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Notes: Allergic asthma is characterized by chronic recruitment of eosinophils in the airways. Once activated, eosinophils release toxic products, including eosinophil cationic protein (ECP), able to damage airway epithelial cells. To test the hypothesis that also in mild-moderate stable asthma, a significant eosinophil activation could occur, we studied 25 asthmatic patients (34 ± 19 years old), of whom 18 were allergic (27 ± 12 years) and seven nonallergic (42±10 years), with FEV1 values ±70% of predicted, and eight normal volunteers (controls, 33 ±11 years). All subjects underwent methacholine (MCh) challenge on the first visit, and bronchoalveolar lavage (BAL) on the second visit (approximately 3–4 days later). BAL cells were counted and albumin (Alb) (as index of protein dilution in BAL fluid) and ECP levels (as index of eosinophil activation) in BAL fluid were measured. As compared to controls, a significant increase in BAL eosinophil and in BAL epithelial cell numbers was observed in asthmatic patients (P〉0.05, each comparison), with no differences between the two asthmatic patient subgroups. Detectable ECP levels (〉2 μg/1) were found in BAL of 18 asthmatic patients (14 allergic and four nonallergic asthmatic patients), while Alb levels were measurable in 25 BAL fluids and found to be similar in controls and asthmatic patients, and in the two asthmatic patient subgroups (P〉0.05, each comparison). In BAL of asthmatic patients, positive correlations were found between eosinophil numbers and 1) ECP/Alb levels (r= 0.50, P = 0.020); 2) epithelial cell numbers (r = 0.S0, P = 0.014). In asthmatic patients, a significant negative correlation was found between bronchial reactivity to MCh (log Pd15) and ECP/Alb levels in BAL fluid (r=-0.6, P= 0.005), whereas no correlation was found between log Pd15 MCh and BAL eosinophil or epithelial cell number (P〉0.1, each correlation). These data suggest that bronchial eosinophil recruitment and activation may occur also in mild-moderate stable asthma and that bronchial epithelium damage and airway responsiveness may be partially associated with the eosinophilic inflammatory reaction.

Notes: Blood levels of inhaled corticosteroids are significantly lower than those measured in the lung, but their concentration could still have anti-inflammatory effects. To determine whether budesonide, at concentrations similar to those obtained in blood after drug inhalation (10 −9 M), could downregulate the allergen-induced activation of mononuclear cells, we studied 21 atopic patients, sensitized to Dermatophagoides pteronyssinus (Der p). On blood mononuclear cells, isolated from these patients, incubated with Der p allergen extract and with or without budesonide, we evaluated: 1) the proliferative response of T cells; 2) the expression of two surface activation markers, the HLA-DR antigens and the interleukin (IL)-2 receptors; and 3) the release of cytokines known to modulate the allergic processes. Allergen-induced T-cell proliferation was associated with increased HLA-DR antigen and IL-2 receptor expression (P 〈 0.001), and with increased release of IL-2, interferon-gamma (IFN-γ), IL-1β, tumor necrosis factor-alpha (TNF-α), and granulocyte/macrophage colony-stimulating factor (GM-CSF). The addition of budesonide at the beginning of the cell cultures induced a dose-dependent inhibition of T-cell proliferation, still significant (P 〈 0.05) at the lowest concentrations tested (10 −9 and 10−10 M). A significant inhibitory effect on T-cell proliferation was also present when budesonide (10 −9 M) was added to the cell cultures 3 or 5 days after the beginning of the cell cultures. In addition, budesonide 10−9 M significantly decreased the expression of IL-2 receptors (P 〈 0.05), but not of HLA-DR antigens, and significantly reduced the release of IL-1β and GM-CSF (JP 〈 0.05), but not of IL-2, IFN-γ, and TNF-α. Thus, the blood concentrations of budesonide, after drug inhalation, may exert some anti-inflammatory effect, downregulating both “local” (through the bronchial circulation) and “systemic” allergen-specific immune reactions.

Notes: Abstract. To characterize the cellular inflammation at the bronchial and bronchoalveolar levels, we evaluated 43 patients with asthma who were sensitized to house dust mites. On 2 consecutive days patients underwent methacholine challenge and allergen bronchial challenge. In addition, 6, 24, or 72 h after allergen challenge, fiberoptic bronchoscopy with bronchial lavage (BL) and bronchoalveolar lavage (BAL) was performed. Patients belonging to the 6-h, 24-h, or 72-h group were divided further into two subgroups: those with isolated early response to allergen (LAR−), and those with dual response to allergen (LAR+). The percentage of eosinophils and of epithelial cells in BAL fluid was significantly higher in LAR+ than in LAR− patients in the 6-h group (p 〈 0.05, each comparison), but not 24 or 72 h after (p 〉 0.05, each comparison). Similarly, the proportion of BL eosinophils was also higher in LAR+ than in LAR− patients, both in the 6-h and in the 24-h group (p 〈 0.05, each comparison). In addition, increased proportions of BL neutrophils were present in the LAR+ patients belonging to the 24-h group (p 〈 0.05). Comparing ``proximal'' = BL vs ``distal'' = BAL data, we found a significantly higher proportion of epithelial cells in BL compared with BAL, in both LAR− and LAR+ subjects, either 6, or 24, or 72 h after challenge (p 〈 0.01, each comparison) and increased percentages of BL neutrophils and eosinophils in LAR+ patients (p 〈 0.05, each comparison), but not in LAR− patients, in the 24-h group. The percentages of BL or BAL macrophages and lymphocytes did not differ significantly among the different patient groups. These data indicate that the development of LAR after allergen inhalation challenge is associated with an early recruitment of eosinophils and with epithelial desquamation in the airways. In addition, after allergen challenge epithelial desquamation is more pronounced in the proximal than in the distal airways, independently of the type of bronchial response.

Notes: Abstract. Recently, much attention has been given to the possible role of lymphocytes and their soluble products in causing and maintaining allergic inflammation. The aim of this study was to assess the production of mRNAs for interleukins (IL) in bronchoalveolar lavage (BAL) cells obtained from allergic asthmatics after challenge with the relevant allergen in the period between early and late reactions. We evaluated BAL fluid cells obtained from six asthmatic subjects and four nonatopic controls. Challenge was performed with the relevant allergen. BAL fluid cells were obtained by fiberoptic bronchoscopy and bronchoalveolar lavage. To detect mRNA encoding each cytokine in BAL cells we used a reverse transcriptase polymerase chain reaction method. We evaluated IL-1α, -2, -4, -5, -6, -13, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-γ (IFN-γ). mRNAs for IL-1α, -2, -4, -5, and IFN-γ were detected in all of the atopic subjects; mRNAs for IL-6 and GM-CSF were found in five asthmatics; and mRNA for IL-13 was found in one patient only. In contrast, no mRNAs for IL-2, -4, -5, -6, -13, and GM-CSF were detected in the nonatopic healthy controls; mRNA for IL-1α was found in one out of four normal subjects; and mRNA for IFN-γ was evidenced in two of four subjects. The cellular environment in BAL fluids from allergic asthmatics before the clinical appearance of the late airway reaction shows an unrestricted expression of mRNA for cytokines. The local cytokine milieu could have an important role in the modulation of bronchial inflammation and in the appearance of allergic symptoms.

Notes: Abstract. Allergen exposure in atopic asthmatic patients is associated with recruitment and activation of eosinophils in the airways. Once activated, eosinophils release toxic products, including the eosinophil cationic protein (ECP), able to damage bronchial structures and to increase bronchial hyperresponsiveness. With this background, the present study was designed to evaluate whether ECP levels in bronchoalveolar lavage (BAL) fluid could reflect, better than BAL eosinophil counts, the cellular activation that follows allergen exposure in atopic asthmatics. Twenty-two atopic patients attended the laboratory on two separate days. On the 1st day, they underwent methacholine (MCh) inhalation challenge to detect the degree of nonspecific bronchial hyperresponsiveness. On the 2nd day, they underwent fiberoptic bronchoscopy and BAL, at baseline or 4–6 h after allergen inhalation challenge. In this latter patient group, MCh challenge was repeated 3–5 h after allergen challenge, 1 h before fiberoptic bronchoscopy. The analysis of the mean baseline FEV1 values and the degree of bronchial reactivity to MCh (MCh Pd20) on the 1st study day did not demonstrate differences between the two patient groups (p 〉 0.1, each comparison). In addition, in the allergen-challenged group, MCh Pd20 was decreased significantly after allergen challenge (151.4 μg/ml and 67.6 μg/ml, respectively, before and after challenge; p 〈 0.05). Evaluation of the different BAL cell types demonstrated that the proportions of eosinophils and epithelial cells were increased significantly in the allergen-challenged group compared with the group evaluated at baseline (p 〈 0.01 and p 〈 0.05, respectively). Moreover, ECP levels, corrected by the correspondent albumin levels (ECP/Alb), were higher in the allergen-challenged group compared with the group evaluated at baseline (p 〈 0.05). In addition, although a positive correlation was demonstrated between BAL eosinophil percentages and ECP/Alb values (r= 0.72, p 〈 0.05) in the group evaluated at baseline, no links were found between these parameters in the allergen-challenged group (p 〉 0.1). However, in this latter group, a weak positive correlation was demonstrated between eosinophil percentages and ΔMch, i.e., the increased nonspecific bronchial reactivity, which is observed after allergen challenge (r= 0.55; p 〈 0.05). Thus, in stable asthmatic patients an ongoing activation of eosinophils parallels their migration, but this eosinophilic inflammation is not strictly related to bronchial reactivity to Mch. By contrast, after allergen inhalation challenge, eosinophil recruitment and activation seem to follow different temporal kinetics, and eosinophilic inflammation may be partially associated with the degree of airway hyperresponsiveness.

Notes: Abstract. Treatment of allergic asthma with inhaled corticosteroids results in local down-regulation of proinflammatory cytokine synthesis and in marked decrease in tissue eosinophilia. Blood concentrations of inhaled corticosteroids, although significantly lower than those measured in the lung, may still have antiinflammatory effects on circulating eosinophils, reducing their ability to migrate. The aim of our study was to evaluate in vitro the activity of budesonide on blood eosinophils by measuring their chemotactic response, eosinophil cationic protein (ECP) release, and hydrogen peroxide (H2O2) production in the presence of different drug concentrations similar to those obtained at airway level (10−8 and 10−7 M) and at blood level (10−10 and 10−9 M). Partially purified blood eosinophils, isolated from 23 asthmatic subjects, were used to evaluate the activity of budesonide on: (1) chemotaxis toward the activated fifth component of complement (C5a, 0.1 μg/ml) or recombinant human (rh) interleukin (IL)-5 (200 pg/ml), (2) ECP release by cells stimulated with tetradecanoylphorbol acetate (TPA) and (3) H2O2 production by TPA-activated cells. The chemotactic response to C5a was down-regulated significantly by budesonide only by the highest concentrations tested (10−8 and 10−7 M); differently, budesonide was effective in inhibiting eosinophil migration toward rhIL-5, at all concentrations tested (p 〈 0.01, each comparison). By contrast, no drug-induced modifications were observed in ECP release or in H2O2 production (p 〉 0.05, each comparison). We conclude that concentrations of budesonide similar to those obtained in vivo are effective in inhibiting eosinophil locomotion but not in down-regulating the release of reactive oxygen species and granule-associated proteins.