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.

Mechanisms of Microtubule Disassembly in Vivo: Studies in Normal and Chronic Granulomatous Disease Leucocytes

1977 ◽  
Vol 37 (3) ◽  
pp. 311-322 ◽  
Author(s):  
Janet M. Oliver ◽  
Richard D. Berlin ◽  
Robert L. Baehner ◽  
Lawrence A. Boxer
Keyword(s):  
Chronic Granulomatous Disease ◽  
In Vivo Studies ◽  
Granulomatous Disease

scholarly journals Dextromethorphan Reduces Oxidative Stress and Inhibits Uremic Artery Calcification

2021 ◽  
Vol 22 (22) ◽  
pp. 12277
Author(s):  
En-Shao Liu ◽  
Nai-Ching Chen ◽  
Tzu-Ming Jao ◽  
Chien-Liang Chen
Keyword(s):  
Oxidative Stress ◽  
Nadph Oxidase ◽  
Vascular Calcification ◽  
Wistar Rats ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Oxidase Inhibitor ◽  
In Vivo Studies ◽  
Ros Production

Medial vascular calcification has emerged as a key factor contributing to cardiovascular mortality in patients with chronic kidney disease (CKD). Vascular smooth muscle cells (VSMCs) with osteogenic transdifferentiation play a role in vascular calcification. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors reduce reactive oxygen species (ROS) production and calcified-medium–induced calcification of VSMCs. This study investigates the effects of dextromethorphan (DXM), an NADPH oxidase inhibitor, on vascular calcification. We used in vitro and in vivo studies to evaluate the effect of DXM on artery changes in the presence of hyperphosphatemia. The anti-vascular calcification effect of DXM was tested in adenine-fed Wistar rats. High-phosphate medium induced ROS production and calcification of VSMCs. DXM significantly attenuated the increase in ROS production, the decrease in ATP, and mitochondria membrane potential during the calcified-medium–induced VSMC calcification process (p < 0.05). The protective effect of DXM in calcified-medium–induced VSMC calcification was not further increased by NADPH oxidase inhibitors, indicating that NADPH oxidase mediates the effect of DXM. Furthermore, DXM decreased aortic calcification in Wistar rats with CKD. Our results suggest that treatment with DXM can attenuate vascular oxidative stress and ameliorate vascular calcification.

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.

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.

Molecular Characterization of Autosomal Recessive Chronic Granulomatous Disease Caused by a Defect of the Nicotinamide Adenine Dinucleotide Phosphate (Reduced Form) Oxidase Component p67-phox

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2505-2514 ◽  
Author(s):  
Pablo J. Patiño ◽  
Julie Rae ◽  
Deborah Noack ◽  
Rich Erickson ◽  
Jiabing Ding ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Molecular Characterization ◽  
Nicotinamide Adenine Dinucleotide ◽  
Autosomal Recessive ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Splice Mutation ◽  
Reduced Form ◽  
Granulomatous Disease

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 (C304 → 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 (A728); 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.

Reconstitution of bactericidal activity in chronic granulomatous disease cells by glucose-oxidase–containing liposomes

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 3097-3105 ◽  
Author(s):  
Claudia E. Gerber ◽  
Gernot Bruchelt ◽  
Ulrike B. Falk ◽  
Andrea Kimpfler ◽  
Oliver Hauschild ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Glucose Oxidase ◽  
Whole Blood ◽  
Fungal Infections ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Confocal Laser ◽  
Granulomatous Disease ◽  
Life Threatening

Abstract Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by phagocytes devoid of a functioning nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The failure of CGD phagocytes to produce reactive oxygen species (ROS) results in a marked increase in the susceptibility of affected patients to life-threatening bacterial and fungal infections. This study investigated whether loading of CGD phagocytes with glucose oxidase (GO)–containing liposomes (GOLs) could restore cellular production of bactericidal ROS (eg, H2O2 and HOCl) in vitro. Results indicate that GO encapsulated in liposomes enabled NADPH oxidase-deficient phagocytes to use H2O2 for the production of highly bactericidal HOCl. The intracellular colocalization of bacteria and liposomes (or liposome-derived ferritin) was demonstrated by confocal laser microscopy and electron microscopy. After uptake of GOLs (approximately 0.2 U/mL at 1 mM total lipid concentration, size approximately 180 nm), CGD granulocytes produced HOCl levels comparable to those of normal phagocytes. Remarkably, after treatment with GOLs, CGD phagocytes killed Staphylococcus aureus as efficiently as normal granulocytes. Moreover, treated cells retained sufficient motility toward chemotactic stimuli as measured by chemotaxis assay. Side effects were evaluated by measuring the H2O2 concentrations and the production of methemoglobin in whole blood. These studies revealed that H2O2 produced by GOLs was degraded immediately by the antioxidative capacity of whole blood. Elevated methemoglobin levels were observed only after application of extremely high amounts of GOLs (2 U/mL). In summary, the application of negatively charged GOLs might provide a novel effective approach in the treatment of patients with CGD at high risk for life-threatening infections.

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 A novel mutation in NCF2 resulting in very-early-onset colitis and juvenile idiopathic arthritis in a patient with chronic granulomatous disease

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Suzan AlKhater
Keyword(s):  
Juvenile Idiopathic Arthritis ◽  
Nadph Oxidase ◽  
Chronic Granulomatous Disease ◽  
Early Onset ◽  
Novel Mutation ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Granulomatous Disease ◽  
Recurrent Infections ◽  
Protein Subunit ◽  
Primary Immunodeficiency Disorder

Abstract Background Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder caused by a defect in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. The disease primarily presents with recurrent infections, and patients may also present with inflammatory conditions, including noninfectious colitis, and an increased frequency of autoimmunity. We report here a patient with CGD in whom the presentation, unlike the classical presentation of CGD, was predominantly of an inflammatory and autoimmune phenotype. Case presentation A 3-year-old Pakistani female presented with bloody diarrhea since the age of 7 days, followed by the development of perianal abscesses and fistula. There was no other history of recurrent infections. The patient subsequently developed joint pain and stiffness with persistently elevated inflammatory markers and elevated anti-cyclic citrullinate peptide (anti-CCP) antibody titer. She was diagnosed with oligoarticular juvenile idiopathic arthritis and colitis. The diagnosis of CGD was later made and was based on the absence of NADPH oxidase activity in the patient’s neutrophils upon phorbol myristate acetate (PMA) stimulation using the dihydrorhodamine-1,2,3 (DHR) flow cytometry test. Targeted next-generation sequencing revealed an unreported deletion mutation in exon 10 as a homozygous loss-of-function variant of the human neutrophil oxidase factor 2 (NCF2) (NCF2: NM_001190789, nucleotide change: c.855_856del:p.T285fs). The gene encodes a protein subunit, p67phox, in the NADPH enzyme complex. Conclusions The case emphasizes the importance of maintaining high clinical suspicion of immunodeficiency and CGD in patients with very-early-onset colitis and autoimmune disorders. This case is important due to its rarity and because it might represent a previously undiscovered mutation, which is possibly more common in the patient’s ethnic group. Other mutations in NCF2 have been linked to inflammatory bowel disease and autoimmunity, but without CGD, suggesting similarities in the pathogenesis.

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