased because the rise in CD8+ cells was more pronounced than the increase in CD4+ cells. Changes in lymphocyte counts showed two well-defined periods: the first, from day 14 to day 28, during which the inflammation of the joints reached a maximum and changes in lymphocyte subsets were more pronounced, that is, there was a threefold increase in CD8+ lymphocytes over normal counts, and the second, from day 42 to day 70, in which modified parameters improved considerably but remained different from controls. CONCLUSION--Alterations were detected in the phenotype of peripheral blood lymphocytes in AA, which provides an additional marker of disease activity.

Mometasone and desloratadine additive effect on eosinophil survival and cytokine secretion from epithelial cells

Although antihistamines and topical corticosteroids are used in combination to treat allergic rhinitis, their additive effect has not been yet demonstrated. The aim was investigate the antiinflammatory additive effect of mometasone and desloratadine on cytokine and sICAM-1 secretion by epithelial cells, and on eosinophil survival stimulated by human epithelial cells secretions from nasal mucosa and polyps. Methods Epithelial cells obtained from nasal mucosa or polyps were stimulated with 10% fetal bovine serum in presence of mometasone (10-11M-10-5M) with/without desloratadine (10-5M). Cytokine and sICAM-1 concentrations in supernatants were measured by ELISA. Peripheral blood eosinophils were incubated during 4 days with epithelial cell secretions with (10-11M-10-5M) and/or desloratadine (10-5M) and survival assessed by Trypan blue. Results are expressed as percentage (mean ± SEM) compared to control. Results Fetal bovine serum stimulated IL-6, IL-8, GM-CSF and sICAM-1 secretion. In mucosa and polyp epithelial cells, mometasone inhibited this induced secretion while desloratadine inhibited IL-6 and IL-8. The combination of 10-5M desloratadine and 10-9M mometasone reduced IL-6 secretion (48 ± 11%, p < 0.05) greater extent than mometasone alone (68 ± 10%) compared to control (100%). Epithelial cell secretions induced eosinophil survival from day 1 to 4, this effect being inhibited by mometasone. At day 4, the combination of mometasone (10-11M) and desloratadine (10-5M) provoked an increased inhibition of eosinophil survival induced by cell secretions (27 ± 5%, p < 0.01) than mometasone (44 ± 7%) or desloratadine (46 ± 7%) alone. Conclusions These results suggest that the combination of desloratadine and mometasone furoate have a greater antinflammatory effect in an in vitro model of eosinophil inflammation than those drugs administered alone.