Form Of Chronic Granulomatous Disease Research Articles

First Report on Chronic Granulomatous Disease from Nepal and a Review of CYBC1 Deficiency.

Chronic granulomatous disease (CGD) primarily results from inherited defects in components of the nicotinamide adenine dinucleotide phosphate oxidase enzyme complex. These include gene defects in cytochrome B-245/558 subunit α/β and neutrophil cytosolic factors 1, 2, and 4. Recently, homozygous loss-of-function variants in cytochrome B-245 chaperone 1 gene (CYBC1) have been discovered to cause CGD (CYBC1-CGD). Data on variant-proven CGD from low-income countries, the most underprivileged regions of the world, remain sparse due to numerous constraints. Herein, we report the first cohort of patients with CGD from Nepal, a low-income country in the Himalayas' challenging terrain. Our report includes a description of a new case of CYBC1 deficiency who was first diagnosed with CGD at our center. Only a dozen cases of CYBC1-CGD have been described in the literature thus far which have been reviewed comprehensively herein. Most of these patients have had significant infections and autoimmune/inflammatory manifestations. Pulmonary and invasive/disseminated bacterial/fungal infections were the most common followed by skin and soft-tissue infections. Inflammatory bowel disease (IBD) was the most common inflammatory manifestation (median age at diagnosis: 9 years) followed by episodes of recurrent/prolonged fever. Other autoimmune/inflammatory manifestations reported in CYBC1-CGD include acute pancreatitis, hemophagocytic lymphohistiocytosis, systemic granulomatosis, interstitial lung disease, arthritis, autoimmune hemolytic anemia, uveitis, nephritis, and eczema. Our analysis shows that patients with CYBC1-CGD are at a significantly higher risk of IBD-like illness as compared to other forms of CGD which merits further confirmatory studies in the future.

A novel mutation in EROS (CYBC1) causes chronic granulomatous disease

Chronic Granulomatous Disease (CGD) is an inborn error of immunity characterised by opportunistic infection and sterile granulomatous inflammation. CGD is caused by a failure of reactive oxygen species (ROS) production by the phagocyte NADPH oxidase. Mutations in the genes encoding phagocyte NADPH oxidase subunits cause CGD. We and others have described a novel form of CGD (CGD5) secondary to lack of EROS (CYBC1), a highly selective chaperone for gp91phox. EROS-deficient cells express minimal levels of gp91phox and its binding partner p22phox, but EROS also controls the expression of other proteins such as P2X7. The full nature of CGD5 is currently unknown. We describe a homozygous frameshift mutation in CYBC1 leading to CGD. Individuals who are heterozygous for this mutation are found in South Asian populations (allele frequency = 0.00006545), thus it is not a private mutation. Therefore, it is likely to be the underlying cause of other cases of CGD.

An unusual presentation of mycetoma around knee joint as a subcutaneous mass – A case report

Introduction: Mycetoma is a form of chronic granulomatous disease which involves subcutaneous tissues and causes bone destruction in advanced stages. The characteristic features are sinus formation, granules formation, and mass in subcutaneous region. Case Report: A 19-year-old male presented to our out-patient clinic with complaint of a painless swelling around the medial aspect of the right knee joint for 8 months with no sinus or discharge of granules. Pes anserinus bursitis was thought of as differential diagnosis for the present condition. “Staging-classification of mycetoma” is commonly used for classifying mycetoma and as per classification, the present case had Stage-A. Conclusion: Single-stage local excision was performed and supplemented with anti-fungal agent for 6 months, which showed good outcome at the final follow-up of 13 months. Keywords: Eumycetoma, wide excision, antifungal agents, knee.

Hematologically important mutations: The autosomal forms of chronic granulomatous disease (third update)

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. CGD patients suffer from severe, recurrent bacterial and fungal infections. The disease is caused by mutations in the genes encoding the components of the leukocyte NADPH oxidase. This enzyme produces superoxide, which is subsequently metabolized to hydrogen peroxide and other reactive oxygen species (ROS). These products are essential for intracellular killing of pathogens by phagocytic leukocytes (neutrophils, eosinophils, monocytes and macrophages). The leukocyte NADPH oxidase is composed of five subunits, four of which are encoded by autosomal genes. These are CYBA, encoding p22phox, NCF1, encoding p47phox, NCF2, encoding p67phox and NCF4, encoding p40phox. This article lists all mutations identified in these genes in CGD patients. In addition, cytochrome b558 chaperone-1 (CYBC1), recently recognized as an essential chaperone protein for the expression of the X-linked NADPH oxidase component gp91phox (also called Nox2), is encoded by the autosomal gene CYBC1. Mutations in this gene also lead to CGD. Finally, RAC2, a small GTPase of the Rho family, is needed for activation of the NADPH oxidase, and mutations in the RAC2 gene therefore also induce CGD-like symptoms. Mutations in these last two genes are also listed in this article.

Lentiviral gene therapy rescues p47phox chronic granulomatous disease and the ability to fight Salmonella infection in mice

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder characterised by recurrent and often life-threatening infections and hyperinflammation. It is caused by defects of the phagocytic NADPH oxidase, a multicomponent enzyme system responsible for effective pathogen killing. A phase I/II clinical trial of lentiviral gene therapy is underway for the most common form of CGD, X-linked, caused by mutations in the gp91phox subunit of the NADPH oxidase. We propose to use a similar strategy to tackle p47phox-deficient CGD, caused by mutations in NCF1, which encodes the p47phox cytosolic component of the enzymatic complex. We generated a pCCLCHIM-p47phox lentiviral vector, containing the chimeric Cathepsin G/FES myeloid promoter and a codon-optimised version of the human NCF1 cDNA. Here we show that transduction with the pCCLCHIM-p47phox vector efficiently restores p47phox expression and biochemical NADPH oxidase function in p47phox-deficient human and murine cells. We also tested the ability of our gene therapy approach to control infection by challenging p47phox-null mice with Salmonella Typhimurium, a leading cause of sepsis in CGD patients, and found that mice reconstituted with lentivirus-transduced hematopoietic stem cells had a reduced bacterial load compared with untreated mice. Overall, our results potentially support the clinical development of a gene therapy approach using the pCCLCHIM-p47phox vector.

Granulomatous inflammation in the manifestation of chronic granulomatous disease: a clinical case report

Chronic granulomatous disease is a primary immunodeficiency, characterized by a violation of the oxygen-dependent mechanisms of phagocytosis. Mutations in the genes encoding proteins of the NADPH-oxidase complex lead to a violation of the respiratory burst. Clinical manifestations are recurrent bacterial and fungal infections, and the development of granulomatous complications due to a defect in autophagy, accompanied by an increase in the level of interleukin-1 inthe blood. The treatment of this disease is continuous preventive antimicrobial therapy, and specific therapy for the treatment of granulomatous complications. Hematopoietic stem cell transplantation from an allogeneic or unrelated donor is currently considered to be only radical treatment for CGD. This article presents a clinical case of the manifestation of an X-linked form of chronic granulomatous disease with granulomatous manifestations in the absence of an infectious history. Parents patient agreed to use personal data in research and publications.

Inherited p40phox Deficiency Differs From Classic Chronic Granulomatous Disease

To characterize clinical and laboratory aspects of p40phox deficiency, a unique autosomal recessive form of chronic granulomatous disease (CGD) previously described in only 1 patient.The population included 24 patients with p40phox deficiency from 12 families in 8 countries. Both male and female patients are reported, with age ranging from 1 to 47 years.Clinical history of each case was collected, including autoinflammatory manifestations and infection history. Laboratory analysis included both gene sequencing and numerous functional studies of the immune system.Mean age at diagnosis for symptomatic patients was 15 years (range: 1–46 years). Twenty patients were symptomatic. Four were asymptomatic (age 1–10 years, recognized by family history). Of the symptomatic patients, skin inflammation was present in 50%, and granulomatous gastrointestinal manifestations were present in 50%, with varied presentation from oral ulcers to severe Crohn-like inflammatory bowel disease. Cutaneous infections were reported in 42%. The phorbol myristate acetate–induced dihydrorhodamine (DHR) oxidation test gave normal or just below normal results for all patients.Excessive inflammatory lesions of the skin (lupuslike) and gastrointestinal tract (Crohn-like) are common in patients with p40phox deficiency. Compared with classic CGD, it is often diagnosed later in life and demonstrates a normal DHR laboratory test result (the laboratory test widely used for diagnosis of CGD). Patients experience peripheral bacterial infections, but fungal infections and invasive infections of any type are not common. Identification of 4 asymptomatic children demonstrates that there is incomplete clinical penetrance, at least until adolescence.It is essential for pediatricians to be aware of this disease and how it differs from classic CGD. Patients may be under their care who have been managed by other subspecialists (dermatology, rheumatology, gastroenterology) but have never been recognized to have an underlying p40phox deficiency. Additionally, immunologists are only recently learning that this form of CGD is not detectable by routine DHR. Therefore, if a patient has clinical signs and symptoms that fit the diagnosis, immunology consultation is warranted even if the immune system has been evaluated in the past. Genetic testing should be considered to confirm the diagnosis.

Cell-Free NADPH Oxidase Activation Assays: A Triumph of Reductionism.

The superoxide (O2·-)-generating NADPH oxidase complex of phagocytes comprises a membrane-associated heterodimeric flavocytochrome, known as cytochrome b 558 (consisting of NOX2 and p22phox) and four cytosolic regulatory proteins, p47phox, p67phox, p40phox, and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2·- generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome, a process known as NADPH oxidase assembly. A consequent conformational change in NOX2 initiates the electron flow along a redox gradient, from NADPH to molecular oxygen (O2), leading to the one-electron reduction of O2 to O2·-. Historically, methodological difficulties in the study of the assembled complex derived from stimulated cells, due to its lack of stability, led to the design of "cell-free" systems (also known as "broken cells" or in vitro systems). In a major paradigm shift, the cell-free systems have as their starting point NADPH oxidase components derived from resting (unstimulated) phagocytes, or as in the predominant method at present, recombinant proteins representing the components of the NADPH oxidase complex. In cell-free systems, membrane receptor stimulation and the signal transduction sequence are absent, the accent being placed on the actual process of assembly, all of which takes place in vitro. Thus, a mixture of the individual components of the NADPH oxidase is exposed in vitro to an activating agent, the most common being anionic amphiphiles, resulting in the formation of a complex between cytochrome b 558 and the cytosolic components and O2·- generation in the presence of NADPH. Alternative activating pathways require posttranslational modification of oxidase components or modifying the phospholipid milieu surrounding cytochrome b 558. Activation is commonly quantified by measuring the primary product of the reaction, O2·-, trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of rates of O2·- production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount role in the identification and characterization of the components of the NADPH oxidase complex, the performance of structure-function studies, the deciphering of the mechanisms of assembly, the search for inhibitory drugs, and the diagnosis of various forms of chronic granulomatous disease (CGD).

Analysis of Chronic Granulomatous Disease in the Kavkazi Population in Israel Reveals Phenotypic Heterogeneity in Patients with the Same NCF1 mutation (c.579G>A).

Chronic granulomatous disease (CGD) is an innate immune deficiency disorder of phagocytes, resulting from mutations in the components of the NADPH oxidase complex that impair the synthesis of oxygen radicals, thus rendering patients susceptible to recurrent infections and excessive hyperinflammatory responses. The most common autosomal recessive form of CGD is p47phox deficiency, which is often clinically milder than the more common X-linked recessive form. Here, we report data on genetics, clinical and biochemical findings in 17 CGD patients of Kavkazi origin with the nonsense mutation c.579G>A in the NCF1 gene, leading to p47phox deficiency. Diagnosis was based on detailed clinical evaluation, respiratory burst activity by cytochrome c reduction and dihydrorhodamine-1,2,3 (DHR) assay by flow cytometry, expression of p47phox by immunoblotting and molecular confirmation by DNA sequence analysis. Twelve male and five female patients with median age at onset of 2.5years (range 1day to 9years) were included in the study. The present cohort displays an encouraging 88% overall long-term survival, with median follow-up of 17years. Clinical manifestations varied from mild to severe expression of the disease. Correlation between genotype and phenotype is unpredictable, although the Kavkazi patients were more severely affected than other patients with p47phox deficiency. Kavkazi CGD patients harbor a common genetic mutation that is associated with a heterogeneous clinical phenotype. Early diagnosis and proper clinical management in an experienced phagocytic leukocyte center is imperative to ensure favorable patient outcome. New treatment strategies are ongoing, but results are not yet conclusive.

299. Efficient Functional Oxidase Correction after CRISPR-Oligomer Gene Repair in X-CGD Patient Hematopoietic Stem Cells (HSC) Using Non-Viral, cGMP Compliant, Scalable, Closed System

Gene therapy using integrating viral vectors in hematopoietic stem cells (HSC) has shown clinical benefit in genetic diseases. The safety concerns associated with random integration and the lack of gene expression regulation remain key challenges with use of viral vectors. Efficient and target-site specific correction of mutation(s) in patient HSC genomic DNA using non-viral methods may improve safety and gene expression regulation. We and others have previously reported that loading of Nucleases in the form of messenger RNA (mRNA) results in low toxicity and efficient functional gene editing when using MaxCyte's rapid, automated, cGMP and regulatory compliant, and closed cell loading platform. This platform has been cleared for use in human clinical trials for multiple applications of targeted gene editing in stem cells and immune cells.We report herein that transfecting CRISPR (Cas9+gRNA) mRNA along with donor oligomer specific to a hotspot mutation in the gp91phox gene (CYBB) located on exon 7 associated with the X-linked form of Chronic Granulomatous Disease (X-CGD), using MaxCyte GT System, results in efficient repair of the 676T (stop codon) to 676C (Arginine) mutation, resulting in gp91 protein expression and associated oxidase activity.Plasmids encoding Cas9 and gRNA were purchased from the Genomic Engineering Center at Washington University (St. Louis, MO). The mRNA encoding Cas9 and gRNA were synthesized at MaxCyte using mMESSAGE mMACHINE® T7 Ultra kit, (Ambion, Austin, TX). Patient EBV-transformed B cell line (B-LCL) and patient HSC were obtained under NIAID IRB approved protocol after patient written informed consent. The cells were cultured in RPMI-1640+10% FBS+2mM L-glutamine and StemSpan medium (Stem Cell Technologies, BC, Canada) supplemented with 100ng/ml SCF, Flit3L and TPO, respectively. We first optimized transfection conditions with the X-CGD patient B-LCL. It was found that B-LCL can be efficiently transfected with CRISPR and donor oligomer. Transfected B-LCL exhibit 80±6% viability, and maintained cell proliferation rate identical to control cells. Cel-1 assay shows 30-40% (N=8) efficiency of specific-site gene editing. Using donor oligomer containing a HindIII restriction site, it was determined that the DNA integration efficiency in Exon 7 was 35±8% (N=3). Using donor oligomer with the removal of HindIII restriction site, the expression of functional gp91 protein as determine by flow cytometry was 9±3% (N=3). These developed protocols were used to transfect HSC obtained from the same CGD patient (N=1). Following in vitro myeloid differentiation of gene mutation-repaired CGD HSC, 6.3% of the differentiating cells containing neutrophils and monocytes were oxidase positive in the dihydrorhodamine (DHR) flow cytometry oxidase function assay. These observations demonstrated restoration of phagocyte oxidase activity from the gene repair. Work is in progress to optimize and achieve clinical scale-up of the process which may have broad applicability to the treatment of monogenic diseases.

An AAVS1-Targeted Minigene Platform for Correction of iPSCs From All Five Types of Chronic Granulomatous Disease

There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.

Chronic Granulomatous Disease Carrier With Recurrent Poor Obstetric Outcome

Chronic granulomatous disease is a primary immunodeficiency disorder characterized by severe recurrent bacterial and fungal infections. Female carriers of the X-linked form of the disorder usually are unaffected and rarely have serious infections. A 22-year-old pregnant patient known to be a carrier of the X-linked form of chronic granulomatous disease had a history of chorioamnionitis during her two previous pregnancies. During her third pregnancy, she presented again with the same diagnosis, which resulted in delivery at 25 weeks of gestation. Carriers of chronic granulomatous disease should be monitored closely during pregnancy, as if they have the disease. To decrease the risk of infectious morbidity and mortality, obstetricians should have a low threshold for starting prophylactic antibiotics early during pregnancy, even if the patient is asymptomatic.

A Platform Minigene AAVS1 Targeted Safe Harbor Approach For Genetic Correction Of iPSC Derived From Patients With Each Of The 5 Genetic Forms Of Chronic Granulomatous Disease

There are 5 genetic forms of chronic granulomatous disease (CGD), resulting from mutations in one of the CYBB, CYBA, NCF1, NCF2, or NCF4 genes encoding, respectively, the subunits of the phagocyte oxidase (phox) gp91phox, p22phox, p47phox, p67phox and p40phox. Phox is required for blood neutrophils and monocytes to produce microbicidal superoxide and hydrogen peroxide. We previously demonstrated zinc finger nuclease (ZFN) targeting of a corrective minigene encoding gp91phox to the AAVS1 safe harbor site of iPSC from a patient with X-linked CGD (XCGD), which restored phox activity in neutrophils differentiated from the corrected iPSC (Blood 2011, 117:5561). Subsequently, we have generated iPSC from the CD34+ hematopoietic stem cells (HSC) present in small volumes of peripheral blood from additional CGD patients to include those representing the other genetic forms of CGD (Blood 2013, 121:e98). We used our previously reported gp91phox AAVS1 targeting plasmid to create CGD genotype-specific targeting plasmids for correction of the other 4 genetic forms of CGD by replacing gp91 cDNA with cDNA for each of the autosomal recessive CGD genes. Using these genotype specific targeting plasmids with AAVS1 targeting ZFNs, this approach resulted in safe harbor minigene correction of iPSC for each of the 5 genetic forms of CGD, routinely achieving minigene insertion with constitutive expression of the specific desired transgene phox subunit protein in 70-80% percent of selected iPSC clones. Neutrophils or macrophages were generated from iPSC by first differentiating in STEMdiff APEL medium for 13 days to generate CD34+CD45+ cells, which were further expanded in HSC medium for 7 days. The expanded cells were differentiated either to neutrophils with G-CSF in the presence or absence of OP9 mouse stromal cells for 7 days, or to macrophages with M-CSF for 10 to 14 days. Full restoration of oxidase activity was observed in neutrophils or macrophages derived from minigene corrected CGD iPSC representing all the 5 genetic types of CGD. Furthermore, we demonstrated significant correction of bacterial growth inhibition function in corrected CGD iPSC derived neutrophils (Staphylococci) and macrophages (Granulibacter). Of particular note, the M-CSF differentiated macrophages derived from iPSC showed a significant increase in IL-10 production (p < 0.01) in response to UV-inactivated Listeria, with comparable levels to M-CSF derived macrophages differentiated from peripheral blood monocytes. This type of iPSC gene-correction approach with ex vivo production of autologous functional neutrophils or macrophages could be used for supportive autologous cell therapy in CGD patients with infections not responding to conventional treatment. Using a universal platform approach that combines iPSC generation from small volumes of peripheral blood and ZFN mediated AAVS1 safe harbor minigene targeting, we show it is possible to efficiently go from peripheral blood sample to genetically corrected iPSC to generation of functionally corrected neutrophils and macrophages using an identical approach for all 5 genetic forms of CGD. This platform approach is broadly applicable to a wide range of inherited single gene metabolic disorders. Disclosures:No relevant conflicts of interest to declare.

A Rare Case of Malakoplakia Presenting as a Polypoid Lesion

Purpose: Malakoplakia, a rare form of chronic granulomatous disease, typically involves the genitourinary tract and is defined by its characteristic histopathological findings. It is usually associated with an immunocompromised state or with a concurrent chronic infection. The second most common location is the gastrointestinal tract and most colonic cases have been diagnosed with a concurrent adenocarcinoma. We report a case of a 64 y/o healthy African American male who was referred to our clinic for a screening colonoscopy. Four nodular appearing polyps, all less than 5 mm in size were found and excised using standard biopsy forceps. Histological examination of three of the four polyps was consistent with tubular adenomas. The fourth polyp, located in the ascending colon, had focal aggregates of granular histiocytes with targetoid Michaelis-Gutmann bodies consistent with Malakoplakia. This is a distinct case because it was diagnosed on endoscopic samples of typical appearing polypoid lesions, as prior described reports were found in surgical specimens. In addition, there was no evidence to suggest an immunocompromised state or infection. Furthermore, with the association regarding adenocarcinoma, a screening protocol may be warranted in these types of patients.Figure: No Caption available.

P47phox Directs Murine Macrophage Cell Fate Decisions

Macrophage differentiation and function are pivotal for cell survival from infection and involve the processing of microenvironmental signals that determine macrophage cell fate decisions to establish appropriate inflammatory balance. NADPH oxidase 2 (Nox2)-deficient chronic granulomatous disease (CGD) mice that lack the gp91(phox) (gp91(phox-/-)) catalytic subunit show high mortality rates compared with wild-type mice when challenged by infection with Listeria monocytogenes (Lm), whereas p47(phox)-deficient (p47(phox-/-)) CGD mice show survival rates that are similar to those of wild-type mice. We demonstrate that such survival results from a skewed macrophage differentiation program in p47(phox-/-) mice that favors the production of higher levels of alternatively activated macrophages (AAMacs) compared with levels of either wild-type or gp91(phox-/-) mice. Furthermore, the adoptive transfer of AAMacs from p47(phox-/-) mice can rescue gp91(phox-/-) mice during primary Lm infection. Key features of the protective function provided by p47(phox-/-) AAMacs against Lm infection are enhanced production of IL-1α and killing of Lm. Molecular analysis of this process indicates that p47(phox-/-) macrophages are hyperresponsive to IL-4 and show higher Stat6 phosphorylation levels and signaling coupled to downstream activation of AAMac transcripts in response to IL-4 stimulation. Notably, restoring p47(phox) protein expression levels reverts the p47(phox)-dependent AAMac phenotype. Our results indicate that p47(phox) is a previously unrecognized regulator for IL-4 signaling pathways that are important for macrophage cell fate choice.

NADPH oxidase complex and IBD candidate gene studies: identification of a rare variant in NCF2 that results in reduced binding to RAC2

ObjectiveThe NOX2 NADPH oxidase complex produces reactive oxygen species and plays a critical role in the killing of microbes by phagocytes. Genetic mutations in genes encoding components of the complex...

Genetic correction of X-linked chronic granulomatous disease with novel foamy virus vectors

The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the CYBB gene encoding gp91(phox), the larger subunit of the oxidase flavocytochrome b(558). Affected individuals suffer from recurrent life-threatening infections due to impaired superoxide production by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Novel foamy virus vectors expressing the human codon-optimized gp91(phox) were evaluated for the genetic correction of the disease in the X-CGD cell line and in X-CGD mouse model. The vectors were evaluated in vitro, in the human X-CGD PLB-985 cell line and in the X-CGD bone marrow Lin(-) cells. Transplantation of transduced Lin(-) cells was performed in X-CGD mice after busulfan conditioning. Real-time polymerase chain reaction was used for chimerism and vector copy number determination. Restoration of reduced NADPH oxidase production was assessed by nitrobluetetrazolium and dihydrorhodamine assays. High and stable gp91(phox) expression, as well as reconstitution of reduced NADPH activity, was achieved in the human X-CGD PLB-985 cell line and in primary murine X-CGD hematopoietic stem cells ex vivo. Transplantation of transduced bone marrow hematopoietic stem cells in the murine model of X-CGD, even with low multiplicities of infection (MOI), reconstituted the levels of oxidase-producing neutrophils and provided enzymatic activity that reached 70% of normal. Foamy virus vectors expressing the human gp91(phox) transgene constitute potential candidates for the gene therapy of CGD because they combine lack of pathogenicity with efficacy even at low MOI.

Intact indoleamine 2,3-dioxygenase activity in human chronic granulomatous disease

Chronic granulomatous disease (CGD) is characterized by a disability to produce reactive oxygen intermediates (ROI) caused by a defect of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. A hyperinflammatory response to immune activation has been reported to contribute to the pathology of CGD. The tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is considered critical for regulating immune responses and suppression of inflammation. IDO is generally believed to require ROI for enzymatic activity and was found to be inactive in murine CGD. Here, we report that, strikingly, in human CGD IDO metabolic activity is intact. Monocyte-derived dendritic cells generated from CGD patients, harbouring X-linked and autosomal recessive forms of CGD, and from healthy controls produced similar amounts of the tryptophan metabolite kynurenine upon activation with lipopolysaccharide and interferon-γ. Thus, in humans, ROI apparently are dispensable for IDO activity. Hyperinflammation in human CGD cannot be attributed to disabled IDO activation.

Signed outside: a surface marker system for transgenic cytoplasmic proteins

Chronic granulomatous disease is a primary immunodeficiency, comprising five molecular defects, characterized by an impaired respiratory burst activity of myeloid cells. We are currently developing a gene therapy vector for the p47phox-deficient form of chronic granulomatous disease. Classic intracellular immunostaining of the cytoplasmic p47phox transgene product, however, interferes with respiratory burst activity. In this study we report a new system for measuring p47phox expression: A single open reading frame encoding the surface marker protein ΔLNGFR (truncated low-affinity nerve growth factor receptor) linked to the p47phox transgene by the 2A oligopeptide coexpression technology. Translation generates two discrete products: p47phox localizing to the cytoplasm and 'ΔLNGFR-2A' localizing to the cell surface. Six weeks after transplantation of transduced autologous hematopoietic stem cells into p47-/- mice, the intracellular p47phox fluorescence-activated cell sorting (FACS) signal intensities corresponded to surface ΔLNGFR staining in monocytes, B cells, T cells and Sca I+ bone marrow cells in vivo. The p47phox cleavage product restored nicotinamide adenine dinucleotide phosphate-oxidase activity in granulocytes differentiated from transduced p47phox-/- murine hematopoietic stem cells ex vivo, in murine granulocytes/monocytes in vivo, and in transduced human monocyte derived macrophages from p47phox-deficient chronic granulomatous disease patients. In conclusion, this new marker system allows highly efficient, indirect detection of cytoplasmic transgene products by FACS surface staining.

Hematologically important mutations: The autosomal recessive forms of chronic granulomatous disease (second update)

Chronic granulomatous Disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by mutations in the genes encoding the components of the leukocyte NADPH oxidase. This enzyme produces superoxide, which is essential in the process of intracellular pathogen killing by phagocytic leukocytes. Four of the five genes involved in CGD are autosomal; these are CYBA, encoding p22- phox, NCF2, encoding p67- phox, NCF1, encoding p47-phox, and NCF4, encoding p40- phox. This article lists all mutations identified in these genes in the autosomal forms of CGD. Moreover, polymorphisms in these genes are also given, which should facilitate the recognition of future disease-causing mutations.