Endotyping of IgE-Mediated Polyethylene Glycol and/or Polysorbate 80 Allergy.

BACKGROUND: Polyethylene glycol (PEG) and polysorbate 80 (PS80) allergy preclude from SARS-CoV-2 vaccination. The mechanism(s) governing cross-reactivity and PEG molecular weight dependence remain unclear. OBJECTIVES: To evaluate PEGylated lipid nanoparticle (LNP) vaccine (BNT162b2) tolerance and explore the mechanism of reactivity in PEG and/or PS80 allergic patients. METHODS: PEG/PS80 dual- (n = 3), PEG mono- (n = 7), and PS80 mono-allergic patients (n = 2) were included. Tolerability of graded vaccine challenges was assessed. Basophil activation testing on whole blood (wb-BAT) or passively sensitized donor basophils (allo-BAT) was performed using PEG, PS80, BNT162b2, and PEGylated lipids (ALC-0159). Serum PEG-specific IgE was measured in patients (n = 10) and controls (n = 15). RESULTS: Graded BNT162b2 challenge in dual- and PEG mono-allergic patients (n = 3/group) was well tolerated and induced anti-spike IgG seroconversion. PS80 mono-allergic patients (n = 2/2) tolerated single-dose BNT162b2 vaccination. Wb-BAT reactivity to PEG-containing antigens was observed in dual- (n = 3/3) and PEG mono- (n = 2/3), but absent in PS80 mono-allergic patients (n = 0/2). BNT162b2 elicited the highest in vitro reactivity. BNT162b2 reactivity was IgE mediated, complement independent, and inhibited in allo-BAT by preincubation with short PEG motifs, or detergent-induced LNP degradation. PEG-specific IgE was only detectable in dual-allergic (n = 3/3) and PEG mono-allergic (n = 1/6) serum. CONCLUSION: PEG and PS80 cross-reactivity is determined by IgE recognizing short PEG motifs, whereas PS80 mono-allergy is PEG-independent. PS80 skin test positivity in PEG allergics was associated with a severe and persistent phenotype, higher serum PEG-specific IgE levels, and enhanced BAT reactivity. Spherical PEG exposure via LNP enhances BAT sensitivity through increased avidity. All PEG and/or PS80 excipient allergic patients can safely receive SARS-CoV-2 vaccines.

Alpha-amylase as the culprit in an occupational mealworm allergy case.

Background: Occupational allergy has been described in employees working in contact with mealworms in pet stores, live fish bait or infested stored grains and recently, in mealworm farming for animal feed and human consumption. Mealworm allergens linked to occupational allergy are troponin C, cockroach-like allergen, tropomyosin, arginine kinase, early-staged encapsulation inducing- and larval cuticle proteins. Objective: We report a case of occupational mealworm allergy and studied the culprit component. Methods: Diagnosis was done by skin prick, specific IgE, basophil activation and lung function testing. Allergen purification was performed by anion-exchange chromatography and immunoblotting with patient IgE. Allergens were identified by in-gel trypsin digest and tandem mass spectrometry. Allergenicity and specificity further confirmed by IgE inhibition and passive basophil activation experiments. Results: We describe a new case of occupational mealworm allergy in a laboratory worker, with sensitization to different developmental stages and derivates of the mealworm. In basophil activation tests, the majority of patient's basophils (69%-91%) degranulated upon stimulation with the lowest concentration of mealworm extracts (0.16 µg/ml). Despite strong sensitization to mites, the patient did not show cross-reactivity to other insects. We were able to identify alpha-amylase as the main allergen and through inhibition experiments, we demonstrated that low amounts (0.1 µg/ml) of this allergen could strongly inhibit mealworm specific IgE by 79.1%. Moreover, passive BAT experiments demonstrated the IgE-alpha-amylase interaction to be functional, inducing up to 25.5% degranulation in healthy donor basophils. Conclusion: Alpha-amylase can be identified as the responsible allergen in this specific case of occupational mealworm allergy.

Guided Gradual Egg-Tolerance Induction in Hen's Egg Allergic Children Tolerating Baked Egg: A Prospective Randomized Trial.

Background: Over the last few years, studies have shown that the majority of egg allergic children tolerate baked egg (e.g., cake), and that consuming baked egg accelerates the resolution of egg allergy. However, few prospective studies demonstrate the step-wise reintroduction of egg at home after developing baked egg tolerance. Although this could have a positive impact on the children's quality of life and nutrition. Additionally, research supporting the theoretical concept that heating in the presence or absence of wheat causes reduced allergenicity of egg proteins is limited. Objective: To investigate the clinically most favorable duration of gradual egg-tolerance induction in baked egg tolerant children at home, with regard to complete raw egg tolerance. Methods: Baked egg tolerant children above 12 months of age were randomly assigned to a short- or long arm protocol. In the short arm, egg-tolerance induction was studied over 18 months compared to 30 months in the long arm. Children were guided through this protocol involving the step-wise introduction of increasingly allergenic forms of egg starting with baked egg offered as cake, followed by hard-boiled egg, omelet/waffle/pancake, soft-boiled egg, and finally raw egg. We hereby designed this protocol based on the influence of thermal processing in the presence or absence of wheat on egg proteins, as investigated by ELISA, SDS-PAGE, and immunoblotting. At inclusion, children either passed an in-hospital cake challenge or had ovomucoid sIgE ≤1.2 kUA/L, which was considered safe for introduction at home. Results: Gel electrophoresis revealed that the ovalbumin band became weaker with heating, while the ovomucoid band remained stable. In accordance, the IgE-binding to ovalbumin decreased with extensive heating, as opposed to ovomucoid. However, heating in the presence of wheat led to a decreased IgE reactivity to ovomucoid. Of the 78 children in the intention-to-treat group, 39 were randomized to each arm. Fifty-eight children reached the raw egg tolerance endpoint, of which 80% were in the short arm and 69% in the long arm. Within the short arm, the median time to raw egg tolerance was 24 months (95% CI, 21-27 months) compared to 30 months (95% CI, 28-32 months) in the long arm (p = 0.005). No grade IV reactions or cases of eosinophilic esophagitis were observed. The short arm was considered to be non-inferior to the long arm. Conclusion: Our gradual short arm protocol appears to be safe and allows clinicians to guide baked egg tolerant children toward raw egg tolerance at home. The allergenicity of the egg proteins was affected by heating temperature and duration, as well as the presence of wheat.

Reciprocal changes of Foxp3 expression in blood and intestinal mucosa in IBD patients responding to infliximab.

BACKGROUND: Immune therapies may act in inflammatory bowel diseases (IBD) by modulating regulatory T cells (Tregs). Therefore, we investigated the effect of infliximab (IFX) therapy on Forkhead box protein3 (Foxp3) T cells in blood and intestinal mucosa from Crohn's disease (CD) and ulcerative colitis (UC). METHODS: Forty patients with active IBD (23 CD / 17 UC) were treated with IFX 5 mg/kg intravenously at weeks 0, 2, 6, and each 8 weeks thereafter. Blood samples were obtained before every infusion and T-lymphocyte subsets were characterized by flow cytometry. Foxp3 expression in intestinal biopsies from 43 patients with active IBD (19 CD / 24 UC) before and after IFX infusion and from 6 controls were assessed by quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. Plasma C-reactive protein (CRP), clinical response, and endoscopic healing data were collected in parallel. RESULTS: IFX therapy resulted in a significant and sustained relative increase of CD4(+)CD25(+)Foxp3(+) Treg and of CD4(+)CD25(-)Foxp3(+) Treg cells in peripheral blood (both P < 0.0001 compared to baseline), particularly in responders (both P < 0.05 compared to nonresponders). The change in CRP over time inversely correlated with the increase of CD25(+)Foxp3(+) cells (P < 0.001, r = -0.39) and durable clinical response was associated with a sustained increase of circulating Foxp3(+) cells. Surprisingly, IFX therapy downregulated mucosal mRNA and protein expression of Foxp3 in UC and CD responders (both P < 0.001) but not in nonresponders. CONCLUSIONS: IFX therapy has opposite effects in Foxp3(+) Treg cells in blood and gut mucosa, which suggests a redistribution of this important T-cell subset.

Detection of basophil-activating IgG autoantibodies in chronic idiopathic urticaria by induction of CD 63.

BACKGROUND: Approximately 40% to 50% of patients with chronic idiopathic urticaria (CIU) have functional IgG autoantibodies against FcepsilonRIalpha or IgE, which induce histamine release from basophils and cutaneous mast cells. A positive autologous serum skin test response is believed to reflect the presence of these autoantibodies. OBJECTIVE: We sought to further define the functional properties of and develop a sensitive functional assay for detection of autoantibodies in patients with CIU. METHODS: Sera from patients with CIU (n=61) and sera from healthy control subjects (n=23) were incubated with donor basophils. Activation of basophils was determined on the basis of CD 63 surface expression, as analyzed on a FACScan flow cytometer. RESULTS: A positive basophil activation test result was found in 51% of patients with CIU, and basophil-activating properties were present in the IgG fractions of sera. When both the in vitro test and the autologous serum skin test were considered, basophil/mast cell-activating autoantibodies were present in 62% of the patients. Patients with a positive basophil activation test result had a significantly higher prevalence of other autoantibodies, had more severe urticaria, and were more likely to have angioedema. CONCLUSION: The results demonstrate the presence of basophil-activating autoantibodies in about 50% of patients with CIU. The data support the autoimmune cause of the disease and provide a simple test for detection of these autoantibodies.

Blockade of CTLA-4 enhances allergic sensitization and eosinophilic airway inflammation in genetically predisposed mice.

CTLA-4 (CD152) expression is restricted to subsets of activated T lymphocytes and shares homology with CD28. CTLA-4 and CD28 molecules both bind to B7 molecules on antigen-presenting cells. Whereas CD28-B7 interaction enhances T cell activation, cytokine production and survival, CTLA-4 signaling down-regulates T cell responses. Here, we studied the involvement of CTLA-4 triggering in the pathogenesis of allergen-induced airway inflammation in mice. Anti-CTLA-4 mAb were injected during i.p. sensitization with ovalbumin (OVA). This treatment favored OVA-specific IgE production and augmented blood eosinophilia in BALB/c mice. In BALB/c mice, enhanced Th2 sensitization after anti-CTLA-4 mAb injections resulted in more severe airway inflammation, and increased airway hyperresponsiveness to metacholine, bronchial eosinophilia and IL-4 and IL-5 levels in broncho-alveolar lavage (BAL) fluid following repeated allergen inhalations. Importantly, aggravation of airway inflammation and enhancement of Th2 responses were accompanied by a significant reduction of pulmonary TGF-beta levels at protein level in BAL fluid as well as on mRNA level in inflamed lung tissue. In contrast to BALB/c mice, blockade of CTLA-4 did not alter IgE production nor the phenotype of airway inflammation or TGF-beta production in C57BL/6 mice. Our data suggest that CTLA-4 triggering represents an important regulatory mechanism for Th2 sensitization in genetically predisposed mice by modulating TGF-beta production.