Chronic Granulomatous Disease Defect Research Articles

High Titer Lentivector from a Stable Lenti-Producer Efficiently Corrects CD34+ Hematopoietic Stem Cells from Patients with p47phox-Deficient Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to mutations in the genes for the NADPH subunits (the genes for p47phox, p22phox, p67phox, p40phox autosomal chronic granulomatous disease), or gp91phox (X-linked chronic granulomatous disease). This results in a failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs), the major defect in chronic granulomatous disease causing recurrent infections and granulomatous complications. Hematopoietic stem cell transplantation (HSCT) with a suitable donor is potentially curative. However, in the absence of HLA-matched donor, gene therapy using autologous gene-corrected HSC offers potential for significant clinical benefit. To date, despite myeloid conditioning, gene therapy for CGD patients using gamma-retroviral vectors have achieved either minimal long-term gene marking and engraftment, or has been associated with insertional mutagenesis. In contrast, lentivector-mediated gene therapy has successfully treated patients with Wiskott-Aldrich syndrome and Metachromatic Leukodystrophy without any dysregulated clonal expansion.We used a lentivector construct which incorporates an MND internal promoter, a modified self-inactivating MoMuLV LTR U3 region with myeloproliferative sarcoma virus enhancer, and a 650bp single chicken b-globin insulator encoding codon-optimized p47phox gene. Mutations in p47phox accounts for the majority of AR-CGD. The production of large-scale, consistently-high-titer lentivector using a transient 4-plasmid transfection system however, is labor- and cost-prohibitive. To address this, we applied concatemeric array transfection of pCL20cW650 MND-p47-OPT into a stable packaging cell line (GPRTG) for HIV-based lentiviral vectors to create a stable producer of VSV-G pseudotyped pCL20cW650 MND-p47OP. The concatemer array of HIV lentiviral vector construct and bleomycin selectable gene cassette showed 10 copies of lentiviral vector in a stable producer line, capable of producing vector at 10^7 IU/ml.Hematopoietic CD34+ stem cells from p47phox- CGD were transduced with pCL20cW650 MND-p47-OPT vector (MOI 10) with 2 overnight transductions following 24 hours pre-activation with SCF, FLT-3L and TPO (100ng/ml). Following three weeks in vitro culture, non-transduced or transduced p47 CGD HSC versus normal HSC were 0%, 42% and 20% p47phox positive, respectively. To determine functional correction, PMA stimulated oxidant production was measured using the dihydrorhodamine assay, confirmation similar levels of oxidant generation in transduced patient cells compared with normal controls. More than 90% of CFU were vector positive, indicating a high level of gene marking. Transduced and control naïve p47phox-patient CD34+ HSC were transplanted into 20 immunodeficient Nodscid-gc deficient (NSG) mice, and at 13 weeks post-transplant the CD13+ human neutrophils arising in mouse bone marrow were assessed for p47phox expression. Over 40% CD13+ neutrophils expressed p47phox protein from NSG mice transplanted with transduced p47-patient CD34 HSC, compared with 74% or 0% in mice transplanted with normal CD34 or p47 patient naive CD34 cells respectively. Detailed histopathology of each transplanted mice confirmed the absence of vector insertion-related myeloid tumors, and deep sequencing of bone marrow CD45+ human cells from each mouse also demonstrated polyclonal distribution of vector integration sites. In conclusion, we provide preclinical data demonstrating the efficacy and safety of high titer VSVg-pseudotyped lentivector (CL20cW650 MND-p47-OPT) generated by our stable GPTRG p47 lenti-producer for correction of p47phox-deficient human CD34 HSC. DisclosuresNo relevant conflicts of interest to declare.

298. Transfection of Neutrophils from X-Linked Chronic Granulomatous Disease Patients with gp91phox mRNA Restores Oxidase Activity with High Efficiency and Viability

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to defective phagocyte NADPH oxidase required for production of antimicrobial oxidants. Fungal or bacterial infections in CGD may persist despite intensive and prolonged antibiotic therapy. In settings where conventional antibiotic therapy and/or surgical debridement do not control infection, use of allogeneic unmatched donor granulocyte transfusions has been therapeutic. However, CGD patients treated with granulocyte transfusions may develop high titers of anti-HLA antibodies, restricting use of allogeneic granulocyte transfusions and further complicating potentially curative bone marrow transplant. Using the CGD patient as an autologous donor for granulocyte transfusions would be a helpful solution.Here we demonstrate highly efficient functional correction of the functional defect in CGD by transfection of patient's own granulocytes with mRNA while retaining viability using a scalable, cGMP-compliant MaxCyte® GT™ electroporation system capable of handling clinically relevant numbers of cells (>1010cells).Freshly collected granulocytes from normal volunteers were electroporated using the MaxCyte electroporation system with eGFP-mRNA on the day of collection. Flow cytometry of demonstrated that >90% of neutrophils expressed eGFP with viability exceeding 90% at 24 hrs after electroporation. Phagocyte NADPH oxidase activity of the normal neutrophils was retained as well as bactericidal activity against Burkolderia cepacia, a CGD bacterial pathogen. Antibacterial activity of electroporated granulocytes was comparable to unmanipulated granulocytes confirming retention of cellular function following the electroporation. Finally, electroporated eGFP positive human neutrophils injected into CGD mice were shown to remain in circulation as well as migrate to the peritoneal cavity following thioglycollate IP injection and there were no adverse effects observed in the mice.Electroporation of gp91phox mRNA into human X-linked CGD (gp91phox-deficient) patient neutrophils resulted in expression of gp91phox protein in >70% of cells with >90% viability at 24 hours after treatment. Using the dihydrorhodamine assay of phagocyte NADPH oxidase activity, >70% of neutrophils remained functionally corrected 2 days after transfection. Other studies of cellular function in vitro and in vivo with electroporated corrected CGD neutrophils are in progress. In summary, we show the feasibility of restoring the function of CGD patient-autologous neutrophils via electroporation of mRNA encoding the defective gene product. This approach may have potential clinical utility in the management of severe chronic bacterial and fungal infections in CGD but also short-term correction of other hematopoietic gene diseases.

Influence of Neutrophil Defects on Burkholderia cepacia Complex Pathogenesis

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacteria that are ubiquitous in the environment and have emerged as opportunistic pathogens in immunocompromised patients. The primary patient populations infected with Bcc include individuals with cystic fibrosis (CF), as well as those with chronic granulomatous disease (CGD). While Bcc infection in CF is better characterized than in CGD, these two genetic diseases are not obviously similar and it is currently unknown if there is any commonality in host immune defects that is responsible for the susceptibility to Bcc. CF is caused by mutations in the CF transmembrane conductance regulator, resulting in manifestations in various organ systems, however the major cause of morbidity and mortality is currently due to bacterial respiratory infections. CGD, on the other hand, is a genetic disorder that is caused by defects in phagocyte NADPH oxidase. Because of the defect in CGD, phagocytes in these patients are unable to produce reactive oxygen species, which results in increased susceptibility to bacterial and fungal infections. Despite this significant defect in microbial clearance, the spectrum of pathogens frequently implicated in infections in CGD is relatively narrow and includes some bacterial species that are considered almost pathognomonic for this disorder. Very little is known about the cause of the specific susceptibility to Bcc over other potential pathogens more prevalent in the environment, and a better understanding of specific mechanisms required for bacterial virulence has become a high priority. This review will summarize both the current knowledge and future directions related to Bcc virulence in immunocompromised individuals with a focus on the roles of bacterial factors and neutrophil defects in pathogenesis.

Residual NADPH Oxidase and Survival in Chronic Granulomatous Disease

Failure to generate phagocyte-derived superoxide and related reactive oxygen intermediates (ROIs) is the major defect in chronic granulomatous disease, causing recurrent infections and granulomatous complications. Chronic granulomatous disease is caused by missense, nonsense, frameshift, splice, or deletion mutations in the genes for p22(phox), p40(phox), p47(phox), p67(phox) (autosomal chronic granulomatous disease), or gp91(phox) (X-linked chronic granulomatous disease), which result in variable production of neutrophil-derived ROIs. We hypothesized that residual ROI production might be linked to survival in patients with chronic granulomatous disease. We assessed the risks of illness and death among 287 patients with chronic granulomatous disease from 244 kindreds. Residual ROI production was measured with the use of superoxide-dependent ferricytochrome c reduction and flow cytometry with dihydrorhodamine oxidation assays. Expression of NADPH oxidase component protein was detected by means of immunoblotting, and the affected genes were sequenced to identify causal mutations. Survival of patients with chronic granulomatous disease was strongly associated with residual ROI production as a continuous variable, independently of the specific gene affected. Patients with mutations in p47(phox) and most missense mutations in gp91(phox) (with the exception of missense mutations in the nucleotide-binding and heme-binding domains) had more residual ROI production than patients with nonsense, frameshift, splice, or deletion mutations in gp91(phox). After adolescence, mortality curves diverged according to the extent of residual ROI production. Patients with chronic granulomatous disease and modest residual production of ROI have significantly less severe illness and a greater likelihood of long-term survival than patients with little residual ROI production. The production of residual ROI is predicted by the specific NADPH oxidase mutation, regardless of the specific gene affected, and it is a predictor of survival in patients with chronic granulomatous disease. (Funded by the National Institutes of Health.).

Genetic Analysis of 10 Unrelated Korean Families with p22-phox-deficient Chronic Granulomatous Disease: An Unusually Identical Mutation of the CYBA Gene on Jeju Island, Korea

Chronic granulomatous disease (CGD) is a rare hereditary disorder characterized by recurrent life-threatening bacterial and fungal infections. The underlying defect in CGD is an inability of phagocytes to produce reactive oxygen species as a result of defects in NADPH oxidase. Considering that CGD generally affects about 3-4 in 1,000,000 individuals, it is surprising that the prevalence of CGD on Jeju Island is 20.7 in 1,000,000 individuals. We performed genetic analysis on 12 patients from 10 unrelated families and found that all patients had an identical homozygous single-base substitution of C to T in exon 1 (c.7C>T) of the CYBA gene, which was expected to result in a nonsense mutation (p.Q3X). Because Jeju Island has long been a geologically isolated region, the high prevalence of CGD on Jeju Island is presumably associated with an identical mutation inherited from a common ancestor or proband.

Drug-selected complete restoration of superoxide generation in Epstein-Barr virus-transformed B cells from p47phox-deficient chronic granulomatous disease patients by using a bicistronic retrovirus vector encoding a human multi-drug resistance gene (MDR1) and the p47phox gene.

Chronic granulomatous disease (CGD) is a group of disorders characterized by the failure of phagocytes to produce superoxide. One-third of the cases of CGD in the USA and Europe results from defects in the gene encoding p47phox, a cytoplasmic component of NADPH oxidase for superoxide generation. In this study, we constructed the bicistronic retrovirus vector Ha-MDR-IRES-p47, which carries cDNAs for a human multi-drug-resistance gene (MDR1) and p47phox. The amphotropic retroviral producer cells were generated, and the supernatant of the producer cells was used to transduce Epstein-Barr virus-transformed B (EBV-B) cells, established from B cells of p47phox-deficient CGD patients, as an in vitro model of gene therapy for p47phox-deficient CGD. The transduced cells expressed both P-glycoprotein and p47phox protein, and the expression levels were increased after appropriate vincristine selection. The levels of superoxide production in the vincristine-selected cells were increased to a level similar to normal EBV-B cells. This result suggests that it is possible to achieve 100% correction of the CGD defect in p47phox-deficient EBV-B cells by using the bicistronic vector. This strategy could be employed not only in vitro, but also in vivo, in the gene therapy of a number of inherited diseases.

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.

CD34+ Peripheral Blood Progenitors as a Target for Genetic Correction of the Two Flavocytochrome b558 Defective Forms of Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) can result from any of four single gene defects involving components of the superoxide (O2-.)-generating phagocyte NADPH oxidase (phox). The phox transmembrane flavocytochrome b558 is composed of two peptides, gp91phox and p22phox. Mutations of gp91phox cause X-linked CGD, whereas mutations of p22phox cause one of the three autosomal recessive forms of CGD. We used the Maloney leukemia virus-based MFG retrovirus vector to produce replication defective retroviruses encoding gp91phox or p22phox. To maximize viral titer MFG retroviruses do not contain internal promoter or resistance elements. Epstein-Barr virus transformed B-lymphocyte cell lines (EBV-B) derived from normal individuals contain phox components and produce O2-., whereas those derived from CGD patients show the CGD defect. Transduction of gp91phox or p22phox-deficient CGD EBV-B lines resulted in correction of O2-. production from a barely detectable baseline to an average 7.2% and 13.8% of normal control, respectively, without any selective regimen to enrich for transduced cells. CD34+ hematopoietic progenitor cells, the therapeutic target for gene therapy of CGD, were isolated from peripheral blood of CGD patients, transduced with MFG-phox retroviruses, and differentiated in culture to mature phagocytes. Transduction of progenitors corrected the gp91phox (seven patients) and p22phox (two patients) CGD phagocyte oxidase defect to 2.5% and 4.9% of normal O2-. production, respectively, representing an 87-fold and 161-fold increase. These studies show correction of flavocytochrome b558-deficient CGD in primary hematopoietic progenitors, providing a basis for development of gene therapy for the X-linked gp91phox and autosomal p22phox-deficient forms of CGD.

CD34+ peripheral blood progenitors as a target for genetic correction of the two flavocytochrome b558 defective forms of chronic granulomatous disease

Chronic granulomatous disease (CGD) can result from any of four single gene defects involving components of the superoxide (O2-.)-generating phagocyte NADPH oxidase (phox). The phox transmembrane flavocytochrome b558 is composed of two peptides, gp91phox and p22phox. Mutations of gp91phox cause X-linked CGD, whereas mutations of p22phox cause one of the three autosomal recessive forms of CGD. We used the Maloney leukemia virus-based MFG retrovirus vector to produce replication defective retroviruses encoding gp91phox or p22phox. To maximize viral titer MFG retroviruses do not contain internal promoter or resistance elements. Epstein-Barr virus transformed B-lymphocyte cell lines (EBV- B) derived from normal individuals contain phox components and produce O2-., whereas those derived from CGD patients show the CGD defect. Transduction of gp91phox or p22phox-deficient CGD EBV-B lines resulted in correction of O2-. production from a barely detectable baseline to an average 7.2% and 13.8% of normal control, respectively, without any selective regimen to enrich for transduced cells. CD34+ hematopoietic progenitor cells, the therapeutic target for gene therapy of CGD, were isolated from peripheral blood of CGD patients, transduced with MFG- phox retroviruses, and differentiated in culture to mature phagocytes. Transduction of progenitors corrected the gp91phox (seven patients) and p22phox (two patients) CGD phagocyte oxidase defect to 2.5% and 4.9% of normal O2-. production, respectively, representing an 87-fold and 161- fold increase. These studies show correction of flavocytochrome b558- deficient CGD in primary hematopoietic progenitors, providing a basis for development of gene therapy for the X-linked gp91phox and autosomal p22phox-deficient forms of CGD.

Clonal growth of functionally normal and deficient neutrophils from the bone marrow of a patient with variant chronic granulomatous disease

To evaluate the effect of colony-stimulating factors and interferon gamma on the oxidative burst capacity of neutrophils in chronic granulomatous disease (CGD) we studied the neutrophils of a patient with variant CGD both from peripheral blood and from bone marrow culture on day 7 and 14. The results revealed that preincubation of peripheral neutrophils for 24 h in medium containing recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), and recombinant human interferon gamma (rhIFN-gamma) alone or in combination did not improve the maximal oxidative burst activity measured by MTT assay. The colonies of this patient formed in agar assay were either composed of predominantly nitroblue tetrazolium (NBT)-positive cells or completely unable to reduce NBT. Despite variable colony numbers in the presence of different cytokines, the rate of NBT-positive colonies was less than 17% of the total number of colonies. However, more than 72% of the colonies were NBT positive in controls. In liquid culture, bone marrow cells yielded a comparable rate of NBT-positive and -negative populations at day 7. These data indicate that rhG-CSF, rhGM-CSF, and rhIFN-gamma alone or rhG-CSF and rhGM-CSF in combination with rhIFN-gamma are not able to reconstitute the oxidative burst defect in CGD in vitro. Indeed, regarding colony-forming capacity, the bone marrow cells from the patient responded to CSFs as well as those from control donors did. This fact may warrant the administration of hematopoietic growth factors, at least in variant CGD, in order to enhance the absolute number of functionally normal neutrophils.

Human granulocytes lack red cell Kx antigen.

We have investigated the presence or absence of the red cell Kx antigen on human granulocytes by measuring specific uptake of anti-Kx using three techniques: direct measurement by 125I-staphylococcal protein A (125I-SPA) and avidin-biotin-complex (ABC) immunoperoxidase staining and also, an indirect measurement using granulocyte adsorption of anti-Kx. Our results with all three methods indicate that the Kx antigen is not present on normal human granulocytes. Prior to adsorption of the anti-Kx serum with purified, pooled, normal human granulocytes, 11 of 21 (53%) of normal granulocytes were non-reactive by 125I-SPA and 16 of 20 (80%) by ABC. This pattern of reactivity was shown to be due to contamination of our anti-Kx serum with an antibody to a granulocyte-specific antigen unrelated to the Kx antigen. After adsorption, there was no diminution in the reactivity of the adsorbed anti-Kx compared to the unadsorbed antiserum against red cells which express strong Kx antigen, i.e. Ko and DTT-modified normal human red cells, by either serologic or 125I-SPA techniques. Likewise, reactivity with McLeod red cells, which have weak expression of the Kx antigen, was not changed using either the unadsorbed or adsorbed anti-Kx. The adsorbed anti-Kx was nonreactive with all 12 normal donors' granulocytes tested by 125I-SPA and with 10 normal donors' granulocytes tested by ABC. Furthermore, granulocytes from a Ko individual were nonreactive using either unadsorbed or adsorbed anti-Kx. These studies indicate that Kx antigen is not present on normal human granulocytes. Further, additional adsorption studies using granulocytes from a boy with X-linked chronic granulomatous disease (CGD) indicated that these granulocytes also do not possess the Kx antigen. In contrast to previous reports, these data suggest that Kx antigen is most probably a red cell-specific antigen and that the red cell Kx antigen has no direct relationship to the biochemical defect in CGD.

Absence of cytochrome b-245 in chronic granulomatous disease. A multicenter European evaluation of its incidence and relevance.

The heme-containing protein cytochrome b-245 has been proposed as a primary component of the microbicidal oxidase system of phagocytes that normally generates superoxide-free radicals but when defective is associated with chronic granulomatous disease. We measured this cytochrome in granulocytes from 27 patients with chronic granulomatous disease and from 64 members of their families. It was undetectable in all 19 of the men in whom the defect appeared to be located on the X chromosome. Female relatives who were heterozygous carriers had reduced concentrations of the cytochrome and variable proportions of cells that were unable to generate superoxide; these two characteristics were closely related (r = 0.93 in the 16 mothers and 0.85 in all 24 carriers, P less than 0.001). In contrast, in all eight patients (seven women) with a probable autosomal recessive inheritance the cytochrome was present but nonfunctional. The properties tested, including midpoint potential, carbon monoxide binding, and organelle distribution, were normal, but the cytochrome did not undergo reduction on cellular stimulation. Thus, absence or malfunction of the cytochrome b-245 may be the causal molecular defect in chronic granulomatous disease, implicating it in the microbicidal oxidase system.

Allosteric transformation of reduced nicotinamide adenine dinucleotide (phosphate) oxidase induced by phagocytosis in human polymorphonuclear leukocytes.

We used sensitive isotopic and fluorometric assay procedures to investigate reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]oxidation in a particulate fraction derived from normal and chronic granulomatous disease leukocytes. Granules isolated from normal resting cells showed allosteric kinetics with regard to oxidation of either NADH or NADPH, so that no enzyme activity was observed at physiological concentrations of substrate. If the granules were isolated from cells that had previously phagocytized zymosan, normal hyperbolic kinetics were obtained, so that activity could now be observed at low levels of substrate. The activity towards NADPH was always substantially greater than that towards NADH at any given concentration of substrate. This alteration in kinetics with phagocytosis was not observed with the other granule enzymes, acid phosphatase or beta-glucuronidase, and thus appeared to be specific for the reduced pyridine nucleotide oxidase(s). In contrast, granules isolated from cells of patients with chronic granulomatous disease showed allosteric kinetics regardless of whether they were obtained from resting or phagocytizing cells, so that NADPH oxidation was not measurable at physiological concentrations of substrate. This defect in the oxidation of NADPH by granules isolated from phagocytizing chronic granulomatous disease cells was observed over the pH range of 4.0 to 7.0. These data suggest that initiation of the respiratory burst by pahgocytosis normally requires an allosteric transformation in a reduced pyridine nucleotide oxidase, which in turn allows expression of enzymatic activity at physiological concentrations of substrate. The defect in chronic granulomatous disease appears to lie in an inability to achieve this transformation, and the enzyme remains in the inactive, allosteric form.

Metabolism of Thyroxine and Triiodothyronine in Patients with Chronic Granulomatous Disease

Normal human leukocytes can degrade thyroid hormones in vitro especially during phagocytosis. To test whether the leukocyte system plays a significant role in hormone economy in vivo, studies of the metabolism of labeled T4 and T3 granulomatous disease, whose leukocytes had earlier been shown to have a reduced ability to degrade the thyroid hormones. Hormone turnover rates in the 3 subjects were similar to those reported for normal children by various investigarors. Also, normal ratios between fecal and urinary excretion of radioactivey were observed, suggested that deiodination was the major route of hormone breakdown, as it is in normal subjects. Our interpretations of these findings are: 1) an intact leukocyte hormone degrading system is not essential for normal rates of thyroid hormone metabolism in vivo; 2) the leukocyte defect in chronic granulomatous disease is not accompanied by a general defect in mechanism for thyroid hormone degradation in other tissues.

TUBULAR RETICULAR STRUCTURES IN PERIPHERAL MONONUCLEAR CELLS OF MALES WITH CHRONIC GRANULOMATOUS DISEASE, THEIR MOTHERS, AND SISTER CARRIERS

Chronic granulomatous disease (CGD) is an intrinsic defect of phagocytic cells resulting in decreased capacity to kill certain bacteria and is a consistent clinical syndrome. Defective phagocytosis is present in a milder form in female carriers who are, in general, asymptomatic. Tubular reticular structures (TRS) have been demonstrated in peripheral lymphocytes and monocytes in patients with discoid lupus erythematosus (DLE). Recently we became aware of 4 boys with CGD who had mothers with DLE. Therefore, we examined the peripheral mononuclear cells by electron microscopy from a series of males with CGD, their carrier sisters, and their mothers, both with and without DLE, to ascertain the significance of TRS in these two disease These studies suggest that patients with CGD and DLE share an abnormality, which may be an additional abnormality to the established phagocytic defect in CGD. The CGD carrier state may predispose to DLE. Further investigation of phagocytosis in patients with DLE and electron microscopic examination of peripheral mononuclear cells in CGD should be performed.

Role of peroxide in phagocytic killing of pneumococci.

Two mutants of a pneumococcus type I with diminished peroxide production were selected from a population of nitrosoguanidine-treated cells. White cells of normal patients killed the mutant pneumococci as well as the otherwise isogenic wild-type strain. In patients studied with chronic granulomatous disease, however, the peroxide-poor strain was killed far less well than the wild type. These studies indicate that the removal of a peroxide-generating system in the phagocytic vacuole specifically brings forth the killing defect in chronic granulomatous disease.