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1.  Invariant Natural Killer T cells in children with Eosinophilic Esophagitis 
Background
Eosinophilic esophagitis (EoE) is an atopic disease characterized by eosinophilic inflammation in which dietary antigens (in particular, milk) play a major role. EoE is most likely a mixed IgE and non-IgE food-mediated reaction in which over-expression of Th2 cytokines, particularly IL-13, play a major role; however, the cells responsible for IL-13 over-expression remain elusive. Th2-cytokines are secreted following the ligation of invariant natural killer T cell receptors to sphingolipids (SL). Sphingolipids (SL) are presented via the CD1d molecule on the INKT cell surface. Cow’s milk-derived SL has been shown to activate iNKTs from children with IgE-mediated food allergies to milk (FA-MA) to produce Th2 cytokines. The role of iNKTs and milk-SL in EoE pathogenesis is currently unknown.
Objective
To investigate the role of iNKTs and milk-SL in EoE.
Methods
Peripheral blood mononuclear cells (PBMCs) from 10 children with active EoE (EoE-A), 10 children with controlled EoE (EoE-C), and 16 healthy controls (Non-EoE) were measured ex-vivo and then incubated with α-galactosylceramide (αGal) and milk-SL. INKTs from peripheral blood (PB) and esophageal biopsies were studied.
Results
EoE-A-children had significantly fewer peripheral blood iNKTs with a greater Th2-response to αGal and milk-SM compared to iNKTs of EoE-C and Non-EoE children. Additionally, EoE-A children had increased iNKT levels in esophageal biopsies compared to EoE-C children.
Conclusion
Milk-SLs are able to activate peripheral blood iNKTs in EoE-A children to produce Th2 cytokines. Additionally, iNKT levels are higher at the site of active esophageal eosinophilic inflammation.
Clinical Relevance
This study suggests that sphingolipids (SL) contained in milk may drive the development of EoE by promoting an iNKT cell-mediated Th2-type cytokine response that facilitates eosinophil-mediated allergic inflammation.
doi:10.1111/cea.12201
PMCID: PMC3926503  PMID: 24118614
Eosinophilic esophagitis; invariant natural killer T cells; sphingolipids
2.  Role of maternal elimination diets and human milk IgA in development of cow’s milk allergy in the infants 
Background
The role of maternal avoidance diets in the prevention of food allergies is currently under debate. Little is known regarding the effects of such diets on human milk (HM) composition or induction of infant humoral responses.
Objective
To assess the association of maternal cow’s milk (CM) avoidance during breastfeeding with specific IgA levels in HM and development of cow’s milk allergy (CMA) in infants.
Methods
We utilized HM and infant serum samples from a prospective birth cohort of 145 dyads. Maternal serum and HM samples were assessed for casein and beta-lactoglobulin (BLG)-specific IgA and IgG by ELISA; 21 mothers prophylactically initiated a strict maternal CM avoidance diet due to a sibling’s history of food allergy and 16 due to atopic eczema or regurgitation/vomiting seen in their infants within the first 3 months of life. Infants’ sera were assessed for casein and BLG-specific IgG, IgA and IgE; CMA was confirmed by an oral food challenge. The impact of HM on BLG uptake was assessed in transcytosis assays utilizing Caco-2 intestinal epithelial cell line.
Results
Mothers avoiding CM had lower casein- and BLG-specific IgA in HM than mothers with no CM restriction (p=0.019 and p=0.047). Their infants had lower serum casein- and BLG-specific IgG1 (p=0.025 and p<0.001) and BLG-specific IgG4 levels (p=0.037) and their casein- and BLG-specific IgA levels were less often detectable than those with no CM elimination diet (p=0.003 and p=0.007). Lower CM-specific IgG4 and IgA levels in turn were associated with infant CMA. Transcytosis of BLG was impaired by HM with high, but not low levels of specific IgA.
Conclusions
Maternal CM avoidance was associated with lower levels of mucosal specific IgA levels and development of CMA in infants.
Clinical relevance
HM IgA may play a role in preventing excessive, uncontrolled food antigen uptake in the gut lumen and thereby in the prevention of CMA.
doi:10.1111/cea.12228
PMCID: PMC4038099  PMID: 24164317
Breast feeding; breast milk; human milk; cow’s milk; avoidance; restriction diet; infants; cow’s milk allergy; IgA; secretory IgA; epithelium
3.  Bakery flour dust exposure causes non-allergic inflammation and enhances allergic airway inflammation in mice 
Background
Baker’s asthma is one of the most commonly reported occupational lung diseases in countries where fresh bread is baked daily in large quantities, and is characterized by rhinitis, bronchial hyperresponsiveness, and reversible airflow obstruction. Epidemiological studies have identified pre-existing atopy as an important risk factor for developing baker’s asthma, yet the etiology and pathogenesis of baker’s asthma remain poorly understood.
Objective
We sought to develop a mouse model of baker’s asthma that could be used to characterize the development and progression of baker’s asthma.
Methods
We were unable to sensitize mice to bakery flour dust or flour dust extract. We assessed total inflammatory cells, cellular differential, total serum IgE and the pro-inflammatory cytokine response to oropharyngeally instilled bakery flour dust or flour dust extract by itself or in the context of OVA sensitization and challenge.
Results
Both bakery flour dust and flour dust extract consistently elicited a neutrophilic inflammation in a tlr4-independent manner; suggesting that endotoxin is not playing a role in the inflammatory response to flour dust. Moreover, bakery flour dust and dust extract significantly enhance the inflammatory response in OVA sensitized and challenged mice.
Conclusions
Bakery flour dust and flour dust extract are strongly pro-inflammatory and can cause non-allergic airway inflammation and can enhance allergen-mediated airway inflammation.
doi:10.1111/j.1365-2222.2008.03038.x
PMCID: PMC4278578  PMID: 18564331
LPS; endotoxin; toll-like receptor 4; occupational airways disease; baker’s asthma; flour dust; allergic asthma
4.  Exhaled Nitric Oxide Predicts Persistence of Wheezing, Exacerbations, and Decline in Lung Function in Wheezy Infants and Toddlers 
Background
There are limited data assessing the predictive value of fraction of exhaled nitric oxide (FENO) for persistence of wheezing, exacerbations, or lung function change over time in infants/toddlers with recurrent wheezing.
Objectives
In an ongoing longitudinal cohort of infants and toddlers with recurrent wheezing compare predictive values of single-breath FENO (SB-FENO), tidal-breathing mixed-expired FENO (tidal-FENO), bronchodilator responsiveness (BDR), and the Castro-Rodriquez asthma predictive index (API) for persistence of wheezing, exacerbations, and lung function change through age 3 yrs.
Methods
Enrollment forced expiratory flows and volumes (iPFTs) were measured in 44 infants/toddlers using the raised-volume rapid thoracoabdominal compression method. SB-FENO was measured at 50 mL/sec, and tidal-FENO was measured during awake tidal breathing. Clinical outcomes were assessed at age 3 yrs. in 42 infants. Follow-up iPFTs were completed between ages 2.5-3 yrs. in 32 subjects.
Results
An enrollment SB-FENO concentration ≥30 ppb predicted persistence of wheezing at age 3 years with a sensitivity of 77%, a specificity of 94%, and an area under the curve (AUC) of 0.86 (95% CI: 0.74 – 0.98). The sensitivity, specificity, positive predictive, and negative predictive values of SB-FENO for persistence of wheezing and exacerbations were superior to tidal-FENO, BDR, and the API. SB-FENO ≥30 ppb and tidal FENO ≥7 ppb measured at enrollment was associated with a decline in both FEV0.5 and FEF25-75 between enrollment and age 3 years.
Conclusions
In wheezy infants/toddlers SB-FENO was superior to tidal-FENO, BDR, and the API in predicting future exacerbations and persistence of wheezing at age 3 years. Both SB-FENO and tidal FENO were associated with lung function decline over time.
doi:10.1111/cea.12171
PMCID: PMC3839057  PMID: 24261945
exhaled nitric oxide; FENO; recurrent wheezing; infants; pulmonary function; raised-volume rapid thoracic compression
5.  The relationship between eosinophilia and airway remodelling in mild asthma 
Background
Eosinophilia is a marker of corticosteroid responsiveness and risk of exacerbation in asthma; while it has been linked to submucosal matrix deposition, its relationship to other features of airway remodelling is less clear.
Objective
The aim of this study was to investigate the relationship between airway eosinophilia and airway remodelling.
Methods
Bronchial biopsies from subjects (n=20 in each group) with mild steroid-naïve asthma, with either low (0 – 0.45 mm−2)) or high submucosal eosinophil (23.43 – 46.28 mm−2) counts and healthy controls were assessed for in vivo epithelial damage (using EGFR staining), mucin expression, airway smooth muscle (ASM) hypertrophy and inflammatory cells within ASM.
Results
The proportion of in vivo damaged epithelium was significantly greater (p=0.02) in the eosinophil-high (27.37%) than the eosinophil-low (4.14%) group. Mucin expression and goblet cell numbers were similar in the two eosinophil groups; however, MUC-2 expression was increased (p=0.002) in the eosinophil-high group compared to controls. The proportion of submucosa occupied by ASM was higher in both asthma groups (p=0.021 and p=0.046) compared to controls. In the ASM, eosinophil and T lymphocyte numbers were higher (p<0.05) in the eosinophil-high group than both the eosinophil-low group and the controls, whilst the numbers of mast cells were increased in the eosinophil-high group (p=0.01) compared to controls.
Conclusion
Submucosal eosinophilia is a marker (and possibly a cause) of epithelial damage and is related to infiltration of airway smooth muscle with eosinophils and T lymphocytes but is unrelated to mucus metaplasia or smooth muscle hypertrophy.
doi:10.1111/cea.12156
PMCID: PMC4040270  PMID: 24261944
Asthma; eosinophil; remodelling; epithelium; goblet cell; smooth muscle; inflammation
6.  γδT cells regulate chronic airway inflammation and development of airway remodelling 
Clinical and Experimental Allergy  2014;44(11):1386-1398.
Background
γδT cells play a crucial immunoregulatory role in the lung, maintaining normal airway tone and preventing hyperresponsiveness to innocuous allergen. During acute inflammatory episodes, γδT cells promote resolution of acute inflammation. However, their contribution to inflammation-associated airway remodelling remains unexplored. Here we investigate the effects of γδT cell blockade on established allergic airway inflammation and development of remodelling.
Methods
Sensitised mice were exposed to prolonged ovalbumin challenge or continuous house-dust mite exposure to induce chronic inflammation and remodelling. Functional blocking anti-TCRδ antibody was administered therapeutically, and parameters of airway inflammation and remodelling were examined.
Results
Therapeutic blockade of γδT cells prevented the typical resolution of acute airway inflammation characterised by elevated eosinophil and Th2 cell numbers. Moreover, the lung displayed exacerbated airway remodelling, typified by excess peribronchiolar collagen deposition.
Conclusions
These results demonstrate a unique role for γδT cells in constraining allergen-induced airway remodelling. Manipulating the γδT cell compartment may therefore contribute to strategies to prevent and treat remodelling.
doi:10.1111/cea.12395
PMCID: PMC4238847  PMID: 25146585
airway remodelling; allergic inflammation; Th2 cells; γδT cells
7.  The regulatory T cells induction by epicutaneous immunotherapy is sustained and mediates long-term protection from eosinophilic disorders in peanut-sensitized mice 
Clinical and Experimental Allergy  2014;44(1):867-881.
Summary
Background
Allergen-specific immunotherapy favours immune deviation from a Th2 to a Th1 response and increases the number of regulatory T cells (Tregs). Epicutaneous immunotherapy (EPIT) of sensitized mice decreases the clinical and the allergen-specific Th2 responses and increases local and peripheral Foxp3+ Tregs.
Objective
To investigate the role of Tregs in EPIT and characterize their phenotype and maintenance following EPIT.
Methods
Tregs were investigated using in vivo depletion or adoptive transfer into BALB/c mice. Tregs were depleted using anti-CD25 antibody injection during EPIT, and allergen-specific responses were compared with Sham, EPIT alone and naïve mice. To demonstrate that Tregs can mediate protection by their own, and to study their maintenance following the end of EPIT, CD25+CD4+ Tregs isolated from mice just after or 8 weeks after EPIT were transferred into peanut-sensitized mice. Foxp3-IRES-mRFP mice were transferred with EPIT-induced Tregs to analyse the induction of host Tregs.
Results
The anti-CD25 antibody injection to EPIT mice abrogated the induction of Tregs in spleen and the expression of Foxp3 in oesophagus. This resulted in levels of peanut-induced eosinophilic infiltration in oesophagus similar to Sham and significantly higher than EPIT. Whereas the transfer of Tregs from Sham-treated mice demonstrated no effect, the transfer of Tregs isolated just after EPIT prevented peanut-induced eosinophil infiltration and eotaxin expression and induced Foxp3 in oesophagus. The transfer of Tregs isolated 8 weeks after EPIT suppressed allergen-specific responses as efficiently as did Tregs isolated just after EPIT and increased spleen Foxp3+ CD25+ CD4+ cells similarly. The use of reporter mice demonstrated an increase in host Tregs.
Conclusions
These results confirm the Tregs-mediated mechanism of EPIT and demonstrate the persistence of efficient Tregs during a long period of time after treatment cessation. This suggests that EPIT induces long-term tolerance in peanut-sensitized mice.
doi:10.1111/cea.12312
PMCID: PMC4233996  PMID: 24666588
epicutaneous immunotherapy; peanut allergy; regulatory T cells
8.  Efficacy and safety of 5-grass pollen sublingual immunotherapy tablets in patients with different clinical profiles of allergic rhinoconjunctivitis 
Clinical and Experimental Allergy  2009;39(3):387-393.
Summary
Background
The optimal dose of grass pollen tablets for sublingual immunotherapy (SLIT) in allergic rhinoconjunctivitis patients was previously established in a multinational, randomized, double-blind, placebo-controlled study in 628 adults. Patients were randomized to receive once-daily 5-grass pollen sublingual tablets of 100 IR (index of reactivity), 300 IR or 500 IR, or placebo starting 4 months before the pollen season.
Objective
The aim of this complementary analysis was to determine whether 300 IR 5-grass pollen SLIT-tablets is effective in different subtypes of patients who are allergic to grass pollen.
Methods
Different subgroups could be identified regarding comorbidities (with or without asthma during the grass-pollen season), sensitization (mono/polysensitization) and symptom severity. An additional exploratory analysis was performed within four subgroups based on pre-treatment assessment: Group 1=high specific IgE; Group 2=high symptom scores; Group 3=high skin sensitivity; Group 4=any of Group 1, 2 or 3.
Results
Asthma and sensitization status were not significant covariates as the average Rhinoconjunctivitis Total Symptom Score (RTSS) was identical for patients with and without grass-pollen asthma, as well as for mono- and polysensitized patients. Across the four subgroups, average RTSSs (± SD) for the optimal dosage (300 IR) were 3.91 ± 3.16, 3.83 ± 3.14, 2.55 ± 2.13 and 3.61 ± 2.97, for subgroups 1, 2, 3 and 4, respectively. ancova showed that in Group 1 average RTSS did not differ significantly with different doses of SLIT. In Groups 2, 3 and 4, doses of 300 IR and 500 IR were significantly more effective than 100 IR and placebo (P0.035). All doses of SLIT administered in this study can be considered safe in the patients investigated.
Conclusions
The risk-benefit ratio validates the use of 300 IR tablets in clinical practice in all of these patient subgroups, regardless of severity profile, sensitization status and presence of asthma.
doi:10.1111/j.1365-2222.2008.03152.x
PMCID: PMC4233960  PMID: 19134019
allergic asthma; monosensitized; polysensitized; rhinitis; rhinoconjunctivitis; safety; severity; sublingual grass pollen tablets
9.  Nasal cytokine responses to natural colds in asthmatic children 
Background
The mechanisms by which viruses induce asthma exacerbations are not well-understood.
Objective
We characterized fluctuations in nasal aspirate cytokines during naturally-occurring respiratory viral infections in children with asthma.
Methods
Sixteen children underwent home collections of nasal aspirates when they were without cold symptoms and again during self-reported respiratory illnesses. The presence of viral infection was ascertained by multiplex PCR. Cytokines were measured by multiplex immune assay. mRNA expression for selected markers of viral infection was measured by RT-PCR. A cumulative respiratory symptom score was calculated for each day of measurement. Generalized estimated equations were used to evaluate associations between viral infection and marker elevation, and between marker elevation and symptom score.
Results
The 16 patients completed a total of 37 weeks of assessment (15 “well” weeks; 22 self-assessed “sick” weeks). Viral infections were detected in three of the “well” weeks and 17 of the “sick” weeks (10 rhinovirus, 3 coronavirus, 2 influenza A, 2 influenza B, 2 respiratory syncytial virus, 1 parainfluenza). Compared to virus-negative well weeks, nasal aspirate IFN-γ, CXCL8/IL-8, CXCL10/IP-10, CCL5/RANTES, CCL11/eotaxin-1, CCL2/MCP-1, CCL4/MIP-1β, CCL7/MCP-3 and CCL20/MIP3α protein levels increased during virus-positive sick weeks. Only a subset of cytokines (IFN-γ, CXCL8, CCL2, CCL4, CCL5 and CCL20) correlated with self-reported respiratory tract symptoms. While many aspirates were dilute and showed no mRNA signal, viral infection significantly increased the number of samples that were positive for IFN-λ1, IFN-λ2/3, TLR3, RIG-I and IRF7 mRNA.
Conclusions & Clinical Relevance
We conclude that, in children with asthma, naturally-occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, IFNs and IFN-responsive genes.
doi:10.1111/cea.12005
PMCID: PMC4219353  PMID: 23181789
asthma; children; cytokines; IRF7; respiratory infection; rhinovirus; virus
10.  High proportions of FOXP3+CD25high T cells in neonates are positively associated with allergic sensitization later in childhood 
Clinical and Experimental Allergy  2014;44(7):940-952.
Background
The role of FOXP3+ regulatory T cells in the prevention against sensitization and allergy development is controversial.
Objective
We followed 65 newborn Swedish children from farming and non-farming families from birth to 3 years of age and investigated the relation between CD4+ T cell subsets in blood samples and development of sensitization and allergic disease.
Methods
The proportions of FOXP3+CD25high, CTLA-4+CD25+, CD45RO+, HLA-DR+, CCR4+ or α4β7+ within the CD4+ T cell population were examined by flow cytometry of blood samples at several time-points. Mononuclear cells were isolated from blood and stimulated with birch allergen, ovalbumin or the mitogen PHA, and the levels of IL-1β, IL-6, TNF, IFN-γ, IL-5 and IL-13 were measured. A clinical evaluation regarding the presence of allergen-specific IgE and allergy was performed at 18 and 36 months of age.
Results
Multivariate discriminant analysis revealed that children who were sensitized at 18 or 36 months of age had higher proportions of FOXP3+CD25high T cells at birth and at 3 days of life than children who remained non-sensitized, whereas allergy was unrelated to the neonatal proportions of these cells. The proportions of CTLA-4+CD25+ T cells were unrelated to both sensitization and allergy. The association between higher proportions of FOXP3+CD25high T cells and sensitization persisted after exclusion of farmer's children. Finally, a farming environment was associated with lower proportions of FOXP3+CD25high T cells in early infancy and to a more prominent T cell memory conversion and cytokine production.
Conclusion & Clinical Relevance
Our results indicate that high proportions of FOXP3+CD25high T cells in neonates are not protective against later sensitization or development of allergy.
doi:10.1111/cea.12290
PMCID: PMC4215110  PMID: 24528482
allergic sensitization; children; farm; FOXP3+CD25high T cells; neonates; T cell development
12.  Sensitization to Food and Inhalant Allergens in Relation to Age and Wheeze Among Children with Atopic Dermatitis 
Background
Atopic dermatitis (AD) is common in children; however, persistence of AD with or without asthma, is less common. Longitudinal studies remain limited in their ability to characterize how IgE antibody responses evolve in AD, and their relationship to asthma.
Objective
To use a cross-sectional study design of children with active AD to analyze age-related differences in IgE antibodies and relation to wheeze.
Methods
IgE antibodies to food and inhalant allergens were measured in children with active AD (5 months to 15 years of age, n=66), with and without history of wheeze.
Results
Whereas IgE antibodies to foods persisted at a similar prevalence and titer throughout childhood, IgE antibodies to all aeroallergens rose sharply into adolescence. From birth, the chance of sensitization for any aeroallergen increased for each 12-month increment in age (OR≥1.21, p≤0.01), with the largest effect observed for dust mite (OR=1.56, p<0.001). A steeper age-related rise in IgE antibody titer to dust mite, but no other allergen, was associated with more severe disease. Despite this, sensitization to cat was more strongly associated with wheeze (OR=4.5, p<0.01), and linked to Fel d 1 and Fel d 4, but not Fel d 2. Comparison of cat allergic children with AD to those without, revealed higher titers to Fel d 2 and Fel d 4 (p<0.05), but not Fel d 1.
Conclusions and Clinical Relevance
Differences in sensitization to cat and dust mite among young children with AD may aid in identifying those at increased risk for disease progression and development of asthma. Early sensitization to cat and risk for wheeze among children with AD may be linked to an increased risk for sensitization to a broader spectrum of allergen components from early life. Collectively, our findings argue for early intervention strategies designed to mitigate skin inflammation in children with AD.
doi:10.1111/cea.12169
PMCID: PMC3801418  PMID: 24074334
Atopic dermatitis; asthma; wheeze; IgE antibodies; age; food allergy; aeroallergens; cat; dust mite
13.  Longitudinal Relationship of Early-Life Immunomodulatory T-cell Phenotype and Function to Development of Allergic Sensitization in an Urban Cohort 
Background
Immunomodulatory T-cells are thought to influence development of allergy and asthma, but early-life longitudinal data on their phenotype and function are lacking.
Objectives
As part of the Urban Environment and Childhood Asthma (URECA) study, we investigated the development of immunomodulatory T-cell phenotype and function, and characterized their relation to allergic disease progression from birth through to age two years.
Methods
Immunomodulatory T-cell phenotype and function in cord blood mononuclear cells and peripheral blood mononuclear cells at age 1 and 2 years were characterized by analyzing CD25bright and FoxP3+ expression; proliferative responses and cytokine production. The relation of immunomodulatory T-cell characteristics to allergic sensitization and disease at 1- and 2-years was investigated.
Results
The proportion of CD4+CD25bright and CD4+CD25+FoxP3+ T-cells (n=114, 83, 82 at birth, 1- and 2-years respectively) increased significantly, while there were no significant changes in the suppressive function of CD25+ T-cells (n=78, 71, 81 at birth, 1- and 2-years respectively). Birth immunomodulatory T-cell characteristics were not related to subsequent allergic sensitization or disease. However, increases in the numbers of CD4+CD25bright cells and their ability to suppress lymphoproliferative responses at 1 year of age were associated with reduced allergic sensitization at ages 1 (p<0.03) and 2 (p<0.02) years. Production of the anti-inflammatory cytokine IL-10 by CD25+ T-cells appeared to mediate this protective suppressive function. In contrast, by two years of age, we observed the emergence of a positive association of CD4+CD25+FoxP3+ T-cell numbers with allergic sensitization (p=0.05) and eczema (p=0.02).
Conclusions and Clinical Relevance
These findings suggest that the relationship between immunomodulatory T-cell subsets, allergic sensitization, and eczema is developmentally regulated. In the first year of life CD4+CD25+ IL-10 producing T-cells are associated with a reduced incidence of allergic sensitization. Once allergic sensitization or eczema are established, CD4+CD25+FoxP3+ T-reg cell expand to potentially counteract the allergic inflammatory response. Understanding the relationship between development of immunoregulatory T cells and early-onset atopy could lead to new preventive strategies for allergic diseases.
doi:10.1111/j.1365-2222.2011.03882.x
PMCID: PMC4162345  PMID: 22092655
T regulatory cells; asthma; allergy; IL-10; newborn; suppressive index; toddler; CD4+CD25+FoxP3+
14.  Perceived Triggers of Asthma: Key to Symptom Perception and Management 
Adequate asthma management depends on an accurate identification of asthma triggers. A review of the literature on trigger perception in asthma shows that individuals vary in their perception of asthma triggers and that the correlation between self-reported asthma triggers and allergy tests is only modest. In this paper, we provide an overview of psychological mechanisms involved in the process of asthma triggers identification. We identify sources of errors in trigger identification and targets for behavioral interventions that aim to improve the accuracy of asthma trigger identification and thereby enhance asthma control.
doi:10.1111/cea.12138
PMCID: PMC3748392  PMID: 23957335
15.  Elevated levels of leukotriene C4 synthase mRNA distinguish a subpopulation of eosinophilic oesophagitis patients 
Background
Cysteinyl leukotrienes contribute to Th2-type inflammatory immune responses. Their levels in oesophageal tissue, however, do not distinguish patients with eosinophilic oesophagitis (EoE) from controls.
Objective
We asked whether mRNA levels of leukotriene C4 synthase (LTC4S), a key regulator of leukotriene production, could serve as a marker for EoE.
Methods
Digital mRNA expression profiling (nCounter® Technology) was performed on proximal and distal oesophageal biopsies of 30 paediatric EoE patients and 40 non-EoE controls. Expression data were confirmed with RT-qPCR. LTC4S mRNA levels were quantified in whole blood samples. Leukotriene E4 was measured in urine.
Results
LTC4S mRNA levels were elevated in proximal (2.6-fold, p<0.001) and distal (2.9-fold, p<0.001) oesophageal biopsies from EoE patients. Importantly, increased LTC4S mRNA transcripts identified a subpopulation of EoE patients (28%). This patient subgroup had higher serum IgE levels (669 U/ml vs. 106 U/ml, p=0.01), higher mRNA transcript numbers of TSLP (1.6-fold, p=0.009) and CD4 (1.4-fold, p=0.04) but lower IL-23 mRNA levels (0.5-fold, p=0.04). In contrast, elevated levels of IL-23 mRNA were found in oesophageal biopsies of patients with reflux oesophagitis. LTC4S mRNA transcripts in whole blood and urinary excretion of leukotriene E4 were similar in EoE patient subgroups and non-EoE patients.
Conclusion & Clinical Relevance
Elevated oesophageal expression of LTC4S mRNA is found in a subgroup of EoE patients, concomitant with higher serum IgE levels and an oesophageal transcriptome indicative of a more-pronounced allergic phenotype. Together with TSLP and IL-23 mRNA levels, oesophageal LTC4S mRNA may facilitate diagnosis of an EoE subpopulation for personalized therapy.
doi:10.1111/cea.12146
PMCID: PMC3728707  PMID: 23889244
eosinophilic gastrointestinal diseases; arachidonic acid metabolism; Th2 immune response; leukotriene; reflux oesophagitis
16.  Allergen hybrids – next generation vaccines for Fagales pollen immunotherapy 
Summary
Background
Trees belonging to the order of Fagales show a distinct geographical distribution. While alder and birch are endemic in the temperate zones of the Northern Hemisphere, hazel, hornbeam and oak prefer a warmer climate. However, specific immunotherapy of Fagales pollen-allergic patients is mainly performed using birch pollen extracts, thus limiting the success of this intervention in birch-free areas.
Objectives
T cells are considered key players in the modification of an allergic immune response during specific immunotherapy (SIT), therefore we thought to combine linear T cell epitope-containing stretches of the five most important Fagales allergens from birch, hazel, alder, oak and hornbeam resulting in a Fagales pollen hybrid (FPH) molecule applicable for SIT.
Methods
A Fagales pollen hybrid was generated by PCR-based recombination of low IgE-binding allergen epitopes. Moreover, a structural-variant FPH4 was calculated by in silico mutagenesis, rendering the protein unable to adopt the Bet v 1-like fold. Both molecules were produced in Escherichia coli, characterized physico-chemically as well as immunologically, and tested in mouse models of allergic sensitization as well as allergy prophylaxis.
Results
Using spectroscopic analyses, both proteins were monomeric, and the secondary structure elements of FPH resemble the ones typical for Bet v 1-like proteins, whereas FPH4 showed increased amounts of unordered structure. Both molecules displayed reduced binding capacities of Bet v 1-specific IgE antibodies. However, in a mouse model, the proteins were able to induce high IgG titres cross-reactive with all parental allergens. Moreover, prophylactic treatment with the hybrid proteins prevented pollen extract-induced allergic lung inflammation in vivo.
Conclusion
The hybrid molecules showed a more efficient uptake and processing by dendritic cells resulting in a modified T cell response. The proteins had a lower IgE-binding capacity compared with the parental allergens, thus the high safety profile and increased efficacy emphasize clinical application for the treatment of Fagales multi-sensitization.
doi:10.1111/cea.12250
PMCID: PMC4041320  PMID: 24330218
allergen-specific immunotherapy; birch pollen allergy; Fagales pollen allergy; hybrid protein; immunomodulation; protein remodelling
17.  Vitamin D Supplementation Reduces Airway Hyperresponsiveness and Allergic Airway Inflammation in a Murine Model 
Background
Asthma is a chronic disease associated with airway hyperresponsiveness (AHR), airway obstruction, and airway remodeling. NF-κB is a transcriptional factor that regulates and co-ordinates the expression of various inflammatory genes. The NF-κB subunits, p50 and Rel-A, are translocated to the nucleus by importin α3 and importin α4. There is growing evidence that vitamin D is a potent immunomodulator. However, the evidence for beneficial or adverse effects of vitamin D in asthma is still unclear.
Objective
In this study, we examined the effect of vitamin D status on AHR, airway inflammation, and cytokines in the bronchoalveolar lavage fluid (BALF) in a murine model of allergic asthma.
Methods
Female BALB/c mice were fed with special vitamin D-deficient or vitamin D-sufficient (2,000 IU/kg) or vitamin D-supplemented (10,000 IU/kg) diet for 13 weeks. Mice were sensitized and challenged with ovalbumin. The effect of vitamin D on lung histology, AHR, T-regulatory cells and BALF cytokines was examined. The expression of importin-α3 and Rel-A in the lung of OVA-sensitized mice was analyzed using immunofluorescence.
Results
Vitamin D deficiency was associated with higher AHR in OVA-sensitized and challenged mice than those in vitamin D-sufficient mice. This was accompanied with marked signs of airway remodeling, high BALF eosinophilia, increased BALF pro-inflammatory cytokines, reduced BALF IL-10 levels, reduced blood T-regulatory cells, increased expression of importin-α3 and Rel-A in the lung tissue. Vitamin D supplementation attenuated the pro-inflammatory effects, but did not completely reverse the features of allergic airway inflammation.
Conclusion and Clinical Relevance
Vitamin D could be beneficial as an adjunct therapy in the treatment of allergic asthma.
doi:10.1111/cea.12102
PMCID: PMC3671499  PMID: 23711130
18.  ALOX5 Polymorphism Associates with Increased Leukotriene Production and Reduced Lung Function and Asthma Control in Children with Poorly Controlled Asthma 
Background
Identification of risk factors for reduced asthma control could improve the understanding and treatment of asthma. A promoter polymorphism in the 5-lipoxygenase gene affects gene expression and response to asthma therapy but its impact on disease control remains unclear.
Objective
We sought to determine if the ALOX5 promoter SP1 tandem repeat polymorphism was associated with changes in cysteinyl leukotriene production, lung function, airway inflammation and asthma control score.
Methods
We analyzed 270 children 6-17 years old with poorly controlled asthma enrolled in a 6-month clinical trial (NCT00604851). In secondary analysis, we associated the ALOX5 promoter SP1 tandem repeat polymorphism genotype (rs59439148) with asthma outcomes using both additive and recessive genetic models. We evaluated FEV1 percent predicted, symptom control, exhaled nitric oxide and urinary LTE4 levels.
Results
14.8% (40/270) of all children (and 28% (38/135) of African Americans) carried 2 non-5 repeat variant alleles of rs59439148. Children who were homozygous for variant alleles had significantly higher urinary LTE4 levels (38 versus 30 nmol/mol creatinine, p=.0134), significantly worse FEV1% predicted (84 versus 91, p=.017), and a trend toward worse asthma control. FEV1% predicted values were significantly negatively correlated with urinary LTE4 (r = -0.192, p=.009).
Conclusion and Clinical Relevance
Carrying two copies of a minor variant ALOX5 promoter SP1 tandem repeat allele contributes to increased cysLT exposure as determined by urinary LTE4 levels, reduced lung function, and potentially worse asthma control. ALOX5 promoter SP1 tandem repeat genotype may be a risk factor for worse asthma outcomes.
doi:10.1111/cea.12076
PMCID: PMC3633142  PMID: 23600541
Asthma; Children; ALOX5; 5-Lipoxygenase; Leukotriene; Asthma Control; FEV1; Exhaled Nitric Oxide; Genetic Association
19.  Glutathione S-transferase P1 Ile105Val polymorphism modulates allergen-induced airway inflammation in human atopic asthmatics in vivo 
Background
Glutathione S-transferase P1 is a Phase II cytoprotective and detoxifying enzyme that is widely expressed in human airways. The glutathione S-transferase P1 Ile105Val polymorphism has been linked with atopic disorders and asthma. Yet, little is known about the regulation of allergic inflammation by glutathione S-transferase P1 in human asthmatics.
Objective
To establish the effect of the glutathione S-transferase P1 Ile105Val polymorphism on allergen-induced airway inflammation and oxidant stress, and nonspecific bronchial hyperresponsiveness to methacholine and reactivity to specific allergen in mild human atopic asthmatics in vivo.
Methods
Five Val105/Val105, twelve Val105/Ile105, and twenty Ile105/Ile105 mild atopic asthmatics underwent methacholine challenge, inhaled allergen challenge, and endobronchial allergen provocation through a bronchoscope. A panel of inflammatory cytokines and chemokines, F2-isoprostanes and isofuranes, markers of oxidative stress, thromboxane B2, and immunoglobulin E were measured in bronchoalveolar lavage fluid at baseline and 24 hrs after allergen instillation.
Results
Asthmatics with glutathione S-transferase P1 Val105/Val105 compared to asthmatics with the glutathione S-transferase P1 Val105/Ile105 and Ile105/Ile105 had greater generation of acute phase cytokines (TNF-α, IL-6, CXCL8), IL-12, CCL11, thromboxane B2, and immunoglobulin E at 24 hrs after local allergen challenge. The GSTP1 genotype had no effect on airway hyperresponsiveness to methacholine and the reactivity to specific allergen.
Conclusion
The glutathione S-transferase P1 Ile105Val polymorphism markedly modifies allergen-provoked airway inflammation in atopic asthmatics in vivo. Modulation of the biochemical milieu in response to allergen provides a mechanistic explanation for regulatory effects of glutathione S-transferase P1 polymorphism on airway pathophysiology, and may guide improvement of future therapeutic methods in human atopic asthmatics. These findings must me confirmed in a larger study population of asthmatics with various ethnicities.
doi:10.1111/cea.12086
PMCID: PMC3633143  PMID: 23600543
Glutathione S-transferase P1; Ile105Val polymorphism; atopic asthma; allergen challenge; methacholine challenge; allergic inflammation; oxidant stress
20.  Age-dependent interaction between atopy and eosinophils in asthma cases: Results from NHANES 2005–2006 
Background
Atopy is an established risk factor for asthma, and an elevated eosinophil level is a hallmark of atopic and non-atopic asthma. Whether atopy and eosinophils act independently or interact to influence asthma has clinical and public health implications.
Objective
To investigate the relationship between atopy and eosinophils in asthma.
Methods
Data on current asthma, atopy (IgE positive to ≥ 1 allergen), and blood eosinophil percent (dichotomized at the median) were obtained for persons aged ≥ 6 years from NHANES 2005–2006. Interaction on an additive scale was evaluated by estimating the prevalences of asthma for combinations of atopy (yes or no) and eosinophil percent (high or low) and calculating the excess prevalence due to interaction.
Results
For all ages combined, the adjusted prevalences of asthma were 4.6%, 7.6%, 6.9%, and 17.2% for persons with neither factor, atopy alone, a high eosinophil percent alone, and both factors, respectively. The excess prevalence of asthma due to interaction was 7.2%, indicating synergism. The excess prevalence was greatest in children aged 6–17 years (15.3%), and it decreased with each older age category until it was absent in adults aged ≥ 55 years (−0.2%). In children, 94% of asthma cases attributable to the 2 factors were attributable to their interaction, whereas in the oldest adults, no cases were attributable to their interaction.
Conclusions and Clinical Relevance
Interaction between atopy and an elevated eosinophil level in asthma cases was very strong in children but absent in the oldest adults, which suggests different mechanistic pathways for these factors by age and supports the notion that asthma is a heterogeneous disease. In addition, the age-dependent interaction between the factors has potential implications for the selection of asthma patients for treatments that would target either IgE or a high eosinophil level.
doi:10.1111/cea.12069
PMCID: PMC3633144  PMID: 23600545
Asthma; atopy; eosinophils; epidemiology; immunoglobulin E; survey
21.  Interleukin-13: prospects for new treatments 
Summary
IL-13 is a T-helper type 2 cytokine. Animal models have implicated IL-13 as a critical cytokine in the development of asthma and chronic obstructive pulmonary disease (COPD). In vitro IL-13 exerts important effects on both structural and inflammatory cells within the airway and has the capacity to drive the clinical features of airways disease. In asthma, this view is strongly supported by associations with IL-13 genetic polymorphisms and increased mRNA and protein expression in blood, sputum and bronchial submucosa. In particular, IL-13 up-regulation is associated with severe disease. Current evidence in COPD is conflicting, with some reports supporting and others refuting a role for IL-13. Early clinical trials of anti-IL-13 therapies in asthma have shown promise, and the results of further efficacy studies are eagerly awaited.
doi:10.1111/j.1365-2222.2009.03383.x
PMCID: PMC3992377  PMID: 19878194
22.  Association of polymorphisms in CASP10 and CASP8 with FEV1/FVC and bronchial hyperresponsiveness in ethnically diverse asthmatics 
Summary
Background
Several chromosomal regions have been identified using family-based linkage analysis to contain genes contributing to the development of asthma and allergic disorders. One of these regions, chromosome 2q32-q33, contains a gene cluster containing CFLAR, CASP10 and CASP8. These genes regulate the extrinsic apoptosis pathway utilized by several types of immune and structural cells that have been implicated in the pathogenesis of asthma.
Objective
To assess the role of genetic variation in CFLAR, CASP10 and CASP8 in asthma and related phenotypes in individuals of diverse ethnic backgrounds.
Methods
We tested 26 single nucleotide polymorphisms (SNPs) in the CFLAR, CASP10 and CASP8 gene cluster for association with asthma and related phenotypes in African-American, non-Hispanic whites, and Hispanic case–control populations (cases, N = 517, controls, N = 644).
Results
Five CASP10 SNPS were associated with forced expiratory volume in 1 s (FEV1)/forced expiration volume capacity (FVC) in the African-American subjects with asthma (P = 0.0009–0.047). Nine SNPs, seven in CASP10 and two in CASP8, were also associated with the degree of bronchial hyperresponsiveness (BHR) (as determined by PC20) in race-specific analysis, predominately in the Non-Hispanic white cases. Two SNPs, rs6750157 in CASP10 and rs1045485 in CASP8 were modestly associated with asthma in the African-American (P = 0.025) and Hispanic (P = 0.033) populations, respectively.
Conclusion
These data suggest a role for CASP10 as a potential modifier of the asthma phenotype, specifically with measures of airway obstruction and BHR.
doi:10.1111/j.1365-2222.2008.03095.x
PMCID: PMC3979622  PMID: 18823309
asthma; bronchial hyperresponsiveness; CASP10; chromosome 2q; FEV1/FVC
23.  Genome-wide association study of body mass index in 23,000 individuals with and without asthma 
Background
Both asthma and obesity are complex disorders that are influenced by environmental and genetic factors. Shared genetic factors between asthma and obesity have been proposed to partly explain epidemiological findings of co-morbidity between these conditions.
Objective
To identify genetic variants that are associated with body mass index (BMI) in asthmatic children and adults, and to evaluate if there are differences between the genetics of BMI in asthmatics and healthy individuals.
Methods
In total, 19 studies contributed with genome-wide analysis study (GWAS) data from more than 23,000 individuals with predominantly European descent, of whom 8,165 are asthmatics.
Results
We report associations between several DENND1B variants (p=2.2×10−7 for rs4915551) on chromosome 1q31 and BMI from a meta-analysis of GWAS data using 2,691 asthmatic children (screening data). The top DENND1B SNPs were next evaluated in seven independent replication data sets comprising 2,014 asthmatics, and rs4915551 was nominally replicated (p<0.05) in two of the seven studies and of borderline significance in one (p=0.059). However, strong evidence of effect heterogeneity was observed and overall, the association between rs4915551 and BMI was not significant in the total replication data set, p=0.71. Using a random effects model, BMI was overall estimated to increase by 0.30 kg/m2 (p=0.01 for combined screening and replication data sets, N=4,705) per additional G allele of this DENND1B SNP. FTO was confirmed as an important gene for adult and childhood BMI regardless of asthma status.
Conclusions and Clinical Relevance
DENND1B was recently identified as an asthma susceptibility gene in a GWAS on children, and here we find evidence that DENND1B variants may also be associated with BMI in asthmatic children. However, the association was overall not replicated in the independent data sets and the heterogeneous effect of DENND1B points to complex associations with the studied diseases that deserve further study.
doi:10.1111/cea.12054
PMCID: PMC3608930  PMID: 23517042
Association; Asthma; BMI; Genetics; Genome-wide; Obesity
24.  Copy number variation prevalence in known asthma genes and their impact on asthma susceptibility 
Background
Genetic studies have identified numerous genes reproducibly associated with asthma, yet these studies have focused almost entirely on single nucleotide polymorphisms (SNPs), and virtually ignored another highly prevalent form of genetic variation: Copy Number Variants (CNVs).
Objective
To survey the prevalence of CNVs in genes previously associated with asthma, and to assess whether CNVs represent the functional asthma-susceptibility variants at these loci.
Methods
We genotyped 383 asthmatic trios participating in the Childhood Asthma Management Program (CAMP) using a competitive genomic hybridization (CGH) array designed to interrogate 20,092 CNVs. To ensure comprehensive assessment of all potential asthma candidate genes, we purposely used liberal asthma gene inclusion criteria, resulting in consideration of 270 candidate genes previously implicated in asthma. We performed statistical testing using FBAT-CNV.
Results
Copy number variation in asthma candidate genes was prevalent, with 21% of tested genes residing near or within one of 69 CNVs. In 6 instances, the complete candidate gene sequence resides within the CNV boundaries. On average, asthmatic probands carried 6 asthma-candidate CNVs (range 1–29). However, the vast majority of identified CNVs were of rare frequency (< 5%), and were not statistically associated with asthma. Modest evidence for association with asthma was observed for 2 CNVs near NOS1 and SERPINA3. Linkage disequilibrium analysis suggests that CNV effects are unlikely to explain previously detected SNP associations with asthma.
Conclusions
Although a substantial proportion of asthma-susceptibility genes harbor polymorphic CNVs, the majority of these variants do not confer increased asthma risk. The lack of linkage disequilibrium (LD) between CNVs and asthma-associated SNPs suggests that these CNVs are unlikely to represent the functional variant responsible for most known asthma associations.
doi:10.1111/cea.12060
PMCID: PMC3609036  PMID: 23517041
25.  Anti-IL-5 attenuates activation and surface density of β2-integrins on circulating eosinophils after segmental antigen challenge 
Background
IL-5 activates αMβ2 integrin on blood eosinophils in vitro. Eosinophils in bronchoalveolar lavage (BAL) following segmental antigen challenge have activated β2-integrins.
Objective
To identify roles for IL-5 in regulating human eosinophil integrins in vivo.
Methods
Blood and BAL eosinophils were analyzed by flow cytometry in ten subjects with allergic asthma who underwent a segmental antigen challenge protocol before and after anti-IL-5 administration.
Results
Blood eosinophil reactivity with monoclonal antibody (mAb) KIM-127, which recognizes partially activated β2-integrins, was decreased after anti-IL-5. Before anti-IL-5, surface densities of blood eosinophil β2, αM, and αL integrin subunits increased modestly post-challenge. After anti-IL-5, such increases did not occur. Before or after anti-IL-5, surface densities of β2,αM, αL, and αD and reactivity with KIM-127 and mAb CBRM1/5, which recognizes high-activity αMβ2, were similarly high on BAL eosinophils 48 h post-challenge. Density and activation state of β1-integrins on blood and BAL eosinophils were not impacted by anti-IL-5, even though anti-IL-5 ablated a modest post-challenge increase on blood or BAL eosinophils of P-selectin glycoprotein ligand-1 (PSGL-1), a receptor for P-selectin that causes activation of β1-integrins. Forward scatter of blood eosinophils post-challenge was less heterogeneous and on the average decreased after anti-IL-5; however, anti-IL-5 had no effect on the decreased forward scatter of eosinophils in post-challenge BAL compared to eosinophils in blood. Blood eosinophil KIM-127 reactivity at the time of challenge correlated with the percentage of eosinophils in BAL post-challenge.
Conclusion and Clinical Relevance
IL-5 supports a heterogeneous population of circulating eosinophils with partially activated β2-integrins and is responsible for upregulation of β2-integrins and PSGL-1 on circulating eosinophils following segmental antigen challenge but has minimal effects on properties of eosinophils in BAL. Dampening of β2-integrin function of eosinophils in transit to inflamed airway may contribute to the decrease in lung inflammation caused by anti-IL-5.
doi:10.1111/j.1365-2222.2012.04065.x
PMCID: PMC3579563  PMID: 23414537
eosinophils; integrins; PSGL-1; forward scatter; IL-5; blood; bronchoalveolar lavage; segmental lung antigen challenge

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