Human Bronchial Epithelial Cell-derived Factors from Severe Asthmatic Subjects Stimulate Eosinophil Differentiation.

Activated bronchial epithelial cells (BEC) release various alarmins, including thymic stromal lymphopoietin (TSLP), that drive type 2 inflammation. We hypothesize that BEC-derived factors promote in situ eosinophil differentiation and maturation, a process that is driven by an IL-5-rich microenvironment in asthmatic airways. To assess the eosinophilopoietic potential of epithelial-derived factors, eosinophil/basophil colony forming units (Eo/B-CFU) were enumerated in 14-day methylcellulose cultures of blood-derived nonadherent mononuclear cells incubated with BEC supernatants (BECSN) from healthy nonatopic controls (n = 8), mild atopic asthmatics (n = 9), and severe asthmatics (n = 5). Receptor-blocking antibodies were used to evaluate the contribution of alarmins. Modulation of the mRNA expression of transcription factors that are crucial for eosinophil differentiation was evaluated. BECSN stimulated the clonogenic expansion of eosinophil progenitors in vitro. In the presence of IL-5, Eo/B-CFU numbers were significantly greater in cocultures of BESCN from severe asthmatics compared with other groups. This was attenuated in the presence of a TSLP (but not an IL-33) receptor-blocking antibody. Recombinant human TSLP (optimal at 100 pg/ml) stimulated Eo/B-CFU growth, which was significantly enhanced in the presence of IL-5 (1 ng/ml). Overnight culture of CD34+ cells with IL-5 and TSLP synergistically increased GATA-binding factor 2 and CCAAT/enhancer-binding protein α mRNA expression. The eosinophilopoietic potential of factors derived from BEC is increased in severe asthma. Our data suggest that TSLP is a key alarmin that is produced by BECs and promotes in situ eosinophilopoiesis in a type 2-rich microenvironment.

IL-25 and IL-33 induce Type 2 inflammation in basophils from subjects with allergic asthma.

BACKGROUND: The alarmin cytokines IL-25 and IL-33 are key promoters of type 2 inflammation. Basophils respond to alarmin cytokines, however the relationship of these cytokines with basophil activation and recruitment in human studies of allergic asthma has not been well characterized. This study investigated the effect of IL-25 and IL-33 on basophils in a model of allergic asthma. METHODS: 10 mild allergic asthmatics underwent allergen and diluent inhalation challenges. Bone marrow aspirates were collected at pre-challenge and 24 h (h) post challenge. Peripheral blood and sputum samples were collected at pre-challenge, 7 h, and 24 h post-challenge to measure basophil expression of IL-17RB, ST2, and intracellular IL-25. Freshly isolated peripheral blood basophils from allergic donors were incubated overnight with IL-25 and IL-33, or sputum supernatant collected post-allergen to assess pro-inflammatory effects of mediators released in the airways. RESULTS: There were increased percentage of basophils expressing IL-17RB, ST2, and intracellular IL-25 collected from bone marrow, peripheral blood, and sputum after allergen inhalation challenge. In vitro stimulation with IL-25 and IL-33 increased the percentage of basophils expressing intracellular type 2 cytokines and surface activation markers, and primed eotaxin-induced migratory potential of basophils, which was mediated directly through IL-17RB and ST2, respectively. Stimulation of basophils with sputum supernatants collected post-allergen challenge up-regulated the percentage of basophils expressing markers of activation and intracellular type 2 cytokines, which was reversed following blockade of the common ß chain (ßc). CONCLUSIONS: Our findings indicate that the alarmin cytokines IL-33 and IL-25 increase basophil activation and migratory potential, and may pose as a novel therapeutic targets for the treatment of allergic asthma.

Allergen-Induced Increases in Interleukin-25 and Interleukin-25 Receptor Expression in Mature Eosinophils from Atopic Asthmatics.

BACKGROUND: Interleukin (IL)-25 plays a pivotal role in type 2 immune responses. In a baseline cross-sectional study, we previously showed that IL-25 plasma levels and IL-25 receptor (IL-25R: IL-17RA, IL-17RB, and IL-17RA/RB) expression on mature blood eosinophils are increased in atopic asthmatics compared to normal nonatopic controls. This study investigated allergen-induced changes in IL-25 and IL-25R expression in eosinophils from asthmatics. METHODS: Dual responder atopic asthmatics (n = 14) were enrolled in this randomized diluent-controlled crossover allergen challenge study. Blood was collected before and 24 h after the challenge. The surface expression of IL-25R was evaluated by flow cytometry on eosinophils and Th2 memory cells. In addition, plasma levels of IL-25 were measured by ELISA, and functional responses to IL-25 including type 2 cytokine expression, degranulation, and the migrational responsiveness of eosinophils were evaluated in vitro. RESULTS: Following the allergen but not the diluent inhalation challenge, significant increases in the expression of IL-17RB and IL-17RA/B were found on eosinophils but not on Th2 memory cells. IL-25 plasma levels and the number of eosinophils but not of Th2 memory cells expressing intracellular IL-25 increased significantly in response to the allergen but not the diluent challenge. Stimulation with physiologically relevant concentrations of IL-25 in vitro caused (i) degranulation of eosinophils (measured by eosinophil peroxidase release), (ii) enhanced intracellular expression of IL-5 and IL-13, and (iii) priming of eosinophil migration to eotaxin. IL-25 stimulated intracellular cytokine expression, and the migration of eosinophils was blocked in the presence of a neutralizing IL-25 antibody. CONCLUSIONS: Our findings suggest that the IL-25/IL-25R axis may play an important role in promoting the recruitment and proinflammatory function of eosinophils in allergic asthma.

Thymic stromal lymphopoietin activation of basophils in patients with allergic asthma is IL-3 dependent.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) released after antigenic stimulation of allergic asthmatic airways is a key initiator of type 2 inflammation. Basophils are important effectors of allergic inflammation in the airways. Murine basophils have been shown to respond to TSLP independently of IL-3 by increasing functional thymic stromal lymphopoietin receptor (TSLPR) expression. OBJECTIVE: The purpose of this study was to investigate the effect of TSLP stimulation on human basophil function. METHODS: Ten patients with mild allergic asthma underwent diluent and allergen inhalation challenges. Peripheral blood and sputum samples were collected at baseline and 7 and 24 hours after challenge, and bone marrow samples were collected at baseline and 24 hours after challenge to measure basophil TSLPR expression. In vitro experiments were conducted on purified human basophils to measure the effect of TSLP on degranulation, expression of activation markers and TH2 cytokines, and eotaxin-induced shape change. RESULTS: Allergen inhalation increased basophil numbers in the airways and significantly upregulated the expression of activation markers, TH2 intracellular cytokines, and receptors for TSLP, IL-3, and eotaxin in blood, bone marrow, and sputum basophils. In vitro stimulation with TSLP primed basophil migration to eotaxin and induced rapid and sustained basophil activation mediated directly through TSLPR and indirectly through an IL-3-mediated basophil autocrine loop. Basophils responded to TSLP at a similar magnitude and potency as the well-described basophil-activating stimuli IL-3 and anti-IgE. CONCLUSION: Our findings indicate that basophil activation during early- and late-phase responses to inhaled allergen might be driven at least in part by TSLP.

Inhibition of allergen-induced basophil activation by ASM-024, a nicotinic receptor ligand.

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) were identified on eosinophils and shown to regulate inflammatory responses, but nAChR expression on basophils has not been explored yet. OBJECTIVE: We investigated surface receptor expression of nAChR α4, α7 and α1/α3/α5 subunits on basophils. Furthermore, we examined the effects of ASM-024, a synthetic nicotinic ligand, on in vitro anti-IgE and in vivo allergen-induced basophil activation. METHODS: Basophils were enriched from the peripheral blood of allergic donors and the expression of nAChR subunits and muscarinic receptors was determined. Purified basophils were stimulated with anti-IgE in the presence of ASM-024 with or without muscarinic or nicotinic antagonists for the measurement of CD203c expression and histamine release. The effect of 9 days of treatment with 50 and 200 mg ASM-024 on basophil CD203c expression was examined in the blood of mild allergic asthmatics before and after allergen inhalation challenge. RESULTS: nAChR α4, α7 and α1/α3/α5 receptor subunit expression was detected on basophils. Stimulation of basophils with anti-IgE increased CD203c expression and histamine release, which was inhibited by ASM-024 (10(-5) to 10(-)(3) M, p < 0.05). The effect of ASM-024 was reversed in the presence of muscarinic and nicotinic antagonists. In subjects with mild asthma, ASM-024 inhalation significantly inhibited basophil CD203c expression measured 24 h after allergen challenge (p = 0.03). CONCLUSION: This study shows that ASM-024 inhibits IgE- and allergen-induced basophil activation through both nicotinic and muscarinic receptors, and suggests that ASM-024 may be an efficacious agent for modulating allergic asthma responses.

Starch or Saline After Cardiac Surgery: A Double-Blinded Randomized Controlled Trial.

BACKGROUND: Despite decades of investigation, the balance of clinical risks and benefits of fluid supplementation with starch remain unresolved. Patient-centered outcomes have not been well explored in a "real-world" trial in cardiac surgery. OBJECTIVE: We sought to compare a starch-based fluid strategy with a saline-based fluid strategy in the cardiac surgery patient. DESIGN: A pragmatic blinded randomized controlled trial comparing starch-based with saline-based fluid strategy. SETTING: A large tertiary academic center in London Ontario between September 2009 and February 2011. PARTICIPANTS: Patients undergoing planned, isolated coronary revascularization. MEASUREMENTS: Serum creatinine and patient weight were measured daily postoperatively. METHODS: Patients were randomized to receive 6% hydroxyethyl starch (Voluven) or saline for perioperative fluid requirements. Fluid administration was not protocolized. Co-primary outcomes were incidence of acute kidney injury (AKI) and maximum postoperative weight gain. Secondary outcomes included bleeding, transfusion, inotropic and ventilator support, and fluid utilization. RESULTS: The study was prematurely terminated due to resource limitations. A total of 69 patients (19% female, mean age = 65) were randomized. Using RIFLE criteria for AKI, "risk" occurred in 12 patients in each group (risk ratio [RR] = 1.0; 95% confidence interval [CI] = 0.5-1.9; P = 1.00), whereas "injury" occurred in 7 of 35 (20%) and 3 of 34 (9%) of patients in the starch and saline groups, respectively (RR = 2.3; 95% CI = 0.6-8.1; P = .31). Maximum weight gain, bleeding and blood product usage, and overall fluid requirement were similar between groups. LIMITATIONS: The study had to be prematurely terminated due to resource limitations which led to a small sample size which was not sufficiently powered to detect a difference in the primary outcomes. CONCLUSIONS: This pragmatic double-blinded randomized controlled trial revealed a number of interesting hypothesis-generating trends and confirmed the feasibility of undertaking a logistically complex trial in a pragmatic fashion.

CONTEXTE: L'équilibre entre les avantages et les risques cliniques d'une supplémentation en fluides à base d'amidon n'est toujours pas établi malgré des décennies d'études. Les résultats des patients subissant une chirurgie cardiaque n'ont pas été explorés suffisamment dans le cadre d'un essai concret. OBJECTIF: Comparer deux stratégies de supplémentation liquidienne, une solution à base d'amidon et une solution saline, chez des patients subissant une chirurgie cardiaque. TYPE D'ÉTUDE: Un essai pragmatique, contrôlé, à répartition aléatoire et mené en double insu comparant deux stratégies de supplémentation liquidienne une solution à base d'amidon et une solution saline. CADRE: Un grand centre universitaire de soins tertiaires de London (Ontario) entre septembre 2009 et février 2011. SUJETS: Des patients subissant une revascularisation coronarienne planifiée et isolée. MESURES: La créatinine sérique et le poids du patient ont été mesurés quotidiennement à la suite de l'intervention. MÉTHODOLOGIE: Les patients ont été répartis aléatoirement pour recevoir du Voluven (solution d'amidon hydroxyéthylé à 6 %) ou une solution saline pour les fluidiques périopératoires. L'administration ne s'est pas faite selon un protocole établi. L'incidence d'insuffisance rénale aiguë (IRA) et un gain pondéral maximal après l'intervention constituaient les deux principaux résultats mesurés. Les résultats secondaires incluaient une hémorragie, l'utilisation de transfusion sanguine, d'inotrope, d'assistance respiratoire et l'administration de fluides. RÉSULTATS: L'étude a été interrompue prématurément par manque de ressources. Les 69 patients (19 % de femmes) répartis aléatoirement avaient en moyenne 65 ans. La classification RIFLE avait permis de détecter un « risque ¼ d'IRA chez 12 patients dans chacun des groupes (RC: 1,0; IC 95 %: 0,5-1,9; p=1,00) et une « insuffisance ¼ chez 7 patients sur 35 (20 %) du groupe « amidon ¼ et 3 patients sur 34 (9 %) du groupe « saline ¼ (RC: 2,3; IC 95 %: 0,6-8,1; p=0,31). Le gain pondéral maximal, le nombre d'hémorragies, l'utilisation de produits sanguins et les besoins liquidiens étaient similaires dans les deux groupes. LIMITES: L'étude a été interrompue prématurément en raison d'un manque de ressources. Par conséquent, le faible échantillon de patients s'avère insuffisamment puissant pour détecter des différences significatives entre les deux groupes. CONCLUSIONS: Cette étude a mis en lumière quelques tendances permettant d'émettre des hypothèses intéressantes. L'étude a également confirmé la possibilité d'entreprendre un essai logistique complexe de manière pragmatique.