Jenny Thiele

Nasal allergen challenge studies of allergic rhinitis: a guide for the practicing clinician

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A post-hoc qualitative analysis of real time heads-up pollen counting versus traditional microscopy counting in the environmental exposure unit (EEU)

Background A custom digital imagery method for real time identification and counting of pollen was qualitatively evaluated in the Environmental Exposure Unit (EEU). Methods Airborne grass pollen was collected in the EEU via a Rotorod ® impact sampler. The pollen grains on each sampling rod were counted using both traditional and headsup microscopy. The heads-up technique incorporated a microscope camera to create an on-screen image of the sampling rod. Firstly, unique images were created by manually advancing the stage, without duplicating previously captured pollen grains. Well-defined, sharp images were obtained by fine focus and zoom combinations to enhance certainty and recognition speed. Secondly, using a custom application, each pollen grain was identified and counted on-screen by “point and click” or “screen touch”, simultaneously counting and permanently anchoring opaque dots to the pollen grain locations. Counts were stored in real time on a central database. Results Increased clarity of the polle ng rains resulted in higher counting accuracy. Duplicate counting of pollen grains was eliminated by digitally labelling counted grains. Additional need for manual counting devices, commonly associated with mechanical and human errors, was eliminated. Error free counts can be obtained with increased speed, therefore, improving the overall efficiency of the process and the EEU system as a whole. Conclusions This validated heads-up counting technique will allow for an increased response time to changes in the EEU pollen levels. This advancement could also enhance pollen counting processes followed by others using direct microscopy pollen counting techniques.

Repeatability of nasal allergen challenge results – further validation of the allergic rhinitis clinical investigator collaborative (AR-CIC) protocols

BACKGROUND Nasal allergen challenge (NAC) models have been used to study allergic rhinitis and new therapies. Symptoms and biological samples can be evaluated at time points after allergen exposure. OBJECTIVE To verify protocol repeatability and adequate interval between allergen exposures. METHODS Ten ragweed allergic participants were exposed to incrementally increasing dosages of ragweed allergen intranasally until they achieved a total nasal symptom score (TNSS) of 8 of 12 and a peak nasal inspiratory flow (PNIF) of 50% reduction or more from baseline. Three weeks later, participants were challenged with a cumulative dose equal to the sum of all the allergen doses received at screening. TNSS and PNIF were recorded at regular intervals, including a 24-hour assessment. A subsequent visit was conducted after a further 3 weeks. Nasal secretion samples were collected for cytokine and eosinophil quantification. RESULTS Nine participants completed all visits. TNSS and PNIF responses followed previous patterns, with an initial peak at 30 minutes followed by a gradual decline. Most participants reported similar patterns at both NAC visits, although some did not demonstrate the same phenotype at both visits. Some experienced a secondary symptom increase 24 hours after NAC. Eosinophil and cytokine sections followed a similar pattern at both NAC visits. CONCLUSION NAC is an adequate method for modeling AR in humans, demonstrating appropriate repeatability of symptoms, nasal mucosal eosinophil, and cytokines. The 24-hour time point, previously not studied in our model, may be beneficial in evaluation of long-acting medications. This three-week interval NAC model will be beneficial for studies in which before and after treatment comparisons are desired.

Peripheral group 2 innate lymphoid cells are decreased following nasal allergen challenge in allergic rhinitis

To the Editor, Allergic rhinitis (AR) is an upper airway inflammatory disorder involving IgE‐mediated inflammation of the nasal mucosa. Inflammatory responses are triggered following inhalation of sensitized aeroallergens and are characterized by Th2 inflammation and the rapid migration of eosinophils into the nasal cavity. Group 2 innate lymphoid cells (ILC2s) represent an alternative source of Th2 cytokines, potentially augmenting pre‐existing Th2‐driven inflammation seen in AR. Lacking a specific antigen receptor and lineage surface markers for B and T cells, ILC2s are activated by IL‐25, IL‐33, and TSLP and can drive Th2 inflammation via production of IL‐5, IL‐13, and modest amounts of IL‐4. In AR patients, elevated levels of peripheral blood ILC2s were reported during the grass pollen season and following a nasal allergen challenge (NAC) with cat allergen extract. This suggests a possible role for these cells in driving allergic symptoms. However, participants from these studies may have been primed (reversible increase in reactivity of the nasal tissue) due to repeated low‐dose allergenic stimulation during pollen season or at home (ie, owning a cat). Sensitized individuals who are also primed can experience more severe allergic symptoms and changes in systemic immunity after allergen exposure. To date, few studies have assessed the effects of acute allergen exposure on circulating ILC2s in non‐primed AR individuals. Thus, the aim of the current study was to evaluate the frequency of peripheral blood ILC2s in AR individuals after a NAC performed outside of the local birch pollen season. We also evaluated ILC2 frequencies in nasal lavage (NL) samples collected pre‐ and post‐challenge. Eleven individuals with birch pollen–induced AR and eight non‐ allergics were recruited into the study (Table S1). The study was reviewed and granted ethical clearance by the Queens University and Affiliated Teaching Hospitals Research Ethics Board, and all participants provided written informed consent. All participants underwent a NAC with a pre‐titrated dose of birch pollen extract (ALK‐Abello) (Figure S1A). Total nasal symptom score (TNSS; sum of rhinorrhea, nasal congestion, sneezing, and nasal itching) and peak nasal inspiratory flow (PNIF) were recorded during the challenge. Peripheral blood and NL samples were collected at baseline and 4 hours post‐challenge. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and cryopreserved. The 4‐hour time point was selected to remain consistent with the cat‐NAC study. Details of the NAC methodology can be found in the Methods S1 section. Thawed PBMCs and freshly obtained NL samples were stained with a fixable viability dye (PBMCs only, eBioscience), a lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56; BD Biosciences), and antibodies to CD4, CD11b, CD235a, FcεRI, CD45, CRTH2, and CD127 (all from eBioscience). ILC2s were identified as CD45 lymphocytes that were also lineage negative and expressed CRTH2 and CD127 (Figure 1A). Compared to non‐allergics, birch‐allergic participants experienced a significantly higher TNSS at 15 minutes (P < 0.0001) through 8 hours (P = 0.0048) and a larger PNIF reduction at 15 (P = 0.0016) and 30 minutes (P = 0.0004) post‐allergen challenge (Figure S1B,C). The mean frequency of peripheral blood CD45 cells remained unchanged after allergen challenge in both groups (Figure 1B). The mean frequency (±SEM) of peripheral blood ILC2s at baseline (0.019 ± 0.003% vs 0.018 ± 0.006%, P = 0.60) and post‐challenge (0.014 ± 0.002% vs 0.015 ± 0.004%, P = 0.90) was comparable between birch‐allergic and non‐allergic participants. Contrasting previous studies, peripheral blood ILC2s were significantly decreased following allergen challenge in birch‐allergic participants (P = 0.0344) (Figure 1C). This was not detected in non‐allergic participants (P = 0.25). A reduction in ILC2s was noted in this group; however, it was likely driven by one non‐allergic participant. In birch‐allergic participants, percentage changes in peripheral blood ILC2s after challenge significantly correlated with TNSS at 4 hours (r = 0.73, P = 0.01) (Figure 1D). This was not observed for 4‐ hour percentage PNIF reduction (r = −0.28, P = 0.41) (Figure 1E). In contrast, peripheral ILC2s at 4 hours alone did not correlate with 4‐ hour TNSS (r = −0.16, P = 0.63) and 4‐hour percentage PNIF reduction (r = −0.13, P = 0.70) (Figure 1F,G). A borderline correlation was observed between percentage changes in peripheral ILC2s and percentage PNIF reduction at 8 hours (late‐phase response, r = 0.61, P = 0.0491) (Table S2). Finally, correlations were not observed for 15‐minute (peak symptom severity) symptom scores (Table S2). These findings suggest that in pollen‐sensitized asymptomatic individuals, the change in peripheral ILC2s after a high‐dose pollen challenge may better reflect symptom severity compared to post‐challenge measurements alone, as observed in symptomatic individuals during pollen season. To investigate local ILC2 responses following allergen challenge, a similar gating strategy was used. We were unable to detect nasal ILC2s in NL samples collected pre‐ and post‐NAC (Figure 2A). Only a very small proportion of NL cells expressed CD45 (median frequency, 3.2%‐6.5% of all cells), which remained unchanged after allergen challenge (Figure 2B). The nasal lymphocyte population was also less defined, with 15/19 participants having less than 100 DOI: 10.1111/all.13614

Investigating Immune Gene Signatures in Peripheral Blood from Subjects with Allergic Rhinitis Undergoing Nasal Allergen Challenge

Nasal allergen challenge (NAC) is a human model of allergic rhinitis (AR) that delivers standardized allergens locally to the nasal mucosa allowing clinical symptoms and biospecimens such as peripheral blood to be collected. Although many studies have focused on local inflammatory sites, peripheral blood, an important mediator and a component of the systemic immune response, has not been well studied in the setting of AR. We sought to investigate immune gene signatures in peripheral blood collected after NAC under the setting of AR. Clinical symptoms and peripheral blood samples from AR subjects were collected during NAC. Fuzzy c-means clustering method was used to identify immune gene expression patterns in blood over time points (before NAC and 1, 2, and 6 h after NAC). We identified and validated seven clusters of differentially expressed immune genes after NAC onset. Clusters 2, 3, and 4 were associated with neutrophil and lymphocyte frequencies and neutrophil/lymphocyte ratio after the allergen challenge. The patterns of the clusters and immune cell frequencies were associated with the clinical symptoms of the AR subjects and were significantly different from healthy nonallergic subjects who had also undergone NAC. Our approach identified dynamic signatures of immune gene expression in blood as a systemic immune response associated with clinical symptoms after NAC. The immune gene signatures may allow cross-sectional investigation of the pathophysiology of AR and may also be useful as a potential objective measurement for diagnosis and treatment of AR combined with the NAC model.

Retraction Note: Meeting Abstract: A post-hoc qualitative analysis of real time heads-up pollen counting versus traditional microscopy counting in the environmental exposure unit (EEU)

Retraction This meeting abstract [1], published in the Canadian Society of Allergy and Clinical Immunology and AllerGen Abstracts 2014 supplement, has been retracted from Allergy, Asthma & Clinical Immunology by the authors. The meeting abstract has previously been published in Journal of Allergy and Clinical Immunology [2] and was inadvertently submitted to Allergy, Asthma & Clinical Immunology as part of the supplement. The authors apologise for any inconvenience that this may have caused.

The Allergic Rhinitis - Clinical Investigator Collaborative (AR-CIC) - optimizing the Nasal Allergen Challenge (NAC) model

Background We sought to optimize the Nasal Allergen Challenge (NAC) model to ensure reliability and repeatability of results by modifying the qualifying criteria and allergen concentration during the challenge. Methods 20 Allergic Rhinitis (AR) participants underwent NAC to determine the concentration at which a Total Nasal Symptom Score (TNSS) of 10/12 OR a Peak Nasal Inspiratory Flow (PNIF) reduction of 50 % was achieved. 4-fold increases in allergen concentration were administered every 15 minutes until qualification criteria were met. The Qualifying Allergen Concentration (QAC) reached was used as a single challenge dose at the subsequent NAC visit. 10 additional ragweed allergic and 4 non-allergic participants were qualified at a TNSS of 8/12 AND a PNIF reduction of 50%. Cumulative Allergen Concentration (CAC) of all incremental doses was used during the subsequent NAC visit. Participants recorded TNSS and PNIF at baseline, 15 minutes, 30 minutes, 1 hour and hourly afterwards up to 12 hours post-challenge during the NAC visit. Results QAC study participants qualifying only based on PNIF reduction had significantly lower TNSS scores than those qualifying on TNSS only or TNSS+PNIF (p<0.01). Participants in both studies’ NAC visit reached peak TNSS at 15 minutes post-challenge followed by a gradual symptom decline, while the “PNIF only” group had significantly lower TNSS compared to others. All 3 groups experienced a decline in peak TNSS following NAC compared to screening, although groups qualifying on TNSS and TNSS+PNIF maintained their PNIF scores. Conclusion The NAC model is well-suited to study AR symptoms. TNSS and PNIF are complementary and must be integrated in the qualifying criteria. Further protocol modifications, such as with multiple allergen challenges during the NAC visit, may produce even more repeatable results. Through optimizing the NAC protocol, the model achieves reproducible results and becomes more reliable; suitable for testing new medications in clinical trials.

The Allergic Rhinitis Clinical Investigator Collaborative – nasal allergen induced eosinophilia

Background The Allergic Rhinitis Clinical Investigator Collaborative (AR-CIC) is a Canadian multi-center initiative with the primary goal of performing standardized nasal allergen challenge (NAC) to study the anti-allergic effects of novel therapeutic agents for allergic rhinitis (AR). The model further allows identification of potential mechanisms of allergic disease and biomarkers. In this study we examined differential counts, more specifically eosinophil numbers, in nasal lavage samples before, 1 hour (1H) and 6 hours (6H) after direct nasal allergen challenge.

Mediators of allergic rhinitis: optimization of RNA isolation, reverse transcription, and qPCR protocols

Background Optimizing methods for the study of allergic rhinitis (AR), especially when using samples likely containing small amounts of material for analysis, ensures the integrity of results that may potentially enhance the understanding of AR disease mechanisms. In order to conduct future mRNA expression analysis, examining the differential expression of AR mediators such as IL33, TSLPR, HPGDS ,a ndCRTH2 at baseline and 6 hours following Nasal Allergen Challenge (NAC) in allergic individuals, this study aims to optimize the RNA isolation, reverse transcription (RT), and qPCR protocols used for the study of nasal mucosal samples. Methods Several RNA isolation and RT kits were evaluated using nasal scrapings from healthy individuals, similar to those collected from allergic participants. These kits were evaluated based on the yield and purity of RNA and cDNA, assessed using spectrophotometry, qPCR amplification, and gel electrophoresis. Reference gene analysis using cDNA isolated from allergic participants was conducted using qPCR and the statistical software GenEx (MultID). Primer design and evaluation of primers for the targets of interest—IL33, TSLPR, HPGDS ,a nd CRTH2—was also pursued. Results RNA isolation and RT kit optimization determined that the Life Technologies-Qiagen (LT-Q) kit combination produced cDNA with maximal purity and qPCR efficiency compared with the other kit combinations evaluated. Reference gene analysis demonstrated that expression of ubiquitin C (UBC) showed limited variability among the differing conditions (time point and study) of nasal sample collection. Primer evaluation yielded inconsistent results. Conclusions Future processing of nasal scraping samples should use the optimal LT-Q kit combination. Following successful primer evaluation, the expression levels of the targets of interest in the allergic nasal mucosal cDNA samples at both baseline and 6h post-NAC will be conducted via the optimized qPCR reaction, using UBC as a reference gene.

Cytokine profiling of umbilical cord blood plasma

Background Cytokines have been shown to be important signal molecules in the development of allergy and asthma, especially the regulatory cytokine IL-10, which has been shown to correlate with higher risk of allergy development in children [1]. Research results regarding cytokine levels in umbilical cord blood plasma vary greatly; some studies find the concentration of cytokines detectable whereas other studies do not. The purpose of this pilot study was to determine if cytokines could be measured from cord blood plasma using IL-10 ELISAs and xMAP Luminex assay.