Reduced in vitro adherence of Staphylococcus species to feline corneocytes compared to canine and human corneocytes.

It is apparent that in-contact humans and animals exchange commensal staphylococci. Previous in vitro studies, however, indicate that staphylococci preferentially adhere to corneocytes from host species. This study compared adherence of meticillin-sensitive and -resistant Staphylococcus aureus (MSSA/MRSA), S. intermedius, S. felis and S. hominis to feline, canine and human corneocytes acquired from 10 healthy subjects using adhesive tape discs. Adherent bacteria were counted using an image processing and analysis programme. Mean adherence of MSSA (P = 0.0009), MRSA (P = 0.0162) and S. intermedius (P = 0.0117), but not S. felis or S. hominis, to feline corneocytes was significantly lower than that to canine and human corneocytes. All the isolates had similar adherence to both human and canine corneocytes. S. felis was the most adherent species to feline corneocytes followed by S. intermedius, and then MSSA, MRSA and S. hominis. For dogs and humans, S. intermedius and S. felis were the most adherent, followed by MRSA and MSSA, and then S. hominis. These results do not reveal any preferential adherence of staphylococci to canine or human corneocytes. Poor adherence to feline corneocytes could suggest that cats are relatively resistant to pyoderma and cross-species transmission of staphylococci.

Induced sputum eosinophil cationic protein (ECP) measurement in asthma and chronic obstructive airway disease (COAD)

BACKGROUND: Induced sputum is a useful way to monitor airway inflammation in asthma, but cell counts are time-consuming and labour intensive. OBJECTIVE: The aim of this study was to evaluate a novel processing method using eosinophil cationic protein (ECP) as a biochemical marker of sputum eosinophil number and activation in subjects with asthma and other airway diseases. METHODS: Sputum was dispersed with dithiothreitol and centrifuged to yield cell free supernatant and a cell pellet. The pellet was treated with a cellular lysis buffer to release cell-associated ECP. ECP was measured in sputum supernatant and in the lysed cell pellet and was compared with sputum eosinophil counts in 31 adults with asthma, chronic obstructive airway disease (COAD), bronchiectasis and healthy controls. The ratio of supernatant to pellet ECP was evaluated as an index of eosinophil degranulation. The effect of sputum processing reagents and storage time on ECP measurement was also evaluated. RESULTS: ECP measured in the cell pellet lysate correlated closely with sputum absolute eosinophil counts across a range of subject groups (r = 0.72, P = 0.004). Sputum eosinophil counts were less well correlated with supernatant ECP levels (r = 0.54, P < 0.05). Incubation with dithiothreitol or lysis buffer did not influence ECP measurement and sputum ECP levels were stable over a 6-9 month period. Sputum supernatant and pellet lysate ECP concentrations were increased in stable asthma, asthma exacerbations and COAD/bronchiectasis (P < 0.05). The ratio of supernatant to pellet ECP was used as an index of eosinophil degranulation and found to be elevated in asthma exacerbations, COAD and bronchiectasis, but not in stable asthma. CONCLUSION: The measurement of ECP in the sputum cell pellet provides a reliable and efficient estimate of sputum eosinophil counts which can potentially be used in clinical trials and epidemiological surveys. The ECP ratio may be a useful marker of eosinophil activation, and was increased in asthma exacerbation and COAD. The increased ECP in COAD reflects a non-selective accumulation of eosinophils in this condition.

Assessment of airway inflammation in children with acute asthma using induced sputum.

The aims of this study were: to assess the safety of a sputum induction method using inhaled normal saline in children with acute asthma; and to investigate changes in sputum cell counts between acute exacerbations of asthma and its resolution. Ultrasonically nebulized normal saline was used to induce sputum from children (n = 8) presenting with acute asthma within 1 h of arrival and again at least 14 days later, after resolution of the exacerbation. Children received pretreatment with bronchodilator, and peak expiratory flow (PEF) was monitored throughout the procedure. Samples were analysed for total cell count, differential cell counts, and for eosinophils and neutrophils using specific immunochemical stains. Sputum induction was performed without adverse effect in each child with acute asthma. The mean fall in PEF from baseline during sputum induction was 5.3% during the acute attack and 3.4% at resolution. A shorter nebulization time was required to induce sputum in acute asthma than at follow-up (7.8 vs 13.9 min; p = 0.04). During acute asthma, there was an intense cellular infiltrate (mean total cell count 34 x 10(6) cells.mL-1), which resolved after recovery (1.9 x 10(6) cells.mL-1) (p = 0.04). The infiltrate was heterogenous, comprising eosinophils (6.7 x 10(6) cells.mL-1), neutrophils (5.4 x 10(6) cells.mL-1) and mast cells (0.47 x 10(6) cells.mL-1). Resolution of the exacerbation was accompanied by a significant fall in eosinophils and neutrophils (p < or = 0.04). Normal saline induction of sputum can be used to assess airway inflammation in acute asthma. Children with acute asthma have intense airway inflammation that is heterogeneous and involves neutrophils, eosinophils and mast cells.

Eosinophil apoptosis and the resolution of airway inflammation in asthma.

Asthma is accompanied by the accumulation of potentially damaging eosinophils within inflamed airways. How eosinophils may be removed from the airways is not clear. The phagocytic removal of eosinophils in vitro requires that they undergo apoptosis, a form of cell death. We postulated that eosinophil apoptosis may occur in vivo, promoting the removal of airway eosinophils and the resolution of inflammation in asthma. We examined eosinophil apoptosis in sputum samples obtained from 11 subjects during an asthma exacerbation and 2 wk after corticosteroid treatment of the exacerbation. Airway function improved following corticosteroid treatment, and eosinophilic inflammation subsided, with significant decreases occurring in the number of airway eosinophils and the percentage of activated eosinophils. The proportion of apoptotic airway eosinophils increased significantly following corticosteroid treatment, and eosinophil products were apparent within macrophages. Our findings indicate that eosinophil apoptosis is clinically relevant in asthma. Apoptosis may represent a mechanism that promotes the resolution of eosinophilic inflammation in asthma.

Interleukin-3 in bronchial biopsies from nonasthmatics and patients with mild and allergen-induced asthma.

Cytokines, such as interleukin-3 (IL-3), have been suggested to play an important role in mediating the increased number of airway eosinophils and metachromatic cells in patients with even mild asthma. We used immunohistochemistry to determine the presence of IL-3 protein in bronchial biopsies from nonasthmatics (n = 10) and subjects with mild (n = 8) and allergen-induced (n = 7) asthma. We also examined whether IL-3 was related to airway eosinophil number and activation, the number of airway metachromatic cells, or airway function. We found that the number and activation of eosinophils and the number of metachromatic cells were increased in the airways of asthmatics, compared with nonasthmatics, with further increases evident after allergen challenge. IL-3 protein was localized primarily to the epithelium in nonasthmatic and asthmatic subjects, with no difference apparent between groups or after allergen inhalation challenge. The extent of staining for IL-3 in the tissue was not correlated with eosinophil number or activity, metachromatic cell number, airway responsiveness, or the severity of the late asthmatic response. This study provides the first demonstration of IL-3 protein localization in bronchial tissue from human airways. The results suggest that the increases in eosinophils and metachromatic cells associated with mild and allergen-induced asthma occur independent of IL-3.

Role of airway eosinophils in the development of allergen-induced airway hyperresponsiveness in dogs.

The role of the eosinophil in the development of allergen-induced airway hyperresponsiveness is uncertain. We examined whether the development of airway hyperresponsiveness in 17 dogs after inhalation of Ascaris suum allergen (10(-6) to 10(-2) weight/volume [w/v]) was associated with increases in the number and level of activation of eosinophils before and after allergen inhalation. Airway responsiveness to inhaled acetylcholine was measured before and 24 h after Ascaris inhalation. Eosinophil number was assessed by bronchoalveolar lavage performed 1 wk before allergen inhalation and 15 min after the 24 h acetylcholine challenge. Dogs that developed Ascaris-induced airway hyperresponsiveness (n = 8) had a significantly greater number of bronchoalveolar lavage eosinophils before allergen inhalation (mean +/- SEM: 4.6 +/- 1.94 x 10(4) cells/ml) than dogs that did not become hyperresponsive (n = 9) (1.2 +/- 0.81 x 10(4) cells/ml) (p = 0.03). Ascaris-induced airway hyperresponsiveness, measured 24 h after allergen inhalation, was not associated with increases in eosinophil number after allergen challenge. These results suggest that the presence of airway eosinophils before allergen inhalation is necessary for the development of allergen-induced airway hyperresponsiveness.

Effects of allergen challenge on eosinophils, eosinophil cationic protein, and granulocyte-macrophage colony-stimulating factor in mild asthma.

Allergen inhalation challenge is associated with increases in eosinophil number and activation, and provides a useful model for investigating airway inflammation in asthma. Limited information, however, is available on the effect of allergen challenge on cytokines regulating eosinophil function. We investigated allergen-induced changes in eosinophil number and activation and in granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine known to regulate eosinophil function in vitro. Seven subjects with mild atopic asthma and late asthmatic responses completed diluent- and allergen-inhalation challenges. Blood, bronchoalveolar lavage fluid (BALF), and biopsy samples were collected 24 h after challenge. Allergen inhalation caused a significant increase in eosinophils in BALF and biopsy samples. Eosinophil activation, as assessed by secretion of eosinophil cationic protein, and GM-CSF levels were significantly increased in BALF and bronchoalveolar lavage (BAL) cells. Allergen inhalation did not cause a significant change in eosinophil activation in biopsy tissue but did result in a significant decrease in GM-CSF in the tissue. Significant correlations were shown between the concentration of GM-CSF in BALF and the percentage of BAL eosinophils (Rs = 0.75, p = 0.05), severity of the late asthmatic response, and number of BAL eosinophils (Rs = 0.82, p = 0.02). A trend was seen between the late response and the concentration of GM-CSF in BALF. These results are consistent with the hypothesis that eosinophils, regulated by GM-CSF, contribute to allergen-induced decreases in airway function.

Granulocyte-macrophage colony-stimulating factor, eosinophils and eosinophil cationic protein in subjects with and without mild, stable, atopic asthma.

Increasing evidence implicates the eosinophil as an important effector cell in asthma, but little is known regarding its regulation in vivo. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to regulate eosinophil function in vitro. We investigated the in vivo role of eosinophils and GM-CSF in mild asthma. We compared the number and function of eosinophils and the presence of GM-CSF in blood, bronchoalveolar lavage (BAL) and biopsy tissue obtained from eight mild, stable, atopic asthmatics and 10 nonasthmatics, five of whom were atopic and five nonatopic. Eosinophils were significantly increased in the blood, BAL and biopsy tissue from asthmatics. Activated eosinophils, assessed by immunostaining for the secreted form of eosinophil cationic protein (EG2), were also increased in asthmatic BAL cells and biopsy tissue. Significant increases in GM-CSF in BAL cells and biopsy tissue from asthmatics were also evident. Significant positive correlations existed between GM-CSF in BAL and EG2, and GM-CSF in biopsy tissue and BAL and biopsy eosinophils. Airway responsiveness was also significantly positively correlated with eosinophil number and activation, and with GM-CSF. These results demonstrate that there are increased numbers of activated eosinophils and GM-CSF is increased in patients with mild asthma. Furthermore, GM-CSF is correlated with eosinophil number and function in vivo and these indices are significantly correlated with airway function. These findings emphasize the importance of eosinophils, potentially regulated in vivo by GM-CSF, in contributing to the disordered airway function evident even in mild asthma.

Effect of an inhaled corticosteroid on airway eosinophils and allergen-induced airway hyperresponsiveness in dogs.

The presence of airway eosinophils before allergen inhalation may contribute to the development of allergen-induced airway responses. We examined whether a reduction in airway eosinophil numbers before allergen inhalation as a result of inhalation of the corticosteroid budesonide would prevent allergen-induced airway hyperresponsiveness in seven dogs. Acetylcholine airway responsiveness was measured before and 24 h after inhalation of Ascaris suum allergen (10(-6)-10(-2) wt/vol) or its diluent on 4 test days separated by > or = 4 wk. Dogs were pretreated for 7 days before and on the morning of each test day with inhaled budesonide (2.69 mg/day) or a placebo (lactose). Airway eosinophil numbers were assessed by bronchoalveolar lavage. Inhaled budesonide significantly reduced the number of airway eosinophils before allergen inhalation from 3.6 +/- 2.38 x 10(4) (SE) cells/ml after inhaled lactose to 0.3 +/- 0.21 x 10(4) cells/ml after inhaled budesonide (P = 0.028). The decrease in eosinophil number was associated with a significant reduction in allergen-induced airway hyperresponsiveness (P = 0.005). These results support the hypothesis that the number of eosinophils in the airways before allergen inhalation is an important determinant in the development of allergen-induced airway hyperresponsiveness in dogs.

Effect of inhaled PGE2 on exercise-induced bronchoconstriction in asthmatic subjects.

Previous studies have suggested that the endogenous release of inhibitory prostanoids limits the bronchoconstrictor response to repeated exercise. The aim of our study was to determine whether inhaled prostaglandin (PG)E2 attenuates exercise-induced bronchoconstriction or methacholine airway responsiveness in asthmatic subjects. Eight subjects with mild stable asthma and exercise bronchoconstriction were studied on 4 separate days, 48 h apart. Subjects inhaled PGE2 or placebo in a randomized, crossover, double-blind fashion, 30 min prior to an exercise challenge or a methacholine challenge. PGE2 inhalation significantly attenuated exercise bronchoconstriction. The mean maximal %fall in FEV1 after exercise was 26% (SEM 3.7%) after placebo, and was 9.7% (SEM 2.7%) after PGE2 (p < 0.001). PGE2 also significantly reduced the duration of exercise bronchoconstriction (p = 0.034). However, PGE2 did not significantly attenuate methacholine airway responsiveness. The geometric mean methacholine provocative concentration causing a 20% fall in FEV1 (PC20) was 0.77 (%SEM 1.48) after placebo day, and 1.41 (%SEM 2.20) after PGE2 (p = 0.30). These results demonstrate that inhaled PGE2 markedly attenuates exercise bronchoconstriction in asthmatic subjects and suggest that this effect is not occurring through functional antagonism of airway smooth muscle.