Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway.

BACKGROUND: Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely. METHODS: Eight healthy smokers aged 40-65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1ß), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis. RESULTS: A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL. CONCLUSIONS: The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression. TRIAL REGISTRATION: NHS Health Research Authority (13/LO/0256).

The effects of the novel SHIP1 activator AQX-1125 on allergen-induced responses in mild-to-moderate asthma.

BACKGROUND: SH2-containing inositol-5'-phosphatase 1 (SHIP1) is an endogenous inhibitor of the phosphoinositide-3-kinase pathway that is involved in the activation and chemotaxis of inflammatory cells. AQX-1125 is a first-in-class, oral SHIP1 activator with a novel anti-inflammatory mode of action. OBJECTIVE: To evaluate the effects of AQX-1125 on airway responses to allergen challenge in mild-to-moderate asthmatic patients. METHODS: A randomized, double-blind, placebo-controlled, two-way crossover study was performed in 22 steroid-naïve mild-to-moderate asthmatics with a documented late-phase response to inhaled allergen (LAR). AQX-1125 (450 mg daily) or placebo was administered orally for 7 days. Allergen challenge was performed on day 6 (2 h postdose), followed by methacholine challenge (day 7), and induced sputum collection and fractional exhaled nitric oxide (FeNO). RESULTS: AQX-1125 significantly attenuated the late-phase response compared with placebo (FEV1 4-10 h: mean difference 150 mL, 20%; P = 0.027) and significantly increased the minimum FEV1 during LAR (mean difference 180 mL; P = 0.014). AQX-1125 had no effect on the early-phase response. AQX-1125 showed a trend in reduction of sputum eosinophils, neutrophils and macrophages although this did not achieve significance as there were only 11 paired samples for analysis. There was no effect on methacholine responsiveness or FeNO. Pharmacokinetic data showed AQX-1125 was rapidly absorbed with geometric mean Cmax and AUC0-24 h values of 1417 ng/mL and 16 727 h ng/mL, respectively. AQX-1125 was well tolerated, but mild GI side-effects (dyspepsia, nausea and abdominal pain) were described in 4/22 subjects on active treatment. These side-effects were mild self-limiting, required no further treatment and did not lead to discontinuation of therapy. CONCLUSION AND CLINICAL RELEVANCE: AQX-1125, a novel oral SHIP1 activator, significantly reduces the late response to allergen challenge, with a trend to reduce airway inflammation. AQX-1125 was safe and well tolerated and merits further investigation in inflammatory disorders.

Setipiprant, a selective CRTH2 antagonist, reduces allergen-induced airway responses in allergic asthmatics.

BACKGROUND: CRTH2 is a G-protein-coupled receptor on T helper2 cells that mediates pro-inflammatory effects of prostaglandin D2 in allergic responses. OBJECTIVE: To investigate the tolerability and pharmacokinetics of setipiprant (ACT-129968), a selective orally active CRTH2 antagonist, in allergic asthmatics and to assess the protective effects of multiple doses of this drug against allergen-induced airway responses. METHODS: In this 3-centre, double-blinded, placebo-controlled, cross-over study, 18 allergic asthmatic males were randomized to setipiprant 1000 mg or matching placebo b.i.d. for 5 consecutive days. Study periods were separated by a washout of ≥ 3 weeks. On study day 4, subjects underwent a standardized allergen challenge and airway response was recorded by FEV1 until 10 h post-allergen. Airway responsiveness to methacholine and exhaled nitric oxide (eNO) were measured pre- and post-dosing. The effects of both treatments on the allergen-induced airway responses were compared by a paired Student's t-test. RESULTS: Fifteen subjects completed the study per-protocol and were included in the analysis. Overall, setipiprant was well tolerated and no clinically relevant adverse events occurred. Trough plasma concentrations showed a high inter-subject variability. Compared with placebo, setipiprant significantly reduced the allergen-induced late asthmatic response (LAR), inhibiting the area under the response vs. time curve (AUC(3-10 h) ) by on average 25.6% (P = 0.006) and significantly protected against the allergen-induced airway hyperresponsiveness (AHR) to methacholine (P = 0.0029). There was no difference in the early asthmatic response (EAR) or in allergen-induced changes in eNO between treatments. CONCLUSION AND CLINICAL RELEVANCE: Setipiprant at multiple oral doses was well tolerated and reduced both the allergen-induced LAR and the associated AHR in allergic asthmatics. Our findings confirm that CRTH2 may be a promising target for the treatment of allergic disorders.

The 5-lipoxygenase-activating protein inhibitor, GSK2190915, attenuates the early and late responses to inhaled allergen in mild asthma.

BACKGROUND: GSK2190915, a potent 5-lipoxygenase-activating protein inhibitor, prevents the synthesis of leukotrienes and 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). OBJECTIVE: To assess the effect of GSK2190915 on the allergen-induced asthmatic responses. METHODS: Nineteen eligible male subjects with mild asthma were enrolled in and completed this four-centre, double-blind, two-way crossover study (ClinicalTrials.gov NCT00748306). Subjects took GSK2190915 100 mg and placebo orally once daily for 5 days in randomized order. On Day 1 and 4 they had a methacholine challenge, on Day 3 they had an inhaled allergen challenge, and on Days 4 and 6 they had sputum induction. RESULTS: GSK2190915 attenuated the early (0-2 h) and late (4-10 h) asthmatic responses to inhaled allergen compared with placebo. There was a statistically significant attenuation of the early asthmatic response (EAR) by GSK2190915; treatment difference of GSK2190915 vs. placebo for the minimum FEV(1) EAR was 0.408 L (0.205, 0.611). There was a statistically significant attenuation of the late asthmatic response (LAR) by GSK2190915; the treatment difference of GSK2190915 vs. placebo for the minimum FEV(1) LAR was 0.229 L (0.041, 0.417). There was a statistically significant attenuation of allergen-induced sputum eosinophil count on Day 4 following GSK2190915: mean treatment difference (95% CI) between GSK2190915 and placebo was -9.95% (-18.15%, -1.77%). Compared with placebo, GSK2190915 100 mg reduced median sputum LTB(4) by > 90% on Days 4 and 6. There was no effect on methacholine PC(20) post allergen. GSK2190915 was generally well tolerated. CONCLUSION AND CLINICAL RELEVANCE: GSK2190915 shows potential as a treatment for patients with asthma.

A novel technique to explore the functions of bronchial mucosal T cells in chronic obstructive pulmonary disease: application to cytotoxicity and cytokine immunoreactivity.

Bronchial mucosal CD8(+) cells are implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, but there are few data on their functional properties. We have developed a novel technique to outgrow these cells from COPD patients in sufficient numbers to examine effector functions. Endobronchial biopsies from 15 COPD smokers and 12 ex-smokers, 11 control smokers and 10 non-smokers were cultured with anti-CD3/interleukin (IL)-2 ± IL-15. Outgrown CD3(+) T cells were characterized in terms of phenotype (expression of CD4, 8, 25, 28, 69 and 56), cytotoxicity and expression of COPD-related cytokines. Compared with IL-2 alone, additional IL-15 increased the yield and viability of biopsy-derived CD3(+) T cells (12-16-day culture without restimulation) without alteration of CD4(+) /CD8(+) ratios or expression of accessory/activation molecules. Biopsy-derived T cells, principally CD8(+)/CD56(+) cells, exhibited statistically significantly greater cytotoxic activity in current or ex-smokers with COPD compared with controls (P < 0·01). Elevated percentages of CD8(+) T cells expressed interferon (IFN)-γ, tumour necrosis factor (TNF)-α and IL-13 (P < 0·01) in current COPD smokers compared with all comparison groups. It is possible to perform functional studies on bronchial mucosal T cells in COPD. We demonstrate increased CD8(+)CD56(+) T cell cytotoxic activity and expression of remodelling cytokines in smokers who develop COPD.

Heparin and structurally related polymers attenuate eotaxin-1 (CCL11) release from human airway smooth muscle.

BACKGROUND AND PURPOSE: The glycosaminoglycan heparin has anti-inflammatory activity and is exclusively found in mast cells, which are localized within airway smooth muscle (ASM) bundles of asthmatic airways. Interleukin (IL)-13 induces the production of multiple inflammatory mediators from ASM including the eosinophil chemoattractant chemokine, eotaxin-1. Heparin and related glycosaminoglycan polymers having structurally heterogeneous polysaccharide side chains that varied in molecular weight, sulphation and anionic charge were used to identify features of the heparin molecule linked to anti-inflammatory activity. EXPERIMENTAL APPROACH: Cultured human ASM cells were stimulated with interleukin (IL)-13 in the absence or presence of heparin and related polymers. Eotaxin-1 was quantified using chemokine antibody arrays and ELISA. KEY RESULTS: Unfractionated heparin attenuated IL-13-dependent eotaxin-1 production and this effect was reproduced with low molecular weight heparins (3 and 6 kDa), demonstrating a minimum activity fragment of at least 3 kDa. N-desulphated, 20% re-N-acetylated heparin (anticoagulant) was ineffective against IL-13-dependent eotaxin-1 production compared with 90% re-N-acetylated (anticoagulant) or O-desulphated (non-anticoagulant) heparin, suggesting a requirement for N-sulphation independent of anticoagulant activity. Other sulphated molecules with variable anionic charge and molecular weight exceeding 3 kDa (dextran sulphate, fucoidan, chondroitin sulphate B) inhibited IL-13-stimulated eotaxin-1 release to varying degrees. However, non-sulphated dextran had no effect. CONCLUSIONS: Inhibition of IL-13-dependent eotaxin-1 release by heparin involved but did not depend upon sulphation, though loss of N-sulphation reduced the attenuating activity, which could be restored by N-acetylation. This anti-inflammatory effect was also partially dependent on anionic charge, but independent of molecular size above 3 kDa and the anticoagulant action of heparin.

Superiority of "triple" therapy with salmeterol/fluticasone propionate and tiotropium bromide versus individual components in moderate to severe COPD.

BACKGROUND: The combination of salmeterol and fluticasone propionate (SFC) and tiotropium bromide (TIO) are commonly used treatments in chronic obstructive pulmonary disease (COPD) but there are few data on their effectiveness when used together. We compared the effects of SFC 50/500 microg twice daily in addition to TIO 18 microg once daily with the individual treatments alone. METHODS: 41 patients with COPD participated in a randomised, double blind, double dummy, three way crossover study with 2 week washout periods between treatments. Lung function assessment included plethysmography and spirometry. The primary end point was post-dose specific airways conductance (sGaw) area under the curve (AUC(0-4 h)) on day 14. RESULTS: AUC(0-4 h) sGaw was significantly higher on day 14 after SFC+TIO compared with TIO (22%) or SFC alone (27%) (both p<0.001). SFC+TIO significantly improved trough forced expiratory volume in 1 s compared with TIO alone (212 ml, p<0.001) and SFC alone (110 ml, p = 0.017) on day 14. Inspiratory capacity measurements also showed significant benefits for triple therapy over individual components on day 14. Subjects receiving SFC+TIO had clinically relevant improvements in Transition Dyspnoea Index (TDI) total score of 2.2 compared with TIO alone (p<0.001) (but not SFC alone, 0.7; NS) and used significantly less rescue medication (1.0 occasion less daily than TIO (p<0.001) and 0.6 less than SFC (p = 0.01)). CONCLUSION: SFC+TIO triple therapy led to greater improvements in bronchodilation compared with TIO and SFC alone. The advantages of triple therapy are observed across a range of physiologically important parameters, including airway conductance and lung volumes. Triple therapy also led to patient related benefits by improving TDI and use of rescue medication.

Elevated expression of adenosine A1 receptor in bronchial biopsy specimens from asthmatic subjects.

Asthmatics, unlike healthy subjects, experience bronchoconstriction in response to inhaled adenosine, and extracellular adenosine concentrations are elevated in the bronchoalveolar lavage fluid and exhaled breath condensate of asthmatic subjects. However, little is known about the location and expression of adenosine receptors in asthmatic airways. The aim of the present study was to investigate the distribution of adenosine A(1) receptors in bronchial biopsy specimens from mildly asthmatic steroid-naïve subjects and then compare the degree of expression with that of healthy subjects. Biopsy sections were immunostained using an adenosine A(1) receptor antibody, the selectivity of which was validated in specific experiments. Image analysis was then performed in order to determine differences in immunostaining intensity. Immunostaining of biopsy sections from the asthmatic subjects revealed strong expression of the A(1) receptor, located predominantly in the bronchial epithelium and bronchial smooth muscle. In comparison, very weak immunostaining was observed in biopsy specimens obtained from healthy subjects. Image analysis revealed that the intensity of positive staining of the asthmatic bronchial epithelium and smooth muscle regions was significantly greater than that observed for the healthy epithelium and smooth muscle. In conclusion, the sensitivity of asthmatics to inhaled adenosine coupled with increased adenosine A(1) receptor expression implies that these receptors play a role in the pathophysiology of this disease.

Phosphodiesterase (PDE) 7 in inflammatory cells from patients with asthma and COPD.

In inflammatory cells, the low K(m) cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase (PDE) 4 subtype is predominant in terms of expression and function, although more recently it has been suggested that PDE 7 may also play a role in regulating inflammatory cell activity. In the present study, PDE 4 and PDE 7 subtype messenger ribonucleic acid (mRNA) transcripts in CD4 and CD8 lymphocytes from healthy (n=10) and asthmatic (n=10) subjects and polymorphonuclear neutrophils (PMNs) and CD8 lymphocytes obtained from healthy (n=10) and chronic obstructive pulmonary disease (COPD) (n=7) subjects were identified and quantified. PDE 4A, PDE 4B, PDE 4D and PDE 7A mRNA were present in similar quantities in both CD4 and CD8 lymphocytes obtained from healthy and asthmatic subjects and in CD8 lymphocytes obtained from healthy and COPD subjects. Expression of PDE 4C and PDE 7B mRNA was also observed, although transcript levels were low and variable between individuals. In addition, the effects of selective PDE 7 inhibition on both phytohaemagluttinin (PHA)-induced human peripheral blood mixed mononuclear cell (HPBMNC) proliferation and fMLP-induced neutrophil elastase (NE) release were studied. HPBMNC and human neutrophils, isolated from the venous blood of healthy volunteers (n=6) were treated with either a novel selective PDE 7 inhibitor PF 0332040 alone or in combination with rolipram. Proliferation of HPBMNC was stimulated by PHA (2microgml(-1)) and assessed by [(3)H]-thymidine incorporation, while fMLP-induced (100nM) NE release was determined using a chromogenic substrate. Both rolipram (0.003-10microM) and PF 0332040 (0.003-10microM) significantly inhibited PHA-stimulated proliferation of HPBMNC ((**)P<0.01). Co-administration of rolipram (0.3-10microM) and PF 0332040 (0.003-10microM) significantly increased the degree of inhibition observed, compared to when either drug was administered alone ((**)P<0.01). PF 0332040 (0.003-10microM) had no inhibitory effect on NE release from human peripheral blood neutrophils stimulated with fMLP (100nM), while rolipram (0.003-10microM) significantly inhibited neutrophil degranulation ((**)P<0.01). These findings suggest no evidence of altered PDE 4 or PDE 7 mRNA transcript levels in inflammatory cells isolated from the peripheral venous blood of mild asymptomatic asthmatic subjects or stable COPD subjects, however, inhibition of PDE 7 may influence mononuclear cell function.

Lack of neutrophil degranulation in low-dose endotoxin inhalation based on a novel intracellular assay.

To study the nature of endotoxin or lipopolysaccharide (LPS) induced inflammation, we developed a method of quantifying intracellular human neutrophil elastase (HNE) in lysed sputum polymorphs as a means to study the degranulation status of LPS-recruited neutrophils. Induced sputum, blood and exhaled nitric oxide (NO) were collected from 10 healthy non-atopic human subjects after inhaling a single 15 microg dose of Escherichia coil LPS in an open study. At 6 hours, LPS inhalation caused significant increase of sputum and blood neutrophils but without parallel increase in myeloperoxidase, HNE or interleukin-8 (IL-8) in sputum sol and blood, or exhaled NO. Intracellular HNE in lysed sputum polymorphs or purified blood neutrophils did not show any significant changes between inhaled LPS and saline, nor was there any appreciable change in percentage HNE release induced by N-Formyl-Met-Leu-Phe (fMLP) in vitro. We concluded that in healthy humans, the transient neutrophilic inflammation induced by a single dose of inhaled 15 microg LPS is mainly characterized by cell recruitment, not enhanced secretion of granular mediators or increased exhaled NO based on our experimental conditions.

Inhaled endotoxin in healthy human subjects: a dose-related study on systemic effects and peripheral CD4+ and CD8+ T cells.

BACKGROUND: Inhaled endotoxin or lipopolysaccharide (LPS) is implicated in the pathogenesis of pulmonary diseases. We investigated the inhalation effects of two different doses of LPS in healthy human subjects. METHODS: Eighteen healthy non-atopic human subjects inhaled either 15 microg (n=10) or 50 microg (n=8)Escherichia coli LPS in an open study. As control, each subject had isotonic saline inhalation 1 week before (baseline) and after LPS inhalation. Data collected included those of clinical parameter, induced sputum and peripheral blood CD4+ and CD8+ T cells. RESULTS: Acute flu-like symptoms and pyrexia were significantly greater in the 50 microg than 15 microg LPS group. Similarly, the increase in sputum and blood total cell and neutrophil counts at 6h following inhaled LPS were greater in the 50 microg group. Myeloperoxidase, human neutrophil elastase and interleukin-8 in sputum sol, but not blood, showed a trend towards greater increase following 50 microg LPS. All these changes were resolved at one week. In the 50 microg dose group alone, there was a reduction in the proportion of peripheral blood interferon (IFN)-gamma-producing CD4+ and CD8+ T cells at 6h followed by an increase at 1 week after inhaled LPS. CONCLUSIONS: The airway and systemic effects of inhaled LPS are dose-related and predominantly neutrophilic. The changes in the proportions of circulating CD4+ and CD8+ T cells suggests preferential recruitment of IFN-gamma-producing T cells into tissue from inhaled 50 microg LPS, followed by reappearance of these cells in blood 1 week later.

Sputum induction in corticosteroid-dependant asthmatics: risks and airway cellular profile.

Sputum induction with nebulized hypertonic saline is increasingly being used to evaluate airway inflammation. We investigated the procedure-associated risk in 16 asthmatics that were still symptomatic despite on high doses of regular corticosteroid (CS) therapy (7 on daily inhaled CS > or = 800 microg budesonide or equivalent; 9 on additional daily oral CS) and their sputum cellular profile. For comparison, 12 mild stable asthmatics and 10 normal healthy subjects were included. All subjects inhaled 3%, 4% and 5% hypertonic saline sequentially via ultrasonic nebulizer as a means to induce sputum. Maximal percentage fall of Forced Expiratory Volume on One Second (FEV1) during sputum induction was significantly greater in CS-dependent asthmatics (median % [IQR]: 16.0 [11.0-32.3]) than in mild asthmatics (5.3 [4.2-10.8], p = 0.002] and in normal subjects (4.6 [3.4-6.4]), p = 0.0001). The maximal percentage FEV1 fall was inversely correlated with baseline FEV1 (Rs= -0.69; p < 0.0001). Compared to mild asthmatics, induced sputum from CS-dependant asthmatics had proportionately fewer eosinophils (2.2 [0.8-7.0] versus 23.3% [10.7-46.3], p = 0.003) and greater neutrophils (64.2 [43.9-81.2] versus 28.7 [19.0-42.6], p = 0.009). Sputum neutrophils showed a significant inverse correlation to FEV1 (Rs = -0.51, p = 0.01). We concluded that sputum induction using nebulized hypertonic saline should be performed with caution in CS-dependant asthmatics. The airway cellular profile observed suggests that the immunopathology underlying CS-dependant asthmatics may be different or a consequence of CS therapy.

Contrasting effects of allergen challenge on airway responsiveness to cysteinyl leukotriene D(4) and methacholine in mild asthma.

BACKGROUND: Cysteinyl leukotrienes (cysteinyl-LTs) have been implicated in the pathogenesis of allergen induced airway responses. Airway responsiveness (AR) to inhaled cysteinyl-LTs is dramatically increased following allergen challenge in animal studies. The effect in man has not been evaluated. METHODS: Ten mild steroid-naïve asthmatic subjects with an isolated early asthmatic response (EAR) and 21 with an additional late asthmatic response (LAR) took part in a randomised controlled crossover study to assess AR to inhaled methacholine (MCh) and cysteinyl-LT D(4) (LTD(4)) 22 and 24 hours, respectively, after allergen challenge. Eight subjects had two further LTD(4) challenges separated by a 2 week washout period to assess the reproducibility of inhaled LTD(4) challenge. RESULTS: In subjects with an isolated EAR, non-significant mean (SE) increases in AR of 0.4 (0.4) doubling doses (DD) for MCh and 0.4 (0.5) DD for LTD(4) followed allergen challenge compared with control. A significant correlation between AR to MCh and LTD(4) followed both control (r=0.91, 95% CI 0.67 to 0.98; p=0.0002) and allergen challenge (r=0.79, 95% CI 0.32 to 0.95; p=0.0063). In subjects with an additional LAR there was a significant increase in AR to MCh (1.2 (0.3) DD, p=0.0005) following allergen challenge but no overall effect on AR to LTD(4) (0.69 (0.4) DD, p=0.11). A significant correlation between AR to MCh and LTD(4) was again observed (r=0.70; 95% CI 0.38 to 0.87; p=0.0004) following control, although it was reduced following allergen challenge (r=0.48; 95% CI 0.063 to 0.76; p=0.027). LTD(4) challenge was highly reproducible with a mean difference of 0.2 (0.3) DD between challenges. CONCLUSIONS: Allergen challenge significantly increases AR to inhaled MCh but not to LTD(4) in subjects with LAR. The lack of a comparable increase in AR to LTD(4) is surprising. Endogenous cysteinyl-LTs are produced in abundance following allergen challenge and may enhance AR to MCh or induce a degree of tachyphylaxis to LTD(4).

Dose-related effects of formoterol on airway responsiveness to adenosine 5'-monophosphate and histamine.

Inhaled short-acting beta2-agonists provide greater protection against airway responsiveness (AR) to the mast-cell stimulus, adenosine 5'-monophosphate (AMP), than to histamine, a direct spasmogen. Both terbutaline and albuterol exhibit this mast-cell stabilizing property in a dose-dependent manner. A single dose of the long-acting beta2-agonist formoterol has also been reported to have a mast cell-stabilizing effect, whereas salmeterol has not. To explore the dose-related actions of the long-acting beta2-agonist formoterol on AR, the authors compared the acute effects of three doses of formoterol and terbutaline on AR to AMP and histamine. In a double-blind, randomized, placebo-controlled, cross-over study, 25 mild, steroid naive, asthmatic subjects attended on 10 occasions. At each visit, subjects inhaled either a single dose of terbutaline (500 microg), formoterol (6, 12 or 24 microg) or a matched placebo, administered via Turbuhaler, 30 min prior to challenge with both AMP and histamine. Each dose of beta2-agonist reduced AR to AMP and histamine. The bronchoprotective effects of formoterol (6 microg) and terbutaline (500 microg) were similar in magnitude in reducing AR to histamine (mean +/- SD: 3.6 +/- 0.3 and 3.1 +/- 0.3 doubling doses (DD)) and AR to AMP (3.5 +/- 0.5 and 3.3 +/- 0.4 DD, respectively). Overall, formoterol reduced AR to both spasmogens in a dose-dependent manner. In addition, formoterol (12 and 24 microg) provided a significantly greater protective effect against AMP than against histamine challenge. It decreased AR by 5.7 +/- 0.6 and 6.3 +/- 0.7 DD against AMP and 4.3 +/- 0.4 and 4.8 +/- 0.43 DD against histamine, respectively. The results of this study indirectly demonstrated an in vivo dose-dependent mast-cell stabilizing effect of formoterol, in addition to functional antagonism on airway smooth muscle. This property of beta2-agonists may have clinical benefits in asthma management.

Identification and quantification of phosphodiesterase 4 subtypes in CD4 and CD8 lymphocytes from healthy and asthmatic subjects.

In the present study, for the first time, PDE4 subtypes were identified and semi-quantified in both CD4 and CD8 lymphocytes from healthy and asthmatic individuals. CD4 and CD8 lymphocytes from healthy and mild asymptomatic asthmatic subjects (receiving beta-agonist therapy only) were isolated from peripheral venous blood using appropriate antibody coated paramagnetic beads. PDE4 subtypes and beta-actin were identified by digoxigenin (DIG)-labelling reverse transcriptase-polymerase chain reaction and semi-quantified by DIG-detection enzyme-linked immunosorbance assay. In CD4 and CD8 lymphocytes PDE4A, PDE4B and PDE4D were detected, with no significant differences observed between healthy and asthmatic groups. In CD8 lymphocytes, enzyme subtype expression was lower and showed more intersubject variability. In functional studies investigating the effects of various PDE inhibitors on PHA-induced proliferation of mononuclear cells from healthy and asthmatic subjects, CDP840 (0.03 - 10 microM), rolipram (0.1 - 10 microM) and theophylline (10 microM - 1 mM) inhibited PHA-induced proliferation of mononuclear cells from healthy and asthmatic subjects in a concentration-dependent manner, although no significant difference was observed between the groups investigated. In additional studies, total monocyte cyclic AMP PDE activity was investigated in cells isolated from asthmatic subjects both prior to and 24 h after allergen challenge. Total monocyte cyclic AMP PDE activity remained unaffected following challenge of asthmatic subjects with either house dust mite or cat dander and was inhibited in a concentration-dependent manner by rolipram (0.01 - 100 microM) both before and after allergen challenge.

Hyperventilation and asymptomatic chronic asthma.

BACKGROUND: We have consistently argued that mild asthma is an important underlying aetiological factor in patients with severe symptomatic hyperventilation. While hyperventilation has been demonstrated in acute asthma, there have been few studies in mild chronic asthma, and mechanisms are uncertain. METHODS: Twenty three currently asymptomatic chronically asthmatic patients (occasional use of bronchodilators, normal lung function, hyperresponsive to methacholine) were studied and 17 matched normal subjects acted as controls. Ventilation, pattern of breathing, arterial carbon dioxide and oxygen tensions (PaCO(2), PaO(2)), end tidal PCO(2) (PETCO(2)), standard lung function, airway responsiveness to methacholine, airway inflammation assessed by eosinophils in induced sputum, and psychiatric morbidity (Spielberger STAI-Y and Beck Depression Inventory) were measured. RESULTS: Despite the absence of current asthmatic symptoms, no clinical evidence of hyperventilation, and normal lung function in the patients with asthma, PaCO(2) and PETCO(2) were significantly (p<0.01) lower in the patients than in the control group (mean (SD) PaCO(2) 4.96 (0.43) kPa for patients versus 5.27 (0.38) kPa for controls (mean difference 0.31 kPa, 95% confidence interval (CI) 0.06 to 0.56, p<0.02)). PETCO(2) was very similar to PaCO(2) in both groups (mean (SD) PETCO(2) 4.89 (0.47) kPa for the patients and 5.28 (0.40) for the controls (mean difference 0.39 kPa, 95% CI 0.12 to 0.66, p<0.01)). There was no significant difference in ventilation or respiratory pattern between the two groups. The reduced PaCO(2) in the asthmatic patients correlated significantly with the concentration of methacholine provoking a fall in FEV(1) of more than 20% (PC(20)) (r = 0.56, p<0.01) but not with any aspect of lung function, eosinophil count, or anxiety/depression. CONCLUSION: Mild asymptomatic asthma is not associated with clinically significant hyperventilation but is associated with a significant reduction in both arterial and end tidal PCO(2) which relates to airway hyperresponsiveness rather than to the degree of airway obstruction or mucosal inflammation. Anxiety and depression appear not to be implicated.

A biochemical and functional assessment of monocyte phosphodiesterase activity in healthy and asthmatic subjects.

The aim of this study was to investigate whether cyclic adenosine 3'5-monophosphate (cAMP) phosphodiesterase (PDE) activity is altered in monocytes from mild asthmatic subjects. Total cAMP PDE activity (pmol/min per mg protein) was significantly greater in homogenates prepared from monocytes from asthmatic subjects (68.3 +/- 7.0, n=9) compared to healthy individuals (46.3 +/- 3.3, n=14, P<0.05). The PDE inhibitors siguazodan (PDE3-selective), rolipram (PDE4-selective) and theophylline (non-selective) produced a concentration-dependent inhibition of cAMP PDE activity in homogenates from monocytes from normal and asthmatic subjects. However, siguazodan produced significantly greater (P<0.05), and rolipram significantly less (P<0.05), inhibition of total cAMP PDE activity in monocytes from asthmatics (n=4) than from healthy individuals (n=5). cAMP PDE activity was inhibited with equal potency by theophylline in monocytes from healthy and asthmatic subjects. We also investigated the functional consequences of the changes in PDE activity in mononuclear cells obtained from asthmatic subjects. There was no significant difference in the ability of PDE4 inhibitors to attenuate TNF alpha release from monocytes obtained from asthmatic compared with healthy subjects (P>0.05). Despite a significant increase in the biochemical activity of PDE3 in monocytes from asthmatic subjects, the PDE3 inhibitor siguazodan, failed to significantly reduce TNF alpha release from human monocytes. Thus, total cAMP PDE activity is increased in monocytes taken from mild asymptomatic asthmatics compared to healthy subjects and is reflected by an increase in the proportion of PDE3 and a decrease in the proportion of PDE4. This augmented enzyme activity was not associated with an alteration in the ability of PDE4 inhibitors to attenuate mononuclear cell function from asthmatics compared to healthy individuals.

The role of adenosine receptors in the action of theophylline on human peripheral blood mononuclear cells from healthy and asthmatic subjects.

1. The aim of the present study was to investigate the role of adenosine A2b receptors in the anti-proliferative action of theophylline in human peripheral blood mononuclear cells (HPBMC) from healthy and asthmatic subjects. 2. Theophylline significantly inhibited PHA-induced proliferation of HPBMC from both healthy and asthmatic donors but only at relatively high concentrations at 1 mM (P<0.05). Enprophylline, a drug which also acts as a non-selective phosphodiesterase (PDE) inhibitor and is a selective A2b receptor antagonist, had no significant effect on proliferation of cells from either group at concentrations up to 10 microM (P>0.05; n=6). 3. Adenosine deaminase (2 u ml(-1)), which metabolizes adenosine, had no significant effect on PHA-induced HPBMC proliferation over a range of concentrations (0 - 8 microg ml(-1)) in cells from either healthy or asthmatic subjects. 4. The adenosine receptor agonists N(6)-cyclopentyladenosine (CPA, A1-selective) and 5'-N-ethylcarboxamidoadenosine (NECA, A1/A2) produced a small but significant inhibition of PHA-induced proliferation of HPBMC from healthy and asthmatic subjects (10 microM, P<0.05; n=6). In contrast, 5'-N-ethylcarboxamido-2-[4-(2-]carboxyethyl)phenethyl]adenosine (CGS21680, A2a-selective) was without significant effect (P>0.05; n=6). 5. The adenosine receptor antagonist alloxazine (A2b-selective) had no significant effect, while 8(3-chlorostyryl)caffeine,(CSC, A2a-selective) significantly inhibited PHA-induced proliferation of HPBMC from both groups (P<0.05; n=6). 6. Our results suggest that endogenous or exogenous adenosine has little effect on the proliferation of HPBMC obtained from healthy or asthmatic subjects. Thus it would appear that the effect of high concentrations of theophylline is not related to adenosine receptor antagonism.

A long-term study of the antiinflammatory effect of low-dose budesonide plus formoterol versus high-dose budesonide in asthma.

Adding inhaled long-acting beta(2)-agonists to a low dose of inhaled corticosteroids (ICS), results in better clinical asthma control than increasing the dose of ICS. However, this approach may mask underlying airway inflammation. In a double-blind parallel-group study, we evaluated the effect of adding formoterol to a low dose of budesonide, compared with a higher dose of budesonide, on the composition of induced sputum. After a 4-wk run-in period of treatment with budesonide (800 microg, twice daily), 60 patients with moderate asthma were randomly assigned to a 1-yr treatment with 400 microg of budesonide plus placebo, twice daily (BUD800), or 100 microg of budesonide plus 12 microg of formoterol, twice daily (BUD200+F). All drugs were administered via Turbuhaler. Budesonide (800 microg, twice daily) during run-in significantly reduced median sputum eosinophils from 4.5 to 0.68%. No significant changes in the proportion of eosinophils, EG2(+) cells, other inflammatory cells, or ECP levels in sputum were observed over the ensuing 1-yr treatment with BUD200+F or BUD800. Clinical asthma control was not significantly different between both groups. In conclusion, no significant differences in sputum markers of airway inflammation were observed during a 1-yr treatment with a low dose of inhaled budesonide plus formoterol compared with a higher dose of budesonide.

Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response.

BACKGROUND: Interleukin-5 (IL-5) is essential for the formation of eosinophils, which are thought to have a major role in the pathogenesis of asthma and other allergic diseases. We aimed to assess the effects of monoclonal antibody to IL-5 on blood and sputum eosinophils, airway hyper-responsiveness, and the late asthmatic reaction to inhaled allergen in patients with mild asthma. METHODS: We did a double-blind randomised placebo-controlled trial, in which a single intravenous infusion of humanised (IgG-K) monoclonal antibody to IL-5 (SB-240563) was given at doses of 2.5 mg/kg (n=8) or 10.0 mg/kg (n=8). The effects of treatment on responses to inhaled allergen challenge, sputum eosinophils, and airway hyper-responsiveness to histamine were measured at weeks 1 and 4 with monitoring of blood eosinophil counts for up to 16 weeks. FINDINGS: Monoclonal antibody against IL-5 lowered the mean blood eosinophil count at day 29 from 0.25x10(9)/L (95% CI 0.16-0.34) in the placebo group to 0.04x10(9)/L (0.00-0.07) in the 10 mg/kg group (p<0.0001), and prevented the blood eosinophilia that follows allergen challenge. After inhaled allergen challenge, 9 days after treatment, the percentage sputum eosinophils were 12.2% in the placebo group and lowered to 0.9% (-1.2 to 3.0; p=0.0076) in the 10 mg/kg group, and this effect persisted at day 30 after the dose. There was no significant effect of monoclonal antibody to IL-5 on the late asthmatic response or on airway hyper-responsiveness to histamine. INTERPRETATION: A single dose of monoclonal antibody to IL-5 decreased blood eosinophils for up to 16 weeks and sputum eosinophils at 4 weeks, which has considerable therapeutic potential for asthma and allergy. However, our findings question the role of eosinophils in mediating the late asthmatic response and causing airway hyper-responsiveness.