Bronchial responsiveness to methacholine and adenosine 5''-monophosphate in atopic and non-atopic preschool children with recurrent wheezing

BACKGROUND: It is well known that atopy is a major determinant of bronchial hyper-responsiveness (BHR) in both asymptomatic and asthmatic children. However, the relationship between atopy and BHR has not been well studied in preschool children with wheezing. BHR is usually measured by bronchial challenges using direct and indirect stimuli. OBJECTIVE: The aim of this study was to investigate whether atopic and non-atopic preschool wheezers display similar or different BHR profiles for direct and indirect stimuli. METHODS: Methacholine and adenosine 5'-monophosphate (AMP) bronchial challenges were performed in 4 to 6-year-old children with recurrent wheezing, using a modified auscultation method. The end-point was defined as the appearance of wheezing and/or oxygen desaturation. Atopy was determined to be present when a child had at least one positive reaction to a panel of 13 common airborne allergens in the presence of positive and negative controls. RESULTS: A positive response to methacholine (an end-point concentration < or =8 mg/mL) was observed in 89.3% (50/56) of atopic wheezers and in 83.8% (31/37) of non-atopic wheezers (P=0.44) for the difference. By contrast, the frequency of a positive response to AMP (an end-point concentration < or =200 mg/mL) was significantly higher in the atopic group (47/56, 83.9%) compared with the non-atopic group (12/37, 32.4%; P<0.01). CONCLUSION: While a majority of both atopic and non-atopic preschool wheezers were hyper-responsive to methacholine, atopic subjects were more hyper-responsive to AMP than non-atopic subjects. These findings suggest that atopic and non-atopic wheeze in preschool children are related to distinctive pathophysiologic pathways.     

Airway inflammation and hyperresponsiveness to adenosine 5'-monophosphate in chronic obstructive pulmonary disease

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is often accompanied by bronchial hyperresponsiveness (BHR). Measurement of BHR may give information about airway inflammation. OBJECTIVE: To investigate the role of airway inflammation in hyperresponsiveness to adenosine 5'-monophosphate (AMP) in COPD. METHODS: We investigated inflammatory indices in induced sputum, bronchoalveolar lavage (BAL) fluid and bronchial biopsies in subjects with COPD with and without hyperresponsiveness to AMP. Twelve nonatopic subjects with COPD with hyperresponsiveness to AMP (mean +/- SD, age 63 +/- 8 years, FEV1% predicted 56 +/- 13%), six without BHR (age 60 +/- 6 years, FEV1% predicted 65 +/- 11%) and 11 healthy nonatopic controls without BHR (age 58 +/- 8 years, FEV1% predicted 104 +/- 11%) participated in the study. RESULTS: Subjects with COPD with BHR had significantly higher numbers of mucosal CD8 + and higher percentages of sputum eosinophils than those without BHR (median, 550 cells/mm2; range, 30-1340 vs 280 cells/mm2; range, 110-450, P = 0.045; and median, 2.7%; range, 0.5-8.5 vs 0.6%; range, 0-0.8 %, P = 0.0036, respectively). No differences were observed in BAL fluid. CONCLUSION: We conclude that hyperresponsiveness to AMP in COPD is associated with airway inflammation that is characterized by increased numbers of mucosal CD8 + cells and percentages of sputum eosinophils. Hyperresponsiveness to AMP may be used as a marker of airway inflammation in COPD, but its significance in the clinical course remains to be determined.     

Bronchial responsiveness to methacholine and adenosine 5'-monophosphate in young children with asthma: their relationship with blood eosinophils and serum eosinophil cationic protein

BACKGROUND: Bronchial hyperresponsiveness is a characteristic feature of asthma, and is usually measured by bronchial challenges using direct or indirect stimuli. Blood eosinophil numbers and serum levels of eosinophil cationic protein (ECP) are considered as indirect measures of airway inflammation in asthma. The aim of this study was to investigate whether bronchial responsiveness to adenosine 5'-monophosphate (AMP) is more closely associated with blood eosinophil markers, compared with that to methacholine, in young children with asthma. METHODS: Methacholine and AMP bronchial challenges were performed in 4- to 6-year-old children with asthma (n = 77) and in healthy controls (n = 32), using a modified auscultation method. The end-point was defined as the appearance of wheezing and/or oxygen desaturation. The peripheral blood eosinophil counts and serum ECP concentrations were determined in each subject. RESULTS: A positive response to methacholine (end-point concentration < or =8mg/ml) and to AMP (end-point concentration < or =200 mg/ml) was observed in 74 (96.1%) and 66 asthmatic children (85.7%), respectively. A majority of controls was unresponsive to both challenges. In the asthma group, there was no significant correlation between methacholine end-point concentration and the eosinophil counts (r = -0.111, P = 0.337) or serum ECP levels (r = -0.126, P = 0.274). In contrast, AMP end-point concentration correlated significantly with the eosinophil counts (r = -0.372, P = 0.001) and with serum ECP levels (r = -0.371, P = 0.001). CONCLUSIONS: Our results suggest that bronchial responsiveness to AMP is more closely related to airway inflammation, compared with that to methacholine, and support the potential usefulness of AMP challenges in detecting inflammatory changes in young children with asthma.     

Induced sputum: Validity of fluid-phase IL-5 measurement

Background: IL-5 measurement in the fluid phase of induced sputum is considered to be important in the assessment of asthma, but the validity of these measurements is uncertain. Objective: We investigated the validity of sputum IL-5 measurements through a series of spiking experiments and examined the effect of dithiothreitol (DTT) on these measurements. Methods: Induced sputum from 26 asthmatic subjects was spiked with IL-5 and processed, and the percentage of recovery was measured by means of immunoassay. In 6 of the 26 samples the effect of adding albumin to the processing fluids was studied. In 3 separate samples radiolabeled IL-5 was added, and the recovery measured by means of gamma counting and immunoassay were compared. In addition, the effect of DTT on the immunoassay was examined. Results: The mean ± SD recovery of spiked IL-5 was 26.1% ± 14.6% measured by means of immunoassay; adding albumin increased the recovery to 47.7% ± 8.0% (P < .001). The mean recovery measured by means of gamma counting was 84.8% ± 5.7% (P < .001); adding albumin had no effect on recovery. DTT had no significant effect on IL-5 measurement. Conclusion: The validity of IL-5 measurement by means of current methods is poor. The discrepancy in recovery as measured by gamma counting compared with immunoassay suggests that there is a problem with the recognition of IL-5 epitopes by immunoassay in induced sputum. This cannot be attributed to DTT but may be due to other interfering substances present in sputum, such as sputum proteases, soluble receptors, or autoantibodies. (J Allergy Clin Immunol 2000;105:1162-8.)     

Bronchial responsiveness to adenosine-5''-monophosphate and methacholine as predictors for nasal symptoms due to newly introduced allergens. A follow-up study among laboratory animal workers and bakery apprentices

BACKGROUND: In asthma patients, bronchial hyper-responsiveness (BHR) to adenosine-5'-monophosphate (AMP) reflects bronchial inflammation more closely than BHR to methacholine. In this follow-up study we studied bronchial responsiveness to both stimuli as predictors of new-onset airway symptoms. METHODS: We included 118 laboratory animal workers and bakery apprentices with a work experience of maximally 1 year. The baseline survey comprised a questionnaire, skin prick tests (SPTs) to common and work allergens, blood eosinophil counting, and bronchial challenge with methacholine and AMP. At follow-up, questionnaire and SPTs to work allergens were repeated. Airway symptoms to common allergens and work allergens were defined as nasal symptoms, chest tightness or asthma attack during or after contact with either common or work allergen. Bronchial challenge tests were analysed by BHR at a 15% fall in forced expiratory volume of 1 s (FEV1), and by dose-response-slope (DRS). RESULTS: Fourteen subjects (12%) developed airway symptoms to work allergens, of whom 12 had nasal symptoms. A positive SPT to work allergens occurred in 64%, and was the strongest predictor of airway symptoms [relative risk (RR) 7.5, 95% confidence interval (CI) 2.0-28.6]. Other predictors were airway symptoms to common allergens (RR 4.3, 95% CI 1.4-12.8), blood hypereosinophilia (RR 4.4, 95% CI 1.2-15.4) and BHR, with a slightly higher risk estimate for AMP than for methacholine (RRAMP 3.7, 95% CI 1.1-12.5 and RRmeth 2.8, 95% CI 1.0-8.5). The difference was more distinct analysing airway responsiveness by DRS, for which AMP predicted symptoms better than methacholine (P < 0.05). CONCLUSIONS: Pre-existent bronchial inflammation or a preinflammatory state marked by AMP (hyper)responsiveness increases the vulnerability to develop nasal symptoms.     

Relationships of methacholine and AMP responsiveness with peak expiratory flow variability in children with asthma

BACKGROUND: Both bronchial responsiveness (BR) and peak expiratory flow (PEF) variability are increased in asthma. PEF variability is presumed to reflect the degree of BR in asthma. BR is commonly assessed by bronchial challenges using direct or indirect stimuli. OBJECTIVE: The aim of this study was to compare methacholine and adenosine 5'-monophosphate (AMP) responsiveness with regard to their relationships with PEF variability in children with asthma. METHODS: Methacholine and AMP challenge tests were performed in 79 children with mild to moderate asthma, and a provocative concentration causing a 20% decline in forced expiratory volume in 1 s (PC(20)) was calculated for each challenge. Subjects recorded PEF each morning and each evening for 14 consecutive days. PEF variability was expressed as amplitude percentage mean (amp%mean; high PEF minus low PEF on each day, expressed as a percentage of their mean, averaged over 14 days), and as the lowest percentage highest (low%high; the lowest PEF expressed as a percentage of the highest PEF recorded over the period). RESULTS: Methacholine PC(20) correlated significantly but weakly with both indices of PEF variability (amp%mean: r=-0.285, P=0.011; low%high: r=0.238, P=0.034). However, there was a significant and strong correlation between AMP PC(20) and both amp%mean (r=-0.583, P=0.000) and low%high (r=0.496, P=0.000). For AMP PC(20), the correlations were stronger than for methacholine PC(20) (comparison of correlation coefficients with amp%mean: P=0.021; with low%high: P=0.063). CONCLUSION: Both methacholine PC(20) and AMP PC(20) correlated significantly with PEF variability. However, the stronger correlations for AMP PC(20) than for methacholine PC(20) suggest that PEF variability may be better reflected by BR assessed by AMP than by methacholine.     

Protective effect of oral terfenadine and not inhaled ipratropium on adenosine 5'-monophosphate-induced bronchoconstriction in patients with COPD

BACKGROUND: Inhalation of adenosine 5'-monophosphate (AMP) causes bronchoconstriction in patients with asthma and in many patients with chronic obstructive pulmonary disease (COPD). In asthma, AMP-induced bronchoconstriction has been shown to be determined mainly by release of mast cell mediators, and possibly by vagal nerve stimulation, since oral terfenadine (H1-receptor antagonist) and inhaled ipratropium bromide (muscarinic receptor antagonist) both increase PC20AMP. OBJECTIVE: To investigate the mechanism of AMP-induced bronchoconstriction in COPD. METHODS: We performed a randomized, double-blind, placebo-controlled, crossover trial. Forty-four nonatopic hyperresponsive smokers with COPD (mean age +/- SD: 60+/-7 years, FEV1 61+/-12% of predicted and FEV1/VC 51+/-8%, geometric mean [GM] PC20methacholine 0.62 mg/mL and GM PC20AMP 6.77 mg/mL) participated. PC20methacholine and PC20AMP were assessed on 3 days. Before the challenges they used either 180 mg of oral terfenadine, 120 microg of inhaled ipratropium bromide, or placebo. RESULTS: GM PC20AMP was 5.44 mg/mL after placebo, increasing with 0.9 doubling concentration (P<0.0001) after terfenadine and decreasing 0.3 doubling concentration after ipratropium bromide (NS). GM PC20methacholine was 0.75 mg/mL after placebo, increasing 0.4 doubling concentration after terfenadine (NS) and 3 doubling concentrations after ipratropium bromide (P<0.0001). CONCLUSION: These findings indicate that histamine release is important in the pathophysiology of AMP-induced bronchoconstriction in smokers with COPD, whereas vagal nerve stimulation does not play a role. Therefore, PC20AMP may be a valuable tool in evaluation of treatments which affect airway histamine release.     

Monitoring of seasonal variability in bronchial hyper-responsiveness and sputum cell counts in non-asthmatic subjects with rhinitis and effect of specific immunotherapy

Bronchial hyper-responsiveness (BHR) is documented in a proportion of non-asthmatic individuals with allergic rhinitis (NAAR) and reflects inflammatory events in the lower airways. Natural exposure to allergens is known to modulate BHR and the level of airway inflammation in asthma, but less consistently in NAAR. Specific immunotherapy (SIT) attenuates symptoms possibly by reducing BHR and airway inflammation. The influence of natural exposure to Parietaria pollen on BHR and sputum cell counts of NAAR was investigated and the effect of Parietaria SIT examined. Thirty NAAR, monosensitized to Parietaria judaica, participated in a randomized, double-blind, placebo-controlled, parallel group study of the effects of a Parietaria pollen vaccine on symptoms/medication score, BHR to inhaled methacholine and adenosine 5'-monophosphate (AMP), and cell counts in the sputum collected out of and during the pollen seasons for 36 months. Seasonal variation in BHR to inhaled methacholine and AMP and changes in sputum cell counts were documented. Changes were consistent for AMP, but not methacholine, and invariably associated with modifications in sputum eosinophils and epithelial cells. The clinical efficacy of Parietaria SIT was associated with a decline in the seasonal deterioration of BHR to AMP, whereas no significant effect was observed on BHR to methacholine or sputum cell differentials. Between-groups comparison of the seasonal changes in PC15 methacholine values and sputum cell differentials calculated as the AUC were not statistically significant, whereas a significant difference in PC15 AMP was demonstrated throughout the study (P=0.029), the median (inter-quartile range) AUC values being 2478.5 (1153.3-3600.0) and 1545.5 (755.3-1797.9) for the SIT- and placebo-treated group, respectively. Bronchial airways of NAAR exhibit features of active inflammation that deteriorate during natural allergen exposure, particularly with regard to BHR to AMP. The clinical efficacy of Parietaria SIT was exclusively associated with attenuation in seasonal worsening of PC15 AMP, suggesting that AMP may be useful in monitoring changes in allergic inflammation of the airways.     

Airway mast-cell activation in asthmatics is associated with selective sputum eosinophilia

BACKGROUND: Tryptase is a serine endoprotease selectively released from mast cells. Although mast cells are known to be activated after experimental allergic provocation, their role in naturally occurring asthma is still debated. METHODS: We have investigated the levels of tryptase in the whole induced sputum collected from 51 asthmatics (31 atopic and 20 intrinsic) seen in our outpatient clinic and 22 normal nonatopic healthy volunteers. Tryptase was measured by a new immunoassay based on B12 monoclonal antibody recognition of total tryptase (UniCAP System, Pharmacia) with a sensitivity of 1 ng/ml. RESULTS: While being below the threshold of detection in all normal volunteers, tryptase was detectable in the sputum from 9/51 asthmatics (18%) including five atopic and four intrinsic asthma cases. In these patients, among whom three were asymptomatic asthmatics, the values ranged between 1 and 6.1 ng/ml. The asthmatics with detectable sputum tryptase had greater sputum eosinophil counts (P<0.05) but lower neutrophil counts (P<0.05) than those in whom tryptase was undetectable. When compared to control subjects, asthmatics without tryptase had still greater eosinophil counts (P<0.0001) but also raised neutrophil counts (P<0.05). No significant difference could be found between asthmatics with tryptase and those without tryptase with respect to the age, the baseline lung function, the methacholine bronchial responsiveness, and the frequency of treatment with inhaled steroids. CONCLUSIONS: With the UniCAP System, tryptase was detectable in the sputum from 18% of asthmatics irrespective of atopy and current symptoms. Asthmatics with tryptase appeared to have a selective increase in sputum eosinophil counts while those without tryptase displayed a mixed sputum granulocyte infiltration with raised eosinophil and neutrophil counts.     

Effect of a 4-week treatment with theophylline on sputum eosinophilia and sputum eosinophil chemotactic activity in steroid-naive asthmatics

BACKGROUND: The precise mechanism of action of theophylline in asthma is not fully understood but recent data have drawn attention to its potential anti-inflammatory effect. OBJECTIVE: The purpose of this study was to assess the effect of theophylline on sputum eosinophilia and sputum eosinophil chemotactic activity in steroid-naive asthmatics. METHOD: We performed a 4-week randomized double-blind, placebo-controlled, parallel group study in 21 mild to moderate steroid-naive asthmatics whose sputum eosinophilia was found twice > 5% during the run in period. Eleven subjects received 600 mg/24 h theophylline for the first 2 weeks and 900 mg/24 h for the last 2 weeks while 10 subjects took a placebo for 4 weeks. Sputum was induced after 2 and 4 weeks of treatment and 1 week after stopping the treatment. The sputum samples were compared for their cell counts, eosinophil cationic protein (ECP) levels and eosinophil chemotactic activity using micro-Boyden chambers. RESULTS: Serum theophylline concentrations reached 7 and 11 microg/mL at V3 and V4, respectively. Intragroup comparisons showed that theophylline, but not placebo, caused a significant reduction in sputum eosinophil counts at V3 (62 +/- 10% from baseline, P < 0.01) and a strong trend at V4 (67 +/- 16% from baseline, P = 0.07) when compared to baseline. The intergroup difference obtained after comparing the area under the curve over the 4 week treatment period only approached the statistical significance (P = 0.08). At baseline the fluid phase of the sputum contained a significant eosinophil chemotactic activity which was inhibited after a 4-week treatment by theophylline (P < 0. 01) but not by placebo. The mean sputum theophylline levels after 4 weeks of treament (1.7 microg/mL) was lower than that required to cause significant inhibition of eosinophil chemotaxis in vitro. CONCLUSION: Theophylline decreases the natural sputum eosinophil chemotactic activity present in asthmatics. However, when using a small sample size, the 35% reduction in sputum eosinophilia achieved by theophylline failed to reach statistical significance when compared to that seen after placebo.     

Nasal and pharyngeal eosinophil peroxidase levels in adults with poorly controlled asthma correlate with sputum eosinophilia

The objective of the study was to compare nasal, pharyngeal, and sputum eosinophil peroxidase (EPX) levels with induced sputum eosinophil percentage in 10 adults with poorly controlled asthma and 10 normal controls. EPX was measured using an ELISA and normalized for grams of protein for nasal and pharynx specimens and for mL‐gram of protein for sputum. Sputum EPX levels were statistically different between asthma and control subjects (P = 0.024). EPX levels measured in the nasal and pharyngeal swab samples derived from the same patients were also different between asthma and control subjects, each displaying a high degree of significance (P = 0.002). Spearman's correlation coefficients for nasal EPX and pharyngeal EPX levels compared to induced sputum eosinophil percentage were 0.81 (P = 0.0007) and 0.78 (P = 0.0017), respectively. Thus, there is a strong association in a given patient between both nasal and pharyngeal EPX levels and the eosinophil percentage of induced sputum.     

Effects of adenosine 5'-monophosphate and adenosine 5'-triphosphate on functionally identified units in the cat spinal dorsal horn. Evidence for a differential effect of adenosine 5'-triphosphate on nociceptive vs non-nociceptive units

A study was done of the effects of iontophoretic application of adenosine 5'-monophosphate (AMP) and adenosine 5'-triphosphate (ATP) on functionally identified neurones in the spinal dorsal horn of the cat. AMP depressed nearly two-thirds of the 32 neurones tested regardless of functional type; the remainder were unaffected. ATP, on the other hand, had three types of effect: depression, excitation and a biphasic effect which consisted of excitation followed by depression. A significant difference was found when a comparison was made of the frequency of occurrence of each of these three types of effect in the samples of non-nociceptive (n = 18) and of wide dynamic range neurones (n = 42): of non-nociceptive neurones 61% were excited, 11% were depressed, 6% had a biphasic response and 22% were unaffected; of wide dynamic range neurones 45% had a biphasic response, 19% were depressed, 14% were excited and 21% were unaffected (chi 2 = 16.2, P less than 0.005). The depressant effects of both AMP and ATP and the depressant phase of the biphasic effect of ATP seem to be mediated through activation of P1-purinergic receptors because these effects were blocked by theophylline, a P1-purinergic antagonist [Burnstock (1978) In Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, pp. 107-118]. Thus the biphasic effect appears to consist of excitatory and depressant responses in the same neurone. The differential effects of ATP on non-nociceptive vs wide dynamic-range neurones are similar to the differential effects on these neurones observed during activation of low-threshold primary afferents. This similarity, together with evidence that ATP can be released from primary afferent neurones [Holton and Holton (1954) J. Physiol., Lond. 126, 124-140; Holton (1959) J. Physiol., Lond. 145, 494-504], prompts us to suggest that ATP may be a chemical mediator of effects of low-threshold primary afferent inputs in the spinal dorsal horn.     

Asthma control,quality of life and successful sputum induction

Introduction

Induced sputum is widely used in clinical practice and scientific studies. This technique has become enormously useful in assessment of airway inflammation. However, some asthmatics are unable to expectorate sputum of sufficient quality and quantity necessary for further processing, therefore not providing reliable results. This research study aimed to examine whether asthma control and asthma quality of life influence the results of sputum induction.

Material and methods

Fourty-seven adult subjects, current non-smokers with symptomatic asthma, were studied. All participants underwent clinical assessment, skin prick testing, spirometry and sputum induction. Before sputum induction, subjects were asked to fill in the Mini Asthma Quality of Life Questionnaire (MiniAQLQ) and Asthma Control Questionnaire (ACQ).

Results

Twenty-nine (62%) subjects produced sputum eligible for processing. This group had a significantly lower ACQ score (0.83 ±0.65 vs. 1.37 ±0.77; p = 0.02), higher MiniAQLQ total score (5.67 ±0.99 vs. 4.86 ±1.07; p = 0.011), higher MiniAQLQ symptoms domain score (5.54 ±1.13 vs. 4.63 ±1.24; p = 0.013) and higher MiniAQLQ activity limitations domain score (6.08 ±0.92 vs. 5.07 ±1.37; p= 0.014). The noted differences between groups of patients were not only statistically but were clinically important.

Conclusions

The study results suggest that successful sputum induction may be expected in patients with better asthma control and better quality of life.     

Activation of mast cells by immunoglobulin E-receptor cross-linkage,but not through adenosine receptors,induces A1 expression and promotes survival

BACKGROUND: Mast cells are a potent source of mediators that regulate the inflammatory response in allergy and asthma. Mast cells can be activated through different receptors, for example, via cross-linkage of the high-affinity IgE receptor (Fc epsilon RI) and by adenosine acting on specific receptors. We have recently described mast cell survival of an IgE receptor activation by up-regulation of the anti-apoptotic gene A1. OBJECTIVE: To compare mast cell survival and expression of A1 after activation through the Fc epsilon RI and by an adenosine agonist. METHODS: Bone marrow-derived, cultured mouse mast cells (BMCMC) were activated either with IgE+antigen or with the adenosine receptor agonist 5'-N-ethylcarboxamido adenosine (NECA). Release of beta-hexosaminidase, cell viability, phosphorylation of Akt and IkB-alpha, and expression of pro-survival and pro-apoptotic genes were measured after activation. RESULTS: Activation of BMCMC with NECA caused the release of beta-hexosaminidase, although to a lesser extent than after Fc epsilon RI activation (33% and 98%, respectively). Activation by both NECA and Fc epsilon RI stimulated phosphorylation of Akt (Ser473 and Thr308) and IkB-alpha (Ser32), both of which are implicated in the regulation of cell survival. However, only cells that were activated through Fc epsilon RI, but not by NECA, expressed A1 and exhibited an increased survival rate compared to the control. CONCLUSION: These results show that adenosine receptor activation of BMCMC does not induce the same survival programme in mast cells as does activation through Fc epsilon RI. These findings may be important for understanding the role that mast cells play in asthma provoked by different stimuli.     

Transient contribution of mast cells to pulmonary eosinophilia but not to hyper‐responsiveness

BACKGROUND: We have recently demonstrated that the transfer of interleukin (IL)-5-producing CD4+ T cell clones into unprimed mice is sufficient for the development ofeosinophilic inflammation in the bronchial mucosa upon antigen inhalation. OBJECTIVE: The aim of this study was to elucidate the possible contribution of mast cells in eosinophilic inflammation and bronchial hyper-responsiveness (BHR), and to discriminate between the roles of CD4+ T cells and mast cells. METHODS: Mast cell-deficient mice (WBB6F1-W/Wv) and their congenic normal littermates (WBB6F1-+/+) were immunized with ovalbumin and challenged by inhalation with the relevant antigen. RESULTS: Airway eosinophilia was induced with equivalent intensity in +/+ and W/Wv mice 6, 24, 96 and 216 h after antigen inhalation. In contrast, 48 h after antigen challenge, eosinophilic infiltration into the bronchial mucosa was significantly less pronounced in W/Wv mice than in +/+ mice. Anti-CD4 monoclonal antibody (mAb), anti-IL-5 mAb, and cyclosporin A were administered next, demonstrating that the airway eosinophilia of W/Wv mice induced 48 h after antigen challenge was almost completely inhibited by each of these three treatments, but that of +/+ mice was significantly less susceptible. Bronchial responsiveness to acetylcholine was increased 48 h after antigen challenge and was not significantly different between +/+ and W/Wv mice. Administration of anti-IL-5 mAb completely inhibited the development of BHR in both +/+ and W/Wv mice. CONCLUSION: These results indicate that, in mice, mast cells do have a supplemental role in the development of pulmonary eosinophilia but not BHR. CD4+ T cells totally regulate these responses by producing IL-5.     

Induction of polyploidy in human lymphocytes in vitro by excess adenine,but not by adenosine

It is known that high levels of DNA precursors can be both clastogenic and mutagenic in cultured cell lines and in vivo. The purpose of the present study was to examine at an observational level the cytogenetic effects of adenine and adenosine in primary human cell cultures. Human peripheral blood lymphocytes from four donors were cultured and treated with a range of concentrations of adenine and adenosine. Although no increase in sister chromatid exchange (SCE) frequency was observed with either compound, there was a statistically significant, dose-related increase in the proportion of polyploid cells in cultures treated with adenine, but not in those treated with adenosine. Some of the polyploid metaphases found after adenine treatment contained diplochromosomes, suggesting that endoreduplication might have been involved in polyploid formation in these cells. It is concluded that a high level of adenine can cause genetic changes in human lymphocytes by interfering with mitosis, perhaps by disturbing the balance of DNA precursor pools. © 1995 Wiley-liss, Inc.     

Comparison between hypertonic and isotonic saline-induced sputum in the evaluation of airway inflammation in subjects with moderate asthma

Background Hypertonic saline-indueed sputum has recently been used for the evaluation of airway inflammation in asthma. Objective To assess the effect of hypertonicity on airway inflammation. Methods We compared the inflammatory cell composition of hypertonic saline-induced sputum with that of isotonic saline-induced sputum in 21 asthmatic subjects and, at baseline and 30min after each sputum induction, we measured bronchial hyper-responsiveness to methacholine as an indirect marker to detect increased airway inflammation. On two different days, the patients inhaled hypertonic saline (3–5% NaCl) or isotonic saline (0.9% NaCl) for 30 min via an ultrasonic nebulizer, while monitoring FEV₁. Sputum was collected for inflammatory cell analysis. Results There was no difference in inflammatory cell percentages obtained with the two methods. Eosinophils were >1% in 20 subjects after hypertonic saline and in 16 subjects after isotonic saline, but this difference was not statistically significant. Intraclass correlation coefficients for sputum inflammatory cells obtained with the two methods were +0.642 for eosinophils, +0.644 for neutrophils. +0.544 for lymphocytes and +0.505 for macrophages. Hypertonic saline induced bronchoconstriction in a significantly greater number of subjects than isotonic saline. Also, hypertonic saline increased bronchial responsiveness to methacholine. while isotonic saline did not. Conclusion We conclude that hypertonicity does not affect sputum cell composition, suggesting that inflammatory cells in hypertonic saline-induced sputum are probably preexisting and not acutely recruited in the airways by the hypertonic stimulus. However, the bronchoconstriction and the increase in bronchial hyper-responsiveness after hypertonic saline inhalation may imply the release of inflammatory mediators. This fact must be considered in the evaluation of soluble markers of inflammation in hypertonic salineinduced sputum.