Nitrosative stress in the bronchial mucosa of severe chronic obstructive pulmonary disease

BACKGROUND: Reactive nitrogen species, formed via the reaction of nitric oxide (NO) with superoxide anion and via (myelo)peroxidase-dependent oxidation of NO(2)(-), have potent proinflammatory and oxidizing actions. Reactive nitrogen species formation and nitrosative stress are potentially involved in chronic obstructive pulmonary disease (COPD) pathogenesis. OBJECTIVES: To investigate the expression of markers of nitrosative stress, including nitrotyrosine (NT), inducible NO synthase (iNOS), endothelial NO synthase (eNOS), myeloperoxidase (MPO), and xanthine oxidase (XO) in bronchial biopsies and bronchoalveolar lavage from patients with mild to severe stable COPD compared with control groups (smokers with normal lung function and nonsmokers). METHODS: The expression of NT, iNOS, eNOS, MPO and XO in the bronchial mucosa and bronchoalveolar lavage of patients was measured by using immunohistochemistry, Western blotting, and ELISA and correlated with the inflammatory cell profile. RESULTS: Patients with severe COPD in stable phase had higher numbers of NT(+) and MPO(+) cells in their bronchial submucosa compared with mild/moderate COPD, smokers with normal lung function, and nonsmokers (P < .01). iNOS(+) and eNOS(+) but not XO(+) cells were significantly increased in smokers with COPD or normal lung function compared with nonsmokers (P < .05 and P < .01, respectively). In patients with COPD, the number of MPO(+) cells was significantly correlated with the number of neutrophils (r = +0.61; P < .0025) in the bronchial submucosa. Furthermore, the number of NT(+) and MPO(+) cells was negatively correlated with postbronchodilator FEV(1). CONCLUSION: These data suggest that nitrosative stress, mainly mediated by MPO and neutrophilic inflammation, may contribute to the pathogenesis of severe COPD.     

Effect of topical capsaicin on the cutaneous responses to inflammatory mediators and to antigen in man

Topical capsaicin pretreatment is known to deplete cutaneous sensory nerves of neuropeptides. We have assessed the effect of topical capsaicin pretreatment on the responses to intradermal injections of histamine and platelet-activating factor (PAF) in six normal subjects, and of prostaglandin E2, histamine, and antigen in 10 atopic subjects. Capsaicin pretreatment caused significant inhibition of the immediate flare response to histamine in both normal (19.8 +/- 2.6 to 7.3 +/- 2.9 cm2 at 5 minutes; p less than 0.01) and atopic subjects (16.5 +/- 1.4 to 10.3 +/- 1.9 cm2 at 5 minutes; p less than 0.01). The PAF-induced flare was also inhibited from 12.2 +/- 2.9 to 2.7 +/- 1.6 cm2 at 5 minutes after injection (p less than 0.01). In contrast, capsaicin pretreatment did not significantly alter the flare responses to prostaglandin E2 or antigen in atopic subjects. The acute wheal responses to all stimuli were unchanged, as was the late-phase response to antigen. These results support the hypothesis that the cutaneous vasodilator effect of histamine and PAF may be mediated by a local axon reflex involving the release of neuropeptides from sensory nerves. A consistent effect of capsaicin pretreatment on the flare response induced by endogenous mediators released during a cutaneous IgE-mediated response was not observed. Increases in microvascular permeability and the late-phase response to antigen are independent of neuropeptide release from cutaneous nerves.     

DLA-DRB1 polymorphisms in dogs defined by sequence-specific oligonucleotide probes (SSOP)

To date, DNA sequences for 29 dog DLA-DRB1 alleles have been reported. However, no data exists on the frequencies of these alleles within the general dog population, nor is there any indication of whether there is interbreed variation of allele distribution. We have addressed this by establishing a molecular based sequence-specific oligonucleotide probing (SSOP) method to identify all of the known broad DRB1 types and we have used this to type a random panel of dogs. A series of oligonucleotide probes were designed to detect known polymorphisms in the three DRB1 hypervariable regions, together with two distinctive motifs in other regions of exon 2. This set of probes enabled us to assign broad DRB1 types. Two hundred and eighteen dogs were SSOP typed for DRB1. All but 4 of the published DLA-DRB1 alleles were identified in these animals. Interbreed variation in both allele distributions and allele frequencies were observed, which may be useful in the study of genetic variation between breeds. This variation also has implications for the selection of control groups for studies aimed at identifying MHC associations with disease susceptibility in the dog.     

A design study for a 'spiral staircase' ionization chamber for the quality control of electron beams

In order to verify that the energies of electron beams used for external beam therapy remain constant, IPEM 81 recommends a constancy check based on the ratio of ionization chamber measurements at two depths along the central axis. Such measurements for a range of electron energies can be a time consuming process. The purpose of this study was to design a device that would use several ion chambers simultaneously to measure electron depth dose curves, and hence the electron energy. A design was developed for a device consisting of ten independent ionization chambers, shaped and arranged in a solid phantom like the steps of a spiral staircase, the axis of the staircase being coincident with the axis of the electron beam. Measurements were carried out to test the design of individual chambers and to optimize the radius of the spiral and both the depth intervals and the lateral spacing between adjacent chambers. For ranges of electron energy from 6-12 MeV and 12-20 MeV the radii of the spirals needed were found to be 36.5 mm and 30.9 mm, the angular separations between edges of the chambers were 52 degrees and 30 degrees and chamber depths were found to be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 mm and 20, 40, 45, 50, 55, 60, 65, 70, 75, 80 mm, respectively.     

Correlates of sensitization to Blomia tropicalis and Dermatophagoides pteronyssinus in asthma in Barbados

BACKGROUND: Sensitivity to the mite Blomia tropicalis is related to asthma in tropical climates, but correlates of sensitivity to B. tropicalis and its relationship to Dermatophagoides pteronyssinus sensitivity have not been widely examined in families with asthma. The main objective of this study was to determine prevalence and correlates of sensitivity to these mites in families with asthma and characteristics of persons sensitized to both. METHODS: Antibodies to major antigens (Blo t 5 and Der p 1) of these mites were measured by immunochemiluminescent assay in 481 members of 29 families from Barbados ascertained through two asthmatic siblings. RESULTS: Blo t 5 sensitivity was present in 261 subjects (46%) and was associated with younger age, higher total serum IgE level, and more than a three-fold increased prevalence of asthma (42 vs. 13%). Der p 1 sensitivity was less common (27%) and showed similar associations with age, IgE, and asthma. Of the 261 subjects sensitized to Blo t 5, 116 were also sensitized to Der p 1; they were younger, had higher total and Blo t 5 specific IgE levels, and had more than twice the asthma prevalence as those sensitized to Blo t 5 alone (59 vs. 29%). Der p 1 sensitivity without Blo t 5 sensitivity was uncommon; 90% of those sensitized to Der p 1 were also sensitized to Blo t 5. Geometric mean total IgE levels were lowest in the 207 participants without any mite sensitization (102 U/ml), intermediate in 158 sensitized to either Blo t 5 OR Der p 1 (609 U/ml), and highest in 116 sensitized to both (1,869 U/ml). CONCLUSIONS: Blo t 5 is the predominant sensitizing mite allergen in these Barbadian families with correlates similar to Der p 1. Concomitant sensitization to Der p 1 appears to identify a more reactive subgroup of individuals at a higher risk of asthma.     

Defective neurogenesis resulting from DNA ligase IV deficiency requires Atm

Ataxia telangiectasia results from mutations of ATM and is characterized by severe neurodegeneration and defective responses to DNA damage. Inactivation of certain DNA repair genes such as DNA ligase IV results in massive neuronal apoptosis and embryonic lethality in the mouse, indicating the occurrence of endogenously formed DNA double-strand breaks during nervous system development. Here we report that Atm is required for apoptosis in all areas of the DNA ligase IV-deficient developing nervous system. However, Atm deficiency failed to rescue deficits in immune differentiation in DNA ligase IV-null mice. These data indicate that ATM responds to endogenous DNA lesions and functions during development to eliminate neural cells that have incurred genomic damage. Therefore, ATM could be important for preventing accumulation of DNA-damaged cells in the nervous system that might eventually lead to the neurodegeneration observed in ataxia telangiectasia.     

Inhibition of inducible nitric oxide synthase expression by interleukin-4 and interleukin-13 in human lung epithelial cells.

Oral feeding of proteins causes peripheral T-cell tolerance, as revealed by reduced delayed-type hypersensitivity (DTH) reactivity after immunization. This type of tolerance can be due both to passive T-cell anergy and active immunosuppression. Using ovalbumin-fed mice we studied whether putatively immunostimulatory cytokines could break this state of mucosal tolerance. Cytokines were administered locally at the site of attempted sensitization. It was found that neither interleukin-2 (IL-2), interferon-gamma (IFN-gamma) nor granulocyte-macrophage colony-stimulating factor (GM-CSF) could restore the response to immunization. In contrast, local administration of IL-12 at the site of attempted immunization resulted in full recovery of DTH reactivity. The dichotomy between the two Th1 stimulatory cytokines IFN-gamma and IL-12 was also reflected by different effects on ovalbumin-specific antibody isotypes. Although both IFN-gamma and IL-12 downregulated serum IgG1-levels in tolerant mice, suggesting decreased ovalbumin-specific Th2 function, only local administration of IL-12 led to increased serum IgG2a levels. These results support the view that potentiation of Th1 effector function is critical for reversal of mucosal tolerance.     

Expression of a human cDNA encoding a protein containing GAR synthetase,AIR synthetase,and GAR transformylase corrects the defects in mutant Chinese hamster ovary cells lacking these activities

The isolation of a human cDNA encoding the multifunctional protein containing GAR synthetase, AIR synthetase, and GAR transformylase by functional complementation of purine auxotrophy in yeast has been reported. Chinese hamster ovary (CHO) cell mutant purine auxotrophs deficient in GAR synthetase (Ade^–C) or AIR synthetase plus GAR transformylase (Ade^–G) activities were transfected with this human GART cDNA subcloned into a mammalian expression vector. This restored 49–140% of the activities of GAR synthetase, AIR synthetase, and GAR transformylase in transfected cells when compared to wild-type CHO K1 parental cells. Study of one stably expressing transfectant, AdeC2, revealed that the human GART cDNA was incorporated into the CHO genome. The enzyme activities appear to be associated with an expressed protein of 110 kDa, very similar to that of purified human GART trifunctional enzyme. The Ade^–C mutant shows reduced amounts of GART mRNA compared to CHO K1 and a protein of apparently reduced size, results consistent with the purine requirement and enzyme deficiency observed in the mutant. These experiments provide definitive evidence that the human GART cDNA encodes and can direct the production of active human GART trifunctional protein in mammalian cells. They also provide important evidence that the Ade^–C and Ade^–G mutants of CHO cells are defective in this gene.     

Characterization of vancomycin-resistant Enterococcus faecium isolates from broiler poultry and pig farms in England and Wales

This study aimed to investigate the occurrence and molecular epidemiology of vancomycin-resistant Enterococcus faecium (VREF) isolates on poultry and pig farms in England and Wales. A total of 217 VREF isolates were obtained from fresh feces and environmental swabs collected from conventional and organic farms. A predominant pulsed-field gel electrophoresis (PFGE) profile was found for each VREF-positive farm, together with less frequent types. All isolates presented the vanA genotype and were esp negative. Seventy-six percent of the VREF isolates were additionally resistant to nine or more antimicrobials, presenting a diverse range of resistance phenotypes. The multiresistance traits did not appear to be specific to individual farms or sample types (i.e., environmental or fecal), nor did they correlate with any specific PFGE type. Ninety-three percent of the isolates were resistant to penicillin, 89% were resistant to tetracycline, 87.5% were resistant to erythromycin, and 50% were resistant to quinupristin-dalfospristin (Synercid). The lack of clonality among these populations may suggest the horizontal transfer of resistance genes and/or a dynamic replacement of clonal lines rather than persistence.