Nasal and blood transcriptomic pathways underpinning the clinical response to grass pollen immunotherapy.

BACKGROUND: Allergen immunotherapy (AIT) is a well-established disease-modifying therapy for allergic rhinitis, yet the fundamental mechanisms underlying its clinical effect remain inadequately understood. Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy was a randomized, double-blind, placebo-controlled trial of individuals allergic to timothy grass who received 2 years of placebo (n = 30), subcutaneous immunotherapy (SCIT) (n = 27), or sublingual immunotherapy (SLIT) (n = 27) and were then followed for 1 additional year. OBJECTIVE: We used yearly biospecimens from the Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy study to identify molecular mechanisms of response. METHODS: We used longitudinal transcriptomic profiling of nasal brush and PBMC samples after allergen provocation to uncover airway and systemic expression pathways mediating responsiveness to AIT. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01335139, EudraCT Number: 2010-023536-16. RESULTS: SCIT and SLIT demonstrated similar changes in gene module expression over time. In nasal samples, alterations included downregulation of pathways of mucus hypersecretion, leukocyte migration/activation, and endoplasmic reticulum stress (log2 fold changes -0.133 to -0.640, false discovery rates [FDRs] <0.05). We observed upregulation of modules related to epithelial development, junction formation, and lipid metabolism (log2 fold changes 0.104 to 0.393, FDRs <0.05). In PBMCs, modules related to cellular stress response and type 2 cytokine signaling were reduced by immunotherapy (log2 fold changes -0.611 to -0.828, FDRs <0.05). Expression of these modules was also significantly associated with both Total Nasal Symptom Score and peak nasal inspiratory flow, indicating important links between treatment, module expression, and allergen response. CONCLUSIONS: Our results identify specific molecular responses of the nasal airway impacting barrier function, leukocyte migration activation, and mucus secretion that are affected by both SCIT and SLIT, offering potential targets to guide novel strategies for AIT.

Technical standards in allergen exposure chambers worldwide - an EAACI Task Force Report.

Allergen exposure chambers (AECs) can be used for controlled exposure to allergenic and non-allergenic airborne particles in an enclosed environment, in order to (i) characterize the pathological features of respiratory diseases and (ii) contribute to and accelerate the clinical development of pharmacological treatments and allergen immunotherapy for allergic disease of the respiratory tract (such as allergic rhinitis, allergic rhinoconjunctivitis, and allergic asthma). In the guidelines of the European Medicines Agency for the clinical development of products for allergen immunotherapy (AIT), the role of AECs in determining primary endpoints in dose-finding Phase II trials is emphasized. Although methodologically insulated from the variability of natural pollen exposure, chamber models remain confined to supporting secondary, rather than primary, endpoints in Phase III registration trials. The need for further validation in comparison with field exposure is clearly mandated. On this basis, the European Academy of Allergy and Clinical Immunology (EAACI) initiated a Task Force in 2015 charged to gain a better understanding of how AECs can generate knowledge about respiratory allergies and can contribute to the clinical development of treatments. Researchers working with AECs worldwide were asked to provide technical information in eight sections: (i) dimensions and structure of the AEC, (ii) AEC staff, (iii) airflow, air processing, and operating conditions, (iv) particle dispersal, (v) pollen/particle counting, (vi) safety and non-contamination measures, (vii) procedures for symptom assessments, (viii) tested allergens/substances and validation procedures. On this basis, a minimal set of technical requirements for AECs applied to the field of allergology is proposed.

Differential induction of allergen-specific IgA responses following timothy grass subcutaneous and sublingual immunotherapy.

INTRODUCTION: There is no detailed comparison of allergen-specific immunoglobulin responses following sublingual immunotherapy (SLIT) and subcutaneous immunotherapy (SCIT). OBJECTIVE: We sought to compare nasal and systemic timothy grass pollen (TGP)-specific antibody responses during 2 years of SCIT and SLIT and 1 year after treatment discontinuation in a double-blind, double-dummy, placebo-controlled trial. METHODS: Nasal fluid and serum were obtained yearly (per-protocol population, n = 84). TGP-specific IgA1, IgA2, IgG4, IgG, and IgE were measured in nasal fluids by ELISA. TGP-specific IgA1, IgA2, and Phleum pratense (Phl p)1, 2, 4, 5b, 6, 7, 11, and 12 IgE and IgG4 were measured in sera by ELISA and ImmunoCAP, respectively. RESULTS: At years 2 and 3, TGP-IgA1/2 levels in nasal fluid were elevated in SLIT compared with SCIT (4.2- and 3.0-fold for IgA1, 2.0- and 1.8-fold for IgA2, respectively; all P < .01). TGP-IgA1 level in serum was elevated in SLIT compared with SCIT at years 1, 2, and 3 (4.6-, 5.1-, and 4.7-fold, respectively; all P < .001). Serum TGP-IgG level was higher in SCIT compared with SLIT (2.8-fold) at year 2. Serum TGP-IgG4 level was higher in SCIT compared with SLIT at years 1, 2, and 3 (10.4-, 27.4-, and 5.1-fold, respectively; all P < .01). Serum IgG4 levels to Phl p1, 2, 5b, and 6 were increased at years 1, 2, and 3 in SCIT and SLIT compared with placebo (Phl p1: 11.8- and 3.9-fold; Phl p2: 31.6- and 4.4-fold; Phl p5b: 135.5- and 5.3-fold; Phl p6: 145.4- and 14.7-fold, respectively, all at year 2 when levels peaked; P < .05). IgE to TGP in nasal fluid increased in the SLIT group at year 2 but not at year 3 compared with SCIT (2.8-fold; P = .04) and placebo (3.1-fold; P = .02). IgA to TGP and IgE and IgG4 to TGP components stratified participants according to treatment group and clinical response. CONCLUSIONS: The observed induction of IgA1/2 in SLIT and IgG4 in SCIT suggest key differences in the mechanisms of action.

Ara h 2-specific IgE is superior to whole peanut extract-based serology or skin prick test for diagnosis of peanut allergy in infancy.

BACKGROUND: Screening of high-risk infants for peanut allergy (PA) before introduction is now recommended in the United States, but the optimal approach is not clear. OBJECTIVE: We sought to compare the diagnostic test characteristics of peanut skin prick test (SPT), peanut-specific IgE (sIgE), and sIgE to peanut components in a screening population of infants before known peanut exposure. METHODS: Infants aged 4 to 11 months with (1) no history of peanut ingestion, testing, or reaction and (2) (a) moderate-severe eczema, (b) history of food allergy, and/or (c) first-degree relative with a history of PA received peanut SPT, peanut-sIgE and component-IgE testing, and, depending on SPT wheal size, oral food challenge or observed feeding. Receiver-operator characteristic areas under the curve (AUCs) were compared, and diagnostic sensitivity and specificity were calculated. RESULTS: A total of 321 subjects completed the enrollment visit (median age, 7.2 months; 58% males), and 37 (11%) were found to have PA. Overall, Ara h 2-sIgE at a cutoff point of 0.1 kUa/L discriminated between allergic and nonallergic best (AUC, 0.96; sensitivity, 94%; specificity, 98%), compared with peanut-sIgE at 0.1 kUa/L (AUC, 0.89; sensitivity, 100%; specificity, 78%) or 0.35 kUa/L (AUC, 0.91; sensitivity, 97%; specificity, 86%), or SPT at wheal size 3 mm (AUC, 0.90; sensitivity, 92%; specificity, 88%) or 8 mm (AUC, 0.87; sensitivity, 73%; specificity, 99%). Ara h 1-sIgE and Ara h 3-sIgE did not add to prediction of PA when included in a model with Ara h 2-sIgE, and Ara h 8-sIgE discriminated poorly (AUC, 0.51). CONCLUSIONS: Measurement of only Ara h 2-sIgE should be considered if screening of high-risk infants is performed before peanut introduction.

Clinical standardization of two controlled allergen challenge facilities: The Environmental Exposure Unit and the Biogenics Research Chamber.

BACKGROUND: Controlled allergen challenge facilities (CACF), in disparate geographic regions with dissimilar engineering and base populations, have historically functioned as single, independent sites in clinical allergy trials. We aimed to demonstrate "between-unit reproducibility" to allow controlled challenge trials of participants using 2 CACFs. OBJECTIVE: To compare and standardize 2 CACFs located in Kingston, Ontario, Canada, and San Antonio, Texas, by examining participant-reported symptom severity during qualifying and treatment visits and evaluating response to treatment, while using the same allergen. METHODS: At 2 different CACFs, participants were enrolled in a double-blind, placebo-controlled, crossover intervention trial with cetirizine 10 mg. Different distribution devices delivered common short ragweed pollen via laminar air flow and maintained an airborne concentration of 3500 ± 700 grains/m3 in both facilities. A 1-hour "sham" run with no pollen release preceded a priming exposure of 3 hours and was followed 3 days later by a qualifying/treatment 5-hour exposure. At least 14 days later, another priming exposure was followed by the crossover exposure and treatment. RESULTS: Forty-eight and 43 subjects completed the study at Kingston and San Antonio, respectively. Demographics were similar. Fewer than 10% exhibited symptoms with sham exposure. No significant differences were found between the 2 facilities in maximal total rhinoconjunctivitis symptom score, total nasal symptom score, and total ocular symptom score, nor in areas under the curve. In both facilities, no significant effects of cetirizine 10 mg over placebo were detected. CONCLUSION: The results were equivalent, demonstrating that the 2 CACFs can be used together in dual-center clinical trials and show the possibility of multicenter trials involving multiple CACFs.

Expression of corticosteroid-regulated genes by PBMCs in children with asthma.

BACKGROUND: Variability in response to inhaled corticosteroids (ICSs) can result in less than optimal asthma control. Development of biomarkers assessing the therapeutic efficacy of corticosteroids is important. OBJECTIVE: We sought to examine whether in vitro PBMC responses to corticosteroids relate to the clinical ICS response. METHODS: PBMCs were collected from 125 children with asthma (6-17 years) at enrollment (visit 0 [V0]) and after 1 year of bimonthly guidelines-based management visits (visit 6 [V6]). Difficult-to-control and easy-to-control asthma were defined as requiring daily therapy with 500 µg or more of fluticasone propionate (FLU) with or without a long-acting ß-agonist versus 100 µg or less of FLU in at least 4 visits. mRNA levels of glucocorticoid receptor α and corticosteroid transactivation (FK506-binding protein 5) and transrepression markers (IL-8 and TNF-α) were measured by using RT-PCR in freshly isolated cells and in response to 10-8 mol/L FLU. RESULTS: Compared with PBMCs from patients with easy-to-control asthma, PBMCs from those with difficult-to-control asthma had significantly lower glucocorticoid receptor α levels at V0 (P = .05). A 30% increase in IL-8 suppression by FLU (P = .04) and a trend for increased TNF-α suppression by FLU between V0 and V6 (P = .07) were observed in patients with easy-to-control asthma. In contrast, no changes between V0 and V6 in IL-8 and TNF-α suppression by FLU were observed in patients with difficult-to-control asthma. Corticosteroid-mediated transactivation (FK506-binding protein 5 induction by FLU) increased in the PBMCs of patients with difficult-to-control and easy-to-control asthma between V0 and V6 (P = .05 and P = .03, respectively). CONCLUSIONS: PBMCs of children with difficult-to-control asthma treated with guidelines-based therapy and requiring high-dose ICSs had reduced in vitro responsiveness to corticosteroids.

Synchronous immune alterations mirror clinical response during allergen immunotherapy.

BACKGROUND: Three years of treatment with either sublingual or subcutaneous allergen immunotherapy has been shown to be effective and to induce long-term tolerance. The Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy (GRASS) trial demonstrated that 2 years of treatment through either route was effective in suppressing the response to nasal allergen challenge, although it was insufficient for inhibition 1 year after discontinuation. OBJECTIVE: We sought to examine in the GRASS trial the time course of immunologic changes during 2 years of sublingual and subcutaneous immunotherapy and for 1 year after treatment discontinuation. METHODS: We performed multimodal immunomonitoring to assess allergen-specific CD4 T-cell properties in parallel with analysis of local mucosal cytokine responses induced by nasal allergen exposure and humoral immune responses that included IgE-dependent basophil activation and measurement of serum inhibitory activity for allergen-IgE binding to B cells (IgE-facilitated allergen binding). RESULTS: All 3 of these distinct arms of the immune response displayed significant and coordinate alterations during 2 years of allergen desensitization, followed by reversal at 3 years, reflecting a lack of a durable immunologic effect. Although frequencies of antigen-specific TH2 cells in peripheral blood determined by using HLA class II tetramer analysis most closely paralleled clinical outcomes, IgE antibody-dependent functional assays remained inhibited in part 1 year after discontinuation. CONCLUSION: Two years of allergen immunotherapy were effective but insufficient for long-term tolerance. Allergen-specific TH2 cells most closely paralleled the transient clinical outcome, and it is likely that recurrence of the T-cell drivers of allergic immunity abrogated the potential for durable tolerance. On the other hand, the persistence of IgE blocking antibody 1 year after discontinuation might be an early indicator of a protolerogenic mechanism.

Effect of 2 Years of Treatment With Sublingual Grass Pollen Immunotherapy on Nasal Response to Allergen Challenge at 3 Years Among Patients With Moderate to Severe Seasonal Allergic Rhinitis: The GRASS Randomized Clinical Trial.

Importance: Sublingual immunotherapy and subcutaneous immunotherapy are effective in seasonal allergic rhinitis. Three years of continuous treatment with subcutaneous immunotherapy and sublingual immunotherapy has been shown to improve symptoms for at least 2 years following discontinuation of treatment. Objective: To assess whether 2 years of treatment with grass pollen sublingual immunotherapy, compared with placebo, provides improved nasal response to allergen challenge at 3-year follow-up. Design, Setting, and Participants: A randomized double-blind, placebo-controlled, 3-parallel-group study performed in a single academic center, Imperial College London, of adult patients with moderate to severe seasonal allergic rhinitis (interfering with usual daily activities or sleep). First enrollment was March 2011, last follow-up was February 2015. Interventions: Thirty-six participants received 2 years of sublingual immunotherapy (daily tablets containing 15 µg of major allergen Phleum p 5 and monthly placebo injections), 36 received subcutaneous immunotherapy (monthly injections containing 20 µg of Phleum p 5 and daily placebo tablets) and 34 received matched double-placebo. Nasal allergen challenge was performed before treatment, at 1 and 2 years of treatment, and at 3 years (1 year after treatment discontinuation). Main Outcomes and Measures: Total nasal symptom scores (TNSS; range; 0 [best] to 12 [worst]) were recorded between 0 and 10 hours after challenge. The minimum clinically important difference for change in TNSS within an individual is 1.08. The primary outcome was TNSS comparing sublingual immunotherapy vs placebo at year 3. Subcutaneous immunotherapy was included as a positive control. The study was not powered to compare sublingual immunotherapy with subcutaneous immunotherapy. Results: Among 106 randomized participants (mean age, 33.5 years; 34 women [32.1%]), 92 completed the study at 3 years. In the intent-to-treat population, mean TNSS score for the sublingual immunotherapy group was 6.36 (95% CI, 5.76 to 6.96) at pretreatment and 4.73 (95% CI, 3.97 to 5.48) at 3 years, and for the placebo group, the score was 6.06 (95% CI, 5.23 to 6.88) at pretreatment and 4.81 (95% CI, 3.97 to 5.65) at 3 years. The between-group difference (adjusted for baseline) was -0.18 (95% CI, -1.25 to 0.90; [P = .75]). Conclusions and Relevance: Among patients with moderate to severe seasonal allergic rhinitis, 2 years of sublingual grass pollen immunotherapy was not significantly different from placebo in improving the nasal response to allergen challenge at 3-year follow-up. Trial Registration: clinicaltrials.gov Identifier: NCT01335139; EudraCT Number: 2010-023536-16.

Optimisation of grass pollen nasal allergen challenge for assessment of clinical and immunological outcomes.

Nasal allergen challenge can be used to assess the clinical and immunological aspects of rhinitis due to inhalant allergens. We aimed to develop a reproducible technique for grass pollen nasal allergen challenge and to study biomarkers within nasal secretions. 20 Grass pollen allergic individuals underwent nasal challenges with purified Timothy grass allergen. An initial dose-titration challenge was used to determine dose-response characteristics. Subsequently, volunteers underwent 3 further challenges using individualised threshold doses. Symptom scores, visual analogue scores, and peak nasal inspiratory flow (PNIF) were recorded at baseline and up to 6h after challenge. Nasal secretions were collected at each time point using synthetic filter papers or absorptive polyurethane sponges and analysed for IL-4, -5, -10, -13, IFN-γ, Tryptase and Eosinophil Cationic Protein (ECP). Challenges gave reproducible symptom scores and decreased PNIF. Tryptase levels in nasal fluid peaked at 5 min after challenge and returned to baseline levels at 1h. ECP, IL-5, IL-13 and IL-4 levels were increased from 2-3 h and showed progressive increases to 5-6 h. Sponges proved the superior nasal fluid sampling technique. We have developed a reproducible nasal allergen challenge technique. This may be used as a surrogate clinical endpoint in trials assessing the efficacy of treatments for allergic rhinitis. Tryptase in local nasal secretions is a potential biomarker of the early phase response; ECP and the Th2 cytokines IL-5, -13 and -4 markers of late phase allergic responses. Our model allows correlation between clinical responses and local biomarkers following nasal allergen challenge.

Changes in allergy symptoms and depression scores are positively correlated in patients with recurrent mood disorders exposed to seasonal peaks in aeroallergens.

Although growing evidence supports an association between allergy, allergens and depression, it remains unknown if this relationship is between "states" (possible triggers) or "traits" (possible vulnerabilities). We hypothesized that patients with recurrent mood disorders who are sensitized to tree pollen (as determined by allergen specific IgE antibodies), in comparison to those who are not sensitized, would report larger negative changes in mood during exposure to tree pollen in spring. We also hypothesized that differences between high and low tree pollen periods in self reported allergy symptoms would correlate positively with differences in self reported depression scores. We present 1-year preliminary data on the first 51 patients with unipolar or bipolar disorder (age: 19-63 years, 65% female, twelve patients were tree-pollen IgE positive). Ratings of mood and allergic disease status were performed once during the peak airborne pollen counts and once during the period of low airborne pollen counts, as reported by two local pollen counting stations. Linear regression models were developed to examine associations of changes in depression scores (dependent variable) with tree pollen sensitization, changes in the allergy symptom severity score, adjusted for gender and order of testing. We did not confirm the hypothesized relationship between a specific tree pollen sensitization and changes in mood during tree pollen exposure. We did confirm the hypothesized positive relationship between the changes in allergy symptoms and changes in subjects' depression scores (adjusted p<0.05). This result is consistent with previous epidemiological evidence connecting allergy with depression, as well as our recent reports of increased expression of cytokines in the prefrontal cortex in victims of suicide and in experimental animals sensitized and exposed to tree pollen. A relationship between changes in allergy symptom scores and changes in depression scores supports a state-level rather than only trait-level relationship, and thus lends optimism to future causality-testing interventional studies, which might then lead to novel preventative environmental interventions in mood disorders.