Non-specific airway hyperresponsiveness is a characteristic feature of bronchial asthma. Airway reactivity to cholinergics may change with age during childhood. The present paper describes tests to see if the airway responsiveness induced by acetylcholine (ACh) or capsaicin, which causes a release of endogenous tachykinins from the sensory nerve, changes with age in guinea pigs. Changes in lung resistance (RL) were measured to monitor airflow obstruction caused by intravenous ACh or capsaicin in anesthetized immature guinea pigs (aged 2 weeks), and to compare with those of adult guinea pigs (aged 11 weeks). It was shown that both ACh and capsaicin induced a rapid and dose-dependent increase in RL both in immature and adult animals. The two age groups had similar responsiveness to ACh or capsaicin in terms of potency or sensitivity. The response induced by capsaicin was not affected by atropine. In addition, capsaicin caused no extravasation of Evans Blue dye as an index of airway plasma exudation in both age groups. The airflow obstruction induced by i.v. capsaicin is mainly due to a cholinergic-unrelated smooth muscle contraction. These results suggest that airway smooth muscle responsiveness to cholinergics or capsaicin is comparable in both immature and adult guinea pigs in vivo.

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The purpose of the present study was to determine whether or not inhaled furosemide has any therapeutic benefit in acute asthma in children. Aerosolized furosemide (20 mg) was administered four times per day together with conventional asthma therapy for 2 days in five children with severe asthma exacerbation. The oxygen saturation with a pulse oximeter (SpO₂), and the peak expiratory flow (PEF) were measured before and 10 min after furosemide inhalation. Although a little improvement in SpO₂ and PEF was observed after the furosemide inhalation, the results were inferior to those with an inhaled β₂-agonist (procaterol hydrochloride). Long-term furosemide therapy was performed in two cases; however, amelioration of the SpO₂ levels (> 90%) and PEF (> 80% of personal best) was not observed within 8 days of medication with furosemide. It is concluded that inhaled furosemide is not of additional benefit in children with severe exacerbation of asthma.

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Eosinophil penetration across the basement membrane (BM) is thought to be dependent on the degradation of membrane components. In this process, matrix metalloproteinases (MMP) appear to be primarily responsible for degradation of the BM. Matrix metalloproteinases -2 and MMP-9 degrade type IV collagen, which is a major component of the BM. In the present study, the effects of eotaxin, a selective chemoattractant for eosinophils, on the expression of MMP mRNA were examined. Incubation with chemotactically active concentrations of eotaxin for 24 h enhanced the expression of mRNA for membrane-type 1 MMP (MT1-MMP), but not of mRNA for MMP-2 and MMP-9. An increase in the protein level of MT1-MMP was also detected in the cell lysate of eotaxin-treated eosinophils. These results suggest that up-regulation of MT1-MMP expression may be involved in the eotaxin-induced penetration of eosinophils.

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Buckwheat extract was analyzed by immunoblotting experiments using sera from nine allergic and three non-allergic individuals. Major IgE-reactive bands were 73, 70, 62, 58 and 54 kDa under non-reducing conditions and were detected in allergic subjects, but not in non-allergic ones. Under reducing conditions, the 73, 70, 62 and 58 kDa bands split to 56 and 24, 52 and 24, 45 and 24, and 43 and 24 kDa, respectively. The 24 kDa molecule was the most prominent band recognized with IgE as well as IgG or IgA. The FA02 cDNA clone, encoding the α and β subunits of the legumin-like storage protein, was isolated from a cDNA library made of immature buckwheat seeds. The deduced amino acid sequence of the cDNA clone is substantially identical to the N-terminal amino acid sequence of the 24 kDa molecule, which may be identical to that of BW24KD reported by Urisu et al. Consistent with these results, the translation product of the cDNA encoding the putative β subunit was strongly recognized with serum IgE, IgG and IgA from buckwheat-allergic patients. These results suggested that the 24 kDa molecule may be the β subunit of the legumin-like storage molecule of buckwheat.

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Study objectives were to compare the numbers of lymphocytes and lymphocyte subpopulations in induced sputum from asthmatic patients and from healthy subjects, and to determine the effect of inhaled anti-asthmatic steroid therapy on these cell numbers. Hypertonic saline inhalation was used to non-invasively induce sputum samples in 34 patients with bronchial asthma and 21 healthy subjects. The sputum samples were reduced with dithioerythritol and absolute numbers of lymphocytes and lymphocyte subpopulations were assessed by direct immunofluorescence and flow cytometry. To assess the effect of beclomethasone dipropionate (BDP) on induced sputum, numbers of lymphocytes and lymphocyte subpopulations in sputum also were evaluated after 4 weeks of BDP inhalation treatment in seven asthmatic patients. An adequate sample was obtained in 85.3% of patients with asthma and in 79.2% of the healthy subjects. Induced sputum from patients with asthma had increased numbers of lymphocytes (P = 0.009); CD4⁺ cells (P = 0.044); CD4⁺ cells-bearing interleukin-2 receptor (CD25; P = 0.016); and CD4⁺ cells bearing human histocompatibility leukocyte antigen (HLA)-DR (P = 0.033). CD8⁺ cells were not increased in asthmatic patients. In patients treated with inhaled steroids, numbers of lymphocytes, CD4⁺ cells, CD25-bearing CD4⁺ cells and HLA-DR-bearing CD4⁺ cells in sputum decreased from pretreatment numbers (P = 0.016, 0.002, 0.003 and 0.002, respectively). Analysis of lymphocytes in induced sputum by flow cytometry is useful in assessing bronchial inflammation, and activated CD4⁺ lymphocytes may play a key role in the pathogenesis of airway inflammation in bronchial asthma.

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We have shown that some patients presenting with chronic bronchodilator-resistant non-productive cough have global atopic tendency, airway cough hypersensitivity without non-specific bronchial hyperrespon- siveness and eosinophilic inflammation of the trachea and bronchi, abbreviated as atopic cough (AC). Histamine H₁ receptor antagonists are effective in relieving the cough in some patients with AC but not in others in whom corticosteroids are needed to improve the cough. The aim of the present study was to compare the intensity of eosinophil infiltration in biopsied bronchial submucosa and sequential broncho- alveolar lavage (SBAL) fluids between two subgroups of patients with AC: (i) group A, successfully treated with histamine H₁ receptor antagonists; and (ii) group B, requiring corticosteroids. Sequential BAL was performed using three 50 mL aliquots of physiologic saline solution and then bronchoscopic bronchial biopsy was performed in group A (n = 9) and B (n = 9) patients. Sequential BAL was also performed in normal controls (NC; n = 13). The first SBAL fraction was analyzed as bronchial lavage fluid (BLF) and the mixed fluid of the second and third SBAL fractions as bronchoalveolar lavage fluid (BALF). The number of eosinophils in the bronchial subepithelium was significantly (P = 0.0134) greater in group B patients (median 8.3 cells/mm² ; range 3.6-21.9 cells/mm² ) compared with group A (median 3.6 cells/mm² ; range 0-10.0 cells/mm²). However, There were no significant differences in the number or percentage of eosinophils in BLF or BALF between group A, group B and NC subjects. These findings confirm that eosinophils do not infiltrate the peripheral airways of AC and suggest that corticosteroids are required to relieve the cough in more severe illness of AC, in which submucosal eosinophilic inflammation of the central bronchi is more intensive compared with the milder illness successfully treated with histamine H₁ receptor antagonists.

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Y-24180 ((±)-4-(2-chloro-phenyl)-2-[2-(4-isobuthylphenyl)ethyl]-6,9-dimethyl-6H-thieno[3,2f][1,2,4]triazolo[4,3-a)[1,4]diazepine) is a lipophilic platelet- activating factor (PAF) antagonist. In the present study, we incorporated Y-24180 into lipid microspheres (LM) and its activity was evaluated in vivo. When Lipo Y-24180 was intravenously given 3 min before PAF injection, the maximal bronchoconstriction was reduced. The mean (±SEM) PAF-induced respiratory flow resistance in Lipo Y-24180 (1 µg/kg)-treated animals at 0.5 min was 19.0 ± 8.0%, which was significantly lower than that of the vehicle-treated control (61.5 ± 13.0%; P < 0.05). Three and 10 µg/kg Lipo Y-24180 almost completely inhibited the respiratory flow resistance. Alone, Y-24180 dose-dependently suppressed the bronchoconstriction; however, a significant decrease was only seen in 10 µg/kg-treated animals. There were also significant differences between the Lipo Y-24180 (1 µg/kg)-treated group and Y-24180 (1 and 3 mg/kg) treated groups. Aerosol inhalation of Lipo Y-24180 (10 µg/mL) caused no significant suppression; however, a significant inhibition was observed in guinea pigs that were administered more than 30 µg/mL. Therefore, it would be concluded that Lipo Y-24180 would have a potency as a bronchodilator and would be promising as both an injectable drug and an inhaler.

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Mite allergens Derp1 and Derf1 were tested in 200 house dust samples using a nitrocellulose membrane-based dot-blot assay. The assay enables the mite allergens in a dust extract to be detected with results that can be visually read semiquantitatively within 30 min. Positive results obtained with this method were easily observed as brown dots. The Derp1 and Derf1 allergens were also precisely quantitated by a two-site monoclonal antibody based enzyme-linked immunosorbent assay. Results of Derp1 and Derf1 mite allergens measured from the two methods were compared. The semiquantitative analysis using Spearman's correlation yielded a correlation coefficient of 0.892 (P < 0.01) for Derp1 and 0.872 (P < 0.01) for Derf1. The sensitivity, specificity and accuracy were 94.4%, 76.9% and 86.5% for Derp1 whereas the sensitivity, specificity and accuracy for Derf1 were 95%, 77% and 86%, respectively.

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The intrusion of airborne allergens into airways elicits eosinophilic inflammation, as represented by bronchial asthma. It has been shown that excessive amounts of allergen in murine trachea lead to an unexpected evasion of deleterious eosinophilic inflammation by inducing T cell tolerance. In the present study, the mechanisms of tracheal high-dose tolerance are examined with regard to accessory cell functions and the effects of pro-inflammatory cytokines on tolerance. Antigen-induced tracheal eosinophilia was suppressed on instillation of high doses of antigen into the trachea, while concurrent instillation of granulocyte-macrophage colony stimulating factor (GM-CSF) with the antigen restored the diminished responses. The restoration of eosinophilic infiltration by GM-CSF occurred in parallel with an increase in interleukin (IL)-4 production by CD4⁺ T cells from the mediastinal lymph nodes. This was found to reflect the empowerment of antigen-presenting cells by GM-CSF, because the impaired ability of Ia⁺ cells from the tolerant mice to stimulate IL-4-producing T cells is restored by GM-CSF administration. The prevention of tolerance by up-regulating accessory cell functions is a feature unique to GM-CSF, because another pro-inflammatory cytokine, IL-1β, failed to empower antigen-presenting cells. Thus, besides the induction of transforming growth factor-β-secreting CD4⁺ T cells, high-dose tolerance in the trachea includes an impairment of the accessory cell functions that support IL-4 production from T cells, which was reversed by GM-CSF. This report is the first demonstration that GM-CSF breaks the T cell tolerance of IL-4-producing T helper cells.

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We examined the functional heterogeneity of eosinophils from an adult T-cell leukemia (ATL) patient with eosinophilia. A 63-year-old man was admitted to our hospital because of lymphadenopathy. The leukocyte count was 10400 /mm³ , with 36.0% eosinophils and 3.0% abnormal lymphocytes. The diagnosis of ATL was based on the presence in serum of anti-human T-cell lymphotrophic virus-1 antibody and on histologic demonstration of ATL cells. The mononuclear cells spontaneously produced eosinophil-related cytokines (granulocytemacrophage colony stimulating factor, 5600 pg/mL; interleukin (IL)-5,375 pg/mL). Peripheral eosinophils were fractionated into normodense eosinophils (NE) and hypodense eosinophils (HE) by a Percoll density gradient method, and these cells were compared in terms of several heterogeneous functions. The NE were more chemotactically attracted to IL-5 than the HE. More apoptotic cells appeared among the NE than among the HE and this difference was correlated with the positive rate of Fas antigen on eosinophils. Survival of the HE was longer than that of the NE. Survival of the HE was prolonged by IL-5 stimulation, but survival of the NE was not. These data suggest that functionally heterogeneous eosinophils were present in this ATL patient with eosinophilia and that IL-5 enhanced this heterogeneity. The response of eosinophils to IL-5 may have contributed to the pathogenesis of eosinophilia in this patient.

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