Mesenchymal stem cell-conditioned medium triggers neuroinflammation and reactive species generation in organotypic cultures of rat hippocampus.

Cell therapy using bone marrow-derived mesenchymal stem cells (MSCs) seems to be a new alternative for the treatment of neurodegenerative diseases. Despite several promising results with their use, possible side effects are still unknown. In a previous work, we have shown that MSC-conditioned medium is toxic to hippocampal slice cultures and aggravates cell death induced by oxygen and glucose deprivation. In this work, we investigated whether the inflammatory response and/or reactive species formation could be involved in that toxicity. Rat organotypic hippocampal cultures were exposed for 24 h to conditioned medium from MSCs isolated from rat bone marrow. A marked glial activation was observed after exposure of cultures to MSC-conditioned medium, as evidenced by glial fibrillary acid protein (GFAP) and isolectin B(4) increase. Tumor necrosis factor-α and interleukin-6 levels were increased in the culture medium, and 2,7-dihydrodichlorofluorescein diacetate oxidation (indicating reactive species generation) and inducible nitric oxide synthase (iNOS) immunocontent were also higher after exposure of cultures to MSC-conditioned medium. Antioxidants (ascorbic acid and TROLOX(®)), N(ω)-nitro-l-arginine methyl ester hydrochloride, and anti-inflammatory drugs (indomethacin and dexamethasone) reduced cell death in hippocampal organotypic cultures after their exposure to MSC-conditioned medium. The results obtained here suggest that MSC-secreted factors trigger reactive species generation and neuroinflammation in organotypic cultures of hippocampus, introducing a note of caution in the use of these cells for neurological application.

Lipoteichoic acid from Staphylococcus aureus increases matrix metalloproteinase 9 expression in RAW 264.7 macrophages: modulation by A2A and A2B adenosine receptors.

Peptidoglycan (PEG) and lipoteichoic acid (LTA) are the main constituents of Gram-positive bacteria cell wall and are described to modulate immune functions. Increased levels of matrix metalloproteinases (MMPs) were described in endotoxemia, suggesting that they participate to tecidual damage, multiple organs failure and vascular disfunction. Staphylococcus aureus PEG is described to increase MMPs 2 and 9 levels in plasma from rat and MMP 9 secretion by human neutrophils, however, the effect of LTA on MMPs is unknown. In this work, was evaluated the modulation of MMPs 2 and 9 expression and secretion in RAW 264.7 macrophages by LTA from S. aureus. The role of A2A and A2B adenosine receptors was also investigated. LTA increased MMP 9 expression and secretion at 12h of treatment. The modulation of MMP 9 secretion was dose dependent, with maximal effect above 1microg/ml. The inhibitor of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway (U0126, 10microM) prevented LTA stimulation of MMP 9 secretion; however, the inhibitors of p38 (SB203580, 10microM) and Jun N-terminal kinase (JNK; SP600125, 10microM) presented any effect. A2A and A2B adenosine receptors pharmacological blockade or gene knockdown resulted in exacerbated MMP 9 secretion, while an adenosine receptors agonist inhibited LTA-stimulated MMP 9 secretion. These results suggest that LTA increased MMP 9 secretion in macrophages could be involved in complications associated to S. aureus infections. Moreover, LTA modulation of MMP 9 is dependent on MEK/ERK pathway and is regulated by A2A and A2B adenosine receptors.

High glucose increases RAW 264.7 macrophages activation by lipoteichoic acid from Staphylococcus aureus.

BACKGROUND: Type 2 diabetes mellitus is associated with an increased risk of cardiovascular diseases and accelerated atherosclerosis, which has been associated to hyperglycemia and chronic inflammation. Activated macrophages are described to participate in atherosclerosis due to foam cell formation and pro-inflammatory mediators production. Bacterial infections are described to accelerate atherosclerosis, moreover, gram-positive and negative bacterial DNA was described in atherosclerotic plaques. METHODS: We studied the glucose modulation of RAW 264.7 macrophages activation by the gram-positive bacterial antigen lipoteichoic acid (LTA), evaluating nitrite production, tumor necrosis factor alpha secretion and matrix metalloproteinase 9 activity. RESULTS: High glucose increased macrophages activation by LTA, evidenced by exacerbated nitric oxide and tumor necrosis factor alpha production, as well matrix metalloproteinase 9 secretion. CONCLUSIONS: These effects could contribute to atherosclerotic risk parameters, like atherome plaque instability, and participate in chronic inflammation present in type 2 diabetes.

Regulation of LPS stimulated ROS production in peritoneal macrophages from alloxan-induced diabetic rats: involvement of high glucose and PPARgamma.

An increased occurrence of long term bacterial infections is common in diabetic patients. Bacterial cell wall components are described as the main antigenic agents from these microorganisms and high blood glucose levels are suggested to be involved in altered immune response. Hyperglycemia is reported to alter macrophages response to lipopolysaccharide (LPS) and peroxisome proliferators activated receptor gamma (PPARgamma) expression. Additionally, glucose is the main metabolic fuel for reduced nicotinamide adenine dinucleotide phosphate (NADPH) production by pentose phosphate shunt. In this work, lipopolysaccharide (LPS) stimulated reactive oxygen species (ROS) and nitrite production were evaluated in peritoneal macrophages from alloxan-induced diabetic rats. Cytosolic dehydrogenases and PPARgamma expression were also investigated. LPS was ineffective to stimulate ROS and nitrite production in peritoneal macrophages from diabetic rats, which presented increased glucose-6-phosphate dehydrogenase and malate dehydrogenase activity. In RAW 264.7 macrophages, acute high glucose treatment abolished LPS stimulated ROS production, with no effect on nitrite and dehydrogenase activities. Peritoneal macrophages from alloxan-treated rats presented reduced PPARgamma expression. Treating RAW 264.7 macrophages with a PPARgamma antagonist resulted in defective ROS production in response to LPS, however, stimulated nitrite production was unaltered. In conclusion, in the present study we have reported reduced nitric oxide and reactive oxygen species production in LPS-treated peritoneal macrophages from alloxan-induced diabetic rats. The reduced production of reactive oxygen species seems to be dependent on elevated glucose levels and reduced PPARgamma expression.

Mitochondrial superoxide production is related to the control of cytokine release from peritoneal macrophage after antioxidant treatment in septic rats.

BACKGROUND: Reactive oxygen species are involved in several intracellular pathways that ultimately lead to the activation of the innate immune system. In addition, oxidized proteins and lipids could stimulate cytokine release from macrophages through the activation of membrane receptors. Thus we here describe the effects of antioxidant administration to septic rats on peritoneal macrophage parameters of oxidative stress and cytokine release. MATERIALS AND METHODS: Peritoneal macrophages from Wistar rats subjected to cecal ligation and puncture (CLP). The animals were divided into four groups: sham operated, CLP, basic support (saline plus antibiotics), basic support plus N-acetylcysteine, and deferoxamine. Several times after CLP macrophages were cultured to the determination of thiobarbituric acid reactive species (TBARS), protein carbonyls, mitochondrial superoxide production, catalase, superoxide dismutase activities, and released cytokines. RESULTS: Sepsis increased TBARS, protein carbonyls, and mitochondrial superoxide production in macrophages and this was associated with an increase release of pro-inflammatory cytokines. Basic support reversed TBARS and protein carbonyls content, but not mitochondrial superoxide production. The addition of antioxidants prevented all oxidative parameters in macrophages, and this was associated with lower cytokine release. Catalase and superoxide dismutase were modulated in the basic support group, but not in the antioxidant treated animals. CONCLUSIONS: Mitochondrial superoxide production seemed to be the differential oxidative parameter associated with antioxidant-induced modulation of cytokine release.

Gastrin-releasing peptide receptor antagonist effects on an animal model of sepsis.

RATIONALE: Several new therapeutic strategies have been described for the treatment of sepsis, but to date none are related to alterations in the bombesin/gastrin-releasing peptide (GRP) receptor pathways. OBJECTIVES: To determine the effects of a selective GRP receptor antagonist, RC-3095, on cytokine release from macrophages and its in vivo effects in the cecal ligation and puncture (CLP) model of sepsis and in acute lung injury induced by intratracheal instillation of LPS. METHODS: We determined the effects of RC-3095 in the CLP model of sepsis and in acute lung injury induced by intratracheal instillation of LPS. In addition, we determined the effects of RC-3095 on tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-10, and nitric oxide release from activated macrophages. MEASUREMENTS AND MAIN RESULTS: The GRP antagonist attenuated LPS- or CLP-induced TNF-alpha, IL-1beta, and nitric oxide release in cultured macrophages and decreased the mRNA levels of inducible nitric oxide synthase. The administration of RC-3095 (0.3 mg/kg) 6 h after sepsis induction improved survival in the CLP model, and diminished lung damage after intratracheal instillation of LPS. These effects were associated with attenuation on the circulating TNF-alpha and IL-1beta levels and decreased myeloperoxidase activity in several organs. CONCLUSIONS: We report that a selective GRP receptor antagonist attenuates the release of proinflammatory cytokines in vitro and in vivo and improves survival in "established" sepsis. These are consistent with the involvement of a new inflammatory pathway relevant to the development of sepsis.

Extracellular inosine participates in tumor necrosis factor-alpha induced nitric oxide production in cultured Sertoli cells.

Recent reports have described purinergic modulation of tumor necrosis factor-alpha (TNF-alpha) signaling in neutrophils and astrocytes. In Sertoli cells, both TNF-R1 and TNF-R2 TNF-alpha receptors are present and this cytokine modulates many functions of these cells related to the maintenance of spermatogenesis. Sertoli cells express distinct purinoreceptors and previous work has shown that these cells secrete extracellular nucleotides and their metabolites. In this work, we studied the possible role of extracellular purines in TNF-alpha signaling in cultured Sertoli cells. This cytokine increased inosine concentration from 30 min to 6 h, with no effect at 24 h. Both TNF-alpha and inosine increased nitrite accumulation and nitric oxide synthase activity. Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an adenosine deaminase inhibitor, abolished the TNF-alpha induced inosine increase, nitrite accumulation and nitric oxide synthase activity. These results suggest that extracellular inosine acts as intermediary in TNF-alpha stimulated nitric oxide production in cultured Sertoli cells.

Metabolism of amino acids by cultured rat Sertoli cells.

Sertoli cells support spermatogenesis both spatially and energetically; for this reason, these cells have important adaptations. The energetic metabolism of Sertoli cells was adapted to provide lactate and pyruvate to developing germ cells, because these substrates are essential for spermatocytes and spermatids. In this study, we investigated whether Sertoli cells use alanine, leucine, valine, and glycine as energetic substrates and whether the simultaneous addition of other nutrients, such as glucose and glutamine, might affect the metabolism of these amino acids. Alanine, leucine, valine, and glutamine are almost totally oxidized to CO2 by these cells. In contrast, glycine has been demonstrated to be a poor energetic substrate, being mainly incorporated into proteins, and their metabolism did not change in the presence of palmitic acid, glucose, and/or glutamine. The metabolism of the 3 other amino acids was modified by palmitic acid; besides, glucose changed alanine, leucine, and valine oxidation. Glutamine decreased the oxidation of alanine, leucine, and valine to CO2. Conversely, both alanine and leucine decreased the oxidation of glutamine. Our present findings show that Sertoli cells can adapt its energy metabolism to the oxidative substrates available to fulfill their role in spermatogenic energetic supply.

Extracellular inosine is modulated by H2O2 and protects sertoli cells against lipoperoxidation and cellular injury.

Extracellular purines are involved in the regulation of a wide range of physiological processes, including cytoprotection, ischemic preconditioning, and cell death. These actions are usually mediated via triggering of membrane purinergic receptors, which may activate antioxidant enzymes, conferring cytoprotection. Recently, it was demonstrated that the oxidative stress induced by cisplatin up-regulated A1 receptor expression in rat testes, suggesting an involvement of purinergic signaling in the response of testicular cells to oxidant injury. In this article, we report the effect of hydrogen peroxide on purinergic agonist release by cultured Sertoli cells. Extracellular inosine levels are strongly increased in the presence of H2O2, suggesting an involvement of this nucleoside on Sertoli cells response to oxidant treatment. Inosine was observed to decrease H2O2-induced lipoperoxidaton and cellular injury, and it also preserved cellular ATP content during H2O2 exposure. These effects were abolished in the presence of nucleoside uptake inhibitors, indicating that nucleoside internalisation is essential for its action in preventing cell damage.

Enhanced UV-mediated free radical generation; DNA and mitochondrial damage caused by retinol supplementation.

Retinoid supplementation has been therapeutically used against various human disorders. We and others have demonstrated that retinol treatment causes free radical generation and increased iron uptake, iron storage and oxidative damage, both in vitro and in vivo. Here, we investigate the possible synergistic effect of retinol on UV-mediated free radical generation, oxidative damage to biomolecules and decreased cellular viability in primary cultured mammalian cells. Retinol treatment (7 microM) resulted in a threefold increase in UV-mediated free radical generation and a 40%, increase in lipoperoxidation. DNA fragmentation and mitochondrial oxidative damage also increased significantly in retinol-supplemented UV-irradiated cultured cells as compared to UV-irradiated control cells, which were only treated with the solvent used to deliver the retinol (0.1% ethanol). All measurements were restored to control values when an iron chelator, 1,10-phenanthroline (100 microM), or an OH* scavenger, mannitol (1 mM), was co-administrated. Rather than protecting against free radical generation, retinol seems to enhance UV-mediated oxidative damage and decreases cellular viability in cultured cells. We suggest that retinol-enhanced iron uptake and storage and increased reactive oxygen species generated by the Fenton reaction may act synergistically with UV-irradiation in causing oxidative damage to cells.

Extracellular purines from cells of seminiferous tubules.

It has been long postulated that extracellular purines can modulate the function of the male reproductive system by interacting with different purinergic receptors of Sertoli and germinative cells. Many authors have described the biological changes induced by extracellular ATP and/or adenosine in these cells, and some hypothetical models for paracrine communication mediated by purines were proposed; however, the cellular source(s) of these molecules in seminiferous tubules remains unknown. In this study, we demonstrated for the first time that Sertoli cells are able to release ATP (0.3 nmol/mg protein) and adenosine (0.1 nmol/mg protein) in the extracellular medium, while germinative and myoid peritubular cells are able to secrete adenosine (0.02 and 0.37 nmol/mg protein, respectively). Indeed, all the three types of cells were able to release inosine at significant concentrations (about 0.4 nmol/mg protein). This differential secretion depending on the cellular type suggests that these molecules may be involved in the paracrine regulation and/or control of the maturation processes of these cells.

A laboratory class on metabolism for medical and nursing students

Num curso prático de bioquímica para alunos de enfermagem e medicina seräo realizadas determinaçöes de glicemia, corpos cetônicos sangüíneos (acetoacetato), concentraçäo de glicogênio hepático e síntese de corpos cetônicos em ratos alimentados e em ratos submetidos a um jejum de 24 ou 48 horas. O consumo de glicose e acetoacetato por fatias de córtex cerebral seräo também determinados. Este curso permite uma ampla discussäo das inter-relaçöes metabólicas que ocorrem entre diferentes órgäos e tecidos do organismo. Permite, também, discutir as situaçöes metabólicas que ocorrem no jejum, diabetes mellitus e dieta hipoglicídica. Estas aulas säo exeqüíveis num período de duas horas ou em dois períodos de duas horas e säo economicamente viáveis