Effect of different intravenous iron preparations on lymphocyte intracellular reactive oxygen species generation and subpopulation survival

<p>Abstract</p> <p>Background</p> <p>Infections in hemodialysis (HD) patients lead to high morbidity and mortality rates and are associated with early cardiovascular mortality, possibly related to chronic inflammation. Intravenous (IV) iron is widely administered to HD patients and has been associated with increased oxidative stress and dysfunctional cellular immunity. The purpose of this study was to examine the effect of three commercially available IV iron preparations on intracellular reactive oxygen species generation and lymphocyte subpopulation survival.</p> <p>Methods</p> <p>Peripheral blood mononuclear cells (PBMC) were isolated from healthy donor buffy coat. PBMC were cultured and incubated with 100 μg/mL of sodium ferric gluconate (SFG), iron sucrose (IS) or iron dextran (ID) for 24 hours. Cells were then probed for reactive oxygen species (ROS) with dichlorofluorescein-diacetate. In separate studies, isolated PBMCs were incubated with the 25, 50 or 100 μg/mL iron concentrations for 72 hours and then stained with fluorescein conjugated monoclonal antibodies for lymphocyte subpopulation identification. Untreated PBMCs at 24 hours and 72 hours served as controls for each experiment.</p> <p>Results</p> <p>All three IV iron preparations induced time dependent increases in intracellular ROS with SFG and IS having a greater maximal effect than ID. The CD4+ lymphocytes were most affected by IV iron exposure, with statistically significant reduction in survival after incubation with all three doses (10, 25 and 100 μg/mL) of SFG, IS and ID.</p> <p>Conclusion</p> <p>These data indicate IV iron products induce differential deleterious effects on CD4+ and CD16+ human lymphocytes cell populations that may be mediated by intracellular reactive oxygen species generation. Further studies are warranted to determine the potential clinical relevance of these findings.</p

Fyn Mediates Leptin Actions in the Thymus of Rodents

BACKGROUND:Several effects of leptin in the immune system rely on its capacity to modulate cytokine expression and apoptosis in the thymus. Surprisingly, some of these effects are dependent on signal transduction through the IRS1/PI3-kinase, but not on the activation of JAK2. Since all the well known effects of leptin in different cell types and tissues seem to be dependent on JAK2 activation, we hypothesized that, at least for the control of thymic function, another, unknown kinase could mediate the transduction of the leptin signal from the ObR towards the IRS1/PI3-kinase signaling cascade. METHODOLOGY/PRINCIPAL FINDINGS:Here, by employing immunoblot, real-time PCR and flow citometry we show that the tyrosine kinase, Fyn, is constitutively associated with the ObR in thymic cells. Following a leptin stimulus, Fyn undergoes an activating tyrosine phosphorylation and a transient association with IRS1. All these effects are independent of JAK2 activation and, upon Fyn inhibition, the signal transduction towards IRS1/PI3-kinase is abolished. In addition, the inhibition of Fyn significantly modifies the effects of leptin on thymic cytokine expression. CONCLUSION/SIGNIFICANCE:Therefore, in the thymus, Fyn acts as a tyrosine kinase that transduces the leptin signal independently of JAK2 activation, and mediates some of the immunomodulatory effects of leptin in this tissue

The C242T polymorphism of the p22-phox gene (CYBA) is associated with higher left ventricular mass in Brazilian hypertensive patients

<p>Abstract</p> <p>Background</p> <p>Reactive oxygen species have been implicated in the physiopathogenesis of hypertensive end-organ damage. This study investigated the impact of the C242T polymorphism of the p22-phox gene (CYBA) on left ventricular structure in Brazilian hypertensive subjects.</p> <p>Methods</p> <p>We cross-sectionally evaluated 561 patients from 2 independent centers [Campinas (n = 441) and Vitória (n = 120)] by clinical history, physical examination, anthropometry, analysis of metabolic and echocardiography parameters as well as p22-phox C242T polymorphism genotyping. In addition, NADPH-oxidase activity was quantified in peripheral mononuclear cells from a subgroup of Campinas sample.</p> <p>Results</p> <p>Genotype frequencies in both samples were consistent with the Hardy- Weinberg equilibrium. Subjects with the T allele presented higher left ventricular mass/height^2.7than those carrying the CC genotype in Campinas (76.8 ± 1.6 vs 70.9 ± 1.4 g/m^2.7; p = 0.009), and in Vitória (45.6 ± 1.9 vs 39.9 ± 1.4 g/m^2.7; p = 0.023) samples. These results were confirmed by stepwise regression analyses adjusted for age, gender, blood pressure, metabolic variables and use of anti-hypertensive medications. In addition, increased NADPH-oxidase activity was detected in peripheral mononuclear cells from T allele carriers compared with CC genotype carriers (p = 0.03).</p> <p>Conclusions</p> <p>The T allele of the p22-phox C242T polymorphism is associated with higher left ventricular mass/height^2.7and increased NADPH-oxidase activity in Brazilian hypertensive patients. These data suggest that genetic variation within NADPH-oxidase components may modulate left ventricular remodeling in subjects with systemic hypertension.</p

High-Fat Diet Induces Apoptosis of Hypothalamic Neurons

Consumption of dietary fats is amongst the most important environmental factors leading to obesity. In rodents, the consumption of fat-rich diets blunts leptin and insulin anorexigenic signaling in the hypothalamus by a mechanism dependent on the in situ activation of inflammation. Since inflammatory signal transduction can lead to the activation of apoptotic signaling pathways, we evaluated the effect of high-fat feeding on the induction of apoptosis of hypothalamic cells. Here, we show that consumption of dietary fats induce apoptosis of neurons and a reduction of synaptic inputs in the arcuate nucleus and lateral hypothalamus. This effect is dependent upon diet composition, and not on caloric intake, since pair-feeding is not sufficient to reduce the expression of apoptotic markers. The presence of an intact TLR4 receptor, protects cells from further apoptotic signals. In diet-induced inflammation of the hypothalamus, TLR4 exerts a dual function, on one side activating pro-inflammatory pathways that play a central role in the development of resistance to leptin and insulin, and on the other side restraining further damage by controlling the apoptotic activity

Vitamin E Supplementation Reduces Oxidative Stress in Beta Thalassaemia Intermedia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Objective: The aim of this investigation was to study the effect of vitamin E treatment in oxidative stress of red and white cells of beta-thalassaemia intermedia patients. Methods: Nine patients undergoing occasional transfusions (5 females/4 males), median age 39 years (range 15-74), were recruited for oral daily administration of 400 IU vitamin E for 3 months. Twenty-seven milliliters of peripheral blood was obtained before and after 3 months of treatment, and 3 months after treatment completion. In the case of transfused patients (n = 4), blood was obtained at least 30 days after transfusion. Reactive oxygen species (ROS) was measured by flow cytometry; red blood cell (RBC) reduced glutathione (GSH) was measured by dinitrothiocyanobenzene reduction, serum malondialdehyde was measured in terms of thiobarbituric acid-reactive substances (TBARS), and alpha-haemoglobin-stabilizing protein (AHSP) mRNA expression was measured by real-time polymerase chain reaction of reticulocyte RNA extracts. Results: beta-Thalassaemia patients presented basal levels of RBC ROS, GSH and serum TBARS statistically different compared with healthy controls. However, after vitamin E administration, patients presented a significant reduction in erythrocyte RBC ROS and serum TBARS levels. In parallel, red cell GSH was significantly increased after treatment. Peripheral mononuclear cells and T lymphocytes also demonstrated a reduction in ROS production. Therefore, after treatment, no significant differences were detected comparing patients and normal controls. Three months after treatment completion, all measurements showed a tendency of returning to basal values. A significant reduction in reticulocyte number was observed after vitamin E treatment. Vitamin E treatment did not modify levels of haemoglobin or AHSP mRNA expression. Conclusion: Although vitamin E is not capable of reducing anaemia in these patients, it could be useful for reducing oxidative damage in other target organs of beta-thalassaemic patients. Finally, this is the first study to analyse the effects of vitamin E on ROS production in red and white blood cells and AHSP mRNA expression. Copyright (C) 2009 S. Karger AG, Basel1204225231Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Reactive oxygen species generation in peripheral blood monocytes and oxidized LDL are increased in hyperlipidemic patients

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Objectives: Experimental and in vitro evidences have established that reactive oxygen species (ROS) generated by vascular wall cells play a key role in atherogenesis. Here, we evaluated the rate of ROS generation by testing peripheral monocytes in naive hyperlipidemic subjects. Design and methods: Primary hypercholesterolemic, combined hyperlipidemic, and normopilidemic individuals were studied. ROS generation and the mitochondrial electrical transmembrane potential were estimated by flow cytometry. Plasma oxidized (ox) LDL levels and lipid profile were measured by ELISA and enzymatic colorimetric methods. Results: Both hyperlipidemic groups presented significantly higher rates of monocyte ROS generation and elevated plasma levels of ox-LDL. Combined hyperlipidemic subjects presented increased levels of small dense LDL and insulin. Significant positive correlations between monocyte ROS generation and ox-LDL concentrations were found in pooled data. Conclusions: These data provide evidence that ROS production by circulating monocytes from hyperlipidemic subjects may contribute to the systemic oxidative stress and possibly to atherogenesis. (C) 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.421212221227Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundo de Apoio ao EnsinoPesquisa e A ExtensAo (FAEPEX/FCM/UNICAMP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

High Bcl-2/Bax ratio in Walker tumor cells protects mitochondria but does not prevent H2O2-induced apoptosis via calcineurin pathways

It has been previously shown that Walker 256 tumor cells express a high content of the anti-apoptotic protein Bcl-2 which protects mitochondria against the damaging effects of Ca2+. In the present study, we analyze H2O2-induced apoptotic death in two different types of tumor cells: Walker 256 and SCC-25. Treatment with H2O2 (4mM) increased reactive oxygen species generation and the concentration of cytosolic free Ca2+. These alterations preceded apoptosis in both cell lines. In Walker cells, which show a high Bcl-2/Bax ratio, apoptosis was dependent on calcineurin activation and independent of changes in mitochondrial membrane potential (Delta psi(m)), as well as cytochrome c release. In contrast, in SCC-25 cells, which show a lower Bcl-2/Bax ratio, apoptosis was preceded by a decrease in Delta psi(m), mitochondrial permeability transition, and cytochrome c release. Caspase-3 activation occurred in both cell lines. The data suggest that although the high Bcl-2/Bax ratio protected the mitochondria of Walker cells from oxidative stress, it was not sufficient to prevent apoptosis through calcineurin pathways.39218619

Verapamil-sensitive Ca2+ channel regulation of Th1-type proliferation of splenic lymphocytes induced by Walker 256 tumor development in rats

Recently, we demonstrated that verapamil, an L-type Ca2+ channel blocker, inhibits the activation of splenic lymphocytes during Walker 256 ascitic tumor development in adult rats. In the present study we have analyzed the changes in spleen size, splenic lymphocyte proliferation, white pulp organization and relative size as well as food intake, and levels of blood haemoglobin in Walker 256 tumor bearing rats. These rats displayed a spleen enlargement associated with a significant increase in white pulp area and TCD8(+) lymphocyte proliferation. Levels of interferon-gamma, but not of interleukin-10, were elevated in tumor bearing rats, indicating a Th1-type immune response. These manifestations were accompanied by reduced food intake and anaemia. Treatment of tumor bearing rats with verapamil avoided spleen enlargement and increased expression of cytokines, as well as the splenic TCD8(+) lymphocyte proliferation. In addition, verapamil treatment promoted an exacerbation of the anorexia, and anaemia caused by Walker tumor development. No such effect was observed in control rats treated with verapamil. Taken together, these findings suggest that verapamil inhibits the immune response to cancer, resulting in an increase of the systemic effects induced by Walker 256 tumor. (c) 2006 Elsevier B.V. All rights reserved.5494169917918