Progenitor cell-derived basophils: A novel barcoded passive degranulation assay in allergic diseases.

BACKGROUND: Effector cells assays provide an overall measure of responsiveness to allergen, but the lack of reliable and high-throughput assays limits the clinical utility. We aimed to develop a high-throughput basophil activation test based on human progenitor cell-derived basophils (PCB) and investigate the role of PCB activation test (PCBAT) in allergic diseases. METHODS: Progenitor cell-derived basophils were differentiated from CD34+ progenitor cells and sensitized with sera from subjects sensitized to cat, peanut or atopic controls. Sensitized PCBs were stimulated with increasing concentrations of the corresponding allergens in vitro. Degranulation was assessed by measuring CD63 expression using flow cytometry. The correlations between PCBAT and clinical allergy were assessed. RESULTS: Following passive sensitization of the mature PCBs with serum and allergen stimulation, an allergen specific dose-dependent increase in CD63 expression was observed. Sera from subjects sensitized to cat (n = 35, of which 17 subjects had clinical reactivity quantified using inhaled allergen challenge), peanut allergic (n = 30, of which 15 subjects had clinical reactivity validated using double blind, placebo controlled food challenges [DBPCFC]), peanut-sensitized but tolerant subjects (n = 5) were used to sensitize PCBs. PCBAT area under the curve (AUC) correlated with sIgE (r2  = .49, p = .001) in subjects sensitized to cat (sIgE ≥ 0.35KU/L). The provocation concentration of inhaled cat allergen (PC20 ) correlated with PCBAT AUC (r2  = .33, p = .016). In subjects sensitized to peanut, PCBAT AUC was highly correlated with sIgE to Ara h 2 (r2  = .59, p < .0001). Peanut threshold cumulative dose during DBPCFC was negatively correlated with PCBAT AUC (r2  = .57, p = .001) and IgE to Ara h1 (r2  = .55, p = .007), but not with sIgE to whole peanut or Ara h2. All peanut-sensitized but tolerant subjects showed no reaction to peanut on PCBAT. CONCLUSION: Progenitor cell-derived basophils activation test is a high-throughput assay, which correlates with clinical allergy and may confer a powerful alternative tool in allergy testing.

The Effect of the Food Matrix on the In Vitro Bio-Accessibility and IgE Reactivity of Peanut Allergens.

SCOPE: Factors such as food processing, the food matrix, and antacid medication may affect the bio-accessibility of proteins in the gastrointestinal tract and hence their allergenic activity. However, at present they are poorly understood. METHODS AND RESULTS: Roasted peanut flour was incorporated into either a chocolate dessert or cookie matrix and bio-accessibility were assessed using an in vitro digestion system comprising a model chew and simulated gastric and duodenal digestion. Protein digestion was monitored by SDS-PAGE and immunoreactivity analyzed by immunoblotting and immunoassay. IgE reactivity was assessed by immunoassay using serum panels from peanut-allergic subjects. Roasted peanut flour proteins proved highly digestible following gastro-duodenal digestion even when incurred into a food matrix, with only low molecular weight polypeptides of Mr < 8 kDa remaining. When gastric digestion was performed at pH 6.5 (simulating the effect of antacid medication), peanut proteins are not digested; subsequent duodenal digestion is also limited. IgE reactivity of the major peanut allergens Ara h 1, Ara h 2, and Ara h 6, although reduced, was retained after oral-gastro-duodenal digestion irrespective of digestion conditions employed. CONCLUSION: Peanut allergen bio-accessibility is unaffected by the dessert or cookie matrices whilst high intra-gastric pH conditions render allergens more resistant to digestion.

Insoluble and soluble roasted walnut proteins retain antibody reactivity.

Thermal processing techniques commonly used during food production have the potential to impact food allergens by inducing physical and/or chemical changes to the proteins. English walnuts (Juglans regia) are among the most commonly allergenic tree nuts, but little information is available regarding how walnut allergens respond to thermal processing. This study evaluated the effects of dry roasting (132 or 180°C for 5, 10, or 20min) on the solubility and immunoreactivity of walnut proteins. A dramatic decrease in walnut protein solubility was observed following dry roasting at 180°C for 20min. However, both the soluble and insoluble protein fractions from roasted walnuts maintained substantial amounts of IgG immunoreactivity (using anti-raw and anti-roasted walnut antisera), with similar patterns of reactivity observed for human IgE from walnut-allergic individuals. Thus, walnut proteins are relatively stable under certain thermal processing conditions, and IgE reactivity remains present even when insoluble aggregates are formed.

An eHealth Approach to Reporting Allergic Reactions to Food and Closing the Knowledge Gap.

There is an important knowledge gap in food allergy management in understanding the factors that determine allergic reactions to food, in gathering objective reports of reactions in real time, and in accessing patients' reaction-histories during consultations. We investigate how eHealth methods can close this knowledge gap. We report experiences with an online tool for reporting allergic reactions that we have developed as a web application. This application has been successfully validated by participants from Ireland and the UK, and is currently being used in a pilot where participants report allergic reactions in near-real time.

Characterization of low molecular weight allergens from English walnut (Juglans regia).

Although English walnut is a commonly allergenic tree nut, walnut allergens have been poorly characterized to date. The objective of this work was to characterize the natural, low molecular weight (LMW) allergens from walnut. A protocol was developed to purify LMW allergens (specifically 2S albumins) from English walnuts. In addition to 2S albumins, a series of peptides from the N-terminal region of the 7S seed storage globulin proprotein were also identified and characterized. These peptides comprised a four-cysteine motif (C-X-X-X-C-X10-12-C-X-X-X-C) repeated throughout the 7S N-terminal region. Upon IgE immunoblotting, 3/11 and 5/11 sera from walnut-allergic subjects showed IgE reactivity to the 7S N-terminal fragments and 2S albumin, respectively. The mature 7S protein and the newly described 7S N-terminal peptides represent two distinct types of allergens. Because the proteolytic processing of 7S globulins has not been elucidated in many edible plant species, similar protein fragments may be present in other nuts and seeds.

Genetic variants in endotoxin signalling pathway, domestic endotoxin exposure and asthma exacerbations.

BACKGROUND: We investigated the interaction between genetic variants in endotoxin signalling pathway and domestic endotoxin exposure in relation to asthma presence, and amongst children with asthma, we explored the association of these genetic variants and endotoxin exposure with hospital admissions due to asthma exacerbations. METHODS: In a case-control study, we analysed data from 824 children (417 asthmatics, 407 controls; age 5-18 yr). Amongst asthmatics, we extracted data on hospitalization for asthma exacerbation from medical records. Endotoxin exposure was measured in dust samples collected from homes. We included 26 single-nucleotide polymorphisms (SNPs) in the final analysis (5 CD14, 7LY96 and 14 TLR4). RESULTS: Two variants remained significantly associated with hospital admissions with asthma exacerbations after correction for multiple testing: for CD14 SNP rs5744455, carriers of T allele had decreased risk of repeated hospital admissions compared with homozygotes for C allele [OR (95% CI), 0.42 (0.25-0.88), p = 0.01, False Discovery Rate (FDR) p = 0.02]; for LY96 SNP rs17226566, C-allele carriers were at a lower risk of hospital admissions compared with T-allele homozygotes [0.59 (0.38-0.90), p = 0.01, FDR p = 0.04]. We observed two interactions between SNPs in CD14 and LY96 with environmental endotoxin exposure in relation to hospital admissions due to asthma exacerbation which remained significant after correction for multiple testing (CD14 SNPs rs2915863 and LY96 SNP rs17226566). CONCLUSION: Amongst children with asthma, genetic variants in CD14 and LY96 may increase the risk of hospital admissions with acute exacerbations. Polymorphisms in endotoxin pathway interact with domestic endotoxin exposure in further modification of the risk of hospitalization.

Assessing the association of early life antibiotic prescription with asthma exacerbations, impaired antiviral immunity, and genetic variants in 17q21: a population-based birth cohort study.

BACKGROUND: The relationship between early-life antibiotic use and the development of wheeze and asthma has been reported in several studies but might arise as a consequence of bias rather than causal relationship. We investigated the association between antibiotic prescription and subsequent development of atopy, wheeze, and asthma exacerbations, and the relation of early life antibiotic prescription with anti-infective immunity and genetic variants on asthma susceptibility locus 17q21. METHODS: Children in a population-based birth cohort were followed from birth to age 11 years. Information on antibiotic prescription, wheeze, and asthma exacerbations was extracted from medical records, and the effect of antibiotic prescription assessed with longitudinal analyses. We assessed immune responses of peripheral blood mononuclear cells, taken at age 11 years, to viruses (rhinovirus and respiratory syncytial virus; RSV) and bacteria (Haemophilus influenzae and Streptococcus pneumoniae) in children who either received at least one or no antibiotic prescriptions in infancy. Finally, we assessed the association of 17q21 polymorphisms with antibiotic prescription. FINDINGS: Of 984 families who gave consent, we extracted data for 916 children. We noted significantly higher risk of physician-confirmed wheezing after antibiotic prescription (hazard ratio [HR] 1·71, 95% CI 1·32-2·23; p<0·0001) and severe wheeze or asthma exacerbation after antibiotic prescription (HR 2·26, 95% CI 1·03-4·94; p=0·041). In children who wheezed, the hazards of exacerbations (2·09, 1·51-2·90; p<0·0001) and admissions to hospital (2·64, 1·49-4·70; p=0·0009) were significantly increased in the 2 years after the first antibiotic prescription. Children who received antibiotics in infancy had significantly lower induction of cytokines, which are important in host defence against virus infections to both RSV and rhinovirus; there were no differences in antibacterial responses. Variants in 17q21 were associated with an increased risk of early life antibiotic prescription. INTERPRETATION: The association between antibiotics and asthma might arise through a complex confounding by indication. Hidden factors that may increase the likelihood of both early life antibiotic prescription and later asthma are an increased susceptibility to viral infections consequent upon impaired antiviral immunity and genetic variants on 17q21. FUNDING: Moulton Charitable Foundation and Medical Research Council.

Joint modeling of parentally reported and physician-confirmed wheeze identifies children with persistent troublesome wheezing.

BACKGROUND: Previous studies have suggested the presence of different childhood wheeze phenotypes through statistical modeling based on parentally reported wheezing. OBJECTIVE: We sought to investigate whether joint modeling of observations from both medical records and parental reports helps to more accurately define wheezing disorders during childhood and whether incorporating information from medical records better characterizes severity. METHODS: In a population-based birth cohort (n = 1184), we analyzed data from 2 sources (parentally reported current wheeze at 4 follow-ups and physician-confirmed wheeze from medical records in each year from birth to age 8 years) to determine classes of children who differ in wheeze trajectories. We tested the validity of these classes by examining their relationships with objective outcomes (lung function, airway hyperreactivity, and atopy), asthma medication, and severe exacerbations. RESULTS: Longitudinal latent class modeling identified a 5-class model that best described the data. We assigned classes as follows: no wheezing (53.3%), transient early wheeze (13.7%), late-onset wheeze (16.7%), persistent controlled wheeze (13.1%), and persistent troublesome wheeze (PTW; 3.2%). Longitudinal trajectories of atopy and lung function differed significantly between classes. Patients in the PTW class had diminished lung function and more hyperreactive airways compared with all other classes. We observed striking differences in exacerbations, hospitalizations, and unscheduled visits, all of which were markedly higher in patients in the PTW class compared with those in the other classes. For example, the risk of exacerbation was much higher in patients in the PTW class compared with patients with persistent controlled wheeze (odds ratio [OR], 3.58; 95% CI, 1.27-10.09), late-onset wheeze (OR, 15.92; 95% CI, 5.61-45.15), and transient early wheeze (OR, 12.24; 95% CI, 4.28-35.03). CONCLUSION: We identified a novel group of children with persistent troublesome wheezing, who have markedly different outcomes compared with persistent wheezers with controlled disease.

Association between C677T and A1298C MTHFR gene polymorphism and nonsyndromic orofacial clefts in the Turkish population: a case-parent study.

Two common MTHFR gene polymorphisms (C677T and A1298C) have been implicated in the etiology of nonsyndromic cleft lip/palate (nsCL/P). To investigate the genotype association among nsCL/P in the Turkish population, 56 case-parent trios were recruited into the study. Genotype frequencies were compared to two groups of controls from the same population. A total of 46 case-parent trios were included in transmission disequilibrium test (TDT) analysis. The mothers of the study group had a higher frequency of 677TT genotype, with a three-fold increased risk of having nsCL/P offspring (odds ratio [OR]: 3.14, p=0.03). The combined 677CT/1298AC genotype was also common among these mothers (28%), but it did not reach statistical significance (OR: 2.27, p=0.07). TDT analysis for (C677T) T allele transmission did not reveal a significant association. In conclusion, mothers carrying 677TT genotype or with 677CT/1298AC combined genotype have increased risk of having nsCL/P offspring; therefore, higher periconceptional folic acid supplementation should be advised for decreasing the recurrence risk.

Increased serum-soluble interleukin-5 receptor alpha level precedes the development of eczema in children.

Interleukin-5 receptor α-subunit expression may be implicated in the development of allergic diseases. In a population-based birth cohort, we investigated the relationship between IL-5Rα and the development of allergic phenotypes in childhood, using soluble IL-5Rα (s-IL-5Rα) as a marker. Children (n = 510) were followed from birth and assessed at age 3, 5 and 8. Based on the onset and resolution of symptoms, we assigned children into the following wheeze and eczema phenotypes: never, transient, persistent, intermittent and late-onset. Specific IgE to common allergens, s-IL-5Rα (ELISA) and urinary eosinophilic protein X (U-EPX) levels was measured at age 5. s-IL-5Rα was significantly higher among atopic compared to non-atopic children (pg/ml, geometric means [95% CI], 152.4 [126.0-184.5] vs. 103.4 [94.0-113.9], p < 0.0001). While we found no association between s-IL-5Rα and current eczema at age 5, there was a significant association between eczema phenotypes and s-IL-5Rα (multiple anova model adjusted for gender and atopy, F = 2.56, p = 0.04). After adjustment for multiple comparisons, we found that children with late-onset eczema had significantly higher s-IL-5Rα compared to those who have never had eczema (mean difference [95% CI], 2.41 [1.03-5.62], p = 0.04) and those with intermittent eczema (2.63 [1.08-6.41], p = 0.02), with no difference between children who have never had eczema and other eczema phenotypes. We found no such association for wheeze phenotypes. There was a weak correlation between s-IL-5Rα and U-EPX (r = 0.16, p < 0.0001). Increased serum s-IL-5Rα level at age 5 was associated with contemporaneous atopic sensitization and with subsequent development of eczema by age 8.

Efficacy of long-term sublingual immunotherapy as an adjunct to pharmacotherapy in house dust mite-allergic children with asthma.

Although sublingual immunotherapy (SLIT) is accepted to be a viable alternative of specific-allergen immunotherapy, the efficacy of long-term SLIT in asthmatic children is not well established. The efficacy of 3 yr of SLIT in addition to pharmacotherapy was compared with pharmacotherapy alone in a prospective, open, parallel-group, controlled study. Children with asthma aged 4-16 yr, sensitive to house dust mite (HDM) were followed up for a run-in period of 1 yr and then grouped as those who would receive SLIT + pharmacotherapy (n = 62) or pharmacotherapy alone (n = 28). All patients were evaluated based on symptom-medication scores and lung function tests every 3 months, as well as skin-prick test and serum total immunoglobulin E (IgE) levels annually for 3 yr. Children in the SLIT + pharmacotherapy group demonstrated significantly lower mean daily dose and annual duration of inhaled corticosteroid (ICS) usage when compared with controls. At the end of the 3 yr, within-group comparisons revealed statistically significant decreases in the dose and duration of ICS only in the SLIT group. Furthermore, 52.4% of subjects in the SLIT + pharmacotherapy group were able to discontinue ICS treatment for at least 6 months, which was only 9.1% for the pharmacotherapy group. Three years of SLIT as an adjunct to pharmacotherapy resulted in reduction of both the duration and dose of ICSs and successful discontinuation of ICSs along with improvement in lung functions in HDM-allergic children with asthma.

Staphylococcus aureus sensitization and allergic disease in early childhood: population-based birth cohort study.

BACKGROUND: Staphylococcus aureus-secreted enterotoxins (SEs) may be involved in the pathophysiology of atopic diseases. OBJECTIVE: We investigated the role of SEs in allergic diseases during early childhood (using the mixture of SE-specific IgEs [SE-mix] as a marker). METHODS: Children (N = 510) were followed from birth to age 5 years (repeated questionnaires, IgE to inhalant and food allergens, lung function [spirometry, plethysmography], airway reactivity [dry air challenge]). We measured SE-mix specific IgE (SE-A, SE-C, toxic shock syndrome toxin 1) by using fluorescence immunoassay. RESULTS: We found no association between rhinitis and SE-mix sensitization. Children with eczema were more frequently SE-mix-sensitized than children without (17.4% vs 8.3%; P = .02). SE-mix sensitization rate increased significantly with increasing eczema severity (no eczema, mild, moderate/severe: 8.3%, 14.8%, 42.9%; P = .003) and remained independently associated with eczema in a multivariate model adjusting for total IgE (adjusted odds ratio, 2.19; 95% CI, 1.05-4.56; P = .04). SE-mix sensitization was associated with current wheeze in the univariate but not the multivariate model. Among wheeze phenotypes, persistent wheezers were most commonly sensitized to SE-mix (never, transient, late-onset, persistent: 8.5%, 3.8%, 7.7%, 17.6%; P = .05). Among wheezers, those SE-mix-sensitized had significantly higher airway reactivity compared with those nonsensitized (mean FEV(1) change, mL [95% CI]: -59 [-121, 3] vs 19 [-10.2, 48.9]; P = .04), with little difference after adjusting for atopy. CONCLUSION: We found differences in SE-mix IgE antibodies between healthy 5-year-old children and children with eczema and wheeze. The proportion of patients sensitized to SE-mix increases with increasing disease severity. CLINICAL IMPLICATIONS: Staphylococcal enterotoxins are potential modifiers of childhood wheeze and eczema.

Dust mite allergen avoidance as a preventive and therapeutic strategy.

Asthma is a global health problem with genetic and environmental components. Indoor allergens have a major impact on asthma, and exposure in sensitized subjects can compromise lung function. A reduction in allergen exposure would seem a logical facet to treatment. Methods for reducing mite allergen levels that are effective in the laboratory may not work in the home and may not result in a clinical benefit. Six ongoing studies are investigating the effects of environmental control on the primary prevention of asthma and allergies. Although the Isle of Wight and Canadian studies provide encouraging results at age 8 and 7 years, respectively, it will be some time before a definitive public health message emerges. For secondary prevention, there is little evidence to support the use of mite-proof encasings as a single intervention in adults. In children, however, single or multifaceted interventions appear to be of some benefit.