Role of endogenous IFN-gamma in macrophage programming induced by IL-12 and IL-18.

Besides the established role of interleukin-12 (IL-12) and IL-18 on interferon-gamma (IFN-gamma) production by natural killer (NK), T, and B cells, the effects of these cytokines on macrophages are largely unknown. Here, we investigated the role of IL-12/IL-18 on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by CD11b(+) adherent peritoneal cells, focusing on the involvement of endogenously produced IFN-gamma. C57BL/6 cells released substantial amounts of NO when stimulated with IFN-gamma or lipopolysaccharide (LPS), but failed to respond to IL-12 or IL-18 or both. However, IL-12/IL-18 pretreatment was able to program these cells to release 6-8-fold more NO and TNF-alpha in response to LPS or Trypanosoma cruzi stimulation, with NO levels directly correlating with macrophage resistance to intracellular parasite growth. Analysis of IL-12/IL-18-primed cells from mice deficient in IFN-gamma, IFNGR, and IFN regulatory factor-1 (IRF-1) revealed that these molecules were essential for LPS-induced NO release, but TNF-alpha production was IFN-gamma independent. Conversely, the myeloid differentiation factor 88 (MyD88)-dependent pathway was indispensable for IL-12/IL-18-programmed LPS-induced TNF-alpha production, but not for NO release. Contaminant T and NK cells largely modulated the IL-12/IL-18 programming of LPS-induced NO response through IFN-gamma secretion. Nevertheless, a small population of IFN-gamma(+) cells with a macrophage phenotype was also identified, particularly in the peritoneum of chronically T. cruzi-infected mice, reinforcing the notion that macrophages can be an alternative source of IFN-gamma. Taken together, our data contribute to elucidate the molecular basis of the IL-12/IL-18 autocrine pathway of macrophage activation, showing that endogenous IFN-gamma plays an important role in programming the NO response, whereas the TNF-alpha response occurs through an IFN-gamma-independent pathway.     

Three distinct loci on human chromosome 21 contribute to interferon-α/β responsiveness

The species specificity of interferons (IFNs) depends on restricted recognition of these ligands by multisubunit cell surface receptors. Expression of the human receptor subunit IFNAR in mouse cells conferred sensitivity only to one subtype of human IFN, IFN-B. Other genes on human chromosome 21 were required for responses to other subtypes of type I IFN. In contrast, IFNAR expression in hamster cells did not confer sensitivity to any human IFN tested, including IFN-B. Using human-hamster somatic cell hybrids, we mapped theIfnabr gene, encoding a ligand-binding subunit of the IFN-/ (type I) receptor, to human chromosome 21.Ifnabr colocalized withIfnar to the distal region of q22.1. The presence of a chromosomal fragment encoding IFNABR and IFNAR was also not sufficient to confer sensitivity to human IFN. In contrast, hybrids carrying in addition the region 21q22.2 showed a full response to human IFN-B, suggesting that a gene located in this region encodes a third factor required for type I IFN receptor activity.     

Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.     

Microbial exposure of rural school children, as assessed by levels of N-acetyl-muramic acid in mattress dust, and its association with respiratory health

BACKGROUND: Endotoxin exposure has been shown to be associated with a decreased prevalence of atopic sensitization and symptoms. Yet endotoxin represents only a part of the indoor microbial exposure. Muramic acid, a constituent of peptidoglycan, is present in gram-negative and gram-positive bacteria in the environment and may therefore serve as an additional marker of microbial exposure. OBJECTIVE: To study the factors determining the level of indoor exposure to muramic acid/peptidoglycan, as well as its potential association with respiratory health. METHODS: In 553 farm and nonfarm school children from Austria, Switzerland, and Germany, mattress dust muramic acid concentrations were determined, and health was assessed by using IgE measurements and questionnaire information. RESULTS: The muramic acid concentration was found to be significantly higher in dust from farm children's mattresses than in dust from nonfarm children's mattresses (157 vs 131 ng/mg). Children with higher mattress dust muramic acid concentrations had a significantly lower prevalence of wheezing (odds ratio of highest vs lowest tertile of muramic acid concentration, 0.3; 95% CI, 0.1-0.9), regardless of farming status and endotoxin exposure. The association for asthma was similar, and no association was found with atopic sensitization. CONCLUSION: Next to endotoxin, muramic acid provides us with an independent marker of microbial exposure. Unlike endotoxin, muramic acid was inversely associated with wheezing rather than with atopic sensitization.     

Metabolic polymorphisms and the micronucleus frequency in buccal epithelium of adolescents living in an urban environment

Micronuclei and other biomarkers were evaluated in oral cells from 11- to 16-year-old girls living in a foster home in the central area of México City. Variables analyzed for possible association with these biomarkers include smoking habits, body mass index, metabolic polymorphisms for NAT1 and GSTM1 and whether the cells were obtained from the cheek or pharynx. The results indicated that individuals having the NAT1*10 homozygous genotype showed a significant increase in chromatin buds and binucleated cells. When the damage in the cheek was compared with damage in the pharynx, a significant increase in micronuclei and binucleated cells was found for the latter tissue in all the individuals analyzed.     

Morphological and cytochemical alterations of the Golgi apparatus and GERL in rat parotid acinar cells during ethionine intoxication and recovery

The present electron microscopic cytochemical investigation was undertaken to characterize the alterations in the Golgi apparatus and GERL of rat parotid acinar cells during ethionine intoxication and recovery. Although the Golgi apparatus and GERL were reduced in size, and some broadening of the Golgi saccules occurred as the result of ethionine treatment, the relative localization of thiamine pyrophosphatase (TPPase) activity in the Golgi saccules, and acid phosphatase activity (AcPase) in GERL, remained unchanged. Shortly after ethionine treatment was stopped, a dramatic redistribution of enzyme activities was noted. Within the first 24 hours of recovery, the Golgi apparatus began to enlarge, and the content of secretory granules increased. By day 3 of recovery, cisternae morphologically identifiable as GERL and forming secretory granules possessed TPPase activity, while AcPase activity was virtually undetectable. After seven days of recovery, the Golgi apparatus and GERL appeared both morphologically and cytochemically normal. The enzyme modulation observed during recovery may be correlated with increased secretory granule production. Furthermore, the presence of TPPase activity in GERL and forming secretory granules lends support to the suggestion that GERL may be derived from the trans Golgi saccule.     

Racial differences in the cause-specific prevalence of blindness in east Baltimore

BACKGROUND. Bilateral blindness unrelated to simple refractive error is twice as prevalent among blacks as among whites, although the difference narrows among the elderly. The reasons for this race- and age-related pattern are uncertain. METHODS AND RESULTS. A randomly selected, stratified, multistage cluster sample of 2395 blacks and 2913 whites 40 years of age and older in East Baltimore underwent detailed ophthalmic examinations by a single team. We identified 64 subjects who were blind in both eyes. The leading causes of blindness were unoperated senile cataract (accounting for blindness in 27 of the total of 128 eyes), primary open-angle glaucoma (17 eyes), and age-related macular degeneration (16 eyes). Together, these three disorders accounted for 47 percent of all blindness in this sample. Unoperated cataract accounted for 27 percent of all blindness among blacks, among whom it was four times more common than among whites; whites were almost 50 percent more likely than blacks to have undergone cataract extraction before the age of 80 (P less than 0.002). Primary open-angle glaucoma accounted for 19 percent of all blindness among blacks; it was six times as frequent among blacks as among whites and began 10 years earlier, on average. By contrast, age-related macular degeneration resulting in blindness was limited to whites, among whom it was the leading cause of blindness (prevalence, 2.7 per 1000; 95 percent confidence interval, 1.2 to 5.4); it affected 3 percent of all white subjects 80 years of age or older. CONCLUSIONS. The pattern of blindness in urban Baltimore appears to be different among blacks and whites. Whites are far more likely to have age-related macular degeneration, and blacks to have primary open-angle glaucoma. The high rate of unoperated cataracts among younger blacks and among elderly subjects of both races suggests that health services are underused. Half of all blindness in this urban population is probably preventable or reversible.