RESUMEN
Essentials Anticoagulants prevent venous thromboembolism but may be associated with greater bleeding risks. Bivariate analysis assumes a non-linear relationship between efficacy and safety outcomes. Extended full-dose betrixaban is favorable over standard enoxaparin in bivariate endpoint. Clinicians must weigh efficacy and safety outcomes in decision-making on thromboprophylaxis. SUMMARY: Background Among acutely ill hospitalized medical patients, extended-duration thromboprophylaxis reduces the risk of venous thromboembolism (VTE), but some pharmacologic strategies have been associated with greater risks of major bleeding, thereby offsetting the net clinical benefit (NCB). Methods To assess the risk-benefit profile of anticoagulation regimens, a previously described bivariate method that does not assume a linear risk-benefit tradeoff and can accommodate different margins for efficacy and safety was performed to simultaneously assess efficacy (symptomatic VTE) and safety (major bleeding) on the basis of data from four randomized controlled trials of extended-duration (30-46 days) versus standard-duration (6-14 days) thromboprophylaxis among 28 227 patients (EXCLAIM, ADOPT, MAGELLAN and APEX trials). Results Extended thromboprophylaxis with full-dose betrixaban (80 mg once daily) was superior in efficacy and non-inferior in safety to standard-duration enoxaparin, and showed a significantly favorable NCB, with a risk difference of - 0.51% (- 0.89% to - 0.10%) in the bivariate outcome. Extended enoxaparin was superior in efficacy and inferior in safety (bivariate outcome: 0.03% [- 0.37% to 0.43%]), whereas apixaban and rivaroxaban were non-inferior in efficacy and inferior in safety (- 0.20% [- 0.49% to 0.17%] and 0.23% [- 0.16% to 0.69%], respectively). Reduced-dose betrixaban did not show a significant difference in either efficacy or safety (0.41% [- 0.85% to 1.94%]). Conclusions In a bivariate analysis that assumes non-linear risk-benefit tradeoffs, extended prophylaxis with full-dose betrixaban was superior to standard-duration enoxaparin, whereas other regimens failed to simultaneously achieve both superiority and non-inferiority with respect to symptomatic VTE and major bleeding in the management of acutely ill hospitalized medical patients.
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Anticoagulantes/administración & dosificación , Anticoagulantes/efectos adversos , Hemorragia/inducido químicamente , Hospitalización , Tromboembolia Venosa/prevención & control , Enfermedad Aguda , Benzamidas/administración & dosificación , Benzamidas/efectos adversos , Toma de Decisiones Clínicas , Ensayos Clínicos Fase III como Asunto , Ensayos Clínicos Fase IV como Asunto , Esquema de Medicación , Enoxaparina/administración & dosificación , Enoxaparina/efectos adversos , Humanos , Análisis Multivariante , Dinámicas no Lineales , Pirazoles/administración & dosificación , Pirazoles/efectos adversos , Piridinas/administración & dosificación , Piridinas/efectos adversos , Piridonas/administración & dosificación , Piridonas/efectos adversos , Ensayos Clínicos Controlados Aleatorios como Asunto , Medición de Riesgo , Factores de Riesgo , Rivaroxabán/administración & dosificación , Rivaroxabán/efectos adversos , Factores de Tiempo , Resultado del Tratamiento , Tromboembolia Venosa/sangre , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/etiologíaRESUMEN
Essentials There is currently no approved reversal agent for factor Xa (FXa) inhibitors Andexanet alfa has been developed to reverse the anticoagulant effects of FXa inhibitors Andexanet reduced blood loss and anticoagulation markers in rivaroxaban-anticoagulated rabbits Andexanet was well tolerated in monkeys and rats, with no evidence of prothrombotic activity SUMMARY: Background Andexanet alfa is a recombinant modified form of factor Xa (FXa), designed to bind to and reverse the anticoagulant activity of FXa inhibitors. Objectives To evaluate the ability of andexanet to reverse the anticoagulant activity of rivaroxaban, and assess its pharmacokinetics (PK) and toxicity in animal models. Methods The effects of andexanet on blood loss, anti-FXa activity, rivaroxaban unbound plasma concentrations and other coagulation parameters were assessed in a rabbit liver laceration 'treatment' model. Andexanet was administered 10 min after blood loss was initiated. The toxicity of repeated administration of andexanet (up to 60 mg kg-1 day-1 ) was assessed in cynomolgus monkeys. PK parameters were evaluated in rats and monkeys. Results Excess blood loss due to anticoagulation with rivaroxaban was significantly decreased by a single intravenous bolus administration of andexanet at 35 and 75 mg per rabbit, by 75% and 63%, respectively. This correlated with dose-dependent decreases in the unbound fraction of rivaroxaban and anti-FXa activity. Co-administration of rivaroxaban had no significant impact on the PK parameters of andexanet. Andexanet (up to 60 mg kg-1 day-1 ) was well tolerated in monkeys, with no accumulation of andexanet or rivaroxaban. There was a single occurrence of anaphylaxis, which resolved after treatment with diphenhydramine and epinephrine. There was no histological evidence of prothrombotic activity with high-dose andexanet compared with vehicle control, as measured by clot and fibrin deposition in all major organs. Conclusions These data suggest that andexanet is a promising therapy for the reversal of FXa inhibitor-induced anticoagulation, supporting clinical studies in humans.
Asunto(s)
Antídotos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Inhibidores del Factor Xa , Factor Xa/farmacología , Hemorragia/prevención & control , Proteínas Recombinantes/farmacología , Rivaroxabán , Animales , Antídotos/farmacocinética , Antídotos/toxicidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Factor Xa/farmacocinética , Factor Xa/toxicidad , Hemorragia/sangre , Hemorragia/inducido químicamente , Laceraciones/complicaciones , Hígado/lesiones , Macaca fascicularis , Masculino , Conejos , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/toxicidad , Medición de RiesgoRESUMEN
Gp91-phox is an integral component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex that generates reactive oxygen species (ROS) in activated circulating phagocytes. The authors previously demonstrated that gp91-phox knockout (KO) mice show significant protection from neuronal injury after cerebral ischemia--reperfusion injury, suggesting a pivotal role for this enzyme. Moreover, results from chimeric mice suggested that elimination of gp91-phox from both circulating phagocytes and a putative central nervous system (CNS) source were required to confer neuroprotection. In the current study, the authors demonstrated gp91-phox-specific immunostaining of perivascular cells in the CNS of control rats. However, after transient cerebral ischemia, gp91-phox-positive phagocytes were observed within the core ischemic region and activated microglial cells were positive in the penumbra. Such activated microglial cells were also gp91-phox-positive in the CNS of a chimpanzee with mild meningitis. Finally, in humans, both normal adult CNS tissues and isolated fetal microglial cells expressed gp91-phox mRNA. These microglia also expressed mRNA for the five other known components that comprise the NADPH oxidase complex. These data strongly suggest that microglial cells may contain a functionally active NADPH oxidase capable of generating ROS during CNS inflammation.
Asunto(s)
Encefalitis/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglía/enzimología , NADPH Oxidasas/metabolismo , Fagocitosis/fisiología , Animales , Anticuerpos Monoclonales , Encefalitis/inmunología , Radicales Libres/metabolismo , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/inmunología , Enfermedad Granulomatosa Crónica/metabolismo , Humanos , Inmunohistoquímica , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Noqueados , Microglía/inmunología , NADPH Oxidasa 2 , Neutrófilos/inmunología , Pan troglodytes , Ratas , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/inmunología , Daño por Reperfusión/metabolismo , Especificidad de la Especie , Accidente Cerebrovascular/inmunología , Accidente Cerebrovascular/metabolismoRESUMEN
A fetus diagnosed with X-linked chronic granulomatous disease was transplanted with Thy-1(+)CD34(+) cells of paternal origin. The transplant was performed at 14 weeks gestation by ultrasound guided injection into the peritoneal cavity. The fetus was delivered at 38 weeks gestation after an otherwise uneventful pregnancy. Umbilical cord blood was collected and used to determine the level of peripheral blood chimerism as well as levels of functional engrafted cells. Flow cytometry was used to detect donor leukocytes identified as HLA-A2(-)B7(+) cells, whereas recipient cells were identified as HLA-A2(+)B7(-) cells. No evidence of donor cell engraftment above a level of 0.01% was found. PCR was used to detect HLA-DRB1*15(+) donor cells among the recipient's HLA-DRB1*15(-) cells, but no engraftment was seen with a sensitivity of 1:1000. The presence of functional, donor-derived neutrophils was assessed by flow cytometry using two different fluorescent dyes that measure reactive oxygen species generated by the phagocyte NADPH oxidase. No evidence of paternal-derived functional neutrophils above a level of 0.15% was observed. Peripheral blood and bone marrow samples were collected at 6 months of age. Neither sample showed engraftment by HLA typing using both flow cytometry and PCR. Functional phagocytes were also not observed. Furthermore, no indication of immunological tolerance specific for the donor cells was indicated by a mixed lymphocyte reaction assay performed at 6 months of age. While there appears to be no engraftment of the donor stem cells, the transplant caused no harm to the fetus and the child was healthy at 6 months of age. Analyses of fetal tissues, obtained from elective abortions, revealed that CD3(+) T cells and CD56(+)CD3(-) NK cells are present in the liver at 8 weeks gestation and in the blood by 9 weeks gestation. The presence of these lymphocytes may contribute to the lack of donor cell engraftment in the human fetus.
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Enfermedades Fetales/terapia , Enfermedad Granulomatosa Crónica/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/inmunología , Adulto , Antígenos CD34/sangre , Padre , Femenino , Sangre Fetal/citología , Enfermedades Fetales/sangre , Edad Gestacional , Rechazo de Injerto/inmunología , Enfermedad Granulomatosa Crónica/sangre , Humanos , Subgrupos Linfocitarios , Masculino , NADPH Oxidasas/metabolismo , Embarazo , Estallido Respiratorio , Antígenos Thy-1/sangre , Factores de Tiempo , Quimera por Trasplante/sangre , Trasplante Homólogo/métodosRESUMEN
Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in any one of 4 genes encoding phagocyte NADPH oxidase subunits. Unlike other CGD subtypes, in which there is great heterogeneity among mutations, 97% of affected alleles in patients previously reported with A47(0) CGD carry a single mutation, a GT deletion (DeltaGT) in exon 2 of the p47-phox gene, NCF-1. This unusually high incidence results from recombination events between NCF-1 and its highly homologous pseudogenes, in which DeltaGT originates. In 50 consecutive patients with A47(0) CGD, 4 were identified who were heterozygous for DeltaGT in NCF-1, and for the first time, 2 were identified whose DNA appeared normal at this position. To avoid co-amplification of pseudogene sequence and to enable the identification of mutations in these patients, allele-specific polymerase chain reaction was used to amplify alleles not containing DeltaGT. In each of the 4 patients who were heterozygous for DeltaGT, an additional novel mutation was identified. These were 2 missense mutations, G125 --> A in exon 2 (predicting Arg42 --> Gln) and G784 --> A in exon 8 (Gly262 --> Ser), and 2 splice junction mutations at the 5' end of intron 1, gt --> at and gtg --> gtt. The first of 2 patients who appeared normal at the GT position was a compound heterozygote with the G125 --> A transition on one allele and a deletion of G811 on the other. In the second of these patients, only a single defect was detected, G574 --> A, which predicts Gly192 --> Ser but is likely to result in defective splicing because it represents the final nucleotide of exon 6.
Asunto(s)
Enfermedad Granulomatosa Crónica/genética , Mutación , Fosfoproteínas/genética , Adolescente , Adulto , Alelos , Niño , Preescolar , Análisis Mutacional de ADN , Exones , Salud de la Familia , Femenino , Genotipo , Humanos , Masculino , NADPH Oxidasas , Reacción en Cadena de la Polimerasa , SeudogenesRESUMEN
In humans with sepsis, the onset of multiorgan failure (MOF), especially involving liver, lungs, and kidneys, is a well known complication that is associated with a high mortality rate. Our previous studies with the cecal ligation/puncture (CLP) model of sepsis in rats have revealed a C5a-induced defect in the respiratory burst of neutrophils. In the current CLP studies, MOF occurred during the first 48 h with development of liver dysfunction and pulmonary dysfunction (falling arterial partial pressure of O(2), rising partial pressure of CO(2)). In this model an early respiratory alkalosis developed, followed by a metabolic acidosis with increased levels of blood lactate. During these events, blood neutrophils lost their chemotactic responsiveness both to C5a and to the bacterial chemotaxin, fMLP. Neutrophil dysfunction was associated with virtually complete loss in binding of C5a, but binding of fMLP remained normal. If CLP animals were treated with anti-C5a, indicators of MOF and lactate acidosis were greatly attenuated. Under the same conditions, C5a binding to blood neutrophils remained intact; in tandem, in vitro chemotactic responses to C5a and fMLP were retained. These data suggest that, in the CLP model of sepsis, treatment with anti-C5a prevents development of MOF and the accompanying onset of blood neutrophil dysfunction. This may explain the protective effects of anti-C5a in the CLP model of sepsis.
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Complemento C5a/fisiología , Insuficiencia Multiorgánica/inmunología , Sepsis/inmunología , Acidosis/inmunología , Acidosis/metabolismo , Acidosis/prevención & control , Alcalosis Respiratoria/inmunología , Alcalosis Respiratoria/prevención & control , Secuencia de Aminoácidos , Animales , Ciego , Quimiotaxis de Leucocito , Complemento C5a/genética , Complemento C5a/inmunología , Complemento C5a/metabolismo , Electroforesis en Gel de Poliacrilamida , Sueros Inmunes/farmacología , Radioisótopos de Yodo/metabolismo , Riñón/patología , Riñón/ultraestructura , Ligadura , Masculino , Datos de Secuencia Molecular , Insuficiencia Multiorgánica/sangre , Insuficiencia Multiorgánica/patología , N-Formilmetionina Leucil-Fenilalanina/sangre , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Unión Proteica/genética , Unión Proteica/inmunología , Ratas , Ratas Long-Evans , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sepsis/sangre , Sepsis/patología , TritioRESUMEN
Histoplasma capsulatum is an important fungal pathogen in immunocompromised hosts, including AIDS patients. Experimental evidence suggests interferon-gamma (IFN) plays a role in host defense against H. capsulatum. In these studies we sought to demonstrate the importance of IFN in innate resistance to systemic histoplasmosis. The possible exacerbation of infection in BALB/c mice was assessed by administering 200 microg of hamster anti-IFN antibody prior to infection with H. capsulatum (2 x 10(6) yeasts, i.v.) and by comparing the severity of infection between BALB/c IFN gene knockout mice (GKO) and congenic control animals. In two separate studies, we found that anti-IFN treatment caused a dramatic loss of resistance to lethal infection and resulted in earlier mortality of IFN-depleted animals compared with normal IgG or no treatment (P<0.001). GKO mice were significantly (P<0.001) more susceptible to lethal infection than were control animals, and histological studies corroborated this. These studies clearly demonstrate that IFN is a vital part of the host's innate resistance to systemic infection with H. capsulatum and provide an additional rationale for studying IFN as an immunomodulatory therapeutic for the treatment of this disease.
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Anticuerpos/uso terapéutico , Histoplasma , Histoplasmosis/tratamiento farmacológico , Interferón gamma/inmunología , Animales , Cricetinae , Modelos Animales de Enfermedad , Femenino , Histoplasma/aislamiento & purificación , Histoplasmosis/genética , Histoplasmosis/patología , Interferón gamma/deficiencia , Interferón gamma/genética , Hígado/microbiología , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Bazo/microbiología , Bazo/patología , Coloración y EtiquetadoRESUMEN
Chronic granulomatous disease is a rare inherited disorder caused by nonexistent or severely decreased phagocyte superoxide production that results in a severe defect in host defense and consequent predisposition to microbial infection. The enzyme responsible for generating the superoxide, NADPH oxidase, involves at least 5 protein components. The absence of, or a defect in, any 1 of 4 of these proteins (p22(phox), p47(phox), p67(phox), or gp91(phox)) gives rise to the known types of chronic granulomatous disease. One of the rarest forms of the disease is due to defects in the CYBA gene encoding p22(phox), which together with gp91(phox) forms flavocytochrome b(558), the catalytic core of NADPH oxidase. To date, only 9 kindreds with p22(phox) deficiency have been described in the literature comprising 10 mutant alleles. Four polymorphisms in the CYBA gene have also been reported. Here we describe 9 new, unrelated kindreds containing 12 mutations, 9 of which are novel. In addition, we report 3 new polymorphisms. The novel mutations are (a) deletion of exons 2 and 3, (b) a missense mutation in exon 3 (T155-->C), (c) a splice site mutation at the 5' end of intron 3, (d) a missense mutation in exon 2 (G74-->T), (e) a nonsense mutation in exon 1 (G26-->A), (f) a missense mutation in exon 4 (C268-->T), (g) a frameshift in exon 3 due to the insertion of C at C162, (h) a nonsense mutation in exon 2 (G107-->A), and (i) a missense mutation in exon 2 (G70-->A).
Asunto(s)
Enfermedad Granulomatosa Crónica/etiología , Enfermedad Granulomatosa Crónica/genética , Proteínas de Transporte de Membrana , Mutación , NADPH Deshidrogenasa/genética , Fosfoproteínas/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , NADPH OxidasasRESUMEN
A registry of United States residents with chronic granulomatous disease (CGD) was established in 1993 in order to estimate the minimum incidence of this uncommon primary immunodeficiency disease and characterize its epidemiologic and clinical features. To date, 368 patients have been registered; 259 have the X-linked recessive form of CGD, 81 have 1 of the autosomal recessive forms, and in 28 the mode of inheritance is unknown. The minimum estimate of birth rate is between 1/200,000 and 1/250,000 live births for the period 1980-1989. Pneumonia was the most prevalent infection (79% of patients; Aspergillus most prevalent cause), followed by suppurative adenitis (53% of patients; Staphylococcus most prevalent cause), subcutaneous abscess (42% of patients; Staphylococcus most prevalent cause), liver abscess (27% of patients; Staphylococcus most prevalent cause), osteomyelitis (25% of patients; Serratia most prevalent cause), and sepsis (18% of patients; Salmonella most prevalent cause). Fifteen percent of patients had gastric outlet obstruction, 10% urinary tract obstruction, and 17% colitis/enteritis. Ten percent of X-linked recessive kindreds and 3% of autosomal recessive kindreds had family members with lupus. Eighteen percent of patients either were deceased when registered or died after being registered. The most common causes of death were pneumonia and/or sepsis due to Aspergillus (23 patients) or Burkholderia cepacia (12 patients). Patients with the X-linked recessive form of the disease appear to have a more serious clinical phenotype than patients with the autosomal recessive forms of the disease, based on the fact that they are diagnosed significantly earlier (mean, 3.01 years of age versus 7.81 years of age, respectively), have a significantly higher prevalence of perirectal abscess (17% versus 7%), suppurative adenitis (59% versus 32%), bacteremia/fungemia (21% versus 10%), gastric obstruction (19% versus 5%), and urinary tract obstruction (11% versus 3%), and a higher mortality (21.2% versus 8.6%).
Asunto(s)
Enfermedad Granulomatosa Crónica/epidemiología , Sistema de Registros , Adolescente , Adulto , Anciano , Causas de Muerte , Niño , Preescolar , Enfermedades Transmisibles/epidemiología , Enfermedades Transmisibles/etiología , Femenino , Estudios de Seguimiento , Enfermedad Granulomatosa Crónica/complicaciones , Enfermedad Granulomatosa Crónica/genética , Humanos , Incidencia , Lactante , Masculino , Persona de Mediana Edad , Prevalencia , Grupos Raciales , Análisis de Supervivencia , Estados Unidos/epidemiologíaRESUMEN
The cytosolic factor p47-phox, encoded by the NCF1 gene, is an essential component of the phagocyte NADPH-oxidase system. Upon activation of this multicomponent system, p47-phox translocates to the membrane and participates in the electron transfer from NADPH to molecular oxygen. A deficiency or absence of p47-phox is the most common autosomal form of chronic granulomatous disease (CGD). We have cloned and characterized the NCF1 gene from four bacteriophage clones, a P1 clone and genomic DNA from normal individuals. The gene is 15,236 base pairs long and includes 11 exons. It is 98.6% homologous in sequence to at least one pseudogene that maps to the same region of chromosome 7q11.23. Slightly more than half (50.37%) of the wild-type NCF1 gene consists of repetitive elements. In particular, the density of Alu sequences is high (1.4 Alu/kb); there are 21 Alu repeats interspersed through 10 introns. These findings are consistent with the observation that recombination events between the wild-type gene and its highly homologous pseudogenes account for the majority of potentially lethal mutations in p47-phox-deficient chronic granulomatous disease. Analysis of 1.96 kb of sequence 5' of the start of translation revealed a high homology (99.6%) between wild-type and pseudogene clones. Characterization of NCF1 establishes a foundation for detailed molecular analysis of p47-phox-deficient CGD patients as well as for the study of the regulation of the NCF1 gene and pseudogenes, both of which are present as full-length transcripts in normal individuals.
Asunto(s)
Fosfoproteínas/genética , Regiones no Traducidas 5' , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Exones , Biblioteca de Genes , Enfermedad Granulomatosa Crónica/genética , Humanos , Intrones , Datos de Secuencia Molecular , NADPH Deshidrogenasa/química , NADPH Deshidrogenasa/genética , NADPH Oxidasas , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADNRESUMEN
Chronic granulomatous disease (CGD) is an inherited disease caused by defects in the superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of phagocytes. Genetic lesions in any of 4 components of this antimicrobial enzyme have been detected. Family-specific mutations are found in 3 of 4 forms of CGD due to deficiencies of the gp91-phox, p22-phox, and p67-phox genes. In p47-phox-deficient CGD (autosomal recessive form A47 degrees ) patients, a GT deletion (triangle upGT) at the beginning of exon 2 of the p47-phox gene has been reported in 19 of 20 alleles. This GT deletion is also characteristic for the recently identified p47-phox pseudogenes. To explore a possible link between these findings, a sequence analysis of 28 unrelated, racially diverse A47 degrees CGD patients and 37 healthy individuals was performed. The GT deletion in exon 2 was present on all alleles in 25 patients. Only 3 patients but all healthy individuals contained the GTGT and triangle upGT sequences. A total of 22 patients carried additional pseudogene-specific intronic sequences on all alleles, either only in intron 1 or in intron 1 and intron 2, which lead to different types of chimeric DNA strands. It is concluded that recombination events between the p47-phox gene and its highly homologous pseudogenes result in the incorporation of triangle upGT into the p47-phox gene, thereby leading to the high frequency of GT deletion in A47 degrees CGD patients. (Blood. 2000;95:2150-2156)
Asunto(s)
Enfermedad Granulomatosa Crónica/genética , Fosfoproteínas/genética , Seudogenes/genética , Recombinación Genética , Secuencia de Bases , Exones , Eliminación de Gen , Genes Recesivos , Heterocigoto , Humanos , Intrones , Datos de Secuencia Molecular , NADPH Oxidasas , Polimorfismo Conformacional Retorcido-Simple , Mapeo Restrictivo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido NucleicoRESUMEN
Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency disease that leads to severe recurrent infections. CGD is caused by defects in the phagocyte NADPH oxidase, a multiprotein enzyme that reduces oxygen to superoxide, a precursor of microbicidal oxidants. Less than 6% of CGD patients have an autosomal recessive form of the disease caused by mutations in NCF-2. This gene encodes p67-phox, a cytosolic oxidase subunit that associates with membrane-bound flavocytochrome b558 and regulates electron transfer. We studied six patients from five families with p67-phox deficiency and identified seven different mutant alleles. Patients from three of the kindreds were homozygous for their respective mutation, although the parents of only one family were known to be related. Five of the mutations have not previously been identified: (1) a missense mutation (383C-->T) in exon 5, (2) a nonsense mutation (196C-->T) in exon 3, (3) a missense mutation (230G-->A) in exon 3, (4) a nonsense mutation (298C-->T) in exon 4, and (5) a dinucleotide deletion (835-836 AC) from exon 9. Phagocytes from each of the patients analyzed failed to generate a measurable respiratory burst and had no detectable p67-phox protein. Our results further demonstrate that there is great heterogeneity among the mutations in p67-phox-deficient CGD patients, with no evidence for mutational hot-spots or a founder effect. Our data also support the hypothesis that the stability of p67-phox is particularly sensitive to missense mutations that cause amino acid substitutions within its N-terminal domain. In contrast, mutations predicting single amino acid changes elsewhere in the protein generally represent benign polymorphisms.
Asunto(s)
Enfermedad Granulomatosa Crónica/enzimología , Enfermedad Granulomatosa Crónica/genética , Mutación , NADPH Oxidasas/genética , Fosfoproteínas/genética , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Preescolar , Codón sin Sentido , ADN/genética , Estabilidad de Enzimas/genética , Femenino , Genes Recesivos , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Mutación Missense , NADPH Oxidasas/deficiencia , Fagocitos/enzimología , Fosfoproteínas/deficiencia , Eliminación de SecuenciaRESUMEN
Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytes in which defective production of microbicidal oxidants leads to severe recurrent infections. CGD is caused by mutations in any of 4 genes encoding components of nicotinamide adenine dinucleotide phosphate (reduced form; NADPH) oxidase, the multisubunit enzyme that produces the precursor of these oxidants, superoxide. Approximately 5% of CGD patients have an autosomal recessive form of disease caused by a severe deficiency of p67-phox, a 526-amino acid subunit of the oxidase that appears to regulate electron transport within the enzyme. Here we report the biochemical and molecular characterization of 6 unrelated kindreds with p67-phox deficiency. These studies show that, as in gp91-phox and p22-phox deficiencies, the p67-phox CGD patients show a high degree of heterogeneity in the genetic defects that underlie their disease. Five different mutant alleles were identified: (1) a nonsense mutation in exon 4 (C(304) --> T); (2) a 5-nucleotide (nt) deletion in exon 13 (nts 1169-1173); (3) a splice mutation in the first nucleotide of intron 4 (G --> A); (4) a deletion of 1 nt in exon 9 (A(728)); and (5) a 9-nt in-frame deletion in exon 2 (nts 55-63). The splice mutation was seen in 3 unrelated kindreds, while the 5-nt deletion was seen in 2 apparently unrelated families (both of Palestinian origin). Homozygosity was present in 4 of the kindreds, 2 of which had consanguineous parentage. In the isolated neutrophils of each of the affected patients in the 6 kindreds, there was no measurable respiratory burst activity and no p67-phox protein detected by immunoblot analysis. The level of 67-phox mRNA was less than 10% of normal in the mononuclear leukocytes from 3 of the 4 patients analyzed by Northern blot studies. Thus, this heterogeneous group of mutations in p67-phox all lead to marked instability of mRNA or protein (or both) that results in the complete loss of NADPH oxidase activity.
Asunto(s)
Empalme Alternativo , Genes Recesivos , Enfermedad Granulomatosa Crónica/genética , Mutación Missense , NADPH Oxidasas/genética , Neutrófilos/enzimología , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Eliminación de Secuencia , Adolescente , Alelos , Secuencia de Bases , Niño , Preescolar , Cartilla de ADN , Exones , Femenino , Enfermedad Granulomatosa Crónica/sangre , Enfermedad Granulomatosa Crónica/enzimología , Humanos , Sustancias Macromoleculares , Masculino , NADPH Oxidasas/deficiencia , Núcleo Familiar , Reacción en Cadena de la PolimerasaRESUMEN
Chronic granulomatous disease is a rare inherited disorder caused by non-existent or severely decreased phagocyte superoxide production that results in a severe defect in host defense and consequent predisposition to microbial infection. The enzyme responsible for superoxide production, NADPH oxidase, involves at least five components. An absence of, or a defect in, any one of four of these proteins (p47(phox), p67(phox), p22(phox) and gp91(phox)) gives rise to the known types of chronic granulomatous disease. The most common form of inheritance is X-linked and is due to mutations in the CYBB gene that encodes gp91(phox), the large subunit of flavocytochrome b, the terminal electron donor of the oxidase. We have recently reported a large number of mutations in this gene revealing a broad range of defects, including large and small deletions, and frameshift, nonsense, missense, splice region and regulatory region mutations. Here we report a patient who has an unusual type of mutation that results in the generation of a 'pseudo-exon' in the gp91(phox) mRNA and an unexpected pattern of splicing.
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Enfermedad Granulomatosa Crónica/genética , Glicoproteínas de Membrana/genética , Mutación , Secuencia de Bases , Secuencia de Consenso , ADN/sangre , ADN/química , Electroforesis en Gel de Agar , Exones , Enfermedad Granulomatosa Crónica/sangre , Humanos , Masculino , Glicoproteínas de Membrana/sangre , Persona de Mediana Edad , Datos de Secuencia Molecular , NADPH Oxidasa 2 , NADPH Oxidasas/química , NADPH Oxidasas/genética , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/químicaRESUMEN
It is commonly assumed that activation of the superoxide-generating NADPH oxidase requires the formation of a stable complex between flavocytochrome b-245 (the gp91phox/p22phox heterodimer) and the cytosolic cofactors p47phox, p67phox and Rac2. This association is thought to convert flavocytochrome b-245, which contains the NADPH-binding site, flavin and haem centres, from an inactive into an active state. Here we provide evidence that, in the cell-free system, this activation process does not necessarily require the formation of a stable stoichiometric complex between the phox proteins. To explain this data we propose the hypothesis that p67phox (and possibly Rac2), are capable of activating flavocytochrome b-245 in a catalytic fashion, where a single molecule of p67phox (or Rac2) is capable of activating multiple flavocytochrome b-245 molecules.
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NADPH Oxidasas/metabolismo , NADP/metabolismo , Fosfoproteínas/metabolismo , Animales , Sitios de Unión , Sistema Libre de Células , Activación Enzimática , Humanos , NADPH Oxidasas/química , Neutrófilos/metabolismo , Fosfoproteínas/química , Unión ProteicaRESUMEN
The superoxide-generating neutrophil NADPH oxidase can be activated in cell-free reconstitution systems by several agonists, most notably arachidonic acid and the detergent sodium dodecyl sulfate. In this study, we show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. Stimulation of superoxide generation by these lipids was dependent upon both Mg(2+) and agonist concentration. Activation of NADPH oxidase by phosphatidic acids did not appear to require their conversion to corresponding diacylglycerols by phosphatidate phosphohydrolase, since diacylglycerols were much slower than phosphatidic acids to activate the system and required the presence of ATP. Stimulation of the oxidase by dioctanoylglycerol proved to be by a means other than the activation of protein kinase C. Instead, dioctanoylglycerol was converted to dioctanoylphosphatidic acid by an endogenous diacylglycerol kinase present in the cell-free reaction system. This conversion was sensitive to the diacylglycerol kinase inhibitor R59949 and explains the markedly slower kinetics of activation and the novel ATP requirement seen with dioctanoylglycerol. The level of dioctanoylphosphatidic acid formed was suboptimal for NADPH oxidase activation but could synergize with the unmetabolized dioctanoylglycerol to activate superoxide generation.
Asunto(s)
Diglicéridos/metabolismo , NADPH Oxidasas/metabolismo , Neutrófilos/enzimología , Ácidos Fosfatidicos/metabolismo , Sistema Libre de Células , Diacilglicerol Quinasa/antagonistas & inhibidores , Diglicéridos/farmacología , Sinergismo Farmacológico , Activación Enzimática , Humanos , Cinética , Ácidos Fosfatidicos/farmacología , Piperidinas/farmacología , Quinazolinas/farmacología , Quinazolinonas , Sistemas de Mensajero Secundario , Superóxidos/metabolismoRESUMEN
We have examined the kinetics of NADPH oxidase activation induced by arachidonic acid or SDS in a cell-free system using mixtures of recombinant Phox proteins and purified flavocytochrome b-245. Activation of oxidase activity required the simultaneous presence of p47(phox), flavocytochrome b-245, and the anionic amphiphile. The activation of electron transfer reactions was much more rapid when iodonitrotetrazolium violet was used as electron acceptor than when oxygen alone was the acceptor. We propose that this difference represents an intermediate activation state of NADPH oxidase in which electron flow can proceed from NADPH to enzyme flavin (and hence to iodonitrotetrazolium violet) but not from flavin to heme (or not between the hemes). A model for NADPH oxidase activation is presented that is consistent with these observations.
Asunto(s)
Grupo Citocromo b/metabolismo , NADPH Oxidasas/metabolismo , Fosfoproteínas/metabolismo , Tensoactivos/farmacología , Aniones/farmacología , Ácido Araquidónico/farmacología , Transporte de Electrón , Activación Enzimática/efectos de los fármacos , Flavinas , Flavoproteínas/metabolismo , Hemo , Cinética , NADH NADPH Oxidorreductasas/metabolismo , NADP/metabolismo , Dodecil Sulfato de Sodio/farmacología , Superóxidos/metabolismo , Sales de TetrazolioRESUMEN
The role of magnesium ions in the activation of NADPH oxidase has been investigated using flavocytochrome b-245 and either neutrophil cytosol or mixtures of recombinant p40phox, p47phox, p67phox and Rac2. Purified flavocytochrome b-245 is highly active (turnover number 120-150 mol of O2(-)/s per mol of cytochrome haem) in the absence of Mg2+, in marked contrast to neutrophil membranes or detergent-solubilized membranes, which have an absolute requirement for Mg2+ for NADPH oxidase activity. It was also found that Mg2+ affected the anionic amphiphile requirement for oxidase activation, and this was dependent on whether neutrophil cytosol or mixtures of recombinant cytosolic proteins were used in the assay. Unexpectedly we found that, using purified flavocytochrome b-245 and recombinant cytosolic proteins, NADPH oxidase undergoes spontaneous activation in the absence of anionic amphiphiles under Mg2+-free conditions. The results suggest that Mg2+ ions play an important role in NADPH oxidase function, perhaps stabilizing the 260 kDa complex of cytosolic phox proteins or the regulation of a guanine nucleotide-binding protein. We provide evidence that if the latter explanation is correct, the identity of the guanine nucleotide-binding protein is unlikely to be Rap1a.