Sample size calculations and methacholine challenge tests.
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Bronchial Provocation Tests↗
Biomedical subjects
M D Inman
Publications and source records attributed to M D Inman.
Murine models of asthma.
In vivo animal models can offer valuable information on several aspects of asthma pathogenesis and treatment. The mouse is increasingly used in these models, mainly because this species allows for the application in vivo of a broad range of immunological tools, including gene deletion technology. Mice, therefore, seem particularly useful to further elucidate factors influencing the response to inhaled allergens. Examples include: the role of immunoregulatory mechanisms that protect against T-helper cell type 2 cell development; the trafficking of T-cells; and the contribution of the innate immunity. However, as for other animal species, murine models also have limitations. Mice do not spontaneously develop asthma and no model mimics the entire asthma phenotype. Instead, mice should be used to model specific traits of the human disease. The present task force report draws attention to specific aspects of lung structure and function that need to be borne in mind when developing such models and interpreting the results. In particular, efforts should be made to develop models that mimic the lung function changes characteristic of asthma as closely as possible. A large section of this report is therefore devoted to an overview of airway function and its measurement in mice.
Animals↗
Inhalation of a harmless antigen (ovalbumin) elicits immune activation but divergent immunoglobulin and cytokine activities in mice.
BACKGROUND: Exposure to aerosolized harmless antigen such as ovalbumin (OVA) has previously been shown to induce inhalation tolerance, a state characterized by inhibition of IgE synthesis and airway inflammation, upon secondary immunogenic antigen encounter. Immune events associated with this phenomenon are still poorly understood. OBJECTIVE: The aim of this study was to investigate cellular and molecular mechanisms underlying this state of 'unresponsiveness'. METHODS: After initial repeated OVA exposure, mice were subjected to a protocol of antigen-induced airway inflammation, encompassing two intraperitoneal injections of OVA adsorbed to aluminium hydroxide followed by airway challenge. We assessed immune events in the draining lymph nodes after sensitization, and in the lungs after challenge. RESULTS: In animals initially exposed to OVA, we observed, at the time of sensitization, considerable expansion of T cells, many of which expressed the activation markers CD69 and CD25, as well as increased numbers of antigen-presenting cells, particularly B cells. While these animals produced low levels of IgE, the observed elevated levels of IgG1 signified isotype switching. Splenocytes and lymph node cells from OVA-exposed mice produced low levels of IL-4, IL-5, IL-13 and IFN-gamma, indicating aborted effector function of both T helper (Th)2- and Th1-associated cytokines. Real time quantitative polymerase chain reaction (PCR) (TaqMan) analysis of costimulatory molecules in the lungs after in vivo challenge showed that B7.1, B7.2, CD28 and CTLA-4 mRNA expression was low in animals initially exposed to OVA. Ultimately, these events were associated with abrogated airway inflammation and attenuated airway hyper-responsiveness. The decreased inflammation was antigen-specific and independent of IL-10 or IFN-gamma. CONCLUSION: Initial exposure to OVA establishes a programme that prevents the generation of intact, fully functional inflammatory responses upon secondary antigen encounter. The absence of inflammation, however, is not associated with categorical immune unresponsiveness.
Animals↗
Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia.
Intranasal instillation techniques are used to deliver various substances to the upper and lower respiratory tract (URT and LRT) in mice. Here, we quantify the relative distribution achieved with intranasal delivery of a nonabsorbable tracer, (99m)Tc-labeled sulfide-colloid. Relative distribution was determined by killing mice after instillation and quantifying the radioactivity in dissected tissues using gamma scintigraphy. A significant effect of delivery volume on relative distribution was observed when animals were killed 5 min after instillation delivered under gas anesthesia. With a delivery volume of 5 microl, no radiation was detected in the LRT; this increased to a maximum of 55.7 +/- 2.5% distribution to the LRT when 50 microl were delivered. The majority of radiation not detected in the LRT was found in the URT. Over the course of the following 1 h, radiation in the LRT remained constant, while that in the URT decreased and appeared in the gastrointestinal tract. Instillation of 25 microl into anesthetized mice resulted in 30.1 +/- 6.9% distribution to the LRT, while only 5.3 +/- 1.5% (P < 0.05) of the same volume was detected in the LRT of awake mice. Varying the body position of mice did not affect relative distribution. When using intranasal instillation, the relative distribution between the URT and LRT and the gastrointestinal tract is heavily influenced by delivery volume and level of anesthesia.
Administration, Intranasal↗
Clinical applications of assessment of airway inflammation using induced sputum.
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Adrenal Cortex Hormones↗
The use of induced sputum in clinical trials.
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Asthma↗
Dose-dependent effects of inhaled mometasone furoate on airway function and inflammation after allergen inhalation challenge.
Comparisons of the potency of different inhaled corticosteroids, delivery devices, and treatment regimens in the management of asthma can only be made when outcome measurements display a dose-dependent effect. These outcomes have been difficult to identify. In this study, we compared in a randomized, double-blind, crossover design, the effects of 6 d treatment with placebo and three doses (50, 100, and 400 microg, twice daily) of mometasone furoate delivered by dry powder inhaler (MF-DPI) on responses after allergen inhalation challenge. Twelve mild asthmatic subjects with dual responses after allergen inhalation were studied. Outcome measurements included early and late asthmatic responses, the change in methacholine airway responsiveness 24 h after challenge, and sputum eosinophilia measured 7 and 24 h after challenge. All three doses of MF-DPI demonstrated similar attenuation of early responses and allergen-induced airway hyperresponsiveness relative to placebo (p < 0.05). The late maximal %fall in FEV(1) after placebo treatment was 23.5% and was significantly reduced in a dose-dependent manner to 12.3%, 11.0%, and 5.9% for the 50-, 100-, and 400-microg twice-daily treatments (p = 0.007). The allergen-induced increase in sputum eosinophilia (x10(4) cells/ml) 24 h after challenge during placebo treatment was 60.2 and was significantly reduced to 24.0, 15.3, and 6.2 for the 50-, 100-, and 400-microg twice-daily treatments. MF-DPI is effective at attenuating allergen-induced early and late responses, airway hyperresponsiveness, and sputum eosinophilia, and dose-response effects exist for the attenuation of the late response.
Administration, Inhalation↗
Th type 1-stimulating activity of lung macrophages inhibits Th2-mediated allergic airway inflammation by an IFN-gamma-dependent mechanism.
In the mucosal immune system, resident dendritic cells are specialized for priming Th2-polarized immunity, whereas the Ag-presenting activity of macrophages has been linked with the development of Th1 phenotype. As an immune switch toward Th1 can protect against Th2-mediated allergic response, this study investigated the capacity of lung macrophages to stimulate Th1 responses during the secondary exposure to inhaled allergen, thereby suppressing Th2-mediated allergic airway inflammation in a murine model of allergic asthma. Following airway macrophage depletion in OVA-sensitized mice, lung T cells defaulted to a phenotype that produced less Th1 (IFN-gamma) and more Th2 (IL-4 and IL-5) cytokines, leading to more severe airway hyperreactivity and inflammation after intranasal Ag challenge. After OVA pulsing and adoptive transfer, lung macrophages selectively promoted a Th1 response in Ag-sensitized recipients and did not induce pulmonary eosinophilia. By contrast, OVA pulsing and adoptive transfer of a lung cell preparation, consisting of dendritic cells, B cells, and macrophages, promoted a Th2 response with an associated inflammatory response that was suppressed when macrophages were present and pretreated with IFN-gamma, but exacerbated when macrophages were depleted before IFN-gamma treatment. In addition, Th1-promoting activity of lung macrophages was not related to the autocrine production of IL-12p40. These results suggest that the Th1-promoting APC activity may be an inherent property of the lung macrophage population, and may play an important role, upon stimulation by IFN-gamma, in antagonizing an ongoing Th2 immunity and Th2-dependent allergic responses.
Administration, Intranasal↗
Allergen-induced murine upper airway inflammation: local and systemic changes in murine experimental allergic rhinitis.
The role of inflammatory effector cells in the pathogenesis of airway allergy has been the subject of much investigation. However, whether systemic factors are involved in the development of local responses in both upper and lower airways has not been fully clarified. The present study was performed to investigate aspects of the pathogenesis of isolated allergic rhinitis in a murine model sensitized to ovalbumin (OVA). Both upper- and lower-airway physiological responsiveness and inflammatory changes were assessed, as well as bone marrow progenitor responses, by culture and immunohistological methods. Significant nasal symptoms and hyper-responsiveness appeared after intranasal OVA challenge (P < 0.0001 and P < 0.01, respectively), accompanied with significant nasal mucosal changes in CD4+ cells (P < 0.001), interleukin (IL)-4+ cells (P < 0.01), IL-5+ cells (P < 0.01), basophilic cells (P < 0.02) and eosinophils (P < 0.001), in the complete absence of hyper-responsiveness or inflammatory changes in the lower airway. In the bone marrow, there were significant increases in CD34+ cells, as well as in eosinophils and basophilic cells. In the presence in vitro of mouse recombinant IL-5, IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF), the level of bone marrow eosinophil/basophil (Eo/Baso) colony-forming cells increased significantly in the OVA-sensitized group. We conclude that, in this murine model of allergic rhinitis, haemopoietic progenitors are upregulated, which is consistent with the involvement of bone marrow in the pathogenesis of nasal mucosal inflammation. Both local and systemic events, initiated in response to allergen provocation, may be required for the pathogenesis of allergic rhinitis. Understanding these events and their regulation could provide new therapeutic targets for rhinitis and asthma.
Allergens↗
The trials and tribulations of IL-5, eosinophils, and allergic asthma.
Eosinophils have been suggested to be part of the pathologic process that characterizes asthma, and their recruitment into the upper or lower airways appears to be essential for the clinical manifestations of allergen inhalation. IL-5 is a cytokine necessary for the development, differentiation, recruitment, activation, and survival of eosinophils. Allergen inhalation increases the production of IL-5 in the airways as measured in bronchoalveolar lavage cells and induced sputum. The relationship between IL-5 and the development of airway eosinophilia has been firmly established in IL-5 transgenic mice, with allergen challenge models in IL-5-deficient mice, and in mice treated with blocking anti-IL-5 antibodies. In addition, an accumulation of evidence suggests that treating mice with anti-IL-5 blocking antibodies prevents allergen-induced airway hyperresponsiveness. A recently reported study examined the effects of treatment with a humanized anti-IL-5 mAb (SB-240563) on allergen-induced airway responses and inflammation in atopic subjects. The authors of the study concluded that their results call into question the role of eosinophils in mediating the allergen-induced late asthmatic response and airway hyperresponsiveness; however, because of methodologic limitations, the study cannot be used either to support or to refute the concept of an important role for eosinophils in causing allergen-induced changes in airway function.
Animals↗
The reproducibility and sample size requirements of exercise-induced bronchoconstriction measurements.
Dry air exercise challenges are frequently used to screen medications that have potential utility in the management of exercise-induced bronchoconstriction (EIB). The purpose of this study was to determine the reproducibility of three outcome measurements made using such challenges, and sample size requirements for drug evaluation studies based on these outcomes. Forty adult subjects with asthma, who tested positively on a screening exercise challenge, were subjected to two further identical challenges, separated by 1 to >35 days. Outcome measurements included the maximum per cent fall in forced expiratory volume in one second (FEV1), after exercise (% fallmax), and the area under the per cent fall in FEV1/time curve for 30 min (AUC30) and 60 min (AUC60) after exercise. The reproducibility of these outcomes, as assessed by intraclass correlation coefficients was 0.72, 0.53 and 0.35 for % fallmax, AUC30 and AUC60 measurements, respectively. The sample size requirements to demonstrate an attenuation of EIB equivalent to a 50% reduction in % fallmax was 9, 14 and 19 subjects for the % fallmax, AUC30 and AUC60 responses, respectively (90% power). It is concluded that the maximum percentage fall in forced expiratory volume in one second has greater reproducibility and results in greater power in clinical trials than area under the curve measurements. Sample size calculation curves are provided which may be used in study design and interpretation of published studies.
Adult↗
Effects of once daily dosing with inhaled budesonide on airway hyperresponsiveness and airway inflammation following repeated low-dose allergen challenge in atopic asthmatics.
BACKGROUND: Repeated low-dose allergen challenge increases airway hyperresponsiveness and sputum eosinophils in atopic asthmatics. Inhaled corticosteroids attenuate the airway responses to high-dose allergen challenge, but have not been evaluated against repeated low dose challenge. OBJECTIVE: This study evaluates the effects of once daily treatments of two doses of inhaled budesonide on airway responses to repeated low-dose allergen challenge. METHODS: Eight atopic asthmatics with a dual airway responses to inhaled allergen were recruited into a randomized, double-blind crossover, placebo-controlled study. In the mornings of four consecutive days (day 1-day 4), subjects inhaled budesonide 100 microg, 400 microg, or placebo, 30 min before inhaling a concentration of allergen causing a 5% early fall in FEV1. Airway hyperresponsiveness to methacholine and sputum eosinophils were measured at baseline, on the afternoon of day 2, day 4, and 24 h after the last challenge. There was a 1-week washout between each of the three treatment periods. RESULTS: The repeated low-dose allergen challenge induced increases in the percentage sputum eosinophils from 2.0 +/- 0.7% at baseline to 16.6 +/- 7.1% on day 4 (P = 0.002), and this effect was reduced by once daily budesonide 100 microg to 5.6 +/- 1.8% (P = 0. 01) and by once daily budesonide 400 microg to 3.1 +/- 0.9% (P = 0. 004). Also, the allergen-induced methacholine airway hyperresponsiveness which occurred by day 4 (P = 0.03) of the repeated low dose challenge was inhibited by budesonide 400 microg (P = 0.017). CONCLUSION: Both budesonide 100 microg and 400 microg inhaled once daily significantly reduces allergen-induced sputum eosinophilia after repeated low dose challenge; however, only the higher dose also attenuates the allergen-induced airway hyperresponsiveness.
Adult↗
Bone marrow events in animal models of allergic inflammation and hyperresponsiveness.
Allergic inflammation is associated with the marked infiltration of eosinophils in affected tissues. Eosinophilia, in turn, is a hallmark clinical feature associated with allergic rhinitis, atopic dermatitis, sinusitis, and asthma. There is considerable evidence in animal models and humans that the bone marrow plays an integral role in allergic inflammation. Evidence shows that, in response to allergen exposure in the airway, bone marrow (white blood cell) progenitors proliferate and differentiate, which leads to persistent increases in eosinophil numbers. These observations suggest that there is signaling between the lung and bone marrow after allergen exposure and provide further support for the proposition that allergy is a systemic disease. Although the nature of the signal-mediating activation of bone marrow after airway allergen exposure is unknown, several pathways have been implicated, including allergen-induced hemopoietic growth factors, cell trafficking, and stimulation of resident bone marrow cells. A common thread in all these pathways is the importance of IL-5. Evidence is reviewed in animal models for the role of bone marrow in allergic inflammation within the context of the systemic nature of allergic disease.
Animals↗
Systemic aspects of allergic disease: bone marrow responses.
In patients with allergic diseases, allergen provocation can activate a systemic response that provokes inflammatory cell production by the bone marrow. After release and differentiation of progenitor cells, eosinophils, basophils, and mast cells are typically recruited to tissues in atopic individuals. An understanding at the molecular level of the signaling process that leads to these systemic responses between the target organ, especially the airways, and the bone marrow may open up new avenues of therapy for allergic inflammatory disease. Studies that support the critical involvement of the bone marrow in the development of eosinophilic inflammation of the airways point out the systemic nature of these conditions and their potential for biologic intervention. Hemopoietic events that originate in the bone marrow are potential targets of long-term therapy for rhinitis and asthma. For example, the "beneficial" systemic activity of cortico-steroids through modulation of hemopoietic mechanisms and inflammatory cell recruitment to the airways is essential for the optimal treatment of both upper and lower airway inflammation.
Animals↗
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.
Adrenergic beta-Agonists↗
New considerations about measuring airway hyperresponsiveness.
Measuring airway responsiveness to inhaled bronchoconstrictor stimuli, such as methacholine or histamine, has become an important tool in the diagnosis of asthma. This is measured by patients inhaling increasing doses or concentrations of the bronchoconstrictor stimulus until a given level of bronchoconstriction is achieved. Inhaled allergens initiate processes that increase airway inflammation and enhance airway hyperresponsiveness in asthmatic subjects. Studies using inhaled allergen challenges have provided insight into how changes in airway hyperresponsiveness are regulated by induced inflammatory processes. These changes in airway hyperresponsiveness (1-2 doubling doses) have been shown to be of much smaller magnitude than those demonstrated when asthmatics with stable airway hyperresponsiveness are compared to normals (4-8 doubling doses). These allergen-induced changes would be of little relevance in subjects with normal airway responsiveness, because they would not increase the degree of airway responsiveness into the asthmatic range. They are, however, important in asthmatics who already have airway hyperresponsiveness because they are similar to changes associated with worsening asthma control. It is likely that the mechanisms responsible for the changes in airway hyperresponsiveness following experimental allergen exposure are similar to those producing transient worsening of control in asthmatics. Nevertheless, it is unlikely that the mechanisms of the transient allergen-induced airway hyperresponsiveness will explain the underlying mechanisms of the persistent airway hyperresponsiveness in asthmatic patients when compared with normal individuals.
Allergens↗
Repeatability of allergen-induced airway inflammation.
BACKGROUND: Allergen inhalation challenge is a useful clinical model to investigate the effects of asthma therapies on allergen-induced airway responses; however, the repeatability of allergen-induced airway inflammation is not known. OBJECTIVE: The purpose of this study was to investigate the repeatability of allergen-induced increases in sputum eosinophils. This information will allow the prediction of the number of subjects required in studies evaluating asthma therapies. METHODS: Seventeen subjects completed 2 allergen challenges using the same dose of allergen, at least 3 weeks apart. Allergen-induced airway responses were measured for 7 hours after challenge. Differential cell counts from induced sputum were determined the day before and 7 and 24 hours after challenge; methacholine PC20 was measured the day before and 24 hours after challenge. RESULTS: The intraclass correlation coefficient for maximum percent late fall in FEV1 was 0.32 and for the area of the late response was 0.61. The sample size predicted to be necessary to observe 50% attenuation of the maximum percent late fall in FEV1 and the late area under the curve with a power of 0.95 was 9 subjects. The intraclass correlation coefficient for percent of allergen-induced sputum eosinophils was 0.60 at 7 hours and 0.53 at 24 hours after challenge. With a randomized cross-over study design, the sample size predicted to be necessary to observe 50% attenuation of allergen-induced percent of eosinophils with a power of 0.95 was 5 subjects. CONCLUSION: Allergen inhalation challenge with measurements of sputum eosinophils is a noninvasive and reliable method for evaluating the anti-inflammatory effects of asthma therapies.
Adult↗
The clinical expression of allergy in the lungs.
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Asthma↗