The phagocyte NADPH oxidase (NOX2) is critical for the bactericidal activity of phagocytic cells and plays a major role in innate immunity. We showed recently that NOX2 activity in mouse dendritic cells (DCs) prevents acidification of phagosomes, promoting antigen cross-presentation. In order to investigate the role of NOX2 in the regulation of the phagosomal pH in human DCs, we analyzed the production of reactive oxygen species (ROS) and the phagosomal/endosomal pH in monocytederived DCs and macrophages (MØs) from healthy donors or patients with chronic granulomatous disease (CGD). As expected, we found that human MØs acidify their phagosomes more efficiently than human DCs. Accordingly, the expression of the vacuolar proton ATPase (V-H ؉ -ATPase) was higher in MØs than in DCs. Phagosomal ROS production, however, was also higher in MØs than in DCs, due to higher levels of gp91phox expression and recruitment to phagosomes. In contrast, in the absence of active NOX2, the phagosomal and endosomal pH decreased. Both in the presence of a NOX2 inhibitor and in DCs derived from patients with CGD, the cross-presentation of 2 model tumor antigens was impaired. We conclude that NOX2 activity participates in the regulation of the phagosomal and endosomal pH in human DCs, and is required for efficient antigen crosspresentation. (Blood. 2008;112:4712-4722)

Mycobacterial disease is relatively common in patients with CGD living in countries in which tuberculosis is endemic, BCG vaccine is mandatory, or both. Adverse reactions to BCG and severe forms of tuberculosis should lead to a suspicion of CGD. BCG vaccine is contraindicated in patients with CGD.

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40 phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40 phox -deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMAinduced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40 phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD. phox -deficient patient is compound heterozygous for a premature stop codon and a missense mutation in the PX domain that compromises binding to PI(3)P, which results in the impairment of neutrophil phagocytosis-induced oxidase activity (26). As in classic CGD neutrophils, intracellular oxidant production after stimulation with serum-opsonized zymosan (SOZ), IgG beads, or serumopsonized Staphylococcus aureus is impaired in the patient's neutrophils, and S. aureus killing is also defective (20,26,30). However, in this patient, unlike in classic CGD patients, the production of O 2 -by neutrophils in response to stimulation with PMA or formyl-methionyl-leucyl-phenylalanine (fMLF) is normal (26). The killing of S. aureus by neutrophils is also impaired in p40 phox deficiency and classic CGD are largely unknown. Here, we describe the characteristics of 24 patients from 12 families in 8 countries with biallelic mutations of NCF4. The Journal of Clinical Investigation R E S E A R C H A R T I C L E

Background Germline heterozygous mutations in human STAT1 can cause loss of function (LOF), as in patients with Mendelian susceptibility to mycobacterial diseases (MSMD), or gain of function (GOF), as in patients with chronic mucocutaneous candidiasis (CMC). LOF and GOF mutations are equally rare and can affect the same domains of STAT1, especially the coiled-coil and DNA-binding domains (CCD/DBD). Moreover, 6% of CMC patients with a GOF STAT1 mutation develop mycobacterial disease, obscuring the functional significance of the identified STAT1 mutations. Current computational approaches, such as combined annotation-dependent depletion, do not distinguish LOF and GOF variants Objective Estimate variations in CCD/DBD of STAT1 Method Mutagenized 342 individual wild-type amino acids in CCD/DBD (45.6% of full-length STAT1) to alanine and tested the mutants for STAT1 transcriptional activity. Results Of these 342 mutants, 201 were neutral, 30 LOF, and 111 GOF in a luciferase assay. This assay system correctly estimated all previously reported LOF mutations (100%) and slightly fewer GOF mutations (78.1%) in CCD/DBD of STAT1. We found that GOF alanine mutants occurred at the interface of the antiparallel STAT1 dimer, suggesting that they destabilize this dimer. This assay also precisely predicted the impact of two hypomorphic and dominant-negative mutations, E157K and G250E, in CCD of STAT1 that we found in two unrelated MSMD patients. Conclusion Systematic alanine-scanning assay is a useful tool to estimate the GOF or LOF status and impact of heterozygous missense mutations in STAT1 identified in patients with severe infectious diseases, including mycobacterial and fungal diseases.

Herein, we describe a combination of clinical, microbiologic, and histopathologic findings significantly associated with osteomyelitis in chronic granulomatous disease. When present, these features should raise the suspicion of underlying chronic granulomatous disease. In patients with these findings, anti-infective prophylactic measures aiming to cover highly prevalent microorganisms, as well as aggressive therapeutic measures, should be strongly encouraged.