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Breast cancer (BC) is the first worldwide most frequent cancer in both sexes and the most commonly diagnosed in females. Although BC mortality has been thoroughly declining over the past decades, there are still considerable differences between women diagnosed with early BC and when metastatic BC is diagnosed. BC treatment choice is widely dependent on precise histological and molecular characterization. However, recurrence or distant metastasis still occurs even with the most recent efficient therapies. Thus, a better understanding of the different factors underlying tumor escape is mainly mandatory. Among the leading candidates is the continuous interplay between tumor cells and their microenvironment, where extracellular vesicles play a significant role. Among extracellular vesicles, smaller ones, also called exosomes, can carry biomolecules, such as lipids, proteins, and nucleic acids, and generate signal transmission through an intercellular transfer of their content. This mechanism allows tumor cells to recruit and modify the adjacent and systemic microenvironment to support further invasion and dissemination. By reciprocity, stromal cells can also use exosomes to profoundly modify tumor cell behavior. This review intends to cover the most recent literature on the role of extracellular vesicle production in normal and cancerous breast tissues. Specific attention is paid to the use of extracellular vesicles for early BC diagnosis, follow-up, and prognosis because exosomes are actually under the spotlight of researchers as a high-potential source of liquid biopsies. Extracellular vesicles in BC treatment as new targets for therapy or efficient nanovectors to drive drug delivery are also summarized.
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Neoplasias da Mama , Exossomos , Vesículas Extracelulares , Feminino , Humanos , Neoplasias da Mama/terapia , Neoplasias da Mama/tratamento farmacológico , Vesículas Extracelulares/metabolismo , Exossomos/metabolismo , Sistemas de Liberação de Medicamentos , Biologia , Microambiente TumoralRESUMO
The sharp increase in obesity prevalence worldwide is mainly attributable to changes in physical activity and eating behavior but the metabolic and clinical impacts of these obesogenic conditions vary between sexes and genetic backgrounds. This warrants personalized treatments of obesity and its complications, which require a thorough understanding of the diversity of metabolic responses to high-fat diet intake. By analyzing nine genetically diverse mouse strains, we show that much like humans, mice exhibit a huge variety of physiological and biochemical responses to high-fat diet. The strains exhibit various degrees of alterations in their phenotypic makeup. At the transcriptome level, we observe dysregulations of immunity, translation machinery, and mitochondrial genes. At the biochemical level, the enzymatic activity of mitochondrial complexes is affected. The diversity across mouse strains, diets, and sexes parallels that found in humans and supports the use of diverse mouse populations in future mechanistic or preclinical studies on metabolic dysfunctions.
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In the world, among all type of cancers, colorectal cancer (CRC) is the third most commonly diagnosed in males and the second in females. In most of cases, (RP1) patients' prognosis limitation with malignant tumors can be attributed to delayed diagnosis of the disease. Identification of patients with early-stage disease leads to more effective therapeutic interventions. Therefore, new screening methods and further innovative treatment approaches are mandatory as they may lead to an increase in progression-free and overall survival rates. For the last decade, the interest in extracellular vesicles (EVs) research has exponentially increased as EVs generation appears to be a universal feature of every cell that is strongly involved in many mechanisms of cell-cell communication either in physiological or pathological situations. EVs can cargo biomolecules, such as lipids, proteins, nucleic acids and generate transmission signal through the intercellular transfer of their content. By this mechanism, tumor cells can recruit and modify the adjacent and systemic microenvironment to support further invasion and dissemination. This review intends to cover the most recent literature on the role of EVs production in colorectal normal and cancer tissues. Specific attention is paid to the use of EVs for early CRC diagnosis, follow-up, and prognosis as EVs have come into the spotlight of research as a high potential source of 'liquid biopsies'. The use of EVs as new targets or nanovectors as drug delivery systems for CRC therapy is also summarized.
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Mutations in IDH induce epigenetic and transcriptional reprogramming, differentiation bias, and susceptibility to mitochondrial inhibitors in cancer cells. Here, we first show that cell lines, PDXs, and patients with acute myeloid leukemia (AML) harboring an IDH mutation displayed an enhanced mitochondrial oxidative metabolism. Along with an increase in TCA cycle intermediates, this AML-specific metabolic behavior mechanistically occurred through the increase in electron transport chain complex I activity, mitochondrial respiration, and methylation-driven CEBPα-induced fatty acid ß-oxidation of IDH1 mutant cells. While IDH1 mutant inhibitor reduced 2-HG oncometabolite and CEBPα methylation, it failed to reverse FAO and OxPHOS. These mitochondrial activities were maintained through the inhibition of Akt and enhanced activation of peroxisome proliferator-activated receptor-γ coactivator-1 PGC1α upon IDH1 mutant inhibitor. Accordingly, OxPHOS inhibitors improved anti-AML efficacy of IDH mutant inhibitors in vivo. This work provides a scientific rationale for combinatory mitochondrial-targeted therapies to treat IDH mutant AML patients, especially those unresponsive to or relapsing from IDH mutant inhibitors.
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Resistencia a Medicamentos Antineoplásicos/genética , Isocitrato Desidrogenase/genética , Leucemia Mieloide/genética , Mitocôndrias/genética , Mutação , Doença Aguda , Aminopiridinas/farmacologia , Animais , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Glicina/análogos & derivados , Glicina/farmacologia , Células HL-60 , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxidiazóis/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Piperidinas/farmacologia , Piridinas/farmacologia , Triazinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Mitochondrial dysfunctions that were not discovered during preclinical and clinical testing have been responsible for at least restriction of use as far as withdrawal of many drugs. To solve mitochondrial machinery complexity, integrative methodologies combining different data, coupled or not to mathematic modelling into systems biology, could represent a strategic way but are still very hard to implement. These technologies should be accurate and precise to avoid accumulation of errors that can lead to misinterpretations, and then alter prediction efficiency. To address such issue, we have developed a versatile functional energy metabolism platform that can measure quantitatively, in parallel, with a very high precision and accuracy, a high number of biological parameters like substrates or enzyme cascade activities in essential metabolism units (glycolysis, respiratory chain ATP production, oxidative stress...) Its versatility (our platform works on either cell lines or small animals and human samples) allows cell metabolism pathways fine tuning comparison from preclinical to clinical studies. Applied here to OXPHOS and/or oxidative stress as an example, it allows discriminating compounds with acute toxic effects but, most importantly, those inducing low noise chronic ones.
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Psoriasis vulgaris is a common skin inflammatory disease characterized by recurrent flare episodes associated with scaly well-demarcated skin plaques. Skin biopsies from psoriatic patients with high PASI score (22.67 ± 8.67) and from HD were used to study APN/CD13. APN/CD13 is over-expressed in LP and nLP compare to HD skins and fibroblasts. This over-expression is positively correlated with specific enzymatic activity enhancement. However, discrepancies between APN/CD13 expression in LP and nLP prompt us to focus our study on APN/CD13 modulation. Calcitonin Gene Related Peptide (CGRP), a neuropeptide, positively modulated expression and activity of APN/CD13. CGRP consistently induced IL4 secretion, which is also involved in the increase of APN/CD13 expression and activity, which is significantly reversed using IL-4 blocking antibody. Surprisingly, retinoic acid altered the APN/CD13 enzymatic activity only in nLP fibroblasts without modification of APN/CD13 expression. APN/CD13 is over-expressed on psoriatic fibroblasts and exerted high level of activity compare to HD fibroblasts. Taken together, several factors such as CGRP and IL-4 acted on positive regulation of APN/CD13 expression and activity. This study highlighted the interest of APN/CD13 as a new potential target, which should be investigated in psoriasis.
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Antígenos CD13/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Fibroblastos/efeitos dos fármacos , Interleucina-4/farmacologia , Psoríase/enzimologia , Pele/efeitos dos fármacos , Tretinoína/farmacologia , Adulto , Idoso , Antígenos CD13/genética , Estudos de Casos e Controles , Células Cultivadas , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Psoríase/genética , Psoríase/patologia , Pele/enzimologia , Pele/patologia , Fatores de Tempo , Regulação para CimaRESUMO
Production of abnormal hemoglobin (HbS) in sickle-cell disease (SCD) results in its polymerization in deoxygenated conditions and in sickled-RBC formation. Dense RBCs (DRBCs), defined as density >1.11 and characterized by increased rigidity are absent in normal AA subjects, but present at percentages that vary of a patient to another remaining stable throughout adulthood for each patient. Polymerized HbS has reduced affinity for oxygen, demonstrated by the rightward shift of the oxygen-dissociation curve, leading to disturbances in oxygen transport. Ninety-two SCD patients' total RBCs were separated into LightDRBC (LRBC) (d < 1.11 g/mL) and DRBC fractions. Venous blood partial oxygen pressure and RBC-fraction-deoxygenation and -reoxygenation Hb-oxygen-equilibrium curves were determined. All patients took a 6-minute walking test (6MWT); 10 had results before and after >6 months on hydroxyurea. 6MWT time with SpO2 < 88% (TSpO2 < 88) assessed the physiological impact of exertion. Elevated mean corpuscular hemoglobin (Hb) concentrations, decreased %HbF, and 2,3-bisphosphoglycerates in DRBCs modulated Hb-oxygen affinity. Deoxygenation and reoxygenation Hb-oxygen equilibrium curves differed between normal Hb AA and SS RBCs and between LRBCs and DRBCs, with rightward shifts confirming HbS-polymerization's role in affinity loss. In bivariate analyses, 50% Hb saturation correlated positively with %DRBCs (P < 0.0001, r(2) = 0.34) and negatively with %HbF (P < 0.0001, r(2) = 0.25). The higher the %DRBCs, the longer the TSpO2 88 (P = 0.04). Hydroxyurea was associated with significantly shorter TSpO2 < 88 (P = 0.01). We report that the %DRBCs directly affects SCD patients' SpO2 during exertion; hydroxyurea improves oxygen affinity and lowers the %DRBCs. Am. J. Hematol. 91:1008-1013, 2016. © 2016 Wiley Periodicals, Inc.
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Anemia Falciforme/sangue , Eritrócitos/metabolismo , Oxigênio/sangue , 2,3-Difosfoglicerato , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia Falciforme/fisiopatologia , Eritrócitos Anormais/metabolismo , Eritrócitos Anormais/patologia , Feminino , Hemoglobina Fetal , Hemoglobina Falciforme , Humanos , Hidroxiureia/farmacologia , Masculino , Pessoa de Meia-Idade , Oxigênio/metabolismo , Esforço Físico , Polimerização , Estudos Prospectivos , Adulto JovemRESUMO
Peroxisome proliferator-activated receptor (PPAR)-α deletion induces a profound decrease in MnSOD activity, leading to oxidative stress and left ventricular (LV) dysfunction. We tested the hypothesis that treatment of PPAR-α knockout (KO) mice with the SOD mimetic tempol prevents the heart from pathological remodelling and preserves LV function. Twenty PPAR-α KO mice and 20 age-matched wild-type mice were randomly treated for 8 wk with vehicle or tempol in the drinking water. LV contractile parameters were determined both in vivo using echocardiography and ex vivo using papillary muscle mechanics. Translational and posttranslational modifications of myosin heavy chain protein as well as the expression and activity of major antioxidant enzymes were measured. Tempol treatment did not affect LV function in wild-type mice; however, in PPAR-α KO mice, tempol prevented the decrease in LV ejection fraction and restored the contractile parameters of papillary muscle, including maximum shortening velocity, maximum extent of shortening, and total tension. Moreover, compared with untreated PPAR-α KO mice, myosin heavy chain tyrosine nitration and anion superoxide production were markedly reduced in PPAR-α KO mice after treatment. Tempol also significantly increased glutathione peroxidase and glutathione reductase activities (~ 50%) in PPAR-α KO mice. In conclusion, these findings demonstrate that treatment with the SOD mimetic tempol can prevent cardiac dysfunction in PPAR-α KO mice by reducing the oxidation of contractile proteins. In addition, we show that the beneficial effects of tempol in PPAR-α KO mice involve activation of the glutathione peroxidase/glutathione reductase system.
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Óxidos N-Cíclicos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , PPAR alfa/fisiologia , Disfunção Ventricular Esquerda/prevenção & controle , Animais , Pressão Arterial/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Ecocardiografia , Eletroforese em Gel de Poliacrilamida , Glucosefosfato Desidrogenase/metabolismo , Técnicas In Vitro , Isomerismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Miocárdio/enzimologia , Miocárdio/patologia , Cadeias Pesadas de Miosina/metabolismo , PPAR alfa/genética , Músculos Papilares/efeitos dos fármacos , Marcadores de Spin , Superóxido Dismutase/metabolismo , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Clinical laboratories need to test patient samples precisely, accurately, and efficiently. The latest member of the Roche cobas modular platform family, the cobas 8000 modular analyzer series allows compact and convenient consolidation of clinical chemistry and immunochemistry assays in high-workload laboratories with a throughput of 3 to 15 million tests annually. Here we present the results of studies designed to test the overall system performance under routine-like conditions that were conducted at 14 laboratories over 2 y. Experiments that test analytical performance of the new module were integrated with overall system functionality testing of all modules in different configurations. More than two million results were generated and evaluated for ~100 applications using serum/plasma, urine, or EDTA blood samples. During the workflow studies, eight configurations of the possible 38 combinations were used, covering all available analytical modules. The versatility of the module combinations makes the system customizable to fit the needs of diverse laboratories, allowing precise and accurate analysis of a broad spectrum of clinical chemistry and immunochemistry parameters with short turnaround times. This new system will contribute to the ability of clinical laboratories to offer better service to their customers and support vital clinical decision making.
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Técnicas de Química Analítica/instrumentação , Técnicas de Laboratório Clínico/instrumentação , Técnicas Analíticas Microfluídicas , Austrália , Automação Laboratorial , Técnicas de Química Analítica/normas , Testes Diagnósticos de Rotina , Europa (Continente) , Ensaios de Triagem em Larga Escala , Humanos , Reprodutibilidade dos Testes , Estados UnidosRESUMO
BACKGROUND: Uropathogenic strains of Escherichia coli cause symptomatic infections whereas asymptomatic bacteriuria (ABU) strains are well adapted for growth in the human urinary tract, where they establish long-term bacteriuria. Human urine is a very complex growth medium that could be perceived by certain bacteria as a stressful environment. To investigate a possible imbalance between endogenous oxidative response and antioxidant mechanisms, lipid oxidative damage estimated as thiobarbituric acid reactive substances (TBARS) content was evaluated in twenty-one E. coli belonging to various pathovars and phylogenetic groups. Antioxidant defense mechanisms were also analysed. RESULTS: During exponential growth in urine, TBARS level differs between strains, without correlation with the ability to grow in urine which was similarly limited for commensal, ABU and uropathogenic strains. In addition, no correlation between TBARS level and the phylogroup or pathogenic group is apparent. The growth of ABU strain 83972 was associated with a high level of TBARS and more active antioxidant defenses that reduce the imbalance. CONCLUSIONS: Our results indicate that growth capacity in urine is not a property of ABU strains. However, E. coli isolates respond very differently to this stressful environment. In strain ABU 83972, on one hand, the increased level of endogenous reactive oxygen species may be responsible for adaptive mutations. On the other hand, a more active antioxidant defense system could increase the capacity to colonize the bladder.
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Escherichia coli/fisiologia , Estresse Oxidativo , Urina/microbiologia , Escherichia coli/química , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Feminino , Humanos , Masculino , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
Cystic fibrosis (CF), a multisystem disease caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations, is associated with an abnormal inflammatory response and compromised redox homeostasis in the airways. Recent evidence suggests that dysfunctional CFTR leads to redox imbalance and to mitochondrial reduced glutathione (mtGSH) depletion in CF models. This study was designed to investigate the consequences of mtGSH depletion on mitochondrial function and inflammatory response. mtGSH depletion was confirmed in colonic epithelium of CFTR-null mice and in CFTR-mutated human epithelial cells. GSH uptake experiments performed on isolated mitochondria suggest that mtGSH depletion is not due to a defective GSH transport capacity by CF mitochondria, despite the decreased expression of two mtGSH carriers, oxoglutarate carrier and dicarboxylate carrier. CM-H(2)DCFDA [5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] fluorescence and aconitase activity showed an increase in reactive oxygen species levels in CFTR-defective cells and a pro-oxidative environment within CF mitochondria. The activities of respiratory chain complexes were further examined. Results showed a selective loss of Complex I (CI) function in CF models associated with an altered mitochondrial membrane potential (Δψ(m)). CI analysis showed normal expression but an overoxidation of its NADH-ubiquinone oxidoreductase Fe-S protein 1 subunit. GSH monoethyl ester (GSH-EE) significantly enhanced mtGSH levels in the IB3-1/C38 model and reversed CI inhibition, suggesting that mtGSH depletion is responsible for the loss of CI activity. Furthermore, GSH-EE attenuated Δψ(m) depolarization and restored normal IL-8 secretion by CFTR-defective cells. These studies provide evidence for a critical role of a mtGSH defect in mitochondrial dysfunction and abnormal IL-8 secretion in CF cells and reveal the therapeutic potential of mitochondria-targeted antioxidants in CF.
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Fibrose Cística/tratamento farmacológico , Glutationa/análogos & derivados , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Protetores contra Radiação/farmacologia , Animais , Linhagem Celular , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Glutationa/farmacologia , Interleucina-8/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Mutação , Recuperação de Função Fisiológica/efeitos dos fármacosRESUMO
OBJECTIVE: After out of hospital cardiac arrest, it has been reported that endothelium dysfunction may occur during the postresuscitation syndrome. However, the consequences of the reperfusion phase on endothelial reactive oxygen species production and redox homeostasis have not been explored in out of hospital cardiac arrest patients. DESIGN: Prospective, observational study. SETTING: Medical intensive care unit in a university hospital. PATIENTS: Twenty successfully resuscitated out of hospital cardiac arrest patients, seven septic shock patients, and ten healthy volunteers. INTERVENTION: Plasma was collected from patients at admission and 12, 24, 36, 48, and 72 hrs after cardiac arrest. We studied the production of reactive oxygen species and cell survival during plasma perfusion using perfused endothelial cells (human umbilical vein endothelial cells) as a model. Cell antioxidant response was studied by measuring superoxide dismutase, glutathione peroxidase, and glutathione reductase activities and reduced and oxidized glutathione levels. Mitochondrial respiratory chain activity was assessed by measuring complex I, II, III, and IV activities and anaerobic glycolysis by measuring glucose-6-phosphate dehydrogenase activity. MEASUREMENTS AND MAIN RESULTS: Using perfused endothelial cells as a model, we demonstrate that plasma from out of hospital cardiac arrest patients induced on naive human umbilical vein endothelial cells a significant and massive cell death compared to plasma from septic shock patients and healthy volunteers. An increase of reactive oxygen species production with a decrease in antioxidant defenses (superoxide dismutase, glutathione peroxidase, and glutathione reductase activities, reduced and oxidized glutathione levels) was observed. The metabolic consequence of plasma exposure showed that mitochondrial respiratory chain activity was significantly impaired and anaerobic glycolysis was significantly increased. Inhibiting hydroxyl radical production significantly decreased cell death, suggesting that plasma from out of hospital cardiac arrest induced significant cell death by triggering the Fenton reaction. CONCLUSION: Plasma from out of hospital cardiac arrest induces major endothelial toxicity with an acute pro-oxidant state in the cells and impairment of mitochondrial respiratory chain activity. This toxicity could be due to hydroxyl radical production by activation of the Fenton reaction.
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Células Endoteliais/metabolismo , Radical Hidroxila/metabolismo , Parada Cardíaca Extra-Hospitalar/sangue , Adulto , Idoso , Reanimação Cardiopulmonar/efeitos adversos , Morte Celular , Células Cultivadas , Células Endoteliais/fisiologia , Feminino , Glucosefosfato Desidrogenase/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Glicólise/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Mitocondriais/fisiopatologia , Parada Cardíaca Extra-Hospitalar/fisiopatologia , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/fisiopatologia , Choque Séptico/sangue , Choque Séptico/fisiopatologia , Superóxido Dismutase/metabolismo , Adulto JovemRESUMO
PURPOSE: Kidneys from haemodynamically unstable donors may suffer from renal ischaemia-reperfusion (RIR) injury. RIR is associated with reactive oxygen species production that induces inflammation and activates the arachidonic acid (AA) pathway which converts AA into prostaglandin E(2). Amifostine was investigated for its renoprotective potential in RIR. MATERIALS AND METHODS: The effect of amifostine (25 mg/kg = 910 mg/m(2)) on the COX pathway, enzymatic antioxidant activity, the lipid peroxidation marker MDA, serum creatinine and apoptosis was determined in rats. Kidneys were subjected to 45 min of ischaemia and 1 or 24 h of reperfusion. Control groups (sham: coeliotomy, no ischaemia; r1: 45 min ischaemia/1 h reperfusion; r2: 45 min ischaemia/24 h reperfusion) were administered physiological saline intraperitoneally, and treated groups (E1: 45 min ischaemia/1 h reperfusion; E2: 45 min ischaemia/24 h reperfusion) received amifostine 30 min before reperfusion. RESULTS: Serum creatinine increased in non-treated control rats: r1 vs sham (1.6-fold; P <0.007), r2 vs sham (2-fold; P <0.007). Amifostine decreased serum creatinine levels in treated rats: E1 vs r1 (8%; P <0.0025), E2 vs r2 (44%; P <0.0025). Amifostine reduced acute tubular necrosis (25%) 24 h after reperfusion: E1 vs r1 (P <0.004), E2 vs r2 (P <0.03) and reduced COX-2 and microsomal prostaglandin E synthase expression: E1 vs r1 (P <0.03), E2 vs r2 (P <0.02). Amifostine decreased MDA (P <0.04) and reduced caspase-3 expression but did not alter enzymatic antioxidant activity after RIR. CONCLUSIONS: Amifostine decreased the degree and severity of tubular damage after reperfusion, probably by scavenging oxygen free radicals and attenuating the cytotoxic effects of inflammatory infiltrates and apoptosis.
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Injúria Renal Aguda/prevenção & controle , Amifostina/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Amifostina/farmacologia , Animais , Creatinina/sangue , Ciclo-Oxigenase 2/metabolismo , Oxirredutases Intramoleculares/metabolismo , Túbulos Renais/irrigação sanguínea , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Modelos Animais , Necrose , Prostaglandina-E Sintases , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Resultado do TratamentoRESUMO
BACKGROUND: Evaluation of specific urinary markers with respect to urine creatinine (uCreat) is common. However, as uCreat is a function of both glomerular filtration and tubular secretion, using uCreat for specific tubular markers, suggests that glomerular function is normal, and there is no tubular secretion. Thus, adjusting values of any tubular marker to uCreat, especially in patients with acute or even moderate chronic renal failure, can be misleading. METHODS: Using urine cystatin-C (uCST3) as a model tubular marker for following 120 kidney graft recipients daily, we evaluated the utility of either uCST3 alone or the uCST3/uCreat ratio to detect tubular damage. All positive kidney biopsies were always associated with a uCST3>0.18 mg/L. RESULTS: Using the uCST3/uCreat ratio, discrepancies regarding biopsy status were observed in nine patients (4 false positive, 5 false negative results). In two patients, variability of uCreat appeared to be the most important factor causing inconsistent uCST3/uCreat ratios. With a negative predictive value (NPV) of 85.7%, uCST3/uCreat can lead to errors in clinical interpretation. These errors can be avoided when estimates of tubular damage are based on uCST3 concentrations alone (NPV=100%). CONCLUSIONS: We recommend using the uCST3 value to evaluate the extent of renal tubular damage. Indeed, our conflicting results on uCST3/uCreat can be extended to every marker of tubular function. Evaluating a urine marker specific for renal tubular damage to a second urine marker that is itself strongly dependent upon glomerular or other renal or non-renal conditions, impairs its clinical relevance and may lead to incorrect interpretations. Correction with uCreat can be performed only in pure glomerulopathy, when specific markers of glomerular function are measured (i.e., urinary albumin). In all other cases of renal diseases, such correction is inappropriate and should be avoided. Clin Chem Lab Med 2009;47:1553-6.
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Biomarcadores/urina , Creatinina/urina , Cistatina C/urina , Túbulos Renais/metabolismo , Humanos , Limite de DetecçãoRESUMO
PURPOSE: Renal injury caused by ischemia-reperfusion (IR) can lead to acute renal failure or delayed graft function. Renal ischemia-reperfusion (RIR) induces inflammatory disorders via activation of arachidonic acid metabolism into prostaglandin E(2) (PGE(2)). Two inducible enzymes, COX-2 and microsomal prostaglandin E synthase (mPGES), regulate PGE(2) production. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated during cellular stress. Overexpression of HO-1 is beneficial in transplantation models including antigen-independent IR injury, acute and chronic allograft rejection. MATERIALS AND METHODS: We investigated the effect of HO-1 induction on the COX pathway, antioxidant enzyme activities, malondialdehyde (MDA) levels, and apoptosis in rat kidneys subjected to 45 min ischemia and 1 h or 24 h reperfusion. Rats were injected intraperitoneally with either: 50 mg/kg hemin (HO-1 inducer groups: H1, H2); 50 micromol/kg ZnPP (HO-1 inhibitor groups: Hz1, Hz2); or 0.9% saline (control groups: r1, r2). Sham animals (Sh) did not undergo RIR. RESULTS: Serum creatinine increased significantly after RIR (r vs Sh; p <0.05). Hemin treatment induced a significant decrease in serum creatinine after RIR (H vs r; p <0.05) whereas ZnPP treatment significantly increased serum creatinine levels (Sh vs Hz; p <0.05). Hemin reduced the severity of acute tubular necrosis and significantly reduced COX-2 and mPGES expression (p <0.05). Hemin did not alter depleted antioxidant enzyme activity but did decrease levels of MDA (p <0.05). Hemin also reduced caspase-3 expression. CONCLUSIONS: HO-1 decreased the degree and severity of tubular damage after IR, probably by attenuating the cytotoxic effects of inflammatory infiltrates and apoptosis.
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Caspase 3/metabolismo , Heme Oxigenase-1/metabolismo , Rim/enzimologia , Malondialdeído/metabolismo , Traumatismo por Reperfusão/enzimologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/imunologia , Creatinina/sangue , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Ciclo-Oxigenase 2/metabolismo , Ativação Enzimática/efeitos dos fármacos , Heme Oxigenase-1/genética , Heme Oxigenase-1/imunologia , Hemina/administração & dosagem , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Rim/imunologia , Masculino , Malondialdeído/imunologia , Protoporfirinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologiaRESUMO
The conformational analysis of artemisinin by molecular dynamics and quantum chemistry calculations revealed the existence of seven energy minima with specific interconversion pathways. Among the seven conformers, only , and were able to undergo bond rearrangements upon Fe(2+) interaction. These rearrangements were due to a peculiar puckering of the trioxane ring that brings its three oxygen atoms in an ideal geometrical position for interacting with Fe(2+) ions, promoting an electronic redistribution in the molecule. A rapid molecule rearrangement led to a stable energy minimum structure with an additional ring that is similar to a plant metabolite. Our results suggest an alternative pathway for generating toxic radical chemical species for the malaria parasite, where artemisinin is not toxic by itself but rather is an intermediate for molecular partners that generate radical structures deleterious for the parasite proteins, after electron transfers from the Fe(2+)/artemisinin complex.
Assuntos
Antimaláricos/química , Artemisininas/química , Ferro/química , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Espectrofotometria InfravermelhoRESUMO
OBJECTIVE: Plasma from septic shock patients can induce production of reactive oxygen species (ROS) by human umbilical vein endothelial cells (HUVEC) in vitro. How endothelial cells defend themselves against ROS under increased oxidative stress has not yet been examined. This study investigates the antioxidant defenses of HUVEC exposed to plasma obtained from either septic shock patients or healthy volunteers. DESIGN: Prospective, observational study. SETTING: Medical intensive care unit in a university hospital. PATIENTS: Twenty-five patients with septic shock and 10 healthy volunteers. INTERVENTIONS: Blood samples were collected within the first 24 hrs of septic shock. In vitro HUVEC production of ROS was studied by spectrofluorimetry using 2',7'-dichlorodihydrofluorescein diacetate fluorescent dye. Reactive nitrogen species were also assessed. Intracellular reduced glutathione (GSH) levels were measured using monochlorobimane fluorescent dye. Activity of catalase and superoxide dismutase in HUVEC were also measured. Cell death was assessed using YOPRO fluorescent dye and the MTT assay. MEASUREMENTS AND RESULTS: On admission, the septic shock population's mean age was 55 yrs old, the mean Sequential Organ Failure Assessment score was 12, mean simplified acute physiology score was 50, and intensive care unit mortality rate was 45%. Evaluation of HUVEC antioxidant defenses showed a significantly decreased GSH level, increased catalase activity, and unchanged superoxide dismutase activity. ROS levels and cell death were significantly reduced when cells were pretreated with N-acetylcysteine or GSH, but no changes in reactive nitrogen species were observed. CONCLUSION: This study demonstrates that plasma-induced ROS production by HUVEC is associated with an intracellular decrease in reduced GSH. Both ROS levels and cell death decreased when N-acetylcysteine or GSH were added before exposing the cells to plasma. These data suggest a pivotal role of alterations in GSH in damage caused by sepsis-generated ROS in endothelial cell.
Assuntos
Morte Celular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Glutationa/deficiência , Glutationa/fisiologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Choque Séptico/sangue , APACHE , Acetilcisteína/farmacologia , Adulto , Idoso , Endotélio Vascular/efeitos dos fármacos , Feminino , Sequestradores de Radicais Livres/farmacologia , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Choque Séptico/metabolismo , Choque Séptico/mortalidadeRESUMO
Liver ischemia-reperfusion (I/R) injury occurs in many clinical conditions, including liver surgery and transplantation. Oxygen free radicals generated during I/R reduce endogenous antioxidant systems and contribute to hepatic injury. trans-Resveratrol (trans-3,5,4'-trihydroxystilbene) is reported to have antioxidant properties. We investigated the effect of trans-resveratrol on liver injury induced by I/R. After 1 hour of ischemia, administered 5 minutes before 3 hours of reperfusion, trans-resveratrol was hepatoprotective at a low dose (0.02 mg/kg). It significantly decreased aminotransferase levels by about 40% and improved sinusoidal dilatation. trans-Resveratrol preserved antioxidant defense by preventing total and reduced glutathione depletion caused by I/R. At 0.2 mg/kg, trans-resveratrol significantly increased glutathione reductase, Cu/Zn-superoxide dismutase, and catalase activities. However, at a high dose (20 mg/kg), trans-resveratrol became prooxidant with an aggravation of liver injury evaluated by aminotransferase release and histological analysis and associated with a depletion of total and reduced glutathione levels and a decrease of antioxidant enzyme activities. In conclusion, a prereperfusion treatment by trans-resveratrol only at low doses decreases liver injury induced by I/R by protecting against antioxidant defense failure. This administration protocol could reduce liver damage during surgery or transplantation.
Assuntos
Traumatismo por Reperfusão/prevenção & controle , Estilbenos/uso terapêutico , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Catalase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Artéria Hepática , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Circulação Hepática/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Resveratrol , Superóxido Dismutase/metabolismo , Vasodilatadores/uso terapêuticoRESUMO
BACKGROUND: Detecting and quantifying circulating free DNA in patient serum has become a major challenge. New methods using conventional or automated DNA amplification have been developed. As quantitative real-time PCR (QPCR) remains expensive and requires dedicated automated instrumentation, we questioned whether simple quantification using fluorescent dyes is efficient for determination of free DNA levels in serum. METHODS: Serum samples from 180 cancer patients and 58 healthy volunteers were used for DNA quantification according to three methods: (i) using an exonic part of the beta-globin gene as the amplifying target; (ii) amplifying a 105-bp intron 1 part of the housekeeping cyclophilin A gene, both referring to specific standard curves; and (iii) using a PicoGreen DNA quantification kit without amplification. RESULTS: The 58 samples from healthy controls showed a reference limit of (95th percentile) <160 cyclophilin gene copies/mL. The 180 cancer samples displayed values ranging between 300 and 215,000 copies/mL. The cyclophilin method showed a high level of correlation with both the beta-globin (r=0.911, p<0.0001) and PicoGreen (r=0.915, p<0.0001) methods. CONCLUSIONS: Aside from the disadvantage that the QPCR assays can only be used in clinical biochemistry laboratories that possess QPCR apparatus, the use of direct PicoGreen quantification displays major advantages in a routine context: it is less time-consuming and is quite inexpensive, but is still correlated with QPCR.
Assuntos
DNA/análise , Corantes Fluorescentes , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico/normas , Adulto , Idoso , Estudos de Casos e Controles , Ciclofilinas , Globinas , Humanos , Pessoa de Meia-Idade , Neoplasias/genéticaRESUMO
Diabetic nephropathy (DN) is associated with increased oxidative stress, overexpression and activation of growth factor receptors, including those for transforming growth factor-beta1 (TGF-beta-RII), platelet-derived growth factor (PDGF-R), and insulin-like growth factor (IGF1-R). These pathways are believed to represent pathophysiological determinants of DN. Beyond perfect glycemic control, angiotensin-converting enzyme inhibitors (ACEI) are the most efficient treatment to delay glomerulosclerosis. Since their mechanisms of action remain uncertain, we investigated the effect of ACEI on the glomerular expression of these growth factor pathways in a model of streptozotocin-induced diabetes in rats. The early phase of diabetes was found to be associated with an increase in glomerular expression of IGF1-R, PDGF-R, and TGF-beta-RII and activation of IRS1, Erk 1/2, and Smad 2/3. These changes were significantly reduced by ACEI treatment. Furthermore, ACEI stimulated glutathione peroxidase activity, suggesting a protective role against oxidative stress. ACEI decreased ANG II production but also increased bradykinin bioavailability by reducing its degradation. Thus the involvement of the bradykinin pathway was investigated using coadministration of HOE-140, a highly specific nonpeptidic B2-kinin receptor antagonist. Almost all the previously described effects of ACEI were abolished by HOE-140, as was the increase in glutathione peroxidase activity. Moreover, the well-established ability of ACEI to reduce albuminuria was also prevented by HOE-140. Taken together, these data demonstrate that, in the early phase of diabetes, ACEI reverse glomerular overexpression and activation of some critical growth factor pathways and increase protection against oxidative stress and that these effects involve B2-kinin receptor activation.