Model Of Chronic Granulomatous Disease Research Articles

Virulence and Cellular Interactions ofBurkholderia multivoransin Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens, but not with Escherichia coli. In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1beta. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.

Is TNFα a Potential Target for the Treatment of Colitis in Chronic Granulomatous Disease?

Background: Chronic granulomatous disease (CGD) is an immmunodeficiency and hyperinflammation disorder. Sterile hyperinflammation in the colon, CGD colitis, contributes significantly to the morbidity of patients. Presently available treatment options for CGD colitis include corticosteroids and interferon γ. Overshooting tumor necrosis factor α (TNFα) production in CGD phagocytes has been proposed as a mechanism contributing to hyperinflammation. In this study, we have investigated whether a deletion or suppression of TNFα resulted in a diminution of the hyperinflammation in a murine CGD model. Methods: For genetic deletion experiments, wild-type (N = 6), CGD (=Nox 2 [gp91phox]-deficient, N = 6), and CGD/TNFα–deficient mice (N = 6) were injected intradermally with β-glucan to induce hyperinflammation. For pharmacological experiments, CGD mice were treated with the TNFα scavenger sTNFR2-Fc (Ethanercept) before β-glucan injection. Seven days later, the severity of inflammation was assessed on hematoxylin and eosin–stained slides of ear sections by a histological score (from 0 to 4, with 4 being the most severe inflammation). Results: The β-glucan generated a mild inflammatory score in the wild-type mice (mean score 1.5 ± 0.5), whereas CGD mice showed severe inflammation (mean 3.5 ± 0.2). Genetic deletion or pharmacological inhibition of TNFα did not decrease the severity of inflammation in CGD mice: histological scores for CGD/TNFα-deficient mice and for sTNFR2-Fc-treated CGD mice were 3.7 ± 0.3, and 3.7 ± 0.4, respectively. In both TNFα suppressed mice, a hyperinflammatory syndrome associated with necrotic lesions was observed. Conclusions: Neither genetic deletion of TNFα nor treatment with a TNFα scavenger prevented skin hyperinflammation in CGD mice. Thus, unlike as previously suggested, TNFα probably does not represent a potential target for the treatment of CGD hyperinflammation. However, our results were obtained in skin hyperinflammation and future research will have to investigate the similarities and differences between hyperinflammation in the skin and the colon. The authors report no conflicts of interest.

Nonresolving Inflammation in gp91phox−/− Mice, a Model of Human Chronic Granulomatous Disease, Has Lower Adenosine and Cyclic Adenosine 5′-Monophosphate

In chronic granulomatous disease (CGD), there is failure to generate reactive oxygen metabolites, resulting in recurrent infections and persistent inflammatory events. Because responses to sterile stimuli in murine models of CGD also result in nonresolving inflammation, we investigated whether defects in endogenous counterregulatory mechanisms and/or proresolution pathways contribute to the etiology of CGD. To this end, we conducted a series of experiments finding, in the first instance that adenosine and cAMP, which dampen innate immune-mediated responses, show a biphasic profile in resolving peritonitis; peaking at onset, waning as inflammation progresses, and rising again at resolution. We also found elevations in adenosine and cAMP in resolving human peritonitis. In gp91(phox-/-) mice, an experimental model of CGD, levels of adenosine and cAMP were significantly lower at onset and again at resolution. Corroborating the finding of others, we show that adenosine, signaling through its A(2A) receptor and therefore elevating cAMP, is not only anti-inflammatory, but, importantly, it does not impair proresolution pathways, properties typical of nonsteroidal anti-inflammatory drugs. Conversely, antagonizing the A(2A) receptor worsens acute inflammation and prolongs resolution. Taking this further, activating the A(2A) receptor in gp91(phox-/-) mice was dramatically anti-inflammatory regardless of the phase the inflammatory response A(2A) agonists were administered, i.e., onset or resolution, demonstrating wide and robust pharmacological flexibility that is unlikely to subvert proresolution pathways. Therefore, we describe the biphasic profile of adenosine and cAMP throughout the time course of acute inflammation that is dysregulated in CGD.

Aspergillus fumigatus: Principles of Pathogenesis and Host Defense

Aspergillus fumigatus is a ubiquitous saprophytic mold (67) that forms airborne spores (conidia). Humans inhale, on average, hundreds of these infectious propagules daily. In immune competent hosts, these encounters are of no further significance—conidia are killed and cleared by cells of the pulmonary immune system. However, disease occurs when the host response is either too strong or too weak. Thus, understanding how the host interacts with the organism to define this balance is a critical goal, the successful pursuit of which requires recognition of the dynamic nature of both fungal and host molecular participants. In immunocompromised hosts, A. fumigatus represents a major cause of morbidity and mortality. This patient population is expanding due to the increasing use of transplantation for end organ disease, the development of immunosuppressive and myeloablative therapies for autoimmune and neoplastic disease, and the human immunodeficiency virus/AIDS pandemic (38). A. fumigatus is the most common invasive mold infection in these patients, and mortality rates exceed 50% in high-risk groups, such as leukemic patients and hematopoietic stem cell transplant recipients (74). Sensitivity to A. fumigatus antigens is associated with asthma, the prevalence of which is increasing in the developed world, though proving causation has been difficult (49, 54). Regardless, this increased prevalence brings a parallel rise in the number of individuals predisposed to allergic bronchopulmonary aspergillosis, a disease associated with aberrant responses to Aspergillus antigens in the setting of chronic inflammation. The spectrum of invasive, semi-invasive, and allergic disease caused by A. fumigatus is reviewed in several outstanding articles (9, 94). The study of A. fumigatus molecules involved in virulence has been hampered by the lack of an identifiable sexual cycle, limiting classical genetic analysis (21). A recent study, however, indicates that A. fumigatus encodes distinct mating-type loci and the pheromone machinery required for sexual mating (103). Nonetheless, within the past decade, researchers have developed and refined experimental tools to generate mutant strains by homologous recombination (21, 35, 62, 152, 154), utilized RNA interference to repress endogenous transcripts (95), and expressed heterologous genes in A. fumigatus under the control of drug-inducible regulatory elements (145). The completion of the A. fumigatus genome (98) has accelerated gene structure and function studies and made possible comparative genomic analyses with other sequenced Aspergillus species (Aspergillus oryzae and Aspergillus nidulans), as well as other genera of pathogenic (e.g., Candida albicans and Cryptococcus neoformans) and nonpathogenic (e.g., Saccharomyces cerevisiae) fungi. An important insight from the genomes has been that A. fumigatus does not share a common set of genes with other fungal pathogens (98). The types of hosts that are susceptible to invasive aspergillosis and the lack of unique pathways conserved among pathogenic fungi underscore the importance of the host contribution to pathogenesis. Damage from A. fumigatus can result from fungal growth and tissue invasion or from inflammatory cells recruited to sites of infection (130). Included in the latter are responses that are ineffective in clearing the organism, occur in the process of immune reconstitution, or are associated with allergy. For example, in a murine model of chronic granulomatous disease, in which mice have defective phagocyte oxidase systems, administration of killed hyphae results in chronic inflammation due to persistence of fungal elements (92). From the perspective of the mammalian immune system, A. fumigatus represents an organism with continuous respiratory tract exposure that must be cleared from terminal airways with an immune response calibrated to avoid fungal tissue invasion, as well as inflammation-induced tissue damage. Here, we review our growing understanding of the interface between A. fumigatus and host defense mechanisms, with an emphasis on invasive disease in humans and small animal models.

Gamma-glutamylcysteine synthetase-based selection strategy for gene therapy of chronic granulomatous disease and graft-vs.-host disease

Efficient ex vivo/in vivo selection of genetically modified hematopoietic stem/progenitor cells (HPCs) and T lymphocytes could greatly improve several gene therapy strategies. We have previously reported that primary murine HPCs, transduced with a bicistronic retroviral vector, co-expressing the catalytic subunit of gamma-glutamylcysteine synthetase (gamma-GCSh) and eGFP, could be selected by l-buthionine-S,R-sulfoximine (BSO). Upon ex vivo transduction with a low, defined gene dosage and BSO selection, HPCs were able to repopulate the bone marrow of syngeneic myeloablated hosts, showing multi-lineage expression [Hum Gene Ther, 16 (2005), 711]. We now provide 'proof-of-principle' that the same strategy can be applied to the gene therapy of graft-vs.-host disease (GVHD) subsequent to allogeneic bone marrow transplantation (ABMT), and of chromosome X-associated chronic granulomatous disease (CGD). Transfer of the herpes simplex virus-thymidine kinase (HSV-Tk) 'suicide' gene into donor T lymphocytes is a potential method to control GVHD after ABMT. However, an efficient selection system is required to eliminate non-HSV-Tk-expressing T lymphocytes before administration to the patient. We now report that, upon transduction with a retroviral vector, co-expressing gamma-GCSh and eGFP, and subsequent selection by BSO, over 95% human T lymphocytes were found to express eGFP; moreover, upon transduction with a novel retroviral vector co-expressing gamma-GCSh and HSV-Tk, and subsequent BSO treatment, over 95% of T lymphocytes could be eliminated by ganciclovir. The efficacy of the gamma-GCSh-BSO selection strategy was then tested on an in vitro model of CGD. Upon transduction of gp91 (phox)-deficient PLBKO cells with a novel bicistronic retroviral vector co-expressing human gp91 (phox) and gamma-GCSh, exposure to BSO for 48 h eliminated most non-transduced cells, resulting in selection of gp91 (phox)-expressing cells, and reconstitution of NADPH oxidase activity.

Acid aspiration-induced lung inflammation and injury are exacerbated in NADPH oxidase-deficient mice

Increased reactive oxidant intermediates (ROIs) from primed leukocytes have been implicated in the pathogenesis of acid aspiration lung injury. To evaluate the specific role of the phagocyte NADPH oxidase-derived ROIs in acid lung injury, the p47phox-/- knockout mouse model of chronic granulomatous disease was used. p47phox-/- mice developed a significantly greater alveolar neutrophilic leukocytosis compared with wild-type mice at all time points after acid injury, with the difference between genotypes being most marked at 48 h. In contrast, the p47phox-/- mice had a decreased number of macrophages in bronchoalveolar lavage (BAL) compared with wild-type at 48 h after acid or saline aspiration. Albumin concentration in BAL reflecting capillary leak was also greater in p47phox-/- compared with wild-type mice. BAL concentrations of proinflammatory cytokines and chemokines were greater in p47phox-/- compared with wild-type mice. These findings suggest that NADPH oxidase, directly or indirectly, plays a role in attenuating the acute neutrophilic response after acid lung injury. We speculate that this downmodulating effect may be mediated by promoting the transition from production of cytokines and chemokines involved in neutrophilic infiltration to a less injurious, chronic inflammatory response.

Invasive infection with Trichosporon inkin in 2 siblings with chronic granulomatous disease

A 9-year-old girl with autosomal recessive chronic granulomatous disease (CGD) presented with asymptomatic bilateral pulmonary infiltrates on routine computed tomography. Fine-needle aspirate of the infiltrates was obtained and showed fungal cells resembling Trichosporon inkin . The specimen grew in culture, and testing by means of both API 20C and PCR amplification confirmed the diagnosis of T inkin . The infiltrates increased in size, despite sequential therapy with voriconazole, liposomal amphotericin B, caspofungin, and posaconazole. The patient required resection of the infected lung tissue, after which she recovered completely. While she was undergoing therapy, her 13-year-old brother, also with CGD, was given a diagnosis of bilateral T inkin -induced pulmonary infection. He also required bilateral pulmonary resection for cure. These cases demonstrate the predisposition of patients with CGD to have invasive infections with unusual fungal organisms, such as T inkin . They also illustrate the difficulty of treating invasive T inkin infections with antifungal agents alone. There are 9 previously reported cases of invasive infections caused by T inkin , 3 of which are in patients with CGD. All patients required removal of infected prosthetic devices or surgical resection of infected tissue for cure.

Catalase negative Staphylococcus aureus retain virulence in mouse model of chronic granulomatous disease

Myeloperoxidase-mediated chlorination is thought to be a necessary microbicidal mechanism. The H 2O 2 required for this process is generated by the NADPH oxidase. Staphylococcus aureus can also produce H 2O 2, which is not broken down by catalase negative organisms. It has been thought that this bacterial H 2O 2 can substitute for cellular H 2O 2 in the halogenation reaction in chronic granulomatous disease (CGD) where neutrophils are lacking the NADPH oxidase. We have readdressed this issue in a mouse model of CGD using clinical isolates of catalase positive and negative strains of S. aureus. The results showed these organisms to be equally virulent and that the H 2O 2 they produced is insufficient to cause significant iodination, a marker for chlorination, thereby contradicting the accepted views on this subject.

Enhanced Cutaneous Inflammatory Reactions to Aspergillus fumigatus in a Murine Model of Chronic Granulomatous Disease

Chronic granulomatous disease is the manifestation of genetic defects of the leukocyte NADPH oxidase resulting in the absence of a respiratory burst. Patients with chronic granulomatous disease can develop chronic granulomas in many locations of the body, including the skin. Using an established murine model of X-linked chronic granulomatous disease (X-CGD) created by homologous recombinant disruption of the gene encoding the gp91phox component of the NADPH oxidase, in this study we examined cutaneous reactivity to sterile Aspergillus fumigatus hyphae. Injection of Aspergillus fumigatus into the dorsal ears of X-CGD mice resulted in an enhanced inflammatory response by 24 h, consisting of neutrophils, which developed into suppurative granulomas by 10 d. Intradermal injection of Aspergillus fumigatus into wild-type mice only resulted in a transient inflammatory response that resolved by 10 d. Injection of Aspergillus fumigatus into female carrier mice resulted in an acute inflammatory response that was similar to that of wild-type mice, but, at higher doses of Aspergillus fumigatus, many carriers subsequently developed granulomatous lesions that were qualitatively similar but smaller than those seen in X-CGD mice by 30 d. Consistent with the ability of X-CGD mice to mount an enhanced neutrophil-rich inflammatory response to Aspergillus fumigatus, significant levels of the potent neutrophil activator/chemoattractant leukotriene B4 were measured by mass spectrometry in skin biopsies at 24 and 72 h. In contrast to the exaggerated inflammatory response to intradermal Aspergillus fumigatus in X-CGD mice compared to their wild-type counterparts, similar levels of inflammation were seen in a model of delayed-type hypersensitivity using 2,4-dinitrofluorobenzene. This study represents the first report of a cutaneous granuloma model in mice with X-CGD, which may also prove useful as a functional test to evaluate the efficacy of gene therapy protocols being developed for chronic granulomatous disease.

Thioglycollate peritonitis in mice lacking C5, 5-lipoxygenase, or p47phox: complement, leukotrienes, and reactive oxidants in acute inflammation

Leukotriene B(4) (LTB(4)) is an easily diffusible proinflammatory chemotactic factor that has been posited to prime the initial inflammatory response for the action of other mediators, including C5a. 5-Lipoxygenase-deficient (5LX(-/-)) and C5-deficient mice only generated about 50% as much peritoneal leukocytosis as wild-type mice following intraperitoneal (IP) challenge with the sterile irritant, thioglycollate (P<0.005). Pretreatment of C5- mice with the specific 5-lipoxygenase inhibitor, zileuton, reduced peritoneal leukocytosis to almost unstimulated levels, suggesting that LTB(4) can act independently of C5a. Previously, LTB(4) and C5a have been shown in vitro to be inactivated by metabolites of superoxide. In the current study, we examined the fate of LTB(4) in the p47(phox-/-) mouse model of chronic granulomatous disease (CGD) in which the phagocyte NADPH oxidase is unable to produce superoxide. p47(phox-/-) mice generated more thioglycollate-elicited peritoneal leukocytosis than wild-type mice. Pretreatment with zileuton caused a 76% reduction in peritoneal leukocytosis in p47(phox-/-) mice (P<0.005) and a 54% reduction in wild-type mice (P<0.05), whereas pretreatment with dexamethasone or toradol (a cyclooxygenase inhibitor) had no effect. Following IP LTB(4) (1 microg/mouse), total recovered peritoneal LTB(4) was similar between p47(phox-/-) and wild-type mice at 10 and 30 min, but was approximately fivefold greater in p47(phox-/-) mice at 180 min. These data suggest that LTB(4) and C5a have separate but overlapping roles in thioglycollate-elicited peritonitis, and at least the leukotriene component is, in turn, regulated by reactive oxidants.

Ym1 Is a Neutrophil Granule Protein That Crystallizes in p47phox-deficient Mice

Crystals were discovered within the aged lung and at sites of chronic inflammation in a mouse model of chronic granulomatous disease. Following re-crystallization at neutral pH, the crystals were identified as the chitinase-like protein Ym1, expressed in organs of the lymphoreticular system, the lung, and distal stomach. Ym1 was shown to be a neutrophil granule protein and to have weak beta-N-acetylglucosaminidase activity, indicating that it might contribute to the digestion of glycosaminoglycans. Crystal formation is likely to be a function of excess neutrophil turnover at sites of inflammation in the chronic granulomatous disease mouse. Failure to remove subcutaneous Ym1 crystals injected into knockout mice indicates that a failure of digestion may also contribute to crystallization.

Impaired superoxide production due to a deficiency in phagocyte NADPH oxidase fails to inhibit atherosclerosis in mice.

Superoxide, the reduced form of molecular oxygen, has been implicated in the genesis of vascular disease. One potential mechanism involves oxidation of low density lipoprotein into an atherogenic particle. A second involves reaction with nitric oxide to generate peroxynitrite, a highly oxidizing intermediate. A third involves regulation of signal transduction in artery wall cells. One well-characterized pathway for superoxide production resides in macrophages, the cellular hallmark of the early atherosclerotic lesion. Macrophages contain a membrane-bound NADPH oxidase that reduces oxygen to superoxide. In the current studies, we used mice that are deficient in the gp91-phox subunit of the NADPH oxidase-a model of chronic granulomatous disease (CGD)-to explore the role of superoxide in atherosclerotic vascular disease. Wild-type and CGD mice on the C57BL/6 background received a high-fat diet for 20 weeks to induce hypercholesterolemia. At the end of this period, the 2 strains of mice had comparable plasma lipid levels, and their atherosclerotic lesions were similar in size. We also crossed CGD mice with apolipoprotein E-deficient (apoE-/-) mice to generate spontaneously hypercholesterolemic animals that lacked functional NADPH oxidase. After 24 weeks, the CGD-apoE-/- animals had lower plasma cholesterol and triglyceride levels than did the apoE-/- animals, but there was no difference in the extent of atherosclerotic plaque. Our findings suggest that superoxide generated by the NADPH oxidase of phagocytes does not promote atherosclerosis in mice with either diet-induced or genetic forms of hypercholesterolemia.

Virulence of catalase-deficient aspergillus nidulans in p47(phox)-/- mice. Implications for fungal pathogenicity and host defense in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare genetic disorder in which phagocytes fail to produce superoxide because of defects in one of several components of the NADPH oxidase complex. As a result, patients develop recurrent life-threatening bacterial and fungal infections. The organisms to which CGD patients are most susceptible produce catalase, regarded as an important factor for microbial pathogenicity in CGD. To test the role of pathogen-derived catalase in CGD directly, we have generated isogenic strains of Aspergillus nidulans in which one or both of the catalase genes (catA and catB), have been deleted. We hypothesized that catalase negative mutants would be less virulent than the wild-type strain in experimental animal models. CGD mice were produced by disruption of the p47(phox) gene which encodes the 47-kD subunit of the NADPH oxidase. Wild-type A. nidulans inoculated intranasally caused fatal infection in CGD mice, but did not cause disease in wild-type littermates. Surprisingly, wild-type A. nidulans and the catA, catB, and catA/catB mutants were equally virulent in CGD mice. Histopathological studies of fatally infected CGD mice showed widely distributed lesions in the lungs regardless of the presence or absence of the catA and catB genes. Similar to the CGD model, catalase-deficient A. nidulans was highly virulent in cortisone-treated BALB/c mice. Taken together, these results indicate that catalases do not play a significant role in pathogenicity of A. nidulans in p47(phox)-/- mice, and therefore raise doubt about the central role of catalases as a fungal virulence factor in CGD.

A Bicistronic Retrovirus Vector Containing a Picornavirus Internal Ribosome Entry Site Allows for Correction of X-Linked CGD by Selection for MDR1 Expression

Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections. The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo. Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low. In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production. We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES). As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element. In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor). Using a chemiluminescence assay of O2-. production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity. With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2-. production equalling or exceeding that of normal EBV-B cells. These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene. Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.

A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression

Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections. The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo. Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low. In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production. We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES). As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element. In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor). Using a chemiluminescence assay of O2-. production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity. With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2-. production equalling or exceeding that of normal EBV-B cells. These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene. Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.

The p47phox mouse knock-out model of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is caused by a congenital defect in phagocyte reduced nicotinamide dinucleotide phosphate (NADPH) oxidase production of superoxide and related species. It is characterized by recurrent life-threatening bacterial and fungal infections and tissue granuloma formation. We have created a mouse model of CGD by targeted disruption of p47phox, one of the genes in which mutations cause human CGD. Identical to the case in human CGD, leukocytes from p47phox-/- mice produced no superoxide and killed staphylococci ineffectively. p47phox-/- mice developed lethal infections and granulomatous inflammation similar to those encountered in human CGD patients. This model mirrors human CGD and confirms a critical role for the phagocyte NADPH oxidase in mammalian host defense.