RESUMO
Introducción: La enfermedad granulomatosa crónica es una inmunodeficiencia primaria causada por mutaciones en la enzima NADPH oxidasa. Esta compromete la producción de especies reactivas del oxígeno, que son importantes contra patógenos. La prueba de la oxidación de la dihidrorodamina es un método eficaz para diagnosticar la enfermedad. Objetivo: Demostrar la utilidad de la prueba de la oxidación de la dihidrorodamina y del patrón de herencia en la confirmación del diagnóstico de la enfermedad granulomatosa crónica de un paciente. Métodos: Estudio de caso de una familia con diagnóstico de enfermedad granulomatosa crónica. Se tomó muestra de sangre periférica para citometría de flujo a tres individuos. Se realizó la prueba de la oxidación de la dihidrorodamina bajo estímulo con acetato de forbolmiristato y se evaluaron las subpoblaciones linfocitarias. Las muestras se leyeron en un citómetro GALLIOS, Beckman Coulter. Los datos obtenidos se analizaron en el programa informático Kaluza. Resultados: El paciente masculino tuvo un valor de oxidación de la dihidrorodamina positiva de 0,87 por ciento, que confirmó un patrón de herencia ligado al cromosoma X; mientras que la madre y hermana gemela portadoras tuvieron valores de 46,76 por ciento y 37,32 por ciento, respectivamente. Se encontraron alteraciones en las subpoblaciones linfocitarias. Conclusiones: La prueba de la oxidación de la dihidrorodamina es un método muy efectivo, rápido y sencillo que confirma el diagnóstico de la enfermedad granulomatosa crónica y determina el patrón de herencia y fenotipo de la enfermedad. Además, permite identificar a las mujeres portadoras según la distribución de los neutrófilos normales y los que tienen el gen CYBB mutado(AU)
Introduction: Chronic granulomatous disease is a primary immunodeficiency caused by mutations in the NADPH oxidase enzymes. This compromises the production of oxygen reactive species, which are important against pathogens. The dihydrorhodamine oxidation test is an effective method for diagnosing the disease. Objective: To demonstrate the usefulness of the dihydrorhodamine oxidation test and the inheritance pattern in confirming the diagnosis of chronic granulomatous disease in a patient. Methods: A case study of a family with a diagnosis of chronic granulomatous disease. A peripheral blood sample was taken from three individuals and by flow cytometry. The dihydrorhodamine oxidation test was performed under stimulation with phorbolmyristate acetate, and lymphocyte subpopulations were evaluated. The samples were read on a GALLIOS, Beckman Coulter cytometer. The data obtained were analyzed using the computer program Kaluza. Results: The male patient had a positive dihydrorhodamine oxidation value of 0.87 percent, which confirmed an inheritance pattern linked to the X chromosome; while the carrier mother and twin sister had values 8203;8203;of 46.76 percent and 37.32 percent, respectively. Alterations were found in the lymphocyte subpopulations. Conclusions: The dihydrorhodamine oxidation test is a very effective, fast and simple method that confirms the diagnosis of chronic granulomatous disease and determines the inheritance pattern and phenotype of the disease. In addition, it allows the identification of female carriers according to the distribution of normal neutrophils and those with the CYBB mutation(AU)
Assuntos
Humanos , Masculino , Feminino , Portador Sadio/congênito , NADPH Oxidases/análise , Padrões de Herança/genética , Doença Granulomatosa Crônica/diagnóstico , Relatos de Casos , Cuba , Triagem de Portadores Genéticos/métodos , Anamnese/métodosRESUMO
Lactate modulates the expression of lactate oxidation complex (LOC)-related genes and cardiac blood flow under physiological conditions, but its modulatory role remains to be elucidated regarding pathological cardiac stress. The present study evaluated the effect of lactate on LOC-related genes expression and hemodynamics of hearts submitted to myocardial infarction (MI). Four weeks after MI or sham operation, isolated hearts of male Wistar rats were perfused for 60 min with Na+-lactate (20 mM). As expected, MI reduced cardiac contractility and relaxation with no changes in perfusion. The impaired cardiac hemodynamics were associated with increased reactive oxygen species (ROS) levels (Sham: 19.3±0.5 vs MI: 23.8±0.3 µM), NADPH oxidase (NOX) activity (Sham: 42.2±1.3 vs MI: 60.5±1.5 nmol·h-1·mg-1) and monocarboxylate transporter 1 (mct1) mRNA levels (Sham: 1.0±0.06 vs MI: 1.7±0.2 a.u.), but no changes in superoxide dismutase (SOD), catalase, NADH oxidase (NADox), and xanthine oxidase activities. Lactate perfusion in MI hearts had no additional effect on ROS levels, NADox, and NOX activity, however, it partially reduced mct1 mRNA expression (MI-Lactate 1.3±0.08 a.u.). Interestingly, lactate significantly decreased SOD (MI-Lactate: 54.5±4.2 µmol·mg-1·min-1) and catalase (MI: 1.1±0.1 nmol·mg-1·min-1) activities in MI. Collectively, our data suggest that under pathological stress, lactate lacks its ability to modulate the expression of cardiac LOC-related genes and the perfused pressure in hearts submitted to chronic MI. Together, these data contribute to elucidate the mechanisms involved in the pathogenesis of heart failure induced by MI.
Assuntos
Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ácido Láctico/metabolismo , Ácido Láctico/farmacologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Animais , Catalase/análise , Expressão Gênica , Ácido Láctico/análise , Masculino , Complexos Multienzimáticos/análise , NADH NADPH Oxirredutases/análise , NADPH Oxidases/análise , Oxirredução/efeitos dos fármacos , Perfusão , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Superóxido Dismutase/análise , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Xantina Oxidase/análiseRESUMO
BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.
Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , NADPH Oxidases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus agalactiae/metabolismo , Análise de Variância , Aderência Bacteriana , Western Blotting , Células Endoteliais da Veia Umbilical Humana/microbiologia , Humanos , NADPH Oxidases/análise , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/análise , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.
Assuntos
Humanos , Espécies Reativas de Oxigênio/análise , NADPH Oxidases/análise , NADPH Oxidases/metabolismo , Células Endoteliais da Veia Umbilical Humana/microbiologia , Transdução de Sinais/fisiologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Os pacientes com diabetes mellitus (DM) apresentam maior prevalência de doenças tireoidianas que a população geral. A autoimunidade certamente é um fator-chave na relação entre essas disfunções endócrinas. Entretanto, outros mecanismos, como redução da captação de iodeto, da atividade tireoperoxidase e aumento do estresse oxidativo na glândula tireoide, também parecem contribuir para este fato. O presente trabalho visa rever aspectos importantes na relação entre DM e doenças tireoidianas, com especial ênfase nos mecanismos envolvidos no aumento do estresse oxidativo na glândula tireoide decorrente do DM...
Diabetes mellitus (DM) patients show a greater prevalence of thyroid disorders than general population. Autoimmunity is a key factor in the relation between these endocrine diseases. However, additional mechanisms, such as reduction of iodide uptake and thyroperoxidase activity, besides increased oxidative stress in the thyroid gland seem to contribute for this fact. The present work aims to review important aspects in the relation between DM and thyroid disease, with special emphasis in the mechanisms involved in the increased oxidative stress in the thyroid gland due to DM...
Assuntos
Humanos , Masculino , Feminino , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Doenças da Glândula Tireoide , Autoimunidade , Complicações do Diabetes/metabolismo , Doenças Cardiovasculares/etiologia , Hipertireoidismo/complicações , Hormônios Tireóideos/biossíntese , Hormônios Tireóideos/metabolismo , NADPH Oxidases/análise , NADPH Oxidases/metabolismo , Estresse Oxidativo , Peróxido de Hidrogênio/metabolismoRESUMO
BACKGROUND: Fructose administration rapidly induces oxidative stress that triggers compensatory hepatic metabolic changes. We evaluated the effect of an antioxidant, R/S-α-lipoic acid on fructose-induced oxidative stress and carbohydrate metabolism changes. METHODS: Wistar rats were fed a standard commercial diet, the same diet plus 10% fructose in drinking water, or injected with R/S-α-lipoic acid (35mg/kg, i.p.) (control+L and fructose+L). Three weeks thereafter, blood samples were drawn to measure glucose, triglycerides, insulin, and the homeostasis model assessment-insulin resistance (HOMA-IR) and Matsuda indices. In the liver, we measured gene expression, protein content and activity of several enzymes, and metabolite concentration. RESULTS: Comparable body weight changes and calorie intake were recorded in all groups after the treatments. Fructose fed rats had hyperinsulinemia, hypertriglyceridemia, higher HOMA-IR and lower Matsuda indices compared to control animals. Fructose fed rats showed increased fructokinase gene expression, protein content and activity, glucokinase and glucose-6-phosphatase gene expression and activity, glycogen storage, glucose-6-phosphate dehydrogenase mRNA and enzyme activity, NAD(P)H oxidase subunits (gp91(phox) and p22(phox)) gene expression and protein concentration and phosphofructokinase-2 protein content than control rats. All these changes were prevented by R/S-α-lipoic acid co-administration. CONCLUSIONS: Fructose induces hepatic metabolic changes that presumably begin with increased fructose phosphorylation by fructokinase, followed by adaptive changes that attempt to switch the substrate flow from mitochondrial metabolism to energy storage. These changes can be effectively prevented by R/S-α-lipoic acid co-administration. GENERAL SIGNIFICANCE: Control of oxidative stress could be a useful strategy to prevent the transition from impaired glucose tolerance to type 2 diabetes.
Assuntos
Metabolismo dos Carboidratos/efeitos dos fármacos , Frutose/farmacologia , Fígado/metabolismo , Estresse Oxidativo , Ácido Tióctico/farmacologia , Animais , Glucoquinase/análise , Glucoquinase/genética , Masculino , NADPH Oxidases/análise , NADPH Oxidases/genética , Ratos , Ratos WistarRESUMO
Role of reactive oxygen species (ROS)/nitric oxide (NO) balance and renin-angiotensin system in mediating cardiac hypertrophy in hyperthyroidism was evaluated in an in vivo and in vitro experimental model. Male Wistar rats were divided into four groups: control, thyroid hormone, vitamin E (or Trolox, its hydrosoluble analogue), thyroid hormone+vitamin E. Angiotensin II receptor (AT1/AT2) gene expression, immunocontent of AT1/AT2 receptors, angiotensinogen, NADPH oxidase (Nox2), and nitric oxide synthase isoforms, as well as ROS concentration (hydrogen peroxide and superoxide anion) were quantified in myocardium. Thyroid hormone increased ROS and NO metabolites, iNOS, nNOS and eNOS isoforms and it was accompanied by cardiac hypertrophy. AT1/AT2 expression and the immunocontent of angiotensinogen and Nox2 were enhanced by thyroid hormone. Antioxidants reduced ROS levels, Nox2, AT1/AT2, NOS isoforms and cardiac hypertrophy. In conclusion, ROS/NO balance may play a role in the control of thyroid hormone-induced cardiac hypertrophy mediated by renin-angiotensin system.
Assuntos
Cardiomegalia/patologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sistema Renina-Angiotensina/fisiologia , Hormônios Tireóideos/metabolismo , Angiotensinogênio/análise , Animais , Western Blotting , Cardiomegalia/metabolismo , Células Cultivadas , Cromanos/farmacologia , Hipertireoidismo/metabolismo , Masculino , NADPH Oxidases/análise , Óxido Nítrico/análise , Óxido Nítrico Sintase/genética , Reação em Cadeia da Polimerase , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores de Angiotensina/genética , Hormônios Tireóideos/farmacologia , Vitamina E/farmacologiaRESUMO
We aimed to characterize the relaxation induced by the soluble guanylyl cyclase (sGC) stimulator 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and its pharmacological interactions with nitric oxide (NO) in the corpus cavernosum (CC) from wild-type (WT), endothelial nitric-oxide synthase (eNOS)(-/-), and neuronal (n)NOS(-/-) mice. The effect of BAY 41-2272 on superoxide formation and NADPH oxidase expression was also investigated. Tissues were mounted in myographs for isometric force recording. Enzyme immunoassay kits were used for cGMP determination. sGC activity was determined in the supernatant fractions of the cavernosal samples by the conversion of GTP to cGMP. Superoxide formation and expression of NADPH oxidase subunits were studied using the reduction of ferricytochrome c and Western blot analysis, respectively. BAY 41-2272 (0.01-10 microM) relaxed CC with pEC(50) values of 6.36 +/- 0.07 (WT), 6.27 +/- 0.06 (nNOS(-/-)), and 5.88 +/- 0.07 (eNOS(-/-)). The relaxations were accompanied by increases in cGMP levels. N(omega)-Nitro-L-arginine methyl ester inhibited BAY 41-2272-evoked responses in CC from WT and nNOS(-/-), but not eNOS(-/-).1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one reduced and sildenafil potentiated the relaxations induced by BAY 41-2272 in all groups. BAY 41-2272 enhanced NO (endogenous and exogenous)-induced relaxations in a concentration-dependent manner. Expression and activity of sGC was similar among the different groups. Superoxide formation was reduced by BAY 41-2272 (0.1-1 microM). The compound also inhibited p22(phox) and gp91(phox) expression induced by 9,11-dideoxy-11 alpha,9 alpha-epoxymethanoprostaglandin F(2 alpha (U46619). Our results demonstrated that sGC activation in the penis by BAY 41-2272 directly or via enhancement of NO effects may provide a novel treatment for erectile dysfunction, particularly in the event of an increased intrapenile oxidative stress.
Assuntos
Músculo Liso/efeitos dos fármacos , NADPH Oxidases/efeitos dos fármacos , Óxido Nítrico Sintase/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Guanilato Ciclase , Camundongos , Camundongos Knockout , Tono Muscular/efeitos dos fármacos , Músculo Liso/fisiologia , NADPH Oxidases/análise , Óxido Nítrico/análise , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase Tipo I/análise , Óxido Nítrico Sintase Tipo I/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/análise , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Guanilil Ciclase Solúvel , Superóxidos/análiseRESUMO
The present study was undertaken to identify whether inflammation or oxidative stress is the primary abnormality in the kidney in spontaneously hypertensive rats (SHR). Renal inflammation and oxidative stress were evaluated in 2- and 3-week-old prehypertensive SHR and age-matched genetically normotensive control Wistar-Kyoto (WKY) rats. Blood pressure was similar in WKY and SHR rats at 2 and 3 weeks, of age. Renal inflammation (macrophage and nuclear factor-kappaB) was elevated in SHR at 3 weeks, but not at 2 weeks, of age compared with age-matched WKY rats. Renal oxidative stress (nitrotyrosine, 8-hydroxy-2'-deoxyguanosine and p47phox) was also clearly elevated in 3-week-old SHR compared with age-matched WKY rats. Additionally, NADPH oxidase subunit p47phox was found elevated in 2-week-old SHR compared to age-matched WKY rats. Moreover, antioxidant (N-acetyl-L-cysteine and Tempol) treatment reduced renal inflammation in prehypertensive SHR. We therefore conclude that the oxidative stress appears before inflammation as a primary abnormality in the kidney in prehypertensive SHR.
Assuntos
Nefrite/metabolismo , Estresse Oxidativo , Ratos Endogâmicos SHR/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Acetilcisteína/uso terapêutico , Fatores Etários , Animais , Antioxidantes/uso terapêutico , Pressão Sanguínea , Óxidos N-Cíclicos/uso terapêutico , Dano ao DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Modelos Animais de Doenças , Progressão da Doença , Glutationa/análise , Hipertensão/tratamento farmacológico , Hipertensão/etiologia , Hipertensão/genética , Hipertensão/metabolismo , Córtex Renal/metabolismo , Masculino , NADPH Oxidases/análise , Nefrite/tratamento farmacológico , Nefrite/genética , Nefrite/prevenção & controle , Ratos , Ratos Endogâmicos WKY , Marcadores de Spin , Tirosina/análogos & derivados , Tirosina/análiseRESUMO
No presente estudo avaliamos a atividade geradora de peróxido de hidrogênio (H 2 O 2 ) em frações particuladas de tireóides suínas e humanas. Inicialmente, analisamos as propriedades bioquímicas da NADPH-oxidase - enzima geradora de H 2 O 2 - localizada na membrana apical da célula tireóidea suína. Nossos resultados demonstram que a atividade geradora de H 2 O 2 na tireóide suína ocorre, principalmente, na fração de membrana apical tireóidea (P 3.000g). Entretanto, no P 3.000g a enzima NADPH-oxidase é apenas parcialmente cálcio-dependente, ao contrário do que acontece em frações purificadas de membrana tireóidea suína, nas quais a enzima é completamente dependente de cálcio, conforme estudos anteriores. Nossos resultados confirmam os previamente descritos para a NADPH oxidase tireóidea suína. Em tecidos tireóideos humanos, a geração de H 2 O 2 ocorreu, tanto na fração microsomal (P 100.000g) quanto na fração de membrana apical (P 3.000g). Nossos dados revelam ainda que a NADPH-oxidase humana é completamente cálcio-dependente, ativada por altas concentrações de fosfato e parece ser tão ativa na glândula humana quanto na suína. Além disto, a enzima humana é dependente de adenina-flavina-dinucleotídeo (FAD) no meio de reação, ou seja, parece ser uma flavoproteína, assim como a proteína suína.