News about Medicine - Part 4

Human Peripheral Blood and Pleural Fluid Eosinophils: Results

Human Peripheral Blood and Pleural Fluid Eosinophils: ResultsAbility of Peripheral Blood Eosinophils and Neutrophils from Control Subjects to Generate H2O2
The production of H202 by human peripheral blood eosinophils or neutrophils was studied under both resting and stimulated conditions. Although neither peripheral blood eosinophils nor neutrophils produced spontaneously detectable amounts of H202, the addition of PMA to the culture media was able to generate an intense respiratory burst which was greater for eosinophils than for neutrophils (eosinophils: 7.5±0.8 nmol H202; neutrophils: 4.2 ± 0.5 nmol H202; p<0.05) (Fig 1). Similar results were obtained when cells were stimulated with IgG IC, which induced a more intense H202 generation by eosinophils than by neutrophils (eosinophils: 4.1 ±0.6 nmol H202; neutrophils: 1.1 ±0.3 nmol H202; p<0.01), although the levels of H202 measured were lower when compared with the levels observed after PMA stimulation, both for eosinophils and for neutrophils (p<0.05, each comparison). Interestingly, the presence of Agg IgE was able to induce a detectable H202 release only by eosinophils and not by neutrophils (eosinophils: 0.9 ±0.3 nmol H202; neutrophils: undetectable amounts of although the respiratory burst induced in eosinophils by Agg IgE was lower than that observed after activation with PMA or IgG IC (p<0.001, each comparison). No detectable H202 production was observed when peripheral blood eosinophils or neutrophils were incubated with IgG alone or IgE alone (data not shown). !–more–>alta white teeth whiteningAbility of Pleural Fluid and Peripheral Blood Eosinophils from Patients with Eosinophilic Malignant Pleural Effusions to Generate H202
As observed for circulating eosinophils from control subjects, neither pleural fluid eosinophils nor peripheral blood eosinophils from patients with eosinophilic malignant pleural effusions spontaneously released H202 (Table 1). All stimuli used in this study were able to induce a detectable respiratory burst in pleural fluid and peripheral blood eosinophils isolated from cancer patients, but the generation of H202 was higher for peripheral blood eosinophils than for pleural fluid eosinophils either with PMA (peripheral blood eosinophils: 7.7 ±0.9 nmol H202; pleural fluid eosinophils: 4.9 ±0.7 nmol H202; p<0.05) or with IgG IC (peripheral blood eosinophils: 4.6 ±0.6 nmol H2Oz; pleural fluid eosinophils: 2.4±0.2 nmol H202; p<0.05), or with Agg IgE (peripheral blood eosinophils: 2.1 ±0.2 nmol H202; pleural fluid eosinophils: 0.4 ±0.1 nmol H202; p<0.01). As described for peripheral blood eosinophils from control subjects, H202 release was higher after stimulation with PMA than after stimulation with IgG IC or Agg IgE both for peripheral blood and for pleural fluid eosinophils (p<0.05, each comparison) and more elevated after IgG IC stimulation than after Agg IgE stimulation (p<0.05, each comparison). In addition, in the experimental conditions tested in this study, no significant difference in the H202 release was observed between peripheral blood eosinophils from normal control subjects and peripheral blood eosinophils from patients with malignant pleural effusions (p>0.2, all comparisons).

Figure 1. Hydrogen peroxide production by human peripheral blood eosinophils (open bars) and neutrophils (solid bars) from normal subjects in resting conditions (RESTING) and after stimulation with phorbol myristate acetate (PMAX IgG immune complexes (ZgG IC) or human heat-aggregated myeloma (Agg IgE). Hydrogen peroxide was quantified after 30 minutes’ incubation by recording the oxidation of phenol red in the presence of horseradish peroxidase. The data represent the mean ± SEM of six experiments.

Figure 1. Hydrogen peroxide production by human peripheral blood eosinophils (open bars) and neutrophils (solid bars) from normal subjects in resting conditions (RESTING) and after stimulation with phorbol myristate acetate (PMAX IgG immune complexes (ZgG IC) or human heat-aggregated myeloma (Agg IgE). Hydrogen peroxide was quantified after 30 minutes’ incubation by recording the oxidation of phenol red in the presence of horseradish peroxidase. The data represent the mean ± SEM of six experiments.

Table 1—H2O2 Release by Peripheral Blood E osinophils from Control Subjects and from Peripheral Blood and Pleural Fluid Eosinophils from Patients with Eosinophilic Malignant Pleural Effusions

StudyPopulations Source of Eosinophils Spontaneous H202 Release (nmol/min/106 Cells) H202 Release after Eosinophil Activation
PMA IgG IC (nmol/min/108 Cells) Agg IgE
Controlsubjects Peripheralblood Not detectable 7.5±0.8 4.1 ±0.6 0.9±0.3
Patientswith

eosinophilic

malignant

pleural

effusions

Peripheralblood Not detectable 7.7±0.9 4.6±0.6 2.1 ±0.2
Pleuralfluid Not detectable 4.9±0.7 2.4 ±0.2 0.4 ±0.1

Human Peripheral Blood and Pleural Fluid Eosinophils: Analysis of Data

Spontaneous and PMA or IgG IC or Agg IgE-lnduced H202 Production by Peripheral Blood and Pleural Fluid Eosinophils and by Peripheral Blood Neutrophils
Peripheral blood and pleural fluid eosinophils and peripheral blood neutrophils (the number of pleural fluid neutrophils was too low to perform the functional studies) were resuspended in HBSS at a final concentration of 1 x 10® cells/ml. Leukocytes (5 x 10) were added to 15 x 75-mm plastic test tubes containing 0.28 mM phenol red, 8.5 U/ml horseradish peroxidase (type 2, Sigma Chemical Co, St Louis, MO), superoxide dismutase, 5 p-g/ml (Sigma Chemical Co), as previously described. When indicated, phorbol myristate acetate (PMA), 75 ng/ml (PMA, Sigma Chemical Co), or various amounts of IgG alone or IgE alone or of IgG IC or of Agg IgE (50, 100, 200 and 300 fiL) were added to the test tubes containing neutrophils or eosinophils. Read the rest of this entry »

Human Peripheral Blood and Pleural Fluid Eosinophils: Isolation of Peripheral Blood and Pleural Fluid Granulocytes

Human Peripheral Blood and Pleural Fluid Eosinophils: Isolation of Peripheral Blood and Pleural Fluid GranulocytesIsolation of Peripheral Blood and Pleural Fluid Granulocytes
The blood and pleural fluid samples were diluted with an equal volume of 4.5 percent dextran T-500 (Pharmacia Fine Chemicals, Piscataway, N.J.) and allowed to sediment at 37°C for 40 min. The leukocyte-rich suspensions were removed, centrifuged three times at 200 g to remove platelets and the cells were then pelleted at 500 g for 15 min. The leukocytes were then washed in Hanks’ balanced salt solution (IiBSS) and layered on a 12-ml discontinuous metri-zamide gradient (Nyegaard and Co, Oslo, Norway), 20 to 25 percent weight per volume ratio (w + /vol) in HBSS containing 0.1 percent (w/v) gelatin and 15 p.g*ml_1 DNase I (DN-CL, Sigma Chemical Co), pH 6.8, by centrifugation at 400 g for 45 min, according to the technique described by Vadas et al modified by Oliver et al. Read the rest of this entry »

Human Peripheral Blood and Pleural Fluid Eosinophils: Materials and Methods

Study Populations and Collection of Peripheral Blood and Pleural Fluid Cells
Heparinized (Parke-Davis, Milan, Italy) venous blood was obtained from normal volunteers after appropriate informed consent Routinely 200 to 250 ml of blood was collected from four women and two men (36 ± 3 years of age) who had no history of atopic or parasitic diseases and who had not taken any medication for at least two weeks before. Specimens of pleural fluid were obtained from three patients with hypereosinophilic malignant pleural efiusions due to lung cancer. The patients, one woman and two men (57 ± 5 years of age), had an untreated adenocarcinoma as primary lung tumor and the neoplastic nature of the pleural effusion was ascertained by detection of malignant cells in the pleural fluid. Read the rest of this entry »

Human Peripheral Blood and Pleural Fluid Eosinophils

Human Peripheral Blood and Pleural Fluid EosinophilsEposinophils are non-dividing, granule-containing cells which arise in the bone marrrow and have secretory as well as phagocytic properties. While eosinophils are generally considered to be beneficial cells able to suppress mediators of inflammation in immediate-type hypersensitivity reactions and to damage parasites such as Schistosoma mansoni and Trichinella spiralis, there is increasing evidence that they also have the potential to injure normal body tissues. In this contest, eosinophils not only carry the major basic protein, a highly toxic polypeptide that under appropriate circumstances can clearly damage tissues such as tracheal mucosa, and a collagenase capable of specifically cleaving type 1 and 3 collagen, but also they can generate and release toxic oxygen radicals that can cause cell injury and death. As for other granulocytes, the activation of eosinophil oxidative metabolic burst can be induced by exposure of the cells to ingestible particles, such as latex beads or opsonized zymosan or by soluble membrane activators, such as phorbol myristate acetate (PMA). Read the rest of this entry »

Ventilation and Breathing Pattern during Sleep in Duchenne Muscular Dystrophy: Conclusion

Ventilation and Breathing Pattern during Sleep in Duchenne Muscular Dystrophy: ConclusionSurface abdominal and rib cage motion reflect diaphragmatic and intercostal contributions to breathing, but the relationship of these indexes to diaphragm and intercostal function is more complex. In neuromuscular diseases, the mechanical coupling between muscle activation and rib cage motion may be further distorted. Although the abdominal contribution gives only an indirect index of diaphragm function, more direct measurements such as transdiaphragmatic pressure were avoided in this study to minimize instrumentation of these subjects. Despite these limitations, however, the mean abdominal contributions of our subjects in wakefulness and sleep were well below normal and their relationship in these patients to oxygen desaturation in REM sleep underlines the importance of diaphragm dysfunction in their vulnerability to sleep desaturation. The similarity between the fall in bilateral diaphragm paralysis in dogs lends further support to this view. Read the rest of this entry »

Ventilation and Breathing Pattern during Sleep in Duchenne Muscular Dystrophy: Outcome

Sampling problems are inevitable when selecting representative data from each sleep stage, particularly from periods of wakefulness and REM sleep. The heterogeneous nature of stage REM, comprising tonic and phasic components and often, in these subjects, brief arousal periods associated with oxygen desaturation, further limits interpretation of its ventilatory data. The heterogeneity is reflected in the measured coefficient of variation of all our measurements and is also seen in normal subjects. There have been several attempts to quantify ventilation and its components in normal subjects nonin-vasively during wakefulness and sleep. The RIP data of Stradling et al were obtained by MLR from restrained sleeping adult healthy males and so offer an appropriate comparison to the present results. Patients with Duchenne MD have severe generalized respiratory weakness usually without selective diaphragm involvement and, in our subjects, without obesity or significant scoliosis. Here Read the rest of this entry »

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