scholarly journals p22-phox-deficient chronic granulomatous disease: reconstitution by retrovirus-mediated expression and identification of a biosynthetic intermediate of gp91-phox

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2767-2775 ◽  
Author(s):  
CD Porter ◽  
MH Parkar ◽  
AJ Verhoeven ◽  
RJ Levinsky ◽  
MK Collins ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Cell Lines ◽  
Cytochrome B ◽  
Function Analysis ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Side Chains ◽  
In Vitro Mutagenesis ◽  
Granulomatous Disease ◽  
P22 Phox

Abstract Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91- phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91- phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.

scholarly journals p22-phox-deficient chronic granulomatous disease: reconstitution by retrovirus-mediated expression and identification of a biosynthetic intermediate of gp91-phox

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2767-2775 ◽  
Author(s):  
CD Porter ◽  
MH Parkar ◽  
AJ Verhoeven ◽  
RJ Levinsky ◽  
MK Collins ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Cell Lines ◽  
Cytochrome B ◽  
Function Analysis ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Side Chains ◽  
In Vitro Mutagenesis ◽  
Granulomatous Disease ◽  
P22 Phox

Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91- phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91- phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.

scholarly journals Detection of gp91-phox precursor protein in B-cell lines from patients with X-linked chronic granulomatous disease as an indicator for mutations impairing cytochrome b558 biosynthesis

1996 ◽  
Vol 315 (2) ◽  
pp. 571-575 ◽  
Author(s):  
Colin D. PORTER ◽  
KURIBAYASHI KURIBAYASHI ◽  
Mohamed H. PARKAR ◽  
Dirk ROOS ◽  
Christine KINNON
Keyword(s):  
Nadph Oxidase ◽  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Lines ◽  
Binding Domain ◽  
Granulomatous Disease ◽  
Cytochrome B558 ◽  
Stable Association ◽  
P22 Phox ◽  
Fad Binding

NADPH oxidase cytochrome b558 consists of two subunits, gp91-phox and p22-phox, defects of which result in chronic granulomatous disease (CGD). The nature of the interaction between these subunits has yet to be determined. Absence of p22-phox in autosomal CGD patient-derived B-cell lines results in detectable levels of an incompletely glycosylated gp91-phox precursor. We have detected this same precursor species in four cell lines from patients with the X-linked form of the disease due to mutations in gp91-phox. Such mutations should delineate regions of gp91-phox important for its biosynthesis, including stable association with p22-phox. One mutation mapped to the putative FAD-binding domain, one mapped to a potential haem-binding domain, and two involved the region encoded by exon 3.

scholarly journals NADPH oxidase correction by mRNA transfection of apheresis granulocytes in chronic granulomatous disease

2020 ◽  
Vol 4 (23) ◽  
pp. 5976-5987
Author(s):  
Suk See De Ravin ◽  
Julie Brault ◽  
Ronald J. Meis ◽  
Linhong Li ◽  
Narda Theobald ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Messenger Rna ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Functional Restoration ◽  
In Vivo Studies ◽  
Allogeneic Bone ◽  
Granulomatous Disease ◽  
Primate Model

Abstract Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity. This not only precludes future use of allogeneic granulocytes in an alloimmunized CGD recipient, but increases the risk of graft failure of those recipients who go on to need an allogeneic bone marrow transplant. Here, we provide the first demonstration of efficient functional restoration of CGD patient apheresis granulocytes by messenger RNA (mRNA) electroporation using a scalable, Good Manufacturing Practice–compliant system to restore protein expression and NADPH oxidase function. Dose-escalating clinical-scale in vivo studies in a nonhuman primate model verify the feasibility, safety, and persistence in peripheral blood of infusions of mRNA-transfected autologous granulocyte-enriched apheresis cells, supporting this novel therapeutic approach as a potential nonalloimmunizing adjunct treatment of intractable infections in CGD patients.

Transient Association of the Nicotinamide Adenine Dinucleotide Phosphate Oxidase Subunits p47phox and p67phox With Phagosomes in Neutrophils From Patients With X-Linked Chronic Granulomatous Disease

Blood ◽  
1999 ◽  
Vol 93 (10) ◽  
pp. 3521-3530 ◽  
Author(s):  
Lee-Ann H. Allen ◽  
Frank R. DeLeo ◽  
Annabelle Gallois ◽  
Satoshi Toyoshima ◽  
Kensuke Suzuki ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Nicotinamide Adenine Dinucleotide ◽  
Polymorphonuclear Leukocytes ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Actin Binding ◽  
Nicotinamide Adenine ◽  
Cell Periphery ◽  
Granulomatous Disease ◽  
Flavocytochrome B

Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requires recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized phagocytosis assay and immunofluorescence microscopy (IFM) to examine the association of cytosolic NADPH oxidase subunits with phagosomes containing opsonized zymosan (OpZ). Ingestion of OpZ began within 30 seconds of particle binding and forming phagosomes were enriched for both F-actin and the actin-binding protein p57. NADPH oxidase subunits p47phox and p67phox were also recruited to forming phagosomes and were retained on mature phagosomes for at least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosomes was confirmed by immunoblotting. Translocation of p67phox, but not p57, to forming phagosomes was deficient in PMNs lacking p47phox. Surprisingly, we found that in PMNs from six individuals with X-linked chronic granulomatous disease (CGD), p47phox and p67phox accumulated in the periphagosomal area during ingestion of OpZ. However, in marked contrast to normal PMNs, p47phox and p67phox were shed from nascent phagosomes along with F-actin and p57 once OpZ was internalized (≈5 minutes). These data support a model in which flavocytochrome b is required for stable membrane binding of p47phox and p67phox, but not their association with the cytoskeleton or transport to the cell periphery.

scholarly journals X-linked chronic granulomatous disease: correction of NADPH oxidase defect by retrovirus-mediated expression of gp91-phox

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2196-2202 ◽  
Author(s):  
CD Porter ◽  
MH Parkar ◽  
RJ Levinsky ◽  
MK Collins ◽  
C Kinnon
Keyword(s):  
Gene Therapy ◽  
Nadph Oxidase ◽  
B Cell ◽  
Chronic Granulomatous Disease ◽  
Cell Lines ◽  
Large Subunit ◽  
Granulomatous Disease ◽  
Barr Virus ◽  
Inherited Immunodeficiency ◽  
Epstein Barr

Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X- linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.

scholarly journals Nox2 NADPH oxidase is dispensable for platelet activation or arterial thrombosis in mice

2019 ◽  
Vol 3 (8) ◽  
pp. 1272-1284 ◽  
Author(s):  
Vijay K. Sonkar ◽  
Rahul Kumar ◽  
Melissa Jensen ◽  
Brett A. Wagner ◽  
Anjali A. Sharathkumar ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Platelet Activation ◽  
Arterial Thrombosis ◽  
High Performance ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Granulomatous Disease ◽  
Female Mice ◽  
Chromatography Analysis

Abstract Deficiency of the Nox2 (gp91phox) catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a genetic cause of X-linked chronic granulomatous disease, a condition in which patients are prone to infection resulting from the loss of oxidant production by neutrophils. Some studies have suggested a role for superoxide derived from Nox2 NADPH oxidase in platelet activation and thrombosis, but data are conflicting. Using a rigorous and comprehensive approach, we tested the hypothesis that genetic deficiency of Nox2 attenuates platelet activation and arterial thrombosis. Our study was designed to test the genotype differences within male and female mice. Using chloromethyl-dichlorodihydrofluorescein diacetate, a fluorescent dye, as well as high-performance liquid chromatography analysis with dihydroethidium as a probe to detect intracellular reactive oxygen species (ROS), we observed no genotype differences in ROS levels in platelets. Similarly, there were no genotype-dependent differences in levels of mitochondrial ROS. In addition, we did not observe any genotype-associated differences in platelet activation, adhesion, secretion, or aggregation in male or female mice. Platelets from chronic granulomatous disease patients exhibited similar adhesion and aggregation responses as platelets from healthy subjects. Susceptibility to carotid artery thrombosis in a photochemical injury model was similar in wild-type and Nox2-deficient male or female mice. Our findings indicate that Nox2 NADPH oxidase is not an essential source of platelet ROS or a mediator of platelet activation or arterial thrombosis in large vessels, such as the carotid artery.

scholarly journals A new mutation in exon 12 of the gp91-phox gene leading to cytochrome b- positive X-linked chronic granulomatous disease

Blood ◽  
1995 ◽  
Vol 85 (11) ◽  
pp. 3274-3277 ◽  
Author(s):  
H Azuma ◽  
H Oomi ◽  
K Sasaki ◽  
I Kawabata ◽  
T Sakaino ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Dna Analysis ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Granulomatous Disease ◽  
New Mutation ◽  
Nadph Oxidase Complex ◽  
Polymerase Chain ◽  
O2 Production ◽  
Complete Formation

We have previously reported a patient with cytochrome b-positive X-linked chronic granulomatous disease. Although the O2-production of neutrophils from the patient was completely defective, we presented data suggesting that the patient's cytochrome b was present at a normal level and possibly had normal spectroscopic features. Thus, to look for a mutation in the cytochrome b heavy chain (gp91-phox) gene, DNA analysis of gp91-phox cDNA derived from this patient was performed. As a result, we found that five nucleotides (1521 through 1525) within exon 12 were deleted, and a new sequence of eight nucleotides was inserted. This mutation converted Gln507-Lys508-Thr509 into His-Ile-Trp-Ala. Mismatched polymerase chain reaction showed that the mother has both wild and mutated alleles, confirming that this case was transmitted in an X-linked fashion. This mutation is different from those previously reported by others. The translocation of p47-phox and p67-phox to the membrane fraction occurred, indicating the complete formation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. We conclude that this case suggests that the structure encoded on exon 12 of gp91-phox is important for electron transfer.

Radiographic features of patients with chronic granulomatous disease

2020 ◽  
Vol 7 (2) ◽  
pp. 66-80
Author(s):  
Paria Kashani ◽  
Lara Farras Roca ◽  
David Manson
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Pulmonary Nodules ◽  
Severe Infection ◽  
Pathogenic Mutation ◽  
Lung Abscess ◽  
Granuloma Formation ◽  
Granulomatous Disease ◽  
Radiographic Findings

Introduction: Chronic granulomatous disease (CGD) is one of the most common primary immunodeficiencies of childhood, and is caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Alongside neutrophil dysfunction, dysregulation of the immune system predisposes patients to recurrent life-threatening infections as well as granuloma formation, hyperinflammation, and autoimmunity. Examination by imaging (radiography, ultrasound, computed tomography, magnetic resonance) in conjunction with biopsy and tissue or fluid cultures are essential to identify the extent and severity of infections as well as the microorganisms responsible. These modalities also help to guide the management of inflammatory complications. Aim: We highlight the common radiographic findings in 10 pediatric CGD patients followed at our centre over a period of 10 years. Methods: Medical records of patients with confirmed CGD diagnosis were reviewed retrospectively. All had low neutrophil oxidative burst index (NOBI) and pathogenic mutation in 1 of the 5 subunits of the NADPH oxidase. Three patients had autosomal recessive CGD and 7 had X-linked recessive CGD. All but 1 are male. Results: The most common radiographic presentation was hilar lymphadenopathy and pulmonary nodules. Other lung complications include cavitating lesions, lung abscess, pulmonary nodule, and pleuritic nodules. Lymphatic tissue and lymph nodes were involved in 50% of our cohort of patients, while gastrointestinal manifestations were noted in approximately 35% of our patients. These include the presence of pigmented macrophages, multiple granulomas, liver abscess, or detection of Aspergillus in tissue or fluid culture. Discussion: It is essential for clinicians to keep primary immunodeficiency as one of the differential diagnoses in patients who present with severe infection or inflammation. We encourage physicians to consider CGD in patients with above described findings and consider measuring NOBI in patients with early onset infection, inflammation, or granuloma formation. Statement of novelty: We describe the radiographic findings of a pediatric cohort of patients with CGD.

Histopathological features of patients with chronic granulomatous disease

2019 ◽  
Vol 6 (3) ◽  
pp. 107-112
Author(s):  
Paria Kashani ◽  
Haiying Chen
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Primary Immunodeficiency ◽  
Tissue Injury ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Granulomatous Inflammation ◽  
Severe Infection ◽  
Granulomatous Disease ◽  
Histopathological Features ◽  
Paediatric Cohort

Introduction: Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Affected patients suffer recurrent life-threatening infections due to phagocyte dysfunction and dysregulation of the immune system. Histopathological assessment is important to help identify the extent and severity of infection and tissue injury. Aim: We present pathological findings in 5 patients with CGD who were followed at our centre. Methods: Patient information was reviewed retrospectively in accordance with local institutional guidelines. All patients had confirmed diagnosis of CGD with mutation in one of the 5 subunits of the NADPH oxidase. Results: Histopathological features of the gastrointestinal tract, liver, and spleen are noted, and include the presence of granulomatous inflammation and pigmented macrophages. Discussion: It is essential for clinicians to keep primary immunodeficiency as one of the differential diagnoses in patients with severe infection or inflammation, whether in the absence or presence of granuloma formation. The detection of PAS-positive macrophages, diffuse granulomatous inflammation, and hepatic abscesses should raise strong suspicion of CGD. Statement of novelty: We describe the histopathological findings of a paediatric cohort of patients with CGD.

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