High-dimensional immune profiles correlate with phenotypes of peanut allergy during food-allergic reactions.

BACKGROUND: Food challenges carry a burden of safety, effort and resources. Clinical reactivity and presentation, such as thresholds and symptoms, are considered challenging to predict ex vivo. AIMS: To identify changes of peripheral immune signatures during oral food challenges (OFC) that correlate with the clinical outcome in patients with peanut allergy (PA). METHODS: Children with a positive (OFC+ , n = 16) or a negative (OFC- , n = 10) OFC-outcome were included (controls, n = 7). Single-cell mass cytometry/unsupervised analysis allowed unbiased immunophenotyping during OFC. RESULTS: Peripheral immune profiles correlated with OFC outcome. OFC+ -profiles revealed mainly decreased Th2 cells, memory Treg and activated NK cells, which had an increased homing marker expression signifying immune cell migration into effector tissues along with symptom onset. OFC- -profiles had also signs of ongoing inflammation, but with a signature of a controlled response, lacking homing marker expression and featuring a concomitant increase of Th2-shifted CD4+ T cells and Treg cells. Low versus high threshold reactivity-groups had differential frequencies of intermediate monocytes and myeloid dendritic cells at baseline. Low threshold was associated with increased CD8+ T cells and reduced memory cells (central memory [CM] CD4+ [Th2] T cells, CM CD8+ T cells, Treg). Immune signatures also discriminated patients with preferential skin versus gastrointestinal symptoms, whereby skin signs correlated with increased expression of CCR4, a molecule enabling skin trafficking, on various immune cell types. CONCLUSION: We showed that peripheral immune signatures reflected dynamics of clinical outcome during OFC with peanut. Those immune alterations hold promise as a basis for predictive OFC biomarker discovery to monitor disease outcome and therapy of PA.

Detection of Circulating Peanut Components in Serum after Ingestion.

INTRODUCTION: Detection of specific food allergens brought into circulation following ingestion is complicated by the minute amounts present in serum. We therefore aimed to assess the utility of selective removal of IgE against peanut components using ImmunoCAP combined with the basophil histamine release (HR) assay (BHRA) to identify the absorbed allergen components after ingestion of peanut. METHODS: Serum from six healthy individuals was drawn before and 1 h after ingestion of 100 g peanut. Serum from two peanut allergic patients was depleted for IgE against single allergen components by incubation with ImmunoCAPs coupled with either Ara h 1; 2; 3 or 6 before donor basophils were passively sensitized with the serum preparations. The sensitized cells were challenged with serum obtained from the six individuals before and after ingestion of peanut, and HR was measured after serum provocation. RESULTS: Ara h 2 and Ara h 6 were detected in serum 1 h after ingestion in 6/6 individuals by negative selection. Depletion of specific IgE against Ara h 2 or Ara h 6 almost completely abolished the response to serum provocation, indicating a sequence homology between the two allergen components in serum. Ara h 1 was demonstrated in 5/6 sera and Ara h 3 in 1/6 sera. CONCLUSION: This study is a proof of concept showing that passive sensitization of basophils with sera depleted of component-specific IgE can be used to identify food allergen components present in serum after ingestion.

PACAP38- and VIP-induced cluster headache attacks are not associated with changes of plasma CGRP or markers of mast cell activation.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) and vasoactive intestinal polypeptide can provoke cluster headache attacks in up to half of cluster headache patients in their active phase. At present, it is unknown whether provoked attacks are mediated via calcitonin gene-related peptide or mast cell activation. METHODS: All enrolled patients with cluster headache were randomly allocated to receive a continuous infusion of either PACAP38 (10 pmol/kg/min) or vasoactive intestinal polypeptide (8 pmol/kg/min) over 20 min. We collected clinical data and measured plasma levels of calcitonin gene-related peptide and markers of mast cell activation (tryptase and histamine) at fixed time points: at baseline (T0), at the end of the infusion (T20), 10 min after the infusion (T30), and 70 min after the infusion (T90). RESULTS: Blood was collected from episodic cluster headache patients in active phase (n = 14), episodic cluster headache patients in remission (n = 15), and chronic cluster headache patients (n = 15). At baseline, plasma levels of calcitonin gene-related peptide, tryptase, and histamine were not different among the three study groups. Plasma levels of calcitonin gene-related peptide (p = 0.7074), tryptase (p = 0.6673), or histamine (p = 0.4792) remained unchanged during provoked attacks compared to attack-free patients. CONCLUSION: Cluster headache attacks provoked by either PACAP38 or vasoactive intestinal polypeptide were not accompanied by alterations of plasma calcitonin gene-related peptide, tryptase or histamine. The provoked attacks may not be mediated by calcitonin gene-related peptide or mast cell activation.Trial Registration: The study is registered at ClinicalTrials.gov (NCT03814226).

What Basophil Testing Tells Us About CSU Patients - Results of the CORSA Study.

Basophil testing is the most effective single approach for diagnosing type-IIb autoimmune chronic spontaneous urticaria (TIIbaiCSU). A positive basophil test has been linked to long disease duration, higher disease activity, a poor response to antihistamines and omalizumab, and a better response to cyclosporine and fenebrutinib. As of now it is unclear what other features are connected to a positive basophil test in chronic spontaneous urticaria (CSU). We aimed to identify features of basophil test-positive CSU patients. We performed a cross-sectional study of 85 CSU patients. Basophil testing was done with the basophil activation test (BAT) and the basophil histamine release assay (BHRA). Data were analysed using SPSS: Student's t-test, Chi-square test, Odds Ratio, Spearman's correlation test. Of 85 CSU patients, 44% and 28% tested positive with the BAT and BHRA, respectively. These patients showed higher disease activity and impact, lower levels of disease control and total serum IgE, as well as higher rates of having a positive autologous serum skin test (ASST), angioedema, nocturnal symptoms, symptoms for >5 days/week, and thyroid autoantibodies. The ASST, by itself, was not a good predictor of basophil test results, but it predicted a positive basophil test in up to 100% of cases when combined with angioedema, thyroid autoantibodies or low IgE. In conclusion, a positive basophil test is linked to known features of TIIbaiCSU and novel characteristics including nocturnal symptoms. Further studies on basophil test-positive and -negative CSU patients can help to better understand CSU endotypes and to develop better management approaches.

Autoimmune Diseases Are Linked to Type IIb Autoimmune Chronic Spontaneous Urticaria.

PURPOSE: Patients with chronic spontaneous urticaria (CSU) have an increased risk for comorbid autoimmune diseases. In this retrospective multicenter study of CSU patients, we evaluated clinical and laboratory features of CSU associated with a higher risk of comorbid autoimmune diseases. METHODS: We analyzed records of CSU patients (n = 1,199) for a history or presence of autoimmune diseases. Patients were diagnosed with type IIb autoimmune CSU (aiCSU) if all 3 tests were positive: autologous serum skin test (ASST), basophil histamine release assay (BHRA) and/or basophil activation test (BAT), and IgG autoantibodies against FcεRIα/IgE detected by immunoassay. RESULTS: Twenty-eight percent of CSU patients had at least 1 autoimmune disease. The most prevalent autoimmune diseases were Hashimoto's thyroiditis (HT) (≥ 21%) and vitiligo (2%). Two percent of CSU patients had ≥ 2 autoimmune diseases, most frequently HT plus vitiligo. Comorbid autoimmune diseases, in patients with CSU, were associated with female sex, a family history of autoimmune diseases, and higher rates of hypothyroidism and hyperthyroidism (P < 0.001). Presence of autoimmune diseases was linked to aiCSU (P = 0.02). The risks of having autoimmune diseases were 1.7, 2.9 and 3.3 times higher for CSU patients with a positive ASST, BHRA and BAT, respectively. In CSU patients, markers for autoimmune diseases, antinuclear antibodies and/or IgG anti-thyroid antibodies were associated with non-response to omalizumab treatment (P = 0.013). CONCLUSIONS: In CSU, autoimmune diseases are common and linked to type IIb autoimmune CSU. Our results suggest that physicians assess and monitor all adult patients with CSU for signs and symptoms of common autoimmune diseases, especially HT and vitiligo.

Investigation of transfollicular caffeine penetration using microdialysis on ex vivo porcine ear skin.

The aim of this study was to develop an ex vivo method that allows to quantify the transfollicular penetration of topically applied substances by combining microdialysis and selective follicular closure with varnish. An experimental setup with three skin areas on ex vivo intact porcine ear skin was designed (varnish on hair follicle, varnish next to hair follicle, no varnish). On each area, 10 µl/cm2 caffeine-hydroxyethyl-cellulose-gel was applied. Samples were collected for 22 h by microdialysis. After sampling, the skin layers were separated, homogenized and caffeine was quantified by high pressure liquid chromatography (HPLC) in all samples. Potential impact of the varnish placed next to the follicle by tension on the follicle during the drying process was monitored by a microscopic setup and could be excluded. The microdialysis and homogenization study showed a significantly reduced penetration of caffeine when the hair follicles were closed. In areas with open hair follicles caffeine was detected already in the first ten minutes after application. The reported novel combination of two methods is suitable to investigate ex vivo transfollicular penetration. Possible impact of the closure material in the control area can be ruled out by adjusting the design of the control area in future studies.

The role of eosinophils in chronic spontaneous urticaria.

Chronic spontaneous urticaria (CSU) is considered to be primarily a mast cell-driven disease. However, recent evidence suggests that eosinophils may also have an axial role in symptomology. Histologic studies have demonstrated the presence of both eosinophils and eosinophil granules, indicative of activation, in CSU lesions. Although many allergic and inflammatory conditions are associated with a peripheral blood eosinophilia, the converse appears to be the case in CSU, with a peripheral blood eosinopenia being observed in many patients. Possible mechanisms include the depletion of blood eosinophils by recruitment into the skin during active disease and immunologic destruction in the blood. We also address in some detail the interactions between eosinophils and mast cells, particularly the cytokine cross-talk of these cells and mediator release possibly leading to clinical symptoms. Also, activation by eosinophil proteins of the coagulation pathway leads to the generation of thrombin and increased mast cell degranulation. Finally, treatments aimed at reducing eosinophil accumulation and activation, such as the anti-IL-5 antibodies mepolizumab, reslizumab, and benralizumab, have been reported to reduce CSU symptoms. Clearly, a new picture of an important role of eosinophils in the pathogenesis of CSU is emerging.

IgE-Mediated Peanut Allergy: Current and Novel Predictive Biomarkers for Clinical Phenotypes Using Multi-Omics Approaches.

Food allergy is a collective term for several immune-mediated responses to food. IgE-mediated food allergy is the best-known subtype. The patients present with a marked diversity of clinical profiles including symptomatic manifestations, threshold reactivity and reaction kinetics. In-vitro predictors of these clinical phenotypes are evasive and considered as knowledge gaps in food allergy diagnosis and risk management. Peanut allergy is a relevant disease model where pioneer discoveries were made in diagnosis, immunotherapy and prevention. This review provides an overview on the immune basis for phenotype variations in peanut-allergic individuals, in the light of future patient stratification along emerging omic-areas. Beyond specific IgE-signatures and basophil reactivity profiles with established correlation to clinical outcome, allergenomics, mass spectrometric resolution of peripheral allergen tracing, might be a fundamental approach to understand disease pathophysiology underlying biomarker discovery. Deep immune phenotyping is thought to reveal differential cell responses but also, gene expression and gene methylation profiles (eg, peanut severity genes) are promising areas for biomarker research. Finally, the study of microbiome-host interactions with a focus on the immune system modulation might hold the key to understand tissue-specific responses and symptoms. The immune mechanism underlying acute food-allergic events remains elusive until today. Deciphering this immunological response shall enable to identify novel biomarker for stratification of patients into reaction endotypes. The availability of powerful multi-omics technologies, together with integrated data analysis, network-based approaches and unbiased machine learning holds out the prospect of providing clinically useful biomarkers or biomarker signatures being predictive for reaction phenotypes.

Eosinopenia, in Chronic Spontaneous Urticaria, Is Associated with High Disease Activity, Autoimmunity, and Poor Response to Treatment.

BACKGROUND: Chronic spontaneous urticaria (CSU) is characterized by the degranulation of skin mast cells and the influx of basophils and eosinophils to affected skin sites. Blood basopenia has been linked to severe antihistamine-resistant CSU and type IIb autoimmunity, whereas the role of eosinophils in CSU is largely unknown. OBJECTIVE: To analyze data from 1613 patients with CSU from 2 centers to study the prevalence, role, and relevance of eosinopenia in CSU. METHODS: Peripheral blood eosinophil and basophil counts were measured by automated hematology analyzers. Patient files were screened for clinical characteristics, results of laboratory tests, the autologous serum skin test, the serum-induced basophil histamine release assay, and response to second-generation H1-antihistamines and omalizumab. RESULTS: Ten percent of patients with CSU had eosinopenia. Eosinopenia was associated with the female sex, high disease activity, autologous serum skin test and basophil histamine release assay positivity, low total IgE, and high levels of C-reactive protein and IgG-antithyroperoxidase (P ≤ .007). Nonresponders to second-generation H1-antihistamines or omalizumab had significantly lower eosinophils as compared with responders (P < .05 and P < .01, respectively). Blood eosinophil counts correlated with basophil counts (r = 0.396; P < .001), and 81% of patients with CSU with undetectable eosinophils had basopenia. The combination of eosinopenia and basopenia is a better predictor of nonresponse to second-generation H1-antihistamines than eosinopenia alone (odds ratio of 9.5 vs 4.8). CONCLUSIONS: Eosinopenia in patients with CSU is associated with type IIb autoimmunity, high disease activity, and poor response to treatment. Eosinophils should be explored as biomarkers and investigated for their contribution to the pathogenesis of CSU.

The Skin Reservoir Model: A Tool for Evaluating Microdialysis Sampling of Large Biomarkers from Human Skin.

Microdialysis is a well-established technique for sampling of small molecules from the human skin, but larger molecules are more difficult to recover. Consequently, sampling feasibility must be evaluated before microdialysis is used in vivo. This report presents a tool for estimating the recovery of large biomarkers from human skin by microdialysis, using previously frozen human skin specimens as reservoirs for biomarker reference solutions. Recovery of the following 17 biomarkers was assessed: CCL27/CTACK, CXCL1/GROα, CXCL7/NAP-2, CXCL10/IP-10, EGF, GM-CSF, IFN-γ, IL-1α, IL-6, IL-8, IL-17, IL-22, IL-23, MIF, TNF-α, TSLP and VEGF. The relative skin recoveries of 13/17 biomarkers were successfully determined in the range 4.0-18.4%. Sampling in the skin reservoir model was not associated with probe leakage, as fluid recovery was stable, at between 80% and 110%. Furthermore, the skin reservoir model enabled studies and optimization of different parameters known to affect biomarker recovery, including flow rate and perfusate composition.

Biomarkers and clinical characteristics of autoimmune chronic spontaneous urticaria: Results of the PURIST Study.

BACKGROUND: Autoimmune chronic spontaneous urticaria (aiCSU) is an important subtype of chronic spontaneous urticaria (CSU) in which functional IgG autoantibodies to IgE or its high-affinity receptor (FcεRI) induces mast cell degranulation and subsequent symptom development. However, it has not been tightly characterized. This study aimed to better define the clinical and immunological features and to explore potential biomarkers of aiCSU. METHODS: This was a multinational, multicenter study of 182 CSU patients. The clinical features studied included: urticaria activity and impact (UAS7 and quality of life); autologous serum skin test (ASST); IgG anti-FcεRI and IgG anti-IgE; IgG-anti-thyroperoxidase (IgG anti-TPO); total serum IgE; and basophil reactivity (BASO) using the basophil activation test (BAT) and basophil histamine release assay (BHRA). RESULTS: Of the 182 patients, 107 (59%) were ASST+, 46 (25%) were BASO+, and 105 (58%) were IgG anti-FcεRI+/IgE+. Fifteen patients (8%) fulfilled all three criteria of aiCSU. aiCSU patients appeared more severe (UAS7 21 vs 9 P < 0.016) but showed no other clinical or demographic differences from non-aiCSU patients. aiCSU patients also had markedly lower total IgE levels (P < 0.0001) and higher IgG anti-TPO levels (P < 0.001). Of biomarkers, positive BAT and BHRA tests were 69% and 88% predictive of aiCSU, respectively. CONCLUSIONS: aiCSU is a relatively small but immunologically distinct subtype of CSU that cannot be identified by routine clinical parameters. Inclusion of BHRA or BAT in the diagnostic workup of CSU patients may aid identification of aiCSU patients, who may have a different prognosis and benefit from specific management.

Skin microdialysis: methods, applications and future opportunities-an EAACI position paper.

Skin microdialysis (SMD) is a versatile sampling technique that can be used to recover soluble endogenous and exogenous molecules from the extracellular compartment of human skin. Due to its minimally invasive character, SMD can be applied in both clinical and preclinical settings. Despite being available since the 1990s, the technique has still not reached its full potential use as a tool to explore pathophysiological mechanisms of allergic and inflammatory reactions in the skin. Therefore, an EAACI Task Force on SMD was formed to disseminate knowledge about the technique and its many applications. This position paper from the task force provides an overview of the current use of SMD in the investigation of the pathogenesis of chronic inflammatory skin diseases, such as atopic dermatitis, chronic urticaria, psoriasis, and in studies of cutaneous events during type 1 hypersensitivity reactions. Furthermore, this paper covers drug hypersensitivity, UVB-induced- and neurogenic inflammation, and drug penetration investigated by SMD. The aim of this paper is to encourage the use of SMD and to make the technique easily accessible by providing an overview of methodology and applications, supported by standardized operating procedures for SMD in vivo and ex vivo.

Is a positive intracutaneous test induced by penicillin mediated by histamine? A cutaneous microdialysis study in penicillin-allergic patients.

BACKGROUND: Diagnostic workup of penicillin allergy comprises skin testing with penicillins, and patients are deemed allergic if skin test is positive. However, the literature suggests that skin test-positive patients may be challenge-negative, indicating that the skin test may be falsely positive. OBJECTIVE: To investigate real-time histamine release from a positive intracutaneous test induced by penicillin in patients with positive and negative challenges to penicillin. METHODS: Skin microdialysis was performed in 21 penicillin-allergic patients with positive skin test, 13 non-allergic volunteers serving as negative controls, and 7 grass pollen-allergic patients serving as positive controls. Histamine was measured by microdialysis after skin test with penicillin/grass/NaCl. Penicillin challenge was subsequently performed in 12 of the patients. RESULTS: Only 10/21 patients (47.6%) were skin test positive at microdialysis. During microdialysis 13 single intracutaneous tests were positive and histamine was detected in 4/13 occurring in four challenge positive patients. Thirteen/21 patients (61.9%) were deemed allergic to penicillin; eight had positive skin test. Two patients with positive skin test were challenge negative. In grass pollen allergic patients, 7/7 had a positive intracutaneous test to grass and all released histamine in the wheals. All 13 negative controls had negative intracutaneous test to penicillin and no histamine release. CONCLUSION: Histamine was only detected in the minority of positive intracutaneous tests with penicillin in penicillin-allergic patients. Other mediators may be involved.

Positive Skin Test or Specific IgE to Penicillin Does Not Reliably Predict Penicillin Allergy.

INTRODUCTION: According to guidelines, patients are diagnosed with penicillin allergy if skin test (ST) result or specific IgE (s-IgE) to penicillin is positive. However, the true sensitivity and specificity of these tests are presently not known. OBJECTIVE: To investigate the clinical relevance of a positive ST result and positive s-IgE and to study the reproducibility of ST and s-IgE. METHODS: A sample of convenience of 25 patients with positive penicillin ST results, antipenicillin s-IgE results, or both was challenged with their culprit penicillin. Further 19 patients were not challenged, but deemed allergic on the basis of a recent anaphylactic reaction or delayed reactions to skin testing. Another sample of convenience of 18 patients, 17 overlapping with the 25 challenged, with initial skin testing and s-IgE (median, 25; range, 3-121), months earlier (T-1), was repeat skin tested and had s-IgE measured (T0), and then skin tested and had s-IgE measured 4 weeks later (T1). RESULTS: Only 9 (36%) of 25 were challenge positive. There was an increased probability of being penicillin allergic if both ST result and s-IgE were positive at T0. Positive ST result or positive s-IgE alone did not predict penicillin allergy. Among the 18 patients repeatedly tested, 46.2% (12 of 25) of positive ST results at T-1 were reproducibly positive at T0. For s-IgE, 54.2% (14 of 24) positive measurements were still positive at T0 and 7 converted to positive at T1. CONCLUSIONS: The best predictor for a clinically significant (IgE-mediated) penicillin allergy is a combination of a positive case history with simultaneous positive ST result and s-IgE or a positive challenge result.

Ethyl cellulose nanocarriers and nanocrystals differentially deliver dexamethasone into intact, tape-stripped or sodium lauryl sulfate-exposed ex vivo human skin - assessment by intradermal microdialysis and extraction from the different skin layers.

Understanding penetration not only in intact, but also in lesional skin with impaired skin barrier function is important, in order to explore the surplus value of nanoparticle-based drug delivery for anti-inflammatory dermatotherapy. Herein, short-term ex vivo cultures of (i) intact human skin, (ii) skin pretreated with tape-strippings and (iii) skin pre-exposed to sodium lauryl sulfate (SLS) were used to assess the penetration of dexamethasone (Dex). Intradermal microdialysis was utilized for up to 24h after drug application as commercial cream, nanocrystals or ethyl cellulose nanocarriers applied at the therapeutic concentration of 0.05%, respectively. In addition, Dex was assessed in culture media and extracts from stratum corneum, epidermis and dermis after 24h, and the results were compared to those in heat-separated split skin from studies in Franz diffusion cells. Providing fast drug release, nanocrystals significantly accelerated the penetration of Dex. In contrast to the application of cream and ethyl cellulose nanocarriers, Dex was already detectable in eluates after 6h when applying nanocrystals on intact skin. Disruption of the skin barrier further accelerated and enhanced the penetration. Encapsulation in ethyl cellulose nanocarriers delayed Dex penetration. Interestingly, for all formulations highly increased concentrations in the dialysate were observed in tape-stripped skin, whereas the extent of enhancement was less in SLS-exposed skin. The results were confirmed in tissue extracts and were in line with the predictions made by in vitro release studies and ex vivo Franz diffusion cell experiments. The use of 45kDa probes further enabled the collection of inflammatory cytokines. However, the estimation of glucocorticoid efficacy by Interleukin (IL)-6 and IL-8 analysis was limited due to the trauma induced by the probe insertion. Ex vivo intradermal microdialysis combined with culture media analysis provides an effective, skin-sparing method for preclinical assessment of novel drug delivery systems at therapeutic doses in models of diseased skin.

Positive serum specific IgE has a short half-life in patients with penicillin allergy and reversal does not always indicate tolerance.

BACKGROUND: The positive and negative predictive values of specific IgE to penicillins are not well established for penicillin hypersensitivity. One reason may be that serum IgE levels to penicillin diminish over time. The objective in this study was to investigate variations in serum half-life (T½) for specific IgE to penicillins (s-IgE) and to evaluate the outcome of penicillin challenges in patients with previous but not present specific IgE to penicillins. METHODS: Two subgroups were investigated. All included patients had a history of penicillin allergy with reported symptoms such as urticaria/angioedema or unclassified cutaneous rash. T½ of specific IgE to penicillins was calculated based on sera from 29 patients with repeated measurements of s-IgE. Twenty-two patients with a previous positive s-IgE was followed and challenged with penicillin when IgE had become negative. RESULTS: The T½ for s-IgE varied between the 26 patients with decreasing s-IgE from 1.6 months to 76.4 months and 52% had a T½ of less than a year. The three patients with stable and increasing IgE-values showed T½ approaching infinity A total of 29 challenges with ß-lactams were performed. Four different patterns were seen when evaluating the clinical reaction to challenge (positive/negative) and post-challenge boost of s-IgE (yes/no). Eight (36.4%) had negative challenge and negative post-challenge s-IgE, eight (36.4%) negative challenge, but positive post-challenge s-IgE levels. 3 (13.6%) had positive challenge and positive post-challenge s-IgE whereas 3 (13.6%) were challenge positive, but had negative post-challenge s-IgE. CONCLUSION: Specific IgE to penicillins declines over time stressing the importance of a close time relation between diagnostic work-up and clinical reaction. Reversal of previously positive s-IgE may still be associated with positive penicillin challenges and/or re-boostering of s-IgE to positivity.