T regulatory cell phenotypes in peripheral blood and bronchoalveolar lavage from non-asthmatic and asthmatic subjects.

BACKGROUND: An unresolved issue in T regulatory cells' cell biology is the lack of consensus on phenotypic markers that accurately define the natural Treg (nTreg) population. OBJECTIVES: To examine nTreg frequency and functional capacity in healthy controls and their frequency in asthmatic subjects using three different phenotypic strategies. We hypothesized that phenotypically different nTreg are quantitatively and functionally different. METHODS: Thirty-four healthy, non-asthmatic and 17 asthmatic subjects were studied. Three nTreg phenotypes were defined as follows: nTreg1 (CD4(+) CD25(+) Foxp3(+) ), nTreg2 (CD4(+) CD25(+) CD127(low) Foxp3(+) ), and nTreg3 (CD4(+) CD25(high) Foxp3(+) ). The flow cytometric determination of nTreg frequency in peripheral blood (PB) and bronchoalveolar lavage (BAL) was performed using fluorescently labelled antibodies. Peripheral blood nTreg functional capacity was assessed using a CFSE-based suppression assay. RESULTS: There was a significantly lower frequency of PB nTreg3 compared to nTreg2 and nTreg1 (P < 0.05). Both nTreg2 and nTreg3 had a significantly greater suppressive capacity than nTreg1 at T responder (Tresp) to nTreg ratios of 16 : 1 up to 1 : 1 (P < 0.01). Asthmatics exhibited a significantly lower PB nTreg3 and nTreg1 frequency than healthy controls (P < 0.05). There were no differences between healthy controls and asthmatic subjects when comparing BAL nTreg frequency. CONCLUSIONS AND CLINICAL RELEVANCE: Phenotypically different nTreg subsets are quantitatively and functionally different and are variably observed in asthma. The CD4(+) CD25(high) Foxp3(+) phenotype was the least frequent, but demonstrated the greatest suppression, and was significantly lower in PB of asthmatic subjects. Consequently, it is imperative that nTreg phenotypes be clearly defined and that the interpretation of their frequency and function be phenotype specific.

The effects of inhaled house dust mite on airway barrier function and sensitivity to inhaled methacholine in mice.

Asthma is functionally characterized by increased airway sensitivity and reactivity. Multiple mechanisms are believed to underlie these functional disorders, including impairment of airway wall barrier function. One proposed mechanism of impaired barrier function is through the direct consequence of proteolytic properties of inhaled allergens, including house dust mite (HDM). Here, we have observed the direct effects of HDM on airway barrier function and response to nebulized or intravenous methacholine. HDM naïve BALB/c mice were anesthetized, exposed to intranasal or intratracheal HDM (15 or 100 µg), and allowed to recover for 30 min or 2 h before methacholine challenge. A separate group of mice was exposed to intratracheal poly-L-lysine (PLL; 100 µg) for a duration of 30 min. This group served as a positive control for the presence of impaired barrier function and airway hypersensitivity. Negative control mice received saline challenges. Outcomes included assessment of lung mechanics in response to nebulized or intravenous methacholine as well as clearance of intratracheally instilled technetium-labeled ((99m)Tc) DTPA to evaluate airway epithelial barrier function. We found that PLL produced a leftward shift in the dose-response curve following nebulized but not intravenous methacholine challenge. This was associated with a significantly faster clearance of (99m)Tc-DTPA, indicating impairment in airway barrier function. However, HDM exposure did not produce changes in these outcomes when compared with saline-exposed mice. These findings suggest that direct impact on airway barrier function does not appear to be a mechanism by which HDM produces altered airway sensitivity in airway disease.

Modulating progenitor accumulation attenuates lung angiogenesis in a mouse model of asthma.

Asthmatic responses are associated with the lung homing of bone marrow (BM)-derived progenitors implicated as effectors of disease pathology. Studies have shown that increases in lung extracted vascular endothelial progenitor cells (VEPCs) correlate with airway angiogenesis and declining lung function. We investigated the effect of modulating lung homing of VEPCs on tissue remodelling and airway hyperresponsiveness (AHR). BALB/c mice were sensitised to ovalbumin, subjected to a chronic exposure protocol and given early concurrent or delayed treatment with a modulator of progenitor traffic, AMD3100 (CXC chemokine receptor 4 antagonist; inhibits chemotactic activity of stromal-derived factor-1α on VEPCs). After ovalbumin challenge, early haemopoietic stem cells (HSCs) and VEPCs were enumerated along with indices of airway inflammation, lung morphometry and AHR. Following ovalbumin challenge, there was a decrease in BM and an associated increase in the lung tissue-extracted HSCs and VEPCs, together with increases in airway eosinophilia, microvessel density and AHR. These outcomes were significantly inhibited by early concurrent treatment with AMD3100. Where lung disease was established, delayed treatment with AMD3100 significantly attenuated HSC numbers and lung angiogenesis but only partially reversed sustained AHR compared with untreated ovalbumin-exposed mice. Progenitor lung homing is associated with the development of asthma pathology, and early modulation of this accumulation can prevent airway remodelling and lung dysfunction.

Effects of furosemide on allergic asthmatic responses in mice.

BACKGROUND: The syndrome of allergic asthma features reversible bronchoconstriction, airway inflammation and hyperresponsiveness as well as airway remodelling, including goblet cell hyperplasia. Managing severe asthma is still a clinical challenge. Numerous studies report that furosemide, an inhibitor of Na(+)-K(+)-Cl(-) cotransporter (NKCC) reduces airway hyperresponsiveness (AHR) in asthmatic patients. However, the mechanism by which furosemide exerts anti-asthmatic action remains unclear. OBJECTIVE: This study sought to investigate the cellular profile of NKCC1 expression in the lung and examine the effects of furosemide on several outcome measurements in a mouse model of allergic asthma. METHODS: Mice were sensitized and challenged with ovalbumin (OVA). Before challenge, the OVA-sensitized mice were treated with furosemide (4.0 mg/kg/day, via daily intraperitoneal injection for 5 days). Outcome measurements in naïve, OVA-exposure, furosemide-treated naïve and furosemide-treated OVA-exposed mice included the slope of the relationship between inhaled methacholine (MCh) concentration and respiratory system resistance (Slope·R(RS)), bronchoalveolar lavage (BAL) cell counts and immunohistochemical and immunoblotting assays of lung tissues. RESULTS: NKCC1 immunoreactivity was observed in airway epithelial cells (AECs) and alveolar type II (ATII) cells of the control mice. OVA exposure enhanced the expression of NKCC1 in AECs and ATII cells, and increased the infiltration of NKCC1-expressing T lymphocytes in the lung. NKCC1 immunoreactivity was not detected in the airway smooth muscle (ASM) cells. Furosemide treatment reduced the Slope·R(RS) in both naïve and OVA-exposed mice by about 50%. Furosemide treatment also increased T lymphocyte infiltration to the lung in OVA-exposed mice by approximately 53%, but had no effect on pulmonary goblet cell hyperplasia. CONCLUSIONS AND CLINICAL RELEVANCE: Furosemide decreases basal airway responsiveness, thereby reducing the extent of allergen-induced AHR. However, the same treatment also increases T lymphocytes infiltration in the course of allergic asthma. Further studies are necessary to address the usefulness of furosemide in the clinical treatment of asthma.

The role of interleukin-4Ralpha in the induction of glutamic acid decarboxylase in airway epithelium following acute house dust mite exposure.

Background Asthma is a disease characterized by airway inflammation, remodelling and dysfunction. Airway inflammation contributes to remodelling, a term that is used to describe structural changes including goblet cell metaplasia (GCM), matrix deposition, and smooth muscle hyperplasia/hypertrophy. GCM has been implicated in asthma mortality by contributing to mucus plugs and leading to asphyxiation. In animal models, this process is highly dependent on IL-13. Recently, we have described an IL-13-dependent up-regulation of a GABAergic signalling system in airway epithelium that contributes to GCM. The mechanism by which IL-13 up-regulates GABA signalling in airway epithelium is unknown. Objectives To test the hypothesis that IL-4Ralpha signalling is required for allergen induced up-regulation of GABAergic signalling and GCM. Methods BALB/c mice were exposed to an acute house dust mite (HDM) protocol and received vehicle, anti-IL-4Ralpha-monoclonal antibody, or control antibody. Outcomes included airway responses to inhaled methacholine (MCh), histology for eosinophilia and GCM, phosphorylated STAT6 levels using immunohistochemistry and immunoblot, and glutamic acid decarboxylase (GAD) 65/67 and GABA(A)beta(2/3) receptor subunit expression using confocal microscopy. Results Acute HDM exposure resulted in increased airway responses to MCh, lung eosinophilia, STAT6 phosphorylation, elevations in GAD65/67 and GABA(A)beta(2/3) receptor expression, and GCM that were inhibited with anti-IL-4Ralpha-monoclonal treatment. Control antibody had no effect. Conclusion The IL-4Ralpha is required for allergen-induced up-regulation of a GABAergic system in airway epithelium implicated in GCM following acute HDM exposure.

Effect of asthma treatment on fitness, daily activity and body composition in children with asthma.

BACKGROUND: Although several cross-sectional studies have assessed the daily physical activity in children with asthma, the impact of the level of asthma control remains unknown. AIM: To assess the influence of asthma treatment-induced changes in asthma control on daily physical activity, cardiovascular fitness and body composition in children with asthma. METHODS: Daily accelerometer-measured physical activity, cardiovascular fitness, body composition (percent fat, percent lean tissue and bone mineral density) and a variety of asthma outcomes (to assess the level of asthma control) were measured over 4 weeks in 55 children with newly diagnosed untreated asthma and 154 healthy, sex and age-matched controls. Treatment with inhaled corticosteroids was initiated after the baseline period. All outcome measurements were repeated after 1 year and some also during the year of treatment. RESULTS: Asthma control improved markedly during the year of treatment. The improvement in control was associated with a significant increase in total daily activity of 2.8 h/week compared with the healthy controls (P < 0.001). In addition, significant increases were seen in moderate-vigorous activity (33 min/week; P = 0.01) and in cardiovascular fitness (1.2 ml O2/min*kg) compared with the healthy controls. The improvement in activity was mainly seen during the last 6 month of the study. No difference was seen between the two groups in changes in percent body fat. CONCLUSION: Poorly controlled asthma is associated with reduced physical activity and cardiovascular fitness. Improvement in asthma control is associated with a clinically relevant increase in daily physical activity and cardiovascular fitness.

Gamma-aminobutyric acid nurtures allergic asthma.

Asthma often occurs as a result of immune-based inflammatory responses, which consequently cause pathological changes in airway structural cells. However, the underlying mechanisms of airway pathology in asthma are still not fully understood. Our recent studies revealed a critical role of gamma-aminobutyric acid (GABA) signalling pathway in the airway epithelium of allergic asthma through its ability to stimulate mucus production. This review briefly describes the GABAergic signalling system and its role in the regulation of mucus protein production in bronchial airway epithelial cells.

Role of STAT6 and SMAD2 in a model of chronic allergen exposure: a mouse strain comparison study.

BACKGROUND: Asthma is a disease characterized by variable and reversible airway obstruction and is associated with airway inflammation, airway remodelling (including goblet cell hyperplasia, increased collagen deposition and increased smooth muscle mass) and increased airway responsiveness. It is believed that airway inflammation plays a critical role in the development of airway remodelling, with IL-13 and TGF-beta1 pathways being strongly associated with the disease progression. Mouse models of asthma are capable of recapitulating some components of asthma and have been used to look at both IL-13 and TGF-beta1 pathways, which use STAT6 and SMAD2 signalling molecules, respectively. OBJECTIVES: Using brief and chronic models of allergen exposure, we utilized BALB/c and C57Bl/6 to explore the hypothesis that observed differences in responses to allergen between these mouse strains will involve fundamental differences in IL-13 and TGF-beta1 responses. METHODS: The following outcome measurements were performed: airway physiology, bronchoalveolar lavage cell counts/cytokine analysis, histology, immunoblots and gene expression assays. RESULTS: We demonstrate in BALB/c mice an IL-13-dependent phosphorylation of STAT6, nuclear localized in inflammatory cells, which is associated with indices of airway remodelling and development of airway dysfunction. In BALB/c mice, phosphorylation of SMAD2 is delayed relative to STAT6 activation and also involves an IL-13-dependent mechanism. In contrast, despite an allergen-induced increase in IL-4, IL-13 and eosinophils, C57Bl/6 demonstrates a reduced and distinct pattern of phosphorylated STAT6, no SMAD2 phosphorylation changes and fail to develop indices of remodelling or changes in airway function. CONCLUSION: The activation of signalling pathways and nuclear translocation of signalling molecules downstream of IL-13 and TGF-beta1 further support the central role of these molecules in the pathology and dysfunction in animal models of asthma. Activation of signalling pathways downstream from IL-13 and TGF-beta1 may be more relevant in disease progression than elevations in airway inflammation alone.

Budesonide prevents but does not reverse sustained airway hyperresponsiveness in mice.

Despite the effectiveness of corticosteroids at resolving airway inflammation, they are only moderately effective at attenuating airway hyperresponsiveness (AHR). The extent to which corticosteroids are able to reverse or inhibit the development of sustained AHR is not known. The present study aimed to determine whether budesonide can resolve and or prevent the development of sustained AHR in mice. Mice were chronically exposed to allergen and treated with budesonide either: 1) briefly during the final weeks of exposure to allergen; 2) prolonged concurrently throughout exposure to allergen; or 3) delayed following final exposure to allergen. AHR was assessed 24 h (brief treatment) or 4 weeks (prolonged concurrent and delayed treatments) following final exposure to allergen. Brief budesonide intervention significantly attenuated the inflammation-associated AHR assessed immediately following final exposure to allergen. Similarly, prolonged concurrent budesonide treatment prevented the development of sustained AHR. Delayed budesonide intervention, however, did not resolve sustained AHR. In conclusion, the early introduction and, importantly, the persistence of corticosteroid treatment prevented the development of sustained airway hyperresponsiveness; however, the inability of corticosteroids to reverse established airway dysfunction indicates a limitation in their use for the complete, long-term management of airway hyperresponsiveness.

Effects of allergen on airway narrowing dynamics as assessed by lung-slice technique.

Asthma is characterised by an excessive airway narrowing in response to a variety of stimuli, called airway hyperresponsiveness (AHR). Previous comparisons between mouse strains have shown that increased velocity of airway narrowing correlates with baseline airway responsiveness. These data prompted the investigation into models of induced AHR to see whether airway narrowing dynamics correlated with in vivo responsiveness. In an attempt to reproduce some of the features of asthma, BALB/c mice were sensitised and subjected to either brief or chronic periods of allergen exposure. Brief exposure involved two challenges with intranasal chicken egg ovalbumin (OVA(in)). Chronic exposure involved six 2-day periods of OVA(in) challenges, each separated by 12 days. Control mice received intranasal saline challenges. Outcomes included videomicrometry of lung slices (magnitude and velocity of airway narrowing), in vivo respiratory physiology measurements and histological staining with morphometric analysis. Neither brief nor chronic allergen exposure resulted in greater airway narrowing and increased velocity compared with saline controls. Structural changes in the airway, such as goblet cell hyperplasia, subepithelial fibrosis and increased contractile tissue, were detected in mice chronically challenged with allergen. In conclusion, increased responsiveness to methacholine following allergen challenge may not be due to an intrinsic change to the smooth muscle per se, but rather to other changes in the lung, which ultimately manifest as an increase in respiratory resistance.

Circulating, but not local lung, IL-5 is required for the development of antigen-induced airways eosinophilia.

IL-5 is induced locally in the lung and systemically in the circulation during allergic airways eosinophilic inflammation both in humans and experimental animals. However, the precise role of local and systemic IL-5 in the development of allergic airways eosinophilia remains to be elucidated. In our current study, we demonstrate that compared with their IL-5(+/+) counterparts, IL-5(-/-) mice lacked an IL-5 response both in the lung and peripheral blood, yet they released similar amounts of IL-4, eotaxin, and MIP-1alpha in the lung after ovalbumin (OVA) sensitization and challenge. At cellular levels, these mice failed to develop peripheral blood and airways eosinophilia while the responses of lymphocytes, neutrophils, and macrophages remained similar to those in IL-5(+/+) mice. To dissect the relative role of local and systemic IL-5 in this model, we constructed a gene transfer vector expressing murine IL-5. Intramuscular IL-5 gene transfer to OVA-sensitized IL-5(-/-) mice led to raised levels of IL-5 compartmentalized to the circulation and completely reconstituted airways eosinophilia upon OVA challenge, which was associated with reconstitution of eosinophilia in the bone marrow and peripheral blood. Significant airways eosinophilia was observed for at least 7 d in these mice. In contrast, intranasal IL-5 gene transfer, when rendered to give rise to a significant but compartmentalized level of transgene protein IL-5 in the lung, was unable to reconstitute airways eosinophilia in OVA-sensitized IL-5(-/-) mice upon OVA-challenge, which was associated with a lack of eosinophilic responses in bone marrow and peripheral blood. Our findings thus provide unequivocal evidence that circulating but not local lung IL-5 is critically required for the development of allergic airways eosinophilia. These findings also provide the rationale for developing strategies to target circulating IL-5 and/or its receptors in bone marrow to effectively control asthmatic airways eosinophilia.

A pathogenic role for tumor necrosis factor-related apoptosis-inducing ligand in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory respiratory disorder, often induced by cigarette smoke (CS) exposure. The development of effective therapies is impaired by a lack of understanding of the underlining mechanisms. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with inflammatory and apoptotic properties. We interrogated a mouse model of CS-induced experimental COPD and human tissues to identify a novel role for TRAIL in COPD pathogenesis. CS exposure of wild-type mice increased TRAIL and its receptor messenger RNA (mRNA) expression and protein levels, as well as the number of TRAIL(+)CD11b(+) monocytes in the lung. TRAIL and its receptor mRNA were also increased in human COPD. CS-exposed TRAIL-deficient mice had decreased pulmonary inflammation, pro-inflammatory mediators, emphysema-like alveolar enlargement, and improved lung function. TRAIL-deficient mice also developed spontaneous small airway changes with increased epithelial cell thickness and collagen deposition, independent of CS exposure. Importantly, therapeutic neutralization of TRAIL, after the establishment of early-stage experimental COPD, reduced pulmonary inflammation, emphysema-like alveolar enlargement, and small airway changes. These data provide further evidence for TRAIL being a pivotal inflammatory factor in respiratory diseases, and the first preclinical evidence to suggest that therapeutic agents that target TRAIL may be effective in COPD therapy.

Oxygen uptake kinetics from ramp work tests: variability of single test values.

The variability in the estimation of the mean response time (MRT) of O2 uptake (VO2) kinetics from single ramp work rate exercise tests was examined in six repetitions by five fit subjects. Work rate increased at 50 W/min from a base line of 25 W to a work rate of 120% ventilatory threshold. Breath-by-breath data were analyzed by linear regression from 2 min after the onset of the ramp to the 120% work rate. Individual subjects showed approximately twofold differences in estimates of MRT; the coefficient of variation from individuals ranged from 18.5 to 29.3%. The MRT obtained as the mean from the individual repetitions did not differ from the MRT obtained from pooled within-subject data. Analysis of variance on the individual MRT estimates showed 53.9% of the variability was attributable to the slope of the regression, whereas only 2.4% could be attributed to baseline VO2. It was concluded that several repetitions of the ramp work rate tests should be pooled prior to estimation of kinetics parameters.

Comparison of cardiac output during exercise by single-breath and CO2-rebreathing methods.

Cardiac output (Q) was estimated in supine rest and in upright cycling at several work rates up to 200 W in five male and one female subjects. At least four repetitions of both the CO2-rebreathing plateau method (Collier, J. Appl. Physiol. 9:25-29, 1956) and the Kim et al. (J. Appl. Physiol. 21: 1338-1344, 1966) single-breath method were performed at each work rate, in a steady state of O2 consumption and heart rate. At supine rest and low work rates, estimates of Q were similar by the two methods. However, at higher work rates, the single-breath method significantly (P less than 0.05) underestimated the value obtained by CO2 rebreathing. The reason for the difference in estimates of Q by the two methods was traced to the determination of arterial partial pressure of CO2 (PaCO2) and mixed venous partial pressure of CO2 (PvCO2). The estimate of PaCO2 from the single-breath method was approximately 88.5% of the estimate from end-tidal PCO2 used with the rebreathing method (P less than 0.001). The oxygenated PvCO2 calculated from the single-breath Q averaged approximately 92.5% of the PvCO2 from CO2 rebreathing (P less than 0.0001). The difference in estimates of Q was not eliminated by using a logarithmic form of the CO2 dissociation curve with the single-breath method.

Estimate of mean tissue O2 consumption at onset of exercise in males.

A mathematical model has been developed that permitted the calculation of the flow-weighted mean tissue O2 consumption (VO2T) at the onset of a step increase in work rate. From breath-by-breath measurements of alveolar O2 consumption (VO2A) and cardiac output (Q) by impedance cardiography and assumptions about the site of depletion of O2 stores, the rate of change in O2 stores (VO2s) was determined. The sum of VO2A + VO2s = VO2T. Six very fit males performed six repetitions of each of two step increases in work rate. STlo was a transition from rest to 100-W cycling; SThi was a transition from 100- to 200-W cycling. For each work rate transition, the responses of VO2A and Q were averaged over the six repetitions of each subject and the model was solved to yield VO2T. The responses of VO2A, VO2T, and Q after the increase in work rate were fit with a monoexponential function. This function included a time constant and time delay, the sum of which gave the mean response time (MRT). In the STlo test, the MRT of VO2A (24.9 +/- 1.1 s, mean +/- SE) was longer than that of VO2T (15.3 +/- 1.3 s) and of Q (16.5 +/- 6.5 s) (P less than 0.05). The MRT of VO2T and Q did not differ significantly. Also for SThi, the MRT of VO2A (34.4 +/- 3.3 s) was significantly longer than that of VO2T (30.0 +/- 3.4 s) (P less than 0.05). The MRT of VO2T and Q (30.3 +/- 5.5 s) were not significantly different at this work rate either.(ABSTRACT TRUNCATED AT 250 WORDS)

Inspiratory muscles during exercise: a problem of supply and demand.

The capacity of inspiratory muscles to generate esophageal pressure at several lung volumes from functional residual capacity (FRC) to total lung capacity (TLC) and several flow rates from zero to maximal flow was measured in five normal subjects. Static capacity was 126 +/- 14.6 cmH2O at FRC, remained unchanged between 30 and 55% TLC, and decreased to 40 +/- 6.8 cmH2O at TLC. Dynamic capacity declined by a further 5.0 +/- 0.35% from the static pressure at any given lung volume for every liter per second increase in inspiratory flow. The subjects underwent progressive incremental exercise to maximum power and achieved 1,800 +/- 45 kpm/min and maximum O2 uptake of 3,518 +/- 222 ml/min. During exercise peak esophageal pressure increased from 9.4 +/- 1.81 to 38.2 +/- 5.70 cmH2O and end-inspiratory esophageal pressure increased from 7.8 +/- 0.52 to 22.5 +/- 2.03 cmH2O from rest to maximum exercise. Because the estimated capacity available to meet these demands is critically dependent on end-inspiratory lung volume, the changes in lung volume during exercise were measured in three of the subjects using He dilution. End-expiratory volume was 52.3 +/- 2.42% TLC at rest and 38.5 +/- 0.79% TLC at maximum exercise.(ABSTRACT TRUNCATED AT 250 WORDS)

Management of exercise-induced bronchoconstriction.

Exercise-induced bronchoconstriction (EIB) is a common clinical manifestation of asthma, occurring in 70% to 80% of asthmatics. Evidence suggests that exercise and the ensuing bronchoconstriction do not contribute to a worsening of asthmatic inflammation. Asthmatics should not be discouraged from exercising, and, with adequate management, most patients should be able to exercise regularly with only minor symptoms. The first step in the management of patients with EIB should be to obtain optimum control of the underlying asthma, often requiring regular treatment with inhaled steroids. Regular treatment with inhaled corticosteroids usually reduces the extent of EIB by 50% or more. Frequently, despite optimal management of the underlying asthma, patients develop EIB symptoms requiring additional treatment. Short and long acting inhaled beta2-agonists are highly effective at reducing the magnitude of EIB, although there are concerns that the extent of protection diminishes during periods of regular use of these agents. Inhaled cromolyn and nedocromyl are effective at reducing the extent of EIB in some patients, although this protection does not extend beyond 2 to 3 h after treatment. The recently developed leukotriene receptor antagonists are effective at reducing the extent of EIB by 40% to 70%, and have the advantage that this protection lasts throughout the day and does not appear to diminish with regular use. Other agents, including anticholinergics and antihistamines, have been shown to offer partial protection against EIB, suggesting the possibility of using a combination treatment to manage some patients' symptoms. Finally, there is encouraging evidence suggesting that modifications of the pattern of exercise can markedly reduce the extent of EIB.

Protective effects of fluticasone on allergen-induced airway responses and sputum inflammatory markers.

BACKGROUND: A direct comparison of the protective effects of single and regular doses of inhaled glucocorticoid on allergen-induced asthmatic responses and inflammation has not been made. OBJECTIVE: To compare the effects of pretreatment with fluticasone 250 microg 30 min before allergen inhalation and two weeks of 250 microg twice daily (last dose 24 h before challenge) with single and regular (twice daily) placebo doses on early and late asthmatic responses, induced sputum cell counts and measures of eosinophil activation at 7 h and 24 h, and methacholine airway responsiveness at 24 h. PATIENTS AND METHODS: Ten mild asthmatic patients were studied in a randomized, double-blind, placebo controlled crossover study. RESULTS: Regular fluticasone increased the baseline mean provocative concentration of methacholine to cause a 20% fall (PC20) in forced expiratory volume in 1 s (FEV1) from 2.6 to 6.4 mg/mL (P<0.05) and lowered the eosinophil count from 3.1% to 0.4% (P<0.05) compared with regular placebo. Neither single nor regular fluticasone had any effect on the early asthmatic response. Single fluticasone attenuated the late asthmatic response, the mean +/- SEM maximum percentage fall in FEV1 (10.8+/-3.6 compared with single placebo 18. 8+/-3.5, P=0.03), the allergen-induced increase of airway responsiveness (P<0.05), and the eosinophilia (P<0.005) and activated eosinophils at 7 h (P<0.01) but not at 24 h. Regular fluticasone also attenuated the late asthmatic response (11.1+/-2.5) compared with regular placebo (19.6+/-4.5), but this was not statistically significant and did not protect against the induced increase in airway responsiveness or the sputum eosinophilia. CONCLUSION: Two weeks of regular inhaled fluticasone discontinued 24 h before allergen challenge does not offer any additional protection against the early or late asthmatic responses, increased airway responsiveness or sputum eosinophilia compared with a single dose of 250 microg immediately before allergen challenge, despite increasing baseline PC20 and decreasing sputum eosinophilia prechallenge. The significance of the protective effect of a single dose of inhaled steroid before an allergen inhalation and the duration of the protective effect need further investigation.

Dose-response protective effect of salbutamol on methacholine airway responsiveness using pressurized metered dose inhalers and Turbuhalers.

The purpose of this study was to estimate the relative dose potency of salbutamol Turbuhaler compared with salbutamol pressurized metered dose inhaler (pMDI) with respect to the protective effect against methacholine bronchoconstriction. Twenty-three asthmatic subjects with stable asthma participated in the study. Baseline forced expiratory volume in 1 s (FEV1) was 70% or more of predicted, and baseline methacholine provocative concentration causing a 20% fall in FEV1 (PC20) was 4 mg/mL or less. The design was randomized, double-blind, double-dummy, crossover and placebo controlled and was conducted over seven study days. On each study day, the subjects inhaled 50 microg or 100 microg of salbutamol via Turbuhaler, 100 microg, 200 microg, 400 microg or 800 microg of salbutamol via pMDI, or placebo in randomized order. PC20 was determined 30 mins after inhalation. Increasing doses of salbutamol pMDI increased the PC20 in a dose-dependent fashion from 3.9 mg/mL after placebo to 13.3 mg/mL after pMDI 100 microg, 19.0 mg/mL after 200 microg, 32.6 mg/mL after 400 microg, and 35.1 mg/mL after 800 microg. The half-maximum response dose for pMDI (ED50) was 104 microg. Salbutamol Turbuhaler 50 microg increased the PC20 to 10.0 mg/mL and 100 microg to 12.6 mg/mL. Salbutamol pMDI 200 microg provided significantly greater protection to methacholine than pMDI 100 microg or Turbuhaler 100 microg and significantly less protection than pMDI 400 microg (P<0. 05). This study demonstrates that the relative protective dose potency of inhaled beta-agonists can be determined by comparing their effects on methacholine airway responsiveness. The estimated relative protective dose potency for salbutamol Turbuhaler in comparison with pMDI was 1.38 (95% CI 0.67 to 2.87) at 50 microg and was 0.96 (95% CI 0.56 to 1.64) at 100 microg.

Amelioration of ovalbumin-induced allergic airway disease following Der p 1 peptide immunotherapy is not associated with induction of IL-35.

In the present study, we show therapeutic amelioration of established ovalbumin (OVA)-induced allergic airway disease following house dust mite (HDM) peptide therapy. Mice were sensitized and challenged with OVA and HDM protein extract (Dermatophagoides species) to induce dual allergen sensitization and allergic airway disease. Treatment of allergic mice with peptides derived from the major allergen Der p 1 suppressed OVA-induced airway hyperresponsiveness, tissue eosinophilia, and goblet cell hyperplasia upon rechallenge with allergen. Peptide treatment also suppressed OVA-specific T-cell proliferation. Resolution of airway pathophysiology was associated with a reduction in recruitment, proliferation, and effector function of T(H)2 cells and decreased interleukin (IL)-17⁺ T cells. Furthermore, peptide immunotherapy induced the regulatory cytokine IL-10 and increased the proportion of Fox p3⁺ cells among those expressing IL-10. Tolerance to OVA was not associated with increased IL-35. In conclusion, our results provide in vivo evidence for the creation of a tolerogenic environment following HDM peptide immunotherapy, leading to the therapeutic amelioration of established OVA-induced allergic airway disease.