Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness.

BACKGROUND: Practical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF). METHODS: We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 per protocol). Doses of ultrapure LPS (1, 10, 30 or 100µg/100µl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge. RESULTS: Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1ß, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100µg LPS). At 100µg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10µg and 30µg LPS). CONCLUSIONS: Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa. KEY MESSAGES: Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1ß, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS. TRIAL REGISTRATION: Clinical Trials.gov NCT02284074.

The effects of an anti-IL-13 mAb on cytokine levels and nasal symptoms following nasal allergen challenge.

BACKGROUND: IL-13 is a key T(H)2 cytokine that is implicated in allergic responses. OBJECTIVE: We evaluated the effects of an anti-IL-13-blocking antibody compared with placebo on repeated nasal allergen challenge responses in hay fever patients out of season. METHODS: We performed a parallel group double-blind study of anti-IL-13 (single dose, 6 mg/kg intravenously, n = 16) and placebo (n = 15), with an additional open label group given a topical nasal corticosteroid (n = 5). Subjects received intranasal timothy grass pollen (Phleum pratense P5 allergen), and serial samples of nasal mucosal lining fluid were taken by using synthetic absorptive matrix and by nasal lavage. RESULTS: Administration of anti-IL-13 on day 1 resulted in a significant decrease in IL-13 levels in synthetic absorptive matrix eluates compared with placebo (area under the curve 0-8 hours, change from baseline) during the late phase after nasal allergen challenge on day 5 (P < .05) and day 7 (P < .01). There were no apparent effects of anti-IL-13 treatment on nasal lavage eosinophil numbers or total nasal symptom scores versus placebo. However, in a subgroup with high late-phase IL-13 levels at screening, there was a trend for a decrease in total nasal symptom scores after nasal allergen challenge on day 5, when compared with subjects with low IL-13 levels (P < .10). Nasal fluticasone caused suppression of IL-13 (P < .05 on day 5) as well as IL-5 (P < .01 on day 5) levels in the late phase compared with placebo. CONCLUSIONS: Anti-IL-13 had specific pharmacodynamic action in this nasal allergen challenge model, causing profound inhibition of nasal lining fluid IL-13 responses. In addition, there was a possible effect of anti-IL-13 treatment on total nasal symptom scores in a subgroup with high late-phase nasal IL-13 levels at screening.

Rapid effect of inhaled ciclesonide in asthma: a randomized, placebo-controlled study.

BACKGROUND: Ciclesonide is a novel inhaled corticosteroid for the treatment of asthma, and it is important to measure the onset of effect of this therapy on airway hyperresponsiveness (AHR), exhaled nitric oxide (NO), and levels of eosinophils in induced sputum. METHODS: In a randomized, double-blind, crossover study, 21 patients with mild asthma inhaled ciclesonide 320 microg (ex-actuator) qd, ciclesonide 640 microg (ex-actuator) bid, and placebo for 7 days. Exhaled NO and AHR to adenosine monophosphate (AMP), measured as the provocative concentration of AMP producing a 20% reduction in FEV1 (PC20FEV1), were assessed after inhalation on days 1, 3 and 7. Eosinophil levels in induced sputum were also measured. RESULTS: Ciclesonide 320 microg qd and 640 microg bid produced significantly greater improvements in PC20FEV1 compared with placebo on day 1 (within 2.5 h), and on days 3 and 7 (all p < 0.0001). On day 3, both ciclesonide doses significantly reduced exhaled NO levels by - 17.7 parts per billion (p < 0.0001) and - 15.4 parts per billion (p < 0.003) vs placebo, respectively. Significant reductions were maintained during the study with both ciclesonide doses (p < 0.01). A nonsignificant trend towards a decrease in eosinophil cell numbers was observed after 7 days of ciclesonide treatment, especially in patients receiving the higher dose. CONCLUSIONS: A single dose of ciclesonide decreased AHR to AMP and exhaled NO within 3 h, while FEV, improved at 3 days and 7 days.

Optimized dialysis and protease inhibition of sputum dithiothreitol supernatants.

RATIONALE: Dithiothreitol (DTT) is commonly used to liquefy induced sputum samples before assessment of cytology, but causes reduction of disulfide bonds and denaturation of proteins. OBJECTIVES: To process sputum supernatants containing DTT to enable quantification of cytokines and chemokines. METHODS: A standard solution of 22 pooled chemokines and cytokines was incubated with DTT at the concentrations used during sputum liquefaction and then dialyzed under 20 different denaturant and redox conditions. MEASUREMENTS AND MAIN RESULTS: After incubation of the standard solution with DTT there was loss of detectable protein mediators on immunoassay, but optimized dialysis permitted recovery of chemokines to 96 +/- 4% and cytokines to 91 +/- 6%. Optimized dialysis of DTT supernatants from subjects with asthma covering a range of severities (n = 35) was performed in the presence of a cocktail of protease inhibitors and demonstrated significantly elevated levels of the chemokine CXCL10 (IFN-gamma-inducible protein-10), CXCL8 (IL-8), and CCL3 (macrophage inflammatory protein-1alpha); with lower but significantly elevated levels of CCL2 (monocyte chemotactic protein-1), CCL11 (eotaxin), and CCL5 (regulated on activation, normal T-cell expressed and secreted) in severe asthma. In sputum from subjects with severe asthma there were also significantly elevated levels of IL-4, IL-5, IL-13, tumor necrosis factor-alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and IL-12(p40). CONCLUSIONS: The technique of optimized dialysis and protease inhibition of sputum DTT supernatants aids the detection of chemokines and cytokines. The detection of elevated levels of particular sputum chemokines and cytokines in individual patients may provide a rationale for specific therapies.

The effects of a monoclonal antibody directed against tumor necrosis factor-alpha in asthma.

RATIONALE: Neutralization of tumor necrosis factor-alpha (TNF-alpha) is an effective antiinflammatory therapy for several chronic inflammatory diseases. METHODS AND OBJECTIVES: We undertook a double-blind, placebo-controlled, parallel-group design study in 38 patients with moderate asthma treated with inhaled corticosteroids but symptomatic during a run-in phase. Infliximab (5 mg/kg) or placebo was administered by intravenous infusion at Weeks 0, 2, and 6. We assessed clinical response by monitoring lung function, symptoms, and inhaled beta(2)-agonist usage using hand-held electronic devices. RESULTS: The primary endpoint, change in morning PEF at Days 50-56 compared with the last 7 d of the run-in, was not significantly different on treatment. However, infliximab was associated with a decrease in mean diurnal variation of PEF at Week 8 (p = 0.02; 95% confidence interval [CI], -8.1 to -0.72). Furthermore, there was a decrease in the number of patients with exacerbations of asthma (p = 0.01; 95% CI, 4.4 to 52.7) and an increased probability of freedom from exacerbation with time (p = 0.03) in patients on infliximab (n = 14) compared with placebo (n = 18). In addition, infliximab decreased levels of TNF-alpha (p = 0.01) and other cytokines in sputum supernatants. There were no serious adverse events related to the study agent. CONCLUSIONS: Treatment with infliximab was well tolerated and caused a decrease in the number of patients with exacerbations in symptomatic moderate asthma. The promising preliminary findings underscore the need to evaluate therapy directed against TNF-alpha in larger trials enrolling patients with more severe asthma.

Glycopyrrolate causes prolonged bronchoprotection and bronchodilatation in patients with asthma.

INTRODUCTION: Inhaled anticholinergic drugs are effective bronchodilators in the treatment of COPD, and tiotropium bromide has recently been introduced as a once-daily bronchodilator for use as a maintenance treatment. Racemic glycopyrrolate is an anticholinergic drug that has been used orally to control gastric acidity, parenterally as an antisialogogue and to reverse neuromuscular blockade, and has been studied by inhalation for asthma and COPD. DESIGN AND OBJECTIVE: We investigated the duration of protection against the constrictor effects of inhaled methacholine of a single dose of inhaled nebulized racemic glycopyrrolate (0.5, 1.0, and 2.0 mg) compared with ipratropium bromide (0.5 mg) and placebo in 10 atopic asthmatic volunteers in a double-blind, five-way, crossover study. RESULTS: Protection against methacholine-induced bronchospasm after administering glycopyrrolate was maintained to 30 h, the last time point measured. Both bronchodilatation and bronchoprotection were significantly longer with glycopyrrolate than after ipratropium bromide, and bronchoprotection was significant at all time points from 2 to 30 h compared to placebo. Dryness of the mouth and nose was described in 18% of patients after the highest dose of glycopyrrolate. CONCLUSIONS: The prolonged bronchodilator response and the protection against methacholine-induced bronchospasm demonstrated in asthma suggests that inhaled racemic glycopyrrolate would be superior to ipratropium bromide for treatment of stable COPD.

Theophylline: mechanism of action and use in asthma and chronic obstructive pulmonary disease.

Despite having been recognized for a long time as a cheap and effective therapy for the treatment of asthma and chronic obstructive pulmonary disease (COPD), theophylline is relegated to third-line therapy in the treatment of airway diseases due to the drug's frequent side effects and relatively low efficacy. However, regardless of the current situation, there are reasons for thinking that the use of theophylline, in addition to inhaled steroids, may come back into fashion for the treatment of chronic asthma, as it may have an anti-inflammatory and immunomodulatory effect when given in low doses. At these low doses, the drug is easier to use, side effects are uncommon and the problems of drug interaction are less of an issue, thus making the clinical use of theophylline less complicated. In COPD, low-dose theophylline is the first drug to demonstrate clear anti-inflammatory effects, and thus it may even have a role in preventing progression of the disease. Furthermore, the reversal of the steroid resistance induced by oxidative stress suggests that theophylline may increase responsiveness to corticosteroids.