RESUMO
Phagocytes, such as granulocytes and monocytes/macrophages, contain a membrane-associated NADPH oxidase that produces superoxide leading to other reactive oxygen species with microbicidal, tumoricidal and inflammatory activities. Primary defects in oxidase activity in chronic granulomatous disease (CGD) lead to severe, life-threatening infections that demonstrate the importance of the oxygen-dependent microbicidal system in host defence. Other immunological disturbances may secondarily affect the NADPH oxidase system, impair the microbicidal activity of phagocytes and predispose the host to recurrent infections. This article reviews the primary defects of the human NADPH oxidase leading to classical CGD, and more recently discovered immunological defects secondarily affecting phagocyte respiratory burst function and resulting in primary immunodeficiencies with varied phenotypes, including susceptibilities to pyogenic or mycobacterial infections.
Assuntos
Doença Granulomatosa Crônica/enzimologia , NADPH Oxidases/imunologia , Fagócitos/enzimologia , Explosão Respiratória/imunologia , Infecções Bacterianas/enzimologia , Infecções Bacterianas/imunologia , Doença Granulomatosa Crônica/imunologia , Doença Granulomatosa Crônica/microbiologia , Humanos , Mutação , NADPH Oxidases/genética , Fagócitos/imunologia , Fagócitos/microbiologiaRESUMO
We demonstrate here that neutrophils from chronic granulomatous disease (CGD) patients release larger amounts of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) than neutrophils from control subjects. Incremental cytokine production was observed under both basal and stimulated conditions in neutrophils from two CGD (gp 91phox) patients. The basal production of IL-8 was over seven-fold greater in CGD patients. The two samples assayed showed 3- and 10-fold increases in TNF-alpha. Basically, the same magnitude of increment was observed in lypopolysaccharide (LPS) and serum amyloid A protein (SAA)-stimulated cells. We also found that the levels of SAA and IL-8 were higher in the serum of CGD patients than the levels found in the serum of healthy donors. The increased responsiveness of neutrophils from CGD patients may be closely related with a deficiency in the assembly of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase enzyme system, or it may be due to a frequent inflammatory condition in these patients. In the latter case, the increased serum levels of systemic inflammatory factors, among them SAA, would contribute to the sustained accumulation and activation of phagocytes. Whatever the origin, the excessive production of cytokines may lead to inappropriate activation and tissue injury and even to increased susceptibility to invasive microorganisms, impairing the quality life of CGD patients.
Assuntos
Doença Granulomatosa Crônica/enzimologia , Doença Granulomatosa Crônica/imunologia , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/deficiência , NADPH Oxidases/deficiência , Neutrófilos/imunologia , Proteína Amiloide A Sérica/farmacologia , Adulto , Criança , Feminino , Doença Granulomatosa Crônica/genética , Humanos , Interleucina-8/análise , Interleucina-8/sangue , Masculino , Glicoproteínas de Membrana/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , Neutrófilos/química , Neutrófilos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/análiseRESUMO
Patients with severe leukocyte G6PD deficiency may present with impairment of NADPH oxidase activity and a history of recurrent infections, mimicking the phenotype of chronic granulomatous disease. We report herein a child with recurrent infections who initially received the diagnosis of G6PD deficiency. His erythrocyte G6PD activity was reduced: 1.8 U/g Hb (normal: 12.1 +/- 2.1 U/g Hb). Further studies revealed that G6PD activity in neutrophils, mononuclear leukocytes, and Epstein-Barr virus-transformed B-lymphocytes from the proband was similar to healthy controls. Molecular studies showed that the G6PD deficiency was due a 202 G-->A mutation, the A- variant common in African ethnic groups. The proband also exhibited severely impaired respiratory burst activity, as observed in X-linked CGD. Sequence analysis of genomic DNA showed a 264 G-->A substitution at the 3' splice junction of gp91-phox exon 3. The cDNA sequence showed a deletion of gp91-phox exon 3, giving rise to an unstable or nonfunctional mutant gp91-phox and to the phenotype of X-linked CGD. We propose that clinicians treating a patient with G6PD deficiency during a severe infection episode consider the possibility of temporary or permanent impairment of the phagocytes' microbicidal activity and the eventual association of G6PD deficiency and chronic granulomatous disease.
Assuntos
Anemia/complicações , Deficiência de Glucosefosfato Desidrogenase/genética , Doença Granulomatosa Crônica/genética , Infecções/complicações , Substituição de Aminoácidos , Anemia/enzimologia , Sequência de Bases , Células Sanguíneas/enzimologia , Criança , DNA/genética , DNA Complementar/genética , Expressão Gênica , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Deficiência de Glucosefosfato Desidrogenase/complicações , Doença Granulomatosa Crônica/enzimologia , Humanos , Infecções/enzimologia , Masculino , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples , Superóxidos/metabolismoRESUMO
BACKGROUND: The cytosolic protein p47-phox (phagocyte oxidase) is one of the essential components of the superoxide generating system in phagocytes and its defect causes approximately 30% of the chronic granulomatous disease (CGD) cases. AIM: Two patients were studied, belonging to the same family, without a consanguinous background, in which deficiency or absence of superoxide generation was found together with recurrent and severe infections in one case and benign infections in the second. METHODS: The presence of gp91-, p67- and p47-phox in patients and controls was determined by Western Blot analysis of granulocytes. Sequencing of PCR amplified DNA was performed by an enzymatic method. RESULTS: Western Blot analysis showed normal expression of gp91 and p67 and absence of p47-phox. The molecular genetic study demonstrated a homocygotic dinucleotide GT (GT) deletion at the beginning of exon 2 of the p47-phox gene. The same mutation has been found in European, American and Japanese patients. CONCLUSIONS: The molecular characterization of this pathology done for the first time in Chile is important for diagnostic classification, patient prognosis, and adequate genetic advice and a possible future therapy.
Assuntos
Doença Granulomatosa Crônica/genética , Fosfoproteínas/deficiência , Adolescente , Adulto , Western Blotting , Estudos de Casos e Controles , Análise Mutacional de DNA , Ensaio de Imunoadsorção Enzimática , Granulócitos/enzimologia , Doença Granulomatosa Crônica/enzimologia , Humanos , Masculino , NADPH Oxidases , Linhagem , Reação em Cadeia da Polimerase , Deleção de SequênciaRESUMO
Chronic Granulomatous Disease (CGD) is a primary immunodeficiency characterized by an unusual predisposition to develop bacterial and fungal infections due to a failure of phagocytic leukocytes to generate superoxide, required for the intracellular killing of microorganisms. The lack of superoxide production is secondary to a defect in the NADPH-oxidase enzymatic complex activation, as a result of mutations of any of the components. Both, X-linked and autosomal recessive patterns of inheritance have been demonstrated in this disease, being the X-linked the most frequent and characterized by mutations in gp91phox. Mutations in p47phox, p67phox and p22phox have been shown in the autosomal recessive pattern. The molecular and genetic characteristic of NADPH-oxidase complex and its pathology in CGD are reviewed along with a brief description of the preliminary findings in two families from Mérida, Venezuela.
Assuntos
Doença Granulomatosa Crônica/genética , NADPH Oxidases/metabolismo , Adolescente , Algoritmos , Criança , Pré-Escolar , Doença Granulomatosa Crônica/diagnóstico , Doença Granulomatosa Crônica/enzimologia , Humanos , Masculino , Mutação , NADPH Oxidases/genética , Linhagem , VenezuelaRESUMO
We studied phagocyte reduced nicotinamide adenine dinucleotide phosphate function to evaluate production of reactive oxygen species in both X-linked and autosomal forms of chronic granulomatous disease. We found a consistent and significant difference between the activated granulocyte response of the X-linked (gp91-phagocyte oxidase) form of chronic granulomatous disease (n = 18) and that of the most common autosomal recessive (p47-phagocyte oxidase) form of the disease (n = 17). The data indicate that mutations in the p47-phagocyte oxidase component of the reduced nicotinamide adenine dinucleotide phosphate oxidase component do not completely prevent oxidation despite severe defects in superoxide generation.
Assuntos
Doença Granulomatosa Crônica/enzimologia , Doença Granulomatosa Crônica/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Citometria de Fluxo , Genótipo , HumanosRESUMO
Flow cytometric quantitative analysis of cytochrome b on outer surface membrane and of oxidative product formation in polymorphonuclear leukocytes (PMNLs) from patients with chronic granulomatous disease was carried out with the use of monoclonal antibody against cytochrome b of human neutrophils and 2', 7'-dichlorofluorescin diacetate. Cytochrome b was present on the outer surface membrane of PMNLs in normal individuals, and its absence on the outer surface membrane was of value in the diagnosis and classification of chronic granulomatous disease. This study has shown that a major type of chronic granulomatous disease is one of the monoclonal antibody-defined surface membrane diseases.
Assuntos
Grupo dos Citocromos b/deficiência , Doença Granulomatosa Crônica/classificação , Adolescente , Adulto , Anticorpos Monoclonais , Membrana Celular/enzimologia , Criança , Pré-Escolar , Grupo dos Citocromos b/análise , Feminino , Citometria de Fluxo , Doença Granulomatosa Crônica/enzimologia , Humanos , Masculino , Neutrófilos/enzimologiaRESUMO
NADPH oxidase activity was examined in paired 27,000 x g granule fractions isolated from normal polymorphonuclear leukocytes from patients with chronic granulomatous disease. At 0.17 mM NADPH, the oxidase activity was not measurable in normal resting cells but was activated by phagocytosis. This activation was absent in CGD cells. At higher levels of NADPH, activity was present in cells from patients with CGD, although it was lower than normal, and no difference in activity was found between resting and phagocytizing cells. Granule fractions from phagocytizing normal cells exhibited higher than granule fractions from resting normal cells at all levels of NADPH. These results suggest that NADPH oxidase activity is defective in chronic granulomatous disease, and further that the defect is not the absence of the enzyme but rather a failure to activate it.
Assuntos
Doença Granulomatosa Crônica/enzimologia , NADH NADPH Oxirredutases/deficiência , Neutrófilos/enzimologia , Disfunção de Fagócito Bactericida/enzimologia , Adolescente , Adulto , Criança , Pré-Escolar , Ativação Enzimática , Feminino , Genes Recessivos , Doença Granulomatosa Crônica/genética , Humanos , Lactente , Masculino , NADP , Concentração Osmolar , Fagocitose , Cromossomos SexuaisRESUMO
A male child with chronic granulomatous disease is described in whom glutathione peroxidase deficiency of leukocytes was identified. Stability and activity of G-6-PD and activity of NADPH oxidase were normal. The leukocytes of the parents showed intermediate activities of glutathione peroxidase, suggesting the possibility of autosomal recessive inheritance.