Marie Jose Stasia - Academia.edu (original) (raw)
Papers by Marie Jose Stasia
HAL (Le Centre pour la Communication Scientifique Directe), 2013
International audienc
Revue d'Oncologie Hématologie Pédiatrique, Dec 1, 2016
Revue de Médecine Interne, Dec 1, 2011
PubMed, 1993
It is of great importance to be able to distinguish hyperamylasemia linked to a pathological cond... more It is of great importance to be able to distinguish hyperamylasemia linked to a pathological condition of the pancreas from one due to the presence of the macroamylase in the serum of the patient. We have evaluated both gel filtration chromatography and precipitation by PEG-6000 for the detection of serum macroamylase. Our study has enabled us to define a simple detection protocol, suitable for routine laboratory use. In this protocol, macroamylasemia is detected initially by precipitation with PEG-6000; if less than 60% of amylase activity is precipitated, there is no macroamylasemia. If more than 60% of amylase activity can be precipitated by PEG-6000, macroamylasemia is confirmed by gel filtration chromatography. The incidence of macroamylasemia in hospitalized patients was studied; it was 1.3% in cases where amylasemia was normal, and 4.5% in hyperamylasemic patients.
Clinical Chemistry, Jul 1, 1994
Macromolecular aspartate aminotransferase was found in the serum of an apparently healthy patient... more Macromolecular aspartate aminotransferase was found in the serum of an apparently healthy patient. This complex was composed of aspartate aminotransferase (AST; EC 2.6.1.1) and immunoglobulin. Electrophoresis of the patient's serum showed an abnormal band migrating between mitochondrial (m) and cytosolic (s) AST. The macromolecular complex was purified by gel filtration on Sephacryl S300. The molecular mass of the complex was estimated to be 250 kDa, suggesting that the complex probably consists of one immunoglobulin molecule associated with one AST molecule. By immunoelectrophoresis, the immunoglobulin was found to be an IgG with kappa-lambda type light chain. When we used polyclonal antibodies against human mAST or sAST, the sAST antibodies strongly inhibited the AST activity of the macrocomplex, whereas the mAST antibodies had no effect. Thus the AST molecule of the macrocomplex is an sAST type.
Journal of Allergy and Clinical Immunology, 2019
Inflammatory dendritic cells-not basophils-are necessary and sufficient for induction of Th2 immu... more Inflammatory dendritic cells-not basophils-are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen.
Human Genetics, Nov 6, 2004
Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADP... more Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADPH oxidase and so cannot generate superoxide anions (O(2) (-)). The most common form is caused by mutations in CYBB encoding gp91 phox, the heavy chain of flavocytochrome b(558) (XCGD). We investigated 11 male patients and their families suspected of suffering from X-linked CGD. These XCGD patients were classified as having different variants (X91(0), X91(-) or X91(+)) according to their cytochrome b(558) expression and NADPH oxidase activity. Nine patients had X91(0) CGD, one had X91(-) CGD and one had X91(+) CGD. Six mutations in CYBB were novel. Of the four new X91(0) CGD cases, three were point mutations: G65A in exon 2, G387T in exon 5 and G970T in exon 9, leading to premature stop codons at positions Try18, Try125 and Glu320, respectively, in gp91 phox. One case of X91(0) CGD originated from a new 1005G deletion detected in exon 9. Surprisingly, four nonsense mutations in exon 5 led to the generation of two mRNAs, one with a normal size containing the mutation and the other in which exon 5 had been spliced. A novel X91(-) CGD case with low gp91 phox expression was diagnosed. It was caused by an 11-bp deletion in the linking region between exon 12 and intron 12, activating a new cryptic site. Finally, a new X91(+) CGD case was detected, characterized by a missense mutation Leu505Arg in the potential NADPH-binding site of gp91 phox. No clear correlation between the severity of the clinical symptoms and the sub-type of XCGD could be established.
Human Mutation, Jul 1, 2009
NOX enzymes are reactive oxygen species (ROS)-generating NADPH oxidases. Several members of the N... more NOX enzymes are reactive oxygen species (ROS)-generating NADPH oxidases. Several members of the NOX family depend on the p22(phox) subunit, encoded by the CYBA gene. CYBA is highly polymorphic, and has been widely studied as a potential risk factor for various diseases, with conflicting results. In the present study, we used Epstein-Barr (EBV)-transformed B-lymphocytes from 50 healthy unrelated individuals to analyze their CYBA mRNA sequence and NOX2-dependent ROS generation. Seven single-nucleotide polymorphisms (SNPs) were identified (five previously described, two novel). The combination of these SNPs yielded 11 distinct haplotypes, which could be grouped into seven haplogroups (A-G). Haplogroup C (c.214T>C, c.521T>C, and c.(*)24G>A) showed a significantly lower ROS generation, as compared to the most frequent haplogroup, A. CYBA variants from the seven haplogroups were transduced into p22(phox)-deficient B-lymphocytes. The haplogroup C variant showed significantly lower ROS production. c.214T>C and c.521T>C lead to nonsynonymous codon changes, while c.(*)24G>A lies within the 3'UTR. Using a luciferase/3'UTR construct, we showed that the (*)24A allele led to decreased reporter gene activity. These results help to unravel the complex nature of how genetic variations in CYBA influence NOX2 activity, and indicate that haplotypes, rather than individual SNPs, define the effect on ROS generation.
Gene, Jul 1, 2016
P22 phox is a ubiquitous protein encoded by the CYBA gene located on the long arm of chromosome 1... more P22 phox is a ubiquitous protein encoded by the CYBA gene located on the long arm of chromosome 16 at position 24, containing six exons and spanning 8.5 kb. P22 phox is a critical component of the superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs). It is associated with NOX2 to form cytochrome b 558 expressed mainly in phagocytes and responsible for the killing of microorganisms when bacterial and fungal infections occur. CYBA mutations lead to one of the autosomal recessive forms of chronic granulomatous disease (AR22 0 CGD) clinically characterized by recurrent and severe infections in early childhood. However, p22 phox is also the partner of NOX1, NOX3 and NOX4, but not NOX5, which are analogs of NOX2, the first identified member of the NOX family. P22 phox-NOX complexes have emerged as one of the most relevant sources of reactive oxygen species (ROS) in tissues and cells, and are associated with several diseases such as cardiovascular and cerebrovascular diseases. The p22 phox-deficient mouse strain nmf333 has made it possible to highlight the role of p22 phox in the control of inner ear balance in association with NOX3. However, the relevance of p22 phox for NOX3 function remains uncertain because AR22 0 CGD patients do not suffer from vestibular dysfunction. Finally, a large number of genetic variations of CYBA have been reported, among them the C242T polymorphism, which has been extensively studied in association with coronary artery and heart diseases, but conflicting results continue to be reported.
Redox biology, Oct 1, 2017
There is emerging evidence for the involvement of reactive oxygen species (ROS) in the regulation... more There is emerging evidence for the involvement of reactive oxygen species (ROS) in the regulation of stem cells and cellular differentiation. Absence of the ROS-generating NADPH oxidase NOX2 in chronic granulomatous disease (CGD) patients, predominantly manifests as immune deficiency, but has also been associated with decreased cognition. Here, we investigate the role of NOX enzymes in neuronal homeostasis in adult mouse brain and in neural cells derived from human induced pluripotent stem cells (iPSC). High levels of NOX2 were found in mouse adult neurogenic regions. In NOX2-deficient mice, neurogenic regions showed diminished redox modifications, as well as decrease in neuroprecursor numbers and in expression of genes involved in neural differentiation including NES, BDNF and OTX2. iPSC from healthy subjects and patients with CGD were used to study the role of NOX2 in human in vitro neuronal development. Expression of NOX2 was low in undifferentiated iPSC, upregulated upon neural induction, and disappeared during neuronal differentiation. In human neurospheres, NOX2 protein and ROS generation were polarized within the inner cell layer of rosette structures. NOX2 deficiency in CGD-iPSCs resulted in an abnormal neural induction in vitro, as revealed by a reduced expression of neuroprogenitor markers (NES, BDNF, OTX2, NRSF/REST), and a decreased generation of mature neurons. Vector-mediated NOX2 expression in NOX2-deficient iPSCs rescued neurogenesis. Taken together, our study provides novel evidence for a regulatory role of NOX2 during early stages of neurogenesis in mouse and human.
Journal of Biological Chemistry, Apr 1, 2005
Assembly of cytosolic factors p67 phox and p47 phox with cytochrome b 558 is one of the crucial k... more Assembly of cytosolic factors p67 phox and p47 phox with cytochrome b 558 is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop 191 TSSTKTIRRS 200 and the C-terminal 484 DESQANHFAVHHDEEKD 500 of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47 phox translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop Nox4-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/ R/G mutants of the ␣-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47 phox membrane translocation. This suggests that the ␣-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.
Revue de Médecine Interne, Mar 1, 2009
La granulomatose septique chronique est une maladie familiale rare due à un déficit immunitaire p... more La granulomatose septique chronique est une maladie familiale rare due à un déficit immunitaire primaire du métabolisme oxydatif des cellules phagocytaires. Le défaut de fonctionnement du complexe NADPH oxydase membranaire des phagocytes entraîne un tableau clinique d'infections fongiques et bactériennes sévères à répétition, en général, dès le plus jeune âge. La forme génétique, la plus fréquente et vraisemblablement la plus grave, est celle liée à l'X, affectant le gène CYBB codant l'élément redox de l'oxydase, gp91phox ou Nox2. Cependant, il existe des formes autosomales récessives plus rares, affectant d'autres composants protéiques du complexe oxydase et qui se caractérisent par des manifestations cliniques frustes, pouvant apparaître plus tardivement, même à l'âge adulte. Le traitement de la granulomatose septique chronique repose sur l'antibiothérapie et les antifongiques en prophylactique. L'allogreffe de moelle osseuse, seul traitement radicalement curatif, n'est envisageable que lorsqu'il existe un donneur HLA compatible et dans un contexte clinique précis. La thérapie génétique semble être un traitement d'avenir potentiel après conditionnement myéloablatif des patients.
Human Genetics, Apr 9, 2002
Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot gene... more Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O(2)(-)) and other microbicidal oxidants because of mutations in one of the four components of the O(2)(-)-generating NADPH oxidase complex. A subgroup (approximately 5% of identified CGD patients) has been reported to have mutations in the gene encoding the small p22 phox subunit of the flavocytochrome b (558), the redox element of phagocyte NADPH oxidase. Here, we report the case of an autosomal recessive CGD patient with a defect in the p22 phox subunit. Neutrophils failed to produce O(2)(-) in response to soluble and particulate stimuli, and cytochrome b (558) was absent as measured by immunoblotting and difference absorption spectra. Mutations in the p22 phox mRNA of the patient were detected by reverse transcription/polymerase chain reaction amplification and sequencing. The defect in the mRNA was a 179-bp insertion associated with a 21-bp deletion of the beginning of exon 5 at position 315 from the translation start codon of the p22 phox cDNA. This defect was also detected in the patient's parents. In the genomic DNA of the patient, the molecular defect was a homozygous 36-bp deletion in the linking sequence between intron 4 and exon 5. This genomic deletion corresponded to 15 bp of the 3' extremity of intron 4 and 21 bp of the beginning of exon 5 (the same deletion of exon 5 seen in the corresponding mRNA). The splicing mRNA error is attributable to the loss of the ag acceptor site of intron 4 and the utilization of a cryptic splice site with an ag sequence at position 355-356 of intron 4.
Clinical and Experimental Immunology, Feb 24, 2023
Chronic granulomatous disease (CGD) is a rare primary immune disorder caused by mutations in one ... more Chronic granulomatous disease (CGD) is a rare primary immune disorder caused by mutations in one of the five subunits of the NADPH oxidase complex expressed in phagocytes. Two-thirds of CGD cases are caused by mutations in CYBB that encodes NOX2 or gp91phox. Some rare X91+-CGD point mutations lead to a loss of function but with a normal expression of the mutated NOX2 protein. It is therefore necessary to ensure that this mutation is indeed responsible for the loss of activity in order to make a safe diagnosis for genetic counselling. We previously used the X-CGD PLB-985 cell model of M.C. Dinauer obtained by homologous recombination in the original PLB-985 human myeloid cell line, in order to study the functional impact of such mutations. Although the PLB-985 cell line was originally described by K.A. Tucker et al. in1987 as a distinct cell line isolated from a patient with acute nonlymphocytic leukemia, it is actually identified as a subclone of the HL-60 cells. In order to use a cellular model that meets the quality standard for the functional study of X91+-CGD mutations in CGD diagnosis, we developed our own model using the CRISPR-Cas9 technology in a certified PLB-985 cell line from DSMZ-German Collection of Microorganisms and Cell Cultures. Thanks to this new X-CGD model, we demonstrated that the G412E mutation in NOX2 found in a X91+-CGD patient prohibits access of the electron donor NADPH to its binding site explaining the absence of superoxide production in his neutrophils.
Immuno-analyse & biologie spécialisée, 1996
RBsumk-Actuellement, aucun des critbres diagnostiques ou pronostiques de I'infarctus medical (ECG... more RBsumk-Actuellement, aucun des critbres diagnostiques ou pronostiques de I'infarctus medical (ECG, scintigraphie, CK-MB...) n'est satisfaisant pour I'Bvaluation de I'infarctus pkriopkratoire en chirurgie cardiaque. La myosine, marqueur du nombre de myocytes n&zrosks, apparait intkessante dans cette indication. Nous avons r@!alisk son dosage immunoradiom&rique lors de pontages aorto-coronariens et compare les r&ultats aux donnkes prkopkatoires, perop&atoires et postopkratoires. Bien que les taux de myosine soient corrklk aux signes classiques et aux facteurs predictifs d'infarctus, leur signification est diffkente de celle des CPK-MB. La myosine peut ktre considk-r6e comme un parambtre de quantification de la n&rose myocytaire pkiop&atoire. Si son int&& diagnostique reste limit6 du fait de sa cinbtique lente, le dosage de la myosine semble p&enter un double avantage, soit en tant que facteur pronostique B dbterminer, soit comme outil de comparaison et d'8volution de techniques opkratoires et de protection myocardique. myosine / CPK-MB / infarctus pkriopbratoire / pontage aorto-coronarien Summary-Interest of immunoradiometric assay of the myosin heavy chain in coronary surgery. At the moment, there is no satisfactory criteria for the diagnosis or prognosis of perioperative myocardial infarction (M/) (ECG, scintigraphy, CPK-MB...) in cardiac surgery. Myosin, which reflects the number of necrosed myocyfes, seems of interest for this purpose. We evaluated myosin plasma levels by immunoradiometric assay during coronary artery bypass operations and compared results to the pre, peri and post-operative data. Although myosin levels correlated with the other classical signs and predictive factors of MI, the correlation with CPK-MB was weak. Myosin could be used for the quantification of the surgery myocyfe necrosis. Because of its low kinetics, the application of this parameter for the diagnosis of necrosis is limited. However, myosin determination seems interesting as a prognosis factor for the evaluation of cardiac surgery and myocardial protection techniques.
Methods in molecular biology, 2019
Induced pluripotent stem cells (iPSCs) are pluripotent stem cells that can be established from de... more Induced pluripotent stem cells (iPSCs) are pluripotent stem cells that can be established from dedifferentiation of all somatic cell types by epigenetic phenomena. iPSCs can be differentiated into any mature cells like neurons, hepatocytes, or pancreatic cells that have not been easily available to date. Thus, iPSCs are widely used for disease modeling, drug discovery, and cell therapy development. Here, we describe a protocol to obtain human mature and functional neutrophils and macrophages as ex vivo models of X-linked chronic granulomatous disease (X-CGD). This method can be applied to model the other genetic forms of CGD. We also describe methods for testing the characteristics and functions of neutrophils and macrophages by morphology, phagocytosis assay, release of granule markers or cytokines, cell surface markers, and NADPH oxidase activity.
Acta Diabetologica, Nov 25, 2015
Aims We investigated the association of polymorphisms of three genes implicated in oxidative stre... more Aims We investigated the association of polymorphisms of three genes implicated in oxidative stress: CYBA C242T, RAGE-374T/A and-429T/C, and ALOX12 Arg261Gln, with the delay of microalbuminuria onset in patients with type 1 diabetes mellitus (DT1). Methods A total of 162 T1D patients presenting with diabetes for 32.9 ± 9 years were included in the study; 53 had persistent microalbuminuria ([30 mg/l) and 109 did not. Onset of diabetes, microalbuminuria and end-stage renal disease (ESRD) were recorded as bio-clinical data. We determined polymorphism association of microalbuminuria with a Cox regression model. Results All polymorphisms respected the Hardy-Weinberg equilibrium. The Cox regression model validated four significant variables associated with microalbuminuria: RAGE 374AA (HR 4.19 [1.84-9.58] (p = 0.001)), CYBA TT?TC (HR 2.1 [1.16-3.80], p = 0.015), male sex (HR 1.92 [1.07-3.43], p = 0.028) and diabetes diagnosis at the pediatric stage (HR 1.85 [1.03-3.32], p = 0.039). The same association was found with ESRD (p = 0.028 and p = 0.033 for CYBA TC?TT and RAGE 374AA, respectively). CYBA C242T and RAGE 374T/A were not significantly associated with diabetic retinopathy. Conclusions CYBA C242T and RAGE-374T/A correlate with microalbuminuria onset in the French DT1 cohort. The same correlation with ESRD onset supports the argument for the involvement of a genetic predisposition involving kidney-specific oxidative stress for diabetic nephropathy.
Methods in molecular biology, 2019
Structure-function analysis of specific regions of NOX2 can be carried out after stable expressio... more Structure-function analysis of specific regions of NOX2 can be carried out after stable expression of site-directed mutagenesis-modified NOX2 in the X0-CGD PLB-985 cell model. Indeed, the generation of this human cellular model by Prof. MC Dinauer's team gave researchers the opportunity to gain a deeper understanding of functional regions of NOX2. With this model cell line, the functional impact of X+-CGD or of new mutations in NOX2 can be highlighted, as the biological material is not limited. PLB-985 cells transfected with various NOX2 mutations can be easily cultured and differentiated into neutrophils or monocytes/macrophages. Several measurements in intact mutated NOX2 PLB-985 cells can be carried out such as NOX2 expression, cytochrome b 558 spectrum, enzymatic activity, and assembly of the NADPH oxidase complex. Purified membranes or purified cytochrome b 558 from mutated NOX2 PLB-985 cells can be used for the study of the impact of specific mutations on NADPH oxidase or diaphorase activity, FAD incorporation, and NADPH or NADH binding in a cell-free assay system. Here, we describe a method to generate mutated NOX2 PLB-985 cells in order to analyze NOX2 structure-function relationships.
Biochemical and Biophysical Research Communications, Sep 1, 2014
High glucose (HG) or synthetic advanced glycation end-products (AGE) conditions are generally use... more High glucose (HG) or synthetic advanced glycation end-products (AGE) conditions are generally used to mimic diabetes in cellular models. Both models have shown an increase of apoptosis, oxidative stress and pro-inflammatory cytokine production in tubular cells. However, the impact of the two conditions combined has rarely been studied. In addition, the impact of glucose level variation due to cellular consumption is not clearly characterized in such experiments. Therefore, the aim of this study was to compare the effect of HG and AGE separately and of both on tubular cell phenotype changes in the HK2 cell line. Moreover, glucose consumption was monitored every hour to maintain the glucose level by supplementation throughout the experiments. We thus observed a significant decrease of apoptosis and H 2 O 2 production in the HK2 cell. HG or AGE treatment induced an increase of total and mitochondrial apoptosis as well as TGF-b release compared to control conditions; however, AGE or HG led to apoptosis preferentially involving the mitochondria pathway. No cumulative effect of HG and AGE treatment was observed on apoptosis. However, a pretreatment with RAGE antibodies partially abolished the apoptotic effect of HG and completely abolished the apoptotic effect of AGE. In conclusion, tubular cells are sensitive to the lack of glucose as well as to the HG and AGE treatments, the AGE effect being more deleterious than the HG effect. Absence of a potential synergistic effect of HG and AGE could indicate that they act through a common pathway, possibly via the activation of the RAGE receptors.
HAL (Le Centre pour la Communication Scientifique Directe), 2013
International audienc
Revue d'Oncologie Hématologie Pédiatrique, Dec 1, 2016
Revue de Médecine Interne, Dec 1, 2011
PubMed, 1993
It is of great importance to be able to distinguish hyperamylasemia linked to a pathological cond... more It is of great importance to be able to distinguish hyperamylasemia linked to a pathological condition of the pancreas from one due to the presence of the macroamylase in the serum of the patient. We have evaluated both gel filtration chromatography and precipitation by PEG-6000 for the detection of serum macroamylase. Our study has enabled us to define a simple detection protocol, suitable for routine laboratory use. In this protocol, macroamylasemia is detected initially by precipitation with PEG-6000; if less than 60% of amylase activity is precipitated, there is no macroamylasemia. If more than 60% of amylase activity can be precipitated by PEG-6000, macroamylasemia is confirmed by gel filtration chromatography. The incidence of macroamylasemia in hospitalized patients was studied; it was 1.3% in cases where amylasemia was normal, and 4.5% in hyperamylasemic patients.
Clinical Chemistry, Jul 1, 1994
Macromolecular aspartate aminotransferase was found in the serum of an apparently healthy patient... more Macromolecular aspartate aminotransferase was found in the serum of an apparently healthy patient. This complex was composed of aspartate aminotransferase (AST; EC 2.6.1.1) and immunoglobulin. Electrophoresis of the patient's serum showed an abnormal band migrating between mitochondrial (m) and cytosolic (s) AST. The macromolecular complex was purified by gel filtration on Sephacryl S300. The molecular mass of the complex was estimated to be 250 kDa, suggesting that the complex probably consists of one immunoglobulin molecule associated with one AST molecule. By immunoelectrophoresis, the immunoglobulin was found to be an IgG with kappa-lambda type light chain. When we used polyclonal antibodies against human mAST or sAST, the sAST antibodies strongly inhibited the AST activity of the macrocomplex, whereas the mAST antibodies had no effect. Thus the AST molecule of the macrocomplex is an sAST type.
Journal of Allergy and Clinical Immunology, 2019
Inflammatory dendritic cells-not basophils-are necessary and sufficient for induction of Th2 immu... more Inflammatory dendritic cells-not basophils-are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen.
Human Genetics, Nov 6, 2004
Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADP... more Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADPH oxidase and so cannot generate superoxide anions (O(2) (-)). The most common form is caused by mutations in CYBB encoding gp91 phox, the heavy chain of flavocytochrome b(558) (XCGD). We investigated 11 male patients and their families suspected of suffering from X-linked CGD. These XCGD patients were classified as having different variants (X91(0), X91(-) or X91(+)) according to their cytochrome b(558) expression and NADPH oxidase activity. Nine patients had X91(0) CGD, one had X91(-) CGD and one had X91(+) CGD. Six mutations in CYBB were novel. Of the four new X91(0) CGD cases, three were point mutations: G65A in exon 2, G387T in exon 5 and G970T in exon 9, leading to premature stop codons at positions Try18, Try125 and Glu320, respectively, in gp91 phox. One case of X91(0) CGD originated from a new 1005G deletion detected in exon 9. Surprisingly, four nonsense mutations in exon 5 led to the generation of two mRNAs, one with a normal size containing the mutation and the other in which exon 5 had been spliced. A novel X91(-) CGD case with low gp91 phox expression was diagnosed. It was caused by an 11-bp deletion in the linking region between exon 12 and intron 12, activating a new cryptic site. Finally, a new X91(+) CGD case was detected, characterized by a missense mutation Leu505Arg in the potential NADPH-binding site of gp91 phox. No clear correlation between the severity of the clinical symptoms and the sub-type of XCGD could be established.
Human Mutation, Jul 1, 2009
NOX enzymes are reactive oxygen species (ROS)-generating NADPH oxidases. Several members of the N... more NOX enzymes are reactive oxygen species (ROS)-generating NADPH oxidases. Several members of the NOX family depend on the p22(phox) subunit, encoded by the CYBA gene. CYBA is highly polymorphic, and has been widely studied as a potential risk factor for various diseases, with conflicting results. In the present study, we used Epstein-Barr (EBV)-transformed B-lymphocytes from 50 healthy unrelated individuals to analyze their CYBA mRNA sequence and NOX2-dependent ROS generation. Seven single-nucleotide polymorphisms (SNPs) were identified (five previously described, two novel). The combination of these SNPs yielded 11 distinct haplotypes, which could be grouped into seven haplogroups (A-G). Haplogroup C (c.214T>C, c.521T>C, and c.(*)24G>A) showed a significantly lower ROS generation, as compared to the most frequent haplogroup, A. CYBA variants from the seven haplogroups were transduced into p22(phox)-deficient B-lymphocytes. The haplogroup C variant showed significantly lower ROS production. c.214T>C and c.521T>C lead to nonsynonymous codon changes, while c.(*)24G>A lies within the 3'UTR. Using a luciferase/3'UTR construct, we showed that the (*)24A allele led to decreased reporter gene activity. These results help to unravel the complex nature of how genetic variations in CYBA influence NOX2 activity, and indicate that haplotypes, rather than individual SNPs, define the effect on ROS generation.
Gene, Jul 1, 2016
P22 phox is a ubiquitous protein encoded by the CYBA gene located on the long arm of chromosome 1... more P22 phox is a ubiquitous protein encoded by the CYBA gene located on the long arm of chromosome 16 at position 24, containing six exons and spanning 8.5 kb. P22 phox is a critical component of the superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs). It is associated with NOX2 to form cytochrome b 558 expressed mainly in phagocytes and responsible for the killing of microorganisms when bacterial and fungal infections occur. CYBA mutations lead to one of the autosomal recessive forms of chronic granulomatous disease (AR22 0 CGD) clinically characterized by recurrent and severe infections in early childhood. However, p22 phox is also the partner of NOX1, NOX3 and NOX4, but not NOX5, which are analogs of NOX2, the first identified member of the NOX family. P22 phox-NOX complexes have emerged as one of the most relevant sources of reactive oxygen species (ROS) in tissues and cells, and are associated with several diseases such as cardiovascular and cerebrovascular diseases. The p22 phox-deficient mouse strain nmf333 has made it possible to highlight the role of p22 phox in the control of inner ear balance in association with NOX3. However, the relevance of p22 phox for NOX3 function remains uncertain because AR22 0 CGD patients do not suffer from vestibular dysfunction. Finally, a large number of genetic variations of CYBA have been reported, among them the C242T polymorphism, which has been extensively studied in association with coronary artery and heart diseases, but conflicting results continue to be reported.
Redox biology, Oct 1, 2017
There is emerging evidence for the involvement of reactive oxygen species (ROS) in the regulation... more There is emerging evidence for the involvement of reactive oxygen species (ROS) in the regulation of stem cells and cellular differentiation. Absence of the ROS-generating NADPH oxidase NOX2 in chronic granulomatous disease (CGD) patients, predominantly manifests as immune deficiency, but has also been associated with decreased cognition. Here, we investigate the role of NOX enzymes in neuronal homeostasis in adult mouse brain and in neural cells derived from human induced pluripotent stem cells (iPSC). High levels of NOX2 were found in mouse adult neurogenic regions. In NOX2-deficient mice, neurogenic regions showed diminished redox modifications, as well as decrease in neuroprecursor numbers and in expression of genes involved in neural differentiation including NES, BDNF and OTX2. iPSC from healthy subjects and patients with CGD were used to study the role of NOX2 in human in vitro neuronal development. Expression of NOX2 was low in undifferentiated iPSC, upregulated upon neural induction, and disappeared during neuronal differentiation. In human neurospheres, NOX2 protein and ROS generation were polarized within the inner cell layer of rosette structures. NOX2 deficiency in CGD-iPSCs resulted in an abnormal neural induction in vitro, as revealed by a reduced expression of neuroprogenitor markers (NES, BDNF, OTX2, NRSF/REST), and a decreased generation of mature neurons. Vector-mediated NOX2 expression in NOX2-deficient iPSCs rescued neurogenesis. Taken together, our study provides novel evidence for a regulatory role of NOX2 during early stages of neurogenesis in mouse and human.
Journal of Biological Chemistry, Apr 1, 2005
Assembly of cytosolic factors p67 phox and p47 phox with cytochrome b 558 is one of the crucial k... more Assembly of cytosolic factors p67 phox and p47 phox with cytochrome b 558 is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop 191 TSSTKTIRRS 200 and the C-terminal 484 DESQANHFAVHHDEEKD 500 of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47 phox translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop Nox4-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/ R/G mutants of the ␣-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47 phox membrane translocation. This suggests that the ␣-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.
Revue de Médecine Interne, Mar 1, 2009
La granulomatose septique chronique est une maladie familiale rare due à un déficit immunitaire p... more La granulomatose septique chronique est une maladie familiale rare due à un déficit immunitaire primaire du métabolisme oxydatif des cellules phagocytaires. Le défaut de fonctionnement du complexe NADPH oxydase membranaire des phagocytes entraîne un tableau clinique d'infections fongiques et bactériennes sévères à répétition, en général, dès le plus jeune âge. La forme génétique, la plus fréquente et vraisemblablement la plus grave, est celle liée à l'X, affectant le gène CYBB codant l'élément redox de l'oxydase, gp91phox ou Nox2. Cependant, il existe des formes autosomales récessives plus rares, affectant d'autres composants protéiques du complexe oxydase et qui se caractérisent par des manifestations cliniques frustes, pouvant apparaître plus tardivement, même à l'âge adulte. Le traitement de la granulomatose septique chronique repose sur l'antibiothérapie et les antifongiques en prophylactique. L'allogreffe de moelle osseuse, seul traitement radicalement curatif, n'est envisageable que lorsqu'il existe un donneur HLA compatible et dans un contexte clinique précis. La thérapie génétique semble être un traitement d'avenir potentiel après conditionnement myéloablatif des patients.
Human Genetics, Apr 9, 2002
Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot gene... more Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O(2)(-)) and other microbicidal oxidants because of mutations in one of the four components of the O(2)(-)-generating NADPH oxidase complex. A subgroup (approximately 5% of identified CGD patients) has been reported to have mutations in the gene encoding the small p22 phox subunit of the flavocytochrome b (558), the redox element of phagocyte NADPH oxidase. Here, we report the case of an autosomal recessive CGD patient with a defect in the p22 phox subunit. Neutrophils failed to produce O(2)(-) in response to soluble and particulate stimuli, and cytochrome b (558) was absent as measured by immunoblotting and difference absorption spectra. Mutations in the p22 phox mRNA of the patient were detected by reverse transcription/polymerase chain reaction amplification and sequencing. The defect in the mRNA was a 179-bp insertion associated with a 21-bp deletion of the beginning of exon 5 at position 315 from the translation start codon of the p22 phox cDNA. This defect was also detected in the patient's parents. In the genomic DNA of the patient, the molecular defect was a homozygous 36-bp deletion in the linking sequence between intron 4 and exon 5. This genomic deletion corresponded to 15 bp of the 3' extremity of intron 4 and 21 bp of the beginning of exon 5 (the same deletion of exon 5 seen in the corresponding mRNA). The splicing mRNA error is attributable to the loss of the ag acceptor site of intron 4 and the utilization of a cryptic splice site with an ag sequence at position 355-356 of intron 4.
Clinical and Experimental Immunology, Feb 24, 2023
Chronic granulomatous disease (CGD) is a rare primary immune disorder caused by mutations in one ... more Chronic granulomatous disease (CGD) is a rare primary immune disorder caused by mutations in one of the five subunits of the NADPH oxidase complex expressed in phagocytes. Two-thirds of CGD cases are caused by mutations in CYBB that encodes NOX2 or gp91phox. Some rare X91+-CGD point mutations lead to a loss of function but with a normal expression of the mutated NOX2 protein. It is therefore necessary to ensure that this mutation is indeed responsible for the loss of activity in order to make a safe diagnosis for genetic counselling. We previously used the X-CGD PLB-985 cell model of M.C. Dinauer obtained by homologous recombination in the original PLB-985 human myeloid cell line, in order to study the functional impact of such mutations. Although the PLB-985 cell line was originally described by K.A. Tucker et al. in1987 as a distinct cell line isolated from a patient with acute nonlymphocytic leukemia, it is actually identified as a subclone of the HL-60 cells. In order to use a cellular model that meets the quality standard for the functional study of X91+-CGD mutations in CGD diagnosis, we developed our own model using the CRISPR-Cas9 technology in a certified PLB-985 cell line from DSMZ-German Collection of Microorganisms and Cell Cultures. Thanks to this new X-CGD model, we demonstrated that the G412E mutation in NOX2 found in a X91+-CGD patient prohibits access of the electron donor NADPH to its binding site explaining the absence of superoxide production in his neutrophils.
Immuno-analyse & biologie spécialisée, 1996
RBsumk-Actuellement, aucun des critbres diagnostiques ou pronostiques de I'infarctus medical (ECG... more RBsumk-Actuellement, aucun des critbres diagnostiques ou pronostiques de I'infarctus medical (ECG, scintigraphie, CK-MB...) n'est satisfaisant pour I'Bvaluation de I'infarctus pkriopkratoire en chirurgie cardiaque. La myosine, marqueur du nombre de myocytes n&zrosks, apparait intkessante dans cette indication. Nous avons r@!alisk son dosage immunoradiom&rique lors de pontages aorto-coronariens et compare les r&ultats aux donnkes prkopkatoires, perop&atoires et postopkratoires. Bien que les taux de myosine soient corrklk aux signes classiques et aux facteurs predictifs d'infarctus, leur signification est diffkente de celle des CPK-MB. La myosine peut ktre considk-r6e comme un parambtre de quantification de la n&rose myocytaire pkiop&atoire. Si son int&& diagnostique reste limit6 du fait de sa cinbtique lente, le dosage de la myosine semble p&enter un double avantage, soit en tant que facteur pronostique B dbterminer, soit comme outil de comparaison et d'8volution de techniques opkratoires et de protection myocardique. myosine / CPK-MB / infarctus pkriopbratoire / pontage aorto-coronarien Summary-Interest of immunoradiometric assay of the myosin heavy chain in coronary surgery. At the moment, there is no satisfactory criteria for the diagnosis or prognosis of perioperative myocardial infarction (M/) (ECG, scintigraphy, CPK-MB...) in cardiac surgery. Myosin, which reflects the number of necrosed myocyfes, seems of interest for this purpose. We evaluated myosin plasma levels by immunoradiometric assay during coronary artery bypass operations and compared results to the pre, peri and post-operative data. Although myosin levels correlated with the other classical signs and predictive factors of MI, the correlation with CPK-MB was weak. Myosin could be used for the quantification of the surgery myocyfe necrosis. Because of its low kinetics, the application of this parameter for the diagnosis of necrosis is limited. However, myosin determination seems interesting as a prognosis factor for the evaluation of cardiac surgery and myocardial protection techniques.
Methods in molecular biology, 2019
Induced pluripotent stem cells (iPSCs) are pluripotent stem cells that can be established from de... more Induced pluripotent stem cells (iPSCs) are pluripotent stem cells that can be established from dedifferentiation of all somatic cell types by epigenetic phenomena. iPSCs can be differentiated into any mature cells like neurons, hepatocytes, or pancreatic cells that have not been easily available to date. Thus, iPSCs are widely used for disease modeling, drug discovery, and cell therapy development. Here, we describe a protocol to obtain human mature and functional neutrophils and macrophages as ex vivo models of X-linked chronic granulomatous disease (X-CGD). This method can be applied to model the other genetic forms of CGD. We also describe methods for testing the characteristics and functions of neutrophils and macrophages by morphology, phagocytosis assay, release of granule markers or cytokines, cell surface markers, and NADPH oxidase activity.
Acta Diabetologica, Nov 25, 2015
Aims We investigated the association of polymorphisms of three genes implicated in oxidative stre... more Aims We investigated the association of polymorphisms of three genes implicated in oxidative stress: CYBA C242T, RAGE-374T/A and-429T/C, and ALOX12 Arg261Gln, with the delay of microalbuminuria onset in patients with type 1 diabetes mellitus (DT1). Methods A total of 162 T1D patients presenting with diabetes for 32.9 ± 9 years were included in the study; 53 had persistent microalbuminuria ([30 mg/l) and 109 did not. Onset of diabetes, microalbuminuria and end-stage renal disease (ESRD) were recorded as bio-clinical data. We determined polymorphism association of microalbuminuria with a Cox regression model. Results All polymorphisms respected the Hardy-Weinberg equilibrium. The Cox regression model validated four significant variables associated with microalbuminuria: RAGE 374AA (HR 4.19 [1.84-9.58] (p = 0.001)), CYBA TT?TC (HR 2.1 [1.16-3.80], p = 0.015), male sex (HR 1.92 [1.07-3.43], p = 0.028) and diabetes diagnosis at the pediatric stage (HR 1.85 [1.03-3.32], p = 0.039). The same association was found with ESRD (p = 0.028 and p = 0.033 for CYBA TC?TT and RAGE 374AA, respectively). CYBA C242T and RAGE 374T/A were not significantly associated with diabetic retinopathy. Conclusions CYBA C242T and RAGE-374T/A correlate with microalbuminuria onset in the French DT1 cohort. The same correlation with ESRD onset supports the argument for the involvement of a genetic predisposition involving kidney-specific oxidative stress for diabetic nephropathy.
Methods in molecular biology, 2019
Structure-function analysis of specific regions of NOX2 can be carried out after stable expressio... more Structure-function analysis of specific regions of NOX2 can be carried out after stable expression of site-directed mutagenesis-modified NOX2 in the X0-CGD PLB-985 cell model. Indeed, the generation of this human cellular model by Prof. MC Dinauer's team gave researchers the opportunity to gain a deeper understanding of functional regions of NOX2. With this model cell line, the functional impact of X+-CGD or of new mutations in NOX2 can be highlighted, as the biological material is not limited. PLB-985 cells transfected with various NOX2 mutations can be easily cultured and differentiated into neutrophils or monocytes/macrophages. Several measurements in intact mutated NOX2 PLB-985 cells can be carried out such as NOX2 expression, cytochrome b 558 spectrum, enzymatic activity, and assembly of the NADPH oxidase complex. Purified membranes or purified cytochrome b 558 from mutated NOX2 PLB-985 cells can be used for the study of the impact of specific mutations on NADPH oxidase or diaphorase activity, FAD incorporation, and NADPH or NADH binding in a cell-free assay system. Here, we describe a method to generate mutated NOX2 PLB-985 cells in order to analyze NOX2 structure-function relationships.
Biochemical and Biophysical Research Communications, Sep 1, 2014
High glucose (HG) or synthetic advanced glycation end-products (AGE) conditions are generally use... more High glucose (HG) or synthetic advanced glycation end-products (AGE) conditions are generally used to mimic diabetes in cellular models. Both models have shown an increase of apoptosis, oxidative stress and pro-inflammatory cytokine production in tubular cells. However, the impact of the two conditions combined has rarely been studied. In addition, the impact of glucose level variation due to cellular consumption is not clearly characterized in such experiments. Therefore, the aim of this study was to compare the effect of HG and AGE separately and of both on tubular cell phenotype changes in the HK2 cell line. Moreover, glucose consumption was monitored every hour to maintain the glucose level by supplementation throughout the experiments. We thus observed a significant decrease of apoptosis and H 2 O 2 production in the HK2 cell. HG or AGE treatment induced an increase of total and mitochondrial apoptosis as well as TGF-b release compared to control conditions; however, AGE or HG led to apoptosis preferentially involving the mitochondria pathway. No cumulative effect of HG and AGE treatment was observed on apoptosis. However, a pretreatment with RAGE antibodies partially abolished the apoptotic effect of HG and completely abolished the apoptotic effect of AGE. In conclusion, tubular cells are sensitive to the lack of glucose as well as to the HG and AGE treatments, the AGE effect being more deleterious than the HG effect. Absence of a potential synergistic effect of HG and AGE could indicate that they act through a common pathway, possibly via the activation of the RAGE receptors.