X-Linked chronic granulomatous disease: mutations in the CYBB gene encoding the gp91-phox component of respiratory-burst oxidase.

Chronic granulomatous disease (CGD) is a hereditary disorder of host defense due to absent or decreased activity of phagocyte NADPH oxidase. The X-linked form of the disease derives from defects in the CYBB gene, which encodes the 91-kD glycoprotein component (termed "gp91-phox") of the oxidase. We have identified the mutations in the CYBB gene responsible for X-linked CGD in 131 consecutive independent kindreds. Screening by SSCP analysis identified mutations in 124 of the kindreds, and sequencing of all exons and intron boundary regions revealed the other seven mutations. We detected 103 different specific mutations; no single mutation appeared in more than seven independent kindreds. The types of mutations included large and small deletions (11%), frameshifts (24%), nonsense mutations (23%), missense mutations (23%), splice-region mutations (17%), and regulatory-region mutations (2%). The distribution of mutations within the CYBB gene exhibited great heterogeneity, with no apparent mutational hot spots. Evaluation of 87 available mothers revealed X-linked carrier status in all but 10. The heterogeneity of mutations and the lack of any predominant genotype indicate that the disease represents many different mutational events, without a founder effect, as is expected for a disorder with a previously lethal phenotype.

Susceptibility of mice to bacterial and fungal infections after intragastric administration of ebselen.

The seleno-organic compound ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) has anti-inflammatory activity and exhibits glutathione peroxidase-like activity in-vitro. Ebselen inhibited candidacidal activity over the same range of concentrations as it inhibited the production of microbicidal H2O2 by human neutrophils and macrophage-like cells. Therefore, the long-term administration of ebselen might be expected to induce an immunocompromised state in the host. To examine such a possibility, mice (5-weeks-old ddY, male) were given daily intragastric doses of 0, 10 or 100 mg/kg-1 ebselen for 21 days and then infected intraperitoneally with Candida albicans (10(8) cells/mouse), Pseudomonas aeruginosa (1.5 x 10(7) cells/mouse) or methicillin-resistant Staphylococcus aureus (5 x 10(8) cells/mouse). Ebselen at none of the tested doses affected the increase in body weight of mice during administration of the drug. No evidence was obtained that mice became more susceptible to the various microorganisms after the administration of ebselen at any tested dose.

Appearance of nuclear factors that interact with genes for myeloid calcium binding proteins (MRP-8 and MRP-14) in differentiated HL-60 cells.

Myeloid calcium binding proteins MRP-8 and MRP-14 were induced, and their genes were coordinately expressed, during differentiation of human leukemia HL-60 cells into macrophage-like cells after treatment with 1,25-dihydroxyvitamin D3 (VD3). Both MRP-8 and MRP-14 mRNAs appeared on the day after VD3 treatment. Their level reached a peak on day 2, and then quickly declined. Nuclear factors that interact with the 5'-upstream regions of MRP-8 and MRP-14 genes were studied with gel mobility-shift assays. Two factors (MP8FI and MP8FII) that interacted with 379 bp (426-48 bp upstream from the transcription-initiation site of MRP-8 gene) and 67 bp (-47 - +20) DNA fragments, respectively, were found in the cells treated with VD3 for 1 day. MP8FI and MP8FII were present neither in the nuclei of untreated HL-60 cells, nor in the nuclei of the cells treated with VD3 for 6 days. Human monocytic leukemia THP-1 cells, which constitutively expressed MRP genes, had MP8FII but not MF8FI. MP8FII was found to interact with the 19-mer sequence located just upstream of the TATA box. Also, two factors that bound to the different upstream regions (-400 - -150 and -149 - +50) of MRP-14 gene were detected in the differentiated HL-60 cells. One of these, MP14FI, appeared on day 1, but on day 6 its concentration greatly decreased. The other, MP14FII, was found in greater quantity on day 6 than on day 1. MP14FI, but not MP14FII, was found in THP-1 cells. These factors may be involved in the expression of MRP-8 and MRP-14 genes in VD3-differentiated HL-60 cells.

Myeloid calcium binding proteins: expression in the differentiated HL-60 cells and detection in sera of patients with connective tissue diseases.

A differentiation antigen induced in 1,25-dihydroxyvitamin D3 (VD3)-treated HL-60 cells was identified as being comprised of the myeloid calcium binding proteins CaBP-p8 and -p14 by determining its amino acid and DNA sequence. Northern blot analysis using a DNA fragment of the gene encoding p14 as a probe indicated that the gene was not expressed in undifferentiated HL-60 cells but transcribed starting on day 1 after VD3 treatment. The level of p 14 mRNA reached a peak on day 2, then declined, and little mRNA remained on day 10, indicating that the p14 gene is activated once and then inactivated during HL-60 differentiation due to VD3. In contrast, thymidylate synthase (TSase) mRNA was present in undifferentiated HL-60 cells but disappeared quickly after VD3 treatment. Both p8 and p14 of CaBP were found at elevated levels in sera of some patients with connective tissue diseases [highly elevated in rheumatoid arthritis (RA), Sjogren's syndrome (SjS), systemic lupus erythematosus (SLE), and progressive systemic sclerosis (PSS), and moderately in polymyositis or dermatomyositis (PM/DM) and mixed connective tissue disease (MCTD)]. These results were in sharp contrast with the finding that p14 is always at a highly elevated level but little p8 is present in the sera of cystic fibrosis (CF) patients [Bruggen et al. (1988) Nature 331, 570).