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1.
Nat Genet ; 9(2): 202-9, 1995 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7719350

RESUMO

Chronic granulomatous disease (CGD) is a recessive disorder characterized by a defective phagocyte respiratory burst oxidase, life-threatening pyogenic infections and inflammatory granulomas. Gene targeting was used to generate mice with a null allele of the gene involved in X-linked CGD, which encodes the 91 kD subunit of the oxidase cytochrome b. Affected hemizygous male mice lacked phagocyte superoxide production, manifested an increased susceptibility to infection with Staphylococcus aureus and Aspergillus fumigatus and had an altered inflammatory response in thioglycollate peritonitis. This animal model should aid in developing new treatments for CGD and in evaluating the role of phagocyte-derived oxidants in inflammation.


Assuntos
Doença Granulomatosa Crônica/genética , Camundongos Transgênicos/genética , Fagócitos/metabolismo , Superóxidos/metabolismo , Alelos , Animais , Aspergilose , Aspergillus fumigatus , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Modelos Animais de Doenças , Feminino , Ligação Genética , Doença Granulomatosa Crônica/fisiopatologia , Pneumopatias Fúngicas , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/fisiologia , Neutrófilos/enzimologia , Peritonite/induzido quimicamente , Fagócitos/enzimologia , Fagócitos/patologia , Infecções Estafilocócicas , Staphylococcus aureus , Células-Tronco/fisiologia , Cromossomo X
2.
J Exp Med ; 185(2): 207-18, 1997 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-9016870

RESUMO

Mice with X-linked chronic granulomatous disease (CGD) generated by targeted disruption of the gp91phox subunit of the NADPH-oxidase complex (X-CGD mice) were examined for their response to respiratory challenge with Aspergillus fumigatus. This opportunistic fungal pathogen causes infection in CGD patients due to the deficient generation of neutrophil respiratory burst oxidants important for damaging A. fumigatus hyphae. Alveolar macrophages from X-CGD mice were found to kill A. fumigatus conidia in vitro as effectively as alveolar macrophages from wild-type mice. Pulmonary disease in X-CGD mice was observed after administration of doses ranging from 10(5) to 48 spores, none of which produced disease in wild-type mice. Higher doses produced a rapidly fatal bronchopneumonia in X-CGD mice, whereas progression of disease was slower at lower doses, with development of chronic inflammatory lesions. Marked differences were also observed in the response of X-CGD mice to the administration of sterilized Aspergillus hyphae into the lung. Within 24 hours of administration, X-CGD mice had significantly higher numbers of alveolar neutrophils and increased expression of the proinflammatory cytokines IL-1 beta and TNF-alpha relative to the responses seen in wild-type mice. By one week after administration, pulmonary inflammation was resolving in wild-type mice, whereas X-CGD mice developed chronic granulomatous lesions that persisted for at least six weeks. This is the first experimental evidence that chronic inflammation in CGD does not always result from persistent infection, and suggests that the clinical manifestations of this disorder reflect both impaired microbial killing as well as other abnormalities in the inflammatory response in the absence of a respiratory burst.


Assuntos
Aspergillus fumigatus/patogenicidade , Ligação Genética , Doença Granulomatosa Crônica/metabolismo , NADPH Oxidases , Explosão Respiratória , Cromossomo X , Animais , Citocinas/imunologia , Citocinas/metabolismo , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/imunologia , Inflamação/imunologia , Pneumopatias/genética , Pneumopatias/imunologia , Pneumopatias/metabolismo , Macrófagos Alveolares/imunologia , Glicoproteínas de Membrana/genética , Camundongos , NADPH Oxidase 2 , Fagocitose
3.
J Exp Med ; 178(6): 2047-53, 1993 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-8245781

RESUMO

The respiratory burst oxidase of phagocytes and B lymphocytes is a multicomponent enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2-production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or physiologic stimuli, such as phagocytosis in phagocytes or cross-linking of surface immunoglobulin in B lymphocytes. The activity of this enzyme is greatly diminished or absent in patients with chronic granulomatous disease (CGD), an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every CGD patient studied so far, an abnormality has been found in a gene encoding one of the four components of the respiratory burst oxidase: the membrane proteins p22phox or gp91phox which together form the cytochrome b558 protein, or the cytosolic proteins p47phox or p67phox. Autosomal recessive cytochrome-negative CGD (A22(0) CGD) is associated with mutations in the gene coding for p22phox. We report here that the capacity for O2- production and cytochrome b558 protein expression were restored to Epstein-Barr virus-transformed B lymphocytes from two A22(0) CGD patients by transfection with an expression plasmid containing a p22phox cDNA. No detectable O2- was generated by untransfected p22phox-deficient lymphocytes. The genetic reconstitution of the respiratory burst in A22(0) CGD B lymphocytes by transfer of the wild-type p22phox cDNA represents a further step towards somatic gene therapy for this subgroup of A22(0) CGD. This system will also be useful for expression of genetically engineered mutant p22phox proteins in intact cells, facilitating the structure-function analysis of cytochrome b558.


Assuntos
Grupo dos Citocromos b/genética , Doença Granulomatosa Crônica/enzimologia , Glicoproteínas de Membrana , NADPH Oxidases , Superóxidos/metabolismo , Linfócitos B/metabolismo , Linhagem Celular , Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Técnicas In Vitro , Medições Luminescentes , Oxirredução , RNA Mensageiro/genética , Transfecção
4.
Gene Ther ; 16(12): 1452-64, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19657370

RESUMO

X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency with absent phagocyte NADPH-oxidase activity caused by defects in the gene-encoding gp91(phox). Here, we evaluated strategies for less intensive conditioning for gene therapy of genetic blood disorders without selective advantage for gene correction, such as might be used in a human X-CGD protocol. We compared submyeloablative with ablative irradiation as conditioning in murine X-CGD, examining engraftment, oxidase activity and vector integration in mice transplanted with marrow transduced with a gamma-retroviral vector for gp91(phox) expression. The frequency of oxidase-positive neutrophils in the donor population was unexpectedly higher in many 300 cGy-conditioned mice compared with lethally irradiated recipients, as was the fraction of vector-marked donor secondary CFU-S12. Vector integration sites in marrow, spleen and secondary CFU-S12 DNA from primary recipients were enriched for cancer-associated genes, including Evi1, and integrations in or near cancer-associated genes were more frequent in marrow and secondary CFU-S12 from 300 cGy-conditioned mice compared with fully ablated mice. These findings support the concept that vector integration can confer a selection bias, and suggest that the intensity of the conditioning regimen may further influence the effects of vector integration on clonal selection in post-transplant engraftment and hematopoiesis.


Assuntos
Medula Óssea/efeitos da radiação , Técnicas de Transferência de Genes , Vetores Genéticos , Doença Granulomatosa Crônica/terapia , Hematopoese , Retroviridae/genética , Condicionamento Pré-Transplante/métodos , Animais , Feminino , Doença Granulomatosa Crônica/genética , Transplante de Células-Tronco Hematopoéticas , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Neoplasias/genética , Neutrófilos/metabolismo , Células-Tronco , Transdução Genética , Integração Viral
5.
J Cell Biol ; 86(2): 537-44, 1980 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-6249825

RESUMO

Refinement of a perfusion technique permitted the simultaneous measurement of cAMP-elicited [3H]cAMP secretion and intracellular [3H]cAMP levels in sensitive D. discoideum amoebae. These data were compared with measurements of the rate of [32P]cAMP synthesis by extracts of amoebae sonicated at different times during the cAMP signaling response. cAMP stimulation of intact cells led to a transient activation of adenylate cyclase, which was blocked if 10(-4) M NaN3 was added with the stimulus. During responses elicited by 10(-6) M cAMP, 10(-8) M cAMP, and an increment in cAMP from 10(-8) M to 10(-7) M, the rate of cAMP secretion was proportional to the intracellular cAMP concentration. Removal of a 10(-6) M cAMP stimulus 2 min after the initiation of the response led to a precipitous decline in intracellular cAMP. This decline was more rapid than could be accounted for by secretion alone, suggesting intracellular phosphodiesterase destruction of newly synthesized cAMP. Employing these data and a simple rate equation, estimates of the time-course of the transient activation of adenylate cyclase and the rate constants for cAMP secretion and intracellular phosphodiesterase activity were obtained. The calculated rate of cAMP synthesis rose for approximately 1 to 2 min, peaked, and declined to approach prestimulus levels after 3 to 4 min. This time-course agreed qualitatively with direct measurements of the time-course of activation, indicating that the activation of adenylate cyclase is a major in determining the time-course of the cAMP secretion response.


Assuntos
AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Periodicidade , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Adenilil Ciclases/metabolismo , Azidas/farmacologia , AMP Cíclico/biossíntese , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Espaço Extracelular/metabolismo
6.
J Cell Biol ; 86(2): 545-53, 1980 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-6249826

RESUMO

In dictyoselium discoideum, an increase in extracellular cAMP activates adenylate cyclase, leading to an increase in intracellular cAMP and the rate of cAMP secretion. Cells adapt to any constant cAMP stimulus after several minutes, but still respond to an increase in the concentration of the stimulus. We have now characterized the decay of adaptation (deadaptation) after the removal of cAMP stimuli. Levels of adaptation were established by the perfusion of [(3)H]adenosine-labeled amoebae with a defined cAMP stimulus. After a variable recovery period, the magnitude of the signaling response to a second stimulus was measured; its attenuation was taken as a measure of residual adaption to the first stimulus. The level of adaptation established by the first stimulus depended on both its magnitude and duration. Deadaptation began as soon as the first stimulus was removed. The magnitude of the response to the second stimulus increased with the recovery time in a first-order fashion, with a t(1/2)=3-4 min for stimuli of 10(-8) M to 10(-5) M cAMP. Responses to test stimuli, although reduced in magnitude, had an accelerated time-course when they closely followed a prior response that had not completely subsided. This effect is called priming; we believe it reveals a reversible, rate-limiting step that modulates the onset and termination of the signaling responses of amoebae that have not recently responded to a cAMP stimulus. We have suggested that the cAMP signaling response is controlled by two antagonistic cellular processes, excitation and adaptation. The data reported here imply that both the rate of rise in the adaptation process and the final level reached depend on the occupancy of cAMP surface receptors and that the decay of adaptation when external cAMP is removed proceeds with first-order kinetics.


Assuntos
AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Periodicidade , Adaptação Fisiológica , Adenilil Ciclases/metabolismo , AMP Cíclico/farmacologia , Ativação Enzimática , Cinética , Modelos Teóricos
7.
J Cell Biol ; 86(2): 554-61, 1980 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-6249827

RESUMO

In dictyostelium discoideum, extracellular cAMP activates adenylate cyclase, which leads to an increase in intracellular cAMP and the rate of cAMP secretion. The signaling response to a constant cAMP stimulus is terminated after several minutes by an adaptation mechanism. The time- course of adaptation stimuli of 10(-6) or 10(-7) M cAMP was assessed. We used a perfusion technique to deliver defined cAMP stimuli to [(3)H]adenosine-labeled amoebae and monitored their secretion of [(3)H]cAMP. Amoebae were pretreated with 10(-6) or 10(-7) M cAMP to periods of 0.33-12 minutes, and then immediately given test stimuli of 10(-8) M to 2.5 x 10(-7) M cAMP. The response to a given test stimulus was progressively attenuated and finally extinguished as the duration of the pretreatment stimulus increased. During concentration of the test stimulus. The responses to test stimuli of 10(-8), 5 x 10(-8), 10(-7), or 2.5 x 10(-7) M cAMP were extinguished after approximately 1, 2.25,2.5, and 10 min, respectively. 1.5 min of stimulation with 10(-7) M cAMP was necessary to extinguish the response of a test stimulus of 10(-8) M cAMP. Our data suggest that adaptation begins within 20 s of stimulation, rises rapidly for approximately 2.5 min, and reaches a plateau after approximately 10 min. The absolute rate of rise was faster during pretreatment with 10(-6) than with 10(-7) M cAMP. These results support a working hypothesis in which the occupancy of surface cAMP receptors leads to changes in two opposing cellular processes, excitation and adaptation, that control the activity of D. discoideum adenylate cyclase.


Assuntos
AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Adaptação Fisiológica , Adenilil Ciclases/metabolismo , AMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Estimulação Química
8.
Science ; 287(5458): 1655-8, 2000 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-10698741

RESUMO

A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has been found to be required for virulence and survival within macrophages. Here, SPI2 was shown to allow Salmonella typhimurium to avoid NADPH oxidase-dependent killing by macrophages. The ability of SPI2-mutant bacteria to survive in macrophages and to cause lethal infection in mice was restored by abrogation of the NADPH oxidase-dependent respiratory burst. Ultrastructural and immunofluorescence microscopy demonstrated efficient localization of the NADPH oxidase in the proximity of vacuoles containing SPI2-mutant but not wild-type bacteria, suggesting that SPI2 interferes with trafficking of oxidase-containing vesicles to the phagosome.


Assuntos
Hidróxidos , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/microbiologia , NADPH Oxidases/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Cério/análise , Genes Bacterianos , Macrófagos Peritoneais/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Microscopia de Fluorescência , Peróxidos/análise , Fagossomos/microbiologia , Explosão Respiratória , Salmonelose Animal/microbiologia , Salmonella typhimurium/fisiologia , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Vacúolos/enzimologia , Vacúolos/microbiologia , Virulência
9.
Gene Ther ; 15(18): 1294-8, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18580967

RESUMO

Research in gene therapy involving genome-integrating vectors now often includes analysis of vector integration sites across the genome using methods such as ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR). To help researchers analyze these sites and the functions of nearby genes, we have developed SeqMap (http://seqmap.compbio.iupui.edu/) a secure, web-based comprehensive vector integration site management tool that automatically analyzes and annotates large numbers of vector integration sites derived from LM-PCR experiments in human and model organisms upon a common genome database. We believe the use of this resource will enable better reproducibility and understanding of this important data.


Assuntos
Mapeamento Cromossômico/métodos , Terapia Genética , Integrases/genética , Internet , Integração Viral/genética , Animais , Bases de Dados Genéticas , Genoma , Humanos , Reação em Cadeia da Polimerase/métodos , Pesquisa , Retroviridae/genética , Software
10.
J Clin Invest ; 84(6): 2012-6, 1989 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-2556453

RESUMO

A membrane-bound cytochrome b, a heterodimer formed by a 91-kD glycoprotein and a 22-kD polypeptide, is a critical component of the phagocyte NADPH-oxidase responsible for the generation of superoxide anion. Mutations in the gene for the 91-kD chain of this cytochrome result in the X-linked form of chronic granulomatous disease (CGD), in which phagocytes are unable to produce superoxide. Typically, there is a marked deficiency of the 91-kD subunit and the cytochrome spectrum is absent (X- CGD). In a variant form of CGD with X-linked inheritance, affected males have a normal visible absorbance spectrum of cytochrome b, yet fail to generate superoxide (X+ CGD). The size and abundance of the mRNA for the 91-kD subunit and its encoded protein were examined and appeared normal. To search for a putative mutation in the coding sequence of the 91-kD subunit gene, the corresponding RNA from an affected X+ male was amplified by the polymerase chain reaction and sequenced. A single nucleotide change, a C----A transversion, was identified that predicts a nonconservative Pro----His substitution at residue 415 of the encoded protein. Hybridization of amplified genomic DNA with allele-specific oligonucleotide probes demonstrated the mutation to be specific to affected X+ males and the carrier state. These results strengthen the concept that all X-linked CGD relates to mutations affecting the expression or structure of the 91-kD cytochrome b subunit. The mechanism by which the Pro 415----His mutation renders the oxidase nonfunctional is unknown, but may involve an impaired interaction with other components of the oxidase.


Assuntos
Grupo dos Citocromos b/genética , Doença Granulomatosa Crônica/genética , Mutação , Neutrófilos/análise , Cromossomo X , Sequência de Bases , Humanos , Masculino , Dados de Sequência Molecular , Fagócitos/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Superóxidos/metabolismo
11.
J Clin Invest ; 86(5): 1729-37, 1990 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2243141

RESUMO

A membrane-bound cytochrome b, a heterodimer formed by a 91-kD glycoprotein (heavy chain) and a 22-kD polypeptide (light chain), is an essential component of the phagocyte NADPH-oxidase responsible for superoxide generation. Cytochrome b is absent in two subgroups of chronic granulomatous disease (CGD), an inherited disorder characterized by the lack of oxidase activity. Mutations in the cytochrome heavy chain gene, encoded by the CYBB locus in Xp21.1, result in the X-linked form of CGD. A rare subgroup of autosomal recessive CGD also lacks cytochrome b (A- CGD), but the genetic defect has not previously been identified. In order to search for possible mutations in the cytochrome light chain locus, CYBA, the structure of this gene was characterized. The CYBA locus was localized to 16q24, and the approximately 600-bp open reading frame determined to be encoded by six exons that span approximately 8.5 kb. Three unrelated patients with A- CGD were studied for evidence of mutations in the light chain gene. One patient, whose parents were first cousins, was homozygous for a large deletion that removed all but the extreme 5' coding sequence of the gene. The other two patients had a grossly normal light chain transcript on Northern blot of mononuclear cell RNA. The light chain transcript was amplified by the polymerase chain reaction and sequenced. One patient was a compound heterozygote for two alleles containing point mutations in the open reading frame that predict a frame shift and a nonconservative amino acid replacement, respectively. The second patient, whose parents were second cousins, was homozygous for a different single-base substitution resulting in another nonconservative amino acid change. These results indicate that A- CGD can results from defects in the gene encoding the 22-kD light chain of the phagocyte cytochrome b.


Assuntos
Grupo dos Citocromos b/genética , Doença Granulomatosa Crônica/genética , Mutação , Neutrófilos/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 16 , Clonagem Molecular , Éxons , Genes Recessivos , Humanos , Células Híbridas , Íntrons , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Genético
12.
J Clin Invest ; 97(11): 2680-4, 1996 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-8647963

RESUMO

Mice with chronic granulomatous disease (X-CGD mice) generated by mutating the X-linked gene for a subunit of NADPH oxidase have been analyzed for their ability to respond to intravenous injection of purified cobra venom factor (CVF). This agent in wild-type mice produces a neutrophil-dependent and catalase-sensitive form of lung injury. Lung injury was evaluated by measuring the accumulation of extravascular albumin. Quite unexpectedly, the lungs of X-CGD mice showed no difference in the increased accumulation of extravascular albumin after injection of CVF when compared to wild-type mice. In both X-CGD and wild-type mice, full development of injury required neutrophils. While catalase was highly protective in wild-type mice, its protective effects were completely lost in the X-CGD mice. Furthermore, a competitive antagonist of L-arginine, N(G)-methyl-L-arginine, was protective in X-CGD mice but not in wild-type mice. Allopurinol was protective in both types of mice. Both the basal and the CVF-inducible lung mRNA for inducible nitric oxide synthase and IL-1beta was similar in X-CGD and wild-type mice. These data indicate that oxygen radical production and lung injury in response to injection of CVF occurs through alternative pathways in mice with genetic deletion of NADPH oxidase.


Assuntos
Proteínas do Sistema Complemento/fisiologia , Venenos Elapídicos/toxicidade , Doença Granulomatosa Crônica/fisiopatologia , Lesão Pulmonar , Pulmão/fisiopatologia , NADH NADPH Oxirredutases/deficiência , Análise de Variância , Animais , Catalase/farmacologia , Ciclofosfamida/toxicidade , Indução Enzimática , Doença Granulomatosa Crônica/imunologia , Molécula 1 de Adesão Intercelular/biossíntese , Interleucina-1/biossíntese , Isoenzimas/biossíntese , Pulmão/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Modelos Biológicos , NADH NADPH Oxirredutases/genética , NADPH Oxidases , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Óxido Nítrico Sintase/biossíntese , RNA Mensageiro/biossíntese , Valores de Referência , Albumina Sérica/análise , Transcrição Gênica , Cromossomo X
13.
Curr Opin Immunol ; 3(1): 61-4, 1991 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1905138

RESUMO

The molecular and biochemical characterization of many components of the phagocyte oxidase complex that generates superoxide have greatly advanced our understanding of this important pathway. Genetic defects in one or more of the components of this host defense system result in the chronic granulomatous disease phenotype. Biochemical advances and the results of a clinical trial that established the efficacy of recombinant human interferon-gamma for prophylaxis of infections in chronic granulomatous disease are the highlights of recent achievements in this area of phagocyte biology.


Assuntos
Doença Granulomatosa Crônica/imunologia , Interferon gama/fisiologia , Ensaios Clínicos como Assunto , Humanos , Interferon gama/farmacologia , Fagócitos/metabolismo
15.
Hum Gene Ther ; 9(11): 1561-70, 1998 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-9694155

RESUMO

Chronic granulomatous disease (CGD) is a disorder of the lymphohematopoietic system, whereby phagocytes of affected patients are unable to kill microorganisms. CGD is caused by a functional defect in the phagocytic nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase (phox) enzyme complex, leading to a lack of microbicidal metabolites. As a therapeutic approach toward the predominant X-linked form of CGD, we have developed a bicistronic retroviral vector containing the coding sequences of gp91-phox and a cytoplasmically truncated version of the human low-affinity receptor for nerve growth factor (deltaLNGFR). Full reconstitution of superoxide-generating activity was achieved with this vector in a gp91-phox-deficient cell line. Using an optimized gene transfer protocol, up to 85% of the CD34+ cells obtained from the bone marrow of X-CGD patients were transduced. CD15+ cells differentiated in vitro from transduced X-CGD CD34+ cells showed correction of NADPH oxidase activity to 45-52% of normal levels whereas deltaLNGFR expression was found in 40-67% of the CD15+ cells. Moreover, immunoblots prepared from extracts of transduced CD15+ cells revealed gp91-phox protein levels similar to those found in neutrophils derived from normal CD34+ cells. Taking into consideration that superoxide production in only 5 to 10% of wild-type neutrophils is sufficient to protect X-CGD heterozygotes from serious infections, the results achieved in this study shows that for X-CGD patients a curative approach based on the genetic modification of hematopoietic stem/progenitor cells is feasible.


Assuntos
Antígenos CD34/genética , Células da Medula Óssea/metabolismo , Técnicas de Transferência de Genes , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , Glicoproteínas de Membrana/genética , Explosão Respiratória , Antígenos CD34/metabolismo , Linhagem Celular , Citometria de Fluxo , Ligação Genética , Terapia Genética , Vetores Genéticos , Humanos , Antígenos CD15/imunologia , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Retroviridae/genética , Cromossomo X
16.
Free Radic Biol Med ; 30(1): 82-8, 2001 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-11134898

RESUMO

Intracellular reactive oxygen species (ROS) production by activated murine T lymphocytes was investigated by analyzing intracellular dichlorofluorescin (DCFH(2)) oxidation in lymph node cells (LNC). An increase in DCFH(2) oxidation in LNC induced by phorbol myristate acetate (PMA) was detected by flow cytometry. It was confirmed that this increase was present in Thy1(+) LNC. We examined the contribution to intracellular DCFH(2) oxidation of ROS released by leukocytes other than T cells present in the LNC suspension. Superoxide dismutase, catalase, and glutathione/glutathione peroxidase inhibited the PMA-induced increase in intracellular DCFH(2) oxidation. Furthermore, PMA failed to elicit DCFH(2) oxidation in LNC isolated from mice lacking a functional NADPH oxidase (gp91(phox) gene knockout mice), but this response could be restored in these cells by the addition of T cell-depleted LNC from wild-type litter mates. This study highlights the necessity for caution in using the DCFH(2) assay to demonstrate specific intracellular ROS production in heterogeneous cell populations. It also suggests that cells other than T cells in lymph node populations may, through production of ROS, influence the intracellular redox state of T lymphocytes.


Assuntos
Fluoresceínas/metabolismo , Linfócitos T/metabolismo , Animais , Catalase/farmacologia , Glutationa/farmacologia , Glutationa Peroxidase/farmacologia , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/deficiência , NADPH Oxidases/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/farmacologia , Acetato de Tetradecanoilforbol/farmacologia
17.
Shock ; 11(4): 253-8, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10220301

RESUMO

Two classes of oxidants are thought to play a critical role in tissue damage in septic shock: reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Particular importance has been ascribed to peroxynitrite, a product arising from the reaction of nitric oxide with superoxide. A major source of ROI is the respiratory burst oxidase of neutrophils, eosinophils, monocytes, and macrophages. A major source of RNI is inducible nitric oxide synthase (iNOS), an enzyme expressed in leukocytes, hepatocytes, vascular smooth muscle cells, endothelium, and cardiac myocytes during inflammation. In previous studies using various mouse models of endotoxic shock, genetic deficiency of iNOS as a sole intervention did not consistently alter survival. Here, using Salmonella typhimurium endotoxic bacterial lipopolysaccharide (LPS) as a sole challenge, genetic deficiency of iNOS was associated with no protection or a reduction in survival, depending on the dose of LPS. Further, no protection from lethality was observed when LPS was injected into mice genetically deficient in the 91 kDa subunit of the respiratory burst oxidase (gp91phox) nor in mice genetically deficient in both gp91phox and iNOS (gp91phox-/-/NOS2-/- mice). For the latter experiments, mice were challenged either with S. typhimurium LPS alone or with inactivated bacille Calmette-Guerin (BCG) followed by Escherichia coli LPS. Deficiency of gp91phox impaired the inflammatory response to inactivated Propionobacterium acnes, rendering survival studies following priming with P. acnes difficult to interpret. Thus, in two models of endotoxic shock, major reductions in the ability to form nitric oxide or superoxide, alone or in combination, failed to improve survival.


Assuntos
NADH NADPH Oxirredutases/genética , NADPH Oxidases , Óxido Nítrico Sintase/genética , Choque Séptico/genética , Animais , Modelos Animais de Doenças , Suscetibilidade a Doenças/fisiopatologia , Endotoxinas/toxicidade , Escherichia coli/patogenicidade , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Mutantes , NADH NADPH Oxirredutases/deficiência , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase Tipo II , Salmonella typhimurium/patogenicidade , Taxa de Sobrevida
18.
Bone Marrow Transplant ; 25 Suppl 2: S99-104, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10933200

RESUMO

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder which results from absence or malfunction of the respiratory burst oxidase normally expressed in neutrophils and other phagocytic leukocytes. Two-thirds of the patients are males hemizygous for mutations in the X-linked gene coding for gp91-phox. As a therapeutic approach towards the X-linked form of CGD bicistronic retroviral vectors containing the gp91-phox gene and a selectable marker gene were constructed. The ability of these vectors to restore NADPH oxidase activity was tested in a human myeloid leukemic cell line that is defective in superoxide production, as well as in primary CD34+ cells obtained from X-CGD patients. Under optimal conditions 80% of the CD34+ cells derived from bone marrow of one X-CGD patient were transduced. The level of superoxide production, in phagocytes derived from transduced cells was 68.9% of normal levels. Considering that low levels of superoxide generating activity are sufficient for normal host defense, the present experiments provide the basis for the development of a gene replacement therapy for the X-linked form of CGD.


Assuntos
Terapia Genética/métodos , Doença Granulomatosa Crônica/terapia , Animais , Antígenos CD34/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/virologia , Transplante de Medula Óssea , Linhagem Celular , DNA Complementar/genética , Expressão Gênica , Vetores Genéticos , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/genética , Camundongos , NADPH Oxidase 2 , NADPH Oxidases/genética , Explosão Respiratória , Retroviridae/genética , Superóxidos/metabolismo , Transdução Genética , Cromossomo X/genética
19.
DNA Cell Biol ; 18(3): 253-63, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10098607

RESUMO

Rac2, a member of the Rho family of GTPases, is highly expressed in myeloid cells and is a regulator of the NADPH-oxidase complex. A murine genomic clone was isolated that contains the 5' end and putative promoter region of the Rac2 gene. Ribonuclease protection experiments detected 13 transcription initiation sites scattered 50 to 130 bp upstream of the translation initiation site. Transient transfection studies revealed that -7 kb to +31 bp (relative to the strongest transcription initiation site) of the Rac2 gene 5'-flanking region exhibited strong promoter activity in both RAW 264.7 macrophage cells that express the endogenous Rac2 gene and NIH-3T3 fibroblast cells that do not express the endogenous gene. Truncated Rac2 promoter fragments containing as little as the -74 to +31 bp sequence produced full transcriptional activity. However, a -57 to +31 promoter fragment directed significantly less transcription, and a -39 to +31 promoter fragment was transcriptionally inactive. In vitro binding assays revealed sequence-specific and widely expressed DNA-binding activities that interacted within the -74 to -58 Rac2 promoter cis element. Oligonucleotide competition and antibody disruption studies indicated that these complexes contained the transcription factors Spl and Sp3. Specific ablation of the Sp1/Sp3 binding site significantly decreased Rac2 promoter activity in both RAW 264.7 and NIH-3T3 cells. Additional cis elements may be required to restrict Rac2 promoter activity to hematopoietic cells expressing the endogenous gene.


Assuntos
GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Regiões Promotoras Genéticas , Células 3T3 , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter/genética , Células HeLa , Humanos , Células Jurkat , Células K562 , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Transcrição Gênica , Transfecção , Proteínas rac de Ligação ao GTP
20.
Hematol Oncol Clin North Am ; 2(2): 225-40, 1988 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-3292508

RESUMO

Chronic granulomatous disease is an inherited disorder of microbial killing characterized by the failure of phagocytic cells to produce superoxide due to a lesion in a membrane-associated NADPH-oxidase. The components of the oxidase have been incompletely characterized and, therefore, a genetic approach has been used to identify the gene affected in the common X-linked form of CGD without reference to a specific protein product. The X-CGD gene was first mapped to Xp21.1. A phagocyte-specific RNA transcript derived from Xp21 was identified and shown to be deficient (or disrupted) in patients with X-CGD. Antisera directed toward the predicted protein product of the X-CGD gene have established its identity as a 90-kD membrane glycoprotein and a component of the phagocyte cytochrome b, recently purified as a heterodimer of a 90-kD species and a 22-kD polypeptide. The more recent genetic and biochemical findings now provide an explanation for the consistent absence of the phagocyte cytochrome b spectrum in X-CGD (now termed "X- -CGD"). Both subunits of the cytochrome b heterodimer are absent in X- -CGD, despite a genetic deficiency of only the larger polypeptide, which indicates that a complete understanding of cytochrome biosynthesis and function will require further characterization of the small subunit. We should anticipate that identification of other functionally associated proteins will aid in analysis of the phagocyte oxidase. Molecular reagents prepared from the cloned X-CGD cDNA or gene may prove to be clinically useful in prenatal diagnosis and may provide a basis for somatic gene therapy in the future.


Assuntos
Grupo dos Citocromos b/genética , Doença Granulomatosa Crônica/genética , NADH NADPH Oxirredutases/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Grupo dos Citocromos b/deficiência , DNA/genética , Feminino , Genes , Ligação Genética , Doença Granulomatosa Crônica/enzimologia , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Glicoproteínas de Membrana/fisiologia , Dados de Sequência Molecular , NADH NADPH Oxirredutases/deficiência , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 2 , NADPH Oxidases , Neutrófilos/enzimologia , Fagócitos/enzimologia , Gravidez , Transcrição Gênica , Cromossomo X
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