Avances en el diagnóstico de la enfermedad granulomatosa crónica: primer estudio familiar en Cuba / Advances in the diagnosis of chronic granulomatous disease: the first family study in Cuba

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)

Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells

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.

Diabetes mellitus tipo 1 e disfunções tireoidianas / Diabetes Mellitus type 1 and thyroid dysfunction

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...

Sistema enzimático gerador de peróxido de hidrogênio: NADPH oxidase na tireóide humana / Enzimatic system generator of hydrogen peroxide: NADPH oxidase in human thyroid

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.