CGD is an immunodeficiency caused by deletions or mutations in genes that encode subunits of the leukocyte NADPH oxidase complex. Normally, assembly of the NADPH oxidase complex in phagosomes of certain phagocytic cells leads to a "respiratory burst", essential for the clearance of phagocytosed micro-organisms. CGD patients lack this mechanism, which leads to life-threatening infections and granuloma formation. However, a clear picture of the clinical course of CGD is hampered by its low prevalence (approximately 1:250,000). Therefore, extensive clinical data from 429 European patients were collected and analyzed. Of these patients 351 were males and 78 were females. X-linked (XL) CGD (gp91(phox) deficient) accounted for 67% of the cases, autosomal recessive (AR) inheritance for 33%. AR-CGD was diagnosed later in life, and the mean survival time was significantly better in AR patients (49.6 years) than in XL CGD (37.8 years), suggesting a milder disease course in AR patients. The disease manifested itself most frequently in the lungs (66% of patients), skin (53%), lymph nodes (50%), gastrointestinal tract (48%) and liver (32%). The most frequently cultured micro-organisms per episode were Staphylococcus aureus (30%), Aspergillus spp. (26%), and Salmonella spp. (16%). Surprisingly, Pseudomonas spp. (2%) and Burkholderia cepacia (<1%) were found only sporadically. Lesions induced by inoculation with BCG occurred in 8% of the patients. Only 71% of the patients received antibiotic maintenance therapy, and 53% antifungal prophylaxis. 33% were treated with gamma-interferon. 24 patients (6%) had received a stem cell transplantation. The most prominent reason of death was pneumonia and pulmonary abscess (18/84 cases), septicemia (16/84) and brain abscess (4/84). These data provide further insight in the clinical course of CGD in Europe and hopefully can help to increase awareness and optimize the treatment of these patients.

During the international placebo-controlled trial on the efficacy of interferon-y (IFN-y) in chronic granulornatous disease (CGD), 19 patients entered the study via our Institute. One patient stopped treatment shortly thereafter. RNA was purified from the mono- nuclear cells ofthe remaining 18 CGD patients before and during this placebo-controlled trial. The mRNA levels for the NADPH oxidase components were sub- sequently analyzed. Compared with the placebo-treated CGD patients, the mRNA levels for p47-phox were sig- nificantly increased in the IFN-'y-treated CGD patients (P < 0.002). No significant changes were observed in the mRNA levels of the other oxidase components. These findings are in agreement with observations in vitro and indicate that IFN-y is active on the NADPH oxidase in vivo as well. However, it remains question- able whether these effects in vivo can explain the ob- served reduction of infections in these patients. J. Leukoc. Biol. 60: 716-720; 1996.

In the X-linked form of chronic granulomatous disease (X91-degrees CGD), the genetic defect is linked to the CYBB locus on the X chromosome. We studied a family with a genetic defect in this gene, consisting of a G-->A substitution at the fifth base of the 5' donor splice site of intron 3. This mutation leads to skipping of exon 3 after transcription of the gene. The expectant mother was diagnosed as a carrier. Analysis of polymerase chain reaction (PCR)-amplified genomic DNA from a chorionic villus biopsy (CVB) showed the same mutation in the male fetus. After termination of the pregnancy, the diagnosis was confirmed by conventional methods. This is the first time that PCR has been used for prenatal diagnosis of CGD.

Monoclonal antibodies (MoAbs) were raised against cytochrome b558, a membrane-bound component of the NADPH:O2 oxidoreductase in human neutrophils. This cytochrome consists of a low-molecular-weight (low- mol-wt) subunit of 22 to 23 Kd, probably encoded by an autosomal gene, and a high-mol-wt subunit of 75 to 90 Kd, encoded on the X-chromosome. MoAb 449 reacts with the low-mol-wt subunit and MoAb 48 with the high- mol-wt subunit on Western blots of purified cytochrome b558 and on blots of whole neutrophil extracts. In extracts of neutrophils from patients with chronic granulomatous disease (CGD) in which cytochrome b558 is not detectable by spectrophotometric methods, the low-mol-wt subunit is present, albeit in a much smaller amount. The high-mol-wt subunit is not detected by MoAb 48 in neutrophils of patients with X- linked CGD and in neutrophils of patients with the autosomal, cytochrome-b558-negative form of the disease. These results can be explained by a marked instability of these subunits when the synthesis of either of the two is disturbed. In differentiated HL-60 cells, the high-mol-wt subunit appears to be present in a different form. Cloning of the low-mol-wt subunit with the help of MoAb 449 suggests the presence of a heme-binding site on this subunit. By comparison of the binding characteristics of MoAb 449 to intact and permeabilized neutrophils with those of MoAb 7D5, recently isolated by Nakamura et al (Blood 69:1404, 1987), the low-mol-wt subunit was established as a transmembrane protein.

The NADPH:O2 oxidoreductase (NADPH oxidase) of human neutrophils is converted from a dormant to an active state upon stimulation of the cells. We have studied the soluble fraction that is required for NADPH oxidase activation in a cell-free system. Human neutrophils were separated in a membrane-containing and a soluble fraction. The soluble fraction was separated on carboxymethyl (CM) Sepharose in 10 mM 4-morpholino-ethanesulfonic acid buffer of pH 6.8. Reconstitution of the NADPH oxidase activity, measured as O2 consumption, was only found when the membrane fraction was combined with the flowthrough of the CM Sepharose column as well as with a fraction that eluted at 125 mM NaCl. This result indicates that at least two soluble components are necessary for reconstitution of the NADPH oxidase activity: one that does not bind to CM Sepharose and one that does bind. These components were designated soluble oxidase component (SOC) I and SOC II, respectively. Boiling destroyed the activity in both fractions. In the soluble fraction of human lymphocytes and thrombocytes neither SOC I nor SOC II activity was found. SOC II copurified with a 47-kD phosphoprotein, previously found defective in patients with the autosomal form of chronic granulomatous disease (CGD). Inactive soluble fractions of cells from autosomal CGD patients were reconstituted with a SOC II fraction from control cells. The result of this experiment indicates that autosomal CGD patients are normal in SOC I but defective in SOC II.

The results of a quantitative spectrophotometric enzyme assay, a fluorescent spot test and a cytochemical assay for glucose-6-phosphate dehydrogenase deficiency were compared systematically. The high sensitivity of the spectrophotometric assay and the fluorescent spot test in the detection of severely deficient individuals was confirmed. For the detection of heterozygote females, however both tests were unreliable; the sensitivities of the fluorescent spot test and the spectrophotometric assay being 32% and 11% respectively. Specificities for both tests were high (99%). Introduction of the ratio of glucose-6-phosphate dehydrogenase and pyruvate kinase (G-6-PD/PK ratio) activities increased the sensitivity of the spectrophotometric assay to nearly 100%. It is concluded that the fluorescent spot test should be used for the diagnosis of G-6-PD deficiency in developing countries; whereas if spectrophotometric enzyme assays are available, the G-6-PD/PK ratio should always be performed. In cases where the ratio is less than 0.70, cytochemical analysis is indicated.

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A B S T R A C T We have tried to elucidate the mechanism ofphagosome acidification in human neutrophils. Assuming that phenomena occurring at the plasma membrane reflect reactions in the phagocytic vacuoles, we have stimulated human neutrophils with agents that induce a respiratory burst, and we have measured the release of protons into the extracellular medium. Phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine and serum-opsonized zymosan particles each caused a rapid release ofprotons, concomitant with the increase in oxygen consumption. The stimulated release of protons was strictly coupled to the increased respiration of the cells, because inhibition of the respiration by either anaerobiosis, chlorpromazine, or glycolytic inhibitors also inhibited the release of protons. Also, in the presence of the above-mentioned stimulating agents, neutrophils from three patients with chronic granulomatous disease enhanced neither respiration nor proton release. In normal cells, the ratio of AH+/-AO2 was 1.04+0.19

The production of hydrogen peroxide in phagccytosing leukocytes is essential for the killing of microorganisms. Glutathione is thought to be involved in two ways in this process: 1) in the detoxification of the excess of H2O2; 2) as hydrogen donor in the production of H2O2. We were able to study the actual role of the glutathione redox system in the first known family with a real glutathione reductase deficiency in all blood cells. We found that in contrast to normal cells, the oxygen consumption and H2O2 generation abruptly stopped 5-10 minutes after the onset of phagocytosis. This effect was probably due to cell injury since the patients' granulocytes and monocytes, after pre-incubation with an H2O2-generating system, did not react any longer with phagocytosable particles. The generation of superoxide radicals (O−2), intermediates in the formation of H2O2, was normal in the patients' granulocytes, indicating that glutothione is not involved in the production of these compounds. Most likely, the cytochrome c, used for the detection of O−2, prevented efficient generation of H2O2, thus protecting the cells against its harmful effects. The lack of recurrent infections in these patients and the normal in vitro phagocytosis and killing of Stophylococcus aureus indicate that the short respiratory burst suffices for effective host defense. Moreover, the occurrence of hemolytic crises after eating of fava beans and the development of cataracts in this family indicate that glutathione fulfils an essential role in the protection of many different cells against oxidative stress.