A dual CysLT1/2 antagonist attenuates allergen-induced airway responses in subjects with mild allergic asthma.

BACKGROUND: The cysteinyl leukotrienes (cysLTs) play a key role in the pathophysiology of asthma. In addition to functioning as potent bronchoconstrictors, cysLTs contribute to airway inflammation through eosinophil and neutrophil chemotaxis, plasma exudation, and mucus secretion. We tested the activity of the dual cysLT1/2 antagonist, ONO-6950, against allergen-induced airway responses. METHODS: Subjects with documented allergen-induced early (EAR) and late asthmatic response (LAR) were randomized in a three-way crossover study to receive ONO-6950 (200 mg) or montelukast (10 mg) or placebo q.d. on days 1-8 of the three treatment periods. Allergen was inhaled on day 7 two hours postdose, and forced expiratory volume in 1 s (FEV1 ) was measured for 7 h following challenge. Sputum eosinophils and airway hyperresponsiveness were measured before and after allergen challenge. The primary outcome was the effect of ONO-6950 vs placebo on the EAR and LAR. RESULTS: Twenty-five nonsmoking subjects with mild allergic asthma were enrolled and 20 subjects completed all three treatment periods per protocol. ONO-6950 was well tolerated. Compared to placebo, ONO-6950 significantly attenuated the maximum % fall in FEV1 and area under the %FEV1 /time curve during the EAR and LAR asthmatic responses (P < 0.05) and allergen-induced sputum eosinophils. There were no significant differences between ONO-6950 and montelukast. CONCLUSIONS: Attenuation of EAR, LAR, and airway inflammation is consistent with cysLT1 blockade. Whether dual cysLT1/2 antagonism offers additional benefit for treatment of asthma requires further study.

Treatment with anti-OX40L or anti-TSLP does not alter the frequency of T regulatory cells in allergic asthmatics.

OX40-OX40L interactions and thymic stromal lymphopoietin (TSLP) are important in the induction and maintenance of Th2 responses in allergic disease, whereas T regulatory cells (Treg) have been shown to suppress pro-inflammatory Th2 responses. Both OX40L and TSLP have been implicated in the negative regulation of Treg. The effect of anti-asthma therapies on Treg is not well known. Our aim was to assess the effects of two monoclonal antibody therapies (anti-OX40L and anti-TSLP) on Treg frequency using a human model of allergic asthma. We hypothesized that the anti-inflammatory effects of these therapies would result in an increase in circulating Treg (CD4(+) CD25(+) CD127(low) Foxp3(+) cells) frequency. We measured Treg using flow cytometry, and our results showed that neither allergen challenge nor monoclonal antibody therapy altered circulating Treg frequency. These data highlight the need for assessment of airway Treg and for a more complete understanding of Treg biology so as to develop pharmacologics/biologics that modulate Treg for asthma therapy.

OX40L blockade and allergen-induced airway responses in subjects with mild asthma.

BACKGROUND: The OX40/OX40L interaction contributes to an optimal T cell response following allergic stimuli and plays an important role in the maintenance and reactivation of memory T effector cells. OBJECTIVE: We tested whether treatment with an anti-OX40L monoclonal antibody (MAb) would inhibit allergen-induced responses in subjects with asthma. METHODS: Twenty-eight mild, atopic asthmatic subjects were recruited for a double-blind, randomized, placebo-controlled, parallel-group trial (ClinicalTrials.gov identifier NCT00983658) to compare blockade of OX40L using a humanized anti-OX40L MAb to placebo-administered intravenously in 4 doses over 3 months. Allergen inhalation challenges were carried out 56 and 113 days after the first dose of study drug. The primary outcome variable was the late-phase asthmatic response. Other outcomes included the early-phase asthmatic response, airway hyperresponsiveness, serum IgE levels, blood and sputum eosinophils, safety and tolerability. RESULTS: Treatment with anti-OX40L MAb did not attenuate the early- or late-phase asthmatic responses at days 56 or 113 compared with placebo. In the anti-OX40L MAb treatment group, total IgE was reduced 17% from pre-dosing levels, and sputum eosinophils decreased 75% by day 113 (both P = 0.04). There was no effect of anti-OX40L MAb on airway hyperresponsiveness or blood eosinophils. The frequency of AEs was similar in both groups. CONCLUSION AND CLINICAL RELEVANCE: Pharmacological activity of anti-OX40L MAb was observed by decreases in serum total IgE and airway eosinophils at 16 weeks post-dosing, but there was no effect on allergen-induced airway responses. It is possible that the treatment duration or dose of antibody was insufficient to impact the airway responses.

Allergen inhalation challenge in smoking compared with non-smoking asthmatic subjects.

BACKGROUND: Smoking asthmatics experience more severe symptoms, require more rescue medication and have more asthma-related hospitalizations than non-smoking asthmatics. However, studies in mice suggest that mainstream cigarette smoke may reduce airway inflammation and may attenuate airway hyperresponsiveness. A comparison of allergen-induced airway inflammatory responses of smoking and non-smoking atopic asthmatics has not been examined previously. OBJECTIVES: To determine whether allergen-induced airway responses and inflammatory profiles are attenuated in smoking when compared with non-smoking mild allergic asthmatic subjects. METHODS: Allergen inhalation challenges were performed in 13 smoking and 19 non-smoking mild allergic asthmatic subjects. The forced expired volume in 1 s (FEV(1) ) was measured up to 7 h after allergen inhalation. Methacholine airway responsiveness was measured before and at 24 h after allergen and sputum was induced before and at 7 and 24 h after allergen. RESULTS: Both the smoking and non-smoking groups developed similar allergen-induced falls in FEV(1) during the early and late asthmatic responses and similar increases in allergen-induced airway eosinophils. The mean maximum fall in FEV(1) during the late response was 16.3 ± 4.3% in non-smokers and 12.9 ± 7.2% in smokers. The smoking asthmatics, however, did not develop allergen-induced methacholine airway hyperresponsiveness, whereas the non-smoking controls developed a 1.18 doubling dose shift in methacholine PC(20) (P < 0.05). CONCLUSIONS AND CLINICAL RELEVANCE: Mild allergic asthmatic subjects, who were current smokers with a mean 6-year pack history, develop allergen-induced eosinophilic airway inflammation and late responses, similar in magnitude to non-smoking asthmatics, but do not develop methacholine airway hyperresponsiveness associated with the allergen-induced airway eosinophilia.

Provoked models of asthma: what have we learnt?

Asthma is a chronic inflammatory disease of the airways characterized by physiological abnormalities of variable airflow obstruction and airway hyperresponsiveness (AHR) to a wide variety of physical and inhaled chemical stimuli and the presence of symptoms. AHR is measured by challenging the airways with a variety of agonists and naturally occurring stimuli, which results in constriction of the airway smooth muscle, leading to airway narrowing and airflow limitation. There are two distinct mechanisms by which the airways can narrow to a constrictor stimulus and these are defined by the pathways they take to induce AHR. Direct stimuli are pharmacological agents administered exogenously (such as histamine or methacholine) that act 'directly' on specific receptors on the bronchial smooth muscle to cause constriction. The other mechanism by which the airway can narrow is via the inhalation of indirect stimuli, which include natural stimuli, such as allergen or exercise, and pharmacological agents such as adenosine monophosphate and hyper-osmotic agents (e.g. hypertonic saline or dry powder mannitol). These stimuli induce airway narrowing 'indirectly' by causing the endogenous release of mediators of bronchoconstriction from airway inflammatory cells. Provoked models of asthma have been extremely valuable in understanding the pathobiology of asthma, in aiding diagnosis, in helping to clarify the mechanisms of actions of effective drugs and in the development of new entities to treat asthma. Some provoked models are valuable clinically, particularly those that measure direct AHR, while others, particularly allergen challenge, have been used in animal models and in humans to study the mechanisms of allergen-induced airway inflammation and the associated physiological changes, as well in the development of new drugs for asthma. An emerging role for measurements of AHR is in the evaluation of the optimal treatment for patients with asthma.

Inhaled leukotriene E(4), but not leukotriene D(4), increased airway inflammatory cells in subjects with atopic asthma.

Allergen-induced late airway responses are associated with increased numbers of airway eosinophils and basophils. The purpose of this study was to compare and contrast the effects of inhaled cysteinyl leukotrienes LTD(4) and LTE(4), which are released during allergen- induced airway responses, and allergen, on airway inflammatory cells. Fifteen subjects with atopic, mild asthma inhaled diluent, LTD(4), LTE(4), and allergen. Spirometry was performed for 7 h, and sputum inflammatory cells were measured before, 7 h, and 24 h after challenges. The maximum early percent fall in FEV(1) was 23.6 +/- 1.4%, 21.6 +/- 2.3%, 29.3 +/- 2.4%, and 4.0 +/- 1.1% after LTD(4), LTE(4), allergen, and diluent, respectively. Only inhaled LTE(4) and allergen significantly increased sputum eosinophils at 7 h and 24 h, and sputum basophils at 7 h. Six additional subjects underwent airway biopsies 4 h after inhalation. There were significantly more eosinophils in the lamina propria after inhalation of LTE(4) compared with LTD(4) and diluent (p < 0.05). These results suggest cysteinyl leukotrienes play a role in eosinophil migration into the airways in allergic asthma, and for the same degree of bronchoconstriction, inhaled LTE(4) causes more tissue and airway eosinophilia than LTD(4).

Exercise-induced bronchoconstriction does not cause eosinophilic airway inflammation or airway hyperresponsiveness in subjects with asthma.

The cysteinyl leukotrienes (LT) C(4), D(4), and E(4) may partially mediate eosinophilic airway inflammation in patients with asthma. High- intensity exercise by patients with asthma can result in exercise- induced bronchoconstriction, partly due to leukotriene production, but it is still debated whether this causes airway inflammation. Ten subjects completed a randomized, controlled study to examine the effects of exercise-induced bronchoconstriction on airway inflammatory cells. Subjects completed exercise challenge and methacholine challenge in random order separated by 1 wk. Spirometry was measured for 2 h after challenges, and airway responsiveness was measured the day before and 24 h after each challenge. Blood and sputum samples were obtained before, and 2, 4, 7, and 24 h after each challenge for measurement of inflammatory cells. Nine of the subjects inhaled allergen at least 3 wk before or 1 wk after the study. Sputum samples were collected before, 7 h, and 24 h after challenge. The maximum percentage fall in FEV(1) was 21.3 +/- 1.5% after exercise, 29.9 +/- 1.5% after methacholine, and 28.9+/-2.7% after allergen. Exercise had no effect on airway responsiveness or inflammatory cells measured in blood or sputum, unlike allergen inhalation, which resulted in significant airway hyperresponsiveness and increases in sputum eosinophils (p < 0.05). This study demonstrates that exercise-induced bronchoconstriction does not cause eosinophilic airway inflammation in subjects with asthma who develop airway inflammation with the same degree of allergen-induced bronchoconstriction. We conclude that exercise-induced bronchoconstriction does not cause airway inflammation or airway hyperresponsiveness.

Interaction between haemopoietic regulation and airway inflammation.

Asthma is characterized by reversible airway narrowing, by airway hyperresponsiveness, and by airway inflammation. Inhaled allergens are the most important of the stimuli known to cause asthma. Methods for studying inhaled allergen in the laboratory have been well standardized and extensively used for the investigation of the pathophysiology and the pharmacological modulation of allergen-induced airway responses. Allergen inhalation by a sensitized subject results in an early asthmatic response, and, in the majority of subjects, a late asthmatic response and airway hyperresponsiveness. The late response and airway hyperresponsiveness are associated with increases in airway eosinophils and metachromatic cells. Allergen-induced airway inflammation in dogs (predominantly neutrophilic) is associated with increased granulocyte-macrophage progenitors in bone marrow, which is dependent on the effects of a circulating serum factor stimulating the bone marrow. The newly formed cells traffic to the airways. These increases in granulocyte-macrophage progenitors are blocked by inhaled corticosteroids. In human subjects, allergen-induced eosinophilic inflammation is associated with increases in Eo/B progenitors, mediated through up-regulation if the IL-5 receptor on progenitors and increases responsiveness to IL-5. Inhaled corticosteroids also attenuate all allergen-induced physiological responses and airway inflammation, an effect possibly mediated, in part, through inhibition of eosinophil and basophil maturation or release from the bone marrow.

An inhaled corticosteroid, budesonide, reduces baseline but not allergen-induced increases in bone marrow inflammatory cell progenitors in asthmatic subjects.

We have previously shown that allergen inhalation by asthmatics is associated with increases in bone marrow eosinophil/basophil colony-forming cells (Eo/B-CFU), and increases in CD34(+) hemopoietic progenitors expressing the alpha-subunit of the IL-5 receptor (IL-5Ralpha). This study investigated the effect of inhaled corticosteroid on baseline numbers and allergen-induced increases in these parameters. Nine subjects with mild, stable asthma inhaled budesonide (400 microgram/d) for 8 d in a placebo-controlled, double-blind, randomized crossover study. On Day 7, subjects inhaled allergen, with bone marrow sampling before and 24 h after challenge. Budesonide inhalation significantly attenuated the allergen-induced early and late asthmatic responses, degree of increase in sputum and blood eosinophils, as well as the baseline numbers of total bone marrow CD34(+) cells (p < 0.05), CD34(+)IL-3Ralpha+ cells (p < 0.01) and IL-5-responsive Eo/B-CFU (p < 0.05). Allergen inhalation significantly increased Eo/B-CFU grown in the presence of IL-3, GM-CSF, or IL-5 alone (p < 0.05) and in combination (p < 0.01), as well as the number of CD34(+)IL-5Ralpha+ cells (p < 0.01). However, these increases in Eo/B-CFU and CD34(+)IL-5Ralpha+ cells were not affected by budesonide treatment. These data demonstrate that short-term inhaled budesonide treatment has a systemic effect in inhibiting the turnover of a subpopulation of bone-marrow-derived progenitors, but that inhalation of allergen overcomes this inhibitory effect.

Effect of inhaled leukotriene D4 on airway eosinophilia and airway hyperresponsiveness in asthmatic subjects.

Inhaled cysteinyl leukotrienes may cause recruitment of eosinophils into asthmatic airways. We compared the effects of inhaled leukotriene D4 (LTD4), methacholine, and allergen on airway eosinophils in 10 nonsmoking, atopic, mildly asthmatic subjects in a double-blind, diluent-controlled, randomized crossover study. Concentrations of LTD4, methacholine, and allergen resulting in a 30% decrease in FEV1, and diluent controls (ethanol and saline), were inhaled with at least 7 d between challenges. Spirometry was conducted for 4 h after inhalation challenge, and airway hyperresponsiveness (AHR) to methacholine was measured before and 24 h after challenge. Sputum was induced before and 4 h, 7 h, and 24 h after challenge. The maximum decrease in FEV1 was 31.4 +/- 1.8% with LTD4, 39.4 +/- 2.8% with methacholine, and 30.1 +/- 3.4% with allergen. AHR to methacholine, at the provocative concentration causing a 20% decrease in FEV1 (PC20), was enhanced 24 h after allergen challenge, but remained unchanged 24 h after LTD4 and methacholine (p > 0.05). The percentage of eosinophils in sputum was increased after inhalation of allergen at 7 h and 24 h (p = 0.003), but not after LTD4 or methacholine (p = 0.70). We demonstrated that neither inhalation of LTD4 nor of methacholine at concentrations causing submaximal bronchoconstriction increases the number of sputum eosinophils in the airways of mildly asthmatic subjects. However, LTD4 may still be an important cofactor for eosinophil recruitment in asthma.

Enhanced expression of GM-CSF in differentiating eosinophils of atopic and atopic asthmatic subjects.

Higher numbers of eosinophil/basophil colony-forming units (Eo/B CFU) are observed in blood of atopic individuals, and can be enhanced in atopic asthmatics by allergen-inhalation challenge. It is known that mature basophils and eosinophils synthesize cytokines relevant to allergic inflammation. To investigate the potential role of growth factors in allergic disease we examined the expression of the hemopoietic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-5, in differentiating Eo/B colony cells from normal and atopic individuals, and from atopic asthmatics before and after allergen-inhalation challenge. Peripheral blood was collected from two normal and 12 atopic individuals, and also from 25 atopic asthmatics before and 24 h after allergen challenge. Nonadherent mononuclear cells were isolated and grown in semisolid growth medium. Eo/B colonies were selected and cytospins were prepared for immunocytochemical analysis of colony cells. Eo/B colonies, especially carbol chromotrope 2R+ cells, selected at Days 10, 14, and 18 from atopic donors contained messenger RNA for GM-CSF by combined in situ reverse transcription-polymerase chain reaction and cytochemistry, and demonstrated time-dependent expression of GM-CSF by immunocytochemistry (P = 0.007). Atopic individuals demonstrated a higher percentage of cells expressing GM-CSF than did normal subjects under all growth conditions when examined at Day 14 (P = 0. 04). Atopic asthmatics challenged with inhaled allergen who demonstrated a dual airway response, an increase in the number of blood eosinophils (P = 0.0001), and an increase in the number of Eo/B CFU (P = 0.02) also demonstrated a significant increase in the percentage of colony cells expressing immunostainable GM-CSF (P = 0. 0009), but only a variable effect on those expressing IL-5, 24 h after allergen. These results suggest that GM-CSF expression by differentiating Eo/Bs may provide an additional stimulus in vivo to enhance Eo/B progenitor differentiation in atopic and asthmatic individuals, especially after allergen challenge. The concept of microenvironmental differentiation, where blood progenitor cells may aid in their own differentiation, is supported by these ex vivo findings.

The relationship between respiratory exchange ratio, plasma lactate and muscle lactate concentrations in exercising horses using a valved gas collection system.

A valved gas collection system for horses was validated, then used to examine the relationship between the respiratory exchange ratio (RER), and plasma and muscle lactate in exercising horses. Four healthy Standardbred horses were trained to breathe through the apparatus while exercising on a treadmill. Comparisons of arterial blood gas tensions were made at 3 work levels for each horse, without (control), and with the gas collection system present. At the highest work level, the arterial oxygen tension (PaO2) was significantly lower (P < 0.05), and the arterial carbon dioxide tension (PaCO2) was significantly higher (P < 0.05), than control levels when the apparatus was present; however arterial oxygen content remained unchanged. The horses completed a standardized incremental treadmill test on 4 occasions to determine the repeatability of measurements of oxygen consumption (VO2), carbon dioxide production (VCO2), inspired minute ventilation (VI), respiratory exchange ratio (RER), ventilatory equivalent for oxygen (VI/VO2), tidal volume (VT), and ventilatory frequency (VF). All gas exchange and respiratory measurements showed good reproducibility with the mean coefficient of variation of the 4 horses ranging from 3.8 to 12%. We examined the relationship between 3 indices of energy metabolism in horses performing treadmill exercise: respiratory exchange ratio (RER), central venous plasma and muscle lactate concentrations. A relationship between RER and plasma lactate concentration was established. To compare muscle and plasma lactate concentrations, the horses completed a discontinuous exercise test without the gas collection apparatus present. Significant relationships (P < 0.05), between plasma lactate concentration and RER, and between plasma and muscle lactate concentration, were described for each horse. The valved gas collection system produced a measurable but tolerable degree of interference to respiration, and provided reproducible measurements of gas exchange and ventilatory measurements. It was concluded that measurements of both gas exchange and blood lactate may be used to indicate increased glycolytic activity within exercising skeletal muscle.