scholarly journals Reconstitution of defective respiratory burst activity with partially purified human neutrophil cytochrome B in two genetic forms of chronic granulomatous disease: possible role of Rap1A

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2438-2445 ◽  
Author(s):  
MT Quinn ◽  
JT Curnutte ◽  
CA Parkos ◽  
ML Mullen ◽  
PJ Scott ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Human Neutrophil ◽  
Autosomal Recessive ◽  
Plasma Membranes ◽  
Close Association ◽  
Granulomatous Disease ◽  
Detergent System

Abstract Neutrophil plasma membranes from patients with the X-linked and autosomal recessive forms of chronic granulomatous disease (CGD) that lack cytochrome b are incapable of generating superoxide anion (O2-) in vivo and in vitro. The O2- generating activity of these defective membranes was reconstituted with the addition of partially purified human neutrophil cytochrome b in a detergent-based, cell-free activation system. Depending on the detergent system used, 50% to 100% of the activity of control membranes was recovered, and this activity was directly dependent on the cytochrome b concentration. However, when cytochrome b was purified to 99% homogeneity, the reconstitutive capacity of the cytochrome was lost, possibly because of subtle denaturation of the cytochrome or the removal of an additional required cofactor. Examination of the latter possibility with respect to a protein known to coassociate with the cytochrome, ie, Rap1A, indicated that this ras-like protein was present in the partially purified cytochrome preparation used to reconstitute activity in CGD membranes, but was missing in the highly purified preparation. However, the finding that Rap1A was present in normal amounts in the neutrophil membranes from all four major types of CGD (including those missing cytochrome b) suggested that the conditions required of the reconstitution assay did not favor the reassociation of the membrane- derived Rap1A with exogenously added cytochrome b or that another unidentified membrane component was lost during the final purification step. The normal expression of Rap1A in CGD cell membranes also indicates that this protein is not responsible for the absence of O2- production in the X-linked and autosomal recessive cytochrome b- negative forms of CGD. Finally, these results show that the expression of Rap1A in the plasma membrane is not dependent on the coordinate expression of cytochrome b, despite the close association shown for these two proteins in the normal cell membrane.

scholarly journals Reconstitution of defective respiratory burst activity with partially purified human neutrophil cytochrome B in two genetic forms of chronic granulomatous disease: possible role of Rap1A

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2438-2445 ◽  
Author(s):  
MT Quinn ◽  
JT Curnutte ◽  
CA Parkos ◽  
ML Mullen ◽  
PJ Scott ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Human Neutrophil ◽  
Autosomal Recessive ◽  
Plasma Membranes ◽  
Close Association ◽  
Granulomatous Disease ◽  
Detergent System

Neutrophil plasma membranes from patients with the X-linked and autosomal recessive forms of chronic granulomatous disease (CGD) that lack cytochrome b are incapable of generating superoxide anion (O2-) in vivo and in vitro. The O2- generating activity of these defective membranes was reconstituted with the addition of partially purified human neutrophil cytochrome b in a detergent-based, cell-free activation system. Depending on the detergent system used, 50% to 100% of the activity of control membranes was recovered, and this activity was directly dependent on the cytochrome b concentration. However, when cytochrome b was purified to 99% homogeneity, the reconstitutive capacity of the cytochrome was lost, possibly because of subtle denaturation of the cytochrome or the removal of an additional required cofactor. Examination of the latter possibility with respect to a protein known to coassociate with the cytochrome, ie, Rap1A, indicated that this ras-like protein was present in the partially purified cytochrome preparation used to reconstitute activity in CGD membranes, but was missing in the highly purified preparation. However, the finding that Rap1A was present in normal amounts in the neutrophil membranes from all four major types of CGD (including those missing cytochrome b) suggested that the conditions required of the reconstitution assay did not favor the reassociation of the membrane- derived Rap1A with exogenously added cytochrome b or that another unidentified membrane component was lost during the final purification step. The normal expression of Rap1A in CGD cell membranes also indicates that this protein is not responsible for the absence of O2- production in the X-linked and autosomal recessive cytochrome b- negative forms of CGD. Finally, these results show that the expression of Rap1A in the plasma membrane is not dependent on the coordinate expression of cytochrome b, despite the close association shown for these two proteins in the normal cell membrane.

scholarly journals Absence of both the 91kD and 22kD subunits of human neutrophil cytochrome b in two genetic forms of chronic granulomatous disease

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1416-1420 ◽  
Author(s):  
CA Parkos ◽  
MC Dinauer ◽  
AJ Jesaitis ◽  
SH Orkin ◽  
JT Curnutte
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Autosomal Recessive ◽  
Polyclonal Antibodies ◽  
Granulomatous Disease ◽  
Absorbance Spectrum ◽  
Phagocytic Cells ◽  
Inherited Disorders ◽  
Western Blots ◽  
Respiratory Burst Oxidase

Abstract Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytic cells fail to generate antimicrobial oxidants. The various forms of CGD can be classified in terms of the mode of inheritance (either X-linked or autosomal recessive), and whether the neutrophils display the absorbance spectrum of a unique b-type cytochrome important for the function of the respiratory burst oxidase. The finding that purified neutrophil cytochrome b is a heterodimer consisting of a 91kD glycosylated and a 22kD nonglycosylated polypeptide has raised the question of which subunits are absent (or defective) in the various types of CGD. To address this question we have studied the expression of the cytochrome b subunits in three genetically distinct forms of CGD: X-linked/cytochrome b-negative (X-), autosomal recessive/cytochrome b-negative (A-), and autosomal recessive/cytochrome b-positive (A+). Using polyclonal antibodies to each of the two subunits, we prepared Western blots of lysates of intact neutrophils from ten CGD patients. In the controls and three patients with A+ CGD, both cytochrome subunits were easily detected. Consistent with the previously reported finding in five X- patients, neither subunit could be identified in neutrophils from three additional X- patients. Both subunits were also undetectable in four patients with A- CGD (three females, one male). This latter group of patients most likely bears a normal 91kD gene, since the patients are genetically distinct from the 91kD-defective X- group. The mutation in A- CGD, therefore, probably involves the 22kD gene and the eventual expression of the 22kD subunit. Furthermore, the expression of the 91kD subunit in this group of patients appears to be prevented due to the 22kD mutation in a manner converse to that seen in the X- CGD patients. Based on these studies, we hypothesize that the stable of expression of either of the two cytochrome subunits is dependent upon the other.

scholarly journals Absence of both the 91kD and 22kD subunits of human neutrophil cytochrome b in two genetic forms of chronic granulomatous disease

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1416-1420 ◽  
Author(s):  
CA Parkos ◽  
MC Dinauer ◽  
AJ Jesaitis ◽  
SH Orkin ◽  
JT Curnutte
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Autosomal Recessive ◽  
Polyclonal Antibodies ◽  
Granulomatous Disease ◽  
Absorbance Spectrum ◽  
Phagocytic Cells ◽  
Inherited Disorders ◽  
Western Blots ◽  
Respiratory Burst Oxidase

Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytic cells fail to generate antimicrobial oxidants. The various forms of CGD can be classified in terms of the mode of inheritance (either X-linked or autosomal recessive), and whether the neutrophils display the absorbance spectrum of a unique b-type cytochrome important for the function of the respiratory burst oxidase. The finding that purified neutrophil cytochrome b is a heterodimer consisting of a 91kD glycosylated and a 22kD nonglycosylated polypeptide has raised the question of which subunits are absent (or defective) in the various types of CGD. To address this question we have studied the expression of the cytochrome b subunits in three genetically distinct forms of CGD: X-linked/cytochrome b-negative (X-), autosomal recessive/cytochrome b-negative (A-), and autosomal recessive/cytochrome b-positive (A+). Using polyclonal antibodies to each of the two subunits, we prepared Western blots of lysates of intact neutrophils from ten CGD patients. In the controls and three patients with A+ CGD, both cytochrome subunits were easily detected. Consistent with the previously reported finding in five X- patients, neither subunit could be identified in neutrophils from three additional X- patients. Both subunits were also undetectable in four patients with A- CGD (three females, one male). This latter group of patients most likely bears a normal 91kD gene, since the patients are genetically distinct from the 91kD-defective X- group. The mutation in A- CGD, therefore, probably involves the 22kD gene and the eventual expression of the 22kD subunit. Furthermore, the expression of the 91kD subunit in this group of patients appears to be prevented due to the 22kD mutation in a manner converse to that seen in the X- CGD patients. Based on these studies, we hypothesize that the stable of expression of either of the two cytochrome subunits is dependent upon the other.

scholarly journals Improvement of superoxide production in monocytes from patients with chronic granulomatous disease by recombinant cytokines

Blood ◽  
1993 ◽  
Vol 81 (8) ◽  
pp. 2131-2136
Author(s):  
V Jendrossek ◽  
AM Peters ◽  
S Buth ◽  
J Liese ◽  
U Wintergerst ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Interleukin 1 ◽  
Superoxide Production ◽  
Granulomatous Disease ◽  
Necrosis Factor Alpha ◽  
Ifn Gamma ◽  
Phorbolmyristate Acetate

Cytokines have been shown to modulate the respiratory burst of polymorphonuclear leukocytes and monocytes from normal controls. We have examined whether monocytes from children with chronic granulomatous disease (CGD) can be primed by cytokines other than interferon-gamma (IFN gamma), which has been demonstrated to improve the production of reactive oxygen species in vivo and in vitro. Monocytes isolated from peripheral blood were cultured without and with IFN gamma (500 U/mL), tumor necrosis factor-alpha (500 U/mL), interleukin-1 beta (IL-1 beta) (100 U/mL), and IL-3 (100 U/mL). After 3 days of culture, the phorbolmyristate acetate (2 ng/mL) and the formyl- methionyl-leucyl-phenylalanine (0.1 mumol/L)-stimulated superoxide- production was determined in a microtiter system. In nearly all of the 14 patients examined (5 autosomal, 5 X-chromosomal, and 4 of unknown inheritance), an improvement of superoxide production could be demonstrated. The most impressive effect with the cytokines newly tested was seen with monocytes from autosomal CGD patients treated with IL-3 and stimulated by phorbolmyristate acetate. In single patients cultivation of monocytes with IL-6 and granulocyte-macrophage colony- stimulating factor resulted in only slight improvement of superoxide production. Our findings indicate that cytokines other than IFN gamma can positively modulate the defective respiratory burst in CGD and that each patient reacts with an individual pattern to different cytokines.

scholarly journals The association of FAD with the cytochrome b–245 of human neutrophils

1982 ◽  
Vol 208 (3) ◽  
pp. 759-763 ◽  
Author(s):  
Andrew R. Cross ◽  
Owen T. G. Jones ◽  
Rudolfo Garcia ◽  
Anthony W. Segal
Keyword(s):  
Plasma Membrane ◽  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Membrane Fraction ◽  
Close Association ◽  
Gradient Centrifugation ◽  
Fluorescence Emission ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Two Peaks

A plasma membrane fraction prepared from human neutrophils had a fluorescence resembling that of a fluorescent flavoprotein, with emission maximum near 520nm and excitation maxima near 380 and 460nm. The fluorescence emission and excitation properties of Triton N-101-solubilized membrane fraction resembled those of FAD. FAD was present in the membranes at a concentration of 417pmol/mg of protein and cytochrome b–245 at a concentration of 407pmol/mg of protein. In a 110-fold purified preparation of cytochrome b–245 the ratio of FAD:cytochrome b was 1:1. Analytical gradient centrifugation of neutrophil homogenates shows a coincidence of two cytochrome b peaks and two peaks of fluorescence, corresponding with plasma membrane and specific granule fractions; most of the FAD was non-fluorescent and located in fractions lighter than the plasma membrane. Plasma membrane fractions prepared from neutrophils of patients suffering from the X-linked form of chronic granulomatous disease lacked cytochrome b and contained 194pmol of FAD/mg of protein; plasma membrane fractions prepared from neutrophils of patients with the autosomal recessive form of chronic granulomatous disease contained both cytochrome b–245 and FAD in the normal range of concentrations in a ratio of 1:1. Phagocytic vesicles were prepared from normal neutrophils and found to contain FAD and cytochrome b in a ratio 2.22:1, suggesting that activation of neutrophils many involve the incorporation of an additional flavin into the membrane. Under anaerobic conditions in the presence of EDTA to act as an electron donor to a flavin, the cytochrome b–245 of neutrophil membranes was partly (12%) photoreducible, an effect increased to 100% by the addition of FMN. The extent of reduction of cytochrome b in an anaerobic neutrophil homogenate containing NADH increased from 30% to 70% on illumination. We suggest that these results indicate a close association between FAD and cytochrome b–245 and support a scheme for electron transport thus: [Formula: see text]

In Vivo Interferon-  Therapy Augments the In Vitro Ability of Chronic Granulomatous Disease Neutrophils to Damage Aspergillus Hyphae

1991 ◽  
Vol 163 (4) ◽  
pp. 849-852 ◽  
Author(s):  
J. H. Rex ◽  
J. E. Bennett ◽  
J. I. Gallin ◽  
H. L. Malech ◽  
E. S. DeCarlo ◽  
...  
Keyword(s):  
Chronic Granulomatous Disease ◽  
Interferon Therapy ◽  
Granulomatous Disease

Long-Term Follow-Up of Patients Treated by Gene Therapy for X-Linked Chronic Granulomatous Disease.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 194-194 ◽  
Author(s):  
Marion G. Ott ◽  
Manuel Grez ◽  
Stefan Stein ◽  
Ulrich Siler ◽  
Ulrike Koehl ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Chronic Granulomatous Disease ◽  
Ex Vivo ◽  
Therapeutic Option ◽  
High Morbidity ◽  
Granulomatous Disease ◽  
Phagocytic Cells

Abstract Chronic granulomatous disease (CGD) is a primary immunodeficiency in which phagocytic cells of affected patients have impaired antimicrobial activity due to a defect in the production of reactive oxygen species (ROS). CGD is caused by mutations in any one of four genes encoding for the subunits of the NADPH oxidase complex. Although curable by HSC transplantation, this strategy is usually limited only to patients with HLA-matched sibling or unrelated donors, as mismatched transplantation is associated with high morbidity and mortality due to graft failure and slow immune reconstitution. A therapeutic alternative for CGD patients is the genetic modification of autologous HSC. In January 2004 we initiated a Phase I/II clinical trial for X-CGD patients including conditioning with busulfan (8 mg/kg/total dose) prior to infusion of genetically modified HSC. G-CSF mobilized CD34+ cells from 2 adult patients (25 and 26 years) were transduced ex-vivo with a monocistronic gp91phox retroviral vector. Therapeutically significant gene marking levels were detected in neutrophils of both patients with up to 60% functionally corrected phagocytes 14 months after gene therapy. This high correction resulted from an unexpected but temporarily restricted expansion of gene transduced myeloid cells in vivo. In contrast gene marking levels in B-cells has remained constant at a level of 20%, while gene marking in T-cells is below 5%. Gene marking in bone marrow was detected at levels between 30% and 40% one year after transplantation of gene modified cells. Killing assays in vitro have demonstrated antibacterial and antifungal activity in gene transduced phagocytes and both patients recovered of Staph. aureus and Aspergillus fumigatus infections after gene therapy. Our results suggest that gene therapy in combination with bone marrow conditioning is a therapeutic option for inherited diseases affecting the myeloid compartment and can be successfully used to treat CGD.

scholarly journals Reevaluation of cytochrome b and flavin adenine dinucleotide in neutrophils from patients with chronic granulomatous disease and description of a family with probable autosomal recessive inheritance of cytochrome b deficiency

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1132-1138 ◽  
Author(s):  
Y Ohno ◽  
ES Buescher ◽  
R Roberts ◽  
JA Metcalf ◽  
JI Gallin
Keyword(s):  
Chronic Granulomatous Disease ◽  
Cytochrome B ◽  
Polymorphonuclear Leukocytes ◽  
Autosomal Recessive ◽  
Flavin Adenine Dinucleotide ◽  
Autosomal Recessive Inheritance ◽  
Granulomatous Disease ◽  
Recessive Inheritance ◽  
Generating System ◽  
Normal Adults

Abstract Chronic granulomatous disease (CGD) is a genetically heterogeneous syndrome characterized by a microbial killing defect of polymorphonuclear leukocytes (PMNs) due to lack of superoxide O2-. 2 generation. Recent studies indicate that the neutrophil O2-.-generating system consists of at least two components, flavoprotein--flavin adenine dinucleotide (FAD)--and cytochrome b. We evaluate the cytochrome b and FAD content in PMN from 30 CGD patients. The method for quantitating cytochrome b was modified by using PMN sonicates incubated with azide plus hydrogen peroxide. With this approach, several absorption peaks corresponding to myeloperoxidase and eosinophil peroxidase, which overlap with peaks of cytochrome b, were obliterated from reduced-minus-oxidized spectra, whereas the peaks of cytochrome b were not and could be readily quantitated. Cytochrome b was detected in PMNs from all 24 normal adults (47.4 +/- 2.9 pmol/7.5 X 10(6) cells), was absent in PMNs from 11 male CGD patients and one female CGD patient but was present in normal amounts in PMNs from nine male and nine female CGD patients. Stimulated nitroblue tetrazolium (NBT) tests performed on PMNs from mothers of CGD patients indicated that cytochrome b deficiency was associated with X-linked inheritance, except in one case in which probable autosomal recessive inheritance was demonstrated. The PMN NBT test of the mother of another male patient without cytochrome b deficiency suggested an X-linked form of inheritance. In related studies, the FAD content in PMN particulate fractions was reduced in 4 of 28 CGD patients studied. All four CGD patients with reduced FAD lacked cytochrome b. However, three patients with cytochrome b deficiency had normal FAD. Thus, the results indicate that PMN cytochrome b deficiency is observed in most X-linked and in some autosomal recessive CGD, that cytochrome b deficiency may be associated with FAD deficiency, and that cytochrome b and FAD are normal in most patients with non-X-linked CGD.

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