Dysregulation of intestinal epithelial CFTR-dependent Cl- ion transport and paracellular barrier function drives gastrointestinal symptoms of food-induced anaphylaxis in mice.

Food-triggered anaphylaxis can encompass a variety of systemic and intestinal symptoms. Murine-based and clinical studies have revealed a role for histamine and H1R and H2R-pathway in the systemic response; however, the molecular processes that regulate the gastrointestinal (GI) response are not as well defined. In the present study, by utilizing an IgE-mast cell (MC)-dependent experimental model of oral antigen-induced anaphylaxis, we define the intestinal epithelial response during a food-induced anaphylactic reaction. We show that oral allergen-challenge stimulates a rapid dysregulation of intestinal epithelial transcellular and paracellular transport that was associated with the development of secretory diarrhea. Allergen-challenge induced (1) a rapid intestinal epithelial Cftr-dependent Cl- secretory response and (2) paracellular macromolecular leak that was associated with modification in epithelial intercellular junction proteins claudin-1, 2, 3 and 5, E-cadherin and desmosomal cadherins. OVA-induced Cftr-dependent Cl- secretion and junctional protein degradation was rapid occurring and was sustained for 72 h following allergen-challenge. Blockade of both the proteolytic activity and Cl- secretory response was required to alleviate intestinal symptoms of food-induced anaphylaxis. Collectively, these data suggest that the GI symptom of food-induced anaphylactic reaction, secretory diarrhea, is a consequence of CFTR-dependent Cl- secretion and proteolytic activity.

A Dose-Response Study Examining the Use of Methacholine Challenge to Demonstrate Local Therapeutic Equivalence of the Salmeterol Component of Generic Inhaled Fluticasone Propionate/Salmeterol Combination Products.

Background: Asthma is widely treated using inhaled corticosteroid/long-acting beta agonist (LABA) combinations, for example, fluticasone propionate/salmeterol (FPS) dry powder inhaler, marketed as Advair® Diskus®. Some regulators require generics to demonstrate local (lung) therapeutic equivalence (LTE) for each component of the FPS reference, ideally with a dose-response within the approved FPS dose range. We sought to develop a methacholine challenge (MeCh) LTE methodology for assessing the LABA (salmeterol) component of FPS. Methods: Forty-six patients with asthma received single doses of albuterol (active control; 90 or 180 µg), FPS (100/50 or 200/100 µg), and placebo on 5 separate study days. Spirometry and MeCh were performed 1, 6, and 10 hours after study drug inhalation. Primary endpoint was provocative concentration of methacholine producing a 20% fall in forced expiratory volume in 1 second (PC20). Study entry required screening PC20 ≤8 mg/mL, with a greater than fourfold increase (and PC20 ≤128 mg/mL) after 180 µg albuterol. Results: Both albuterol (90 and 180 µg) and FPS (100/50 and 200/100 µg) significantly increased PC20 compared with placebo (sustained 6 and 10 hours postdose with FPS but not albuterol). The dose-response slopes (95% confidence interval) estimated 1 hour after treatment were 0.374 (-0.068 to 0.815) and 0.310 (-0.135 to 0.754) between low and high doses of albuterol and FPS, respectively, both nonsignificant. Slopes were shallower than those available in the literature for albuterol and formoterol, but similar to those for salmeterol. Conclusions: These data confirm that the bronchoprotective effect of FPS lasts longer than that of albuterol. The shallow dose-response slope we observed for albuterol is contrary to previous reports, probably due to the measurement of PC20 beginning at 1 hour postdose. The results suggest that use of MeCh to assess LTE for salmeterol formulations may be more difficult to accomplish than it is for albuterol and formoterol products.

Effect of Study Design on Sample Size in Studies Intended to Evaluate Bioequivalence of Inhaled Short-Acting ß-Agonist Formulations.

Pharmacodynamic studies that use methacholine challenge to assess bioequivalence of generic and innovator albuterol formulations are generally designed per published Food and Drug Administration guidance, with 3 reference doses and 1 test dose (3-by-1 design). These studies are challenging and expensive to conduct, typically requiring large sample sizes. We proposed 14 modified study designs as alternatives to the Food and Drug Administration-recommended 3-by-1 design, hypothesizing that adding reference and/or test doses would reduce sample size and cost. We used Monte Carlo simulation to estimate sample size. Simulation inputs were selected based on published studies and our own experience with this type of trial. We also estimated effects of these modified study designs on study cost. Most of these altered designs reduced sample size and cost relative to the 3-by-1 design, some decreasing cost by more than 40%. The most effective single study dose to add was 180 µg of test formulation, which resulted in an estimated 30% relative cost reduction. Adding a single test dose of 90 µg was less effective, producing only a 13% cost reduction. Adding a lone reference dose of either 180, 270, or 360 µg yielded little benefit (less than 10% cost reduction), whereas adding 720 µg resulted in a 19% cost reduction. Of the 14 study design modifications we evaluated, the most effective was addition of both a 90-µg test dose and a 720-µg reference dose (42% cost reduction). Combining a 180-µg test dose and a 720-µg reference dose produced an estimated 36% cost reduction.

Airway microbiota across age and disease spectrum in cystic fibrosis.

Our objectives were to characterise the microbiota in cystic fibrosis (CF) bronchoalveolar lavage fluid (BALF), and determine its relationship to inflammation and disease status.BALF from paediatric and adult CF patients and paediatric disease controls undergoing clinically indicated bronchoscopy was analysed for total bacterial load and for microbiota by 16S rDNA sequencing.We examined 191 BALF samples (146 CF and 45 disease controls) from 13 CF centres. In CF patients aged <2 years, nontraditional taxa (e.gStreptococcus, Prevotella and Veillonella) constituted ∼50% of the microbiota, whereas in CF patients aged ≥6 years, traditional CF taxa (e.gPseudomonas, Staphylococcus and Stenotrophomonas) predominated. Sequencing detected a dominant taxon not traditionally associated with CF (e.gStreptococcus or Prevotella) in 20% of CF BALF and identified bacteria in 24% of culture-negative BALF. Microbial diversity and relative abundance of Streptococcus, Prevotella and Veillonella were inversely associated with airway inflammation. Microbiota communities were distinct in CF compared with disease controls, but did not differ based on pulmonary exacerbation status in CF.The CF microbiota detected in BALF differs with age. In CF patients aged <2 years, Streptococcus predominates, whereas classic CF pathogens predominate in most older children and adults.

Safety and Efficacy of a Novel Microbial Lipase in Patients with Exocrine Pancreatic Insufficiency due to Cystic Fibrosis: A Randomized Controlled Clinical Trial.

OBJECTIVE: To evaluate the safety and efficacy of a novel microbial lipase (NM-BL) in a liquid formulation for the treatment of exocrine pancreatic insufficiency (EPI) in patients with cystic fibrosis (CF) in a phase IIa proof-of-concept study. STUDY DESIGN: We conducted a double-blind, randomized, placebo controlled crossover study in patients with cystic fibrosis and exocrine pancreatic insufficiency. Adolescent and adult patients with CF were randomized to receive NM-BL or placebo for 1 week as replacement for their usual pancreatic enzyme formulation. They were subsequently crossed-over to the alternate study treatment. The coefficient of fat absorption was evaluated as the primary endpoint. Symptoms and adverse events were evaluated as secondary endpoints. RESULTS: A total of 35 patients were randomized into the study and 22 patients completed both treatment periods. During treatment with NM-BL, the coefficient of fat absorption was significantly greater (72.7%) compared with placebo (53.8%) with a difference between groups of 18.8% (P < .001). Subjective assessment of stool fat and stool consistency also improved under treatment with NM-BL. Adverse events were mostly gastrointestinal in nature and were more common in the group receiving NM-BL. CONCLUSIONS: Currently available pancreatic enzyme products are limited because of the lack of liquid formulations and being largely porcine based. The novel microbial lipase NM-BL was safe and effective in this short term trial. The trial provided clinical proof-of-concept for this novel microbial lipase as a treatment for EPI in CF. A larger phase 2 dose ranging trial is warranted. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01710644.

Bioassay of salmeterol in children using methacholine challenge with impulse oscillometry.

BACKGROUND: Bronchoprovocation with methacholine (MC) is the most sensitive method of determining bioequivalence of inhaled bronchodilators. FEV1 is used to determine the endpoint, but many children cannot perform spirometry reproducibly. The purpose of this study was to determine whether MC, using impulse oscillometry (IOS) as the endpoint, can differentiate between two doses of salmeterol (SM). METHODS: This was a single-blind, randomized study of 10 subjects with mild stable asthma, ages 4-11 years. None were taking a long-acting ß-agonist but most were on low-dose inhaled corticosteroid. On one study day, MC was performed 1 hr after one inhalation from each of two separate Advair 100/50 Diskus (100 µg salmeterol treatment). On a second day, MC was performed after one inhalation from Advair Diskus and one inhalation from Flovent Diskus 100 (50 µg salmeterol treatment). The provocative concentration of methacholine causing a 40% increase in total airway resistance (PC40 R5 ) was calculated. RESULTS: The reduction in R5 (bronchodilator effect) was 15.5% and 18.4% for 50 and 100 µg, respectively (NS). After MC (bronchoprotective effect), the geometric mean (95%CI) PC40 R5 (mg/ml) was 2.4 (1.3-4.4) during screening, 22.9 (8.5-61.6) after 50 µg SM and 47.0 (25.2-87.8) after 100 µg SM (P = 0.051 for 50 vs. 100 using a linear mixed effects model). No adverse effects were observed. CONCLUSIONS: MC with IOS endpoint will be a useful method for determining bioequivalence of a generic inhaler in children. Seventy-two subjects will be required to achieve 80% power to assess bioequivalence of SM. Pediatr Pulmonol. 2016;51:570-575. © 2015 Wiley Periodicals, Inc.

Efficacy response in CF patients treated with ivacaftor: post-hoc analysis.

Clinical studies in patients with cystic fibrosis and G551D-CFTR showed that the group treated with ivacaftor had improved clinical outcomes. To better understand the effect of ivacaftor therapy across the distribution of individual FEV(1) responses, data from Phase 3 studies (STRIVE/ENVISION) were re-examined. In this post-hoc analysis of patients (n = 209) who received 48 weeks of ivacaftor or placebo, patients were assigned to tertiles according to FEV(1) response. These groups were then used to evaluate response (FEV(1), sweat chloride, weight, CFQ-R, and pulmonary exacerbation). The number needed to treat (NNT) was calculated for specific thresholds for each outcome. Across all tertiles, numerical improvements in FEV(1), sweat chloride, CFQ-R and the frequency of pulmonary exacerbations were observed in ivacaftor-treated patients: the treatment difference versus placebo was statistically significant for all outcomes in the upper tertile and for some outcomes in the lower and middle tertiles. The NNT for a ≥ 5% improvement in %predicted FEV(1) was 1.90, for a ≥ 5% body weight increase was 5.74, and to prevent a pulmonary exacerbation was 3.85. This analysis suggests that the majority of patients with clinical characteristics similar to STRIVE/ENVISION patients have the potential to benefit from ivacaftor therapy.

Loss of IL-4Rα-mediated PI3K signaling accelerates the progression of IgE/mast cell-mediated reactions.

Clinical and experimental evidence indicate that polymorphisms within the interleukin 4 (IL-4) receptor (IL-4R) chain are sufficient for altered strength of IL-4/IL-13 signaling, leading to an exaggerated allergic inflammatory response and increase susceptibility to allergic phenotypes. In the present study, we show that ablation of IL-4Rα-induced phosphatidylinositol 3-kinase (PI3K) activating signal by germline point mutation within the IL-4Rα motif (Y500F) did not alter susceptibility to IgE-mediated, food-induced experimental anaphylaxis. Moreover, diarrhea occurrence, antigen-specific IgE and intestinal mastocytosis were comparable between WT and IL-4Rα(Y500F) mice. However, mice unable to stimulate IL-4Rα-mediated PI3K signaling had accelerated disease progression. Notably, the accelerated anaphylactic response was associated with more rapid histamine-induced hypovolemia. Mechanistic in vitro and in vivo analyses revealed that endothelial IL-4Rα PI3K signaling negatively regulates the histamine-induced endothelial leak response. These results define an unanticipated role for IL-4Rα-mediated PI3K signaling in negative regulation of IgE-mediated anaphylactic reactions.

Nonprescription racemic epinephrine for asthma.

BACKGROUND: Inhaled racepinephrine (RE) (Asthmanefrin) became available in September 2012 as a nonprescription treatment for bronchospasm based on a 1986 US Food and Drug Administration rule. It contains 11.25 mg RE in 0.5 mL and is delivered by a handheld electronic nebulizer. In 2001, we conducted a pilot study that was never published. Now that the product is promoted as a replacement for epinephrine chlorofluorocarbon metered-dose inhaler (Primatene), we provide the results of that study. Methacholine challenge was used as a bioassay. OBJECTIVE: To determine the dose of RE that is equivalent to nebulized albuterol. METHODS: Four subjects, 18 to 45 years old, with mild stable asthma completed the pilot study. Methacholine challenge was performed on the first screening day, without pretreatment, and then on different days, 15 minutes after 1.25 mg albuterol and 2.5, 5, 10, and 20 mg RE delivered by a Pari LC Plus nebulizer. The end point was the provocative concentration of methacholine that caused a 20% decrease in FEV1. Data were log transformed and analyzed by an ANOVA for repeated measures. RESULTS: There was a significant dose response for RE. The geometric mean provocative concentration of methacholine that caused a 20% decrease in FEV1 was 44 mg/mL (95% CI, 23-85 mg/mL) after albuterol, and 10.2 mg/mL (95% CI, 3.5-30 mg/mL) after the 10-mg dose of RE (approximate nonprescription dose) (P = .001). There were no adverse effects. CONCLUSION: RE provides less bronchoprotection from methacholine than does albuterol and may be less effective in treating acute bronchospasm.

CD14+CD33+ myeloid cell-CCL11-eosinophil signature in ulcerative colitis.

This study tested the hypothesis that eotaxins (CCL11, CCL24, and CCL26) and IL-5 contribute to eosinophil recruitment to the intestine in UC and that intestinal macrophages are important producers of CCL11 in this disease. Peripheral blood and rectal biopsy samples were obtained from patients with active (n=18) and quiescent UC (n=9), and control patients (n=7). Eosinophil and macrophage levels and activation were analyzed by flow cytometry. Rectal mRNA levels of CCL11, CCL24, CCL26, and IL-5 were determined by qRT-PCR. The cellular source of CCL11 was visualized by immunofluorescence analyses. Eosinophil numbers were elevated in the blood and rectum of active and quiescent UC patients compared with controls. Levels of activated eosinophils (CD66b(high)) correlated with disease severity. Rectal CCL11, CCL24, and CCL26 mRNA levels were increased in active UC, whereas only CCL11 was elevated in quiescent UC. Levels of CCL11, but not CCL24 and CCL26, positively correlated with eosinophil numbers. Numbers of CD14(+)CD33(+) cells correlated with CCL11 and eosinophil levels. Immunofluorescence analyses revealed the presence of CD14(+)CCL11(+) mononuclear cells in colonic biopsies in UC. These results support the hypothesis that CCL11 contributes to eosinophil recruitment in UC and that intestinal myeloid cells are a source of CCL11. Interestingly, rectal levels of CCL24, CCL26, and IL-5 only increase during active UC, coinciding with further elevation of eosinophil numbers and with the activation of rectal eosinophils. In conclusion, there is a link among CD14(+)CD33(+) myeloid cells, CCL11, and eosinophils in adult UC.

Intestinal CCL11 and eosinophilic inflammation is regulated by myeloid cell-specific RelA/p65 in mice.

In inflammatory bowel diseases (IBDs), particularly ulcerative colitis, intestinal macrophages (MΦs), eosinophils, and the eosinophil-selective chemokine CCL11, have been associated with disease pathogenesis. MΦs, a source of CCL11, have been reported to be of a mixed classical (NF-κB-mediated) and alternatively activated (STAT-6-mediated) phenotype. The importance of NF-κB and STAT-6 pathways to the intestinal MΦ/CCL11 response and eosinophilic inflammation in the histopathology of experimental colitis is not yet understood. Our gene array analyses demonstrated elevated STAT-6- and NF-κB-dependent genes in pediatric ulcerative colitis colonic biopsies. Dextran sodium sulfate (DSS) exposure induced STAT-6 and NF-κB activation in mouse intestinal F4/80(+)CD11b(+)Ly6C(hi) (inflammatory) MΦs. DSS-induced CCL11 expression, eosinophilic inflammation, and histopathology were attenuated in RelA/p65(Δmye) mice, but not in the absence of STAT-6. Deletion of p65 in myeloid cells did not affect inflammatory MΦ recruitment or alter apoptosis, but did attenuate LPS-induced cytokine production (IL-6) and Ccl11 expression in purified F4/80(+)CD11b(+)Ly6C(hi) inflammatory MΦs. Molecular and cellular analyses revealed a link between expression of calprotectin (S100a8/S100a9), Ccl11 expression, and eosinophil numbers in the DSS-treated colon. In vitro studies of bone marrow-derived MΦs showed calprotectin-induced CCL11 production via a p65-dependent mechanism. Our results indicate that myeloid cell-specific NF-κB-dependent pathways play an unexpected role in CCL11 expression and maintenance of eosinophilic inflammation in experimental colitis. These data indicate that targeting myeloid cells and NF-κB-dependent pathways may be of therapeutic benefit for the treatment of eosinophilic inflammation and histopathology in IBD.

Efficacy and safety of ivacaftor in patients aged 6 to 11 years with cystic fibrosis with a G551D mutation.

RATIONALE: Ivacaftor (VX-770), a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, has been shown to improve lung function, pulmonary exacerbation rate, respiratory symptoms, and weight gain compared with placebo in patients with cystic fibrosis aged 12 years or older with a G551D-CFTR mutation. OBJECTIVES: This randomized, double-blind, placebo-controlled trial evaluated ivacaftor in patients with cystic fibrosis aged 6-11 years with a G551D-CFTR mutation on at least one allele. METHODS: Patients were randomly assigned to receive ivacaftor administered orally at 150 mg (n = 26) or placebo (n = 26) every 12 hours for 48 weeks in addition to existing prescribed cystic fibrosis therapies. MEASUREMENTS AND MAIN RESULTS: Despite near-normal mean baseline values in FEV1, patients receiving ivacaftor had a significant increase in percent predicted FEV1 from baseline through Week 24 versus placebo group (treatment effect, 12.5 percentage points; P < 0.001). Effects on pulmonary function were evident by 2 weeks, and a significant treatment effect was maintained through Week 48. Patients treated with ivacaftor gained, on average, 2.8 kg more than those receiving placebo at Week 48 (P < 0.001). The change from baseline through Week 48 in the concentration of sweat chloride, a measure of CFTR activity, with ivacaftor was -53.5 mmol/L (P < 0.001) versus placebo. The incidence of adverse events was similar in the two groups. CONCLUSIONS: In patients who are younger and healthier than those in previously studied populations, ivacaftor demonstrated a significant improvement in pulmonary function, weight, and CFTR activity compared with placebo. Clinical trial registered with www.clinicaltrials.gov (NCT00909727).

Chymase-mediated intestinal epithelial permeability is regulated by a protease-activating receptor/matrix metalloproteinase-2-dependent mechanism.

Mast cells regulate intestinal barrier function during disease and homeostasis. Secretion of the mast cell-specific serine protease chymase regulates homeostasis. In the present study, we employ in vitro model systems to delineate the molecular pathways involved in chymase-mediated intestinal epithelial barrier dysfunction. Chymase stimulation of intestinal epithelial (Caco-2 BBe) cell monolayers induced a significant reduction in transepithelial resistance, indicating decreased intestinal epithelial barrier function. The chymase-induced intestinal epithelial barrier dysfunction was characterized by chymase-induced protease-activated receptor (PAR)-2 activation and matrix metalloproteinase (MMP)-2 expression and activation. Consistent with this observation, in vitro analysis revealed chymase-induced PAR-2 activation and increased MAPK activity and MMP-2 expression. Pharmacological and small interfering RNA-mediated antagonism of PAR-2 and MMP-2 significantly attenuated chymase-stimulated barrier dysfunction. Additionally, the chymase/MMP-2-mediated intestinal epithelial dysfunction was associated with a significant reduction in the tight junction protein claudin-5, which was partially restored by MMP-2 inhibition. Finally, incubation of Caco-2 BBe cells with chymase-sufficient, but not chymase-deficient, bone marrow-derived mast cells decreased barrier function, which was attenuated by the chymase inhibitor chymostatin. Collectively, these results suggest that mast cell/chymase-mediated intestinal epithelial barrier function is mediated by PAR-2/MMP-2-dependent pathways.

Efficacy and safety of a unique enteric-coated bicarbonate-buffered pancreatic enzyme replacement therapy in children and adults with cystic fibrosis.

BACKGROUND: Pancreatic enzyme replacement therapy (PERT) is used to treat exocrine pancreatic insufficiency in cystic fibrosis. RESULTS/METHODS: Efficacy and safety of a unique enteric-coated (EC) bicarbonate-buffered PERT product (PERTZYE®/PANCRECARB®; Digestive Care, Inc., Bethlehem, PA, USA) was studied in a randomized, double-blind, placebo-controlled cross-over design. Subjects were stabilized on EC-bicarbonate-buffered PERT and a high-fat diet. During two treatment periods, subjects were randomized to EC-bicarbonate-buffered PERT or placebo, followed by a 72-h stool collection employing an ingested stool dye marker. Mean coefficient of fat absorption with EC-bicarbonate-buffered PERT was 82.5% compared with 46.3% with the placebo (absolute difference 36.2%; p < 0.001), a 78.2% improvement for active over placebo. Similar improvements in nitrogen absorption were observed. Overall stool frequency and stool weight decreased (p < 0.001). No safety concerns were identified. SUMMARY: EC-bicarbonate-buffered PERT is effective in treating cystic fibrosis-associated exocrine pancreatic insufficiency.

Equivalence considerations for orally inhaled products for local action-ISAM/IPAC-RS European Workshop report.

The purpose of this article is to document the discussions at the 2010 European Workshop on Equivalence Determinations for Orally Inhaled Drugs for Local Action, cohosted by the International Society for Aerosols in Medicine (ISAM) and the International Pharmaceutical Consortium on Regulation and Science (IPAC-RS). The article summarizes current regulatory approaches in Europe, the United States, and Canada, and presents points of consensus as well as ongoing debate in the four major areas: in vitro testing, pharmacokinetic and pharmacodynamic studies, and device similarity. Specific issues in need of further research and discussion are also identified.

Intestinal mast cell levels control severity of oral antigen-induced anaphylaxis in mice.

Food-triggered anaphylaxis can encompass a variety of symptoms that affect multiple organ systems and can be life threatening. The molecular distinction between non-life-threatening and life-threatening modes of such anaphylaxis has not yet been delineated. In this study, we sought to identify the specific immune functions that regulate the severity of oral antigen-induced anaphylaxis. We thus developed an experimental mouse model in which repeated oral challenge of ovalbumin-primed mice induced an FcεRI- and IgE-dependent oral antigen-triggered anaphylaxis that involved multiple organ systems. Strikingly, the severity of the systemic symptoms of anaphylaxis (eg, hypothermia) positively correlated with the levels of intestinal mast cells (r = -0.53; P < 0.009). In addition, transgenic mice with both increased intestinal and normal systemic levels of mast cells showed increased severity of both intestinal and extra-intestinal symptoms of IgE-mediated passive as well as oral antigen- and IgE-triggered anaphylaxis. In conclusion, these observations indicate that the density of intestinal mast cells controls the severity of oral antigen-induced anaphylaxis. Thus, an awareness of intestinal mast cell levels in patients with food allergies may aid in determining their susceptibility to life-threatening anaphylaxis and may eventually aid in the treatment of food-triggered anaphylaxis.

Long-term intersession variability for single-breath diffusing capacity.

BACKGROUND: Characterizing long-term diffusing capacity (DL(CO)) variability is important in assessing quality control for DL(CO) equipment and patient management. Long-term DL(CO) variability has not been reported. OBJECTIVES: It was the aim of this study to characterize long-term variability of DL(CO) in a cohort of biocontrols and to compare different methods of selecting a target value. METHODS: Longitudinal DL(CO) monitoring of biocontrols was performed as part of the inhaled insulin development program; 288 biocontrols were tested twice monthly for up to 5 years using a standardized technique. Variability, expressed either as percent change or DL(CO) units, was assessed using three different target values. RESULTS: The 90th percentile for mean intersession change in DL(CO) was between 10.9 and 15.8% (2.6-4.1 units) depending on the target value. Variability was lowest when the mean of all DL(CO) tests was used as the target value and highest when the baseline DL(CO) was used. The average of the first six DL(CO) tests provided an accurate estimate of the mean DL(CO) value. Using this target, the 90th percentile for mean intersession change was 12.3% and 3.0 units. Variability was stable over time and there were no meaningful associations between variability and demographic factors. CONCLUSIONS: DL(CO) biocontrol deviations >12% or >3.0 units, from the average of the first six tests, indicate that the instrument is not within quality control limits and should be carefully evaluated before further patient testing.

Methacholine challenge as a clinical bioassay of pulmonary delivery of a long-acting ß2-adrenergic agonist.

STUDY OBJECTIVE: To determine whether the methacholine challenge method used for albuterol can be applied to assess long-acting ß2-adrenergic agonist (LABA) bioequivalence, which would require a sufficiently steep dose-response curve. DESIGN: Prospective, unblinded, randomized, 2-way crossover study. SETTING: University medical center clinical research laboratory. PATIENTS: Ten adults, aged 21-58 years, with mild asthma (forced expiratory volume in 1 sec [FEV(1)] ≥ 70% predicted) and a baseline provocational concentration of methacholine required to decrease FEV(1) by 20% (PC(20)) of 4 mg/ml or less completed the study. INTERVENTION: Patients were randomized to receive a single dose of either 12 or 24 µg of formoterol delivered by a dry powder inhaler; 3-7 days later, at the same time of day, they received the other dose. MEASUREMENTS AND MAIN RESULTS: The FEV(1) was measured before and 1 hour after dosing, followed by performance of a methacholine challenge. Statistical analysis was performed by the 2-sample regression method for crossover studies. The dose-response curve for bronchodilatation was flat; the mean ± SD increase in FEV(1) after formoterol 12 and 24 µg was 14 ± 5% and 14 ± 8%, respectively (p>0.05). In contrast, the geometric mean PC20 (95% confidence interval) was 7 mg/ml (2-22 mg/ml) after the 12-µg dose and 16 mg/ml (5-45 mg/ml) after the 24-µg dose (p<0.001). CONCLUSION: Bioassay by methacholine challenge will be useful for bioequivalence studies of LABAs. A sample of at least 28 patients will be required for formoterol when methacholine challenge is performed in an optimal manner. The sample size may differ for other LABAs.

Colonic eosinophilic inflammation in experimental colitis is mediated by Ly6C(high) CCR2(+) inflammatory monocyte/macrophage-derived CCL11.

Recent genome-wide association studies of pediatric inflammatory bowel disease have implicated the 17q12 loci, which contains the eosinophil-specific chemokine gene CCL11, with early-onset inflammatory bowel disease susceptibility. In the current study, we employed a murine model of experimental colitis to define the molecular pathways that regulate CCL11 expression in the chronic intestinal inflammation and pathophysiology of experimental colitis. Bone marrow chimera experiments showed that hematopoietic cell-derived CCL11 is sufficient for CCL11-mediated colonic eosinophilic inflammation. We show that dextran sodium sulfate (DSS) treatment promotes the recruitment of F4/80(+)CD11b(+)CCR2(+)Ly6C(high) inflammatory monocytes into the colon. F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocytes express CCL11, and their recruitment positively correlated with colonic eosinophilic inflammation. Phenotypic analysis of purified Ly6C(high) intestinal inflammatory macrophages revealed that these cells express both M1- and M2-associated genes, including Il6, Ccl4, Cxcl2, Arg1, Chi3l3, Ccl11, and Il10, respectively. Attenuation of DSS-induced F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocyte recruitment to the colon in CCR2(-/-) mice was associated with decreased colonic CCL11 expression, eosinophilic inflammation, and DSS-induced histopathology. These studies identify a mechanism for DSS-induced colonic eosinophilia mediated by Ly6C(high)CCR2(+) inflammatory monocyte/macrophage-derived CCL11.

Interleukin-13 (IL-13)/IL-13 receptor alpha1 (IL-13Ralpha1) signaling regulates intestinal epithelial cystic fibrosis transmembrane conductance regulator channel-dependent Cl- secretion.

Interleukin-13 (IL-13) has been linked to the pathogenesis of inflammatory diseases of the gastrointestinal tract. It is postulated that IL-13 drives inflammatory lesions through the modulation of both hematopoietic and nonhematopoietic cell function in the intestine. To delineate the relevant contribution of elevated levels of intestinal IL-13 to intestinal structure and function, we generated an intestinal IL-13 transgenic mouse (iIL-13Tg). We show that constitutive overexpression of IL-13 in the small bowel induces modification of intestinal epithelial architecture (villus blunting, goblet cell hyperplasia, and increased epithelial proliferation) and epithelial function (altered basolateral → apical Cl(-) ion conductance). Pharmacological analyses in vitro and in vivo determined that elevated Cl(-) conductance is mediated by altered cystic fibrosis transmembrane conductance regulator expression and activity. Generation of iIL-13Tg/Il13rα1(-/-), iIL-13Tg/Il13rα2(-/-), and iIL-13Tg/Stat6(-/-) mice revealed that IL-13-mediated dysregulation of epithelial architecture and Cl(-) conductance is dependent on IL-13Rα1 and STAT-6. These observations demonstrate a central role for the IL-13/IL-13Rα1 pathway in the regulation of intestinal epithelial cell Cl(-) secretion via up-regulation of cystic fibrosis transmembrane conductance regulator, suggesting an important role for this pathway in secretory diarrhea.