Amino terminal recognition by a CCR6 chemokine receptor antibody blocks CCL20 signaling and IL-17 expression via ß-arrestin.

BACKGROUND: CCR6 chemokine receptor is an important target in inflammatory diseases. Th17 cells express CCR6 and a number of inflammatory cytokines, including IL-17 and IL-22, which are involved in the propagation of inflammatory immune responses. CCR6 antagonist would be a potential treatment for inflammatory diseases such as psoriasis or rheumatoid arthritis. The aim of this study is to develop an antagonistic monoclonal antibody (mAb) against human CCR6 receptor (hCCR6). RESULTS: We generate monoclonal antibodies against hCCR6 immunizing Balb/c mice with hCCR6 overexpressing cells. The antibodies were tested by flow cytometry for specific binding to hCCR6, cloned by limiting dilution and resulted in the isolation and purification monoclonal antibody 1C6. By ELISA and flow cytometry, was determined that the antibody obtained binds to hCCR6 N-terminal domain. The ability of 1C6 to neutralize hCCR6 signaling was tested and we determined that 1C6 antibody were able to block response in ß-arrestin recruitment assay with IC50 10.23 nM, but did not inhibit calcium mobilization. In addition, we found in a chemotaxis assay that 1C6 reduces the migration of hCCR6 cells to their ligand CCL20. Finally, we determined by RT-qPCR that the expression of IL-17A in Th17 cells treated with 1C6 was inhibited. CONCLUSIONS: In the present study, we applied whole cell immunization for successfully obtain an antibody that is capable to neutralize hCCR6 signaling and to reduce hCCR6 cells migration and IL-17 expression. These results provide an efficient approach to obtain therapeutic potential antibodies in the treatment of CCR6-mediated inflammatory diseases.

Serum from postmenopausal women treated with a by-product of olive-oil extraction process stimulates osteoblastogenesis and inhibits adipogenesis in human mesenchymal stem-cells (MSC).

Aging may enhance both oxidative stress and bone-marrow mesenchymal stem-cell (MSC) differentiation into adipocytes. That reduces osteoblastogenesis, thus favoring bone-mass loss and fracture, representing an important worldwide health-issue, mainly in countries with aging populations. Intake of antioxidant products may help to retain bone-mass density. Interestingly, a novel olive-pomace physical treatment to generate olive oil also yields by-products rich in functional antioxidants. Thus, diet of postmenopausal women was supplemented for two months with one of such by-products (distillate 6; D6), being rich in squalene. After treatment, serum from such women showed reduced both lipidic peroxidation and oxidized low-density lipoprotein (LDL). Besides, vitamin E and coenzyme Q10 levels increased. Furthermore, culture medium containing 10% of such serum both increased osteoblastogenesis and reduced adipogenesis in human MSC from bone marrow. Therefore, highly antioxidant by-products like D6 may represent a relevant source for development of functional products, for both prevention and treatment of degenerative pathologies associated with aging, like osteoporosis.

Oxidative stress and inflamatory plasma biomarkers in respiratory syncytial virus bronchiolitis.

INTRODUCTION: Oxidative stress (OS) plays a crucial role in the pathogenesis of inflammatory lung diseases. OBJECTIVES: (i) We determined whether acute bronchiolitis (AB) caused by respiratory syncytial virus (RSV) induced OS; (ii) assessed whether OS biomarkers correlated with the severity of RSV-AB; and (iii) studied whether the levels of interleukins are associated with OS biomarkers. METHODS: We performed an observational study by comparing healthy infants (Group 1) with RSV-AB infants, classified as Group 2 (pulse oximetry (SpO2 ) >93%), and Group 3 (SpO2 ≤ 92%), which needed oxygen therapy. Blood samples were collected to determine the levels of lipid peroxidation (LPO) products (LPO), total glutathione (TG), oxidised glutathione (GSSG), reduced glutathione (GSH), glutathione peroxidase (GPx), interleukins (ILs) IL-10, IL-6, IL-8, interferon-gamma (IFNγ), tumour necrosis factor-alpha (TNFα) and macrophage inflammatory proteins (MIP α and MIP ß). RESULTS: Forty-six RSV-AB infants (47% needed oxygen therapy) and 27 healthy infants were included. The GSH/GSSG ratio was lower in RSV-AB infants than in Group 1 (P<0.001). GSSG and GPx were significantly higher in Group 3. GSSG predicted the need for oxygen therapy with an optimal cut-off point of 15 µM/g for haemoglobin. The GSH/GSSG ratio negatively correlated with IL-6 (P: 0.014), IL-8 (P: 0.014) and IL-10 (P: 0.033). Group 3 exhibited a direct correlation between GPx and IL-10 levels (P: 0.024) and between LPO and MIP ß (P: 0.003). CONCLUSIONS: RSV induced OS in AB. An increase in GSSG correlated with the disease severity in the infants. OS may contribute to the pathogenesis of RSV-AB.

Pre-exercise intake of different carbohydrates modifies ischemic reactive hyperemia after a session of anaerobic, but not after aerobic exercise.

The acute ingestion of a supplement with different glycemic carbohydrates, including fructose, is a typical practice for athletes before exercising. Observational evidence suggests that different metabolic responses may modify the exercise-stimulated endothelium-dependent vasodilation. The purpose of the present study was to investigate whether endothelial reactivity, stimulated by anaerobic exercise (AnE) or aerobic exercise (AE), both performed with glycemic supplementation, is modified by the addition of fructose. Twenty physically trained men ingested an oral dose of glucose (G) or glucose plus fructose (F) 15 minutes before starting a 30-minute session of AnE (10 sets of 10 repetitions of half squat) or AE (cycling). The combination resulted in 4 randomized interventions in a crossover design in which all subjects performed all experimental conditions: G+AnE, F+AnE, G+AE, and F+AE. Ischemic reactive hyperemia (IRH), glycemia, plasma lipoperoxides (LPOs), nitric oxide (NO), and lactate were determined at baseline, exercise, and acute recovery time points. Immediately after AnE, IRH was 26.35% higher in F+AnE than in G+AnE (p<0.05); this difference rose to 27.24% at the end of the recovery period (p<0.05). The glycemic peak in F+AnE was lower than in G+AnE (p<0.05), and there was a second peak during recovery (p<0.05). There were no differences observed in LPO between anaerobic trials, but the NO bioavailability increased and was higher in F + AnE than in G+AnE after exercise and recovery (p<0.05). Residual lactate was also higher under the F+AnE condition (p<0.05). During AE, there were no differences in IRH, glycemia, or NO between groups, but LPO was significantly higher after F supplementation. These results suggest that the addition of fructose to a single G supplement ingested before a glycolitic exercise can modify the glucoregulation and increases ischemic reactive hyperemia.

Fructose modifies the hormonal response and modulates lipid metabolism during aerobic exercise after glucose supplementation.

The metabolic response when aerobic exercise is performed after the ingestion of glucose plus fructose is unclear. In the present study, we administered two beverages containing GluF (glucose+fructose) or Glu (glucose alone) in a randomized cross-over design to 20 healthy aerobically trained volunteers to compare the hormonal and lipid responses provoked during aerobic exercise and the recovery phase. After ingesting the beverages and a 15-min resting period, volunteers performed 30 min of moderate aerobic exercise. Urinary and blood samples were taken at baseline (t(-15)), during the exercise (t(0), t(15) and t(30)) and during the recovery phase (t(45), t(75) and t(105)). Plasma insulin concentrations were higher halfway through the exercise period and during acute recuperation (t(15) and t(75); P<0.05) following ingestion of GluF than after Glu alone, without any differences between the effects of either intervention on plasma glucose concentrations. Towards the end of the exercise period, urinary catecholamine concentrations were lower following GluF (t(45); P<0.05). Plasma triacylglycerol (triglyceride) concentrations were higher after the ingestion of GluF compared with Glu (t(15), t(30), t(45) and t(105); P<0.05). Furthermore, with GluF, we observed higher levels of lipoperoxides (t(15), t(30), t(45) and t(105); P<0.05) and oxidized LDL (low-density lipoprotein; t(30); P<0.05) compared with after the ingestion of Glu alone. In conclusion, hormonal and lipid alterations are provoked during aerobic exercise and recovery by the addition of a dose of fructose to the pre-exercise ingestion of glucose.

Comparison of melatonin versus vitamin C on oxidative stress and antioxidant enzyme activity in Alzheimer's disease induced by okadaic acid in neuroblastoma cells.

We demonstrated that exposure of cells to 50 nM okadaic acid for 2 h induced a reduction in cellular glutathione transferase, glutathione reductase and catalase activity. Likewise, this acid prompted an increase in lipid peroxidation. Treatment of cells with 10(-5) M melatonin or 0.5 microg/ml vitamin C prevented the effects of okadaic acid. These results indicate that okadaic acid induces an oxidative stress imbalance, while melatonin and vitamin C prevent the oxidative stress induced by okadaic acid. Likewise, these data indicate the great importance of oxidative stress in both this experimental model and in the development and course of neurodegenerative disease, especially Alzheimer's disease. They show that melatonin is much more efficient than vitamin C in reducing the extent of oxidative stress. This phenomenon was demonstrated by the smaller dose of melatonin needed to obtain effects similar to those obtained with vitamin C on lipid peroxidation and by the protective effect of melatonin on antioxidant enzyme activity.