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.     

A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes

CD8(hi+) cytotoxic T lymphocytes (CTL) are major players in immune defense. In addition, they contribute to the maintenance of immune homeostasis. We now describe a hitherto unavailable, but simple assay to determine ex vivo lytic granule-based cytotoxic functions of human CD8(hi+) CTL subgroups in a clinical setting, under target cell free conditions. Ficoll-isolated peripheral blood lymphocytes from 17 healthy volunteers were stimulated either by phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin or by antibody mediated crosslinking of the CD3 molecule on the T cell surface. Using perforin as a marker for lytic granules, the reduction of CTL granules over time intervals up to 120 min was quantified by FACScan flow cytometry. The kinetics of perforin reduction were compared to the kinetics of NA-CBZ-L-lysine-thiobenzyl ester hydrochloride (BLT)-esterase release and of CD63 upregulation. The reduction in the perforin(+) portion of CD8(hi+) CTLs was correlated inversely with BLT-esterase release and CD63 upregulation. At 30 and 120 min after PMA/ionomycin stimulation, 55 +/- 14% and 42 +/- 14%, respectively, of CD8(hi+) CTLs still stained perforin(+) (time point 0 min = 100%). Perforin-granule release induced by CD3-crosslinking occurred as fast within 30 min (55 +/- 17%), but over the 120 min time interval it was not as complete when compared to PMA/ionomycin-stimulated perforin-reduction. Thus, the combination of an established degranulation assay with the power of immuno flow cytometry allows one to investigate the cytotoxic capability of CTL-subtypes and the kinetics of perforin-granule release. In addition, the assay may prove useful in the elucidation of intracellular signaling cascades governing the perforin-granule release process.     

Mammalian transforming growth factor beta1 activated after ingestion by Anopheles stephensi modulates mosquito immunity

During the process of bloodfeeding by Anopheles stephensi, mammalian latent transforming growth factor beta1 (TGF-beta1) is ingested and activated rapidly in the mosquito midgut. Activation may involve heme and nitric oxide (NO), agents released in the midgut during blood digestion and catalysis of L-arginine oxidation by A. stephensi NO synthase (AsNOS). Active TGF-beta1 persists in the mosquito midgut to extended times postingestion and is recognized by mosquito cells as a cytokine. In a manner analogous to the regulation of vertebrate inducible NO synthase and malaria parasite (Plasmodium) infection in mammals by TGF-beta1, TGF-beta1 regulates AsNOS expression and Plasmodium development in A. stephensi. Together, these observations indicate that, through conserved immunological cross talk, mammalian and mosquito immune systems interface with each other to influence the cycle of Plasmodium development.     

Effects of selective NMDA and non-NMDA blockade in the nucleus accumbens on the plus-maze test

Effect of blocking N-methyl-D-aspartic acid (NMDA) and non-NMDA-glutamatergic receptors on performance in the plus-maze was studied in male rats bilaterally cannulated into the nucleus accumbens (Acc). Rats were divided into seven groups that received either 1 microl injections of saline, (+/-)2-amino-7-phosphonoheptanoic acid (AP-7, 0.2, 0.5, or 1 microg) or 2,3 dioxo-6-nitro-1,2,3,4,tetrahydrobenzo-(f)quinoxaline-7-sulphonamide disodium (NBQX, 0.2, 0.5, or 1 microg) 15 min before testing. Time spent in open arm, time per entry, end arrivals, open, closed, and total arm entries, relationship between open-, closed-, and total arm entries, rearing, face-, head-, and body grooming, and number of fecal boli were recorded. Time spent in the open arm increased under AP-7 (0.5 and 1 microg; P<.01) and NBQX (1 microg; P<.05) treatment, whereas time per entry was increased only with AP-7 (1 microg; P<.05). Open arm entries were increased by the intermediate doses of AP-7 (0.5 microg; P<.01) and NBQX (0.5 microg; P<.05); end arrivals were increased by the intermediate dose of AP-7 (0.5 microg/1 microl, P<.05). The frequency of rearing, grooming, and closed arm entries was not affected by the treatment. We conclude that NMDA and non-NMDA-glutamatergic blockade in the Acc lead to a behavioral disinhibition of cortical influences with the median doses, but that at higher doses the blockers have an anxiolytic-like effect.     

Clinical,immunological, and molecular analysis in a large cohort of patients with X-linked agammaglobulinemia: an Italian multicenter study

A questionnaire-based retrospective clinical and immunological survey was conducted in 73 males with a definite diagnosis of X-linked agammaglobulinemia based on BTK sequence analysis. Forty-four were sporadic and 29 familial cases. At December 2000, the patients' ages ranged from 2 to 33 years; mean age at diagnosis and mean duration of follow-up were 3.5 and 10 years respectively. After the mid-1980s all but 2 were on intravenous immunoglobulin (IVIG) substitution therapy, with residual IgG >500 mg/dl in 94% of the patients at the time of enrollment. Respiratory infections were the most frequent manifestation both prior to diagnosis and over follow-up. Chronic lung disease (CLD) was present in 24 patients, in 15 already at diagnosis and in 9 more by 2000. The cumulative risk to present at diagnosis with CLD increased from 0.17 to 0.40 and 0.78 when the diagnosis was made at the ages of 5, 10, and 15 years respectively. For the 9 patients who developed CLD during follow-up, the duration of follow-up, rather than age at diagnosis; previous administration of intramuscular immunoglobulin; and residual IgG levels had a significant effect on the development of CLD. Chronic sinusitis was present in 35 patients (48%), in 15 already at diagnosis and in 20 by 2000. Sistemic infections such as sepsis and meningitis/meningoencephalitis decreased over follow-up, probably due to optimal protection provided by high circulating IgG levels reached with IVIG.     

Deletion of the mental retardation gene Gdi1 impairs associative memory and alters social behavior in mice

Non-specific mental retardation (NSMR) is a common human disorder characterized by mental handicap as the only clinical symptom. Among the recently identified MR genes is GDI1, which encodes alpha Gdi, one of the proteins controlling the activity of the small GTPases of the Rab family in vesicle fusion and intracellular trafficking. We report the cognitive and behavioral characterization of mice carrying a deletion of Gdi1. The Gdi1-deficient mice are fertile and anatomically normal. They appear normal also in many tasks to assess spatial and episodic memory and emotional behavior. Gdi1-deficient mice are impaired in tasks requiring formation of short-term temporal associations, suggesting a defect in short-term memory. In addition, they show lowered aggression and altered social behavior. In mice, as in humans, lack of Gdi1 spares most central nervous system functions and preferentially impairs only a few forebrain functions required to form temporal associations. The general similarity to human mental retardation is striking, and suggests that the Gdi1 mutants may provide insights into the human defect and into the molecular mechanisms important for development of cognitive functions.     

The effect of anti-IgE treatment on in vitro leukotriene release in children with seasonal allergic rhinitis

BACKGROUND: Binding of allergens with IgE to the IgE receptors on mast cells and basophils results in the release of inflammatory mediators as sulfidoleukotrienes (SLTs), triggering allergic cascades that result in allergic symptoms, such as asthma and rhinitis. OBJECTIVE: We sought to investigate whether anti-IgE (Oma-lizumab), a humanized monoclonal anti-IgE antibody, in addition to specific immunotherapy (SIT) affects the leukotriene pathway. METHODS: Ninety-two children (age range, 6-17 years) with sensitization to birch and grass pollens and with seasonal allergic rhinitis were included in a phase III, placebo- controlled, multicenter clinical study. All subjects were randomized to one of 4 treatment groups. Two groups subcutaneously received birch SIT and 2 groups received grass SIT for at least 14 weeks before the start of the birch pollen season. After 12 weeks of SIT titration, placebo or anti-IgE was added for 24 weeks. The primary clinical efficacy variable was symptom load (ie, the sum of daily symptom severity score and rescue medication score during pollen season). Blood samples taken at baseline and at the end of study treatment after the grass pollen season were used for separation of leukocytes in this substudy. After in vitro stimulation of the blood cells with grass and birch pollen allergens, SLT release (LTC4, LTD4, and LTE4) was quantified by using the ELISA technique. RESULTS: Before the study treatment, SLT release to birch and grass pollen exposure did not differ significantly among the 4 groups. Under treatment with anti-IgE + SIT-grass (n = 23), a lower symptom load occurred during the pollen season compared to placebo + SIT-grass (n = 24, P =.012). The same applied to both groups receiving birch SIT (n = 23 and n = 22, respectively; P =.03). At the end of treatment, the combination of anti-IgE plus grass SIT, as well as anti-IgE plus birch SIT, resulted in significantly lower SLT release after stimulation with the corresponding allergen (416 ng/L [5th-95th percentile, 1-1168] and 207 ng/L [1-860 ng/L], respectively) compared with placebo plus SIT (2490 ng/L [384-6587 ng/L], P =.001; 2489 ng/L [1-5670 ng/L], P =.001). In addition, treatment with anti-IgE was also followed by significantly lower SLT releases to the allergens unrelated to SIT (grass SIT: 300 ng/L [1-2432 ng/L] in response to birch allergen; birch SIT: 1478 ng/L [1-4593 ng/L] in response to grass pollen) in comparison with placebo (grass SIT: 1850 ng/L [1-5499 ng/L], P =.001; birch SIT: 2792 ng/L [154-5839 ng/L], P =.04]. CONCLUSION: Anti-IgE therapy reduces leukotriene release of peripheral leukocytes stimulated with allergen in children with allergic rhinitis undergoing allergen immunotherapy independent of the type of SIT allergen used.